Theory of delocalized ionic binding to polynucleotides: structural and excluded-volume effects. A previously developed theory for the delocalized binding of ions to polyelectrolytes was restricted to point ions and a structurally rigid polyelectrolyte. For the binding of substances like oligolysines and polyamines to DNA, the restriction to point ions would appear not to be realistic. For the binding of ions to flexible chains like single-stranded polynucleotides, the restriction to a rigid polyelectrolyte may not be realistic. In this article, we assess the effect of relaxation of these two restrictions. Excluded volume among bound ions is modeled by a hard-rod potential in the context of the theory of a one-dimensional fluid. The possibility that a flexible chain folds in some manner in the immediate vicinity of a bound ion is modeled by allowing the mean spacing between charged groups on the polymer to become smaller as the number of bound ions increases. We compare our results with recent data on the binding of a series of oligolysines to single-stranded polynucleotides, which conflict with the predictions of the original theory of delocalized binding of point ions to rigid polyelectrolytes. Inclusion of excluded volume among bound ions does not significantly improve agreement with the data. Substantial improvement in the level of agreement is obtained when the polyion chain is assumed to be flexible. One of our conclusions is that the excluded-site description of anticooperativity, which was designed for the binding of ligands to discrete sites on a polymer chain, and which does not include the effect of ionic forces, should not be used in cases of delocalized binding of ions.


Computer modeling of polysaccharide-polysaccharide interactions: an approach to the kappa-carrageenan-mannan case. A computer program SAINT has been developed for the investigation of the structure and for the prediction of minimum-energy structure of polysaccharide-polysaccharide complexes. The energy minimization is carried out on internal geometrical parameters--namely bond angles, torsional angles, and five parameters describing the mutual orientations of polysaccharide chains. For this purpose, the nonderivative method of conjugated directions is used. This procedure was applied to computer modeling of an idealized model of the binary gelling kappa-carrageenan and galactomannan system. It is shown that the interaction between two chains influences the structure of the individual polysaccharide molecule and that in the minimum-energy structures of the complex, the conformation of the chains does not correspond to the lowest energy.


Theoretical determination of conformational paths in citrate synthase. Two methods are developed for the theoretical determination of a conformational path between two well-documented forms, a closed form and the open form [Remington et al. (1982) J. Mol. Biol. 158, 111-152] of pig heart citrate synthase, a dimeric enzyme of 2 x 437 residues. The first method uses the minimization of the sum of the potential energies at a set of equidistant points, according to Elber and Karplus [(1987) Chem. Phys. Lett. 139, 375-380]. The initialization of the algorithm is modified to account for large-angle rotations of many groups by performing the interpolations in the space of internal polar coordinates of a set of generalized Jacobi vectors earlier introduced by Durup [(1991) J. Phys. Chem. 95, 1817-1829] and by carefully testing all choices of directions of rotation for determining the initialized midpoint between the known forms. The path includes intermediate points, created by successive splittings of each interval into two equal parts, with a partial energy minimization performed after each splitting. The minimization encounters the well-known local-minima problem, which here is handled by low-temperature molecular dynamics annealing. It is shown that the best ratio of potential energy decrease to rms deviation is achieved by running the dynamics at 50 K, as compared to 100 K and above. The main character of the path obtained is the occurrence of strong to-and-fro variations of some dihedral angles at specific stages along the path. The second method, which we name directed dynamics, uses only low-temperature molecular dynamics simulations by starting trajectories from each of the two known forms with initial velocities directed toward the other one. The procedure is iterated by restarting trajectory pairs after the points of closest approach of the preceding pair. The two half-paths thus built eventually meet after 70 iterations. This method provides a second path with strong similarities, as well as some differences, with respect to the path obtained by the first method.


Cytokines and proteases in invasive processes: molecular similarities between inflammation and cancer. Tumor-derived serine proteinases and metalloproteinases have been associated with invasion and metastasis of cancer cells. Leukocytes, particularly monocytes/macrophages and neutrophils, actively synthesize and store these proteolytic enzymes. The production by tumor cells of chemotactic factors that attract white blood cells raises questions that are important for the basic researcher as well as the clinical scientist. Are the proteinases, which have the capacity to dissolve the extracellular matrix and by this solubilization promote cell migration, the same in tumor cells as in normal cells? Is the production of chemotactic factors by tumor cells a coincident epiphenomenon of the malignant state or a selective way to parasitize the host? Does the early attraction of leukocytes to the tumor site contribute to early host defense against cancer? Does our knowledge about mechanisms of action of cytokines have implications for therapy of the cancer patient? Recent experimental data give hints to the answers to these questions and make it possible to deduce a fundamental model of cytokine mediated proteolysis in tissue remodelling.


Molecular area involved in the in-vitro dimerization of the murine p55 IL-2 receptor. In order to study structure-function relationships of the M(r) 55,000 subunit of the murine IL-2R (p55 IL-2R) by epitope mapping, we have expressed the p55 IL-2R molecule in a cell-free translation system. Under these in vitro conditions, we detected the expected p55 IL-2R polypeptide initiated at Met 1 and, in addition, two internally initiated molecules at Met 64 and Met 105. In this report we describe that from such a mixture, containing three molecular species of p55 IL-2R, mAb 135D5 immunoprecipitated the polypeptide initiated at Met 105 although this N-terminally truncated form of p55 IL-2R does not contain its epitope located between amino acids 72-88. This observation can be taken as a further evidence for the formation of p55 IL-2R dimers. Finally, we identified the region implicated in the formation of p55 IL-2R dimers close to the transmembrane region of the molecule.


Butylated hydroxyanisole specifically inhibits tumor necrosis factor-induced cytotoxicity and growth enhancement. The effect of commonly used food antioxidants on recombinant tumor necrosis factor alpha (rTNF-alpha)-induced cytotoxicity, growth enhancement and adhesion has been evaluated. Butylated hydroxyanisole (BHA) and 4-hydroxymethyl-2,6-di-t-butylphenol (HBP) were the only two of nine antioxidants that completely inhibited rTNF-alpha-induced cytotoxicity in L929 and WEHI 164 fibrosarcoma cells. Ethoxyquin, propyl gallate and butylated hydroquinone only partially inhibited rTNF-alpha-induced cytotoxicity, while the antioxidants butylated hydroxytoluene (BHT), alpha-tocopherol, ascorbic acid and thiodipropionic acid had minimal effects. The only difference between the molecular structure of the efficient HBP and the non-efficient BHT, is a hydroxymethyl group instead of a hydroxyl group on the phenolic ring. Neither BHA nor BHT inhibited the activation of NF kappa B after 10 or 60 min challenge with rTNF-alpha in L929 cells. BHA also inhibited rTNF-alpha-induced, but not rIL-1 beta-induced growth enhancement in FS-4 fibroblasts. Further, BHA blocked both rTNF-alpha-induced and rIL-1 beta-induced prostaglandin E2 synthesis in FS-4 fibroblasts. BHA inhibited the rTNF-alpha-induced release of arachidonic acid in both FS-4 and L929 cells, suggesting that BHA inhibits cellular phospholipase(s). Neither alpha-tocopherol nor BHA inhibited rTNF-alpha-induced adhesiveness of human endothelial cells. The results indicate that BHA is a specific and potent inhibitor of rTNF-alpha- and rTNF-beta-induced cytotoxicity, as well as of rTNF-alpha-induced growth enhancement.


Biological effects of recombinant vaccinia virus-expressed interleukin 4. Recombinant vaccinia viruses expressing murine interleukin 4 (IL-4), either alone or together with interleukin 2 (IL-2) or gamma interferon (gamma-IFN), were constructed. Unlike IL-2, IL-4 expressing viruses were not cleared from immunodeficient mice and the mice died. As they died more rapidly than immunodeficient mice inoculated with a control virus, it appeared that IL-4 contributed to their death and the IL-4 mediated toxicity was confirmed in normal immunocompetent mice. The toxicity was reversed by co-expression of either IL-2 or gamma-IFN, probably due to virus clearance and therefore lower levels of circulating IL-4. Vaccinia virus-expressed IL-4 did not increase antibody or natural killer cell levels and caused a slight decrease in cytotoxic T lymphocyte levels.


Synergism of glucocorticoids with granulocyte macrophage colony stimulating factor (GM-CSF) but not interferon gamma (IFN-gamma) or interleukin-4 (IL-4) on induction of HLA class II expression on human monocytes. Peripheral blood monocytes from up to 13 normal donors were stimulated with the cytokines interferon gamma (IFN-gamma), interleukin 4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) in the presence or absence of dexamethasone (Dex), and the effects on HLA class II (HLA-DR, DP and DQ) expression studied. Dex markedly augmented HLA-DR, DP and DQ levels induced by GM-CSF, in all samples tested. Particularly striking were the effects on HLA-DQ expression, since stimulation with a combination of Dex and GM-CSF induced markedly higher levels of HLA-DQ antigen than stimulation with IFN-gamma. Northern blot analysis of samples treated for 40 hours with Dex and GM-CSF indicated that levels of DR alpha, DP alpha and DQ alpha mRNA were also increased. In contrast, despite variation between individual donors, in general Dex weakly inhibited both constitutive and IFN-gamma- or IL-4-induced HLA-DR expression. Variability in the responsiveness of monocytes purified from individual donors to each cytokine was also observed. GM-CSF was less potent than IFN-gamma and IL-4, enhancing HLA class II expression in only seven of 13 donors tested, whereas in the presence of Dex all donors responded to GM-CSF. The differential effects of glucocorticoids in vitro suggest that these cytokines induce HLA class II expression by different mechanisms.


Circulating interleukin 6 as a useful marker for predicting postoperative complications. We examined postoperative serial changes in the levels of serum interleukin 6 (IL-6), serum acute phase reactants (APRs) and plasma neutrophil elastase (NE) in patients with various cancers and reviewed these changes in patients who did, and did not, show postoperative complications. Serum IL-6 level was elevated after surgery, peaking on the first postoperative day. Elevation of serum APRs and plasma NE levels also followed. There was a significant correlation between the serum peak level of IL-6 and those of APRs and NE (P less than 0.01). Moreover, there was a significant difference in the serum IL-6 level in patients with and without complications. The relationship between the serum IL-6 greater than 400 pg/ml and the incidence of postoperative complications was also marked. These results suggest that circulating IL-6 is a clinically useful marker for the earliest detection and prediction of postoperative complications.


