Mechanism of action of rifamazine, a member of a new class of (dimeric) rifamycins. 1. Rifamazine (AF/RP) a dimeric rifamycin, is active against bacterial DNA-dependent RNA polymerase and against viral RNA-dependent DNA polymerase. 2. Rifamazine is active also against DNA-dependent RNA polymerase extracted from rifampicin-resistant mutants of Escherichia coli. It does not interfere with enzyme-template interaction or with RNA elongation. It blocks initiation. 3. A comparison is made between the mechanism of action of rifamazine and that of rifampicin, and of AF/013 (octyloxime of 3-formylrifamycin SV), a C-class rifamycin. Our results show that the mechanism of action of rifamazine is more similar to that of rifampicin than to that of the octyloxime derivative. 4. Activity of rifamazine against RNA polymerase from rifampicin-resistant mutants is thought to be due to binding of the dimer to both the rifamycin-specific binding site and to a second weak site.


Non-helical sequences of rabbit collagen. Correlation with antigenic determinants detected by rabbit antibodies in homologous regions of rat and calf collagen. Non-helical peptide fragments were isolated from rabbit skin collagen after cleavage of alpha chains with cyanogen bromide and proteases. Determination of their amino acid sequence indicated a length of 9, 16 and 25 amino acid residues for the non-helical sequences located in the N-terminal region of alpha2 and alpha1 chain and in the C-terminal region of alpha1 chain, respectively. The C-terminal sequence Tyr-Tyr hitherto considered as the genuine end of collagen alpha1 chain is in part of rabbit collagen extended by two residues, alanine and arginine. Rabbit collagen may differ considerably in its non-helical sequences from other vertebrate collagens, particularly in the C-terminal part. Some but not all of these differences are clustered in areas occupied by antigenic determinants which are recognized in the antibody response of rabbits to rat or calf collagen. On the other hand, a high homology to rabbit collagen, e.g. in the N-terminal region of rat collagen alpha1 chain or calf collagen alpha2 chain, probably prevents immunological recognition by the rabbit. The degree of foreignness alone, however, may not necessarily determine whether a particular non-helical area is able to express immunogenic activity.


Serum protein pattern of mice during infection with single and repeated doses of Ancylostoma caninum larvae. Electrophoretic pattern of serum protein in mice during Ancylostoma caninum larval infection with various single and repeated doses were observed to ascertain whether these changes take place according to their increase in dose levels. Comparison between single and repeatedly infected groups were also made statistically.


Demonstration of membrane-bound immunoglobulin determinants and the significance of a quantitative characterization of the antisera used. Lymphocytes from 22 healthy blood donors were tested for the presence of immunoglobulin determinants on their surfaces by indirect membrane immunofluorescence. The proportion of MemIF+ cells was, on the average, 8.7% (3-18) for IgM and 15.8% (8-28) for IgG. The small differences in the proportion of MemIF+ lymphocytes upon use of various anti-IgM sera could be attributed mainly to the different antibody concentrations. The importance of a quantitative characterization of the antisera used was pointed out.


[Influence of hormonal contraceptives upon carbohydrate metabolism in women with abnormal glucose assimilation during previous pregnancy (author's transl)]. Carbohydrate metabolism was examined--by means of the 100-gm-glucose standard tolerance test--in 23 patients showing a decrease in glucose tolerance during pregnancy; the examinations were carried out postpartum and after a three months' intake of a hormonal contraceptive (compound preparation: mestranol 0.1 mg,  lynestrenol 1.0 mg). The peak values and the two-hour-values, being statistically significant, showed a normalization after delivery, and furthermore again a deterioration of glucose tolerance during the intake of the preparation. A remarkable increase in weight, as a possible cause for the dysbolism during contraception, was not observed.


Placental antigens and fetoproteins in the urine of rats - indicators of resorption of conceptuses. Placenta-specific antigens and alpha-fetoprotein were detected in the urine of rats with induced abnormal pregnancies. Moreover, in those cases, in which the presumptive diagnosis of intrauterine fetal death or resorption was made, urinary excretion of placental antigens persisted for at least 5 days.


Effects of antibiotics on the life cycle of Neurospora crassa. Some antibiotics and synthetic inhibitors affect, in several ways, the life cycle of Neurospora crassa (germination of conidia leads to myceliar growth leads to formation of conidia). Bikaverin, cyanein, scopathricin and phenethyl alcohol retard the germination of conidia, without inhibiting it completely. 5-Fluorouracil, ramihyphin A and zygosporin A (cytochalasin D) do not inhibit the germination. Bikaverin brings about a thickening of the hyphae of growing mycelium. Ramihyphin A, cyanein, scopathricin and zygosporin A stimulate the ramification of hyphae while 5-fluorouracil and phenethyl alcohol do not affect the myceliar morphology apart from their inhibitory effect on growth. Actinomycin D, 5-fluorouracil, cycloheximide, ramihyphin A and partially also sodium iodoacetate inhibit to a different degree the photoinduced formation of conidia. The inhibition by 5-fluorouracil is very conspicuous when the agent is present during the photoinduction but considerably weaker when it is applied 2 h after the photoinduction.


Protein polymorphism in the Hanuman langur (Presbytis entellus). 26 specimens of Presbytis entellus were examined for a variety of blood proteins. In contrast to previous studies of other species of leaf monkeys, our P.entellus sample proved to be very heterogeneous. Polymorphisms were found in the third component of complement, group-specific component, glycine-rich beta-glycoprotein, alpha1-antitrypsin, phosphoglucomutase, 6-phosphogluconate dehydrogenase, adenylate kinase, superoxide dismutase, malate dehydrogenase, and phosphohexose isomerase. Variable band strengths that might represent polymorphism were found in acid phosphatase and lactate dehydrogenase. Further analyses of the P. cristatus sample studied by Barnicot and Hewett-Emmett failed to disclose variation. The interpretation of blood protein variability in relation to sample collection and population structure is discussed.


Relevance of three-dimensional reconstructions of stain distributions for structural analysis of biomolecules. The relevance of a three-dimensional model of a protein molecule is discussed in the context of artefacts which inevitably occur during the preparation and imaging process. It is emphasized that even under optimistic assumptions, there-construction of a structure complementary to a given spatial stain distribution cannot, at resolutions below 10-15 A, be regarded as being identical with the real protein structure.


Some hereditary blood factors of the Bengali Muslim of Bangladesh (red cell enzymes, haemoglobins, and serum proteins). In a sample of Bengali Muslems from Dacca, haptoglobin, group-specific component, haemoglobin, adenosine deaminase, adenylate kinase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, acid phosphatase and several other red cell enzyme types were studied. For most serum protein and red cell enzyme systems the gene frequencies are similar to those in other populations to the west of Bangladesh, but others suggest affinity with populations to the east.


Relative position of trypsin banded homologous chromosomes in human (female) metaphase figures. "Generalized distances" between centromeres were statistically analyzed (chi2 test) on 50 normal female trypsin-banded metaphase figures. This study revealed that the homologous chromosomes of the pairs 13, 17, 14, and 21 lie closer together than would be expected by a reference distribution, and this in a statistically significant way. The same relative position was demonstrated for the chromosome groups 13-14, 13-21, 14-21, 15-22, and 14-22. Evidences were collected that also showed that homologous chromosomes of the pairs 1, 19, and 20 and the chromosome groups 15-21, 13-15, and 18-20 tend to lie closer together. Giving a functional interpretation to the phenomenon of non-random distribution of chromosomes in metaphase figures, it may be suggested that the chromosomes 13, 14, and 21 are involved in the organization of the human nucleolar organizers, more frequently than the other D- and G-group chromosomes.


Group-specific component in Macaca.

Group-specific component in Macaca. The group-specific component in 17 Macaca mulatta was examined. All the individuals revealed the same Gc 1--1 pattern.


The human complement after trypsin pretreatment as compared to the Paris standard. A composite G-banding diagram after trypsin pretreatment of metaphase chromosomes from 5 different individuals (2 males and 3 females) is presented and compared with the Paris diagram. The patterns obtained by the present technique were very similar to those previously reported. It was found that the darkly staining bands were much more consistent in appearance than the lightly staining bands and that there was little individual variation.


Variation in trypsin banding at different stages of contraction in human chromosomes and the definition, by measurement, of the "average" karyotype. Ideograms of chromosomes obtained by the trypsin/Leishman method are presented in which the size and position of the bands of chromosomes at different stages of contraction have been defined by measurement. A hypothesis is suggested which could explain the changes observed in the banding pattern between stages of condensation.


Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining. The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.


Relative DNA content of human euchromatin and heterochromatin after G, C and Giemsa 11 banding. Human chromosomes and interphase nuclei labeled with 3H-thymidine and treated with the ASG and trypsin technique for G banding show no DNA loss. However, after G 11 and C banding significantly more DNA is removed from euchromatin than from constitutive heterochromatin.


Interchromosomal DNA-containing fibres in human cells. Interchromosomal fibres were observed by light microscopy in metaphases of HeLa cells and human lymphocytes prepared by spreading on a water surface. Autoradiography after labelling with tritiated thymidine showed that the fibres contained DNA. They were resistant to pronase treatment.


Identification of interspecific translocation chromosomes in human-Chinese hamster hybrid cells. Chromosome material of human and Chinese hamster origin can be stained differentially in human-Chinese hamster translocation chromosomes by use of the Giemsa-11 staining technique.


Lymphocyte stimualtion by a mammary carcinoma assoicated glycoprotein. A glycoprotein was isolated from 3M KCl extracts of mammary carcinoma. Addition of an optimal amount of the carcinoma associated glycoprotein induces proliferation of both peripheral blood lymphocytes from normal donors and from patients with mammary carcinoma. RNA-dependent DNA-polymerase levels were found to be markedly increased in glycoprotein-untreated lymphocytes from patients with mammary carcinoma and in glycoprotein-treated lymphocytes of both normal donors and tumor-bearing patients. A factor ("Blocking Factor") was eluted at pH 3.1 from the cell membrane of the mammary carcinoma cells. Addition of an optimal amount of this factor inhibits the glycoprotein induced stimulation of the RNA-dependent DNA-polymerase activity in glycoprotein-treated lymphocytes of both normal donors and tumor-bearing patients.


