Antioxidants attenuate microvascular changes in the early phase of experimental pneumococcal meningitis in rats. We tested in a rat meningitis model 1) whether pneumococcal cell wall components are capable of producing changes in regional cerebral blood flow, brain water content, and intracranial pressure similar to those we have already observed after intracisternal inoculation of live pneumococci and 2) whether antioxidants would modulate these alterations in the early phase of meningitis. Regional cerebral blood flow as measured by laser Doppler flowmetry and intracranial pressure were monitored continuously for 4 hours after intracisternal challenge. Brain edema formation was assessed by brain water content determinations. We investigated the following groups: rats challenged intracisternally with the whole intact pneumococcal cell wall (n = 7) or the pneumococcal cell wall hydrolyzed by the M1-muramidase (n = 7); rats injected intracisternally with phosphate-buffered saline (n = 6); rats pretreated intravenously with superoxide dismutase conjugated with polyethylene glycol (10,000 units/kg) and injected intracisternally with cell wall components (n = 5) or phosphate-buffered saline (n = 6); rats injected intracisternally with phosphate-buffered saline and pretreated intravenously with polyethylene glycol (10% solution, 1.2 ml/kg, n = 5) or continuously treated with intravenous free superoxide dismutase (22,000 units/kg per hour, n = 6); and rats continuously treated intravenously with deferoxamine mesylate (10 mg/kg per hour) and injected intracisternally with cell wall components (n = 6) or phosphate-buffered saline (n = 7). Both pneumococcal cell wall preparations produced a significant increase in regional cerebral blood flow, intracranial pressure, and brain water content. Conjugated superoxide dismutase as well as deferoxamine prevented the increase in intracranial pressure and brain water content. In addition, the increase in regional cerebral blood flow as observed in untreated, cell wall-challenged rats (baseline, 100%; 183.1 +/- 12.3% after 4 hours, mean +/- SEM) was significantly attenuated by administration of both conjugated superoxide dismutase (136.6 +/- 14.1%) and deferoxamine (149.8 +/- 8.2%) (p < 0.05). Polyethylene glycol-conjugated superoxide dismutase alone produced an increase in regional cerebral blood flow (125.6 +/- 8.7% after 4 hours). We found that polyethylene glycol per se accounts for this action. These data show that pneumococcal cell wall components containing teichoic acid produce changes in regional cerebral blood flow, intracranial pressure, and brain water content and that oxygen radicals contribute to these pathophysiological alterations in the early phase of experimental pneumococcal meningitis.


Effects of retrograde perfusion of the brain with combined drug therapy after focal ischemia in rat brain. The ischemic edema associated with blood-brain barrier permeability changes and the excess production of free radicals are serious complications in prolonged cerebral ischemia. We examined the efficacy of transvenous perfusion of the brain, starting treatment 5 hours after occlusion of the middle cerebral artery for a period of 2 hours in rats with the combined agents mannitol (10 ml/2 hr) and dexamethasone (1 mg/2 hr) to counter edema and verapamil (0.05 mg/kg/2 hr) for vasodilation. In experiment 1, blood-brain barrier permeability changes were examined in five groups with six rats each: group C rats underwent 7 hours of middle cerebral artery occlusion with no treatment; group V, treatment with verapamil alone; group VD, treatment with verapamil and dexamethasone; group VM, treatment with verapamil and mannitol; and group VDM, treatment with verapamil, dexamethasone, and mannitol. In experiment 2, we examined local cerebral blood flow, ischemic tissue damage volume, and water content of cerebral hemispheres in two groups of 16 rats each subjected to the same treatment as groups C and VDM rats in experiment 1. There was a significant reduction of blood-brain barrier permeability changes in the ischemic cortex of rats in group VDM compared with rats in the other groups. In the group undergoing transvenous perfusion of the brain with the three combined agents, there was a significant improvement of cerebral blood flow (39-58%, p < 0.05) in the ischemic cortex and reduction of ischemic cerebral damage volume (22%, p < 0.01) and water content of the ischemic hemisphere (p < 0.05) compared with the control group. The therapeutic approach using combined agents is effective treatment when initiated within 5 hours of focal cerebral ischemia in rats.


Spontaneous cerebral hypothermia diminishes focal infarction in rat brain. Brain temperature during ischemia is known to strongly influence the extent of cellular injury. The objectives of the present study were to determine the effect of severe focal ischemia on brain temperature and to assess the influence of those changes on focal infarction. Severe focal ischemia was produced in rats using permanent occlusion of the distal middle cerebral artery combined with transient (60-minute) bilateral carotid artery occlusion. The temperature of the ischemic focus was measured with a small subdural probe. Three groups of rats were studied. In the first group, brain temperature was permitted to decline spontaneously to 32 degrees C after occlusion. In the second, brain temperature was maintained at 37.5 degrees C during occlusion. In the third group, the brain temperature was maintained at 37.5 degrees C for 40 minutes postocclusion before cooling. After recovery for 24 hours, the volume of infarction was measured in histological sections. In the absence of cranial heating, the brain temperature fell to 33 degrees C by 10 minutes postocclusion, and infarct volume was 19 +/- 9 mm3 (mean +/- SEM; n = 6). Maintaining brain temperature at 37.5 degrees C increased the volume of infarction to 82 +/- 16 mm3 (n = 7; p < 0.001). Delayed cooling did not prevent the increase in infarct volume (75 +/- 16 mm3; n = 6). These results demonstrate that in the present model of transient focal ischemia, spontaneous cooling of the brain during ischemia diminishes the extent of focal infarction, relative to that observed when cerebral hypothermia is prevented or delayed for 40 minutes.


Cerebral infarction in a heterozygote with variant antithrombin III. We report a heterozygous case of familial qualitative deficiency of antithrombin III associated with cerebral infarction. A 33-year-old man had a history of recurrent transient ischemic attacks from the age of 28. Cerebral computed tomography at age 29 disclosed a low-density area in the left frontal lobe, and an internal carotid angiogram showed branch occlusion of the right anterior cerebral artery and stenosis of the left middle cerebral artery. Occlusion of the right middle cerebral artery developed thereafter. The plasma antithrombin III antigen concentration and progressive antithrombin activity were normal, but plasma heparin cofactor activity was low in the patient and his father. Nucleotide sequence analysis of the proband's deoxyribonucleic acid showed no mutation in exons II and VI of antithrombin III. We conclude that abnormal antithrombin III with defective heparin binding, even though heterozygous, may cause ischemic stroke in young adults. We named this antithrombin III variant "Antithrombin III Nagasaki."


Thrombolysis in acute ischemic stroke: does it work? This article is presented to provoke further discussion regarding the use of thrombolytic drugs to treat acute ischemic stroke. Overview analysis of the six randomized trials of thrombolysis in acute ischemic stroke available in the world literature shows a 20% increase in the odds of death and a 30% reduction in the odds of death or deterioration (both with wide confidence intervals, neither result significant) after thrombolytic treatment for acute ischemic stroke. Exclusion of the two trials conducted without the benefit of computed tomographic scanning shows a 37% reduction in the odds of death (95% confidence interval, 74% reduction to 40% excess) and a significant reduction of 56% in the odds of death or deterioration after thrombolytic treatment (95% confidence interval, 20-76% reduction; 2p = 0.007). Analysis of all published studies (randomized and nonrandomized) shows that there does not appear to be an excess risk of hemorrhagic transformation of the cerebral infarct or of severe edema formation. We believe the present evidence is sufficiently encouraging to warrant proper testing of thrombolysis in sufficiently large and well-designed randomized clinical trials to influence clinical practice.


Prediction of unbound serum valproic acid concentration by using in vivo binding parameters. In a previous study, we determined the in vivo binding parameters of valproic acid (VPA) to serum proteins in seven healthy young adults at steady state by using the Scatchard equation. To evaluate the ability of the Scatchard binding equation to predict steady-state unbound serum VPA concentrations (Cf), 39 adult patients receiving VPA monotherapy and ranging in age from 16 to 68 years were studied. The correlation between predicted and observed Cf was high (r = 0.865). Mean prediction error, mean absolute error (MAE), and root mean squared error (RMSE) were calculated, and served as a measure of prediction bias and precision. The MAE and RMSE were low (MAE = 12.9 mumol/L, RMSE = 17.7 mumol/L). It is feasible to use the Scatchard binding equation to predict Cf in patients receiving VPA monotherapy.


Ability of three pharmacokinetic equations to predict steady-state serum theophylline concentrations in pediatric patients. The pharmacokinetic equations of Chiou, Koup, and Kurland are often used in the pediatric setting to predict steady-state theophylline clearance using non-steady serum theophylline concentrations. However, these equations have not been validated or compared in a pediatric population. We evaluated the ability of these equations to predict steady-state serum theophylline concentrations in 61 children (0.21-14.3 years) who received a continuous intravenous theophylline (0.79 +/- 0.12 mg/kg/h) infusion for a minimum of five half-lives. Theophylline concentrations used in the Kurland equation were obtained 10.8 +/- 4.5 h after initiation of therapy and the time between the two concentrations used in the Chiou and Koup equations was 9.2 +/- 3.9 h. Predicted steady-state theophylline concentration values for the three methods were not different from each other (p = 0.91), nor were they different from the observed steady-state concentration values (p = 0.92). The coefficient of determination for predicted vs. observed steady-state concentrations was statistically significant (p less than 0.001) and was comparable for the three methods. There was no difference in mean bias (p = 0.78), precision (p = 0.82), or % error (p = 0.86) values for the three methods. Regardless of the method used, 75 to 82% of all predicted theophylline concentrations were within 20% of the observed steady-state value. However, on average, all methods underpredicted the clearance and hence overpredicted the serum theophylline concentration. The Kurland method did not predict steady-state concentrations any better in patients who had received theophylline prior to admission.(ABSTRACT TRUNCATED AT 250 WORDS)


