Effects of cholinergic muscarinic agents on protein kinase C activity in rat pineal gland.

The role of protein kinase C (PKC) on muscarinic regulation of serotonin release in the pineal gland was investigated by measuring the pineal-PKC activity and serotonin secretion in response to muscarinic agents. Pineal slices, short-term incubated (0-15 min) without additions produced a low serotonin release and 20 to 24 percent PKC activity was found associated with membrane fractions. Prolonged exposure of pineal slices (30-180 min) produced further translocation of PKC activity to the membranes and a significant increase of serotonin release. Short-term treatment with pilocarpine and carbachol, stimulated PKC activity of both cytosolic and particulate fractions and the release of pineal serotonin. The pilocarpine effect was blocked by atropine indicating that it was mediated by muscarinic receptors. The present data support that PKC activation correlates with the increase of serotonin release by muscarinic agonist in pineal gland.     

Change in energy metabolism of ascites cancer cells with a decrease in pH]

In Hank's balanced salt solution EL-4 ascites thymoma cells possessed endogenous respiration which was sufficient for the maintenance of their ATP level: pH decrease down to 6.0 had no effect either on endogenous respiration or the ATP level. Glucose had no influence on the respiration of EL-4 cells but inhibited that of Ehrlich ascites carcinoma (EAC) cells by 40% (Crabtree effect); respiration of the both cell lines was strongly (4-fold) inhibited after simultaneous addition of glucose, lactate and pH decrease. EL-4 cells had no endogenous glycolysis; EAC cells showed a low level of glycolysis only after pH decrease. Glucose addition led to activation of glycolysis (both inhibited 2-fold after a decrease of pH down to 6.0. The respiration inhibition at pH 7.3 and 6.0 caused no decrease of ATP depletion when glucose was present in the medium; this result may be due to suppression of ATP consumption. Incubation of EL-4 cells under respiration and glycolysis deficiency conditions resulted in a sharp ATP depletion; pH decrease delayed this depletion.     

Chromatographic methods for large-scale preparation of immunoglobulin G2a monoclonal antibodies Lym-1 and TNT-1 F(ab')2 fragments

Lym-1 and TNT-1 are two murine immunoglobulin G_2a monoclonal antibodies (MAbs) which have been used for clinical trials in cancer patients. This paper describes methods for large-scale preparation of F(ab')₂ fragments from 50 mg to 4 g of MAbs Lym-1 and TNT-1. Digestion of MAbs with pepsin was optimized and performed at pH 3.8, a pepsin/antibody ratio of 1:250, and 3–4 h of incubation at 37°C. The F(ab')₂ fragments were purified by tandem column procedures using fast protein liquid chromatography. Quality control analyses of the products included protein purity, isoelectric point, immunoreactivity, and endotoxin level. The results revealed that the chromatographic procedures are practical, simple, and effective, and can be used to produce gram quantities of clinical-grade F(ab')₂ fragments for the diagnosis of cancer in patients.     

An immune response defect due to low levels of class II cell surface expression. Analysis of antigen presentation and positive selection.

The effects of quantitative differences in class II cell surface expression have been difficult to address in intact animals. This study uses several lines of H-2s/s mice carrying an A beta k transgene that differ significantly in terms of class II cell surface expression. Due to inefficient chain pairing, mice carrying 60 to 65 copies of this transgene express only low levels of A alpha s/A beta k on the cell surface, and cell surface expression of the endogenous A alpha s/A beta s complex (and total Ia) is severely reduced (to 7-15% control levels). The significant decrease in class II cell surface expression in the thymic cortex of these mice did not affect the frequency of peripheral T cells expressing at least 10 distinct TCR V beta chains. However, T cell proliferative responses to the A alpha s/A beta s-restricted peptide MBP 89-101 were abrogated in high copy number A beta k mice. Experiments using bone marrow chimeras demonstrated that both inefficient Ag presentation and failure to positively select appropriate T cells contributed to this lack of response. Inefficient Ag presentation was clearly the dominant defect, and the density of class II cell surface expression required for positive selection appeared to be quite low.     

Interaction of menadione and duroquinone with Q-cycle during DT-diaphorase function]

The interaction of quinones (menadione and duroquinone) with DT-diaphorase and mitochondrial electron transport chain translocators at low (120 mosM) and high (400 mosM) values of the medium tonicity in the quinone concentration range of 6-90 microM was studied. It was shown that with a rise in menadione (K3) concentration the number of electron transport carriers interacting with it increase. At K3 concentration of 6 microM the latter is reduced by DT-diaphorase and fully oxidized via the Q-cycle. At K3 concentration of 15 microM the latter is also reduced by DT-diaphorase via the Q-cycle, but in this case the oxidation is incomplete (about 30% K3H2 is oxidized by the terminal part of the respiratory chain). At 90 microM K3 50% of quinone is reduced by DT-diaphorase and 50% by the respiratory chain NADH dehydrogenase complex enzymes; about 30% of K3H2 is oxidized via the Q-cycle, about 20%--by the terminal part of the respiratory chain and about 50%--by O2 without cytochrome oxidase. Unlike menadione, duroquinone (6-90 microM) is reduced only by DT-diaphorase and is oxidized in all cases by cytochrome oxidase. It was shown that the increase in the mitochondrial matrix volume in low tonicity media decreases the rate of the DT-diaphorase shunt operation.     