Inhibition of T- and B-lymphocyte functions by normal immunosuppressive protein. Normal immunosuppressive protein (NIP) isolated from human plasma was studied in two well defined systems. (1) Spontaneous rosettes of sheep red blood cells with human peripheral blood lymphocytes and PHA-induced lymphocyte cytotoxicity as indicators for T-cell function. (2) Rosette formation tests of human lymphocytes with antibody-coated erythrocytes or erythrocytes coated with antibody and complement as well as antibody-induced lymphocyte-mediated cytotoxicity represented non-T-cell activity. While NIP did not inhibit the formation of any of the above mentioned rosettes, it practically prevented both PHA-induced and antibody-mediated lymphocyte cytotoxicity. Relatively small amounts of NIP inhibited PHA-induced cytotoxicity while higher doses were required for the inhibition of antibody-mediated lymphocyte cytotoxicity. Possible mechanisms of its suppressive activity are discussed. NIP was found to be heat-stable and did not show any species specificity, as NIP preparations from human plasma were immunosuppressive in human, mouse and guinea-pig systems.


Alteration of the antibody response to Escherichia coli O antigen in mice by prior exposure to various somatic antigens. In the present study in mice we used the Jerne plaque assay to compare the immunity enhancing potential of different Gram-negative bacteria with special regard to their endotoxin. The results confirm the recent finding that injection of Escherichia coli bacteria of various serotypes may enhance the IgG antibody response to the O antigen of a serologically unrelated E. coli strain injected subsequently, but may suppress the IgM antibody formation. The O antibodies formed were of low avidity but were antigen specific. Smaller amounts of antibodies were formed to a serologically unrelated antigen, E. coli O76, which had not been injected. Of the strains tested as primary stimuli E. coli O4 gave considerably greater enhancement than any other serotype including the homologous E. coli O6, when a short interval between the injections was used. The influence of O4 on the serologically unrelated anti-O6 response was stronger than on the response to the cross-reactive E. coli O18 antigen, suggesting that O antigen cross-reactivity is not the basis for the immunomodulation. Formalin-killed bacteria were more effective in this respect than boiled bacteria or purified lipopolysaccharide and rough mutants (E. coli R1--R4) and E. coli O4 were less effective than many of the other smooth E. coli. These findings suggest that shared determinants in the lipid, basic carbohydrate core or Kunin common antigen portions of the endotoxin do not play the major immunomodulating role in this system. Salmonella reading but not Pseudomonas aeruginosa affected the anti-E. coli O6 response in a similar manner. One explanation for the alterations in the immune response observed implies the presence of an antigen determinant shared by many Enterobacteriaceae in such a position in relation to the O antigen that it can be utilized for cellular co-operative events in the O antibody response. The protein portion of the endotoxin protein--lipid--carbohydrate complex is a possible location.


A radioisotopic method to measure delayed type hypersensitivity in the mouse. I. Studies in sensitized and normal mice. Delayed type hypersensitivity (DTH) lesions have been difficult to evaluate objectively in the mouse because they are usually assessed in terms of an increase in footpad swelling or ear thickness. We have developed a radioisotopic method which not only reduces the observer's error but also gives an objective measurement of the cellular activity in the lesions. In brief, 10 mul of the test antigen is injected intradermally into the left pinna and either nothing or the same volume of a control solution into the right. 10 h later, a 2-muCi pulse of 5-iodo-2'deoxyuridine-125I is given intravenously, the ears cut off at the hairline 16 h later and the radioactivity counted in a gamma spectrometer. The following was obtained as evidence that the increased radioactivity of the left pinna over the right was a measure of the extent of a DTH response: (1) the ear reaction was delayed in mice without serum antibodies becoming maximum at 24 h; (2) there was a mononuclear cell infiltration in the left pinna and autoradiographs revealed radioactive label bound to these cells; (3) athymic mice could not develop a 24-hour ear reaction, and (4) antigens known not to activate T cells did not elicit the ear response. Cell transfer studies will be described in a subsequent paper. Different sensitization regimes were required with different antigens in order to obtain the highest levels of DTH as tested by the ear response, and the maximal ear reaction occurred at different days. The ear reaction showed the specificity expected of a DTH response.


Detection in colorectal carcinoma patients of antibody cytotoxic to established cell strains derived from carcinoma of the human colon and rectum. Sera from eight of 15 patients with colonic carcinoma exhibited demonstrable cytotoxicity against an established cell strain derived from adenocarcinoma of the ileocecum, HCT-8. Sera from 12 of 16 patients with rectal carcinoma were cytotoxic for an established cell strain derived from an adenocarcinoma of the rectum, HRT-18. Patients with colonic carcinoma exhibited serum cytotoxicity against only the colonic target cells, whereas patients with rectal carcinoma gave significant cytotoxicity against both cell strains. This cytotoxicity was shown to be complement-dependent and appeared to be specific for colonic and/or rectal carcinoma cells. Although the cells produced carcinoembryonic antigen (CEA) in vitro, the cytotoxic antibody response in these patients did not appear to be directed against CEA. Serum cytotoxicity was not demonstrated against two other cell strains, HCT-48 and HT-29, derived from adenocarcinomas of the human colon, except for a reaction against a blood-group-related antigen. These cell strains had comparable levels of cell-associated CEA. The routine titration of cytotoxic antibody against these established cell cultures may provide meaningful information on the host's immune response to colorectal neoplasms.


Specificity of cell-mediated cytotoxicity against human melanoma lines: evidence for "non-specific" killing by activated T-cells. The specificity of cell-mediated cytotoxicity against melanoma cells in vitro has been analyzed in a large number of studies with cells both from normal and melanoma subjects. As in a number of other, recent, similar human studies, no evidence for tumour specificity was found. Effector cells in peripheral blood responsible for the cytotoxic raction were examined by cell separation methods based on red cell rosette formation and separation through Hypaque-Ficoll mixtures. The evidence suggests that non-specificity results from killing by cells separating largely in the non-sheep red blood cell rosetting fraction and which have cytotoxic specificity directed broadly to cells with abnormal membranes. Further analysis revealed that the cells were non-phagocytic and did not bear receptors for complement. They appear to be activated into cell division and to bear surface receptors for the Fc portion of IgG. Additional evidence is presented suggesting that the cells mainly responsible are activated thymus-dependent cells present in the circulation of both tumour-bearing and normal subjects.


Non-organ-specific and tumour-specific antibodies in children with Wilms' tumour. Sera from groups of children with Wilms' tumour, with non-renal solid tumours, and from control children were studied by immunofluorescence and by absorption techniques. A high proportion of sera from both tumour groups contained non-organ-specific auto-antibodies. Three of the 45 sera in the Wilms' group and one of the 27 sera in the "other" solid tumour group contained antibody reacting with normal kidney cells. Only one of 45 sera in the Wilms' tumour group contained tumour-specific antibody.


Studies of lymphocyte stimulation by intact tumor-cell and solubilized tumor antigen. BALB/c mice were rendered immune to syngeneic SV40-induced sarcoma by subcutaneous injection of mKSA-TU5 tissue-culture adapted cells. Spleen cells from immune mice were examined for tumor-cell neutralization in the Winn assay as well as in in vitro lymphocyte stimulation assays. A microculture (200 mul) lymphocyte stimulation (LS) assay utilizing immune spleen cells was employed with mixed lymphocyte/tumor-cell cultures (MLTC) and the papain crude soluble (CS) extracts from mKSA-TU5 cells. Specificity in the LS assay was determined using spleen cells from mice immune to other syngeneic tumors and by soluble antigenic preparation of normal BALB/c spleen cells. The Winn assay studies demonstrated that spleen cells from mKSA-sensitized mice neutralized mKSA tumor cells and this was corroborated by their resistance to direct tumor challenge. Positive lymphocyte transformation responses in MLTC were observed when mKSA-TU5-sensitized spleen cells were mixed with mitocycin-C-treated intact tumor cells or when papain-solubilized antigens of mKSA cells were employed, but not with non-immune spleen cells or with a soluble antigen from normal cells. Papain-solubilized antigen preparations employed in in vitro assays also immunized against challenge with mKSA tumor cells. Specificity of these lymphocyte transformation reactions was demonstrated with non-sensitized lymphoid cells or lymphoid cells from mice sensitized with a syngeneic Kirsten virus-induced respond. Thus, mKSA tumor surface antigens were recognized on intact tumor cells or with microgram quantities of papain-solubilized extracts from these tumor cells. We believe the lymphocyte stimulation assay affords a method for demonstrating the presence of tumor-specific antigen and for monitoring further purification procedures.


Sequential in vitro reactivity of lymphocytes from melanoma patients receiving immunotherapy compared with the reactivity of lymphocytes from healthy donors. A microcytotoxicity technique was used to determine the sequential in vitro reactivity against melanoma cells of lymphocytes from melanoma patients receiving immunotherapy and from healthy donors. Lymphocytes were collected 2 weeks for 2-3 months and were stored in liquid nitrogen until use. Preliminary studies had indicated that freezing did not effect the reactivity of lymphocytes. Lymphocytes from 10 healthy donors tested against melanoma cells exhibited substantial reactivity which showed no consistent pattern over time. Lymphocytes from 9 melanoma patients exhibited increased reactivity after immunotherapy. Patterns of reactivity against melanoma cells and against bladder carcinoma cells were similar, indicating lack of specificity for melanoma antigens. Correlations with clinical course of the disease were not apparent.