Altered heparin pharmacodynamics in patients with pulmonary embolism. Heparin clearance and pharmacodynamic response were examined in 12 patients being treated for deep venous thrombosis (DVT, 6 patients) or pulmonary embolism (PE, 6 patients). A loading dose of 70 units/kg was administered to DVT patients and 100 units/kg to PE patients followed by an initial infusion rate of 15 or 25 units/kg/h for DVT or PE patients, respectively. Heparin clearance was determined at 4, 12, and 24 h after initiating heparin therapy. The mean heparin clearance in the DVT group was 2,164 +/- 1,024 ml/h at 4 h, 2,591 +/- 1,239 ml/h at 12 h, and 2,795 +/- 1,863 m/h at 24 h. The PE patients had clearances of 1,775 +/- 494, 2,004 +/- 321, and 2,843 +/- 1,000 ml/h at 4, 12, and 24 h, respectively. The difference between the two groups was not statistically significant (p greater than 0.50). The activated partial thromboplastin time (aPTT) was used as a measure of heparin effect. The maximum effect (EMAX) and concentration required to attain 50% of the maximum effect (EC50) were determined for each group using the Lineweaver-Burke linearization method. The mean EMAX and EC50 for the DVT patients were 130 +/- 40.99 s and 1.01 +/- 0.70 units/ml, respectively. For the PE patients, the mean EMAX was 418 +/- 200 s and the mean EC50 was 4.32 +/- 2.81 units/ml. The difference between both groups for each parameter was statistically significant (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Preliminary studies on the in vitro and in vivo effect of salicylate on sperm motility. We have studied the in vitro effect of different concentrations of salicylic acid on the motility of normal human spermatozoa. Sperm motility was evaluated over 48 h at 0, 50, 100, and 200 mg/L of salicylate concentrations. Results are reported as the mean % motility, where 100% motility at each time interval was taken to be the motility of the control (0 mg/L of salicylate). At 48 h, the mean % motility (n = 12) was 61, 43, and 33% at 50, 100, and 200 mg/L of salicylate. Twenty-four-hour values (n = 17) were 75, 65, and 48%. Eosin supravital staining demonstrated that the decrease in sperm motility was caused by direct inhibition of motility rather than by spermatozoal death. Four healthy males (17-51 years of age) gave a semen specimen prior to taking 650 mg of salicylate four times daily, followed by a second semen specimen 72 h later. Each individual, therefore, served as his own control. The mean % motility in the postdrug sample at 2, 24, and 48 h was 49, 53, and 47% of the motility found in the predrug sample (n = 4). We conclude that salicylate significantly decreases sperm motility in vitro and in vivo.


Pattern of 6-mercaptopurine urinary excretion in children with acute lymphoblastic leukemia: urinary assays as a measure of drug compliance. A method for the measurement of 6-mercaptopurine (6MP) in urine using high-performance liquid chromatography is described. Urinary excretion of 6MP was measured in 46 children with acute lymphoblastic leukemia. The proportion of unchanged drug excreted after oral dosage in the morning was greater than after an evening dose (5.6 +/- 3.3% vs. 3.3 +/- 2.4%). Possible reasons for the discrepancy are discussed. In all children receiving 6MP in the morning, the drug was detected in urine at 2 and 4 h after ingestion. At 6 h, 6MP was still detectable in 77% of patients, at 8 h in 54%, at 10 h in 12%, and at 12 h in 8%. The reliability of urinary 6MP determination as a measure of drug compliance was assessed in 39 children accustomed to receiving their medication in the evening. 6MP was detected in 81% of first morning urine samples, indicating compliance with medication the preceding evening. The absence of 6MP in first morning urine samples did not necessarily indicate poor compliance because of the variability in 6MP excretion and unpredictable pattern of night voiding in children. The method was therefore a reliable measure of good short-term compliance. It also directed attention toward possible noncompliance in children with negative samples.


Changes in unbound and total valproic acid concentrations after replacement of carbamazepine with oxcarbazepine. Total and free valproic acid (VPA) concentrations were measured in patients switched from a combined VPA and carbamazepine (CBZ) to a combined VPA and oxcarbazepine (OXC) therapy, both administered in individualized daily doses. Four young epileptic patients (13-17 years old) were studied for a 12-week period, total and free VPA concentrations being analyzed just before and 4 h after the morning dose, at the end of VPA and CBZ therapy, and 2 and 10 weeks after CBZ was replaced by OXC. The expected increase in the level/dose (L/D) ratio of total VPA observed at the end of the study was preceded by a clear-cut increase in the L/D ratio of free VPA, which led to VPA-related side effects and required the retitration of VPA daily doses.


Plasma levels of trimipramine and metabolites in four patients: determination of the enantiomer concentrations of the hydroxy metabolites. A two-step high-performance liquid chromatography method is described, using a CN column and an alpha 1-acid glycoprotein column, which allows the measurement of the enantiomers of the hydroxy metabolites of trimipramine in plasma of trimipramine-treated patients. Of the four patients analyzed, three showed approximately equimolar concentrations of the (D)- and (L)-enantiomers of the hydroxy metabolites (2-hydroxy-trimipramine and 2-hydroxy desmethyltrimipramine), and one was found to have roughly twice as much of the (L)-form and of the (D)-form of 2-hydroxy trimipramine and 2-hydroxy desmethyltrimipramine. From the data available on the pharmacological effects of the enantiomers of trimipramine, it is postulated that this interindividual variability in its pharmacokinetics is another factor that could contribute to the interindividual variability in its pharmacodynamics.


Measurement of phenytoin in serum using in-house reagents employing the Syva enzyme-multiplied immunoassay technique principle. Reagents have been developed for the measurement of phenytoin in serum, using the enzyme-multiplied immunoassay approach first introduced by Syva. The assay uses a commercially available antiserum and phenytoin valeric acid coupled to glucose-6-phosphate dehydrogenase as the tracer. It has been adapted for use on an Eppendorf EPOS analyser, which allows 300 assays/h with high precision. Excellent agreement was achieved with commercial reagents, and costs were reduced markedly.


Determination of lorajmine and its metabolite ajmaline in plasma and urine by a new high-performance liquid chromatographic method. Lorajmine is a monochloroacetyl derivative of ajmaline with electrophysiological properties somewhat different from those of the compound of origin. Since lorajmine is rapidly hydrolyzed to ajmaline by plasma and tissue esterases, it is crucial to measure plasma levels of both drugs separately. A major problem in assaying lorajmine is its chemical instability in plasma both after blood sampling and during the extraction procedure. Furthermore, lorajmine (unlike ajmaline) is not fluorescent and has a very low UV absorbance, so the standard detectors for high-performance liquid chromatography cannot be used. We describe a new method that solves the problems of instability and sensitivity. Plasma esterases are first blocked pharmacologically (neostigmine); ajmaline is then measured by direct on-column injection of samples. Last, lorajmine is completely converted to ajmaline, extracted, and measured with a fluorescence detector. The molar concentration of ajmaline obtained in the last step, minus that found by direct injection, gives the concentration of lorajmine. Some examples of pharmacokinetic applications are also given.


High-performance liquid chromatographic analysis of cyclosporin G (Nva2-cyclosporine) in human blood. A sensitive high-performance liquid chromatographic method for the analysis of the immunosuppressant cyclosporin G (OG 37-325, Nva2-cyclosporine, CsG) in whole blood has been developed. Sample preparation, employing cyclosporin A (CsA) as internal standard, involves organic extraction with methyl t-butyl ether under sequential acidic and basic conditions. Chromatography is performed using a 2 mm inside diameter x 25 cm column packed with 5 microns octyl (C8) material. An isocratic mobile phase comprised of acetonitrile:methanol:water, at a flow rate of 0.4 ml/min, is utilized. Separation is monitored at 230 nm. Data are also presented that demonstrate the use of CsG as an alternative internal standard to cyclosporin D for liquid chromatographic determinations of CsA.


Determination of dextromethorphan and its O-demethylated metabolite from urine. A high-performance liquid chromatography (HPLC) assay for the simultaneous quantitation of dextromethorphan and its O-demethylated metabolite dextrorphan from urine is described. A cyano analytical column was used with a mobile phase consisting of MeOH 16%, acetonitrile 3%, and triethylamine 0.06% at pH 2.8 and a flow rate of 1.0 ml/min. Betaxolol was used as the internal standard. Standard curves from 50 ng/ml to 10,000 ng/ml (dextrorphan), and from 50 ng/ml to 8,000 ng/ml (dextromethorphan) were developed. The peaks eluted at 7.8 min (dextrorphan), 12.2 min (betaxolol), and 17.8 min (dextromethorphan). The coefficients of variance ranged from 1.3 to 4.5% at 250 ng/ml and 0.9 to 2.5% at 5,000 ng/ml. This assay was used to determine dextromethorphan/dextrorphan molar ratios in healthy male volunteers for the purpose of determining phenotype status for the P450IID6 isozyme.


Application of the Empore solid-phase extraction membrane to the isolation of drugs from blood: II. Mexiletine and flecainide. A stabilized therapeutic drug monitoring procedure incorporating the novel Empore solid-phase extraction membrane (SPEM) for isolation of the antiarrhythmic drugs mexiletine (MEX) and flecainide (FLEC) from serum is described. Routinely, serum (0.5 ml), adjusted to pH 4.5, is passed through an octyl (C8) SPEM to extract the drugs. A methanol:water wash follows to remove proteins and interferences. MEX and FLEC are eluted from the membrane with mobile phase and an aliquot is injected directly onto a Zorbax cyanopropyl (CN) high-performance liquid chromatographic column with detection at 214 nm. Evaporating/concentrating techniques that can adversely influence the stability of the volatile MEX are unnecessary. Recovery for both drugs exceeds 90% and the assay is linear from 0.05 mg/L up to at least 6.0 mg/L for MEX and from 0.05 mg/L up to at least 3.0 mg/L for FLEC. Precision (between-run) coefficients of variation range from 2.3 to 3.0% (0.49-1.97 mg/L) for MEX and 3.7 to 5.9% (0.240-0.992 mg/L) for FLEC. Interferences are minimal. When we compared performance of the Empore SPEM and large-particle solid-phase sorbents packed in cartridges, we observed greater capacity per gram of sorbent and smaller elution volume with the membrane. Most important, concentrating steps that adversely affect the stability of MEX are avoided with the SPEM.


Relationship between serum concentration and dose of valproic acid during monotherapy in adult outpatients. The relationship between serum concentration and dose of valproic acid (VPA) is reported to be variable and inconsistent. However, studies evaluating this relationship have included individuals of varying ages and patients receiving potentially interacting medications. In this study, the relationship between VPA serum concentration and dose was evaluated in a homogeneous patient population. Medical records of 60 adult outpatients with epilepsy receiving VPA monotherapy were examined retrospectively for VPA dose (milligrams per kilogram) and corresponding serum VPA concentrations. A significant linear correlation was found in the relationship between VPA dose and serum concentration among all patients (r = 0.63; p less than 0.01). However, considerable interindividual variability in this ratio was demonstrated [coefficient of variation (CV) = 28.9%], and the ratio was significantly dependent on VPA dose. In three selected individual patients, a significant linear correlation was also demonstrated in the VPA serum concentration:dose relationship over time (r = 0.91, 0.94, 0.96; p less than 0.05 for all three patients) with substantially less variability (CV = 10.2-14.6%) and without significant dose dependency, suggesting that this parameter may be useful for guiding VPA dosage adjustment and monitoring patient compliance. Further study is required to evaluate the utility of the serum concentration:dose ratio in monitoring VPA therapy.


