Binding of cGMP to GAF domains in amphibian rod photoreceptor cGMP phosphodiesterase (PDE). Identification of GAF domains in PDE alphabeta subunits and distinct domains in the PDE gamma subunit involved in stimulation of cGMP binding to GAF domains

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Palphabeta) and two identical regulatory subunits (Pgamma). Biochemical studies have shown that amphibian Palphabeta has high affinity, cGMP-specific, non-catalytic binding sites and that Pgamma stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In Pgamma-depleted membrane-bound PDE (20-40% Pgamma still present), a single type of cGMP-binding site with a relatively low affinity (K(d) approximately 100 nm) was observed, and addition of Pgamma increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in Pgamma reduced its ability to stimulate cGMP binding. Among these, the site involved in Pgamma phosphorylation by Cdk5 (positions 20-23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in Pgamma inhibition of the cGMP hydrolytic activity of Palphabeta. In addition, the Pgamma concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the Palphabeta heterodimer contains two spatially and functionally distinct types of Pgamma-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the Palphabeta-Pgamma interaction in which Pgamma, by binding to one of the two sites in Palphabeta, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.     

Quantitative profiling of proteins in complex mixtures using liquid chromatography and mass spectrometry

The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples.     

Role of bacterial toxins in pathogenesis of hemolytic-uremic syndrome, caused by enterohemorrhagic Escherichia

The pathogenesis of the hemolytico-uremic syndrome (HUS) caused by enterohemorrhagic E. coli (EHEC) has been studied previously rather completely. HUS is characterized by the signs of microangiopathic hemolytic anemia, thrombocytopenia with renal lesions and manifestations of transient disturbances in the functions of the central nervous system. The adherence of EHEC to enterocytes was found to occur in the terminal section of the ileum and the large intestine. This process is realized with involvement intimin, EHEC outer membrane protein. Shiga-like toxins (SLT) produced by EHEC are the leading factor of their pathogenicity. The mechanism of the toxin translocation through enterocytes is not yet clear, still there is no doubt that SLT penetrates into the systemic blood stream. This is indicated by the results of histopathological studies it possible to find the toxin traces on the membranes of endothelial cells of blood vessels. The study reveals that the cells of the vascular epithelium are highly sensitive to SLT. These cells carry receptors Gb3, also known as CD77, on their membranes. Enterohemolysin, serine protease, causing disturbances in the barrier function of the intestine, can be regarded in the pathogenesis of hemorrhagic colitis which develops as the result of the damaging action of EHEC and the above-mentioned toxins. This leads to the increased level of blood systemic bacterial lipopolysaccharides, which may play, in combination with the action of SLT, an important role in the development of multi-organ pathology in HUS patients.     

Dependence of immunosuppressive action of lipopolysaccharide on degree of Salmonella pathogenicity

Immunosuppressive activity of Salmonella typhimurium extracellular lipopolysaccharide (LPS) was studied. In this study isogenic S. typhimurium strains with different degree of virulence were used. The attenuation of these strains was linked with mutations on their chromosome (altered synthesis of RNA polymerase or gyrase DNA) or their own virulence plasmid (the insertion of transposon Tn-5). To obtain LPS fraction with different molecular weights, the filtrate of bacterial culture was subjected to gel filtration through a column packed with Sephadex G-200. The immunosuppressive action of LPS fractions was determined on the model of delayed-type hypersensitivity to nonbacterial antigen in experiments on BALB/c mice. The study revealed that transposon-mediated mutation on plasmid, accompanied by the attenuation of salmonellae, led to the loss of immunosuppressive activity of the high-molecular heat-sensitive component of LPS; only the second heat-resistant component with medium molecular weight retained its activity. The presence of two chromosomal attenuating mutations (rifr nalr) was accompanied by the loss of immunosuppressive activity in both components of LPS.     

