Feeder layer and nutritional requirements for the establishment and cloning of human malignant lymphoma cell lines. Cell lines were successfully established in continuous suspension culture from 10 patients with a histopathological diagnosis of diffuse histiocytic lymphoma (SU-DHL-1 to SU-DHL-10), two with North American Burkitt's lymphoma (SU-AmB-1 and SU-AmB-2), and one with acute lymphoblastic leukemia (SU-ALL-1). By screening a variety of parameters, including media, sera, effusion fluids, feeder layers, and chemical supplements, the nutritive growth requirements of lymphoma cells obtained from malignant effusions and lymph node biopsies were determined for each tumor. Most of these cell lines initially required human skin fibroblast or epithelial cell feeder layers from which they could be weaned after one to six weeks in culture and maintained in Roswell Park Memorial Institute Tissue Culture Medium 1640 containing 20% fetal calf serum and 10% pooled human serum. Several of these cell lines were successfully cloned on 0.5% Noble agar substrates. In the presence of human serum and selected feeder monolayers, cloning efficiencies increased significantly from less than 1% to 15 to 25%. In addition, the cloning efficiencies of certain cell lines showed a concentration-dependent increase with specific chemical supplements including L-cysteine and dithiothreitol. Placental colony-stimulating factor, nerve growth factor, epithelial growth factor, and fibroblastic growth factor were ineffective in augmenting the cloning efficiencies of the human lymphoma cell lines. After a single passage on agar, cells subpassaged from visible colonies showed markedly increased cloning efficiencies to levels as high as 50%. Such cloning efficiencies, coupled with the use of replica plating, make this technique applicable to genetic and quantitative radiobiological, immunological, and chemotherapeutic studies. Although these methods have thus far been used only with lymphoreticular tumors, they may also be applicable to the cell culture of other human neoplasms and normal tissues.


Clinical evaluation of peptichemio.

Clinical evaluation of peptichemio. Peptichemio is a peptide complex of m-L-phenylalanine mustard. A clinical evaluation of this agent was conducted in 116 patients, of whom 104 were evaluable for both toxicity and response. The majority of patients had solid tumors. The drug was administered iv daily for 3 days at doses of 20-75 mg/m2/day, with courses repeated at 3-4-week intervals. The optimal dose schedule appears to be 45 mg/m2/day for 3 days. The major side effects were cumulative myelotoxicity, phlebitis, and mild nausea and vomiting. No other major organ toxicity was observed. The partial remission rate was 7%. Most patients had received an alkylating agent as part of their previous therapy. There were seven partial responses and four less than partial responses achieved in patients with melanoma, lymphoma, and gastrointestinal, genitourinary, breast, and head and neck carcinomas. Responses lasted 4-36 weeks.


Phase II study of Leo 1031 (prednimustine) in advanced ovarian carcinoma. Thirty-six patients with advanced ovarian carcinoma, 21 who had not responded to conventional surgery and irradiation therapy and 15 who had not responded to conventional surgery, irradiation therapy, and chemotherapy, were treated in a phase II study with Leo 1031. Objective responses were obtained in ten (28%) of the 36 patients (two complete and eight partial responses). An additional 11 (32%) patients had stable disease. The two complete responders were in remission for 4 months. During remission, all of the patients had an improved quality of life. Myelosuppression of varying severity occurred in 11 patients; three of these patients died from their complications. Two patients developed reversible confusion and one patient became psychotic. Eight patients became euphoric during treatment. Leo 1031 might be worthwhile as a palliative chemotherapy for patients with advanced ovarian carcinoma, but the risk of euphoria must always be considered since it can lead to voluntary overdosage.


Phase I trial of a new form of an oral administration of VP-16-213. A new oral form of VP-16-213 consisting of a hydrophilic soft gelatin capsule containing a VP-16-213 solution was tested for toxicity in 26 patients. The drug was administered at four different dose levels, 350, 500, 650, and 750 mg/m2, in 5-day courses every 21-28 days. A dose-dependent effect was observed with regard to hematologic toxicity and alopecia. It is concluded that the drinking ampules of VP-16-213 can be replaced with the new oral capsules with a recommended initial dose of 100-130 mg/m2 given in 5-day courses every 21-28 days.


Results of a phase II study of maytansine in patients with breast carcinoma and melanoma. During the phase I study of maytansine at our institution, some activity was observed against breast carcinoma and melanoma. A phase II study was thus initiated to more thoroughly investigate the activity of this drug against these two tumors. In 33 evaluable patients with melanoma, no complete or partial responses were observed. Twenty-one evaluable patients with breast cancer were entered and only one response (partial) was seen. The toxicity was similar to that observed in the phase I study and consisted mainly of diarrhea, paresthesias, phlebitis, and flu-like symptoms. Myelosuppression was infrequent and was short-lived when it occurred.


Phase II study of single-agent therapy with megestrol acetate, VP-16-213, cyclophosphamide, and dianhydrogalactitol in advanced renal cell cancer. One hundred and forty-four patients with advanced renal cell cancer were evaluated for objective response to single-agent treatment with megestrol acetate, VP-16-213, cyclophosphamide, or dianhydrogalactitol. In view of the low order of response observed, these agents do not appear to be effective in the treatment of metastatic renal cell cancer. Performance status and a relatively long interval from onset of primary symptoms to study entry were the most favorable prognostic variables affecting time to progression and survival for patients in this study.


Structural studies on the hexose region of the Enterobacteriaceae type R3 core polysaccharide. The structure of the hexose region of the Enterobacteriaceae type R3 core polysaccharide, which is the common core in Shigella flexneri and some Escherichia coli lipopolysaccharides (LPS), has been investigated. The principal method used in these studies involved methylation analysis of the original and modified materials. The polysaccharide was modified by N-deacetylation-deamination and by Smith degradation. As a result of these studies, the following structure is proposed: alpha-D-Glcp-(1 leads to 2)-alpha-D-Glcp-(1 leads to 2)-[alpha-D-GlcNAcp-(1 leads to 3)]-alpha-D-Galp-(1 leads to 3)-alpha-D-Glcp-(1 leads to.


The discrete-time kinetic model analysis of DNA content distributions in experimental tumour cells. A method was developed to analyse and characterize FMF measurements of DNA content distribution, utilizing the discrete time kinetic (DTK) model for cell kinetics analysis. The DTK model determines the time sequence of the cell age distribution during the proliferation of a tumor cell population and simulates the distribution pattern of the DNA content of cells in each age compartment of the cell cycle. The cells in one age compartment are distributed and spread into several compartments of the DNA content distribution to allow for different rates of DNA synthesis and instrument dispersion effects. It is assumed that the DNA content of cells in each age compartment has a Gaussian distribution. Thus, for a given cell age distribution the DNA content distribution depends on two parameters of the cells in each age compartment: the average DNA content and its coefficient of variation. As the DTK model generates the best fit DNA content distribution to the FMF measurement data, it enables one to estimate specific values of these two parameters in each stage of the cell cycle and to determine the fraction of cells in each cycle phase. The method was utilized to fit FMf measurements of DNA content distributions and to analyse their relationship tothe cell kinetic parameters, namely cell loss rate, cell cycle times and grwoth graction of exponentially growing Chinese hamster ovary cells in vitro and, also, with a wide range of coeffficients of variation, of the L1210 ascites tumour during the growth period.


In vivo effects of 5-fluorouracil and ftorafur[1-(tetrahydrofuran-2-yl)-5-fluorouracil] on murine mammary tumors and small intestine. The in vivo anti-tumour and toxic effects of ftorafur (FT) and 5-fluorouracil (FU) were studied in the C3H mouse. On a molar basis, FU was two to three times more potent than FT with respect to growth inhibition of murine mammary adenocarcinomas. However, FT produced less host toxicity than FU when both drugs were compared at dose levels which produced equivalent anti-tumor effects. The differences between FT and FU with respect to tumor growth inhibition and host toxicity were reflected in their ability to suppress deoxyuridine incorporation into tumor cell and intestinal DNA, respectively. Flow cytometry (FCM) studies indicated that FT and FU were capable of producing pertubations in the DNA distribution of tumour cells. Both drugs induced an initial accumulation of cells in S phase following their administration at equivalent anti-tumour dose levels. At later intervals, an apparent block of cell progression at the G1/S boundary was observed. Drug-induced perturbations in the DNA distribution of tumour cells as detected by FCM correlated with results obtained by classical autoradiographic techniques using tritiated thymidine. Both procedures showed that tumor cells were capable of moving through S phase even in the presence of an apparently near complete inhibition of deoxyuridine incorporation into DNA. That such cells were, in fact, capable of synthesizing DNA at moderate rates was shown by their ability to incorporate 32P into DNA. The possible relationship of these findings to the therapeutic and toxic activities of FT and FU is discussed.


DNA flow cytometry of isolated keratinized epithelia: a methodological study based on ultrasonic tissue disaggregation. Ultrasonication of keratinized, stratified, squamous epithelium, which had been separated from underlying tissue by means of acetic acid, resulted in disaggregation of all cellular layers in the epithelium, giving a suspension of single nuclei with mitoses preserved. This suspension was treated with RNAse and ethidium bromide for analysis by flow cytometry. From the resulting DNA histogram the G1, S and G2 + M fractions were estimated using the computer program of Fried (1976). Treatment with dithiothreitol before sonication increased the yield of nuclei in suspension and decreased the amount of debris and clumps, thereby suppressing overestimation of small S fractions. This method of preparation prior to DNA flow cytometry was useful for the study of the hamster cheek pouch epithelium and of normal and pathological human epidermis.