Production and induction of a novel cytotoxin (factor 2) by a human B-cell line. The human B-cell line, Karpas 160, was found to produce Factor 1 and 2 (F1 and F2). F1 was found to be indistinguishable from tumor necrosis factor beta (TNF-beta), while F2 appears to be a new cytotoxin. We used several stimuli alone or in combination to trigger the 160b cells, a subclone of Karpas 160, to produce higher yield of F2. The optimal culture times, concentrations of cells and a range of stimuli were studied. We found that 20-25 ng/ml of phorbol myristate acetate (PMA) effectively induced the higher production of F2. Pretreatment of the cells with sodium butyrate enhanced F2 production. Production of TNF but not F2 was inhibited when the cells were cultured with tunicamycin and PDB. When ciprofloxacin or cycloheximide was added to the medium, F2 production in the presence of PMA was amplified. When 160b cells were cocultured with K562 cells, low levels of F2 induction were observed. We found that most types of human tumor cell lines were highly susceptible to F2, but less sensitive or even resistant to TNF. In contrast, normal human cell lines were not susceptible to F2. Therefore, it appears that F2 could be a new human cytotoxin.


Gastric carcinoma in young Hong Kong Chinese. Gastric carcinomas usually occur in older people. Those occurring in the young are uncommon. The pathological and clinical features of gastric carcinomas were reviewed in 42 Chinese patients who were 35 years of age and younger. The data were obtained from the record files of the University Department of Pathology, Queen Mary Hospital for the period 1976-85. The patients comprised 4% of the total cases of gastric carcinomas in that period. These patients (age range: 20-35 years, mean: 30 years) showed a male to female ratio of 1:2.5 which differs from the usual male preponderance seen in gastric carcinoma. Among the 27 cases with known staging, 22 (81.5%) were stage III or IV. Twenty-five cases had an ulcerative appearance. All were adenocarcinomas and the majority (83.3%) were poorly differentiated. Associated dysplasia was found in 35 (83.3%) cases, although only 14 of these were in association with poorly differentiated adenocarcinomas. Intestinal metaplasia was found in 13 cases and, when present, involved less than 30% of the mucosa. Only two cases were of type III metaplasia. The findings show that gastric carcinoma in young Chinese tended to occur more frequently in females, presented at late stages, showed poor glandular differentiation, was frequently associated with gastric dysplasia and had minimal association with intestinal metaplasia.


Brush border hydrolases in normal and neoplastic colonic epithelium. Previous studies have suggested that abnormal expression of enzymes characteristic of the intestinal brush border might accompany colonic neoplasia and possibly facilitate identification of epithelium at risk of malignancy. To test this possibility, the distribution of the brush border enzymes sucrase-isomaltase (SIM), maltase-glucoamylase (MGA), aminopeptidase-N (APN) and diamino-peptidylpeptidase-IV (DPPIV) were studied by the immunoperoxidase method in biopsies from the rectum and caecum of normal subjects, and neoplastic and non-neoplastic tissues from patients with adenoma or cancer. Brush border enzymes were detected by immunohistochemistry more frequently in the caecum than the rectum (P less than 0.05) of normal subjects. Diamino-peptidylpeptidase-IV and APN were present in highest concentration at the brush border of the most mature colonocytes on the luminal surface with less staining in the crypt, whereas SIM and MGA staining of the brush border was as prominent on crypt cells as surface cells. While all cancers expressed at least one enzyme, there was heterogeneity of staining within tumours and a tendency to lose polarity of enzyme expression in cells, sometimes with dense staining of the cytoplasm. Distally situated adenomas uncommonly expressed a brush border enzyme (25%) and the only enzyme expressed in them was SIM. These finding indicate that these brush border enzymes are not exclusively expressed in the small intestine; DPPIV and APN are markers of the normal mature colonocyte and should prove useful as markers of differentiation. However, the change associated with neoplasia would not appear to be of clinically predictive value.(ABSTRACT TRUNCATED AT 250 WORDS)


Fluoride as a possible aetiological factor in non-ulcer dyspepsia. A prospective case controlled study was conducted to evaluate the role of fluoride as a possible aetiological factor for non-ulcer dyspepsia (NUD). Twenty patients with NUD and 10 age and sex matched healthy controls were subjected to clinical evaluation, upper gastrointestinal endoscopy and biopsies from the gastric antrum and duodenum. The antral and duodenal mucosa was subjected to a rapid urease test for Helicobacter pylori and histological and electron microscopic examinations. Fluoride levels in the drinking water, serum and urine were estimated using a ION 85 ion-analyser. These levels were significantly higher in patients with NUD than in controls (P less than 0.05). Histological abnormalities in the antral and duodenal mucosa were seen in 14 patients (70%) with NUD and 1 control subject (10%) (P less than 0.05). Electron microscopic abnormalities in the mucosal cells were seen in all patients with NUD but in none of the controls (P less than 0.01). The fluoride levels in serum and urine correlated with the symptoms, histological and electron microscopic abnormalities (P less than 0.05). It was concluded that chronic exposure to fluoride may result in NUD and should be considered in patients where other known cause of dyspepsia have been excluded.


C-reactive protein and lactate dehydrogenase isoenzymes in the assessment of the prognosis of acute pancreatitis. The value of serum C-reactive protein, lactate dehydrogenase isoenzymes and erythrocyte sedimentation rate in predicting the outcome of acute pancreatitis was evaluated for 57 episodes in 54 patients. Serum C-reactive protein levels on day 2, 4 and 7 after admission were significantly higher in 19 episodes of severe attacks than in 38 episodes of mild attacks (13.71 +/- 9.68, 9.00 +/- 7.54, 6.02 +/- 3.83 vs 4.78 +/- 3.91, 3.30 +/- 3.61, 1.43 +/- 2.08 mg/dL; P less than 0.0001, P less than 0.005, P less than 0.0001, respectively). The sensitivity, specificity and accuracy of predicting a severe attack were 94, 76 and 82% using C-reactive protein greater than or equal to 8 mg/dL on day 2; 67, 92 and 84% using C-reactive protein greater than or equal to 5 mg/dL on day 7; and 59, 76 and 70% using Ranson's criteria greater than or equal to 3. Increases in LDH-4 and LDH-5 isoenzymes were found in both groups, with LDH-4 being slightly higher in severe attacks than in mild attacks. There was no significant difference of erythrocyte sedimentation rate between both groups. When compared with Ranson's criteria, lactate dehydrogenase isoenzymes and erythrocyte sedimentation rate, C-reactive protein is more valuable in the early assessment of the severity of acute pancreatitis.


Persistent ileitis after ileal salvage from a failed Kock pouch. A case of persistent ileitis following the refashioning of a Kock ileal pouch into a Brooke ileostomy for severe pouchitis is reported. Preserving the terminal ileum by a salvage procedure may not be a safe alternative to pouch excision with the formation of a new end ileostomy in the surgical management of pouchitis refractory to medical treatment.


Fulminant hepatic failure and acute intravascular haemolysis as presenting manifestations of Wilson's disease in young children. The cases of three young children (mean age 5.8 years) in whom fulminant hepatic failure and acute intravascular haemolysis were the presenting manifestations of Wilson's disease are reported. Although diagnosis was made ante-mortem and chelation therapy instituted, the course was relentlessly fatal in all three cases. This presentation of Wilson's disease at such a young age is noteworthy.


Safety and feasibility of percutaneous ultrasound guided puncture of the gall-bladder for crystal analysis. The safety and feasibility of ultrasound guided percutaneous aspiration (USPA) of bile, for bile crystal analysis was assessed critically. Forty-four patients about to undergo elective cholecystectomy underwent intended transhepatic USPA 1 h before their operation. At laparotomy a careful inspection for bile and blood leakage was made and a further sample of gall-bladder bile collected. Successful USPA occurred in 40 of the 44 patients. The four unsuccessful cases each had a gall-bladder packed with stones and contained virtually no bile. Three patients developed bile leak after transperitoneal (n = 2) and transhepatic (n = 1) puncture of the gall-bladder and another patient lost 100 mL of blood. Microscopic analysis of paired bile samples confirmed that gall-bladder bile (as opposed to hepatic bile) had always been aspirated. Ultrasound guided percutaneous aspiration of bile offers a simple and promising alternative to duodenal intubation, but considerable experience is required before it can be recommended as its replacement.


Tattooing and hepatitis B infection.

Tattooing and hepatitis B infection. To determine the prevalence of HBV carrier status in tattooed persons, 200 healthy, tattooed volunteers and 200 non-tattooed volunteers to match the first group for age, sex and ethnic group were screened. Prevalence of HBsAg in tattooed and non-tattooed volunteers was 19.5 and 9% respectively, which was a statistically significant difference (P less than 0.01). There was no difference in the prevalence in various age groups, ethnic groups, or sex, between the two groups.


Duplex-Doppler assessment of cirrhosis in patients with chronic compensated liver disease. Portal venous flow velocity (PFV) was measured with duplex-Doppler equipment in 50 normal subjects and in 117 patients with suspected chronic liver disease who showed no evidence of decompensation such as ascites, hepatic encephalopathy, jaundice or oesophageal bleeding. All the patients underwent percutaneous liver biopsy which demonstrated non-cirrhotic liver disease in 58 cases (CH-patients: steatosis 8, persistent chronic hepatitis 8, active chronic hepatitis 42) and liver cirrhosis in the other 59 cases (LC-patients). The normal subjects and the CH-patients had similar values of max-PFV and mean-PFV (max-PFV 26.7 +/- 3.2 and 25.7 +/- 3.4 cm/s respectively; mean-PFV 22.9 +/- 2.8 and 22.4 +/- 3.8 cm/s respectively). The LC-patients' values (max-PFV 19.3 +/- 3.5; mean-PFV 16.9 +/- 2.9) were significantly lower than those of the normal subjects (P less than 0.001) and of the CH-patients (P less than 0.001). Considering the normal max-PFV to be in the range 20-33.1 cm/s (mean +/- 2 s.d. of the normal subjects, 95% confidence limits), max-PFV was reduced in 0/50 normal subjects, 1/58 CH-patients and 39/59 LC-patients (66.1% sensitivity; 98.2% specificity). In conclusion, the duplex-Doppler measurement of PFV is of great interest in the diagnostic study of patients with suspected chronic compensated liver disease and in the early diagnosis of cirrhosis. A low max-PFV is a reliable pointer to liver cirrhosis, whereas a normal max-PFV indicates a non-cirrhotic liver disease but is less probative. Each centre should standardize normal PFV values in order to establish their own threshold value for diagnosing liver cirrhosis.