Evaluation of instrumental, nonisotopic immunoassays (fluorescence polarization immunoassay and enzyme-multiplied immunoassay technique) for cyclosporine monitoring in whole blood after kidney and liver transplantation. Two new instrumental methods, an enzyme-multiplied immunoassay technique (EMIT) and a fluorescence polarization immunoassay (FPIA), were evaluated for monitoring of cyclosporine (CyA) in whole blood samples of renal and liver transplant patients. They are considered as being specific to the parent drug and they were compared with a specific radioimmunoassay (RIA) and a nonspecific FPIA. The data reveal that the novel procedures provide slightly overestimated CyA levels compared with specific RIA (6-12% for EMIT, 20-25% for FPIA). For both assays, intrarun and interrun reproducibilities were found to be in the 2-8% range. The ease of performance and the possibility of performing approximately 40 assays/h make the two methodologies very attractive for both routine and emergency analyses. These approaches are viewed to be complementary to the only previously available instrumental method, the nonspecific FPIA, which provides three- to fourfold higher CyA levels than those obtained with specific methods. Specific and nonspecific monitoring of CyA levels allowed variations in proportions of metabolites to total CyA and metabolites to be distinguished. A higher percentage and variability of cross-reacting metabolites were found in whole blood samples after liver transplantation compared with those in blood of kidney transplant recipients.


[Effect of Fusarium autolysate on various immunologic and biologic indices of the state of the carp body]. The Fusarium autolysate has been studied for its effect on certain immunobiochemical, parasitological and fishery-biological characteristics of carp fingerlings in the experiment. It is shown that different methods of introduction and treatment of fish with the autolysate induce changes in titres of the compliment and the content of lysozyme in the blood serum of fish as well as growth of its body weight. Treatment of carp spawn before its incubation increases survivability of fish. It is shown advisable to apply the autolysate in food fishery.


[Riboflavin metabolism in diabetes mellitus]. Riboflavin excretion with urine in children suffering from diabetes mellitus is found to be significantly higher in comparison with healthy adults, the riboflavin content in the serum being normal. The total riboflavin concentration in erythrocytes is substantially lower and administration of daily-required riboflavin doses does not increase this parameter. FAD-dependent glutathione reductase in erythrocyte hemolysates is approximately 1.5 times higher than that of healthy children and does not depend on the additional intake of polyvitamins. The seeming affinity of the enzyme with exogenous FAD in sick children is almost 40 times lower in comparison with this value in healthy children, that is why the FAD-effect value in diabetes mellitus children does not exceed 1.2. Peculiarities of the riboflavin metabolism at diabetes mellitus and possibility to use the investigated parameters as criteria of vitamin B2 supply are discussed.


[Methemoglobin-induced lipid peroxidation in model membranes]. Lipid peroxidation induced by methemoglobin in liposomes prepared from lecithin, cardiolipin and their mixtures has been investigated. Using absorption spectroscopy technique it was shown that in the bilayers with low initial oxidation methemoglobin caused the formation of diene conjugates. In the bilayers with high degree of oxidation protein activated cleavage of the available fatty acid hydroperoxides. Hydroperoxides were found to induce the reduction of methemoglobin absorption in the Soret band.


[Change in the intensity of biosynthesis of proteins, lipids and glycogen in animal tissues during long-term administration of morphine]. The intensity of biosynthesis processes in animal organism has been studied as affected by long-term administration of morphine. It was established that morphine administration to rats for five weeks intensified protein biosynthesis in the brain, kidneys, skeletal muscles: specific radioactivity of blood serum proteins also increased. Incorporation of 2-/14C/glycine label to the brain, cardiac and skeletal muscles increased as affected by morphine: the label incorporation to the liver lipids decreased and that to the kidney and spleen lipids did not change. Specific radioactivity of glycogen multiply increased in the rat liver as affected by morphine.


[A model of intra- and intermolecular interactions of peptide chains]. Results of attempts to determine the code of interaction of amino acids in peptide chains proceeding from their coding nucleotide sequences have been summarized. According to the model suggested the G/C and A/U complementarity of codon roots determines the mutual binding of coded amino acid residues. Structures of analogs of the immunoactive peptide, a fragment of IgG1 (336-370) EPQVY have been constructed on the basis of the model.


[Interaction of melittin with a model membrane in the presence of antibodies]. The method of fluorescence spectrofluorimetry has been applied to study the interaction of melittin at high and low ionic strength with the phosphatidylcholine model membrane in the presence of monospecific polyclonal and monoclonal antibodies against this peptide. The formation of antigen-antibody complex with the excess of the antigen is shown to decrease the leakage of calcein, a fluorescence dye. With the excess of antibodies the dye leakage is completely suppressed. This effect does not depend on the state of peptide aggregation before binding to the membrane. It is suggested that melittin antigenic determinants are located on the sites of peptide molecule which are necessary for its interaction with the membrane.


The charge and atherogenicity of low density lipoproteins. It is concluded on the basis of literature data, that apolipoprotein B-100 is the most high-molecular, hydrophobic, and positive charged protein compared to the other apoproteins of the plasma lipoproteins. Low density lipoproteins of healthy subjects, mainly containing apo B-100, have little heterogeneity on both charge and isoelectric point, in spite of heterogeneity on sizes and apolipoprotein composition. The reason of formation of subfraction with elevated negative charge is the damage with the free radicals and/or aldehydes. The reason of formation of more cationized subfraction is unclear. LDL charge changes are noted in some diseases and syndromes (ischemic heart diseases, familial hyper-alpha-lipoproteinemia, Tangier disease, X-bound ichthyosis and, possibly, others). Some IHD patients treatment with antioxidants leads to the disappearance of negative charged LDL subfraction, that shows participation of peroxidation products in their formation. Electrical characteristics of LDL of tissue fluids and of aorta wall differ essentially from those of the same class plasma lipoproteins. Lipid peroxidation and influences of several enzymes play the main role in these differences.


[Catalytic effect of gamma-thrombin on synthetic low molecular weight peptide substrates]. Hydrolysis and respective catalytic parameters of hydrolysis of ester peptide substrates that contain residues of hydrophobic and nonpolar amino acids in P2, P3 subsites have been studied. It is shown that efficiency of hydrolysis by thrombin is determined by the length of polypeptide chains and by the nature of the amino acids in P2, P3 subsites in the substrate. In spite of the fact that gamma-thrombin retains the conformation activity of the catalytic centre the local conformation changes of the second binding region of the enzyme have been discovered.


[Effect of low doses of external and internal ionizing radiation on proteinases of cells in the rat cerebral cortex of rats]. Single external gamma-irradiation in a dose of 0.5-2 Gy as well as the long-term (30 days) internal irradiation caused by the everyday influx of Cs-137 and Sr-85 isotopes to the organism have been studied for their effect on the activity and properties of histone-specific proteinase from the nuclei of the rat brain cortex cells. It is found out that external irradiation induces a dose-dependent increase of the activity during the first 24 hours after irradiation followed by its decrease 7-30 days later. Internal irradiation induces a decrease of the enzyme activity at the 30th day as well. Certain specificity of the studied indices depending on the type of irradiation has been also observed.


[High density lipoproteins of the human aortic wall; size distribution]. The paper presents data on distribution of human aortic high density lipoproteins (A-HDL) according to their size. A-HDL were isolated from the aortic intima (autopsy material) and analyzed by the gradient gel electrophoresis method. The obtained data show that the clear-cut difference exists between size distribution of A-HDL from patients whose death was connected with atherosclerosis and those who died from the diseases not connected with atherosclerosis. In the first case small particles (HDL3B and HDL3C) were predominant. A-HDL with size corresponding to plasma HDL2A and HDL2B slightly predominated in normal intima and A-HDL with size of plasma HDL3A and HDL3C predominated in affected parts of intima. The principal component analysis of data permits suggesting that one of the factors responsible for the accumulation of small A-HDL is a decreased permeability of the affected parts of the vessel wall for the larger HDL particles. Besides, changes of the normal HDL metabolism can also be responsible for the presence of small HDL particles in the intima.


[Effect of phytoecdysteroids and nerobol on parameters of carbohydrate and lipid metabolism and phospholipid spectrum of liver mitochondrial membrane in experimental diabetes mellitus of rats]. Phytoecdysteroids: ecdysterone and turkesterone, introduced orally to male rats with the body mass 180-120 g in a dose of 5 mg/l kg of mass and nerobol in a dose of 10 mg per 1 kg of the mass for 15 days against a background of the developed alloxan diabetes cause a considerable decrease in the content of free fatty acids of the blood serum, sharply increased after the subcutaneous injection of alloxan to the animals (150 mg per 1 kg of the mass). The content of glycogen, malonic dialdehyde, pyruvic acid and calcium transporting function of the liver mitochondria are also normalized. These changes are closely interrelated (and may be mutually conditioned) with the preparation-induced reduction of phospholipid spectrum of the liver mitochondrial membranes pathologically changed owing to insulin insufficiency. In this case phytoecdysteroids in the first turn normalize the fractions of phospholipids which play the structural role in the mitochondrial membranes, and nerobol normalizes the level of minor and monoacylic phospholipids.


[Functional and diagnostic significance of A-, Bbeta 1-42 and Bbeta 15-42 peptide fragments of fibrin(ogen)]. A scheme of in vitro formation and hydrolysis of the fibrin clot is suggested. This scheme considers the data concerning a cofactor role of fibrin in activation of polymerization and fibrinolysis. Such parameters of a number of components as concentration of A-, B beta 1-42-and B beta 15-42 peptides and of other fragments of fibrinogen which allow characterizing a state of hyperfibrinolysis, hyperclotting or dynamic equilibrium of these systems are selected in the scheme.


[Effect of nitrobenzoic acid on methemoglobin content in blood and activity of antioxidative enzymes in erythrocytes]. Intraperitoneal administration of meta-nitrobenzoic, 3,5-dinitrobenzoic, 2,4,6-trinitrobenzoic and 3,5-dinitro-4-methylbenzoic acids to the white mice in a dose equal to LD50 has induced an increase in the methaemoglobin content in their blood. Total activity of dehydrogenases of pentosephosphate pathway, content of 2,3-diphosphoglycerate increase in response to the intoxication evoked by the mentioned acids. The acute intoxication does not practically change the activity of the key enzymes of antioxidant protection: superoxide-dismutase, catalase and glutathione peroxidase.