Colony-forming units in diffusion chambers (CFU-d) and colony-forming units in agar culture (CFU-c) obtained from normal human bone marrow: a possible parent-progeny relationship. An series of experiments was performed to elucidate the relationship between cells that form granulocytic colonies in fibrin clot diffusion chambers implanted into the peritoneum (i.p.) of irradiated mice (CFU-d) and day 7 and day 14 CFU-U which give rise to colonies after 7 and 14 days in agar cultures in vitro, respectively. Normal human bone marrow cells were cultured in suspension in vitro or in diffusion chambers implanted into irradiated or non-irradiated mice. During these culture conditions there was an initial decrease in the number of CFU-c per culture. This was followed by an increase between day 2 and day 7 of culture. No similar increase of neutrophilic CFU-d was observed. When CFU-d, day 14 and day 7 CFU-c in normal marrow were separated by velocity sedimentation and cultured in suspension culture or in diffusion chambers for 7 days, the maximum increase of day 7 and day 14 CFU-c was observed in slowly sedimenting cell fractions which contained the majority of CFU-d. After 3 days in suspension culture, the maximum increase of day 14 CFU-c was found in fractions which also gave rise to maximum numbers of CFU-c after 7 days. However, day 7 CFU-c were found in fractions which initially contained the majority of day 14 CFU-c. No increase in CFU-d was found in fractions initially containing peak numbers of CFU-c. Between 53 and 71% of CFU-c harvested from diffusion chambers in irradiated mice or from suspension cultures were sensitive to pulse incubation with tritiated thymidine, suggesting that the cells were proliferating during these culture conditions. In diffusion chambers implanted into non-irradiated mice, however, CFU-c were found to be relatively resistant to this treatment (3-11% sensitive to tritiated thymidine). Thus marked increases in CFU-c were also observed during experimental conditions, where no significant DNA synthesis was detected. A reproducible time sequence of increase in CFU-c populations in culture was observed. Day 14 CFU-c and cells that gave rise to clusters on day 7 in agar increased between day 2 and day 4, whereas day 7 CFU-c increased between day 4 and day 7. The results suggested that CFU-d gave rise to CFU-c in culture and that day 14 CFU-c were precursors of day 7 CFU-c.


The mutagenic effect of 1,2-dichloroethane on Salmonella typhimurium. II. Activation by the isolated perfused rat liver. In this investigation Salmonella typhimurium strain TA 1530 and TA 1535 were combined with isolated perfused rat liver. Samples of perfusate and bile produced were tested for mutagenicity after treatment with 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE) or 2-chloroethanol. The results are in good agreement with our previous experiments which indicate that both DEC and DBE are activated through conjugation with glutathione (GSH). Most GSH conjugates are normally excreted in bile. Following liver perfusion the bile was highly mutagenic after DCE and DBE treatments, while 2-chloroethanol did not have this effect. The highest mutagenic effect was seen 15--30 min after the addition of DCE or DBE. The production of mutagenic bile also occurred in mice treated in vivo with DCE. One possible metabolic endproduct of a GSH conjugate is the corresponding mercapturic acid. Thus synthetic N-acetyl-S-(2-chloroethyl)-L-cysteine was tested on TA 1535 and found to be as mutagenic as S-(2-chloroethyl)-L-cysteine in the concentration range 0.2--0.6 mumol/plate. Differences and similarities in the metabolism of DCE and vinyl chloride are discussed on the basis of these results.


Phytohemagglutinin (PHA) skin test. Characterization of immunological properties and clinical application. Immunological properties of phytohemagglutinin (PHA) skin reaction were investigated by animal and clinical experiments. In the guinea pigs an intradermal dose of PHA-P produced erythema and induration with a maximal response at 24 hours after the injection. Histologically it was characterized by perivascular infiltration of lymphoid cells in the dermis and subcutis, being similar to that of tuberculin (PPD) skin reaction. PHA skin reaction, however, showed some difference from that of PPD in the initial cellular response in that the former was composed of small mononuclear cells and granulocytes with rapid development and the latter was composed of large mononuclear cells (macrophages) and granulocytes with slow development. Intradermal injection of 1:1000 dilution of PHA-P produced a similar erythema in man. In 39 of 59 patients with connective tissue diseases, the results of the in vivo (skin test) and in vitro (lymphocyte transformation) response to PHA correlated well. In the 59 patients, the incidence of the positive rate of the PHA tests (55.9%) was significantly higher than that of the DNCB test (33.9%) and of the PPD test (23.7%). These observations suggest that the PHA skin test has properties of delayed hypersensitivity and is highly sensitive and that it may be a useful measure of cell-mediated immunity.


Separation of Plasmodium berghei-parasitized rat erythrocytes by means of carrier-free electrophoresis. A carrier-free electrophoresis apparatus was used to isolate rat erythrocytes parasitized with Plasmodium berghei. The region of high electrophoretic mobility yielded noninfected erythrocytes, whereas that of low electrophoretic mobility yielded erythrocytes infected with viable parasites. Over 98% purity of parasitized erythrocytes was obtained when blood at an advanced stage of parasitaemia was used. Merozoite-rich fractions were also observed. This continuous one-step separation method should provide large numbers of parasitized erythrocytes for use in immunological and biochemical studies of malaria.


Concentration and separation of erythrocytes infected with Plasmodium falciparum by gradient centrifugation. Concentration of infected erythrocytes was achieved in cell suspensions derived from long-term culture of Plasmodium falciparum growing asynchronously in human erythrocytes. This new procedure involves the slow centrifugation (at 33 g) of erythrocyte suspensions through 5% Ficoll solutions. Mature asexual erythrocytic forms are preferentially retained in the gradient solution (top fraction). After further gradient centrifugation of these parasitized cells, the concentration of mature forms is increased 15- to 31-fold and a mature form parasitaemia of 71-81% is obtained in the final erythrocyte suspension. Furthermore, at least 75% of the total number of the mature forms can be retrieved by this method. Parasitized cells that are not retained in the gradient are sedimented to the bottom of the tube (bottom fraction) and consist predominantly of ring forms. Parasites from both the top and bottom fractions are viable and have been used to initiate short-term synchronous cultures. By providing purified parasite preparations, this simple procedure will facilitate immunological, chemotherapeutic, and biochemical studies with P. falciparum.


The effects of geometry, elasticity, and external pressures on the diastolic pressure-volume and stiffness-stress relations. How important is the pericardium? The concept of an incremental elastic modulus is applied in the quantification of passive elastic stiffness-stress relations of intact heart muscle, and a transmural pressure-volume relation for the left ventricle is subsequently derived in terms of geometry, muscle elasticity, and external pressures to assess their importance. Physiological and clinical applications of this method indicate that: (1) stiffness-stress relations obtained on the basis of pressure-volume data from dog hearts are not significantly different from those obtained from muscle strips excised from these same hearts; (2) shape and the presence of right ventricular, pericardial, or pleural pressures are of secondary importance in an assessment of passive elastic stiffness; and (3) dramatic shifts in the left ventricular intracavity pressure-volume relations following drug interventions are primarily due to the presence of substantial pericardial pressures; however, the transmural pressure-volume relations are not markedly altered, implying no alteration in the intrinsic ventricular compliance.


Prostaglandins and renin release: the effect of PGI2, PGE2, and 13,14-dihydro PGE2 on the baroreceptor mechanism of renin release in the dog. We compared the ability of the vasodilator prostaglandins PGI2, PGE2, and 13,14-dihydro PGE2 to release renin when infused into the denervated, nonfiltering canine kidney in vivo. Papaverine was used as a nonprostaglandin vasodilator. All the prostaglandins tested were capable of stimulating renin secretion, with the scale of potency being 13,14-dihydro PGE2 greater than PGI2 greater than PGE2; papaverine had no effect on renin secretion. These results indicate that both PGE2 and PGI2 can stimulate renin secretion but that vasodilation per se is not a stimulus. 13,14-Dihydro PGE2 was included because it is a poorer substrate than PGE2, both for transport into cells and catabolism to inactive products, but has comparable potency to PGE2 when tested in systems with limited ability to catabolize PGE2. The fact that 13,14-dihydro PGE2 was the most potent prostaglandin tested suggests that the effects of PGE2 in our system are reduced by the kidneys' recognized ability to extract and catabolize PGE2. Since PGI2 is less avidly metabolized than PGE2 by the kidney, the differences in observed potency between PGE2 and PGI2 could be largely the result of differences in renal catabolism of the two prostaglandins rather than differences in intrinsic potency. Therefore, both PGE2 and PGI2 are candidates for the endogenous prostaglandin responsible for stimulating renin secretion.


Treatment of hypertension with nifedipine, a calcium antagonistic agent. Hemodynamic monitoring after a single dose (10 mg) of nifedipine in 27 primary hypertensive subjects (diastolic pressure greater than 110 mm Hg) documented that this calcium antagonistic agent exerts a potent arteriolar vasodilating action, which results in prompt (-21% of control at 30 minutes) and persistent (-16% of control at 120 minutes) fall in mean arterial pressure associated with a rise in cardiac output and pulse rate. The same patients received oral treatment for 3 weeks. Hourly pressure readings showed that 1) the antihypertensive response to each dose lasts 8--12 hours; and 2) nifedipine every 6 hours significantly reduced blood pressure throughout the 24 hours, without postural hypotension. Side effect were short-lasting (headache in five patients, palpitation without arrhythmias in eight patients, burning sensation in the face and legs in five patients and sporadic extrasystoles in five patients) and tended to disappear with continued treatment. Development of drug resistance, sodium retention, plasma volume expansion, renin release or angina pectoris were not observed during the study. Although these findings seem to differentiate nifedipine from other vasodilators currently used in the treatment of hypertension, broader experience and more prolonged trials with nifedipine as an antihypertensive agent will be needed before conclusions can be drawn on these particular aspects.


Presurgical changes in unilateral cleft palate subjects. This investigation utilizes computerized measuring and statistical techniques, stereophotogrammetry, and a newly developed method of measuring palatal surface areas to study changes in the maxillary arch dimensions at birth and at 4 months in 3 groups of subjects: (1) 30 complete unilateral cleft lip and palate cases who were treated by presurgical maxillary orthopaedics; (2) 15 similar cases who had no such treatment and served as controls; and (3) 30 normal children. The changes occurring in the 3 groups over the 4-month period were compared. In particular, it was noted that presurgical treatment had a constrictive effect on general arch growth and that it also retarded the growth of palatal tissue. The significance of this in relation to other findings and to presurgical treatment in general is discussed.