Duplex-Doppler ultrasonography in the evaluation of cirrhotic patients with portal hypertension and in the analysis of their response to drugs. Sixteen patients (15 males, aged 48-70) affected by liver cirrhosis and oesophageal varices were subjected to duplex-Doppler ultrasonographic study (DDUS). Four patients (three with a portal thrombosis and one with a hepatofugal portal flow) were excluded from the subsequent pharmacological test. The twelve remaining patients took part in a double blind cross-over study that evaluated the variations of heart rate (HR), mean systemic arterial pressure (SAP), portal vein diameter (PVD), maximal and mean portal flow velocity (PFV) after the administration of either 40 mg of propranolol or placebo per os, on two consecutive days. Propranolol caused no significant variation in mean SAP and in PVD, whereas it reduced the HR from 67.7 +/- 8.0 to 58.4 +/- 7.0 beats/min (mean +/- s.d.; P less than 0.001); the maxPFV dropped from 18.2 +/- 5.4 to 14.0 +/- 3.7 cm/s (P less than 0.001) and the meanPFV dropped from 15.3 +/- 4.1 to 13.2 +/- 3.1 cm/s (P less than 0.005). No significant variation was observed with placebo. After propranolol administration eight patients exhibited a significant maxPFV decrease, whereas the other four patients exhibited only a drop in HR, suggesting either drug inefficacy, inappropriate dosage or inadequate duration of treatment. DDUS is the only non-invasive method for the examination of the portal vein system.(ABSTRACT TRUNCATED AT 250 WORDS)


Striking variability of hepatic copper levels in fulminant hepatic failure. Two cases of acute hepatic failure are reported in which the diagnosis of Wilson's disease was considered because of low serum ceruloplasmin, low serum copper levels and high 24 h urinary copper. Case 1 had Kayser-Fleischer rings, haemolysis and a high 24 h urinary copper, and so Wilson's disease was confidently diagnosed. Case 2 had high urinary copper excretion, but [64Cu] study indicated a 24:2 h ratio of 0.7 and made the diagnosis of Wilson's disease uncertain. Both patients underwent orthotopic hepatic transplantation, and multiple biopsies were taken from the resected specimen in order to estimate hepatic copper levels. In both cases, hepatic copper levels revealed considerable variation: 0.8-5.2 mumol/g dry wt (case 1) vs 0.02-12.65 mumol/g dry wt (case 2). In case 1, only two of 14 levels were within the diagnostic range for Wilson's disease (greater than 4 mumol/g dry wt), whereas hepatic copper levels in case 2 were in the Wilsonian disease range in three of 16 specimens. These results were in contrast to uniformly high hepatic copper levels in one patient with established cirrhosis secondary to Wilson's disease and two cases of primary biliary cirrhosis. This report indicates that hepatic copper levels vary greatly in acute liver failure, and that estimates from a single biopsy specimen may be misleading as to the cause of the underlying liver disease.


Effects of a gastric mucosal protecting agent in rats with liver cirrhosis. Male Sprague-Dawley rats were fed a 0.1% ethionine-added choline-deficient diet for 8 weeks to induce liver cirrhosis. At the same time 100 mg/kg/day teprenone was administered orally in order to evaluate its effects on the liver and gastric mucosal blood flow. Blood flow increased not only in gastric mucosa but also in liver tissues in the teprenone group. Serum transaminase levels and histopathologic findings of the liver also improved. These findings suggest that teprenone alleviates hepatocellular injuries. This effect may be partly attributable to cytoprotective effects of the catenoid isoprenoid moiety of teprenone on liver cells.


The interferon-alpha 2b gene in Japanese patients with chronic viral hepatitis who developed antibodies after treatment with recombinant interferon-alpha 2a. DNA was extracted from leucocytes of 23 Japanese patients with chronic viral hepatitis who received treatment with recombinant interferon-alpha 2a (IFN-alpha 2a) and nine healthy controls, as well as eight human cell lines of Caucasian or African origin. A part of the gene encoding IFN-alpha 2 was amplified by polymerase chain reaction and the sequence of nucleotides 1-231 was determined. Interferon-alpha 2a, -alpha 2b and -alpha 2c genes were tested for in five clones each from a patient or control, or a cell line, based on adenine or guanine at nucleotide positions 68 and 101. The IFN-alpha 2b gene was detected in all 160 clones from 23 Japanese patients and nine controls, but the IFN-alpha 2a or -alpha 2c gene was not found in any. Of five cell lines derived from Caucasians, four exhibited only the IFN-alpha 2b gene, while the remaining one exhibited both IFN-alpha 2a and -alpha 2b genes. Of three cell lines derived from Africans, one each showed only the IFN-alpha 2b or -alpha 2c gene, and the remaining one both IFN-alpha 2b and -alpha 2c genes. The 23 patients with the IFN-alpha 2b gene and chronic viral hepatitis included 10 who developed antibodies against IFN after treatment with recombinant IFN-alpha 2a. These results indicated a distinct geographical distribution of the three IFN-alpha 2 genes, and suggested the use of a recombinant IFN-alpha 2 preparation in agreement with the IFN-alpha 2 gene possessed by the recipient to avoid antibody responses.


Morphological correlates of protein kinase C induced potentiation in the chick brain slice. Continuous perfusion of a chick brain slice with 10 microM 4 beta phorbol 12,13 diacetate (PDAc) produces a significant increase in the amplitude of the response to electrical stimulation at 0.1 Hz recorded within the intermediate and medial part of the hyperstriatum ventrale (IMHV). This PDAc-induced potentiation and that induced by tetanising stimulation appear to share similar mechanisms. Quantitative electron microscopy of synapses within the IMHV from slices in which a PDAc induced potentiation had been produced 30 minutes earlier showed that compared with control slices there was a significant increase in the size of the postsynaptic density of spine synapses. The change was greater in magnitude than that observed in spine synapses following tetanically induced potentiation. No other synaptic parameters were affected.


Protein kinase activity and synaptic plasticity in a chick brain slice. In an in vitro slice preparation of the chick brain it is possible to record responses to single electrical stimuli from within the intermediate and medial part of the hyperstriatum ventrale (IMHV), a region known to be involved in learning. The amplitude of such responses is significantly increased by superfusion of the slice with 10 microM 4 beta-phorbol 12,13 diacetate (PDAc), a phorbol ester which stimulates protein kinase activity. The ability of PDAc to induce potentiation is greatest in chicks less than 6 days old. Administration of the kinase antagonist H7 prevents the induction of persistent potentiation and in fact produces a long lasting depression of response amplitude. H7 also produces a short term increase in excitability within the IMHV and results in increased expression of N-methyl-D-aspartate receptor activity.


Duodenal ulcer in China.

Duodenal ulcer in China. The author visited China in 1981 and 1984 and obtained data comparing the incidence of duodenal ulcer in the rice eating districts of the south with the incidence in the wheat, maize and millet eating areas of the north. The evidence suggested a higher prevalence of duodenal ulcer in the rice eating areas than in the wheat eating areas, and a low prevalence in association with millet eating. However, the differences were less marked than between similar rice and wheat eating areas of India. It is suggested that the lower prevalence of duodenal ulcer in the wheat eating areas of north India compared with the rice eating areas of south India may be due in part to the mucosal protective effect of wheat bran in the unrefined wheat that is used in making chappatis. In China white refined flour is used in the making of steamed bread with the loss of any protective effect of wheat bran.


Spontaneous regeneration of adult rat retinal ganglion cell axons in vivo. The superior brachial region of the left optic tract was lesioned in adult rats and foetal tectal tissue was implanted into the lesion site. The retinal projection from the contralateral (right) eye was examined 2 to 8 months later. In the majority of animals, retinal ganglion cell (rgc) axons were found to regenerate through cellular membranes which formed over the lesion. Axon growth could extend for up to 5 or 6 mm. Surviving tectal grafts were identified in all host rats. In animals in which regrowing rgc axons contacted tectal grafts, axons were found to innervate selectively their appropriate target regions within the graft neuropil.


Mono-arthritis in a chronic hepatitis B patient after alpha-interferon treatment. A 28 year old woman with hepatitis B (HB) related chronic active hepatitis was treated with a 12 week course of alpha-interferon (alpha-IFN). She developed acute mono-arthritis 1 week after completion of treatment. Her rheumatoid factor (RF) was positive before alpha-IFN and fell steadily during therapy. This was followed by a rebound of RF level with the associated arthritis occurring 1 week after completion of the course of alpha-IFN. In absence of any medication RF gradually fell and became negative at the end of 1 year. This observation is thought to be related to the immunomodulatory effect of alpha-IFN either directly on RF production or indirectly through the control of hepatitis.


Induction of social dominance by L-dopa treatment in Arctic charr. The effect of L-dopa on social dominance was studied in the juvenile Arctic charr (Salvelinus alpinus). L-dopa is the immediate precursor of dopamine, a neurotransmitter that has been connected with aggressive behaviour in fish as well as mammals. Arctic charr were placed in pairs. One individual in each pair was given L-dopa orally, while the other was given vehicle. The results showed that 18 out of 22 fish given 10 mg L-dopa kg-1 became dominant (p = 0.004, binomial test). A higher dose of L-dopa (200 mg kg-1) induced dyskinesia. L-dopa was found to cause a dose-dependent increase in the brain levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC, a major dopamine metabolite), as well as an increase in the DOPAC/dopamine ratio (an index of dopaminergic activity).


Expression of a single dopamine transporter cDNA can confer two cocaine binding sites. Radiolabeled cocaine analogs can bind to low and high affinity sites on striatal dopamine transporters (DAT). Recently, a cDNA encoding a rat brain dopamine transporter pDAT1 has been cloned. COS cells transfected with the pDAT1 in a eukaryotic expression vector express both a high (KD = 3.4 nM) and low affinity (KD = 163.6 nM) cocaine binding sites, suggesting that both sites are provided by a single gene product.


CaBP D-28k and NADPH-diaphorase coexistence in the magnocellular neurosecretory nuclei. Coexistence of the calcium binding protein calbindin D-28k and NADPH-diaphorase activity was studied in the magnocellular secretory nuclei of the rat hypothalamus using both immunocytochemical and histochemical techniques. Coexistence was found in all the nuclei considered (supraoptic, paraventricular, circularis and fornicals nuclei) with the exception of the hypothalamic area situated between the supraoptic and the paraventricular nuclei. Since both stainings are reliable markers, not based upon the physiological characteristics at a given moment, our study provides a further characterization of the neurons in the magnocellular neurosecretory nuclei.


Variations of pre-proNPY mRNA in the arcuate nucleus during the rat estrous cycle. In order to assess the role of sex steroids on the regulation of neuropeptide Y (NPY), levels of pre-proNPY mRNA were measured by in situ hybridization in the rat arcuate nucleus during the estrous cycle. Pre-proNPY mRNA levels were quite stable during diestrus I, diestrus II and the morning of the proestrus, and were 25-30% higher during the afternoon of the proestrus and during the day of the estrus. These data suggest that NPY synthesis is modulated by variations in the circulating levels of sex steroids. Also the excellent correlation between the variations in the levels of pre-proNPY mRNA and those of luteinizing hormone-releasing hormone (LHRH) mRNA observed throughout the estrous cycle supports the hypothesis that NPY might be involved in the regulation of LHRH secretion.