[Structural organization of the monofilamentary protofibrils and their association into fibrils]. Evidence of the fibrinogen molecule structure which is suggestive of an antiparallel arrangement of polymerization sites is summarized. A three-dimensional model of the structural organization of a monofilamentary protofibril is proposed. A possible role of the "A" and "a" polymerization centers in the lateral association of protofibrils is contemplated. The formation of fibrils in a form of a hollow cylinder is postulated.


[High density lipoproteins in the blood of rats of varying age]. The study revealed age-related changes in the content and ratio of apoproteins which are part of high-density lipoproteins (HDL), as well as the development of dysapolipoproteinemia. The most significant fall (by 46.7%) was found in the content of the key HDL apoprotein--apoA1, and rise (by 107%)--in apoE. The development of dysapolipoproteinemia was accompanied by changes in the lipid composition of HDL and their subfractions (HDL2 and HDL3). HDLs were enriched by cholesterol and its esters. HDL cholesterol/apoA1 ratio increased twice, while the content of phospholipids decreased.


Correlative study of adult respiratory distress syndrome by light, scanning, and transmission electron microscopy. The sequential pulmonary changes occurring in the evolution of adult respiratory distress syndrome (ARDS) were studied in 35 patients by correlative light, scanning, and transmission electron microscopy. The causes of ARDS were diverse, the major ones being sepsis or aspiration. Patient survival ranged from 3 to 51 days. The acute stage in patients surviving 2 to 7 days was characterized by an exudative reaction with a predominance of hyaline membranes. This acute stage merged with and was replaced by a subacute reparative stage in patients surviving 7 to 14 days, which in turn was replaced by a chronic fibroproliferative stage complicated by interstitial pulmonary fibrosis and a deranged acinar architecture. Correlation of findings by scanning electron microscopy with those by light and transmission electron microscopy provided an added dimension to understanding of the evolving stages of ARDS and demonstrated that type 2 pneumocytes contributed to the fibroproliferative stage through organization of hyaline membranes and re-epithelialization of alveoli.


[Ligand spectrum of hemoglobin activity of methemoglobin-reductase and hemolytic resistance of erythrocytes during chronic exposure to nitrates]. It is found that nitrite-ions formed as a result of biotransformation during long term feeding of calves with sodium and potassium nitrates induce changes in some biochemical parameters of blood, including HS-glutathione content in erythrocytes, acid hemolytic resistance of erythrocytes, activity of NAD-dependent methemoglobin-reductase, correlation of ligand forms of hemoglobin and its total content. It is supposed that the observed changes are of an adaptational character and, as a whole, provide for the optimization of both quantitative and qualitative composition of population of erythroid cells at the expense of erythropoiesis intensification.


Immunohistochemical and electron microscopic profiles of cutaneous Kaposi's sarcoma and bacillary angiomatosis. Thirty cases of cutaneous Kaposi's sarcoma (KS) were evaluated and compared with eight cases of acquired immunodeficiency syndrome (AIDS)-related bacillary angiomatosis (BA). The morphologic features of both lesions were studied by light and electron microscopy and by immunohistochemistry with monoclonal endothelial antibodies against CD34, BNH9, and factor VIII-related antigen as well as the lectins Ulex europaeus 1 and Psophocarpus tetragonolobus. Macrophage/monocyte markers used were alpha 1-antitrypsin, lysosome, Kp1 (CD68), and polyclonal factor XIIIa. Electron microscopic studies demonstrated that most of the spindle cells in KS showed a paucity of cell organelles and an absence of Weibel-Palade bodies (WPB), whereas the cells in BA showed activated endothelial cells with WPB. By immunohistochemistry the spindle cells in KS were consistently positive for CD34 only, whereas proliferating cells in BA expressed all endothelial markers used. Numerous cells expressing macrophage/monocyte markers were present surrounding both KS and BA, and a small number of similar cells were entrapped within both lesions. The results demonstrated a restricted immunohistochemical profile for endothelial cell markers in spindle cells of KS (CD34+) distinct from that of endothelial cells in BA. These findings suggest that the spindle cells in KS are poorly differentiated endothelial cells or that they belong to an endothelial cell subset with partial expression of endothelial phenotype.


Membranocystic lesion in sclerosing lipogranuloma of the scrotum: an ultrastructural study. Two cases of intrascrotal sclerosing lipogranuloma are reported in which affected adipose tissues showed peculiar membranocystic lesions (MCL) similar to the features described in Nasu-Hakola disease. On light microscopy, the MCL were seen in the degenerative fatty tissues and within multinucleated giant cells, which were positive for periodic acid-Schiff stain and resistant to diastase digestion. Ultrastructurally, two types of MCL were observed; one type was characterized by thick membranes composed of vesicular to minute tubular or fibrillar substructures, and the other type was characterized by thin membranes composed of only small numbers of tiny vesicles. The substructures in both types of MCL contained lipid droplets similar to those in the inner space. These results support the concept that the membranes in the MCL are derived from degenerated fat droplets.


Ultrastructure and immunohistochemistry of a fetal-type Leydig cell tumor. A symptomless scrotal mass was removed from a 34-year-old man. The lesion was 7 cm in diameter and it was grossly a hemorrhagic cyst with indurated walls. By light microscopy tumor cell clusters and cords were seen infiltrating the testicle, tunica albuginea, and paratesticular tissue. In the immunohistochemical analysis the tumor cells were immunoreactive with anti-S-100 protein and anticarcinoembryonic antigen, but they did not express cytokeratin or alpha-fetoprotein as tested with paraffin sections. Tumor cell clusters were enveloped by a laminin-positive basement membrane. Electron microscopy revealed abundant smooth endoplasmic reticulum, lipid droplets, and membranous whorls in the cytoplasm. Lamellar whorled bodies were also seen in mitochondria, which contained tubulovesicular cristae. The presence of a well-developed, often multilayered basement membrane was confirmed at ultrastructural level. The activity of 3-beta-hydroxysteroid dehydrogenase suggested that the tumor cells were capable of androgen synthesis. The morphological features are reminiscent of fetal-type Leydig cells and are distinctly different from the Leydig cell tumors described so far.


Two-dimensional arrays for medical ultrasound. The design, fabrication and evaluation of two-dimensional transducer arrays are described for medical ultrasound imaging. A 4 x 32, 2.8 MHz array was developed to use new signal processing techniques for improved B-scan imaging including elevation focusing, phase correction and synthetic aperture imaging. Laboratory measurements from typical array elements showed 50 omega insertion loss of -56 dB, -6 dB fractional bandwidth of 43%, interelement crosstalk of -19 dB, and -6 dB pulse-echo angular response of 62 degrees. Simulations of pulse-echo beam plots have shown grating lobes 20 dB below the main lobe at +/- 7 degrees in the elevation direction. The complete 2-D array has been used for measurements of phase aberrations in breast, and the individual 32 element linear arrays have been used to obtain conventional B-scans. Several 16 x 16 arrays have also been developed for high speed volumetric imaging. These include 96 transmit elements and 32 receive channels. With a lambda/4 matching layer, laboratory measurements show 50 omega insertion loss of -72 dB, -6 dB fractional bandwidth of 63%, interelement crosstalk of -29 dB and -6 dB angular response of 25 degrees. Pulse-echo sensitivity was improved by 21 dB through the use of integrated circuit preamplifiers of high impedance mounted in the transducer handle. In vivo cardiac, abdominal, and obstetric B-scans with elevation focusing, as well as high speed C-scans, have been obtained with these 2-D arrays.


Solid and papillary neoplasm of the pancreas. In two cases of solid and papillary neoplasm of the pancreas (SPN), positive staining for argyrophil granules, chromogranin-A, neuron-specific enolase, chymotrypsin, alpha 1-antitrypsin, vimentin, cytokeratin, and estrogen receptors was present. Ultrastructurally, neurosecretory as well as zymogenlike granules were demonstrated. Measurements of mean nuclear volume and volume-corrected mitotic index discriminated between SPN and well-differentiated ductal adenocarcinoma of the pancreas, with notably lower values being seen in SPN. Silver-stained nucleolar organizer region counts showed wide overlaps. The results suggest that SPN is a tumor with mixed endocrine and exocrine features. Its low malignant potential compared to ductal adenocarcinoma is reflected in the mean nuclear volume and volume-corrected mitotic index. The presence of estrogen receptors may prove therapeutically useful.


In vivo imaging using a copolymer phased array. Phased-array images have been obtained in vivo with a steered copolymer array operating at 2.5 MHz. The array was fabricated using 28 microns copolymer film, lambda/4 resonant at 21 MHz, which was bonded to a glass ceramic backing with a thin film bond. The 32 array elements were created by laser ablation of the continuous gold electrode on one side of the film. The transducer was driven by 200 V shock excitation, and the received signal was processed by 32 custom IC preamplifiers that were mounted to the array connector. The amplifiers had a high input impedance compared to that of the array elements and their output impedance matched that of the coaxial cable that connected to the scanner. Because the copolymer transducer had a broad bandwidth, the spectrum of the pulse echo image was modified by the frequency dependent attenuation of tissue and the bandpass of the ultrasound scanner, resulting in a center frequency of 2.5 MHz. The performance of the copolymer array was compared to that of a 3 MHz, PZT array with similar dimensions. On the Duke phased-array scanner, the copolymer sensitivity was 28 dB less than that of PZT. The copolymer elements had a 6 dB pulse-echo angular response of 30 degrees, and the interelement cross-coupling was -35 dB. Phased-array images were made of the heart of a 25 year-old normal male.


Optimum nonlinear signal detection and estimation in the presence of ultrasonic speckle. A unified approach to the design of nonlinear filters for speckle suppression in ultrasound B-mode images is presented. The detection of the (lesion) signal is formulated as a binary hypothesis-testing problem. The structure of the optimal decision rules is derived both in the case where the lesion signal is assumed either a constant or random variable. In the case of a constant signal, the maximum likelihood (ML) estimator and the optimal L-estimator are derived. In the case of a random lesion signal, the maximum a posteriori probability estimator of the lesion signal has also been found. Experimental results verify the superiority of the proposed ML-estimator and the L-estimator over the straightforward choice of an arithmetic mean for speckle filtering in simulated tissue mimicking phantom ultrasound B-mode images.