Leukocyte adherence inhibition and specific immunoreactivity in malignant melanoma. The leukocyte adherence inhibition (LAI) test has been used to assess specific anti-tumour immunoreactivity in 80 patients with malignant melanoma, 21 of whom had apparently been successfully treated by surgery, and 44 control subjects. Reaction with melanoma extracts in vitro enabled the activity of blood leukocytes to be detected by inhibition of their adherence to glass, while serum was tested for factors which modified this inhibition. Of the patients with tumours (ranging from primary melanoma in situ to advanced disseminated disease), 22/24 had active leukocytes and 50/58 has serum blocking factor; two of the sera, from patients with regressing tumours were unblocking. After surgery with no clinical recurrence, leukocytes continued to be active except when tested several years after operation. Blocking factor rapidly disappeared in 16/20 patients tested, and in several patients examined serially the serum became unblocking. In three cases, persistence of serum blocking was followed by clinical diagnosis of metastases. Leukocyte activity was nerver detected in control subjects (0/10), many of whom had other kinds of tumours or skin lesions. Blocking activity in serum was found in only 3/38 controls with no history of melanoma (1 had a fibrosing cellular blue naevus and 2 had liver disease). Thus the LAI test correlated well with clinical and pathological findings, and shows great promise for the reliable, rapid and specific immunodiagnosis of malignant melanoma.


Severe arterial hypertension caused by chronical abuse of a topical mineralocorticoid. A 24-year-old woman was referred to our clinic because of severe hypocalemic hypertension and latent myocardial insufficiency. Plasma renin activity and plasma aldosterone concentration were lowered. Plasma cortisol concentrations and the urinary excretion of vanillin mandelic acid were found within normal range. Some efforts to identify a specific adrenal enzymatic defect were unsuccessful. By combined therapy with spironolactone, methyldopa and clonidine, the blood pressure was significantly lowered, but not normalized. Triamterene had also an expressed hypotensive effect and induced orthostatic phenomena. After six months of hypotensive drug therapy, the patient's mother reported that her daughter consumed for more than seven years high doses of special nasal drops containing, beside ephedrine and naphazoline, the potent mineralocorticoid 9-alpha-fluoroprednisolone. A strong drug-dependence to the naphazoline component could be evaluated. The application of the mineralocorticoid was stopped, and only a slight low-sodium diet had to be added to restore the long-standing elevated blood pressure to normal


The metabolic spectrum of halofenate.

The metabolic spectrum of halofenate. Halofenate is a new antilipemic drug which is an ester of carboxylic acid. In dosages ranging from 10 to 15 mg/kg, halofenate produces prominent decreases in serum triglycerides and uric acid, with only a slight reduction in serum cholesterol. The decrease in serum uric acid is due to augumented uricosuria. Halofenate is less antilipemic, but more antiuricemic than clofibrate. It apparently interferes with the protein binding of some hormones (thyroid) and drugs (tolbutamide), which can produce changes in the laboratory or clinical findings. In general, halofenate was well tolerated, except for occasional gastrointestinal symptoms.


Tumor growth inhibition by ammonium chloride-induced acidosis. Ammonium chloride-induced metabolic acidosis decreases the growth of various experimental tumors. Spleen exhibits the same effects. There is a sex factor which seems to affect the growth of both tumor and spleen. The observed tumor inhibition appears to be related to a systemic impairment of the anabolic mechanisms. The decrease in tumor calcium suggests that this element may play a role in the tissue growth. The possible implication of cell membrane changes and of a block in glycolysis in the acidosis effects are discussed.


The thyrotropin-stimulation test in patients with solitary hot nodules of the thyroid gland. Thyrotropin (TSH)-stimulation tests were performed on 21 patients with solitary hot nodules in the thyroid gland. Twelve of the patients were euthyroid and nine were hyperthyroid. The average age of the euthyroid patients was 38 +/- 14 (SD) years, and that of the hyperthyroid patients, 55 +/- 8 years. Prior to TSH stimulation, the scanograms of the euthyroid patients showed 131I uptake only in the nodules, while after the TSH test, almost equal uptake was observed in both lobes. The scanograms of the hyperthyroid patients that showed 131I uptake only in the area of the nodules prior to the TSH test showed no appreciable change after TSH stimulation.


Relationships between mitochondrial content and glycogen distribution in porcine muscle fibres. Serial sections of longissimus dorsi and rectus femoris muscles from 15 Yorkshire breed pigs (live weights 24-46 and 49-139 kg) were stained for glycogen (PAS) and a mitochondrial enzyme (NAD tetrazolium reductase). Muscle fibres with a low mitochondrial content in both muscles were more frequently PAS-positive than fibres with a high or intermediate mitochondrial content. However, some pigs had all their muscle fibres PAS-positive while one pig with a high post-mortem muscle pH had all rectus femoris fibres PAS-negative. Relative to lighter weight pigs, longissimus dorsi muscles of heavy pigs tended to have less fibres with a high mitochondrial content and less fibres with a positive PAS reaction. Compared to longissimus dorsi muscles, rectus femoris muscles had more fibres with a high mitochondrial content and less with a positive PAS reaction. All fibres in both muscles became PAS-negative with an accompanying decrease in pH by 24 hr post-mortem. Fibres from longissimus dorsi muscles frequently had PAS-positive sarcoplasmic cores between their myofibrils. Heavy pigs tended to have larger cores (up to a mean maximum diameter of 13.4 mum), more fibres with cores, and more cores per fibre. The pigs involved exhibited no other ante- or post-mortem muscle abnormalities.


The establishment of a venereal disease clinic: II. An appraisal of current diagnostic methods in uncomplicated urogenital and rectal gonorrhea. Some of the diagnostic procedures for uncomplicated urogenital and rectal gonorrhea were studied in a university-based venereal disease clinic. On the basic of our results, recommendations are made concerning the types of media to be used, sites to be sampled, methods of obtaining specimens, and other diagnostic considerations. We conclude that ongoing quality control including a review of diagnostic procedures should be carried on in all venereal disease clinics.


How to make the diagnosis of rectal gonorrhea in men. A simple quick method of diagnosing male rectal gonorrhea, applicable to office practice, is described using conventional staining techniques and microscopy. Photomicrographs of clinical smears serve as a guide line to practitioners. Neigon Jembec plates proved superior to Transgrow media for the culturing of isolates from 100 patients who had shown gram-negative diplococci at the initial microscopic investigation. Heretofore, lack of standardized diagnostic techniques have left male rectal gonorrhea a seldom recognized potential reservoir of infection.


Macromolecular synthesis in Streptomyces antibioticus: in vitro systems for aminoacylation and translation from young and old cells. In vitro systems for the aminoacylation of transfer ribonucleic acid (tRNA) and for polypeptide synthesis have been constructed from young (12-h cultures, not producing actinomycin) and old (48-h cultures, producing actinomycin) cells of Streptomyces antibioticus. When Escherichia coli aminoacyl-tRNA synthetases were used to acylate S. antibioticus tRNA's, it was observed that, per absorbance unit of tRNA, the tRNA's from 48-h cells had a lower ability to accept the amino acids, leucine, serine, pheynlalanine, methionine, and valine than did the tRNA's from 12-h cells. Individual differences were observed between aminoacyl-tRNA synthetases from 12-h cells and those from 48-h cells with respect to the rate and extent of aminoacylation of E. coli tRNA with the five amino acids listed above. In vitro systems for the synthesis of polyphenylalanine have been constructed from 12- and 48-h cells. Ribsomes and soluble enzymes from 12-h cells are more efficient than those from 48-h cells in supporting polyphenylalanine synthesis, and, although the activity of both systems can be stimulated by the addition of E. coli tRNA, the higher level of incorporation observed in the unstimulated 12-h system (ribosomes and soluble enzymes) is maintained. Indeed, the difference in capacity for polyphenylalanine synthesis between in vitro systems from 12- and 48-h cells is greater when the systems are maximally stimulated by E. coli tRNA. Cross-mixing experiments reveal that enzymes from 48-h cells support a slightly higher level of polyphenylalanine synthesis than enzymes from 12-h cells with ribosomes from either cell type, and that the ribosomes are the primary agents responsible for the decreased efficiency of the in vito system from 48-h cells are compared with that from 12-h cells. To determine whether ribosome-associated factors were responsible for the relative inefficiency of the ribosomes from 48-h cells in translation, salt-washed ribosomes from 12- and 48-h cells were examined for their abilities to catalyze polyphenylalanine synthesis. Even after salt washing, ribosomes from 12-h cells were about five times higher in specific activity (counts per minute of polyphenylalanine synthesized per absorbance at 260 nm of ribosomes) than equivalent amounts of ribosomes from 48-h cells. Analysis of the proteins of salt-washed ribosomes of the two cell types by acrylamide gel electrophoresis suggests that the relative amounts of individual proteins present on ribosomes from 12-h cells are different from the amounts present on ribosomes from 48-h cells. These results are discussed in terms of the regulation of translation in S. antibioticus.


Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second beta-glucosidase. A second extracellular beta-glucosidase (betalarge) of Aspergillus fumigatus was purified to homogeneity and shown to be a glycoprotein, as determined by polyacrylamide gel electrophoresis followed by staining for protein and for carbohydrate. Its molecular weight was approximately 340,000 by gel filtration, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave an apparent molecular weight of 170,000, suggesting that the enzyme has two subunits. The glucosidase contained covalently bound sugars consisting of about 2 mol of glucosamine and 16 mol of mannose per mol of protein. The carbohydrate was found to be attached to the peptide via glucosaminyl leads to peptide linkage, possibly to asparagine residues. At pH 4.5 this enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (Km = 0.88 mM) and cleaved two glucose disaccharides: gentiobiose (beta,1 leads to 6; Km = 0.75 mM) and cellobiose (beta,1 leads to 4; Km = 0.84 mM). Although its activity is similar to that of a previously purified beta-glucosidase (betasmall), the two enzymes differ with respect to their pH activity profiles, substrate specificities, and molecular weights. Also double diffusion tests with anti-betasmall antiserum and both purified beta-glucosidases revealed a nonidentical cross-reaction. Microcomplement fixation of native and periodate-oxidized betasmall suggested that the oligosaccharide chain(s) was not a major antigenic site.