Noradrenergic inputs enhance the response of neurosecretory cells to osmotic stimulation. Extracellular recordings from neurosecretory cells in the hypothalamic paraventricular nucleus (PVN) displayed either an excitation or no response following electrical stimulation (5 Hz) of the A1 noradrenergic region of the ventrolateral medulla, and exhibited either an increase or no effect in neuronal activity in response to intracarotid injections of hypertonic saline (0.2 M, 0.05 ml). The neurons responded to both the electrical and osmotic stimuli, A1 region stimulation at the subthreshold current (80-90% of the threshold required to evoke the response) significantly enhancing the facilitatory response caused by the osmotic stimulation. These results suggest that the A1 region may serve to potentiate the responsiveness of PVN neurosecretory cells in response to changes in plasma osmolality.


Age-dependent changes in position sense in human proximal interphalangeal joints. Using a position matching paradigm, the ability of subjects to detect displacements at the proximal interphalangeal joint of the index finger was tested. These displacements were imposed at an angular velocity of 2 degrees per minute which is below the threshold for movement detection. An older group of subjects whose mean age (+/- s.e.m.) was 56.6 +/- 3.2 years showed significantly poorer performance in detecting the position of the index finger than a group of younger subjects (23.7 +/- 0.5 years). Analysis of the correlation between age and magnitude of matching error gave a positive correlation coefficient of 0.466 which was statistically significant. However, there was no systematic bias in judgements of finger position by the older group. These results indicate that the sense of position of the fingers decrements with age.


Dopamine fiber detection by [11C]-CFT and PET in a primate model of parkinsonism. Monkeys were treated on two regimens of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injections to achieve dopamine fiber degeneration of differing severities. A rapid treatment regimen produced a severe parkinsonian syndrome, whereas an intermittent regimen did not cause locomotor symptoms to appear up to 25 weeks. High resolution PET scanning of dopamine nerve terminals revealed that the specific binding of the dopamine transporter [11C]-WIN 35,428 ([11C]-CFT) was diminished by 94% (caudate nucleus) and by 93% (putamen) in the symptomatic monkey. Decreases of 65 and 67% were detected in these regions in the non-symptomatic monkey. Post-mortem immunocytochemical evaluation of presumed dopamine fibers by tyrosine-hydroxylase showed similar reductions in the symptomatic animal.


Trophic influences of excitatory and inhibitory synapses on neurones in the auditory brain stem. The effects of a reduction during development of excitatory and inhibitory synaptic input on CNS neurones were studied in the lateral superior olivary nucleus (LSO) of the ferret following neonatal, unilateral cochlear removal. LSO neurones normally receive excitatory input from the ipsilateral ear and inhibitory input from the contralateral ear. After cochlear removal, the ipsilateral LSO was smaller and contained fewer neurones than either the contralateral or the normal LSO. No difference was found between the volume or number of neurones in the latter nuclei. Remaining neurones in the LSO ipsilateral to the removal were smaller than those in the contralateral LSO of the same ferrets. These data show that activity in excitatory pre-synaptic terminals can be sufficient for post-synaptic target maintenance, but that activity in inhibitory terminals alone is not.


P2 purinoceptors stimulate inositol phosphate release in the organ of Corti. We examined the effects of purinoceptor agonists on inositol phosphate (IP) release in the guinea-pig organ of Corti. The P2y receptor agonist ATP-gamma-S (200 microM) increased IPs 4-fold; identical concentrations of alpha, beta-methylene ATP, a P2x agonist, and adenosine, a P1 agonist, did not significantly affect IP release. In calcium-free incubations, simulated IP release decreased by 35% indicating partial dependence of ATP-mediated phosphoinositide hydrolysis on calcium influx. ATP-stimulated IP release was not enhanced by the cholinergic agonist carbachol known to increase IPs via muscarinic receptors in the organ of Corti. This is consistent with the notion that ATP and carbachol have a common target, most likely outer hair cells. P2 purinoceptors coupled to the phosphoinositide cascade suggest ATP as an afferent neuromodulator or efferent neurotransmitter in the cochlea.


M1 and M3 muscarinic receptors: specific roles in sleep regulation. P-fluoro-hexahydro-sila-difenidol hydrochloride (p-F-HHSiD) (15, 30 micrograms) and pirenzepine (7.5, 15, 30 micrograms), which are highly selective M3 and M1 muscarinic antagonists, respectively, were injected intracerebroventricularly into freely moving rats. p-F-HHSiD (30 micrograms) reduced wakefulness (W) (from 34.7 +/- 3.1 to 24.9 +/- 1.3 min) and increased slow wave sleep (SWS) (from 56.7 +/- 2.4 to 67.2 +/- 1.5 min); however, it did not modify desynchronized sleep (DS) latency and percentage in 6 h recordings. W and SWS were not affected by pirenzepine (7.5, 15, 30 micrograms) which decreased significantly DS amount but left unaffected DS latency. The results suggest that each muscarinic receptor subtype may induce different and specific changes in sleep phases and cortical desynchronization processes.


In vivo and in vitro NGF studies on developing cerebellar cells. The biological effect of nerve growth factor (NGF) in the early prenatal cerebellar cell was studied in vivo and in vitro with autoradiographic, tissue culture and immunohistochemical techniques. Iodinated NGF (125I-NGF) injected into the cerebella of 16-day-old rat embryos showed accumulation of this ligand in the Purkinje cell layers. The ability of these cells to accumulate NGF lasted to the 19th day of embryonic life. Cerebellar cells isolated from embryos of the same age, but not older embryos, cultured in vitro for two weeks in the presence of NGF, showed morphological characteristics similar to Purkinje-like cells. These findings suggest that NGF exerts a time-limited trophic effect on immature Purkinje cell precursors.


Multi-phase expiratory inhibition of phrenic motoneurons in the decerebrate cat. We studied the membrane potential changes of 11 phrenic motoneurons (PMs), located in the C5 portion of the cervical spinal cord, in decerebrate, paralyzed and artificially ventilated cats. Intracellular chloride iontophoresis into PMs could reverse the waves of inhibitory postsynaptic potentials (IPSPs) causing membrane hyperpolarization during expiration. In all PMs, we revealed, in addition to the previously described late expiratory augmenting wave of IPSPs, an early expiratory decrementing wave of IPSPs. Moreover, in three PMs, recorded in two cats exhibiting slow respiratory rhythms, an additional wave of IPSPs was interposed between the early and late ones. The possible origins and roles of these three different waves of inhibition are discussed.


Analgesia for tonic pain by self-administered lateral hypothalamic stimulation. The present study was designed to determine if analgesia for tonic pain could be induced with self-administered lateral hypothalamic (LH) stimulation. The majority of studies of LH stimulation employed behaviorally non-contingent stimulation. According to some investigators behaviorally non-contingent LH stimulation might be aversive and consequently yield stress-induced analgesia rather than a primary analgesic effect. Other investigators have reported that non-contingent LH stimulation can be rewarding. Our findings indicate that LH analgesia for tonic pain can be obtained with self-administered LH stimulation. These findings indicate that the analgesia we have obtained in previous work is a primary effect and not dependent on stress induced by behaviorally non-contingent LH stimulation.


Application of testosterone accelerates oligodendrocyte maturation in brains of zebra finches. The effects of exogenously applied hormones on glial cell maturation of telencephalic song motor centers, midbrain and cerebellar structures was studied in juvenile male zebra finches. Testosterone was administered and the development of oligodendrocytes was studied using immunochemistry and computer aided image analysis on silver impregnated brain sections. A testosterone induced acceleration of oligodendrocyte maturation could be detected in several brain areas by using the monoclonal antibody O 10 recognizing an oligodendrocyte-specific cell surface antigen and by the silver impregnation for myelin. The increase in myelin density was higher in the testosterone treated animals than in the control animals in the forebrain and in the cerebellum, whereas two regions in the midbrain showed no difference between treated and controls.


Two classes of potassium currents in Xenopus muscle cells in young cultures. The aim of the present study was to determine if both inactivating and non-inactivating potassium currents occur in skeletal muscle cells at early times in culture. Whole cell current was recorded from embryonic muscle cells within 4-10 h of plating. We found that in the cells which had an outward current, half had a current that did not inactivate and half had a current that inactivated during a maintained depolarization (100 ms). The two groups of cells did not differ in their age in culture or resting membrane potential. The results indicate that Xenopus skeletal muscle cells can acquire both types of outward potassium current early in culture life.


First isolation of gamma-L-glutamyl-gamma-aminobutyric acid from bovine brains. We had obtained some immunohistochemical indications which suggested that peptide(s) containing GABA at the C-terminal could exist in specific neurons in brains. We therefore sought such GABA conjugate(s) by isolating an acidic GABA-dipeptide from bovine brains and determined its structure as gamma-L-glutamyl-gamma-aminobutyric acid (gamma-L-Glu-gamma Abu:GluGABA). Its gamma-linkage was determined by a mass-spectrometric (B/E linked scan SIMS) analysis; its chirality was established by a chiral column HPLC technique. This peptide is the first acidic GABA-peptide recognized in brain and the first brain peptide containing GABA at the C-terminal.


Biocytin injections produce selective neuronal labeling in the rat CNS. Large injections of biocytin into the lateral ventricle or brain resulted in the labeling of particular neuronal subpopulations in the rat CNS. Localization was accomplished using the avidin-biotin-peroxidase technique. In many cases the staining of neurons was totally complete and resembled that obtained with the Golgi technique. Regions containing labeled cells included the olfactory bulb, cerebral cortex, hippocampus, amygdala, striatum, hypothalamus, superior and inferior colliculi, cerebellar cortex, and dorsal horn of the spinal cord. Only particular cell types were labeled in each of these regions. The results of this study suggest that there is selective uptake and/or retention of biocytin, or a biotinylated metabolite of biocytin, by subpopulations of CNS neurons.


Chol-1 is a cholinergic marker in the human central nervous system. The cholinergic-specific gangliosides Chol-1 alpha and beta were detected in human brain and spinal cord by immune staining of thin-layer chromatography (TLC) plates on which ganglioside extracts had been separated. The colocalization of choline acetyltransferase (ChAT) and the Chol-1 antigens in ventral horn motoneurons was demonstrated immunocytochemically. In analytical studies, Chol-1 was found to be more concentrated in dorsal cord than ventral but the reverse was true of ChAT. This difference was explained by differences in the subcellular location of the two markers. Within each region of the thoracic cord the levels of ChAT and Chol-1 in different cords showed covariance. The expected fall of ChAT and Chol-1 in amyotrophic lateral sclerosis (ALS) cords was not seen and possible reasons for this are discussed.


The effects of two different inhibitors of PNMT and their interactions with ethanol. The effects of two structurally different inhibitors of phenylethanolamine-N-methyltransferase, LY 134046 and CGS 19281A were investigated in a holeboard test of directed exploration and locomotor activity. Both compounds dose-dependently reduced exploratory head-dipping without affecting locomotor activity. The interaction of each drug with ethanol was also studied by testing the ataxia. Administration of these compounds had differential effects in a test of ethanol-induced ataxia. LY 134046 significantly attenuated ethanol-induced ataxia whereas CGS 19281A was without effect or (at 50 mg kg-1) potentiated ethanol's effect. These results suggest that the ethanol attenuating properties of LY 134046 may not solely be due the inhibition of PNMT and that its alpha 2-adrenoceptor blocking properties may be playing a role.