Large-transducer measurements of wavefront distortion in the female breast. Ultrasonic waves propagating through soft tissue experience wavefront distortion. Refraction occurs at boundaries between tissue beds having different sound speeds; scattering occurs within a tissue bed, caused by local impedance variations. This paper describes measurements of wavefront distortion in the human female breast that indicate that refraction is the dominant distortion mechanism when the ultrasonic phased array is very large. The determination that refraction dominates the wavefront distortion is based upon studies of multiple image artifacts that result from a single source radiated through in vivo breasts and breast phantoms. The receiving apertures used were 4.65 and 9.6 cm. Such image artifacts are repeatedly observed in the 10 young subjects reported in this paper, and also in older subjects. An understanding of the in vivo observations is obtained by phantom studies.


Ultrastructural and immunocytochemical study of a primary gastrinoma of the liver. A primary hepatic gastrinoma found in a 13-year-old boy was studied by light microscopy, immunohistochemistry, electron microscopy, and immunoelectron microscopy. Results were consistent with a neuroendocrine neoplasm with abundant gastrin-immunoreactive cells. Unlike all previously reported cases of primary hepatic neuroendocrine tumors, which have been endocrinologically asymptomatic, the patient had a Zollinger-Ellison syndrome apparently cured by surgical resection of the tumor.


Adaptive imaging in aberrating media: a broadband algorithm. An adaptive algorithm is derived for imaging in an aberrating medium based on wideband (time-domain) linear array data. Image reconstructions using simulated scattering data are presented. An analogy is also drawn between broadband array imaging and limited-angle tomography. This analogy suggests some tomographic-like filtering schemes that can be applied to improve the quality of the image. The algorithm is designed for imaging in composite material, although the imaging of other heterogeneous media such as tissue may be feasible under certain conditions.


Pigmented intracerebral neuroepithelial cyst: report of a case. A 35-year-old man with seizure disorder was found to have a parietal lobe neuroepithelial cyst. The lining of the cyst was made up of ependymal cells containing neuromelanin, which was confirmed ultrastructurally. This appears to be the first report of a pigmented neuroepithelial cyst in the brain parenchyma.


Role of Helicobacter felis in chronic canine gastritis. Five gnotobiotic Beagle dogs were orally inoculated with a pure culture of Helicobacter felis. The remaining two littermates served as contact controls. Thirty days after infection, all animals were euthanatized and specimens were collected for evaluation. In infected dogs, H. felis was recovered from all areas of the stomach. Colonization was heaviest in the fundus and antrum. H. felis was not cultured from any segment of the gastrointestinal tract distal to the duodenum. Two weeks after infection, all five infected dogs had detectable IgM and IgG serum antibody to H. felis, whereas control dogs had no measurable H. felis serum antibody throughout the study. Histopathologic changes in the stomachs of infected dogs included large numbers of lymphoid nodules throughout all regions of the gastric mucosa and were most numerous in the fundus and body. A mild, diffuse lymphocytic infiltrate with small numbers of plasma cells and eosinophils was also present in the subglandular region of all portions of the gastric mucosa. Electron microscopic examination revealed large numbers of spiral-shaped H. felis in gastric mucus adjacent to or superimposed over the areas of inflammation. Occasionally, however, H. felis was observed within the canaliculi of gastric parietal cells. Histopathologic changes in the stomachs of the contact control dogs were limited to focal infiltrates of eosinophils and small aggregates of lymphocytes in the subglandular portions of the gastric mucosa in one animal. Infection with H. felis is a likely cause of naturally occurring lymphofollicular gastritis.


Rift Valley fever virus-induced encephalomyelitis and hepatitis in calves. Three calves (Nos. 1, 2 = 7 days old; No. 3 = 21 days old) were inoculated subcutaneously with virulent Rift Valley fever (RVF) virus. All calves became viremic and clinically ill, but the two 7-day-old calves were moribund and were euthanatized subsequently on post-inoculation day (PID) 3. Highest viral titers were measured in the serum, with lesser concentrations in the brain, heart, spleen, and liver of these animals. Viral antigens were detected by immunohistochemical analysis only in the livers, where positive staining was localized in coalescing foci of hepatocellular necrosis. The 21-day-old calf appeared to recover after viremia and pyrexia but became lethargic and ataxic and was euthanatized on PID 9. The calf was no longer viremic, and RVF virus was isolated only from the brain. Microscopic examination of the central nervous system revealed diffuse perivascular infiltrates of lymphocytes and macrophages, multifocal meningitis, and focal areas of neuronal necrosis and aggregates of macrophages, lymphocytes, and neutrophils throughout all regions of the brain and cervical spinal cord. There was positive immunohistochemical staining for viral antigens within the cytoplasm of neurons and glial cells throughout the central nervous system. Thus, RVF virus can cause encephalomyelitis in calves, and the specific virologic diagnosis can be made by immunohistochemical localization of viral antigens in formalin-fixed tissues.


Flow cytometric analysis of feline reticulocytes. Hemolytic anemia was induced in five Domestic Shorthair cats (four adult males and one spayed female obtained from a breeding colony at Colorado State University, CO), and blood samples were analyzed from five other cats (two castrated male Domestic Shorthairs, one castrated male Domestic Longhair, one castrated male Persian, and one spayed female Siamese presented to the Veterinary Teaching Hospital at Colorado State University for miscellaneous problems). Blood samples taken from these cats had percentages of aggregate reticulocytes that ranged from 0% to 14.5% as determined by manual counting and were used to identify the best technique for staining cat reticulocytes for flow cytometric analysis. The best technique was mixing a blood sample (1/2,000 dilution) with 0.2 micrograms thiazole orange in 1 ml of diluent and incubating the mixture in the dark at room temperature for 30 to 60 minutes. The percentage of reticulocytes determined by flow cytometry correlated well (r = 0.88) with manually determined aggregate reticulocyte percentages; no significant differences were observed between the two techniques (P > 0.05). For the conditions used, punctate reticulocytes were not detected by flow cytometry. Samples with very high platelet numbers and very low packed cell volumes may show falsely elevated percentages of reticulocytes as determined by flow cytometry. The reproducibility of the flow cytometric technique was good; the coefficient of variation ranged from 4.8% to 17.9% in two samples with two different times of incubation. Staining of cat aggregate reticulocytes with thiazole orange and use of flow cytometric quantification is a reproducible technique that has a good correlation with the manual reticulocyte counting method.


Experimental oral administration of canine adenovirus (type 2) to raccoons (Procyon lotor). Canine adenovirus type 2 (CAV2) has been proposed for recombinant vaccines to control rabies in wild animals. To evaluate the suitability of CAV2 as a safe vector for the genetically engineered vaccines, seven wild-caught raccoons (three males and four females) were administered CAV2 per os. Two of the animals were euthanatized on each of post-infection days 3, 6, and 14, and one was euthanatized on day 21. Two other control raccoons (a male and a female) were also euthanatized on day 21. Microscopic pulmonary lesions of multifocal necrotizing bronchiolitis with basophilic intranuclear inclusions were seen in 3/4 raccoons euthanatized on post-infection days 3 and 6. Ultrastructural examination of lungs with pulmonary lesions revealed hexagonal viral particles characteristic of adenoviruses. CAV2 is potentially pathogenic for raccoons, and this susceptibility should be of concern to developers of recombinant vaccines who intend to use CAV2 as a vaccine vector.


Immunohistochemical localization of matrix proteins in the femoral joint cartilage of growing commercial pigs. The immunocytochemical localization of several matrix macromolecules, including collagen type II and proteoglycans, in the distal femoral articular-epiphyseal cartilage complex of 15 commercial pigs between the age of 6 and 18 weeks was studied. Early osteochondrotic lesions, i.e., chondronecrosis in the resting region of the growth cartilage, as well as extensions of necrotic cartilage into the subchondral bone, were present in all animals, except those 6 weeks old. A battery of antibodies were used for identification of macromolecules in the matrix at different stages of the disease. Chondrocyte involvement in the process could be studied by identifying the sequence of alterations in matrix macromolecules as the lesion developed. The immunostaining for aggrecan (large aggregating proteoglycans), cartilage oligomeric matrix protein, fibronectin, collagen type II, fibromodulin, and biglycan was more prominent in the areas of chondronecrosis, extending into the subchondral bone, than in the normal resting region. This altered pattern of matrix macromolecules resembled that of the matrix of the proliferative chondrocytes and suggests that the chondrocyte maturation had stopped in the proliferative zone. The matrix in the areas of chondronecrosis in the resting region resembled that in the normal resting region. Thus the chondronecrosis appears to have preceded alterations of the matrix composition. The antibody reactivity pattern was, however, altered in the matrix of the clustered chondrocytes in areas of chondronecrosis. Staining in these regions suggested a more prominent appearance of fibronectin and collagen type II than in the normal matrix of the resting region. These changes are suggestive of attempt to repair.(ABSTRACT TRUNCATED AT 250 WORDS)


Early sequential ultrastructural renal alterations induced by 2-bromoethylamine hydrobromide in the Swiss ICR mouse. Thirty-two male Swiss ICR mice were injected intraperitoneally with 300 mg 2-bromoethylamine hydrobromide/kg body weight, anesthetized, and perfused with glutaraldehyde-paraformaldehyde solution at 5, 15, 30, 60, 90, 120, 150, and 180 minutes after treatment. Eight control mice were injected intraperitoneally with sterile diluent, and one was perfused at each of the same time periods as the treated mice. Proximal tubule epithelial alterations progressed over time from increased secondary lysosome and myeloid body formation to cellular and mitochondrial swelling and eventually cell necrosis. The glomerular, peritubular, and vasa recta capillaries had endothelial cell swelling and desquamation and platelet aggregation. Bromoethylamine nephrotoxicosis in the male Swiss ICR mouse is an ischemic necrosis of the proximal tubules and papilla initiated by endothelial cell damage and makes an excellent model of chemically induced damage to endothelial cells and tubular necrosis.


Hepatic lesions in rabbits infected with Encephalitozoon cuniculi administered per rectum. Microsporidia have been recognized recently as opportunistic pathogens in acquired immunodeficiency syndrome patients. In an attempt to develop an animal model of enteric microsporidiosis, adult (5 to 6 months old) male Flemish Giant rabbits from a closed New York colony were administered 5 x 10(3), 5 x 10(5), and 5 x 10(7) Encephalitozoon cuniculi per rectum. Rabbits given 5 x 10(5) and 5 x 10(7) E. cuniculi had moderate granulomatous periportal infiltrates, characterized by the presence of numerous macrophages, epithelioid cells and a few multinucleated giant cells, lymphocytes, and plasma cells. Inflammatory cells also were seen infiltrating the tunica adventitia and tunica media of hepatic portal veins and branches of the hepatic artery. This study demonstrates that administration of E. cuniculi per rectum to rabbits results in infection that is characterized by high frequency and severity of hepatic lesions.


