Role of CLASP2 in microtubule stabilization and the regulation of persistent motility

In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.     

Effect of homocysteine on properties of neutrophils activated <Emphasis Type="Italic">in vivo</Emphasis>

We have found that neutrophils begin to express NMDA receptors on their membranes after in vivo activation. These receptors are the target for action of homocysteine (HC). After incubation of activated neutrophils with HC, the degranulation process is stimulated and generation of reactive oxygen species is increased. We conclude that expression of NMDA receptors on neutrophil membrane makes neutrophils sensitive to HC. Thus, hyperhomocysteinemia may induce additional stimulation of immune competent cells.     

Diagnostic screening of the human myocardium during the bicycle exercise test

The diagnostic and prognostic potentialities of Kardio-Vizor-06 (KV) in early detection of deviations in the myocardial electrophysiological parameters were assessed using the standard functional bicycle stress exercise test. Ten apparently healthy subjects from different age groups were examined. The data obtained with KV (baseline, loading, and in the recovery period) were compared with the results of the standard ECG (12 leads) and blood pressure (BP) methods. Data agreement was observed in 90% of cases; KV underestimated the test tolerance in 10% of cases. Detailed examination of ECG verified the presence of incipient alterations in the myocardium. Thus, unlike other methods, this technology provides unique information and predicts transient functional disorders that are precursors of pathology. The high noise resistance of the automated analysis of the dispersion of the ECG signal makes the device applicable directly during functional loading tests.     

Use of basidiomycetes in industrial waste processing and utilization technologies: fundamental and applied aspects (review)

This review provides an analysis of recent data on the mechanisms of degradation of lignocellulosic materials and xenobiotics by basidiomycetes. Special attention is given to the analysis of the current state of research of ligninolytic enzymes and their involvement in the degradation ofxenobiotics. Data on the practical use of basidiomycetes for bioconversion of industrial wastes are systematized. The most promising areas of bioconversion technologies are considered, such as contaminated water purification (including wastewater), cleanup of soils contaminated with heavy metals and xenobiotics, and degradation of difficult-to-degrade substrates (lignin and lignocellulose wastes, low-energy coal, and synthetic polymers).     

Proteasomes on thyroid tissue allotransplantation under induction of donor-specific tolerance in rats

Proteasomes in the liver of August rats (RT1^c) were investigated 30 days after allotransplantation of Wistar rat (RT1^u) thyroid tissue under renal capsule with/without induction of donor-specific tolerance by donor splenocyte intraportal administration. The levels of total proteasome pool, immune proteasomes containing subunits LMP2 and/or LMP7, and proteasome regulators 19S and 11S were defined. Intact and sham-operated August rats were used as control groups. The level of all immune proteasome forms and 11S regulator increased while the level of the total proteasome pool and 19S regulator decreased in the liver of experimental animals compared to the control groups, which indicated changes of liver functional state after transplantation. The 19S/11S ratio increased in the liver of nontolerant rats compared to tolerant animals. In the liver of tolerant rats with accepted grafts, the number of mononuclear cells expressing the immune subunit LMP2 greatly increased in comparison with control and nontolerant animals. Study of accepted grafts showed an increase in the ratio of LMP2/LMP7 immune subunits and 19S/11S regulators in them, compared to the tissue replacing the rejected grafts. Immune proteasomes were almost completely absent from the control intact thyroid tissue, while 19S/11S ratio was maximal in it. Thus, the development of the immune reaction or its suppression are accompanied by a change in the balance between different proteasome forms. Immune subunit LMP7 and 11S regulator are associated with the response against donor tissue. On the contrary, immune subunit LMP2 and 19S regulator are likely to be important for the development of immune tolerance and surviving tissue functioning. Immunofluorescence assay revealed a low content of the immune proteasomes in the follicle cells. Probably, formation of antigens for the major histocompatibility complex class I molecules was impaired by the low content of immune proteasomes, which led to immunological tolerance of hormone-producing follicle cells.     

Native structure of rat liver immune proteasomes

Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αβ activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of “rotated” ubiquitinated proteins.     

Effect of vitamin A on the adhesive interaction of cells in epithelial tissue

Both the increasing and decreasing contents of vitamin A in rats induced a lowering of cell adhesion in the skin multilayer epithelium, and that of their packing density. However, the same stimulus did not alter cell adhesion in the small intestinal monolayer epithelium. The data obtained show that vitamin A may serve as a factor modulating intercellular adhesive interactions with tissue specificity.     

Expression of DNA-Encoded Antidote to Organophosphorus Toxins in the Methylotrophic Yeast <Emphasis Type="Italic">Pichia Pastoris</Emphasis>

A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia рastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day).     

Fluid transport in isolated rabbit eye lens

Fluid transport in the isolated rabbit eye lens was assayed gravimetrically, and was found to be, at least partly, an active process involving Na,K-ATPase. Thereby the pressure inside the lens proved to be elevated by 6 mm Hg. The energy required for this process was estimated at (1.5–6)·10^?2 J. The movement of fluid in vivo (monitored with fluorescein) proceeds from the anterior to the posterior surface and out into the vitreous body.     

Stretch affects phenotype and proliferation of vascular smooth muscle cells

The focus of this study was to identify the molecular basis for the hypersensitive response of glycogen phosphorylase activation to epinephrine stimulation in alloxan diabetic-derived cardiomyocytes. Cyclic AMP levels were found not to be significantly different between normal and diabetic-derived cells while cGMP concentrations were found consistently to be significantly lower in diabetic-derived cells than in normal cells. Treatment with cyclic GMP analogues did not affect phosphorylase activation by epinephrine in normal cardiomyocytes whereas, IBMX, a nonselective phosphodiesterase inhibitor, had a significant effect on basal and agonist-stimulated phosphorylase activity in both normal and diabetic-derived cardiomyocytes. Differences in the time course for the rate of decay of phosphorylasea from agonist-stimulated to basal levels were observed between normal and diabetic cells. After 3 h in primary culture, phosphorylasea activity returned to basal levels more quickly in normal than in diabetic-derived cells while after 24 h in culture, the time for phosphorylasea decay was not significantly different between normal and diabetic myocytes and was longer than the 3 h response. After 3 h in primary culture, no significant difference in phosphorylase kinase activity was observed between normal and diabetic-derived cells exposed to epinephrine whereas, after 24 h in culture, phosphorylase kinase activity was significantly decreased in diabetic cells under basal and agonist-stimulated conditions. These data collectively suggest that the hypersensitive response of glycogen phosphorylase to epinephrine stimulation in diabetic-derived cardiomyocytes is not due to a defect present at the level of phosphorylase kinase but may, in part, result from an alteration in cardiac phosphodiesterase activity resulting from diminished intracellular cyclic GMP concentrations.