The microflora of ulzerous zone mucosa in patients with duodenal ulcer

Bacteriological study of the biopsies taken from gastric and duodenal mucosa of 10 healthy volunteers and 74 patients with duodenal ulcer, was carried out. In the gastroduodenal zone of healthy subjects microorganisms of 6 genera (Streptococcus, Candida, Staphylococcus, Bacillus, Helicobacter and Lactobacillus) were detected. H. pylori was isolated in 20% of cases only in biopsy specimens taken from the antral section of the stomach of healthy as monoculture or in combination with C. albicans. In patients with duodenal ulcer activation of opportunistic microflora was observed in the periulcerous zone. More often H. pylori occurred in associations with fungi of the genus Candida, streptococci, staphylococci, enterobacteria, Pseudomonas and other microorganisms (of more than 30 genera). Quantitatively the dominating microorganisms (3.8-5.7 lg CFU/g) were H. pylori, fungi of the genus Candida, bacteria of the genera Streptococcus, Peptostreptococcus, Bacteroides, Gemella, Prevotella, Veillonella, Peptococcus, Bacillus, different species of opportunistic enterobacteria, as well as bacteria of the genera Staphylococcus, Micrococcus, Corynebacterium, Neisseria, Pseudomonas, etc. Opportunistic bacteria detected in the ulcerous zone, as a rule, expressed hemolytic, lecithinase, RNAase, caseinolytic, catalase and urease activity. Sonicated filtrates of such cultures produced a cytotoxic effect on cells HEp-2. Ulcer is an infected wound that needs sanitation.     

Expression of a fragment of the env gene, coding for the GP46 surface glycoprotein of the human T-cell leukemia virus, in Escherichia coli cells

Designing of recombinant plasmids pSB2 and pSB3 with the 932 bp HTLV-II env gene inserts encoding the full-length surface membrane glycoprotein gp46 is described. Vectors pGOmpF and pET32a expressing genes cloned under control of the late bacteriophage T7 promoter were used. Western blot analysis of cellular proteins derived from E. coli B834/pSB2 and E. coli B834/pSB3 revealed that 34 kD and 31 kD polypeptides corresponding to the full-length gp46 and its processed form without signal peptide were synthesized under control of these recombinant plasmids. Cytotoxic activity of the recombinant proteins towards bacterial cells was demonstrated. Both polypeptides specifically reacted to sera from humans infected with HTLV-II. High antigenic specificity of P34-HTLV-II proteins makes a promising candidate for diagnostic confirmation tests.     

Model of "standing water" of Baikalian phytoplankton interannual dynamics

A "standing waves" model is presented which is based on the idea that an energy impulse accumulated from the spring bloom outburst of phytoplankton moves along a food chains. When its "echo" comes back, it generates a new bloom outburst, and a next cycle occurs. It is supposed that the resonance of self-supporting oscillations in populations with periodic solar influences led to standing waves, which provide for biosystem stability and cyclic recurrence of phytoplankton bloom. To test the model, an analysis of the interannual dynamics (from 1941 to 1998) of the pelagic phytoplankton in the southern basin of the lake Baikal was carried out. The discovered 11-year cycles with 3 2/3-year cycles within them conform well to the model, which made it possible to explain not only the cyclic recurrence but also a number of other obscure aspects of phytoplankton life in the lake Baikal. The next bloom burst is predicted to take place in 2001.     

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. I. Identification of the kinase and its role in the turnoff of phosphodiesterase in vitro

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H. , Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024-15029), we showed that Pgamma is phosphorylated by a previously unknown kinase (Pgamma kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pgamma loses its ability to interact with GTP/Talpha, but gains a 10-15 times higher ability to inhibit GTP/Talpha-activated PDE than that of nonphosphorylated Pgamma. Thus, we propose that the Pgamma phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Talpha-activated PDE. Here we demonstrate that all known Pgammas preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pgamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pgamma indicates that all known Pgammas contain a P-X-T-P-R sequence and that this sequence is required for the Pgamma phosphorylation by Pgamma kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pgamma kinase activity exactly coelutes with Cdk5 and p35. The Pgamma kinase activity ( approximately 85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pgamma. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pgamma in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pgamma complexed with GTP/Talpha in mature vertebrate retinal photoreceptors.     

Selective inhibition of inducible NO-synthase by nonselective inhibitor

The study has shown that Nw-nitro-L-arginine, a nonselective nitric oxide (NO) inhibitor, in low non-vasoactive doses (10 mg/kg) exerted a protective effect in heat shock as demonstrated by a decrease in the mortality rate and prevention of acute hypotension in rats. The L-NNA in the same dose inhibited the basal NO production but left unaffected a carbachol-activated NO production. The findings suggest a possibility in principle of preferential inhibition of inducible NO-synthase in pathological conditions related to the NO overproduction using non-vasoactive doses of L-NNA the nonselective NO-synthase inhibitor.