Identification of a binding protein to the X gene promoter region of hepatitis B virus. The X protein of hepatitis B virus (HBV) is a transactivator to homologous and heterologous viral and cellular transcriptional regulatory elements. One sequence-specific binding protein, whose binding site located from nt 1102 to nt 1117 of HBV DNA, was identified by mobility shift assay and DNase I foot-printing analysis. A CAT assay experiment demonstrated this 16-bp binding site to have a promoter activity in the X gene transcription. The 58-bp DNA fragment (nt 1085 to nt 1142), which contains the above binding site, could be enhanced by the HBV enhancer. Mobility shift assay using the mutated 58-bp DNA fragments as probes, showed that the mutation, which damaged the palindrome structure between nt 1105 and nt 1112, resulted in loss of the binding activity. This mutation also remarkably reduced the promoter activity. The binding site differed from the target sequences of known transcriptional factors. This factor was thus concluded to be a binding protein to the X gene promoter (X-PBP) of HBV. A homology search demonstrated the binding site to be highly homologous to the promoter elements of human laminin receptor (2H5epitope) and lipoprotein receptor-related protein (LRP) genes.


Influence of amantadine resistance mutations on the pH regulatory function of the M2 protein of influenza A viruses. Mutations in the influenza M2 membrane protein which confer resistance to the antiviral drug amantadine are exclusively located within the transmembrane region of the molecule. The influence of specific amino acid substitutions on the activity of the M2 protein in influenza A virus-infected cells is assessed in this report by their effects upon haemagglutinin (HA) stability and virus growth. A number of amino acid substitutions, e.g., L26H, A30T, S31N and G34E reduced the activity of the M2 protein of A/chicken/Germany/34 (Rostock) and caused a substantial increase in expression of the low-pH form of HA. The adverse effects of the mutations on virus replication were evident from changes selected during subsequent passage of the mutant viruses in the presence or absence of amantadine: reversion to wt, the acquisition of a second suppressor mutation in M2, or the appearance of a complementary mutation in HA which increased its pH stability. In contrast, 127T and 127S, mutations which were most readily selected following passage of the wt virus in the presence of drug, caused an increase in M2 activity. Furthermore, in double mutants the 127T mutation suppressed the attenuating effects of the A30T and S31N mutations on M2 activity. The influence of primary structure on the consequences of particular amino acid changes was further emphasized by the contrasting effects of the G34E mutation on the activities of two closely related proteins, causing an increase in the activity of the M2 of A/chicken/Germany/27 (Weybridge) as opposed to the decrease in activity of the Rostock protein. Estimates of differences in trans Golgi pH based on the degree of conversion of HA to the low-pH form, or complementation of differences in pH stability of mutant HAs, indicate that changes in M2 may influence pH within the transport pathway by as much as 0.6. The results thus provide further evidence that M2 regulates transmembrane pH gradients in the trans Golgi. Incompatibility between particular HA and M2 components and the selection of M2 mutants with suboptimal activity stresses the essential relationship between the structures and functions of these two virus proteins.


Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Hepatitis E virus (HEV) is the major causative agent of hepatitis E or what was formerly known as enterically transmitted non-A, non-B hepatitis. The disease has a worldwide distribution but occurs principally in developing countries in any of three forms: large epidemics, smaller outbreaks, or sporadic infections. Genetic variation of different HEV strains was previously noted and it will be important to determine the extent to which this variation may pose problems in the diagnosis and treatment of HEV infection. To analyze differences at the genetic level between HEV(Mexico; M) and the previously characterized HEV(Burma; B) and HEV(Pakistan; P) isolates, overlapping cDNAs were cloned from samples obtained from an infected human and an experimentally inoculated cynomolgus macaque. These cDNA clones, representing the nearly complete (7185-bp) genome of HEV(M), confirmed an expression strategy for the virus that involves the use of 3 forward open reading frames (ORFs). The HEV(M) strain has an overall 76 and 77% nucleic acid identity with the HEV(B) strain and HEV(P) strain, respectively; however, the degree of sequence variation was not uniform throughout the viral genome. A hypervariable region was identified in ORF1 that exhibited a 58 and 54% nucleic acid sequence and 13% amino acid similarity with the Burma strain and the Pakistan strain, respectively. A large number of the nucleotide differences occurred at the third codon position, with the deduced amino acid sequences similarity of 83, 93, and 87% between HEV(M) and HEV(B) isolates in ORF1, ORF2, and ORF3, respectively, and with 84, 93, and 87% amino acid identities between HEV(M) and HEV(P) isolates in ORF1, ORF2, and ORF3, respectively. The nucleotide sequences derived from the highly conserved regions of HEV genome will be useful in developing polymerase chain reaction-based tests to confirm the viral infection. Knowledge of the extent of the sequence variation encountered with HEV will not only aid in the future development of diagnostic and vaccine reagents but also further our understanding of how HEV strain variation might impact the pathological outcome of infection.


Two strains of SIVmac show differential transactivation mediated by sequences in the promoter. Two infectious molecular clones of simian immunodeficiency virus, SIVmac251 and SIVmac239, have very different in vivo properties, SIVmac239 being much more pathogenic than SIVmac251. To assess whether the in vivo differences between the two viruses would be reflected in transcriptional rates in vitro, transcriptional activity in the presence of the transactivation protein tat was analyzed by transient transfection assays in HUT-78 and U937 cells. Whereas the two promoters had similar basal activities (Anderson and Clements, 1991, J. Virol. 65, 51-60) the promoter of SIVmac239 was transactivated to a greater extent. Removal of sequences 5' to -225 and 3' to +18 maintained the basal activity, yet made the promoter unresponsive to tat. Addition of bases +19 to +149 reconstituted transactivation and decreased basal activity. Analysis of deletion mutants with reconstituted transactivation response region determined that differences between the two strains were maintained even when only the proximal sequences, -225 to +18 of the U3 and R region were placed upstream of the TAR sequences. This region contains four nucleotide differences and the potential Sp-1-binding sites, where there are an additional 11 bases in SIVmac239 that create a third potential Sp-1 site, compared to only 2 in SIVmac251. Transactivation in this assay system was found to correlate better to RNA differences shortly after transfection (12 hr) than later (46 hr).


Genetic evidence that epizootic Venezuelan equine encephalitis (VEE) viruses may have evolved from enzootic VEE subtype I-D virus. An important question pertaining to the natural history of Venezuelan equine encephalitis (VEE) virus concerns the source of epizootic, equine-virulent strains. An endemic source of epizootic virus has not been identified, despite intensive surveillance. One of the theories of epizootic strain origin is that epizootic VEE viruses evolve from enzootic strains. Likely enzootic sources of VEE virus occur in Colombia and Venezuela where many of the epizootic outbreaks of VEE have occurred. We have determined the nucleotide sequences of the entire genomes of epizootic VEE subtype I-C virus, strain P676, isolated in Venezuela, and of enzootic VEE subtype I-D virus, strain 3880, isolated in Panama. VEE subtype I-D viruses are maintained in enzootic foci in Panama, Colombia, and Venezuela. The genomes of P676 and 3880 viruses differ from that of VEE subtype I-AB virus, strain Trinidad donkey (TRD), by 417 (3.6%) and 619 (5.4%) nucleotides, respectively. The translated regions of P676 and 3880 genomes differ from those of TRD virus by 54 (1.4%) and 66 (1.8%) amino acids, respectively. This study and the oligonucleotide fingerprint analyses of South American I-C and I-D viruses (Rico-Hesse, Roehrig, Trent, and Dickerman, 1988, Am. J. Trop. Med. Hyg. 38, 187-194) provide the most conclusive evidence to date suggesting that equine-virulent strains of VEE virus arise naturally from minor variants present in populations of I-D VEE virus maintained in enzootic foci in northern South America.


Characterization of TH3, an induction-specific protein interacting with the interferon beta promoter. We report the purification and characterization of a unique DNA-binding protein termed TH3 that interacts with the positive regulatory domain (PRD) I and PRDIII domains of the interferon (IFN) beta promoter. In cells treated with poly rl:rC and cycloheximide, appearance of TH3 DNA-binding activity was inversely proportional to the disappearance of a constitutive complex TH1 and coincided temporally with induction of IFN-beta gene transcription. The TH3 DNA-binding protein is a small 14-kDa polypeptide that appears to be derived from the TH1 complex; TH1 in turn is related to interferon regulatory factor (IRF) 2 by immunological cross-reactivity. The TH3 protein appeared to lack the epitope required for recognition by anti-IRF-2 antisera; however, a short microsequence obtained for TH3 overlapped a sequence from the IRF-2 protein. Although TH3 binds to multimers of the AAGTGA hexamer and to PRDI, the TH3 protein alone had a predominantly neutral phenotype on PRDI-dependent transcription in vitro and lacked the negative transcriptional effect attributed to IRF-2. These results raise the possibility that specific proteolysis of a negative regulatory protein involved in silencing the IFN-beta promoter may be an important event leading to transcriptional activation of the interferon gene.


Cloned DNA copies of cowpea severe mosaic virus genomic RNAs: infectious transcripts and complete nucleotide sequence of RNA 1. Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member. Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter. Previously, similar clones of CPSMV RNA 2 had been obtained. A 5'-rUAUUAAAAUUUU sequence is common to RNA 1 and RNA 2. From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences. Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions. The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831. The calculated molecular weight of the polyprotein is 208,000. Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5'-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites. Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24K/87K junction has a distinct aa pair in the CPSMV polyprotein. Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively. Published mutational analysis of the CPMV 24K proteinase and alignment of aa sequences from three comoviruses suggest that cysteine-168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase. Results are discussed in the context of the resistance that some cowpea (Vigna unguiculata) lines exhibit against CPMV but not against CPSMV.


In situ immunogold labeling analysis of the rice hoja blanca virus nucleoprotein and major noncapsid protein. Ribonucleoprotein particles (RNPs) of rice hoja blanca virus (RHBV) were purified and used for electron microscopic analysis and antibody production. Antibodies made to RNPs specifically decorated purified RNPs. The RNPs typically showed characteristic tenuivirus morphologies. They were approximately 8 nm in diameter, mostly circular in nature, and exhibited branching and a high degree of superhelicity. When the RNP antibodies were used for in situ immunogold labeling analysis of RHBV-infected tissues, no specific structures were identified, but gold particles were distributed throughout the cytosol of RHBV-infected but not healthy plants. However, amorphous semi-electron opaque inclusion bodies (ASO-IBs) were abundant in cells of RHBV-infected plants. While the ASO-IBs were not labeled with the anti-RNP antiserum, they were specifically labeled with antibodies to the RHBV major noncapsid protein (NCP) and with antibodies to the NCP of another tenuivirus, maize stripe virus.