The contribution of phosphorylation and loss of COOH-terminal arginine to the microheterogeneity of myelin basic protein. Two guinea pig myelin basic protein preparations which differed markedly in their contents of high pH electrophoretic or chromatographic forms were studied in an attempt to elucidate the causes of their microheterogeneity. Both total preparations and components isolated therefrom were examined for their amino acid compositions, NH2-terminal and COOH-terminal residues, total phosphorus contents, amd contents of phosphamino acids. The results showed that the five components differed sequentially by a single charge and that the microgeterogeneity arose as a result of secondary modifications of a single secies (Component 1) Of basic protein. Two modifications were demonstrated; viz. phosphorylation of serine and threonine and loss of COOH-terminal arginine. These two modifications were insufficient to account completely for the observed microheterogeneity; an additional cause, deamidation, was postulated. From the relationship between the number of components present in the total basic protein, the phosphorus and phosphoamino acid contents of the components, and the changes in relative electrophoretic mobility of the components which accompanied their phosphorylation and dephosphorylation we conclude that in the native basic protein no more than two sites in any polypeptide chain are phosphorylated.


Cell adhesion to polymeric materials: implications with respect to biocompatibility. The adhesion of radio-labeled chick embryo muscle cells to the surfaces of radiation grafted hydrogels and other polymeric materials was measured in vitro. The degree of adhesion was determined by measuring the percentage of cells which remained adherent to the surfaces after 180 min of contact time (plating efficiency). Plating efficiency was found to vary between 2 and 94% depending on the nature of the surface. Preadsorption of albumin, gamma-globulin or fibrinogen markedly affected subsequent adhesion of cells. Radiation grafted poly(2-hydroxyethyl methacrylate) and poly(N-vinyl-2-pyrrolidone) hydrogels on silicone rubber demonstrated exceptionally low adhesiveness in this assay. The potential for using this cell adhesion assay as a general screening test for biomaterials is discussed.


Cyclic amp and cell morphology in cultured fibroblasts. Effects on cell shape, microfilament and microtubule distribution, and orientation to substratum. The change in shape of 3T3 and L929 cells due to Bt2cAMP treatment is accompanied by altered intracellular distribution of microfilaments and microtubules. Bt2cAMP added to cells in low density culture causes (a) microfilaments to accumulate in bundles near the plasma membrane, mainly at the cell periphery, and (b) microtubules to accumulate beneath these microfilament bundles. In narrow cell processes that form characteristically in Bt2cAMP-treated L cells, microtubules accumulate in parallel arrays near the center of these processes. A new simple method for evaluating the relative distance of the cell from its underlying substratum is desribed. In normal medium, 3T3 cells attach to their substratum near the nucleus and at the tips of cell processes, bridging irregularities in the plastic surface. With Bt2cAMP treatment, attachment occurs at the cell edge and at many isolated points under the cytoplasm, and the cells conform more closely to irregularities of the underlying substratum. A model of the mechanism by which cAMP modulates cell shape is presented.


Ribonucleoprotein components in liver cell nuclei as visualized by cryoultramicrotomy. The interphase nucleus of the normal rat hepatocyte has been studied in ultrathin frozen sections after glutaraldehyde fixation and the modification of various staining procedures known to be specific for DNA structures (Moyne's thallium stain, Gautier's osmium-ammine) or preferential for RNP carriers and basic proteins (regressive stains based on the use of EDTA or citrate, negatively charged colloidal iron). The results are comparable to those obtained after classical dehydration and embedding. Particular attention has been paid to the nucleolus and extranucleolar RNP components, such as perichromatin fibrils and granules, as well as interchromatin granules. A striking observation was the uneven size and the strongly increased number of perichromatin granules, and the appearance of a contiguous interchromatin net, containing nucleoproteins. Cryoultramicrotomy without embedding appears to be very useful for the exploration of the nucleus in thick sections which remain sufficiently transparent even with the usual accelerating voltages.


Androgen-binding proteins in human benign prostatic hypertrophy. Prostatic samples were surgically removed from 7 patients suffering from benign prostatic hypertrophy. High-speed supernatants (cytosol) containing 20-25 mg of protein/ml were prepared. Glycerol gradient ultracentrifugations were performed, using cytosol labeled at 0 C with 2-5 nM 3H-17beta-hydroxy-androstan-3-one (androstanolone or dihydrotestosterone) alone, or in the presence of 50-250-fold excess of androstanolone, estradiol, or androstane-3alpha, 17beta-diol (androstanediol). Two high-affinity saturable binding components were observed. One binding component was the androgen receptor. Its sedimentation coefficient was 8 S in low-salt medium. It had a high affinity for androstanolone. The binding of 3H-androstanolone was strongly completed by androstanolone itself, less by estradiol, and not by androstanediol. In one case, endogenous androstanolone found in the 8 S region of glycerol gradients was measured by radioimmunoassay, and it was calculated that more than 90% of the cytosol receptor binding sites might be occupied by this steroid while the total binding capacity of the 8 S receptor was estimated to approximate 2.6 pmol of androstanolone/g of prostate. No testosterone was found in the receptor fraction. The second binding component was attributable, at least in part, to the sex steroid-binding plasma protein (SBP), as indicated by its sedimentation coefficient (congruent to 4 S in low salt medium), its high affinity for androstanolone and androstanediol and its lower affinity for estradiol, and finally, its migration on polyacrylamide gel electrophoresis. In one instance, the concentration of the SBP-like protein in prostate cytosol was measured by equilibrium dialysis, and it was calculated that the binding capacity of the prostate SBP-like component corresponded to 4 pmol of androstanolone/g of prostate, a small (less than 5%) value with regard to SBP concentration in the plasma of the same patient. The blood contamination of the cytosol, as obtained from the measurement of hemoglobin, did not account for the amount of SBP found in the prostate sample. Since SBP-like protein is probably of plasma origin, to determine whether SBP was located in the extracellular space or inside the prostate cells, BPH slices from another patient were incubated in the presence of 3H-testosterone, the cytosol was prepared, and was fractionated by Sephadex G-150 column chromatography. The androstanolone/testosterone ratio in the receptor-containing peak was high (1.7), whereas in the incubation medium it was very low (0.08). In the peak containing the SBP-like protein, the ratio was 0.74, which may suggest that all or part had been exposed to the predominant androstanolone environment inside the prostatic cell.


Use of electron microscopy for detection of viral and other microbial contaminants in bovine sera. A total of 25 lots of bovine serum samples were pelleted in Beem capsules for thin sectioning and were examined by electron microscopy. These included 17 lots of fetal bovine serum pools and five lots of calf serum pools obtained from commercial sources, and three lots of adult bovine serum from local dairy farms. Virus-like particles, 50 to 300 nm in diameter, were detected in 17 of 25 (68%) of the sera. Five of 25 serum samples showed the presence of mycoplasma-like agents. Incubation of bovine serum at 35 C for 1 or 2 weeks appeared to destroy some of these agents, but in certain instances it enhanced bacteria and bacteriophage contaminants. The advantages of electron microscopy using the thin-sectioning technique for detection of microbial contamination in bovine sera are illustrated.


Improved recognition and quantitation technique for oncornaviruses. A technique to recognize and quantitate oncornaviruses using perforated pointed BEEM capsules has been developed in our laboratory. Virus samples that presented problems in counting, or those which could not be evaluated at all by negative staining, could be clearly defined and counted using the thin-sectioning BEEM capsule technique. Perforation of the BEEM capsule allowed rapid infiltration of reagents into the tip of the virus pellet and made further manipulation and orientation unnecessary. The sensitivity of this technique, determined by making serial dilutions of viral concentrates, allows observation of as few as 5 times 10(5) virus particles per ml. Precision in counting by this technique varied only +/- 0.3 log in repeat aliquots of identical concentrated virus samples quantitated, making this a highly useful and reliable system.


Screen for type-C ribonucleic acid viruses in vaccines using the ribonucleic acid-dependent deoxyribonucleic acid polymerase assay. The ribonucleic acid-dependent deoxyribonucleic acid polymerase assay was used to detect type-C viruses in live virus vaccines. Conditions were first established to maximize the sensitivity of the assay. Vaccines tested included live poliomyelitis, rubella, measles, mumps, and yellow fever. Only yellow fever and measles vaccines known to have been produced in avian leukosis-contaminated cells showed evidence of type-C viruses using the assay. The result of the survey show that the assay has direct practical application to the problem of detecting latent agents in biological products intended for human use.


New method for the removal of extraneous proteins from purified oncornaviruses. Conventional methods (i.e. gradient centrifugation) for the purification of oncornaviruses are usually not effective in complete removal of nonviral proteins. Such contaminants often prove to be a nuisance in subsequent immunological or biochemical studies. Hyperimmune sera prepared from these viruses must be absorbed to assure specificity; cell-derived proteins can be shown to interfere with studies of virus structural proteins, nucleic acids, or viral enzymes. Herein is described a method for removal of most of these contaminants. Viruses are diluted in a high concentration of NaCl to achieve a final concentration of 15%, incubated for 30 min, sedimented, and resuspended in buffer. This procedure results in reductions of up to 48% of the protein without affecting particle count. Immunological, biochemical, and biological properties are not adversely affected. Of the proteins removed, fetal calf serum components and a ribonuclease (presumably cell-derived) were identified. This technique differs significantly from other high-salt methods in that the virus is not precipitated from suspension. It is believed that absorbed proteins are desorbed and left in solution (or suspension) as the virus is sedimented by centrifugation.


Effect of decalcifying agents on the staining of Mycobacterium tuberculosis. Lymph nodes from guinea pigs inoculated with Mycobacterium tuberculosis were fixed in buffered formalin, then treated for the recommended times in Gooding and Stewart's fluid, EDTA, aqueous nitric acid, von Ebner's fluid, and rapid decalcifier (RDC). The blocks were processed to paraffin wax and sections were stained by the Ziehl-Neelsen technique. Only in sections of the blocks treated with RDC were no acid alcohol fast bacilli demonstrable. Hydrochloric acid is a known constituent of RDC and it was found that Myco. tuberculosis is altered by treatment with 2-5M solutions of hydrochloric acid and above and cannot subseuqently be demonstrated by the Ziehl-Neelsen stain. From these results it is recommended that calcified tissue from patients in whom there is a suspicion of tuberculosis should be decalcified with an agent other than RDC.