Changes in pituitary gonadotropins during the amenorrhoea-galactorrhoea syndrome due to sulpiride. Sulpiride, a dopamine receptor blocker which raises prolactin, was given to six women with idiopathic amenorrhoea for 10 days and to eleven women with regular cycles for 3 to 6 months. The latter group developed galactorrhoea and amenorrhoea during treatment. Basal LH AND FSH concentrations showed no significant changes. At 3 months but not at 10 days (amenorrhoea group) or 6 months (regular menses group) the area-under-the-curve of LH response to LHRH was increased. FSH responses to LHRH was not altered. The significance of these results to the hyperprolactinaemia-amenorrhoea syndromes are discussed.


Further studies on the effects of dihydrotestosterone on gonadotrophin release induced by LH-RH in men. In order to determine the effect of dihydrotestosterone on the feed-back mechanism of the hypothalamo-pituitary testis axis, seven men were subjected to LH-RH tests before and after treatment with dihydrotestosterone. Our results clearly show that DHT at least in pharmacological doses, inhibits directly basal LH-RH and gonadotrophin secretion, without affecting the pulsatile gonadotrophin release or impairing the pituitary response to LH-RH. The physiological significance of these observations remains however to be proven.


Acromegaly: insensitivity of the pancreatic alpha cell to hyperglycaemia. Plasma glucagon levels were determined after 50 g of oral glucose loading in eleven acromegalics and fourteen normal subjects. Basal plasma glucagon levels were significantly elevated in patients with acromegaly, as compared with those in normal subjects. Oral glucose loading caused a decrease in plasma glucagon in normal subjects but not in acromegalics. Since this non-suppressibility of plasma glucagon by orally administered glucose was observed even in acromegalics without diabetes, it is concluded that insensitivity of the pancreatic alpha cell to hyperglycaemia exists in patients with acromegaly as well as in diabetics.


Pharmacological immunosuppression in clinical organ grafting. Observations on four agents: cyclosporin A, Asta 5122 (cytimun), lambda carrageenan and promethazine hydrochloride. In this article I have attempted to summarize experiments on four agents investigated for immunosuppressive activity in experimental and clinical organ grafting. The difficulty of finding a suitable laboratory model relevant to man has been demonstrated in this experience. A cyclophosphamide-derivative, Asta-5122 (cytimun), has only a marginally superior immunosuppressive activity in a dog with a renal allograft compared with cyclophosphamide and is much inferior to azathioprine. This agent, however, appears to be valuable in clinical practice in patients with liver grafts. A combination of lambda carrageenan, promethazine hydrochloride and imuran has profound immunosuppressive activity in the same canine model, but proved to be both ineffective and potentially toxic in a limited trial in man. The fungal cyclic peptide, cyclosporin A, has been shown to be an extremely powerful immunosuppressive agent and remarkably non-toxic in dogs with renal allografts and pigs with orthotopic heart grafts. This agent is currently being investigated as an immunosuppressant in patients with organ grafts.


Miconazole prolongs murine skin graft survival. The effects of miconazole, a broad spectrum anti-fungal compound, on the immune response was studied in mice. At the non-toxic dose of 300 mg/kg per day, there was minimal suppression of the antibody response to sheep red blood cells. In contrast, there was a marked prolongation of skin graft survival from 13.6 +/- 1.8 days (mean +/- s.d.) in untreated mice to 18.6 +/- 3.5 dyas in the treated group. Miconazole may be of use in clinical transplantation.


Possible C1q bypass loop activation in the haemolytic uraemic syndrome. Ultrastructural and immunofluorescent microscopic studies were performed on renal tissue obtained from nine patients during the acute and convalescent phase of the haemolytic uraemic syndrome (HUS). All had glomerular deposits of IgM in the absence of circulating immune complexes. This was associated with deposition of C1q during the acute phase, and properdin and C3 during the convalescent phase. C4 was consistently absent. Since such a pattern of complement deposition does not fulfil criteria either for alternate or classical pathway activation, the possibility of C1q bypass loop activation by IgM is suggested.


Direct Coombs antiglobulin reactions in Gambian children with Plasmodium falciparum malaria. I. Incidence and class specificity. Gambian children with past or present Plasmodium falciparum malaria were investigated for the incidence of Coombs positivity using monospecific antisera. Approximately 50% were positive and the most frequent form of erythrocyte sensitization was with C3d. Other specificities, EIgG, EIgGC3d and EIgGC4bC3d were less common. Erthyrocytes were never found sensitized with IgA or IgM. There was no correlation between a positive test and age, tribal status or level of parasitaemia at presentation, although a positive test was often found in association with anaemia. Sensitized erythrocytes were present in the circulation for a period of up to 6 weeks following initial observation. The mechanism of erythrocyte sensitization is not known, but the results suggest a Type III complex-mediated hypersensitivity involving parasite antigen-antibody complexes. It is likely that these reactions contribute to the pathogenesis of the anaemia in falciparum malaria.


Antibody-dependent cell-mediated cytotoxicity (ADCC) and cell-mediated cytotoxicity (CMC) to HBsAg-coated target cells in patients with hepatitis B and chronic hepatitis (CAH). A new technique using HBsAg-coated Chang cells as target cells was developed in order to measure cell-mediated immune reactions to HBsAg. The specificity of cytotoxic reactions was tested in experiments using Chang cells conjugated with human serum albumin. Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for the HBsAg-coated target cells was demonstrated up to dilutions of anti-HBsAg serum of 10,000 : 1, when lymphocytes from the peripheral blood of normal individuals were added to the target cells. Spontaneous cell-mediated cytotoxicity (CMC) to HBsAg-coated target cells was demonstrated for lymphocytes from patients with hepatitis B and from patients with chronic active hepatitis (CAH), but not for lymphocytes from healthy controls. The CMC of hepatitis B lymphocytes to HBsAg-coated target cells was inhibited in the presence of antiserum to HBsAg. In experiments using purified lymphocyte populations evidence is presented that the CMC is T-cell dependent. HLA-restriction of the CMC was not observed. The described cytotoxicity test system has the advantage that target cells conjugated with defined antigens are used and that relevant control target cells are available.


Facilitation reaction (enhancing antibodies and suppressor cells) and rejection reaction (sensitized cells) from the mother to the paternal antigens of the conceptus. Humoral and cellular immune agents of a maternal reaction were investigated during pregnancy. Fluorescence studies performed on mouse placentae at 14 days detected maternal immunoglobulins of mainly IgG1 but also IgG2 subclasses. These immunoglobulins, after acid elution, can rebind the placenta and the thymocytes of the relevant paternal strain in case of allogeneic pregnancies, demonstrating an antibody activity towards both placenta specific and paternal strain antigens. They can specifically enhance a paternal strain tumour allograft on a maternal strain recipient. Spleen cells from an allogeneically pregnant mother can reduce or promote paternal strain tumour allograft on a maternal strain recipient. The aggressive effect is shown with small doses of transferred cells, whereas large doses promote enhancement. The suppression of the cytotoxic response of the recipient was ascribed to T cells by use of anti-theta plus complement. Thus, both the rejection reaction (immune cytotoxic cells) and the facilitation reaction (enhancing antibodies and suppressor cells) were demonstrated during pregnancy.


Antibodies to maize in patients with Crohn's disease, ulcerative colitis and coeliac disease. The incidence of antibodies to maize using an immunofluorescent technique has been found to be 14% in controls, 33% in Crohn's disease, 50% in ulcerative colitis and 44% in coeliac disease. This result indicates that humoral immunity to maize is probably unimportant in the pathogenesis of Crohn's disease. The similar incidence of antibodies in the inflammatory bowel disease and coeliac groups suggests absorption of dietary antigen secondary to an increased mucosal permeability.


Allergy in cystic fibrosis.

Allergy in cystic fibrosis. Forty-four patients with cystic fibrosis, sixty-seven parents, and thirty-nine controls were compared with regard to their immediate cutaneous hypersensitivity, total and specific serum IgE and serum IgG4 concentrations. The patients had increased prick test reactions and specific IgE to Aspergillus fumigatus. The development of reactions to A. fumigatus correlated with the severity of the disease. There were no other significant differences when serum total IgE, specific IgE and IgG4 concentrations or skin test reactivity were compared in the patients, parents and controls.


A method for the ultrastructural examination of cell monolayers cultured in plastic microtitre plates. A method is presented by which cells growing as monolayers cultured in microtitre plates can be embedded for electron microscopy. The technique has the following advantages: numerous specimens may be prepared with relatively small numbers of cells, cell-cell interactions remain undisturbed and may be enumerated, and morphology can be studied under circumstances identical with those used in cell-mediated cytotoxicity assays.


Measurement of anti-thyroid antibodies in dried blood spots. The method for determination of anti-thyroid antibodies in extracts of dried whole blood on filter paper was described. Antibodies in dried blood were stable at room temperature for at least 1 month and were extracted well by overnight elution. This simple method requires only a minimal 30 microliter of venous blood and would enable large-scale screening for autoimmune thyroid diseases.


A placebo controlled clinical trial of transfer factor in lepromatous leprosy. The effects of repeated injections of transfer factor over a period of 20 weeks were investigated in fourteen bacteriologically positive patients at the lepromatous side of the leprosy spectrum. All patients showed negative (0 mm induration) skin tests to M. leprae antigens (i.e. leprolin and lepromin). Of these patients, seven were treated with transfer factor with a total of 9 units (1 unit being equivalent to 5 x 10(8) lymphocytes) and seven with a placebo. Maintenance treatment with clofazimine was continued. Transfer factor was prepared from the lymphocytes of donors who showed positive skin tests to M. leprae antigens (i.e. leprolin greater than or equal to 12 mm induration, average 15.5 mm or lepromin greater than or equal to 8 mm induration, average 13.6 mm), as well as a positive lymphocyte transformation in vitro to M. leprae (the average transformation being higher than the average transformation of lymphocytes of tuberculoid leprosy patients). No differences were found between the two groups as regards the clinical course of the disease, the histopathological and bacteriological evaluation of skin biopsies, changes in skin test reactivity to various antigens (i.e. lepromin, leprolin, PPD, Mumps, C. albicans, Tr. rubrum and Varidase), as well as the lymphocyte transformation in vitro to various mitogens (i.e. PHA, PWM, Con A) and antigens (i.e. M. leprae, leprolin, PPD, BCG, Mumps, C. albicans, Trichophyton and Varidase). No evidence was found to suggest that transfer factor is a valuable adjuvant in the treatment of lepromatous leprosy patients or that it increases cell-mediated immune reactivity towards M. leprae.