Regulation of the efficiency and thermodependence of murine sarcoma virus MuSVts110 RNA splicing by sequences in both exons. Efficient splicing of MuSVts110 RNA is restricted to temperatures of 33 degrees or lower. Previously, we have shown that this conditional splicing event is mediated, in part, by cis-acting intronic sequences. We have now examined the role of exon sequences in MuSVts110 RNA splicing. We found that deletion of all but 36 nucleotides of the gag exon (E1) yielded a transcript incapable of supporting splicing. However, inefficient, growth temperature-dependent splicing was recovered after restoration of the 300 nucleotides of E1 proximal to the 5' splice site (5' ss). Increasingly efficient splicing was observed as more E1 was restored. Hence, although MuSVts110 E1 sequences were required for splicing, they were not involved in its thermodependence. Similarly, removal of all but 88 nucleotides of the mos exon (E2) abolished splicing at the usual 3' splice site (3' ss). In contrast to E1, restoration of the 200 nucleotides of E2 adjacent to the 3' ss reactivated efficient, temperature-independent splicing. Thermodependent splicing, however, reappeared with the replacement of E2 sequences located more than 400 nucleotides distal to the 3' splice site. In MuSVts110 mutants containing the minimum amounts of both E1 and E2 which would support splicing, splicing was both far more efficient than predicted and temperature-independent, suggesting that cooperation between E1 and E2 may help to regulate MuSVts110 splicing.


Analysis of proviruses integrated in Fli-1 and Evi-1 regions in Cas-Br-E MuLV-induced non-T-, non-B-cell leukemias. The DNAs of the Cas-Br-E MuLV-induced leukemias always contain somatically acquired mink cell focus-forming (MCF) recombinant proviruses. MCF recombinants could be involved during leukemogenesis at both preleukemic times and in late-stage tumors. Among the Cas-Br-E-induced non-T-, non-B-cell leukemias, viral integrations were found in the Fli-1 and Evi-1 region in 71% (36 out of 51) and 22% (16 out of 72) of the tumors analyzed, respectively. As an approach to evaluate the contribution of Cas-Br-E MCF recombinant formation in cis-activation of proto-oncogenes, we analyzed the structure of the Fli-1- and Evi-1-associated proviruses by Southern blot hybridization. In Fli-1, we found that the proviruses, ecotropic as well as MCF, are all integrated within a very short DNA region immediately upstream of the initiator ATG, toward the 3' end of a 5' exon (Ben-David, Giddens, Letwin, and Bernstein, 1991, Genes Dev. 5, 908-918). All proviruses are oriented the same way, in the 5' to 3' transcriptional sense. Both provirus types are able to direct the Fli-1 expression to the same extent presumably via a promoter insertion mechanism. Most of the proviruses had no detectable deletion and contained both 5' and 3' LTR sequences with similar U3 sequences. MCF recombinants did not show any selective advantage over ecotropic proviruses for the Fli-1 locus since the frequency of ecotropic to MCF-recombinant virus at the Fli-1 locus was identical to that observed at any other locus. This suggests that the formation of these MCF recombinants is not essential for activation of Fli-1 and that ecotropic Cas-Br-E already possesses the required sequences for full cis-activation of Fli-1. On the other hand, in Evi-1, there is a strict selection for ecotropic proviruses. Presumably, viral genetic elements outside of the U3 region could be critical for the Evi-1 cis-activation.


Interaction of cellular factors with intragenic cis-acting repressive sequences within the HIV genome. Expression of the human immunodeficiency virus (HIV) structural gene products is suppressed in the absence of the Rev protein. The block to expression reflects, in part, nuclear retention of those mRNAs which encode the structural proteins. The presence of intragenic cis-acting repressive sequences (CRS) and inefficient splicing of the primary viral transcript are thought to contribute to nuclear entrapment of viral RNA. To elucidate the mechanism for repression of HIV gene expression, the ability of a 270-bp segment of the pol gene shown previously to repress gene expression to interact with cellular factors was investigated. Incubation of RNA corresponding to the 270-bp CRS element with nuclear extract prepared from human T-cells revealed a strong and specific interaction with several cellular factors. Covalent cross-linking of the RNA-protein complex demonstrated the presence of at least three proteins, the predominant one having a molecular weight of approximately 42 kDa. A monoclonal antibody raised against hnRNP C, a component of the splicing machinery, recognized the CRS-protein complex, suggesting that hnRNP C or a closely related gene product interacts with CRS-containing RNA. Consistent with this conclusion, addition of RNA corresponding to a beta-globin intron sequence in the binding reaction completely blocked formation of the CRS-protein complex. These findings raise the possibility that the CRS elements elicit nuclear entrapment of viral RNA through formation of RNA-protein complexes that are not accessible to nuclear export pathways.


The E3-11.6K protein of adenovirus is an Asn-glycosylated integral membrane protein that localizes to the nuclear membrane. The 11,600 MW (101 amino acids; 11.6K) protein of adenovirus 2 (Ad2) is a protein of unknown function which is synthesized in low amounts during early stages of infection but in very high amounts at late stages. The 11.6K protein migrates as three major groupings of diffuse bands of ca. 14K, 21K, and 31K on SDS-PAGE, indicating that 11.6K undergoes post-translational modification. We show here that 11.6K is Asn-glycosylated with complex (endo H-resistant) oligosaccharides and that 11.6K is an integral membrane protein. Immunofluorescence indicated that 11.6K initially is associated with the endoplasmic reticulum and Golgi apparatus and that it ultimately localizes to the nuclear membrane. The 11.6K protein is predicted to have a single signal-anchor sequence at residues 41-62 and only one potential Asn-linked glycosylation site at residue 14; thus, 11.6K must be oriented in the membranes with its NH2-terminus in the lumen and its COOH-terminus in the cytoplasm. The signal-anchor and glycosylation features of 11.6K are preserved in Ad2 and Ad5 (group C), and in Ad3 and Ad7 (group B), but the sequence of 11.6K is more diverged among these serotypes than is the sequence of most other adenovirus proteins.


Adenovirus type 12 early region 1B proteins and metabolism of early viral mRNAs. Early region 1B (E1B) of human adenoviruses encodes two major proteins. The 19-kDa polypeptide appears to prevent E1A-induced cytolysis and DNA degradation. The larger E1B product of approximately 55 kDa, which is essential for viral replication, plays a role in the accumulation and stability of viral mRNAs and the late shutoff of host metabolism. For serotype 12 (Ad12), this 482-residue (482R) protein is essential for viral DNA replication. In the present report we have used a series of mutants to examine the roles of Ad12 482R and the 19-kDa, 163R protein in the metabolism of early viral mRNAs. No specific effects on the accumulation of early (or late) mRNAs were detected with any of the mutants affecting 163R. With mutant dl42, which encodes an altered 482R product that lacks residues 114-155, both viral DNA replication and late viral protein synthesis were defective. Accumulation of E1A transcripts in the nucleus and cytoplasm resembled wt. The levels of mRNAs from early regions E1B, E2A and E3 at later times during infection were somewhat lower than those of wt, but this decrease may have been due to the absence of progeny viral DNA in dl42-infected cells. However, the accumulation of both E2B and E4 mRNAs at all times was severely reduced. These data suggested that the requirement of 482R for Ad12 DNA replication may be related to its specific role in the metabolism of E2B and E4 mRNAs that encode products necessary for viral DNA synthesis.


Vaccinia virus gene B1R encodes a 34-kDa serine/threonine protein kinase that localizes in cytoplasmic factories and is packaged into virions. Vaccinia virus open reading frame B1R was expressed in E. coli and shown to encode a serine/threonine protein kinase which phosphorylated casein and calf thymus histones in vitro. A polyclonal rabbit antiserum was raised against a TrpE-B1R bacterial fusion protein and used to characterize the B1R gene product. Immunoprecipitation and immunoblotting analyses detected a 34-kDa polypeptide that was synthesized early during vaccinia virus infection and which was apparently stable since it was easily detectable 18 hr postinfection. Immunofluorescence demonstrated that this protein localizes in cytoplasmic virus factories, the sites of virus DNA replication. Immunoblotting of vaccinia virions showed that the enzyme is packaged into virus particles.


The restricted nature of HIV-1 tropism for cultured neural cells. Infection of the central nervous system by HIV-1, the agent of AIDS, is characterized by the presence of infected and giant microglial cells as well as astrocytosis, demyelination, and neuronal loss. To determine whether cells of neuroectoderm origin can be infected by HIV-1, we have inoculated primary cultures derived from adult human brain with a lymphotropic virus (LAV) or a neurotropic virus (Jr-FL) isolated from a patient with AIDS dementia. While Jr-FL invariably causes productive infection of cultured brain microglia, neither astrocytes nor oligodendrocytes became productively infected by these viral strains. Moreover, the cultured oligodendrocytes develop a normal network of processes and express differentiation antigens in the presence of an ongoing lytic infection of microglial cells. No HIV-1 proviral DNA was detected in primary astrocyte cultures devoid of microglial after inoculation of either HIV-1 strain. Similarly, the neuronal cell line HCN-1 in its differentiated state did not allow the virus to go through cycles of reverse transcription and replication. LAV, however, was able to replicate in undifferentiated HCN-1 cells. Thus, tropism of HIV-1 appears tightly restricted to only one type of differentiated cell in the CNS, the microglia.


Expression and secretion of Japanese encephalitis virus nonstructural protein NS1 by insect cells using a recombinant baculovirus. The nonstructural protein NS1 of Japanese encephalitis virus (JEV) was expressed at a high level under the control of the polyhedrin promoter in Spodoptera frugiperda (Sf9) insect cells using a recombinant baculovirus. Recombinant NS1 was designed to contain its natural signal sequence at its N-terminus and no C-terminal hydrophobic domain that could act as a membrane anchor. This recombinant protein exhibited similar size to native NS1 expressed in Aedes albopictus (C6/36) insect cells infected with wild-type JEV. The signal sequence of NS1 allowed translocation of the protein into the endoplasmic reticulum where it underwent glycosylation. A small fraction of synthesized NS1 was able, in the absence of any other viral protein, to associate as a homodimer, showing similar characteristics to the native dimer. Interestingly, this recombinant dimeric form seemed to be exported and released in the extracellular medium of infected cell culture. During its transport, one of the two N-linked oligosaccharides of the polymannose type was processed to an endoglycosidase H-resistant form, suggesting that the protein had passed through the Golgi compartment before reaching the cell surface. Moreover, Triton X-114 partitioning analysis showed that monomeric NS1 behaved essentially as a hydrophilic protein, whereas both intracellular and extracellular dimeric NS1 were either free of or associated to membraneous components.