Effects of ipsilateral and contralateral counterirritation on experimentally produced itch in human beings. The experimental study of itch was reviewed, and an experiment performed to test the effects of ipsilateral and contralateral counterirritation distal to the itching wrist. Each of the 18 subjects served in all experimental conditions, receiving cowage as the itch stimulus and a placebo. Counterirritation consisted of a 10-sec immersion of the fingers into a 2 degrees C water bath. Lateral differences both in response to cowage and to counterirritation were obtained. In general, counterirritation reduced itch significantly more than the control procedure during the treatment period and the first three intervals following treatment. The results of the present experiment suggest a central mechanism attenuating the sensation of itch.


Glycogenolysis and control of anaphylactic histamine release by cyclic adenosine monophosphate--related agents. The relationship of glycogen and glucose to anaphylactic histamine release from chopped sensitized guinea pig lung in vitro was studied. A parallelism was observed between the total amount of glycogen in the sensitized lung and the total amount of histamine released from the lung by antigen-antibody reactions. Removal of glucose from the medium for tissue suspension resulted in reduction in histamine release. Depletion of glycogen and/or glucose from the system was associated with (1) abolition of the inhibition of histamine release by isoproterenol and high concentrations of dibutyryl cyclic adenosine monophosphate (AMP) and (2) increase in the rate of enhancement of histamine release by lower concentrations of dibutyryl cyclic AMP. The results indicate that (1) glycogen may be one of the ultimate energy sources for anaphylactic histamine release, and (2) the presence of adequate amounts of glycogen and/or glucose in the sensitized tissue is necessary for the normal beta adrenergic effects on the histamine release in vitro from sensitized lung fragments.


Glycogenolysis and control of anaphylactic histamine release by cyclic adenosine monophosphate-related agents. II. Modification of histamine release by glycogenolytic metabolites. D-glucose-6-phosphate (less than or equal to 5 x 10(-3) M), pyruvate, lactate (less than or equal to 1 X 10(-2) M), and dibutyryl cyclic AMP (less than 5 X 10(-3) M) were capable of inhibiting anapylactic histamine release in vitro from chopped guinea pig lung. In lower concentrations, pyruvic acid and lactate, as well as dibutyryl cyclic AMP, enhanced the release. Significant synergism was observed betweenpyruvate (5 X 10(-3) M) and isoproterenol (1 X 10(-8) M) in the inhibition of histamine release. The inhibitory actions of isoproterenol, glucose-6-phosphate, and pyruvate were influenced by calcium ion concentration. However, beta blockade, which diminished the isoproterenol effect, was without efect on pyruvate (1 X 10(-2) M) to the release system. Glucose-6-phosphate and isoproterenol did not have this effect. The results, together with a prevouus study, suggest that glycogenolysis may possess a role in the anaphylactic istamine release in vitro from sensitized lung fragments...


Histamine release from human leukocytes: modulation by cadmium ion. The effect of cadmium on histamine release was studied in relation to the role of calcium. The antigenic histamine release from peripheral leukocytes of ragweed-sensitive patients was inhibitied in the presence of cadmium (greater than 10(-5) M). Spontaneous histamine release was not affected by cadmium except for an enhancement observed in high concentrations (greater than or equal to 10(-3)M). Cadmium did not seem to affect the first stage of histamine release but to act mainly on the calcium-dependent second stage. Increasing the level of calcium in the medium could antagonize the inhibitory effect of cadmium. The effect of cadmium could be totally abolished by deuterium oxide and partially by dibutyryl cyclic adenosine monophosphate (AMP) in certain concentrations. These results would indicate that: (1) cadmium acts as an antagonist of calcium and can be used for the study of the role of calcium in histamine release, (2) the action of calcium in the histamine release reaction seems to be related to the microtubular system, (3) cyclic AMP may potentiate histamine release when the action of calcium is inhibited.


The isolation of allergens from the green pea. The aqueous extract of green peas was separated into 3 fractions (albumin, legumin, and vicilin) by dialysis against distilled water and isoelectric precipitation. The major antigenic and all of the allergenic activity of the pea extract was associated with the albumin fraction. The albumin fraction retains its allergenicity upon heating at 60 degrees C for 30 min or boiling at 100 degrees C for 5 min, but becomes partially inactivated by autoclaving at 120 degrees C for 15 min. The allergenic determinant expressed by the albumin fraction appears to be common to several other members of the legume family. In addition, the pea dialysate fraction was shown to specifically inhibit precipitin and passive cutaneous anaphylaxis (PCA) reactions involving rabbit antipea serum and the pea albumin fraction, and histamine release from passively sensitized monkey lung tissue using the serum of pea-sensitive patients.


Applying concepts of visual perception to formats of hospital menus. Standardized printed menu formats for all diets utilizing concepts of visual perception were evaluated in a machine-paced hospital tray-assembly process. Formats of existing menus differed among the various diets. On the redesigned menus, all menu items were arranged in basic groups which were assigned specific positions; groups were accentuated by white strips across the various color-coded selective menus; and accessory items were placed in specific, standard positions on all menus. Criteria for evaluating the effect of using the redesigned menu in tray assembly operations were: overall productivity, individual productivity, and error rate per tray. Data were charted (a) during a control period when the existing menu formats were used to provide baseline data and (b) during an experimental period when the redesigned menu formats were used. Overall productivity was measured by man-minutes per tray. Video tapes of five station operators servicing selected trays were made to study individual productivity. Station operator and checker accuracy were measured in terms of ratio of error-free trays, errors per tray, and errors to possibility of errors per tray. Man-minutes per tray decreased significantly in the experimental period from 2.44 to 2.17--a productivity increase of 11.1 per cent. The individual productivity analysis revealed no significant changes from control to experimental periods. Accuracy of the tray assembly station operators improved significantly. Decreases in ratio of mean number of errors to possibility of errors per tray were recorded in the experimental period. The error rate per tray decreased 44.9 per cent from 0.48 to 0.26, and the ratio of errors to possibility of errors per tray decreased from 6.3 to 3.5 per cent. The percentage of error-free trays rose from 69.9 to 80.9 per cent. Checkers' errors per tray did not change significantly from control to experimental period when data for the two periods were compared. This study provides a practical means of increasing productivity and improving accuracy of the machine-paced tray assembly process.


Stoichiometry of the aldehyde fuchsin staining reaction for proteins. Model systems of agar films containing known concentrations of bovine serum albumin and alpha-chymotrypsinogen were stained with aldehyde fuchsin after oxidation with acidified permanganate solution. These films were scanned in a scanning microphotometer to determine the mean extinction and the total extinction of predetermined areas. Results indicate that the dye binds quantitatively to the proteins. Blocking the acidic side groups of the proteins inhibited the binding of the dye. The degree of inhibition was directly related to the number of sulfhydryl or carboxyl groups that were blocked. Similar blocking reactions performed on the type "A" neurosecretory cells of the pars intercerebralis of the insect Oncopeltus fasciatus gave similar results. Analysis of the dye protein complexes gave a dye to acidic group ratio of 1:1.


Use of KT 50 for orientative evaluation (screening) of sensitivity of flies to insecticides. The author describes a modification of the method of determining the median knock-down time (KT50) in flies and its utilization for orientative determination of the current sensitivity of flies under field conditions. The method and its reliability were tested both in the laboratory and in field trials and were found to be entirely adequate and sufficiently reliable for the required purpose.


Genetic variation in number of pituitary PAS-purple cells in the house mouse. A genetic variation in the number of pituitary PAS-purple cells in the house mouse is described. C57BL/K1 mice have low and DBA/2/K1 mice have high numbers of these cells in both peripheral and central parts of the anterior pituitary gland. The variation may be due to a single locus with incomplete dominance for the low-number allele. There is no correlation with glucuronidase or galactosidase activities and no association with the b and d loci.


G-band identification of the chromosomes of sheep. Peripheral blood cultures were made on 20 Dorset sheep including 16 ewes and 4 rams. Photomicrographs of 100 cells were examined for trypsin-induced G-bands, and the length of each chromosome was measured. The banding patterns of each of the 27 pairs of chromosomes were identified and described, and the results observed in this study were compared with those previously reported. Measurements of all chromosomes in which both members of a homologous pair were identifiable were used to calculate mean relative length. Karyotypes, in which chromosomes were arranged to mean relative length, were prepared and an idiogram illustrating major bands was constructed.


A contribution to immunological specificity of DNA in leukosis. Active antisera containing antibodies to deproteinized DNA preparations of normal tissue and the spleen of cows suffering from myeloleukosis were obtained. The anti DNA sera to DNA preparations contained complement fixing antibodies related to gamma M globulins. In the study of leukosis and normal anti DNA sera in quantitative CFR with the corresponding test antigens, immunological specificity of DNA preparations isolated from the organs of cows affected with myeloleukosis was established. Immunological specificity of leukosis DNA was confirmed in tests with the absorption of leukosis anti DNA sera by DNA preparations of homologous normal tissues. This specificity is an inherent quality of not only the native but also the heat-denatured DNA molecule.


Aleutian disease of mink: the antibody response of sapphire and pastel mink to Aleutian disease virus. The specific antiviral antibody response of sapphire and pastel mink to Pullman strain of ADV has been examined. Sapphire mink inoculated with from 300,000-3 LD50 developed high levels of specific antibody and AD. Pastel mink inoculated with parallel doses of ADV also produced antibody but did not develop AD. The low incidence of AD in pastel mink inoculated with Pullman strain of ADV is probably related to factors other than antiviral antibody.