Recovery of nucleus basalis cholinergic neurons by grafting NGF secretor fibroblasts. The effects of nerve growth factor (NGF) secreted from genetically modified fibroblasts was studied in vitro, using dissociated septal cells and in vivo, in rats bearing unilateral cortical devascularizing lesions. Transfected fibroblasts expressing nerve growth factor (NGF) were co-cultured with rat embryonic cholinergic cells of the septal region. This in vitro system showed that NGF secretor cells produce biologically active NGF, as determined by increasing choline acetyltransferase (ChAT) activity in septal culture after seven days. The potential therapeutic value of applying grafts of transfected fibroblasts expressing NGF in the model of retrograde atrophy of cholinergic neurons of the nucleus basalis magnocellularis (NBM) was assessed following partial devascularizing lesions of the cerebral cortex. We observed an increase in ChAT activity in the remaining cortex and a partial protection of the ipsilateral NBM, as determined by morphometric and biochemical studies.


Presynaptic calcium transients evoked by paired-pulse stimulation in the hippocampal slice. The fluorescent dye Calcium Green and optical recording techniques were used to record intracellular Ca2+ transients resulting from paired-pulse stimulation in stratum moleculare of area CA1 in guinea-pig hippocampal slices. Presumed presynaptic calcium transients were recorded while glutamatergic synaptic transmission was blocked by kynurenic acid. Peak responses to paired-pulses (10-160 ms interval) were higher than responses to single pulses of same stimulation strength (42-23% increase). The isolated response to the second pulse, however, was of smaller magnitude in comparison to the first one; the difference in magnitude depended on the interstimulus interval. Thus, the residual presynaptic free calcium concentration may be responsible for paired-pulse facilitation of synaptic transmission in hippocampus. At the same time, a use-dependent inactivation of presynaptic calcium channels may occur.


Respiration-related neurons recorded in the deep cerebellar nuclei of the alert cat. The activity of deep cerebellar nuclei neurons was recorded in the alert cat and related to the respiratory cycle. Respiration-related neurons (RRNs, n = 29), located in the rostral fastigial and interpositus nuclei, were classified as inspiratory (24%) or expiratory (76%). Nine RRNs were antidromically activated from the red nucleus, but none from the inferior olive. Half of the RRNs showed well defined proprioceptive inputs of a rather broad origin. Other RRNs (27%) showed a respiration-related pattern independent of respiratory movement performance. Repeated electrical stimulation of the inferior olive exerted a synchronizing effect on the firing rate of 24% of the RRNs. It is proposed that cerebellar nuclear RRNs are involved in locomotor re-adjustments of the respiratory musculature.


Oscillatory activity is not evident in the primate temporal visual cortex with static stimuli. It has been suggested in studies in the visual system of anaesthetized cats that oscillatory activity with a frequency of 40-60 Hz occurs during the presentation of moving visual stimuli and reflects a synchronization process between neurons that could implement the binding together of related neurons into different sets. We found no evidence for such oscillations in the inferior temporal visual cortex and related areas of awake macaques fixating effective static visual stimuli, which for the neurons analysed were faces. We put forward the possibility that temporal synchronization between neurons to implement binding may not be generally used in the visual system as a solution to the binding problem, at least when static objects are being processed and recognised in higher parts of the visual system.


A T-DNA transfer stimulator sequence in the vicinity of the right border of pRi8196. An 8 bp sequence repeated 6 times is present to the right of the mannopine type pRi8196 T-DNA right-border sequence. Experiments were designed to test whether these repeats have a role in T-DNA transfer. Several constructs in which different lengths of pRi8196 right-border region were linked to the cucumopine synthesis gene on an Agrobacterium-Escherichia coli shuttle vector were made. The recombinant plasmids were tested for their efficiency to act as a source of T-DNA in a binary system in which a wild-type Ri plasmid provided virulence and root-inducing functions. The T-DNA transfer efficiency of the constructs was assessed by computing the relative frequency of roots containing cucumopine. Depending on the Ri plasmid used as source of virulence functions, a high level of T-DNA transfer was observed only if 6 (pRi8196) or 5 (pRiA4) repeats were present. These results were confirmed by looking for single-stranded T-DNA molecules (T-strands) in bacteria induced for virulence. The repetition of the 8 bp unit was named 'T-DNA transfer stimulator sequence' (TSS).


Transfection of heteroduplexes containing uracil.guanine or thymine.guanine mispairs into plant cells. We have compared the fate of U.G mispairs or analogous T.G mispairs in DNA heteroduplexes transfected into tobacco protoplasts. The heteroduplex DNA consisted of tomato golden mosaic virus DNA sequences in the Escherichia coli vectors pUC118 or pUC119. After transfection, the mismatched U residues were lost with an efficiency of greater than 95%, probably as a result of the uracil-DNA glycosylase pathway for excision of U residues in any sequence context. In contrast to the preferential removal of the mispaired U residues, biased removal of T residues from analogous heteroduplexes was not seen in the transfected plant cells. Also, we investigated the effect of extensively methylating one strand of the heteroduplex DNA used for transfection. Surprisingly, such methylation resulted in highly biased loss of the mismatched base from the 5-methylcytosine-rich strand of T.G-containing heteroduplexes.


Nucleotide sequence and temporal regulation of a seed-specific Brassica napus cDNA encoding a stearoyl-acyl carrier protein (ACP) desaturase. The nucleotide sequence is reported for a cDNA containing the entire coding region of a stearoyl-ACP desaturase (EC 1.14.99.6) from Brassica napus L. cv. Jet neuf. The cDNA was obtained from a library constructed from poly(A)+ RNA purified from embryo tissue. The derived amino acid sequence demonstrates substantial similarity with those from other plant delta 9-desaturases. Comparative RNA-dot blot analyses using the delta 9-desaturase cDNA and a rapeseed oleosin cDNA as probes showed that although both these transcripts were seed-specific, they exhibited distinct patterns of temporal regulation. The desaturase message was induced by 25 days after anthesis (DAA), peaking at 45 DAA but decreasing considerably thereafter. In contrast, the oleosin transcript did not increase until 45-50 DAA, reaching a peak much later at about 70 DAA.


Potato granule-bound starch synthase promoter-controlled GUS expression: regulation of expression after transient and stable transformation. Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the beta-glucoronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosum Solanum brevidens, Nicotiana tabacum and Arabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.


The cab-m7 gene: a light-inducible, mesophyll-specific gene of maize. Southern blot analysis has revealed the existence in maize of perhaps 12 members of the nuclear cab multigene family encoding the chlorophyll a- and b-binding proteins of the photosystem II light-harvesting complex. Hybridization with 3' probes derived from unsequenced cDNA clones showed that six members of this family differ from one another with respect to expression in mesophyll and/or bundle sheath cells and regulation by light. An additional member of this family, designated cab-m7, that encodes a 28 kDa primary translation product has now been identified. It has been cloned from a maize genomic library and sequenced to begin to define the bases for differences in the expression of these genes. This cab gene is shown to be strongly preferentially expressed in the mesophyll (vs. bundle sheath) cells of maize. Furthermore, the gene is photo-responsive; although small amounts of cab-m7 mRNA are present in etiolated leaves, the mRNA pool is 8-fold larger after six hours of illumination. DNA sequences upstream of the cab-m7 gene resemble those found in the 5'-flanking regions of some other plant genes.


Arabidopsis thaliana small subunit leader and transit peptide enhance the expression of Bacillus thuringiensis proteins in transgenic plants. The expression of the modified gene for a truncated form of the cryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein from Bacillus thuringiensis var. kurstaki (B.t.k.) HD73, under control of the Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit ats1A promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants. Examination of leaf tissue revealed that the ats1A promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10-fold to 20-fold increase in cryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-En35S) was used to express the same cryIA(c) gene. Transient expression assays in tobacco protoplasts and the whole plant results support the conclusion that the transit peptide plus untranslated sequences upstream of that region are both required for the increase in expression of the CryIA(c) protein. Furthermore, the CaMV-En35S promoter can be used with the Arabidopsis ats1A untranslated leader and transit peptide to increase expression of this protein. While subcellular fractionation revealed that the truncated CryIA(c) protein fused to the ats1A transit peptide is located in the chloroplast, the increase in gene expression is independent of targeting of the CryIA(c) protein to the chloroplast. The results reported here provide new insight into the role of 5' untranslated leader sequences and translational fusions to increase heterologous gene expression, and they demonstrate the utility of this approach in the development of insect-resistant crops.


Peptide growth factors and their interactions during chondrogenesis. Peptide growth factors have been implicated in three aspects of cartilage growth and metabolism; the induction of mesoderm and differentiation of a cartilaginous skeleton in the early embryo, the growth and differentiation of chondrocytes within the epiphyseal growth plates leading to endochondral calcification, and the processes of articular cartilage damage and repair. Three peptide growth factor classes have been strongly implicated in these processes, the fibroblast growth factor family (FGF), the insulin-like growth factors (IGFs) including insulin, and transforming growth factor-beta (TGF-beta) and related molecules. Each of these peptide groups are expressed in the early embryo. Basic FGF, TGF-beta and the related activin have been shown to induce the appearance of mesoderm from primitive neuroectoderm. TGF-beta and related bone morphometric proteins can induce the differentiation of cartilage from primitive mesenchyme, and together with basic FGF and IGFs promote cartilage growth. Each class of growth factor is expressed within the epiphyseal growth plate where their autocrine/paracrine interactions regulate the rate of chondrocyte proliferation, matrix protein synthesis and terminal differentiation and mineralization. Basic FGF may prove useful in articular cartilage repair, while basic FGF, IGFs and TGF-beta are among a number of growth factors and cytokines that have been implicated in cartilage disease.


[Damage to the remaining teeth from displaced upper canines]. 25 patients with 35 retained upper canines were examined by high resolution CT; only 5 patients did not show evidence of damage to the remaining teeth. 8 patients showed resorption related to medial and lateral incisors. 19 teeth were in contact with the crowns of the displaced canines and were therefore liable to damage. The average extent of the resorption was 4.5 mm. Contact between teeth was demonstrable by means of two consecutive CT cuts.


[The magnetic resonance tomography of intracranial ependymomas. Their clinical appearance and comparison with computed tomography]. The MRT and CT images of histologically confirmed intracranial ependymomas in 10 patients have been compared. All tumours were demonstrated by CT and MRT. The solid portions of the tumours showed variation in density on CT (hypodense, isodense, hyperdense, or mixed), whereas there was uniform signal intensity on MRT (T1 weighted: hypointense, T2 weighted: hyperintense). Cystic portions of the tumours (6 patients) were shown equally well by both procedures, calcifications were only shown by CT. All ependymomas were related to the ventricles. On unenhanced CT only 3 tumours were clearly delineated, whereas MRT defined all tumours satisfactorily. The significant advantage of MRT is better topographic demonstration of the tumour.