Genomic investigation of phenotypic variation in Campylobacter jejuni flagellin.

Western blots of whole-cell sonicates of 10 different clones of a faecal isolate of Campylobacter jejuni 533 detected the expression of flagella antigens of either 59 or 62 kDa. Other antigenic proteins appeared identical both in the parent and all the clones. The mechanism for this phenotypic variation was studied using Southern blotting with a flagellin-specific gene probe and products of a polymerase chain reaction (PCR) using flagellin-gene primers. Restriction-enzyme digestion and Southern blotting did not detect any genomic rearrangements in the flagellin genes of the different phenotypes nor did restriction-enzyme analysis of the PCR products.     

Mechanism of inhibition of peptide chain initiation by amino acid deprivation in perfused rat liver. Regulation involving inhibition of eukaryotic initiation factor 2 alpha phosphatase activity.

In previous studies, initiation of protein synthesis was shown to be inhibited in perfused rat livers deprived of single essential amino acids. In the present study, histidinol, a competitive inhibitor of histidinyl-tRNA synthetase, was used to amplify the effects of histidine deprivation on protein synthesis in perfused liver to facilitate investigation of mechanisms involved in the inhibition of peptide chain initiation. Protein synthesis was reduced to 77% of the control rate in livers deprived of histidine and to 13% of the control rate in livers deprived of histidine and exposed to 2.0 mM histidinol. The inhibition of protein synthesis caused by histidine deprivation alone was accompanied by a 2-fold increase in the number of free ribosomal particles, a 29% decrease in Met-tRNA(i) binding to 43 S preinitiation complexes, and a 31% reduction in activity of eukaryotic initiation factor 2B (eIF-2B). By comparison, histidine deprivation combined with histidinol addition resulted in a 3-fold increase in free ribosomal particles, a 66% decrease in Met-tRNAi binding, and a 78% reduction in eIF-2B activity. The proportion of the alpha-subunit of eukaryotic initiation factor two (eIF-2) in the phosphorylated form increased from 8.9 +/- 0.8% in control livers to 52.4 +/- 5.5% in response to histidinol. The increase in the amount of eIF-2 alpha in the phosphorylated form apparently was not due to an increase in kinase activity, because there was no change in eIF-2 alpha kinase activity in extracts of liver perfused with medium containing histidinol compared to controls. Instead, the increased phosphorylation of eIF-2 alpha was associated with an inhibition of eIF-2 alpha phosphatase activity. Thus, in contrast to other systems that have been examined, the mechanism involved in the increase in the phosphorylation state of eIF-2 alpha appears to involve an inhibition of eIF-2 alpha phosphatase activity rather than activation of an eIF-2 alpha kinase.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

Three saponins from Oxytropis species.

Three flavonoids and three saponins have been isolated from Oxytropis species. Their structures were determined as isorhamnetin-3-O-beta-D-glucoside, rhamnetin-3-O-beta-D-galactoside, apigenin, 3-O-[alpha-L-rhamnopyranosyl (1----2)-beta-D-glucopyranosyl(1----4)-beta-D-glucuronopyranosyl]+ ++soyasapogenol B, 3-O-[beta-D-glucopyranosyl(1----2)-beta-D-glucuronopyranosyl] azukisapogenol and a new saponin 3-O-[beta-D-glucopyranosyl(1----2)-beta-D-glucopyranosyl]-25-O-alpha-L- rhamnopyranosyl-(20S,24S)-3 beta,16 beta, 20,24,25-pentahydroxy-9,19-cycloanostane.     

Development and use of striped bass-specific RFLP probes

We sought to develop nuclear DNA (nDNA) probes which could be used to complement mtDNA and DNA fingerprinting markers in distinguishing striped bass, Morone saxatilis (Walbaum), from discrete spawning systems. Restriction endonuclease-generated single copy, 10–20-kb striped bass nuclear nDNA fragments were cloned into the bacteriophage vector Lambda Dash II and tested in Southern blot analyses for their abilities to reveal population-specific polymorphisms. Three of the I7 nDNA sequences tested exhibited polymorphisms which potentially could be used to delineate striped bass populations. One probe, DSB 22, revealed significant genotypic frequency differences between Gulf of Mexico and Atlantic striped bass and among striped bass representative of some Atlantic systems. These preliminary results suggest that single copy nDNA sequences may provide sufficient polymorphisms to aid in stock identification of species which proved genetically monomorphic using other approaches.