The effect of thiols on immunologic release of slow reacting substance of anaphylaxis. I. Human lung. Preincubation of human lung fragments with cysteine for 2.5 to 5.0 min resulted in dose-dependent, selective enhancement of the antigen-induced or anti-IgE-induced formation and release of slow reacting substance of anaphylaxis (SRS-A). Comparable effects were observed with sodium sulfide and thioglycolate but not with other more potent reducing agents or metabolites of cysteine. Sulfhydryl alkylated derivatives of cysteine were ineffective. The effects observed with the active thiols were easily reversed and could not be attributed to an action in the bioassay or on SRS-A itself. The physicochemical characteristics of the contractile activity were identical to those described for SRS-A.


Immunologic properties of mast cells from rats infected with Nippostrongylus brasiliensis. The concentration of IgE in the serum of Sprague-Dawley rats increased after infection with Nippostrongylus brasiliensis (NB). The IgE concentration in normal rats was less than 1 mug/ml. After re-infection with NB, the concentration increased in 100 to 300 mug/ml. Mast cells were purified from peritoneal cells of both normal and NB-infected animals. Purified mast cells from the infected animals released histamine upon exposure to NB antigen. The antibody specific for IgE released histamine from purified mast cells of both normal and infected animals. Dose-reponse curves of histamine release suggested that mast cells from NB-infected animals bear more IgE molecules than normal mast cells. Binding of 125I-labeled rat E myeloma protein with normal mast cells was demonstrated by autoradiography. Under the same experimental conditions, mast cells of infected animals were not labeled with 125I-IgE. Mast cells from both normal and infected animals failed to combine 125I-labeled IgG. The number of IgE molecules bound per mast cell was determined by incubating 125I-labeled IgE with purified mast cells. When mast cells were incubated incubated in 0.6 to 2 mug/ml of IgE, the number of IgE molecules combined with the mast cells from infected animals was about 10% of that bound with normal mast cells. The results indicated that a large proportion of IgE receptors on mast cells of infected animals was occupied by their own IgE. No significant difference was observed between normal mast cells and those of infected animals with respect to histamine content and intracellular levels of cyclic nucleotides.


Carrier-induced tolerance to nucleic acid antigens. BALB/c and SJL mice were treated with nucleosides-IgG1 as a tolerogen, before either primary or secondary immunization with nucleosides-keyhole limpet hemocyanin. Nucleoside-specific responses were measured serologically by a modified Farr assay, with either 14C-labeled denatured DNA or nucleosides-131-I-labeled BSA as test antigen. Specificity of the response was tested by hapten inhition experiments. Multiple doses of nucleosides-IgG1 tolerogen given before the primary or secondary immunization effectively suppressed the secondary and tertiary anti-nucleoside responses. The tolerogen did not suppress the response to an unrelated hapten-KLH conjugate. The IgG alone did not suppress the anti-nucleoside response of BALB/c mice to nucleosides-KLH. Single doses of tolerogen before the primary or secondary immunization were less effective. Residual antibody in partially suppressed BALB/c mice showed changes in specificity as compared to controls. Suppression of the secondary response of SJL mice was measured much more readily by binding of nucleosides-131-I-BSA than by binding of denatured DNA. This reflected an altered specificity of the residual antibody; in control animals, antibodies were directed against all four nucleosides, whereas the antibodies of partially suppressed animals were directed only against guanosine. Suppression of anti-nucleic acid antibody responses may have therapeutic application in the management of systemic lupus erythematosus.


Comparative studies on monotypic IgMlambda and IgGkappa from an individual patient. I. Evidence for shared VH idiotypic determinants. A comparative idiotypic antigenic analysis of an IgMlambda and an IgGkappa paraprotein obtained from sera of an individual patient, Br, revealed the presence of very similar idiotypic determinants associated with the VH regions of the Br mu- and gamma-chains. In addition, the IgGkappa protein expressed light (L) chain-associated idiotypic determinants which were not evident on the IgMlambda protein or its isolated L chains. Extensive tests of specificity revealed that the shared VH idiotypic determinants were not present in large weight amounts of heterologous nonspecific IgM and IgG, nor on Ig molecules contained in a large number of normal and myeloma sera.


The antibody responses to myelin basic protein (BP) in Lewis rats: the effects of Bordetella pertussis. A time-course study was made of the systemic humoral immune response of Lewis rats to myelin basic protein (BP) as influenced by the dosage of ancillary pertussis adjuvant. Peak activities were observed 5 to 7 weeks after injection. When injected proximal to BP and Mycobacterium butyricum in complete Freund's adjuvant (CFA), Bordetella pertussis at the level of 4 billion organisms doubled the antibody-binding activity of rat sera for 125I-labeled BP as compared to activities obtained with 0, 2, 6, or 8 billion. The severity of clinical symptoms of experimental allergic encephalomyelitis (EAE) at the end of the 2nd week was greatest in rats receiving 64 billion organisms, the very same rats that displayed a severely dampened humoral immune response to BP 5 weeks later. When pertussis was injected i.p. rather than proximal to the CFA mixture, the time-course of the humoral immune response displayed a different profile--unusually high binding activities at the time of onset of EAE that fluctuated back and forth from high to low and that eventually dampened to an intermediate level.


Synergy between subpopulations of normal mouse spleen cells in the in vitro generation of cell-mediated cytotoxicity specific for "modified self" antigens. Responding lymphoid cells cultured in vitro with irradiated trinotrophenyl (TNP)-modified syngeneic spleen cells develop direct cell-mediated cytotoxicity which is specific for target cells bearing both the TNP moiety and histocompatibility determinants of the modified sensitizing cell. Two subpopulations of normal mouse spleen cells have been shown to synergize in the in vitro generation of specific cell-mediated cytotoxicity to these "modified self" antigens. The synergizing populations are nylon wool column-adherent and column-nonadherent fractions of normal mouse spleen. When mixtures of these two cell populations are cultured in vitro with irradiated TNP-modified syngeneic spleen cells, greater cytotoxicity is generated in the two populations sensitized separately. The synergizing cell in the column-adherent population is resistant to lysis by rabbit anti-mouse brain serum, is distinct from the cytotoxic effector T lymphocyte, and is unresponsive to phytohemagglutinin; its synergizing function could not be replaced by peritoneal cells. These results suggest that it is a non-T cell which may be distinct from the macrophage.


Inhibition of macrophage-lymphocyte interaction by cytochalasin B during antigen recognition by T lymphocytes. The effects of cytochalasin B on functional and physical macrophage-lymphocyte interaction have been examined. Cytochalasin B, an inhibitor of a variety of membrane activities blocks antigen-dependent bindings of immune lymphocytes to macrophages and antigen-triggered lymphocytes proliferation if added at the initiation of culture. Cytochalasin B becomes progressively less inhibitory if addition is delayed by increasing intervals from the onset of culture. Under these conditions neither antigen handling by macrophages nor the proliferative response of lymphocytes to PHA is inhibited by cytochalasin B. These data are interpreted to suggest that cytochalasin B inhibits antigen-specific macrophage-lymphocyte interaction either by inhibition of an initial antigen-independent phase of macrophage-lymphocyte interaction or by interfering with a lymphocyte membrane event necessary for the interaction of the antigen-specific lymphocyte receptor with the macrophage-bound antigenic signal.


Molecular restriction of anti-DNP antibodies induced by (DNP)2-gramicidin S. The molecular properties of anti-DNP antibodies were studied in 80 rabbits hyperimmunized with (DNP)2-gramicidin S. Unimmunized animals or those previously skin-painted with DNFB were subjected to biweekly injections of (DNP)2-gramicidin S. Isoelectric focusing revealed that the spectrotypes of the anti-DNP antibodies fell into three classifications; monoclonal, 18 to 21%; biclonal, 21 to 30%; and restricted, 14 to 26%. In each immunization protocol 35% of the rabbits did not respond. Longer time periods and more intensive immunization were necessary to achieve and anti-DMP response than with conventional DNP-protein conjugates, with skin painting tending to shorten the overall response time. The anti-DNP responses were invariant with respect to affinity and isoelectric spectra regardless of the immunization protocol or degree of restriction. Chain reassociation studies on antibodies purified from the sera of rabbits displaying monoclonal spectrotypes provide additional evidence that such antibodies are biosynthetically homogeneous. These data support the concept that carrier complexity influences the heterogeneity of the anti-hapten response but strongly suggests that the genetics of an outbred population may be an overriding factor in controlling response heterogeneity.


Antigenic determinants of the 70,000 molecular weight glycoprotein of woolly monkey type C RNA virus. The 70,000 molecular weight glycoprotein (gp70) of a type-C RNA virus originally isolated from a woolly monkey has been partially purified and immunologically characterized. Evidence that this viral protein is viral coded was derived from studies showing its antigenic properties to be unaltered by virus passage in cells of different species. A broadly reactive competition immunoassay was developed utilizing antiserum prepared against feline leukemia virus to precipitate 125I-labeled woolly monkey virus gp70. Gibbon and woolly viruses, as well as feline and several mouse type-C viruses, all reacted with equal efficiency in this assay. In contrast, an endogenous virus of the baboon failed to cross-react, suggesting that viruses of this latter group are less immunologically related to the others. In a homologous competition immunoassay for the woolly viral glycoprotein, the woolly virus was readily distingusihed from otherwise colsely related viruses of gibbon apes. These findings demonstrate the pronounced type-specific antigenic dterminants possessed by this viral protein. The antigenic determinants of gp70 responsible for neutralization have also been investigated.


Antigenic analysis of Chlamydiae by two-dimensional immunoelectrophoresis. II. A trachoma-LGV-specific antigen. Two-dimensional immunoelectrophoresis was utilized to study precipitins in hyperimmune rabbit serum made against chlamydiae and from patients with chlamydial infections. An antigen of Triton X-100-solubilized L2/434/Bu organisms with an electrophoretic mobility of 0.65 relative to bovine serum albumin at pH 8.6 was excised from the agarose gel of electrophorograms as antigen-antibody complexes and used to immunize rabbits. A monospecific antiserum to antigen 0.65 was obtained that reacted with Trachoma-LGV strains L2/434/Bu, B/TW-5/OT, and K/UW-31/Cx, but not with the mouse pneumonitis (Nigg) strain or the psittacosis strain meningopneumonitis (Cal-10). The Trachoma-LGV specificity of antigen 0.65 was further shown by indirect immunofluorescence straining with the monospecific antiserum of chlamydial inclusions in infected HeLa cells. Precipitins with a specificity for antigen 0.65 were indentified in 15 of 18 sera from patients with diagnosed Chlamydia trachomatis infections LGV, trachoma, nongonococcal urethritis, and nongonococcal cervicitis by using monospecific antiserum to antigen 0.65 in the peak suppression test. Thus, antigen 0.65 appears to be a Trachoma-LGV-specific antigen that has considerable promise for serodiagnosis.