[Intracranial aneurysms. A comparison between magnetic resonance tomography and arteriography]. 42 MRI of 36 patients with cerebral aneurysms were analysed retrospectively and compared with selective angiography. 92% of aneurysms proven by angiography were also detected by MRI. The smallest and surgically confirmed aneurysms had a diameter of 2-3 mm on MRI. 3 aneurysms were only demonstrated by selective angiography (8%). This concerned 5 mm sized aneurysms located at the anterior communicating artery (2) and middle cerebral artery (1). On the other hand 2 thrombosed aneurysms were only delineated by MRI (6%). Because partial thrombosis could not be demonstrated by angiography extensions of aneurysms were underestimated in 44% (n = 16) angiographically. A 2.5 cm sized lobulated aneurysm of the internal carotid artery was initially evaluated by MRI as being thrombosed subtotally, due to flow-phenomena. Angiography though showed a wide perfused lumen without visible thrombosis. A progressive thrombosis of a mycotic aneurysm of the internal carotid artery was shown by MRI during septic thrombosis of the cavernous sinus. In this case the number of angiographic controls could be reduced by use of MRI. Nevertheless, MRI was not able to substitute selective angiography, especially to exclude under 6 mm sized cerebral aneurysms. MRI could not demonstrate clearly the vessels in the neighbourhood of saccular aneurysms preoperatively.


[Primary and secondary tethered cord syndromes. Their nuclear magnetic tomographic diagnosis and comparison with the surgical findings in 40 patients]. The MRT findings in 40 patients with the tethered cord syndrome (TCS) have been compared with the operative findings. In cases of primary TCS (17 patients), MRT showed a low conus medullaris in 15 cases and this was confirmed surgically in 14. Other abnormal findings were lipomas (MRT 12/operatively 13), diastematomyelia (MRT 2/operatively 2) and thickened filum terminale (MRT 6/operatively 3). In secondary TCS (23 patients) postoperatively adhesions were prominent (MRT 16/operatively 23) while low position of the conus was found by MRT in 19 and surgically in 18 patients. Other findings were lipomas (MRT 5/operatively 6), epidermoids (MRT 2/operatively 2) and diastematomyelia (MRT 2/operatively 0). Agreement between MRT and operative findings regarding fixation of the spinal cord is closer in primary TCS (15/17, 88%) than in secondary TCS (16/23, 69%).


[The CT-controlled treatment possibilities in intervertebral disk prolapses]. A CT-controlled method for the treatment of lumbar and cervical disc prolapse is described. Compared with fluoroscopically controlled percutaneous lumbar discectomy, percutaneous treatment of the L5/S1 disc by a dorsal approach is possible even in the presence of high iliac crests, as is described in this paper. In the cervical region the risk of iatrogenic damage is reduced. In a small group of 42 patients, an attempt has been made to remove disc material which is intraforaminal, intraspinal, subligamentous or sequestered. Amongst 79 patients with lumbar and 15 patients with cervical disc prolapse treatment was successful in 85% (after 17 months of observation) and 93% (after 12 months of observation). These figures refer to the early results from a new method. The success of the method will have to be assessed after a larger number of patients has been treated and there has been a longer period of observation.


[Automated percutaneous lumbar diskectomy (APLD): the early and late changes in the CT with a clinical correlation]. In 52 out of 162 patients treated, CT changes were measured at early and late stages and correlated with clinical findings. Reduction in height of discs was measured by digital radiography. During early stages 9.6% showed gas collections peripherally and 3.8% centrally. Early and late examination showed uniform reaction in the area of the prolapse: no changes (type I), return to normal findings (type II), diffuse reduction of a broadly based lesion (type III), reduction in focal changes based on a broad lesion (type IV). Early results: 94.3% type I, 3.8% type III, 1.9% type IV. Late results: 55.7% type I, 3.8% type II, 21.2% type III, 19.3% type IV. Maximal reduction of the height of the disc of 10% occurred in 32.6% of cases. There was no meaningful correlation between CT appearances and clinical status.


[Quantitatively evaluated dynamic magnetic resonance tomography in chronic polyarthritis of the knee joint. A therapeutic follow-up after intra-articular cortisone application]. 32 patients with chronic polyarthritis were examined by MRT. Following intra-articular application of 25 mg prednisolone, 18 patients were re-examined after two of three weeks and 7 out of 18 patients were re-examined after 8 to 16 weeks, both by MRT and clinically. After an initial double echo sequence, we carried out a dynamic series of FLASH measurements (TR 30, TE 10 ms, 70 degrees) following an intravenous bolus of 0.1 mmol gadolinium-DTPA. Single intensity-time curves were treated quantitatively in relation to the pannus, muscles and effusions. Two or three weeks after treatment the curve parameters showed a reduction in pannus vascularisation which remained constant for the 8 to 16 weeks of further observation. Contrast diffusion in the pannus is initially reduced but approaches the early measurements at the time of the second examination.


[Comparative studies with quantitative computed tomography and dual-energy x-ray absorptiometry on bone density in renal osteopathy]. Measurements of bone density were carried out in 25 patients on dialysis for terminal renal insufficiency, using quantitative computed tomography (QCT) and dual-energy X-ray absorptiometry (DXA). Unlike in subjects with normal kidneys, there was no significant correlation between these methods in this series. Ten patients showed an increase in bone density of the vertebral spongiosa on QCT measurements, which was interpreted as due to osteosclerotic bone changes in renal osteopathy. QCT showed advantages over DXA in demonstrating these changes.


[CT-guided large-bore biopsy of solid non-organ-bound space-occupying lesions in the retroperitoneum]. Seventy-eight solid, non-organic retroperitoneal tumours were biopsied with a 14-gauge Tru-Cut needle in 73 consecutive patients with the exception of one. Adequate material was not obtained in only one patient with the primary known (1.3%). In the adequate material (98.7%), the dignity of all lesions was accurately determined and 93.1% of lesions were accurately classified. Typing accuracy reached 100% in histologically known primaries (n = 31) and 87.8% in histologically unknown primaries (n = 41). A hypertensive crisis and a small haematoma following biopsy of an inadvertent extra-adrenal phaeochromocytoma was the single complication found in this series. An arterial bleeding following biopsy was prophylactically embolised through the biopsy needle. CT-guided large-bore biopsy of solid non-organic retroperitoneal tumours is a safe, non-invasive procedure with a high diagnostic yield that obviates the need for open diagnostic procedures in a large number of cases.


The clinical value of computed tomography and lymphography in detecting lymph node metastases from epithelial ovarian cancer. We investigated the accuracy of both lymphography and computed tomography (CT) in detecting lymph node metastases in 58 patients with primary epithelial ovarian cancer (group a) and subsequently submitted to surgery including pelvic and/or lumbo-aortic lymphadenectomy. CT accuracy was also investigated in 41 patients with clinically suspected relapse of ovarian cancer (group b). In the first group (a) overall results in the pelvis were, respectively, for lymphography and CT: 94.8 vs 89.6% accuracy, 85.7 vs 57.1% sensitivity, 97.7 vs 100% specificity, 97.7 vs 100% positive predictive value and 95.5 vs 88% negative predictive value. In the lumbo-aortic region, we had: 88.8 vs 86.1% accuracy, 71.4 vs 64.2% sensitivity, 100% specificity and positive predictive value for both techniques, and 84.6 vs 81.6% negative predictive value. In the second group (b) CT accuracy, sensitivity and specificity were 90.2, 80 and 100%, respectively. CT, thanks to its high specificity and positive predictive value, can represent the method of choice for the evaluation of pelvic and lumbo-aortic lymph node metastases in untreated and relapsing ovarian cancer. CT demonstration of lymph node metastases can affect not only lesion staging, but also chemotherapy; different indications for lymphadenectomy may also depend on CT.


[Computed tomography: the TNM staging of laryngeal carcinoma]. In 64 patients with malignant lesions of the larynx, CT and microlaryngoscopy (ML) were evaluated in terms of T-staging. Histological information was present in all cases following operation. CT showed an accuracy of 90% in T-staging. ML showed an accuracy of only 71% of correct T-staging. CT is superior to ML particularly in distinguishing between stages T3 and T4 and T1 and T2. CT tends to overestimate tumour stage whereas ML tends to underestimate it. CT was also compared with palpation and sonography as regards N-staging. In this respect sonography has the highest accuracy (94%) followed by palpation (76%) and CT (74%).


[The potentials and limits of blood-flow quantification in the peripheral arteries with MRT using a phase-mapping procedure]. MRT is able to demonstrate arteries while MR angiography can quantify blood flow by a noninvasive method. In the present paper blood flow measurements were carried out in four selected cases on the basis of phase mapping. In 3 patients with lesions in the pelvis or thigh, angiography was performed in order to localise the stenosis or occlusion and this was followed by quantitative blood flow measurements. The results showed that angiography may not always provide all necessary information concerning a haemodynamically significant stenosis. The method may also be used for quantifying blood flow in the renal arteries and that it has significant advantages over the colour Doppler method.


[Magnetic resonance tomography of the liver with TurboFLASH and segmented acquisition. Fast imaging with flexible image contrast]. The value of a fast imaging sequence in diagnosing focal liver lesions was assessed in 43 patients. The sequence (TurboFLASH with segmented acquisition) allows the acquisition of T1- and T2-weighted images within a breath-hold interval. Lesion-liver contrast was compared with T2-weighted spin-echo, T1-weighted gradient-echo, and single shot TurboFLASH sequences. All lesions that were seen with the latter sequences were also visible on the segmented TurboFLASH images. Contrast on the segmented TurboFLASH images was similar to that of T2-weighted spin-echo sequences and superior to T1-weighted gradient-echo sequences. Signal of fat could be nulled at an inversion time of 10 ms, signal from liver tissue could be nulled at an inversion time of 300 ms. By minimising the liver signal, the lesions could be "highlighted". This was not possible with the other imaging sequences. The segmented TurboFLASH technique allows breath-hold imaging of the liver with arbitrary image contrast. The lesion-liver contrast is comparable to time-consuming T2-weighted spin-echo sequences.