An evaluation of the immunoperoxidase technique in renal biopsy diagnosis. An evaluation of the use of horseradish peroxidase labelled antisera against IgG, IgM, IgA and C3 compared with corresponding antisera labelled with fluorescein isothiocyanate has been carried out on renal biopsy material from 200 patients with renal disease. A disparity between the two methods was observed in 10-19% of the readings according to the antigen being demonstrated. The majority of differences recorded would not have influenced the executive diagnosis. Where the immunological results could have influenced the executive diagnosis, 5 probable false results were obtained using innumofluorescence and 4 with immunoperoxidase. Immunoperoxidase has the advantage over immunofluorescence in that it provides a permanent preparation which can be reviewed months or years later. In addition it provides more accurate localization of the immune deposits since the section can be counterstained with Mayer's hemalum and viewed by conventional light microscopy. In view of these advantages we recommend that serious consideration should be given to using immunoperoxidase in place of immunofluorescence for the examination of renal biopsy material.


Acute disseminated aspergillosis during the neonatal period. Report of an instance in a 14-day-old infant. We describe an infant who died with extensive lesions of disseminated aspergillosis on the 18th day of postnatal life. Aspergillus fumigatus was recovered from blood cultures. Initial clinical manifestations were suggestive of hepatitis, and steroids and antibiotics were used in the treatment. This therapy may have fostered the onset of an opportunistic mycosis. Advanced multisystemic aspergillotic lesions were seen at autopsy, especially prominent in lungs and gastrointestinal tract. The extent and magnitude of the lesions observed suggest inception of the disease very early in life, although no case of human aspergillosis has been known to be congenital. Neonatal aspergillosis is poorly characterized. Only four previously reported cases came to our notice in which the disease could be diagnosed in the first month of life. The ubiquitous nature of pathogenic Aspergillus, joined to aggresive treatments designed to achieve increased survivals in neonatology, could potentially result in greater numbers of cases of this and other uncommon mycoses.


Once-daily metoprolol in primary hypertension. The antihypertensive effect of the selective beta-1-adrenoceptor blocker, metoprolol, administered once daily was evaluated in 32 patients with primary hypertension. After a 4-wk placebo period, the patients were treated with either 150 mg or 300 mg of metoprolol, once daily, for 8 wk. Initially and during the treatment periods blood was drawn for analysis of metoprolol in plasma, plasma renin activity (PRA), and electrolytes, and urine was collected for determination of the urinary aldosterone excretion. Metoprolol reduced the blood pressure measured up to 26 hr after the last dose. The percentage of responders to metoprolol (decrease of mean arterial pressure greater than or equal to 10% over placebo) was 40% for patients on 150 mg and 71% for patients on 300 mg. Except in the standing position, heart rates were reduced for 26 hr after a 150-mg dose. There was a correlation between pretreatment PRA levels and antihypertensive effect of metoprolol in patients on 300 mg metoprolol but not in patients on 150 mg. Urinary aldosterone decreased equally during treatment in responders and nonresponders. Antihypertensive effects and side effects did not correlate with plasma metoprolol concentrations.


The calcium carbimide-ethanol interaction: effects of ethanol dose. In a double-blind, placebo-controlled study involving five male alcoholic volunteers, oral administration of 0.7 mg/kg of calcium carbimide (CC) 12 hr before ingestion of ethanol (0.125, 0.25, and 0.5 gm/kg) produced an interaction consisting of increased blood acetaldehyde level, tachycardia, and decreased diastolic blood pressure. The order of intensity of the interaction with regard to ethanol dose was 0.5 greater than 0.25 greater than 0.125 k gm/kg. The subjects were aware of a CC-ethanol interaction only for 0.25 and 0.5 gm/kg of ethanol, for which heart rate was elevated above 100 bpm. With the criterion of heart rate above 100 as indicative of the CC-ethanol interaction, the onset was 0.25 and 0.38 hr for the 0.5 and 0.25 gm/kg ethanol doses and the duration of the interaction was 1.0 and 0.38 hr, respectively. There were positive linear correlations between blood acetaldehyde level and both heart rate and pulse pressure. There was appreciable individual variability in the intensity and duration of the interaction. Pretreatment with CC reduced the rate of ethanol metabolism at the 0.5 gm/kg ethanol dose.


Tooth loss, prosthetics and dental treatment habits in a group of Swedish men. Dental history, dental status, the need for prosthetic treatment and dental treatment habits were studied in 389 men aged 21--54 years. The men were selected from a group undergoing military refresher training in the south of Sweden. One percent of the men were toothless. About 3% had full dentures in one or both jaws and an equal percentage had partial dentures. About 3% of the men had bridges in the maxilla and 2% in the mandible. Tooth loss was greatest in the mandibular molar segment, followed by the maxillary molar segment. By means of an index for dental status about 8% of the men were judged to be in great need of prosthetic treatment for aesthetic reasons and/or to improve occlusion. Age, place of birth, educational level and smoking habits were among the factors which seemed to be related to dental status. Seventy percent of the men reported that they went to the dentist once a year, while about 10% seemed rarely or never to seek dental treatment. Dental treatment habits were correlated to place of birth, number of brothers and sisters, edentulousness and the need for prosthetic treatment, among other factors.


A method of utilizing the subjects' initial caries experience to increase efficiency in caries clinical trials. A problem which often occurs in caries clinical trials is the existence of an imbalance by group in the initial DMFS scores among subjects completing the study. Four methods of incorporating the study subjects' initial caries experience were identified and compared in six recently conducted clinical trials to see which of the methods was the most efficient in making treatment group comparisons of study subjects' caries increment scores. The consistent method of choice was one which utilized an adaptation of Grainger's caries severity index as a stratification factor in a blocking analysis of caries increments. The results suggest that more information regarding future caries exist in knowing where decay is present in the dentition than in knowing the subjects' total DMFS counts.


Effect of the LRF-antagonist [D-pGlu1, D-Phe2, D-Trp3,6]-LRF on pregnancy in the rat. The potent LRF-antagonist [D-pGlu1, D-Phe2, D-Trp3,6]-LRF (Antag.) has been examined for its effect in mated female rats. While 1.25 mg Antag. given b.i.d. for 7 days after mating prolongs pregnancy, the administration of 2.5 mg Antag. b.i.d. for the same period results in the complete absence of developing fetuses.


Cancer metastasis: a product of tumor-host interactions. Metastasis continues to be the most devastating event for the patient with an established primary cancer. The most significant therapeutic problems are: (1) treatment of patients with established macrometastases, (2) identification of patients who have micrometastases and (3) the development of adequate adjunctive therapies for micrometastases. It is hoped that our evolving understanding of the biology of experimental metastasis and the high level of premium quality laboratory research ongoing in this area will result in further resolution of this clinical problem or, at least, a better understanding of this most extreme expression of the malignant phenotype.


Ascites: its correction by peritoneovenous shunting. Patients with refractory ascites and HRS should be considered to present an urgent indication for peritoneovenous shunting. The shunt offers a method of continuous reinfusion of ascitic fluid which corrects avid sodium retention, oliguria and azotemia. Severe encephalopathy, jaundice or peritoneal sepsis--common complications of cirrhosis--contraindicate installation of the shunt before improvement occurs. Associated cardiac disease does not contraindicate the use of the shunt provided that ascitic fluid is removed at the time of operation and large amounts of diuretics are used. This operation has also proved useful in ascites attributed to causes other than cirrhosis. The main complications include disseminated intravascular coagulopathy, hepatic coma and sepsis in a few patients. Results of a randomized prospective study indicate that the shunt should probably be considered in patients with diet-resistant massive ascites even before they prove to be refractory to diuretic therapy.


A simple procedure for obtaining autoradiographs of G-banded chromosomes. Chromosomal preparations from cells flash-labeled with 3H-dT were Giemsa-banded following trypsin digestion, allowed to air-dry, and then were coated with a layer of 1% Formvar. The slides were subsequently coated with radiographic emulsion (NTB2) and processed for autoradiography. The resulting chromosomes had distinct G-banding and radiographic labeling patterns. Chemographic grain formation in the emulsion, normally caused by Giemsa stain, was prevented by the film of Formvar, allowing a very low background grain level.


A probe into nuclear events during the cell cycle of Saccharomyces cerevisiae: studies of folded chromosomes in cdc mutants which arrest in G1. The sedimentation behavior of folded chromosomes from cell-division-cycle (cdc) mutants which arrest in G1 was examined. At the restrictive temperature the folded genome of cdc 7, which arrests after spindle pole body (SPB) separation and spindle formation, cosediments with a standard g1 structure, indicating that by the cdc 7 step the g1 form of the folded genome has been assembled. In the mutant, cdc 4, which arrests before SPB separation but after SPB duplication, a standard g1 structure is not formed. cdc 4 cells, however, are able to enter G0 at the restrictive temperature, and the corresponding go structure is stable. These results indicate that the cdc 4 gene product may be involved in the development of folded genome conformation which leads to the g1 structure. Since the cdc 4 gene product is required for SPB separation, the g1 structure may be defined by an association between chromosomes and spindle components. The folded chromosomes of the "start" mutants cdc 25 and cdc 28 are unstable at the restrictive temperature. In contrast to cdc 4, neither cdc 25 nor cdc 28 are able to enter the G0 stage in a normal manner, i.e., the g0 structure is unstable at the restrictive temperature. The inference is that both the cdc 25 and cdc 28 gene products are required for the functional integrity of the folded genome at both a stage early in G1 and in the pathway to G0.


A sequential analysis of meiosis in the male mouse using a restricted spermatocyte population obtained by a hydroxyurea/triaziquone treatment. A method is described to restrict the spermatocyte population in mice and other rodents using hydroxyurea (HU) and triaziquone (T). HU affects cells in S-phase, whereas T is an agent especially active on spermatogonia and not on spermatocytes. An application of three i.p. HU injections with 12 h intervals, followed about nine days later by one i.p. T injection creates two large gaps in the spermatogenic line. The two gaps enclose a small, well-defined group of primary spermatocytes in meiotic interphase. - The development of the restricted spermatocyte population is followed day by day. The analysis of meiosis in male mice has revealed the correct sequence of meiotic, and especially prophase I stages. On account of clearly visible differences in chromosome morphology the diplotene stage could be divided into three periods. It is suggested to use the following nomenclature: pre-diffuse diplotene, diffuse diplotene and post-difuse diplotene. The experiment was also informative about the timing of the stages in spermatocyte development by correlating the days at which the successive stages were observed with the corresponding stage of the epithelial cycle. The calculation of the position and duration of the diffuse diplotene, enables us to put forward a proposal about the significance of the diffuse diplotene. - A combination of the HU/T method with cell separation techniques provides good perspectives for detailed biochemical studies on processes taking place during meiosis.