The properties of plaque-forming cells from autoimmune and normal strains of mice with specificity for autologous erythrocyte antigens. Treatment of mouse erythrocytes with the proteolytic enzymes, bromelain, reveals antigenic determinants not normally exposed on the erythrocyte surface. It was found that not only NZB mice, a known autoimmune strain, but also several normal strains of mice contain cells in small numbers in their spleens and in larger numbers in their peritoneal cavities which will form plaques against bromelain-treated MRBC. During in vitro culture the number of anti-BR-MRBC PFC increases slightly in the spleen cell populations whereas the number of these PFC in peritoneal cells increases dramatically to as many as 100,000 PFC/10(6) cells. The plaques detected in this assay contain a central lymphoid cell and their development, which requires the presence of complement and protein synthesis, is inhibited by anti-mouse immunoglobulin.


Lymphoid cell subpopulations. I. Synergy between lymph node cells and thymocytes in response to alloantigens and mitogens. Mixtures of isogenic thymocytes (TC) and lymph node cells (LNC) were shown to exhibit synergistic responsiveness to M and H-2 alloantigens in the mixed lymphocyte interaction (MLI). With respect to the kinetics and magnitude of proliferation and effector cell generation, the response occurring in synergizing cultures closely resembled that of optimal numbers of LNC or spleen cells (SC). In addition, the antigen specificity of effector cells generated by synergizing cultures was similar to that of effectors derived from cultures containing optimal numbers of responding SC. LNC-TC mixtures also exhibited synergy in response to the phytomitogens concanavalin A and pokeweed mitogen but not to phytohemagglutinin. Weakly positive synergy was observed in response to bacterial lipopolysaccharide. It is proposed that the phenomenon of synergy is not restricted to cultures containing mixtures of LNC and TC but also occurs in cultures containing optimal numbers of LNC or SC as a result of interactions between subpopulations of lymphocytes contained within these tissues.


Anomalous reactions of mouse alloantisera with cultured tumor cells. I. Demonstration of widespread occurrence using reference typing sera. M0use alloantisera produced against different specificities of the K, I, and D regions of the H-2 gene complex reacted as immunogenetically anticipated with normal lymphoid target cells of different haplotypes in cytotoxicity and indirect immunofluorescence tests. These same alloantisera, however, produced anomalous positive reactions when tested on cultured MCA-induced sarcoma cells from B10 background H-2 congenic mice. Absorption experiments demonstrated that the anomalous activity in these sera was directed against a tumor membrane antigen(s) which was distinct from H-2 region specificities against which the reference alloantisera were produced, and which was shared in common by multiple cultured sarcoma lines. Similar anti-tumor antibody activity could be demonstrated in the serum of older (greater than 12 weeks) but not younger normal unimmunized mice of the strains used as recipients for alloantiserum production. It is suggested that the observed anamalous anti-tumor activity in these alloantisera may be due to the presence of antibodies reactive with envelope antigens of murine leukemia virus which are expressed on sarcoma cells maintained in culture.


Immune response to a syngeneic mammary adenocarcinoma. II. In vitro generation of cytotoxic lymphocytes. A method is described for the consistent in vitro generation cytotoxic cells by incubating Fischer 344 rat spleen cells on monolayers of a syngeneic mammary adenocarcinoma. Significant cytotoxicity by in vitro culture is generated as early as 3 days after initiation and effector cells are cytolytic only toward target cells of the sensitizing monolayer. Reciprocal sensitization with allogeneic fibroblasts as the immunizing monolayer yielded effector cells cytolytic for the fibroblasts but without effect on the mammary tumor. The consistency in the generation of cytotoxic cells by in vitro culture should permit its standardized use in following other related immune phenomena such as blocking by serologic factors and suppression, recritment of memory for cytotoxic function.


The xenogeneic effect. I. Antigen and mitogen-stimulated human lymphocytes produce a non-antigen-specific factor which reconstitutes the antibody response of T cell-deficient mouse spleen cells. Modified Marbrook culture vessels with two chambers separated by a 0.2-mu porosity membrane have been utilized to show that antigen-stimulated human lymphocytes produce a soluble factor(s) which restores the ability of thymectomized, irradiated, and bone marrow-protected mice to mount a primary IgM plaque-forming cell response in vitro. In the initial experiments, the human lymphocytes plus antigen (sheep erythrocytes) were cultured in the lower chambers of the Marbrook vessels and the T cell-deficient mouse spleen cells plus sheep erythrocytes were cultured in the upper chambers. The response of the spleen cells was shown to be enhanced as a function of the number of human lymphocytes in the lower chambers. In subsequent experiments, the human lymphocytes were challenged with allogeneic lymphocytes or activated with a variety of T cell mitogens. Supernatants from these cultures, when placed in the lower chambers of the Marbrook vessels, were also capable of reconstituting the antibody-forming cell response of the mouse B cells. The results of the experiments are discussed in relation to a model of B cell induction which incorporates a non-antigen-specific "helpher" T cell.


Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens. Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.


T lymphocyte-enriched murine peritoneal exudate cells. I. A reliable assay for antigen-induced T lymphocyte proliferation. The in vitro activation of murine thymus-derived (T) lymphocytes by soluble protein and synthetic antigens has been difficult to assess because of the lack of a specific and reliable proliferation assay. The present report describes the development of an assay system which overcomes these problems by making use of a population of nylon wool column-purified T lymphocytes obtained from thioglycollate-induced peritoneal exudates of immunized mice. PETLES (peritoneal exudate, T lymphocyte-enriched cells) were composed  mainly of T lymphocytes, eosinophils and small numbers of macrophages. Contamination with bone marrow-derived (B) lymphocytes averaged only 2%. When PETLES from immunized mice were stimulated in microtiter cultures with the immunizing antigen, large degrees of proliferation ensued as measured by incorporation of 3H-methyl-thymidine 5 days after initiation. As few as 1.25 x 10(4) cells and as little as 50 ng/ml of antigen gave significant stimulation. Maximum responses were obtained witn a series of 10 experiments under these optimal conditions, gave a mean incorporation of 70,900 cpm while the controls cultured without antigen showed only 3,600 cpm. PETLES from nonimmunized mice or from mice immunized to other antigens did not respond to DNP5OVA although they did respond to mitogens. The antigen-induced proliferation was shown to require the presence of immune T lymphocytes by two criteria: elimination of the response by treatment with anti-Thy 1.2 serum plus complement and failure to reconstitute the response when the few remaining immune B lymphocytes left after anti-Thy 1.2 treatment were added to nonimmune T lymphocytes. In addition, the system exhibited carrier specificity. Because of the paucity of B lymphocytes in the population, their contribution to the overall magnitude of the proliferative response was negligible as demonstrated by the small response to B cell mitogens. Thus, the assay appears to be a quantitative as well as a qualitative assay for one aspect of T lymphocyte function. This technique should prove useful for the study of murine T lymphocytes in vitro.


Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole. Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.


The immune response against an encephalitogenic fragment of guinea pig basic protein in the Lewis and Brown Norway strains or rat. Lewis rats which have Ir-EAE gene develop EAE after challenge with either GPBP or GPEF, a 42-residue fragment. BN rats which lack the Ir gene do not develop EAE after challenge with these proteins. The immune response against GPEF was compared in these two strains of rats. It was found that Le rats produce small amounts of antibody  and a cell-mediated immune response against GPEF; in contrast BN rats did neither. These findings support the concept that the Ir-EAE gene exerts its effect by controlling the immune response against GPEF.


Measuring ventricular extrasystoles.

Measuring ventricular extrasystoles. Uniform ventricular extrasystoles can be divided into three types: fixed coupling, parasystole, and variable non-parasystolic coupling. Frequency distributions of coupling intervals of ventricular extrasystoles were determined from long electrocardiographic recordings of 51 patients with variable coupling. These distributions were of five types: (a) two distinct coupling intervals, (b) a preponderance of extrasystoles with short coupling intervals and less frequent extrasystoles with progressively longer coupling, (c) a preponderance of long coupling intervals and less frequent extrasystoles with progressively shorter coupling, (d) an even distribution and (3) a central distribution. Interectopic intervals were measured in these electrocardiograms (ECGs). Parasystole was found only in three recordings. It is recommended that the diagnosis of parasystole be made only if the degree of variation of the ectopic cycle (both when it is manifest and when it is concealed) is less than the variation of the coupling intervals. When the ECGs with variable non-parasystolic coupling were compared with 44 ECGs with fixed coupling, it was found that variable coupling was associated with abnormalities of the basic electrocardiographic contour, multiformity of extrasystoles and repetitive extrasystoles.


An immunofluorescent study of cornea development in the chick. The developmental pattern of appearance of a structural corneal antigenic determinant was studied by immunofluorescent methods. It was first detected in the primary corneal stroma at stage 23, and thereafter spread throughout the primary and secondary stroma. This antigen was absent from Bowman's membrane at its initial stage of development, suggesting that this membrane is a new structure in corneal morphogenesis, and not simply a modification of the primary stroma. This study also suggests that the antigenic determinant is a carbohydrate and is secreted by the corneal epithelial cells.


Developmental changes in the composition and amount of mouse fetal fluids. Fetal fluid was studied in mice of the Q, C57BL/McL, C3H/Bi/McL and JU/Fa strains. The amount increases to day 16 of gestation, and then decreases. Amniotic fluid and fetal plasma contain three transferrins, five alpha-fetoproteins and albumin. Protein concentration in both increases during gestation; glucose decreases in amniotic fluid although it rises in fetal plasms; urea levels remain constant. The regulation of volume and biochemical composition of amniotic fluid, and its relation to the yolk-sac membrane, are discussed.