Complex intrapatient sequence variation in the V1 and V2 hypervariable regions of the HIV-1 gp 120 envelope sequence. Human immunodeficiency virus (HIV) quasi-species fluctuate both in time and in space. In general intrapatient variation is less extensive than interpatient variation. The V1 and V2 hypervariable regions of the envelope gene were analyzed for patient samples harboring highly divergent genomes in other loci. Proviral sequences were amplified by PCR, cloned, and sequenced. It was concluded that intrapatient variation may exceed interpatient variation but the presence of two clearly distinct populations was not confirmed. The env hypervariable regions appeared to be evolving independently of one another, cautioning against extrapolation of data, particularly from the V3 region. Virus typing and the assessment of HIV superinfection by genetic methods will prove difficult and experimental approaches will have to be carefully designed.


The trypsin-sensitive RVER domain in the capsid proteins of minute virus of mice is required for efficient cell binding and viral infection but not for proteolytic processing in vivo. Analysis of a series of mutations in the trypsin-sensitive RVER region of the amino terminal domain in the capsid proteins (VP1 and VP2) of the autonomous parvovirus, minute virus of mice (MVM), demonstrates that this sequence is not essential for proteolytic processing of VP2 into VP3 in vivo, but specific amino acids within this domain are important for viral infection. Analysis of the most deficient of these mutants, VP(delta 2842-2863), a 7-aa deletion of aa 159-165 in VP1 and 17-23 in VP2, has identified at least two steps in MVM infection in which this domain is important. VP(delta 2842-2863) was 3-fold defective in binding to murine A9(2L) cells and, when an equivalent amount of virus was bound to cells, additionally 10-fold deficient compared to wild-type in initiating a productive infection. However, in those cells effectively infected, VP(delta 2843-2863) replicated similar to wild-type. These results suggest that these seven amino acids constitute a region important for both binding and a subsequent step prior to the start of DNA replication such as viral uptake or transport to the nucleus.


Identification of the active site residues in the nsP2 proteinase of Sindbis virus. The nonstructural polyproteins of Sindbis virus are processed by a virus-encoded proteinase which is located in the C-terminal domain of nsP2. Here we have performed a mutagenic analysis to identify the active site residues of this proteinase. Substitution of other amino acids for either Cys-481 or His-558 completely abolished proteolytic processing of Sindbis virus polyproteins in vitro. Substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. These results suggest that nsP2 is a papain-like proteinase whose catalytic dyad is composed of Cys-481 and His-558. Since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the C-terminal half of nsP2 and found that all could be substituted without total loss of activity. Among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of Trp-559 in alphaviruses is unusual. A mutant enzyme containing Ala-559 was completely inactive, implying that Trp-559 is essential for a functional proteinase. All of these mutations were introduced into a full-length clone of Sindbis virus from which infectious RNA could be transcribed in vitro, and the effects of these changes on viability were tested. In all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for Sindbis replication.


Constitutive expression of the nef gene suppresses human immunodeficiency virus type 1 (HIV-1) replication in monocytic cell lines. In order to study the effect of nef gene expression on viral replication in monocytic cells, we established monocytic (U937 and THP-1) cell transfectants constitutively expressing the human immunodeficiency virus type 1 nef gene. We constructed a plasmid expressing the nef gene derived from an infectious clone, NL432, under the control of SR alpha promoter which can drive a high level of gene expression. We found suppressed viral replication in nef-expressing monocytic cells, although a negative effect of nef was observed, with some variation depending on the virus strain and the cell. We also observed that the expression of the surface CD4 molecule is inversely related to the expression of the nef gene, especially in the U937 transfectants. These results indicate that the suppression of viral replication and the down-modulation of CD4 molecule by nef gene expression occur in monocytic cell lines as in T cell lines.


Natural variants of the HIV-1 long terminal repeat: analysis of promoters with duplicated DNA regulatory motifs. Sequence variation in the long terminal repeat (LTR) region of HIV-1 was analyzed in viral isolates of 17 infected individuals. Two classes of LTR size variants were found. One HIV-1 variant was detected containing an additional binding site for the transcription factor Sp1. Another LTR size variation was observed in four patients in a region just upstream of the NF-kappa B enhancer. This variation was the result of a duplication of a short DNA sequence (CTG-motif). Cell culture experiments demonstrated that the natural variant with four Sp1 sites had a slightly higher promoter activity and viral replication rate than the isogenic control LTR with three Sp1 sites. No positive effect of the duplicated CTG-motif could be detected. In order to measure small differences in virus production more accurately, equal amounts of a size variant and the wild-type plasmid were cotransfected into T-cells. The virus with four Sp1 sites did outgrow the three Sp1 virus in 35 days of culture and CTG-monomer virus outcompeted the CTG-dimer virus in 42 days. Based on these results we estimate a 5-10% difference in virus production of the LTR variants when compared to that of wild-type.


Rice dwarf phytoreovirus segment S12 transcript is tricistronic in vitro. Sequence analysis revealed that rice dwarf phytoreovirus segment S12 is 1066 nucleotides long with a small out-of-phase, overlapping open reading frame (ORF) as well as a major ORF. The large ORF (positions 42 to 980) encodes 312 amino acids, while the small one (bases 313 to 591) encodes 92 amino acids with an additional in-frame AUG codon (positions 337-339) 24 nucleotides downstream from the first one. Transcripts from a full-length cDNA directed the in vitro synthesis of three polypeptides of 33 (considered to be translated from the long ORF), 8, and 7 kDa. Alteration of each of the two ATG codons on the small ORF demonstrated their involvement in the generation of the 8- and 7-kDa polypeptides. Although it is still unknown whether these proteins are expressed in vivo, the small ORF is shown to be conserved in S9s of two other members of the genus Phytoreovirus, rice gall dwarf virus and wound tumor virus, suggesting its common, important function.


The genetic evolution of the envelope gene of simian immunodeficiency virus in cynomolgus macaques infected with a complex virus pool. Two cynomolgus macaques were infected with a complex, but characterized, challenge stock of simian immunodeficiency virus (SIVmac251 32H). The polymerase chain reaction was applied in a temporal sequence analysis to determine the sequences of the gp120 region of the SIV env gene, which were present in the blood of both macaques at 1, 6, and 15 months postinfection (p.i.). At 1 month p.i. selected sequences, which had been present in the original virus challenge stock, were reisolated. At later times, new sequences emerged, which had not been detected in the original virus challenge stock. Changes in sequence were restricted to specific regions of gp120, notably those equivalent to V1, V2, V4, and V5 of HIV-1, but not V3. The diversity and the rate of appearance of new sequences in the V1 region suggest that genetic evolution occurs by mechanisms in addition to nucleotide substitutions. These results are discussed in relation to the role of the envelope protein in the generation of protective immunity against infection with immunodeficiency viruses.


[Changes in the lymphatic system in the crush syndrome]. In vivo indirect electrolymphoroentgenography was conducted to study the reaction of lymphatics during prolonged compression syndrome (PCS). The studies were made on white rats, Wistar-line, males. It was found out that the early period of clinical course of PCS was characterized by lymphostasis. The intermediate period of PCS corresponds to the stage of the collateral lymphokinesis. At the edge of intermediate and late period of PCS the electrolymphoroentgenogram shows the removal of lymphostasis and recovery of main lymphokinesis.


[The deontological aspects of the legal regulation of the activities of military medical commissions]. Practically, the questions of deontology are not working out in the sphere of military medical rating (expertise). Finally this fact leads to methodical errors, defects in medical examinations, and thus to a considerable number of requests and complaints. The authors examine the basic questions of deontology from the point of view of the following aspects: collegiality, obligation to render the verdict on medical examination in the presence of examinee, medical secrecy and strict legal regulation of this norm, deontological questions in drawing up, filling in, storing and giving out medical rating documents.


[The prospects for the use of a Soviet apparatus for the removal of gallstones with focused shock waves]. Clinico-experimental substantiation was conducted for extracorporal crushing of the stones in biliary ducts in patients who suffered from gallstone disease. The "Urat-II" national lithotriptor and "Urat-B" biostand were used to crush in experimental conditions 140 gallstones (0.5-2.5 cm in size) obtained during cholecystectomies. Experimental researches on 56 white rats were conducted to study the liver tissue and adjacent organs. Optimal regimes for the percussive wave generator were found out in which the best crushing effect was obtained (with the dimensions of fractions up to 1.6 mm) and at the same time the minimal disturbing factor was produced. The data obtained during these researches are used in clinical pilot testings on "Urat-II"--a prototype of the national cholelithotriptor.


[Regional dehydration in the combined treatment of fractures of the bones of the extremities]. The article makes a theoretical substantiation of regional dehydration in the treatment of the posttraumatic swellings in bone fractures of the extremities. The state of regional hemodynamics in 226 patients with the affections of bones and vessels of the extremities was studied using clinical, functional and angiographical methods. The authors make a conclusion concerning the efficiency of the proposed method of treatment for posttraumatic swellings of the extremities, which is based on the rapid enhancing of the mole-factor in the blood plasma in order to recover optimal conditions for the exchange process and microcirculation. This method of regional dehydration is proposed by the authors as an alternative to the fasciotomy which is based on mechanical influence upon extravasal squeezing of vessels.


[The epidemiological, clinical, diagnostic and treatment aspects of Legionella infection]. The article studies the modern problems of legionellosis infection and gives data concerning epidemiological aspects of this disease, especially clinical course of its types (pneumonia, acute respiratory viral infection syndrome, alveolitis, pleuritis), as well as methods of bacteriological and serological diagnostics, and etiotropic treatment. The authors use their own materials to describe the restricted outbreak of legionellosis infection and sporadic cases of this disease. The article makes clinic classification of legionellosis taking into account various publications and the proper experience of the authors.


[The assessment of body functional systems by the indices of their nonspecific reactions]. The analysis of long standing researches in the military operators' procedure has shown some regularities in the changes of indices which were examined. Varying in time and magnitude the non-specific reactions at first manifest itself in unstable vegetative regulations, then proliferate its influence upon visual and motor coordination, and finally cause negative shifts in professional activities (worsening of accuracy and precision, appearance of errors, etc). It is obvious, that specific reactions impose their own features on the development of this program without changing its basic trend.