Telescoping anastomosis of the colon: a comparative study. The incidence of large-intestinal anastomotic leaks remains unacceptably high. For this reason, we studied the resistance to dehiscence of a telescoping type of anastomosis and compared it with the conventional one-layer, two-layer, and stapled anastomoses in a canine model. These experiments demonstrated that the telescoping anastomosis provided a more resistant suture line during the early postoperative phase and did not cause encroachment of the lumen by the diaphragm, which was consistently produced when an inverting suture line was used. Three days postoperatively the mean bursting pressures of the one-layer, two-layer, and stapled anastomoses were 31 +/- 12, 120 +/- 46, and 52 +/- 21 mm Hg, respectively. The telescoping anastomosis had a mean bursting pressure of 210 +/- 44 mm Hg, which was significantly (P less than 0.01) higher than those of all the other anastomoses tested. However, seven and 14 days after operation, there was no statistically significant difference among the bursting pressures of the various anastomoses. When the different types of anastomoses were examined histologically, it was found that there was considerably more suture-line inflammation, edema, micro-abscess formation, mucosal ulceration and pericolic inflammation of the fat in the one-layer, two-layer, and stapled anastomoses than in the telescoping anastomosis.


Antimitochondrial antibodies: reagent variables may lead to diagnostic error. Laboratory-prepared and commercially obtained fluorescein-labeled rabbit antihuman IgG were compared in performing the antimitochondria antibody (AMA) assay. Identical results were obtained using either of the fluorescent antisera at protein concentrations of 1.5 mg/ml and 1:10 dilutions of patients' sera. Positive AMA tests with either antisera were observed in each of 7 patients with primary biliary cirrhosis (PBC), 2 of 83 patients with miscellaneous hepatic diseases, and in 1 of 24 patients with extrahepatic biliary obstruction (EBO) of 2-24 weeks duration. However, when undiluted commercial fluorescent antiserum (15.8 mg protein/ml) was substituted in the assay, sera from 11 of 23 AMA-negative patients with EBO and 12 of 15 with miscellaneous liver diseases demonstrated an atypical fluorescence located primarily along the periphery of the rat renal tubules. Thus, if the conjugated antibody is not adjusted to an optimal protein concentration, this atypical fluorescence could be interpreted as a positive AMA test and lead to diagnostic error.


Different gastric, pancreatic, and biliary responses to solid-liquid or homogenized meals. We have compared responses to an ordinary solid-liquid (S) meal and to a homogenized (H) meal of identical composition (sirloin steak, bread, butter, ice cream with chocolate syrup, and water) by measuring simultaneously postprandial gastric, pancreatic, and biliary functions by marker-perfusion techniques. Responses to each (S or H) meals differed strikingly both in magnitude and pattern. S meals elicited a stronger early gastric secretory response (acid, pepsin, and volume) which compensated for faster initial emptying and resulted in higher gastric acidity and volume than after H meals. Further, nutrients ingested with S meals were emptied at a slower rate than H (as evidenced by a more gradual decline in intragastric buffer and osmolality, as well as time required for complete emptying of the meal). This, in turn, prolonged pancreatic and biliary responses since stimulation of these organs continued for as long as meal was delivered into the duodenum. However, early biliary outputs (gallbladder response) were less after S than H, probably because nutrients entered the duodenum more slowly and were initially diluted by rapidly emptying water. The physical characteristics of each meal (encompassing appearance, taste, and form of ingestion) probably accounted for early differences in digestive responses. Later, interactions between gastric (motor and secretory), pancreatic, and biliary functions played a major role. Our findings suggest that gastric, pancreatic, and biliary responses to liquid test meals introduced into the stomach may differ substantially from the presumably more physiological response to ordinary solid-liquid meals.


A review of upper-gastrointestinal effects of the newer nonsteroidal antiinflammatory agents. Newer nonsteroidal antiinflammatory agents (NSAI's) such as ibuprofen, neproxen, fenoprofen, and tolmetin have broadened the therapeutic choice and increased the chances of providing optimum arthritis control, but require careful assessment of the possibilities for unwanted drug effects when long-term therapy is required. A review of the literature on the gastrointestinal effects of the promising newer NSAIs, as compared with the older agents, aspirin, indomethacin, and phenylbutazone, is presented, highlighting animal toxicology and human adverse reaction surveillance data and the evidence for various suggested pathophysiological mechanisms.


[Pre-orthodontic surgical treatment of retained or ectopic teeth in juvenile dentitions]. For orthodontic and gnathologic reasons, impacted teeth in adolescents should not be extracted but rather placed in their appropriate positions. This article deals with the surgical aspects of a combined surgical and orthodontic methods for incorporating these teeth into the dental arch. The procedure for lifting the gingival tissue to expose the impacted tooth and the application of a plastic attachment via "direct bonding" were described.


[Defect and filling therapy by means of dental enamel etching in the deciduous dentition]. The cauterization of enamal for treating defects and filling decidious teeth was studied clinically and with the raster electron microscope. Results up to now tend to indicate that this procedure is particularly well suited for decidious teeth and that it will be an increasingly important procedure in the future.


[The change of dimensions in bonded crowns]. The dimension of the crown is changed when the ceramic mass is fased. The reduction in dimension in the mesiodistal direction is greater than in the buccolingual direction when the crown is faced on one side. This shrinkage influences the exact fit of the crown faced with ceramic material. The form of the crown faced with plastic also changed; this change however was a deformation rather than a shrinkage.


[Evaluation and habits of wearing model cast prostheses--results of an inquiry among patients]. An evaluation was made of 1246 questionnaires. A difference could be established between patients wearing prostheses for the first time and patients wearing prostheses over a long period of time. No differences were demonstrable between men and women. Age-related differences could not be determined, the treated jaw did not influence the evaluation, and there was no relationship between the type of prosthesis and the subjective statements given by the patients.


[Gnathologic aspects in total prostheses]. Using a combination of the all-oral procedure (M. Hofmann), the Lauritzen technique, and the TMJ system, it is possible to construct dental prostheses geared to the individual patient for individuals whose teeth have all been extracted. This rational procedure was introduced and discussed.


[The anatomical course of the mandibular canal--a computer assisted trigonometric radiographic interpretation]. The mandibular canal was demonstrated on two stereoscopic radiographs which correspond radiographically to isolated films of the mandible. The central ray forms 20 degrees and a 50 degrees angle with the camperian plane on the radiograph. A reference pointer attached to the teeth is also reproduced on the film. The angle and direction of the central ray is computed on the basis of the diagram of the reference pointer. A defined adjustment therefore is unnecessary when the radiograph is being taken. The coordinates of the mandibular canal and the surrounding root tips are accurately computed with a programmable computer.


Developmental changes in the response of male and female rat pituitary to LH-RH in vitro. Changes in the binding of 125I-LH-RH and the sensitivity of male and female rat pituitary of LH-RH were measured in vitro from the 5th to 60th day of age. Pituitaries of all the age groups were responsive to LH-RH. Two phases of pituitary sensitivity to LH-RH could be detected in both the male and female rat. The first phase or the period of low sensitivity extended up to the day 14, while the second one or the period of high sensitivity started from the day 15 onwards. During the second period the sensitivity of female pituitary to LH-RH was significantly higher than that of male one. In the female pituitary the changes in the sensitivity to LH-RH were parallel to changes in 125I-LH-RH binding. However, in the male pituitary 125I-LH-RH binding was higher while the sensitivity of pituitary to LH-RH was compatively lower than in the female rat. It is postulated that two different types of receptors may be involved in the stimulation of LH-RH induced LH release from the male and female pituitary.


Insulin secretion and the morphological and metabolic characteristics of pancreatic islets of hyperthyroid ob/ob mice. Thyroxine treatment induced experimental hyperthyroidism in ob/ob mice, inhibited glucose-induced insulin secretion from the isolated perfused ob/ob mouse pancreas, and reduced total pancreas insulin content. In contrast, glucose-induced insulin release from incubated pancreatic islets and insulin content of pancreatic islets from ob/ob mice isolated by freehand microdissection were not reduced after thyroxine treatment when expressed per microgram dry islet. Histological examination of the ob/ob mouse pancreas revealed islets without degenerative lesions of islet cells. Granularity of beta cells was well preserved. The average number of pancreatic islets was unchanged. However, the beta cell area was significantly decreased in relation to the total pancreatic parenchyma after thyroxine treatment. This implies that insulin release and content per pancreatic islet was half of that of the controls. ATP content of islets was slightly reduced. Glucose oxidation and glucose utilization by islets from treated mice were slightly increased. Thyroxine treatment of the animals did not abolish the stimulation of 45Ca2+ uptake by glucose, but it did suppress the potentiating effect of fasting on the stimulatory effect of glucose on 45Ca2+ uptake. The metabolic characteristics of islets from experimentally hyperthyroid mice are those of all hyperthyroid tissues. The results provide no evidence for the view that the effects of thyroxine treatment may be due to disturbed metabolic function or energy deprivation of pancreatic islets. Inhibition of insulin secretion from the pancreas after thyroxine administration is apparently due to a reduction in pancreas insulin content and a diminished pancreatic islet volume. Reduced pancreatic islet volume represents most probably a reduction of individual islet cell volume.