High-dose chemoradiotherapy followed by autologous bone marrow transplantation as consolidation therapy during first complete remission in adult patients with poor-risk aggressive lymphoma: a pilot study. Twenty consecutive patients with poor-risk aggressive lymphoma who at presentation either had elevated serum lactic dehydrogenase level (LDH) and any one of the other poor-prognostic features: bulky mass greater than or equal to 10 cm, advanced stage III or IV, and greater than or equal to 2 extranodal sites, or normal LDH level and all other three features, underwent high-dose chemo/radiotherapy followed by unmanipulated autologous bone marrow transplantation (BMT) during their first complete remission. Eighteen had B-cell lymphoma and 2 had T-cell lymphoma. Eleven patients had high-grade (7 immunoblastic, 3 small noncleaved, non-Burkitt's, and 1 Burkitt's) and 9 had diffuse large cell lymphoma. All patients had achieved a complete remission following conventional chemotherapy. Four patients had also received involved field radiotherapy to areas of bulky disease. The preparative regimen consisted of high-dose etoposide 60 mg/kg and cyclophosphamide 100 mg/kg in combination with fractionated total body irradiation (FTBI) 1,200 cGy (15 patients), or single-dose TBI 750 cGy (2 patients), or carmustine 450 mg/m2 (3 patients). All patients tolerated the treatment well and achieved complete hematologic recovery. Three patients have relapsed at days 79, 196, and 401 after transplantation. Seventeen patients (84%) are alive and relapse-free with a median follow-up of 34 months (range 2 to 54). We conclude that high-dose chemo/radiotherapy followed by autologous BMT can be given as consolidation therapy during first remission in these patients with minimal transplant-related toxicity.


Disposition of recombinant human granulocyte-macrophage colony-stimulating factor in patients receiving high-dose chemotherapy and autologous bone marrow support. Recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) produces dose-related therapeutic and toxic effects; however, relationships between its pharmacokinetics and pharmacodynamics have not been extensively evaluated. The following studies were undertaken to investigate patterns in the disposition of rHuGM-CSF administered after high-dose chemotherapy (cyclophosphamide, cisplatin, carmustine) and autologous bone marrow support. Continuous 14 or 21 day intravenous infusions or daily 4-hour infusions were studied at doses of 1.2 to 19.2 micrograms/kg/d. GM-CSF was measured by an enzyme-linked immunosorbent assay from serum and urine samples collected throughout drug administration. Pharmacokinetic parameters were determined by compartmental (4-hour infusions) or noncompartmental methods (continuous infusions). GM-CSF was rapidly eliminated from the serum. Average systemic exposure increased with dose, although wide interpatient variability was evident. Approximately one half of the patients receiving continuous infusions demonstrated increasing GM-CSF clearance that corresponded to the appearance of white blood cells in the periphery. Conversely, clearance decreased in those experiencing renal dysfunction during the infusion. The percentage of a GM-CSF dose found in 24-hour urine collections was substantially reduced in the latter group. A subset of patients who developed renal dysfunction also experienced significant hypotension. Rapidly increasing serum GM-CSF concentrations corresponded to the hypotensive episodes. GM-CSF serum concentration monitoring may be useful for evaluation of therapeutic and toxic effects in patients receiving high-dose chemotherapy with autologous bone marrow support.


Effects of interleukin-3 after chemotherapy for advanced ovarian cancer. To define the maximum tolerated dose and to study whether recombinant human interleukin-3 (rhIL-3) reduced chemotherapy-induced neutropenia and thrombocytopenia, 20 chemotherapy-naive patients with advanced ovarian cancer eligible for treatment with 6 cycles of carboplatin-cyclophosphamide every 4 weeks (day 1) were entered in a phase I/II open, single-center trial. Cohorts of five patients received during 7 days 1, 5, 10, or 15 micrograms/kg/d rhIL-3 (days 5 through 11) in cycles 1, 3, and 5 by continuous intravenous (IV) infusion or once daily subcutaneous (SC) administration. In control cycles 2, 4, and 6, no rhIL-3 was administered. rhIL-3 significantly increased the recovery of leukocyte, neutrophil, and platelet counts, especially at 5, 10, and 15 micrograms/kg rhIL-3. rhIL-3 also increased basophil, eosinophil, monocyte, and lymphocyte counts at this dose steps. Effects on reticulocytes were limited. No difference in efficacy between SC and IV rhIL-3 treatment was found. Chemotherapy postponement for insufficient bone marrow recovery was necessary in 22 of 45 control cycles versus 2 of 49 rhIL-3 cycles (P less than .001). Platelet transfusions were required in 7 of 45 control cycles versus 3 of 50 rhIL-3 cycles (P less than .5). rhIL-3 up to 10 micrograms/kg/d could be administered without severe side effects. At 15 micrograms/kg/d, rhIL-3 headache was dose-limiting. Other side effects were fever, flu-like symptoms, nausea, skin rash, flushing, facial erythema, and urticaria. Liver toxicity occurred in rhIL-3 and control cycles. rhIL-3 slightly increased tumor necrosis factor alpha, C-reactive protein, and serum amyloid A plasma levels, whereas no effect on IL-6 plasma levels was observed. rhIL-3 administered SC appears to be an interesting hematopoietic growth factor for reduction of chemotherapy-induced myelotoxicity.


Recombinant human granulocyte-macrophage colony-stimulating factor after high-dose chemotherapy and autologous bone marrow transplantation with unpurged and purged marrow in non-Hodgkin's lymphoma: a double-blind placebo-controlled trial. The toxicity of autologous bone marrow transplantation (ABMT) is correlated to neutropenia. Although recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) seems to hold promise in accelerating neutrophil recovery, few analyses from randomized studies are presently available. Ninety-one patients with non-Hodgkin's lymphoma receiving high-dose ablative chemotherapy followed by ABMT with unpurged or purged marrow were included in a randomized, double-blind, placebo-controlled trial. Forty-four patients received 250 micrograms rhu GM-CSF (Escherichia coli)/m2 and 47 patients received placebo. Treatment was administered daily as continuous infusion from day of ABMT until the absolute neutrophil count (ANC) reached 0.5 x 10(9)/L for 7 days or until day 30, whichever was first. With rhu GM-CSF, 50% of the patients reached an ANC count greater than 0.5 x 10(9)/L at day 14 as opposed to day 21 with placebo (P less than .0001). Patients transplanted with marrow purged by mafosfamide also recovered earlier when treated with rhu GM-CSF (16 v 20.5 days, P = .013). The hospitalization duration was shorter in the rhu GM-CSF group (median, 23 v 28 days, P less than .05). No difference was observed in fever, number of infections, and antibiotic administration between the two groups. The major adverse event ascribed to rhu GM-CSF was a capillary leak syndrome in three patients graded as severe in two patients, moderate in one, and reversible in all three patients. In addition, one patient in the rhu GM-CSF group died suddenly with no explanation. In long term follow-up, the relapse rate was identical in both groups and there was no significant difference in the number of deaths at 1 year (12 with rhu GM-CSF v 9 with placebo), although deaths seemed to occur slightly earlier in the rhu GM-CSF group. We conclude that after ABMT with purged or unpurged marrow, rhu GM-CSF (E coli) significantly reduces neutropenia duration and hospitalization stay. A positive causative relation between the study drug and/or its mode of application with an increased toxicity as compared with GM-CSF from other sources and/or other modes of application cannot be deduced from the experiences in this study. Additional randomized trials would be necessary for an appropriate answer.


Effect of adrenergic agents on hematopoiesis after syngeneic bone marrow transplantation in mice. We show that adrenergic agents modulate hematopoietic reconstitution after syngeneic bone marrow transplantation (BMT) in mice. Chemical sympathectomy by 6-hydroxydopamine (6-OHDA) significantly increased the number of peripheral blood leukocytes after syngeneic BMT. The alpha 1-adrenergic antagonist prazosin mimicked and extended the effect of 6-OHDA, with an additional rapid and significant increase of platelets, marrow granulocyte-macrophage colony-forming units, and nucleated spleen cells. Differential leukocyte counts and spleen histology from prazosin-treated mice confirmed that myelopoiesis was greatly enhanced and accelerated. In contrast, the beta-adrenergic blocker propranolol abolished the prazosin-induced increase of platelets. The detailed mechanisms by which prazosin exerts these interesting effects remain to be elucidated.


Insulin-like growth factor-I regulates pro-B cell differentiation. Progression of B-lymphocyte development in the bone marrow of postnatal mammals is marked by progressive rearrangement and expression of immunoglobulin (Ig) heavy- and light-chain genes. Following productive VHDJH gene rearrangement in the Ig heavy-chain gene complex, mu-heavy chain is the first Ig gene product expressed in cells committed to the B-lymphoid differentiation pathway. Interleukin (IL)-7 has been shown to stimulate proliferation of pre-B cells following c mu expression and this proliferative stimulus is potentiated by kit ligand (KL). However, it appears that neither of these cytokines contributes to differentiation of pro-B cells or initiation of expression of Ig gene products. We previously demonstrated that differentiation of pro-B cells and expression of mu-heavy chain is stimulated by either bone marrow stromal cell line S17 or cell-free supernatants from that line. This biological activity was attributed to molecules with an apparent M(r) of less than 10 Kd and approximately 40 to 60 Kd. We now report that this biological activity resides with stromal cell-derived insulin-like growth factor-I (IGF-I). Recombinant IGF-I stimulated the expression of cytoplasmic mu-heavy chain in short-term bone marrow cultures and this stimulus was abrogated in the presence of anti-IGF-I antibody. We also demonstrate that either anti-IGF-I antibody or pretreatment of S17 cells with antisense oligonucleotide for IGF-I abrogated the pro-B cell differentiation activity of S17 stromal cell supernatants. Although IGF-I did not directly stimulate proliferation of B-lineage cells, like KL, it potentiated the proliferative stimulus provided by IL-7. Taken together, these data strongly suggest that IGF-I produced by bone marrow stromal cells in the hematopoietic microenvironment plays a key role in regulating primary B lymphopoiesis.


Quantification of a novel dense granule protein (granulophysin) in platelets of patients with dense granule storage pool deficiency. An antigen-capture sandwich enzyme-linked immunosorbent assay (ELISA) was developed for a novel protein granulophysin, a constituent of the platelet dense granule (DG) membrane and used to characterize patients with dense granule storage pool deficiency (delta-SPD). The assay uses two monoclonal antibodies against the protein, one of which is conjugated to peroxidase. Purified DGs, an enriched source of the protein, were used for the standard curve. Granulophysin levels were only low in forms of delta-SPD associated with albinism. Granulophysin levels in platelet homogenates of 30 patients with the Hermansky-Pudlak syndrome form of delta-SPD were 1/4 to 1/5 of levels in controls or obligate heterozygotes. Two patients with the Chediak-Higashi form of delta-SPD syndrome also had markedly reduced levels of granulophysin. Patients with other forms of delta-SPD had normal levels of granulophysin. Two sisters with delta-SPD in one family had normal granulophysin present in empty dense granule membrane vesicles. Three members of another family with delta-SPD had low DG counts but normal granulophysin levels, indicating that in this group the level of granulophysin was maintained despite the reduction in granule formation. Thus, granulophysin quantitation facilitates characterization of delta-SPD patients and may provide clues to the nature of defective granules in delta-SPD subtypes.