Inverse relationship between net electric charge on the antigen and that on the sensitized cell in cellular immune response: demonstration with basic encephalitogen of the brain. An inverse relationship exists between the net-electrical charge of immunogens and the antibodies elicited (1). The cellular basis of the net charge phenomenon has been established for both positively and negatively charged immunogens, by cell separation techniques over columns of opposite charge (7, 8). To establish whether this phenomenon can be extended to include cell-mediated immunity, the response to basic encephalitogenic protein (BE) which induces experimental allergic encephalomyelitis (EAE) was now investigated. Lymph node cells from sensitized strain 13 guinea pigs were fractionated over positively and negatively charged columns and compared to unfractionated cell populations in two assay systems: (a) in vitro response to BE in terms of lymphocyte transformation and (b) the passive transfer of EAE to unsensitized syngeneic recipients. The response was found to be confined to the fraction of cells eluted from glass bead columns, namely, the more negative cells. Cells eluted from poly-L-lysine-coated glass bead columns (i.e., positive cells) were devoid of the capacity to respond to this antigen either in vivo or in vitro. It was previously established that thymocytes rather than bone marrow cells account for the inverse charge phenomenon as assayed by T-helper-cell function in in vivo antibody production (8). We have now extended the inverse charge effect to include cell-mediated immune response of the delayed hypersensitivity type.


Antigen-binding T cells as helper cells. Separation of helper cells by immune rosette formation. The spleen T cells from mice immunized 6 days earlier with either chicken gamma globulin (CGG) or with donkey erythrocytes (DRC) were rosetted with CGG-coated sheep erythrocytes or with DRC. The immune rosettes (RFC) (antigen-binding cells) were separated from the bulk of nonrosette-forming cells (non-RFC) by 1-g velocity sedimentation and the RFC and non-RFC tested for helper activity in cooperative antihapten responses in vitro. RFC or non-RFC were mixed with normal or hapten-primed spleen cells, challenged with the appropriate hapten-carrier conjugate and cultured for 4 days in Marbrook tissue cultures. The helping activity was quantitated from the numbers of antihapten antibody-producing cells generated per culture. The results show that specific helper cell activity could be selectively recovered in the immune rosette-forming cell population whereas the non-RFC population was depleted of help. These findings indicate that the helper T cells express specific antigen binding receptors.


Collaboration of histoincompatible T and B lymphocytes using cells from tetraparental bone marrow chimeras. T-B collaboration has been studied in a secondary response to sheep erythrocytes using either syngeneic or allogeneic T- and B-cell combinations. T cells prepared from tetraparental bone marrow chimeras (TBMC), carrying H-2 determinants of one parental strain only, cooperated with syngeneic, as well as with allogeneic B cells carrying the alloantigens to which the T cells had been tolerized in the chimeric environment. When TBMC-derived cells of a single H-2 specificity were transferred with a mixture of TBMC-derived B cells of both H-2 types of the parental strains, no preference for syngeneic cooperation was found. The data therefore suggest that the presence of differing H-2-complex determinants on the allogeneic T- and B-cell populations of the two different strain combinations tested do not interfere with T-B collaboration when the cell populations studied are mutually tolerant.


Alteration of immunoglobulin phenotype in cell culture-adapted lines of two mouse plasmacytomas. Murine plasmacytomas can be adapted to continuous in vitro culture by alternate passage between culture and animal. We have found that the kinetics of adaptation reflect a selection for the growth of variant plasmacytoma cells. The inclusion of an altered immunoglobulin phenotype in such variant cells could explain the Ig-producing variants that we observed in two of six transplantable lines of plasmacytomas that were adapted to culture. The first variant, an IgM-producing cell line (104-76), was adapted from a transplanted line of MOPC 104E that had stopped producing IgM with binding specificity for alpha1-3 Dextran. Unlike MOPC 104E, the IgM of 104-76 contains kappa- instead of lambda-light chains and probably contains an altered or different mu-heavy chain. A second variant (352-57) was found in an IgG2b-producing tumor (MOPC 352) which was induced in a BALB/c mouse strain (CB-6) that carried Ig genes of the C57BL/Ka allotype. This cell line apparently switched from producing IgG2b molecules of the C57BL allotype (H9) and of a known idiotype to IgG1 molecules of the BALB/c allotype (F19) without the idiotype marker. The propagation of a biclonal plasmacytoma from the time of original tumor induction does not appear as a likely explanation for these results. Rather, we seem to be dealing with plasmacytoma variants or with the possible induction of secondary tumors of host origin.


Feulgen microspectrophotometric analysis of deoxyribonucleoprotein organization in larval and adult freshwater sponge nuclei. Microspectrophotometric measurements of nuclei of adult choanocytes and archeocytes and larval archeocytes and flagellated ectodermal epithelia from stages III and IV were undertaken on tissues from the freshwater sponge, Eunapius fragilis (Leidy). The condensed nuclei of differentiated choanocytes and state IV flagellated epithelial cells presented integrated extinction values about 64% of those obtained for either adult or larval archeocytes. This suggests that such measurements represent sensitive indicators of deoxyribonucleoprotein-complex organization; thus, nuclear differentiation. Further, no large population of G2 (4C) nuclei was found among these populations as has been reported in two classes of coelenterates.


Some properties of the polysaccharide from cell cultures infected with TRIC agent (Chlamydia trachomatis). The polysaccharide, elaborated in trachoma-inclusion conjunctivitis (TRIC) agent inclusions, was isolated from baby hamster kidney (BHK) cells grown and infected in suspension cultures. It was characterized by physical, chemical and enzymic methods as a glycogen with an average chain length of 14 to 16 glucose units.


[Steinert's syndrome and the myocardium. Total gene expression by the myocardium]. As revealed by a very thorough cardiological study of Steinert's disease in 13 cases, it would appear that the incidence of cardiac involvement observed in this disease is not merely frequent; it is, in fact, usual, systematic, and forms an integral part of the pathological picture in the same way as peripheral muscular dystrophy. One is aware of the diagnostic interest of this fact in the juvenile or abortive forms of the disease. The cardiac involvement is an indication of the complete expression of the gene towards striated muscle tissue, whether skeletal or myocardial. Finally, it opens new perspectives on the prophylactic attitude to be adopted for such patients; regular and systematic cardiological checks, moderation in the prescription of digitalis and anti-arythmia drugs, implantation of a cardiac pacemaker before an auriculo-ventricular syncopal block occurs.


Morphological effects of osmolarity on purified noradrenergic vesicles. Large dense core vesicles (LDV) were purified from bovine splenic nerve homogenates by the sucrose-D2O density gradient method. Vesicles were subjected to a 50% increase and decrease in osmolarity from the control 330 mosmol 1(-1) by adjusting sucrose or potassium phosphate buffer during pre- and/or postfixation. Control vesicles with a mean diameter of 717 A readily swelled to approximately 1050 A and shrunk to approximately 600 A in the hypotonic and hypertonic media, respectively, with either sucrose or phosphate buffer. The dense core responded similarly but to a lesser degree. Prefixation in glutaraldehyde had little effect on vesicle sensitivity to subsequent tonicity change, not did the fixative per se exert an obvious osmotic effect. Thus, final vesicle size was largely determined by the OsO4 postfixation medium and principally by the vehicle rather than the fixative. In controls there was a mixture of spherical to oblate vesicles mostly filled with an electron-dense matrix. Upon swelling, more vesicles became spherical and nearly all had a prominent translucent halo between core and membrane. Upon shrinking, more vesicles became oblate, the halo was obliterated and the electron-density of the matrix increased. Frequency distributions of vesicle diameters at different tonicity clearly indicated that the diameter of LDV could overlap the 400-500 A range characteristic of small dense core vesicles; however, there was no suggestion of a population of the latter in the purified LDV fraction. Implications are discussed concerning the biochemical and morphological identification of 'light' and 'heavy' density peaks of noradrenaline and dopamine beta-hydroxylase from mixed vesicle populations and the possible relevance of changes in vesicle shape to a functional state in situ.


[Correlation between sensory action potentials and vibratory perception in uremic polyneuropathy (author's transl)]. 1. The sensory action potentials of the tibial nerve at the medial malleolus were studied by averaging in 51 patients with chronic renal failure treated by  hemodialysis. Vibratory sense was also tested quantitatively on the dorsum of the foot with a pallesthesiometer. 2. Good correlation was found between sensory tibial nerve potentials and vibration sense in subclinical as well as in clinical uremic polyneuropathy. A biphasic potential correlated with unaffected vibration sense in 18 out of 23 patients, and impaired vibratory sense with a polyphasic response in 20 of 28 patients. Maximal nerve conduction of sensory fibres was faster (mean 37.4 m/sec) in cases with normal vibratory sense, but slower (mean 31.3 m/sec), when vibratory sense was impaired. Furthermore there was a correlation between the threshold of vibratory perception and sensory nerve conduction. 3. Sensory function, tested with conventional methods, was impaired only 5 times in 28 patients with altered vibratory perception. 4. The earlier impairment, especially of the vibratory sense, may be explained by the following neurophysiological mechanisms: a) Because of the polyphasic prolonged response of the sensory potentials, no rhythmical groups of impulses reach the central nervous system, but only a continual stream of small peaks arrives, so that vibration perception does not develop. b) A multiplication of the frequency of discharges caused by alternating firing of different sensory fibres is impossible due to the reduction of the number of axons. c) The prolongation of the relatively refractory period due to demyelinization of the surviving fibres prevents the transmission of frequent impulses. 5. Alterations of the sensory action potentials of the tibial nerve, as well as of vibratory perception tested quantitatively, are earlier signs of uremic polyneuropathy than the prolonged motor nerve conduction velocity. Since not all patients give accurate information when tests of vibratory sense are performed both methods should be applied. Physiological polyphasia of sensory action potentials and diminishing vibration perception in advanced age must be taken into account.