The effects of vitamin A on insulin release and glucose oxidation in isolated rat islets. We tested the effects of vitamin A, a membrane surface-active agent, on glucose (16.7 mM)-induced biphasic insulin release from collagenase-isolated rat islets. Also, efforts were made to correlate the effects of vitamin A with glucose oxidation. Vitamin A (10(-4) M) inhibited first- and second phase insulin release; 10(-5) M vitamin A inhibited second phase release only and to a lesser extent than that observed with 10(-4) M vitamin A; and 10(-6) M vitamin A had no effect. Vitamin A (10(-7) M) stimulated biphasic insulin release. Exposure to high glucose (27.8 mM) overcame the effects of 10(-4) M vitamin A on first phase release, but not on second phase release of insulin. Exposure to 10(-5) M hydrocortisone opposed the effects of 10(-4) M vitamin A on both phases of insulin release. Vitamin A (10(-4) and 10(-5) M) inhibited glucose oxidation by islets, as measured by the production of 14CO2 from [14C]glucose. The effects of vitamin A on insulin release were dissociated in part from those effects on glucose oxidation, in that hydrocortisone opposed the effect of vitamin A on insulin release but not on glucose oxidation. The effects of vitamin A on insulin secretion can best be explained by the interaction of vitamin A at multiple sites affecting the membrane and intracellular glucose oxidation.


Secretion of "inhibin" by rat granulosa cells in vitro. Rat granulosa cells secrete a substance in vitro that acts directly upon cultured rat pituitary cells to preferentially suppress FSH production. It is inferred from these results that the granulosa cell is a source of ovarian "inhibin" and that the pituitary gonadotroph is a site of "inhibin" action.


On the biochemical nature of triose- and hexose-stimulated insulin secretion. The differential effects of several specific inhibitors of intermediary metabolism, mannoheptulose, 2-deoxylucose, and iodoacetate, were studied with isolated perifused pancreatic islets stimulated with glucose, mannose, glyceraldehyde, dihydroxyacetone, or alpha-ketoisocaproate. Insulin release rates and/or capacities to metabolize these caloric stimuli served as indicators of the inhibitors' actions. Mannoheptulose and 2-deoxyglucose blocked hexose-stimulated hormone release and hexose metabolism concomitantly, but left the functional and metabolic actions of trioses unaltered. Iodoacetate blocked hexose- and triose-stimulated hormone release as well as their metabolism in a parallel fashion. The action of alpha-ketoisocaproate was not affected by any of these three inhibitory agents. The data are most easily explained by a theory that incorporates metabolic signals, arising during the degradation of insulin-releasing fuel molecules, as an integral component in the process of beta-cell stimulation.


Chronic effects of [D-Trp6-Pro9-NEt]luteinizing hormone-releasing factor on reproductive processes in the female rat. LRF and one of its highly potent analogs, [D-Trp6-Pro9-NEt]LRF (A), were tested for their effects on pregnancy. Daily doses of A prevented implantation and/or caused resorption of implanted blastocysts in mated female rats. This effect was dose related and was most evident when given in regimens including days 5-7 or days 9-10 or pregnancy. LRF also has antigonadal effects, but at much higher doses than A. The use of corn oil vs. 0.1% gelatin-saline improved the effectiveness of low doses of A. Termination of pregnancy in A- or LRF-treated rats is most probably due to the markedly diminished progesterone secretion, which could result from the secretion of biologically inactive gonadotropins, reduced pituitary responsiveness to endogenous LRF, or down-regulation of gonadal receptors for LH.


Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli. Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II). It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site. The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.


Purification and characterization of the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae. 1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation. 2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51,000. 3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1. 5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 microns, 18 microns, and 50 microns respectively.


Defect in the split proteins of 30-S ribosomal subunits and under-methylation of 16-S ribosomal RNA in a polyamine-requiring mutant of Escherichia coli grown in the absence of polyamines. Polyphenylalanine synthesis was carried out with Escherichia coli Q13 50-S ribosomal subunits and reconstituted 30-S particles containing different combinations of 23-S core particles and 30-S subunit split proteins obtained from a polyamine-requiring mutant of E. coli during its growth in the presence or absence of putrescine. It was concluded that the defect in the amount of some kinds of 30-S subunit split proteins was responsible for the decrease of polypeptide synthesis in a polyamine-requiring mutant of E. coli grown in the absence of polyamines. The methylation of 16-S RNA during growth in the absence of putrescine was decreased, while the degree of methylation of 23-S RNA did not change significantly. The decrease in methylation of 16-S RNA in the absence of putrescine was due mainly to a decrease of methylation of adenine. The relationship between the decrease of polypeptide synthetic activity of 30-S ribosomal subunits obtained from a polyamine-requiring mutant of E. coli grown in the absence of polyamines and the decrease of methylation of 16-S RNA is discussed.


The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic. Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome. The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho- deletion mapping and recombination analysis. The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence. 1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2 leads to OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type. 2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red. 3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown. 4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).


Translational fidelity and specificity of ribosomes cleaved by cloacin DF13. The effect of cloacin DF13 cleavage on several functional properties of the ribosome has been studied in a translational system in vitro. Cleaved ribosomes synthesize relatively shorter polypeptide chains on synthetic and natural templates. Their translational specificity is, however, unchanged as judged by the read-out of MS2 RNA. Here, cleaved as well as control ribosomes start translation only on the coat cistron of the phage RNA. Cloacin cleavage of ribosomes increases their fidelity of translation. Differential inhibition of translation of synthetic and natural template was not observed.


Late prognosis in acute posttransplant renal failure in 102 patients. An ivestigation was made of the effect of the duration of posttransplant renal failure on the late prognosis of graft as well as patient survival and on the frequency of complications in 102 patients who had a functioning graft at three months after transplantation. A direct correlation was found between duration of acute tubular necrosis (ATN) and the late prognosis of the graft. The creatinine clearance was significantly higher and the frequency of complications was lower in the group with immediate resumption of renal function. The mortality rate increased with the duration of ATN.


Thymic involvement in control of bone marrow growth. Use of T-cell-depleted hybrid mice. Because of mounting evidence of involvement of thymus-derived cells in blood formation we have studied the growth of transplanted bone marrow in mice extensively depleted of T-lymphocytes (TCD). Poor growth of parental marrow was found not to be appreciably altered in TCD hybrid recipients. However, parental thymic lymphocytes even in massive doses were not able to augment hemopoiesis in TCD hosts, in contrast to findings from sham-thymectomized or age control mice. This indication that a third (host) cell takes part in the thymocyte-marrow stem cell interaction was reinforced by the finding that isogenic (hybrid) thymocytes administered to TCD mice 5 weeks before the final irradiation restored their ability to support thymocyte-induced augmentation of parental marrow growth. Data were obtained from theta-poor sham-thymectomized irradiated controls which are interpreted as evidence for a suppressor T cell. Thymocytes administered with marrow produced a shift toward granulopoiesis in TCD mice as well as in controls. From this finding we infer that although thymus-derived cells are intimately involved in regulation of myelopoiesis, the effect of administered thymic lymphocytes on the differentiative pathway does not depend on host T cells.


Terminal deoxynucleotidyl transferase as a leukemic marker in bone marrow transplant recipients. Terminal deoxynucleotidyl transferase was measured in bone marrow samples from five patients with acute lymphoblastic leukemia in relapse before bone marrow transplantation and serially thereafter. This enzyme seems to be a sensitive marker which can serve as an early indicator of recurrence of the disease. It is not influenced by clinical graft-versus-host reaction.


The collection, preservation and function of peripheral blood hematopoietic cells in dogs. Semicontinuous flow centrifugation (SFC) was employed in canines to obtain adequate numbers of cells for autologous marrow repopulation following supralethal cyclophosphamide administration (100 mg per kg). Four cycles using a 225 ml bowl and 30 ml per minute flow rate were carried out for procurement. Collections averaged 8.4 +/- 0.4 x 10(9) (n = 30) leukocytes. Mononuclear cells (MNC) comprised 75 +/- 2% of the population and granulocyte colony-forming units (CFU-C) totaled 1.9 +/- 0.09 x 10(5) colonies per collection. Eighty-six percent of mononuclear cells were "T" cells in the 30 to 90 second fraction compared to 49% at 150 to 120 seconds. CFU-C fractionation revealed a peak at 30-210 seconds and a second peak at 120-210 seconds. Following programed freezing and rapid thawing 77.7 +/- 11.4% of CFU-C were recovered. Using a dose of 1 x10(9) MNC per kg for reinfusion, marrow repopulation and clinical recovery occurred in four out of four dogs. It was concluded that 1) SFC was effective for obtaining adequate numbers of peripheral blood stem cells for autologous marrow repopulation. 2) A rapid thawing and direct transfusion technique appears satisfactory for administration. 3) Some separation of "T", "B" and CFU-C peripheral blood components is possible by centrifugation.


Chelation therapy in beta-thalassemia major: a one-year double blind study of 2,3-dihydroxybenzoic acid. A year-long double-blind study of 2,3-dihydroxybenzoic acid (2,3-DHB) given orally at a dose of 25 mg/kg four times per day was undertaken in 15 patients with beta-thalassemia major. 2,3-DHB and placebo (mannitol) were tolerated to an equal degree and there were no signs of drug toxicity at the end of 1 year. Efficacy in terms of retardation of iron accumulation could be documented using serial liver biopsies, serum ferritin determinations, or clinical laboratory assessment. Serum iron values increased, as did the iron binding capacity, in the group receiving 2,3-DHB. The increase in iron binding capacity was due to drug interference with the method of determination. Because of the greater efficacy of slow infusions of desferrioxamine in chelating iron when administered slowly, the clinic has shifted its emphasis toward further evaluation of that compound. Nevertheless, in view of the minimal toxicity of 2,3-DHB, further work appears warranted to define its role in the treatment of iron-overload.


Elimination of platelets from mononuclear cell preparations using heat-killed yeast. A new method for reducing platelet contamination in mononuclear cell preparations using heat killed yeast is presented. Yeast cells are added to the buffy coat of peripheral blood, incubated at 37 degrees C for 1 hour, layered over a Ficoll-Hypaque gradient, and centrifuged at 700 g for 30 minutes at room temperature. The cells at the interface are collected and evaluated for platelet count, lymphocyte surface markers, and mononuclear cell recovery. With increasing yeast concentration, the platelet count decreases to 93% over control values. No detectable lymphocyte subset is removed by this method. Cell recovery varies, but seems to decrease with the addition of higher concentrations of yeast cells. Normal plasma is required for the successful removal of platelets. Heat-inactivated plasma significantly reduces the efficiency of platelet removal.