Antithrombotic properties of L-cysteine, N-(mercaptoacetyl)-D-Tyr-Arg-Gly-Asp-sulfoxide (G4120) in a hamster platelet-rich femoral vein thrombosis model. Platelet aggregation plays an important role in the pathogenesis in arterial thrombotic disorders. The binding of fibrinogen via the Arg-Gly-Asp (RGD) recognition sequence to the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is an essential step of platelet aggregation induced by various physiologic agonists, and RGD-containing peptides that bind to the GPIIb/IIIa receptor inhibit thrombus formation in vivo. L-cysteine, N-(mercaptoacetyl)D-tyrosyl-L-arginylglycyl-L alpha-aspartyl-cyclic (1----5)-sulfide, 5-oxide (G4120), a cyclic RGD-containing synthetic pentapeptide, inhibits adenosine diphosphate (ADP)-induced platelet aggregation with 50% inhibition (IC50) at a concentration of 0.05 microgram/mL in human plasma, 0.12 microgram/mL in hamster plasma, and 11 micrograms/mL in rat plasma. Corresponding values for the linear tetrapeptide Arg-Gly-Asp-Phe (RGDF) were 7 and 100 micrograms/mL in human and hamster plasma. The antithrombotic effects of G4120 and RGDF were evaluated in a hamster model consisting of a mural platelet-rich femoral vein thrombus induced by standardized endothelial cell damage. Bolus intravenous injection of G4120 was followed by a biphasic disappearance of G4120 from plasma with t1/2 alpha of 3.7 minutes and t1/2 beta of 63 minutes, corresponding to a plasma clearance of 5.2 +/- 0.68 mL/min. Bolus intravenous injection of G4120 inhibited ex vivo platelet aggregation with 0.5 mumol/L ADP and in vivo thrombus formation in a dose-dependent manner, with ID50 of 11 and 11 micrograms/kg, respectively. Bolus injection of RGDF inhibited in vivo thrombus formation; 43% inhibition was obtained at a dose of 30 mg/kg. Thus, this hamster platelet-rich femoral vein thrombosis model may be useful for the investigation of the antithrombotic properties of platelet GPIIb/IIIa antagonistic peptides. The cyclic synthetic peptide G4120 appears to have a very potent antithrombotic activity in vivo.


Atypical Wiskott-Aldrich syndrome in a girl. Wiskott-Aldrich syndrome (WAS) is a fully penetrant X-linked recessive disorder characterized by thrombocytopenia with small platelets, eczema, and defects of both T-cell and B-cell immunity. Obligate carriers of this disorder show no signs of the gene defect because in the cell lineages primarily affected by the disorder they demonstrate preferential use of the normal, nonmutant X as the active X. This can be explained by the selective disadvantage in proliferation and/or survival experienced by the cells with the mutant X as the active X. We have recently evaluated an 8-year-old girl with a disorder phenotypically identical to WAS. Cytogenetic studies did not show any structural abnormalities of the X chromosome and X chromosome inactivation analysis showed that both of her X chromosomes could function as the active X. These findings suggest that there is an autosomal recessive disorder that is very similar to classic WAS.


Growth and spread of human malignant T lymphoblasts in immunosuppressed nude mice: a model for meningeal leukemia. Previous work has shown that nude (nu/nu) mice additionally immunosuppressed by splenectomy, sublethal irradiation, and treatment with antiasialo GM1 antiserum (SIA-nu/nu mice) have no detectable natural killer activity and allow the growth of human malignant lymphoblasts. We show here that all SIA-nu/nu mice engrafted intravenously with 5 x 10(6) malignant lymphoblasts originally derived from a child with a T-cell acute lymphoblastic leukemia (PF382) and from a boy with a T-cell lymphoma (ST-4) develop lethal meningeal leukemia and die within 35 days. Histologic examination of moribund SIA-nu/nu mice showed that vertebral and skull bone marrow was always replaced by proliferating human T lymphoblasts. From the spinal canal, lymphoblasts spread to the meninges, causing hind leg paralysis. Leaving the skull, they permeated the meninges and then invaded the nervous parenchyma. This efficient and reproducible experimental model may be suitable for experimental studies on the pathogenesis of meningeal leukemia.


Follicular dendritic cells inhibit human B-lymphocyte proliferation. In germinal centers, B lymphocytes are intimately associated with follicular dendritic cells (FDCs). It has been hypothesized that FDCs are involved in the regulation of B-cell growth and differentiation through cell-cell interactions. In this study, highly enriched preparations of FDCs were isolated by cell sorting using the FDC restricted monoclonal antibody DRC-1. When irradiated FDCs were cultured with mitogen stimulated B cells, B cell 3H-TdR uptake was inhibited by up to 80%. This inhibitory effect was not seen when paraformaldehyde fixed FDCs were added to B-cell cultures, suggesting that the FDCs needed to be metabolically active. Moreover, supernatants from cultured FDCs were similarly able to inhibit B-cell proliferation. These results demonstrate that FDCs may downregulate the clonal expansion of B cells that occurs within lymphoid follicles as part of the normal physiologic immune response. Potentially, the loss of the inhibitory role of FDCs in vivo may be of importance in certain infectious and neoplastic processes in which germinal centers are affected.


Sensitivity of fresh acute myeloid leukemia cells to etoposide: relationship with cell growth characteristics and DNA single-strand breaks. In this study, we evaluated the individual in vitro sensitivity of fresh acute myeloid leukemia (AML) cells to VP-16, and attempted to correlate VP-16 cytotoxicity with AML cell growth characteristics and drug-induced DNA single-strand breaks (SSB). Primary (PE1) colony inhibition assays allowed us to characterize two distinct groups of AML: group I (patients 1 through 6), which displayed sensitivity to VP-16 similar to that of normal CFU-GM (IC90 of 20.52 +/- 2.44 micrograms/mL v 20.48 +/- 2.23 micrograms/mL after 1 hour drug exposure, respectively); and group II (patients 7 through 11), which was more sensitive to VP-16 (IC90 of 7.26 +/- 2.93 micrograms/mL, P = .004). Subsequently, groups I and II were termed normosensitive and hypersensitive, respectively. This objective VP-16 sensitivity classification, as determined by PE1, remained unaltered when assessed by secondary (PE2) colony inhibition assay (evaluating the self-renewal fraction of AML progenitors), or by cytofluorometric viability assay (evaluating the ultimately differentiated blast cell population). These findings would suggest that individual sensitivity to VP-16 of a particular cell population is maintained throughout CFU-AML differentiation. Finally, we report that sensitivity of AML cells to VP-16 did not correlate either with cell growth characteristics or with SSB generation. Indeed, AML cell sensitivity to VP-16 appeared more closely related to DNA repair kinetics after drug removal, ie, hypersensitivity being essentially characterized by a prolonged retention of SSB during the posttreatment period. Interestingly, the established HL-60 cell line, which presented greater sensitivity to VP-16 cytotoxicity than KG1, HEL, and K562, was also found to exhibit delayed DNA SSB repair kinetics, as compared with the other AML cell lines. These results suggest that hypersensitivity to VP-16 of some AML cells may be related to a deficient DNA-repair mechanism.


Effect of herbimycin A, an antagonist of tyrosine kinase, on bcr/abl oncoprotein-associated cell proliferations: abrogative effect on the transformation of murine hematopoietic cells by transfection of a retroviral vector expressing oncoprotein P210bcr/abl and preferential inhibition on Ph1-positive leukemia cell growth. Herbimycin A, a benzoquinoid ansamycin antibiotic, was demonstrated to decrease intracellular phosphorylation by protein tyrosine kinase (PTK). In Philadelphia chromosome (Ph1)-positive leukemias such as chronic myelogenous leukemia (CML) and Ph1-positive acute lymphoblastic leukemia (ALL), both of which express bcr-abl fused gene products (P210bcr-abl or P190bcr-abl protein kinase) with augmented tyrosine kinase activities, herbimycin A markedly inhibited the in vitro growth of the Ph1-positive ALL cells and the leukemic cells derived from CML blast crisis. However, the same dose of herbimycin A did not inhibit in vitro growth of a broad spectrum of Ph1-negative human leukemia cells, and several other protein kinase antagonists also displayed no preferential inhibition. Furthermore, we demonstrated that herbimycin A has an antagonizing effect on the growth of transformed cells by a transfection of retroviral amphotrophic vector expressing P210bcr/abl into a murine interleukin (IL)-3-dependent myeloid FDC-P2 cell line. This inhibition was abrogated by the addition of sulfhydryl compounds, similar to the reaction previously described for Rous sarcoma virus transformation. The inhibitory effect of herbimycin A on the growth of Ph1-positive cells was associated with decreased bcr/abl tyrosine kinase activity, but no decrease of bcr-abl mRNA and protein, suggesting that the inactivation of bcr-abl tyrosine kinase activity by herbimycin A may be induced by its binding to the bcr-abl protein portion that is rich with sulfhydryl groups. The present study indicates that herbimycin A is a beneficial agent for the investigation of the role of the bcr-abl gene in Ph1-positive leukemias and further suggests that the development of agents inhibiting the bcr-abl gene product may offer a new therapeutic potential for Ph1-positive leukemias.


Delta-thalassemia caused by disruption of the site for an erythroid-specific transcription factor, GATA-1, in the delta-globin gene promoter. Delta-thalassemia is a complex group of inherited disorders of globin genes characterized by impaired synthesis of the delta-globin chain. The T-C substitution was detected at position -77 of the delta-globin gene isolated from three independent Japanese individuals who were homozygotes for delta-thalassemia. To elucidate the significance of the mutation in delta-globin gene expression, we investigated the genotype of three delta-thalassemia homozygotes and 58 normal individuals using dot blot hybridization of the polymerase chain reaction (PCR)-amplified DNA. The mutation was observed in six alleles of three homozygotes, while no mutation was detected in 116 alleles of normal individuals, thereby indicating the close association of this mutation with the thalassemia phenotype. Since the mutation (TTATCT-TCATCT) is located within the inverted binding motif of GATA-1 (T or A-G-A-T-A-G or A), an erythroid cell-specific transcription factor, we did gel retardation assays using nuclear extracts from the erythroid cells. We found that GATA-1 binds the oligonucleotide spanning positions -61 to -90, but does not bind to the oligonucleotide with the mutation at position -77. Competition gel retardation assays showed that GATA-1 binding can be competed out by the fragment with the GATA-1 motif, but not with the mutant oligonucleotide. Analysis of the transient expression of the CAT gene linked to the delta-globin gene promoter region demonstrated that the construct with the mutant promoter region was expressed about 20-fold less compared with the normal one. Thus, the mutation at position -77 impairs delta-globin gene expression by abolishing GATA-1 binding to the AGATAA sequence of the promoter region of the delta-globin gene. This provides a good example of involvement of tissue-specific transacting factors in the molecular pathogenesis of hereditary diseases.