S-Nitrosoglutathione in Rat Cerebellum: Identification and Quantification by Liquid Chromatography-Mass Spectrometry

Abstract: Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S -nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S -nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [³⁵S]cysteine-prelabeled slices suggested that S -nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography-mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using ¹⁵N-labeled GSNO ([¹⁵N]-GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 ± 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.     

Human immunodeficiency virus drug therapy and virus load.

Analysis of the short-term dynamics of human immunodeficiency virus (HIV) type 1 infection in response to drug therapy has elucidated crucial kinetic properties of viral dynamics in vivo (D. D. Ho et al., Nature 373:123-126, 1995; A. S. Perelson et al., Science 271:1582-1586, 1996; X. Wei et al., Nature 373:117-122, 1995). Here we investigated long-term changes in virus load in patients treated with a combination of lamivudine and zidovudine to identify principal factors responsible for the observed 10- to 100-fold sustained suppression of virus load in vivo. Interestingly, most standard accounts of virus dynamics cannot explain a large sustained reduction without shifting the virus very close to extinction. The effect can be explained by taking into consideration either (i) the immune response against HIV, (ii) the killing of uninfected CD4 cells, or (iii) the differential efficacies of the drugs in different cell populations.     

Automata,repeated games and noise

We consider two-state automata playing repeatedly the Prisoner's Dilemma (or any other 2 × 2-game). The 16 × 16-payoff matrix is computed for the limiting case of a vanishingly small noise term affecting the interaction. Some results concerning the evolution of populations of automata under the action of selection are obtained. The special role of win-stay, lose-shift-strategies is examined.     

An analysis of the relative activities of a number of promoter constructs from genes which are expressed during late pollen development as determined by particle bombardment

Summary The promoters of a tobacco actin gene, a tobacco pectate lyase, a tobacco and maize polygalacturonase and aBrassica S-locus related gene have been fused to the-glucuronidase reporter gene and their activities determined by biolistic transient assay in tobacco pollen. In stably transformed tobacco all the transgenes with the exception of Cauliflower Mosaic Virus-35S--glucuronidase appear to express efficiently in maturing pollen. Transient assay analysis showed that the tobacco pectate lyase and the polygalacturonase constructs were 8x more active than the tobacco actin construct, and that the tobacco polygalacturonase construct was some 33x more active than the maize polygalacturonase construct. Constructional manipulations that altered the lengths of the 5-untranslated leaders including one which resulted in the removal of a 490 bp leader intron had little effect on the observed level of expression. However, the alteration of the context of the ATG from A/TnnATGG to CnnATGT resulting in a 70% reduction in the observed levels of activity, was obtained with the pectate lyase and polygalacturonase promoters. An identical reductional was also observed in transgenic plant populations transformed with the polygalacturonase transgenes.Abbreviations GUS -glucuronidase - LUC luciferase - NosTer nopaline synthase terminator - CaMV Cauliflower Mosaic Virus - UTL untranslated leader - PCR polymerase chain reaction - PG polygalacturonase - Npg tobacco polygalacturonase - Pl pectate lyase - Ac actin     

Progress in fold recognition

The prediction experiment reveals that fold recognition has become a powerful tool in structural biology. We applied our fold recognition technique to 13 target sequences. In two cases, replication terminating protein and prosequence of subtilisin, the predicted structures are very similar to the experimentally determined folds. For the first time, in a public blind test, the unknown structures of proteins have been predicted ahead of experiment to an accuracy approaching molecular detail. In two other cases the approximate folds have been predicted correctly. According to the assessors there were 12 recognizable folds among the target proteins. In our postprediction analysis we find that in 7 cases our fold recognition technique is successful. In several of the remaining cases the predicted folds have interesting features in common with the experimental results. We present our procedure, discuss the results, and comment on several fundamental and technical problems encountered in fold recognition. © 1995 Wiley-Liss, Inc.     

HPLC shadowing: Artifacts in peptide characterization monitored by RIA

High performance liquid chromatography (HPLC), a valuable tool for characterization of peptides, is frequently used in combination with sensitive radioimmunoassays (RIA). The shadow phenomenon, representing carry-over of the peptide from previous application of the standard, can appear to result in the presence of endogenous peptide in the test sample when none is actually there. With delta sleep-inducing peptide (DSIP), we found the shadowing to be as high as 10%, although it was only 1% with ¹²⁵I-Tyr-DSIP. Thus, when HPLC-RIA systems are used for identification of peptides, caution must be used to avoid false positive results.     

Homology between the ribosomal DNA of Escherichia coli and mitochondrial DNA preparations of maize is principally to sequences other than mitochondrial rRNA genes

E. coli ribosomal DNA has been used to probe maize mitochondrial DNA. It hybridizes primarily with chloroplast ribosomal DNA sequences and with fungal and bacterial sequences which may contaminate the mtDNA preparations. It also hybridizes to the chloroplast 16S ribosomal RNA gene sequence present in the mitochondrial genome (1) as well as to the mitochondrial 18S ribosomal RNA gene sequence. Weak sequence homology was detected between E. coli rDNA and the mitochondrial 26S ribosomal RNA gene.