Clomiphene citrate in the management of infertility associated with shortened luteal phases. Repetitively short luteal phases were found in eight infertile women. The short luteal phase was defined as 10 days or less from the presumed time of ovulation (as assessed by basal body temperature recording) to the onset of menses. Clomiphene citrate (Clomid) therapy resulted in pregnancy in two patients and lengthened the luteal phase in the other six. Ultimately, seven of eight patients conceived during Clomid therapy. Clomid therapy can lengthen the luteal phase in patients with luteal temperature elevation of 10 days or less. The occurrence of short luteal phases may be associated with infertility.


Contraceptive properties of endotoxin in rabbits. Endotoxin derived from Salmonella enteritidis-Boivin at a dose of 20 micrograms/kg intravenously interfered with follicular rupture normally induced by human chorionic gonadotropin (hCG) in rabbits. This action was greatest when the endotoxin was given 5 to 6 hours after the administration of hCG. The failure of follicular rupture resulted in entrapment of ova. Endotoxin (5 micrograms/kg) given intravenously to rabbits on day 4 of pregnancy resulted in failure of implantation. Indomethacin (2 micrograms/kg) given intramuscularly concomitantly was unable to reverse this action. Endotoxin (5 micrograms/kg) given intravenously to rabbits on day 8 of pregnancy had an immediate lethal action on embryonic development, and this effect was inhibited by concomitant indomethacin treatment. Nevertheless, most fetuses died after the indomethacin treatment. Whether this was due to a direct toxic action of indomethacin or to a secondary action of endotoxin not blocked by indomethacin is not clear. Endotoxin did not exert its antifertility actions through a luteolytic mechanism.


Effect of native and modified bull seminal ribonuclease on tumour and testicular cells and phytohaemagglutinin-stimulated pig lymphocytes. The carboxymethylated, oxidized and reduced forms of AS RNase inhibited transplantability and DNA synthesis of tumour cells BP-8 and EL-4 incubated in vitro. With tumour cells EL-4 the results under in vitro conditions did not not correspond to those obtained under the conditions in vivo. The survival of mice given injections of EL-4 cells and of the native and carboxymethylated AS RNase was only slightly prolonged. Mice that received intra-abdominally BP-8 cells and both carboxymethylated and oxidized and reduced forms of AS RNase survived two or three times longer than the controls. Succinylation and maleylation of AS RNase eliminated any antitumoral effect. Aspermatogenic activity of AS RNase was abolished by any modification of the molecule which had substantially reduced, or removed, the RNase activity. Neither native nor modified forms of AS RNase had an inhibitory effect on unstimulated pig lymphocytes. The DNA synthesis of PHA-stimulated lymphocytes was inhibited by the native and carboxymethylated AS RNase only. Bovine pancreatic A RNase had any inhibitory effect on neither tumour nor testicular cells.


Detection of thymosin 5 in calf and mouse thymus and in nude mouse dysgenetic thymus. Thymosin 5 was traced in calf and mouse thymuses by fluorochrome-labelled rabbit anti-calf thymosin. The presence of thymosin 5 or its individual components was found 1. in groups of cortical epithelial cells in calf thymuses and in single cortical epithelial cells in mouse thymuses. 2. In some marginal (blastema) cells of the thymus cortex of calves and 14-day-old mice. 3. In perivascular epithelial cells of the calf thymus. 4. In occasional medullary epithelial cells of the calf and mouse thymus. In all cases there was a marked alternation of entirely negative and positive cell-containing thymic lobuli. In 4 of 13 cases comparatively strong positivity was found in tightyly arranged epithelial cells in an individual acinus in the dysgenetic thymus of nude mice, the positive cases being concentrated among the youngest mice studied.


The failure to induce allotransplantation tolerance with serum in mice. The possibility of allotransplantation tolerance induction with serum in newborn and adult mice was investigated. In comparison with the previous findings in pigs and rats, not even a prolongation of skin graft survival was obtained in the treated recipient mice.


Family formation and the life cycle.

Family formation and the life cycle. Mathematical expressions are developed for certain life cycles when only the age-specific birth and death rates are known. The probability at birth that a woman will have a specified number of children and the expected length of time spent before the first birth, between the first and the last child, and between the last child and the time of the woman's death are shown to be calculable. Expressions for the probability of at least one child outliving the mother and for the expected number of children outliving the mother are also developed and are evaluated for three selected countries with different birth and death rates to show how these life cycles depend on birth and death rates.


Validation of an inferential approach for the measurement of illegitimacy. An inferential model which utilizes surname comparisons in inferring legitimacy status is developed and validated. Criterion validation is employed to assess the level of agreement between the inferential approach and the conventional reporting procedure. Data for the analysis are based on a sample of birth certificates contained in files of the National Center for Health Statistics for calendar year 1973. Results indicate that despite excessive error within some categories of the control variables, the inferential method is generally adequate to obtain reliable estimates of illegitimacy.


Social background composition and educational growth. This paper examines the impact of intercohort changes in social background composition on changes in grade progression rates at selected schooling levels. It presents formal arguments that the relative and absolute effects of background composition on grade progression rates should decline over levels of schooling, and using data for white males born beteen 1907 and 1951, offers empirical support for these arguments. Whereas twentieth century increases in average educational attainment are primarily due to increases in grade progression rates at the elementary and secondary levels, future growth must occur through increases in transition rates beyond high school, given the near universality of high school graduation for cohorts born at midcentury. Our analysis shows that postsecondary progression rates are much less responsive to changes in family background composition than rates in the schooling process. Despite intercohort changes in background composition that are increasingly favorable to educational attainment, future educational growth may be slower than past growth because compositional effects on average attainment will be through progression rates where the effects are weak.


Influence of the base on the results of clinical trials with topical corticosteroids. A comparative trial between betamethasone valerate and halcinonide has shown the former to be superior, thus contradicting the results from other trials. It is thought that the discrepancy is due to differences in the nature of the preparations used in the various studies. It is suggested that note is made of the base used in trials before final conclusions about the efficicacy of steroids are made.


Cryogenic preservation of isolated rat Islets of Langerhans: effect of cooling and warming rates. Isolated rat Islets of Langerhans have been frozen to and stored at -196 degrees. After thawing, these islets were capable of secreting near normal levels of insulin in response to graded glucose challenge. Maximal retention of functional viability as measured by the ability of the islets to secrete insulin in response to a glucose challenge was obtained after freezing islets at a cooling rate of approximately 75 degrees per minute in the presence of 1.0 mol/1 dimethyl sulfoxol followed by warming at rates of greater than 3.5 degrees/minute. The critical freezing parameters include the time and temperature of exposure to dimethyl sulfoxide, the rate of cooling, the temperature of the post-thaw dilution from the freezing medium and the presence of serum in the dilution medium.


Alloxan-induced alteration of insulin release, rubidium efflux and glucose metabolism in rat islets stimulated by various secretagogues. Insulin release and 86Rb efflux were studied in perifused rat islets exposed in vitro to alloxan (2 mmol/l) for 5 min. At a low glucose concentration, alloxan transiently increased 86Rb efflux. Alloxan immediately and completely abolished the secretory response to glucose (15 mmol/l) and markedly delayed the reduction in 86Rb efflux normally produced by the sugar. 3-O-methylglucose (20 mmol/l) provided complete protection against the alteration of 86Rb efflux and partial protection against the inhibition of insulin release. Immediately after alloxan treatment, glyceraldehyde, alpha-ketoisocaproic acid and tolbutamide still induced a rapid release of insulin, but the late phase normally stimulated by glyceraldehyde and alpha-ketoisocaproic acid was inhibited. If islets were exposed to glyceraldehyde or tolbutamide 15 min after alloxan treatment, the rapid insulin release was also markedly impaired. Alloxan failed, however, to affect the ability of these three stimuli to reduce 86Rb efflux from islet cells. Glucose oxidation and utilization were decreased in alloxan-treated islets and 3-O-methylglucose protected against this effect. The results show that the glucose recognition system in B-cells is the most rapidly and severely affected by alloxan. The drug also alters the response to other secretagogues, the insulin releasing properties of which can be impaired without alteration of their ability to reduce 86Rb efflux.


Long loop Roux-Y esophagojejunostomy following total gastrectomy. Excellent results were obtained with total gastrectomy in 42 patients having received one-layer esophagojejunostomy by hand-suturing. This is a safe and easy technique to use and therefore we use it from 1971 in all patients undergoing total gastrectomy.


Genetic analysis of multiple drug cross resistance in Saccharomyces cerevisiae: a nuclear-mitochondrial gene interaction. A mutant of the yeast Saccharomyces cerevisiae, cross resistant to several antibiotics, was isolated in our laboratory and subjected to genetic analysis. Tetrad analysis of diploids obtained from crosses between the resistant mutant and a sensitive wild-type strain suggest that the multiple resistance to the five agents, oligomycin (OLI), rhodamine 6G (RHG), tetracycline (TCN), chloramphenicol (CAP) and cycloheximide (CHX) is determined by a single nuclear gene, ant1, and requires several cytoplasmic genes for expression of resistance to oligomycin, rhodamine 6G and tetracycline. --Vegetatively growing diploid clones derived from the cross ant1 [RHO+] X +[RHO+] show mitotic segregation of two phenotypic classes for the drugs OLI, RHG TCN. Diploids derived from the two reciprocal crosses, ant1 [RHO+] X +[RHO-] and ant1 [RHO-] X +[RHO+], fail to exhibit mitotic segregation. These results are consistent with our hypothesis concerning the involvement of cytoplasmic loci. They suggest, in addition, that these loci are associated with mitochondrial DNA (mtDNA). --Evidence for this association is provided by the demonstration of genetic linkage between the cytoplasmic loci involved in the interaction, RHG-1, TCN-1 and OLI-5, and two well-characterized mitochondrial loci, ERY and CAP. --We have mapped the nuclear ant1 locus 3.3 cM from the centromere-linked gene, leu1, on the same side of the centromere of chromosome VII as leu1. --In the light of these findings, we discuss the claims made by several authors of the episomal nature of mutations similar to the one described here, as well as of the possible involvement of yeast 2 mu DNA in such mutations.


