Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase.

Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7-REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7-REP complex. For this interaction we determined a Kd value of about 120 nM. Association of RabGGTase with the Rab7-REP complex occurs with a rate constant of approximately 108 M-1 x s-1. We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7-REP-1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C-terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C-terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7-REP-1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C-terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then 'scans' the flexible C-terminus to position the proximal cysteines into the active site.     

Expression of mammalian geranylgeranyltransferase type-II in Escherichia coli and its application for in vitro prenylation of Rab proteins

Mammalian geranylgeranyltransferase type II (GGTase-II) is a 100-kDa heterodimer that catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab GTPases. This modification is essential for the biological activity of Rab proteins. Geranylgeranylation can be performed in vitro using recombinant GGTase-II but so far large-scale production of the enzyme was challenging. We report here the design of a two plasmid expression system that will produce GGTase-II at levels as high as 15 mg/L in Escherichia coli. The protein was produced as a heterodimer with the alpha subunit bearing a cleavable tandem 6His-glutathione S-transferase (GST) tag that was used for two-step purification of the enzyme. Purified enzyme was functionally active as determined by in vitro prenylation and phosphoisoprenoid binding assay. Furthermore, the GST-tagged GGTase-II was used for preparative in vitro prenylation of the Rab7:REP-1 complex. Using this procedure, 10 mg of doubly prenylated Rab7:REP-1 complex were obtained.     

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.     

Dependence of plasticity of set to emotional facial expression on working memory load

The effect of increasing working memory load (by introduction of an additional cognitive task into the experimental context) on the recognition of emotional facial expression in a visual set paradigm was studied in healthy adult subjects. The link between plasticity of the cognitive set to emotional facial expression and the working memory was revealed. It was found that an increase in the working memory load was associated with a delay of set shifting in a modified situation. The set became more rigid which appeared as increasing number of trials with erroneous assessments of facial expression in the form of contrast or assimilative illusions. The significance of inner states and priming for the insight into psychophysiological mechanisms of erroneous assessments under conditions of the working memory loading is discussed in terms of the concept of the integration of bottom-up and top-down streams.     

Stages in the improvement of drug therapy at a polyclinic]

Within 1968-1997 the authors studied the steps of introduction of the achievements of the medical science, technology and pharmacology to therapy of exacerbations and complications of peptic ulcer (PU). The scientific and practical value of endoscopic, histological, biochemical and bacteriological examinations in the improvement of the methods of pharmacotherapy of exacerbations and complications of PU was shown. Three phases of the PU development were indicated by the clinical signs and results of esophagogastroduodenoscopy, target biopsy and histological examinations. These data and available scientific achievements were assumed as a basis for the design of optimal drug combinations and their introduction to the medical practice. The use of such combinations made it possible to prevent relapses and life-threatening complications of the disease in the overwhelming majority of the patients. The best results of the pharmacotherapy were recorded in the years (1988-1997) when the drug combinations began to be used. The combinations provided eradication of Helicobacter pylori in the gastroduodenal mucosa and it was proved that in all the patients with PU and the relapsing lesions in the duodenum and in the overwhelming majority of the patients with gastric ulcer the disease developed at the background of chronic active gastroduodenitis associated with H.pylori. The success of the pharmacotherapy in the patients with PU was due to the use of the rational combinations of antibacterial and antisecretory agents.     

Paralytic poliomyelitis in Russian Federation in 1998-2005

From 1998 through 2005 3,294 cases of acute flaccid paralysis (AFP) including 93 cases with clinical picture of poliomyelitis were registered in Russian Federation. From the latter cases 91 were classified as vaccine-associated paralytic poliomyelitis (VAPP): 66 were VAPP cases in oral poliomyelitis vaccine (OPV) recipients and 25--VAPP cases in contacts. VAPP rate was 1 case per 1.6 million of distributed OPV doses, 1 case per 2.2 million doses for OPV recipients, and 1 case per 186,000 doses for recipients of 1st OPV dose in children aged < 1 year. Majority of VAPP cases in recipients occurred after 1st dose (89.4%) and in contacts--in non-vaccinated children (76%). Mean interval between OPV administration and onset of VAPP in recipients was 21 days. Children aged < 1 year were predominant among VAPP cases (92.4% among recipient VAPP cases, and 80% among contact VAPP cases). Majority of the patients had unfavorable health status including defects of immunity. Most of poliovirus strains isolated from VAPP cases belonged to type 3 (52.9%) whereas to type 2 and 1--29.8% and 17.4% of strains respectively. All VAPP cases were associated with vaccine-derived polioviruses. A highly diverged poliovirus type 1 (2.65% of nucleotide substitutions in VP1 region) was isolated from patient with contact VAPP. Formation of poliovirus-neutralizing serum antibodies in children with VAPP including persons with immunodeficiency reflects the ability of the organism to produce specific antiviral immune response.     

Role of working memory in forming the cognitive visual set

It was found in healthy adult subjects (n = 90) that under conditions of complication of cognitive tasks (increasing number of relevant stimuli), the stability (rigidity) of the unconscious cognitive sets markedly rises. These changes in both verbal and nonverbal sets depend on the working memory loading. The revealed dependence of the motor reaction time to the probe stimulus on the sequence of the set stimuli in a context and on the set stage suggests that unconscious cognitive sets can regulate selective attention.     

Evaluation of the results of arterial reconstructions supplemented by the creation of an arteriovenous anastomosis

Arterial reconstructions supplemented with arteriovenous anastomosis were studied experimentally. The ischemic changes in the operated dog limb were evaluated by various methods, including electronic diagnostic technique. When the diameter of arteriovenous anastomosis was equal to 40-60 per cent of arterial graft diameter, the best postoperative results were observed.