The CAN1 locus of Saccharomyces cerevisiae: fine-structure analysis and forward mutation rates. A system of strains and growth media was developed to allow efficient detection of forward mutation, reversion, complementation, and suppression at the canavanine-resistance (CAN1) locus of Saccharomyces cerevisiae. Genetic fine-structure analysis revealed that the map length is at least 40, and possibly as much as 60 X-ray map units; this is the longest gene map yet reported in S. cerevisiae. Allelic complementation was not observed, despite testing of a large number of allele pairs, and alleles suppressible by the ochre suppressor SUP11 were absent from a sample of 48 spontaneous mutants and occurred infrequently (7%) among a sample of ultraviolet-induced mutants. Infrequent mutant types included canavanine-resistant mutants capable of arginine uptake and alleles thought to represent deletions or inversions. In contrast to previous reports in the literature, the spontaneous forward mutation rate at CAN1 did not increase during meiosis.


Expression of radiation-induced mutations at the arginine permease (CAN1) locus in Saccharomyces cerevisiae. In the yeast Saccharomyces cerevisiae, the expression of resistance to the L-arginine analog, L-canavanine, after mutagenesis, is strongly dependent on the metabolic state of the cell. The frequency of mutations recovered after exposure to ultraviolet light or X rays was measured under a variety of culture conditions. The results indicate that the frequency of mutants recovered is determined by the following three factors: (1) The potential mutants still possess enough permease activity to take up some of the cell poison, and some are therefore killed before they can express the mutant genotype. The sensitivity is strongly influenced by the endogenous free arginine, which is in turn influenced by the growth medium. (2) The rapid decay of the permease molecules and the inability of the potential mutants to resynthesize this protein results in a rapidly increasing change of expression when selection is delayed. (3) During the time when the permease activity is decaying, repair of the mutagen-induced damage appears to occur.


Restriction map of the region surrounding the EcoRI site in the pCR1 plasmid and analysis of an inserted ovalbumin gene. We have determined a restriction map of a 1650 base pair region surrounding the EcoRI site of the bacterial plasmid, pCR1. We have used pCR1 as a vector in cloning synthetic ovalbumin double-stranded cDNA. Using the pCR1 restriction map, we have characterized the ovalbumin sequences inserted in one recombinant plasmid, pOvE12. POvE12 appears to contain all, or nearly all, of the sequences found in full length, double-stranded cDNA synthesized in vitro.


The physical localization of the gene for ribosomal protein S20. As a prerequisite to the examination of the structure and properties of the promoter for ribosomal protein S20 of Escherichia coli, I have determined the cleavage sites in lambdadapB2cI857S7 DNA for four restriction endonucleases. Subsequently, purified fragments obtained after digestion of this DNA with BamI or HindIII have served as templates for coupled transcription and translation. This has permitted the localization of the structural gene for S20 within a 1000 base pair segment of lambdadapB2cIB57S7 DNA . Cleavage of this DNA with HindIII partially inactivates the expression of S20 in vitro, implying that one HindIII site lies in or near a region essential for the expression of S20.


Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter. Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, have been constructed that carry four different single restriction sites down-stream from the phage lambda promoter pL. The promoting activity of pL is switched off at low temperature in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction. cIts was located either on the host chromosome, or on a second plasmid pRK248 that is compatible with the cloning vehicle, or on the vehicle itself. Three different restriction fragments, each carrying the gene trpA of Salmonella typhimurium or Shigella dysenteriae, have been inserted into the EcoRI, BamHI and SalI sites, respectively, of these plasmids and pL dependent expression of the inserted gene in Escherichia coli was determined by measuring the enzymatic activity of the trpA gene product. Heat induction resulted in a level of expression of trpA corresponding to 1 to 6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.


Effects of centrophenoxine on the monovalent electrolyte contents of the large brain cortical cells of old rats. The effect of centrophenixine has been studied on the monovalent electrolyte contents of the brain cortical cells of old female Wistar rats by means of energy dispersive x-ray microanalysis. The drug was administered in form of an intraperitoneal injection of 100 mg/kg body weight for 20 days. The treatment decreased the potassium content in both the nucleus and cytoplasm in terms of dry mass fraction, whereas the sodium concentration remained unchanged. The Na+/K+ ration increased significantly in both cell compartments. The chloride content of the nuclei decreased slightly. If one supposes just 1--2% rehydration of the old cytoplasm, the decrease of the total ionic strength may be of considerable extent, it may even reach the values obtained in young adult animals. The results are interpreted in terms of a recently developed 'membrane hypothesis' of aging.


Double-blind comparison of cimetidine and placebo in the maintenance of healing of chronic duodenal ulceration. Patients suffering from chronic duodenal ulceration were allocated at random to treatment with either cimetidine (400 mg twice daily) or matching placebo for six months. Before entry to the trial all patients were shown to have healed ulcers on endoscopy. Most of the patients had participated in a one-month trial of cimetidine during which their ulcers healed. The trial showed that four of 29 patients relapsed on maintenance treatment with cimetidine, which therefore did not confer complete immunity from relapse. However, cimetidine treatment was very much better than placebo treatment, on which 18 of 31 patients relapsed. Of the 22 patients who relapsed clinically, 20 were submitted to endoscopy and 19 of these were shown to have ulcerated again. Endoscopy at the end of the trial showed that ulcers had also redeveloped in five of 28 asymptomatic patients. Length of previous dyspeptic history had no bearing on the results of the trial but there was evidence that relapse on placebo was less likely if the ulcer had originally healed on a high dose of cimetidine. Clinical relapse was associated with worsening duodenitis. Symptoms, clinical observation, and laboratory tests showed no important abnormalities in the patients.


[Malignant cutaneous lymphoma. Histology and clinical aspects with special reference to plastic-embedded tissue specimens]. The basis of this paper is the terminology of Non-Hodgkin's lymphomas based on Gerard-Marchant, Hamlin, Lennert and others, and known as the "Kiel Classification". We have come to the opinion through histological studies of 70 malignant skin lymphomas, that this concept can also be used for malignant lymphoreticular proliferations of the skin, especially since advances in cell differentiation have shown lymphatic origin of different skin tumors.


[Cefazedone in infectious diseases in gynecology. Clinical tests on efficacy and tolerance]. A clinical trial on efficacy and tolerance of Cefazedone, a new semisynthetic Cephalosporine-derivate, was carried out in a gynaecological clinic on 75 patients. In 38 of these cases, besides the gynaecological affection, urinary tract infections were determined microbiologically (E. coli, P. mirabilis, klebsiella or enterococci). Inflammation of the lower abdominal area was established in the remaining 37 patients (adnexitis, pelvic peritonitis, endometritis, salpingitis). The average duration of treatment was 10 to 11 days. Cefazedone was injected intravenously twice a day, the daily dose was 2 to 4 g. The clinical assessment of the treatment was very good in 28 cases, good in 43 cases, moderate in 3 patients and poor in 1 case. All initially identified pathogens were eliminated after treatment. A slight transitory diarrhea was observed in 5 cases. There were no further side-effects. The values of gamma-GT, serum-creatinin, hemoglobin and erythrozytes were controlled before and after medication and showed no detremental effect of the drug. The local tolerance of Cefazedone was good in more than 90% of the cases treated.


[Lipoid A, a factor in the pathogenesis of chronic pyelonephritis. Experimental and clinical studies of a lipoid A dependent pathological immune reaction]. Lipid A injected into the temporarily occluded renal pelvis of adult dogs, persisted in the kidney tissue and induced an abacterial interstitial nephritis with positive anti-lipid A titers. This reaction was increased by a single dose of lipid A vaccine, reduced by four consecutive immunisations prior to the lipid A injection and absent in puppies. The presence of IgG, IgM and complement complexes in the kidney was demonstrated by immunofluoroscopy. Lipid A antibody titers were measured by the passive hemolysis test in 349 humans. In two out of 20 healthy adults and 16 out of 18 children with recurrent urinary tract infection anti-lipid A antibodies were present. In contrast, no titers were found in 23 newborn babies. In a group of 156 patients with acute urinary tract infection, 28% revealed positive titers, whereas in a group of 132 patients with recurrent urinary tract infection titers occurred in 81%. Selected from this group of 132 patients 61 suffered from an acute infection of the upper tract. 59 oft these (96%) showed definite titers. There was no difference in the development of anti-lipid A antibodies between men and women and the height of the titers did not correlate with the clinical picture of the disease (acute or chronic). The combination of proteinuria and anti-lipid A antibodies indicates the pressure of reccurrent urinary tract infection or chronic pyelonephritis with about 90% accuracy. The titers are caused by immunogenically active lipid A in the body. Since lipid A has the ability to remain in the renal tissue for a long period of time and thereby to maintain the inflammatory response, long-term antimicrobial prophylaxis (six months) should be given to patients with a high risk of recurrent urinary tract infection.


Ethology and veterinary science.

Ethology and veterinary science. After a brief introduction on the scope and methods of ethology, this review analyses the development of the concepts of normal and pathological behaviour in accordance with the ethological approach. Finally there is a discussion on the possible functions of ethology in the veterinary field.


The identification of gonadotropin-releasing hormone (GnRH) in hypothalamic and extrahypothalamic loci of the human nervous system. Immunoreactive-like GnRH activity has been identified in 24 of 26 separate loci of the human central nervous system. Tissues, secured from 5 brains at autopsy, were dissected, extracted sequentially with 2N and glacial acetic acid, lyophilized, and eluted in buffered saline for GnRH determinations by specific radioimmunoassay. GnRH concentrations (ng/mg protein) ranged from 8.96 (infundibulum) to 0.001 (cerebellum. middle lobe). Highest extrahypothalamic concentrations of GnRH were found in mamillary body (0.076) and thalamus (0.002). Extrahypothalamic GnRH was identical to synthetic and hypothalamic GnRH by criteria of immunoidentity. No post-mortem GnRH peptidolysis, evaluated experimentally in rats, was evident between 0 and 16 hrs in intact tissues maintained at 4 degrees C. These data suggest that GnRH is distributed throughout regions of the human brain outside the hypothalamus and suggest new, non-endocrine functions for GnRH in the human CNS, analogous the those reported recently for GnRH in experimental animals.