Sulfated Derivatives of Arabinogalactan and Their Anticoagulant Activity

Russian Journal of Bioorganic Chemistry - A comparison of IR spectra and molecular weight distribution of arabinogalactan sulfates as sodium and ammonium salts obtained using various sulfating...     

The bidirectional depolymerizer MCAK generates force by disassembling both microtubule ends

During cell division the replicated chromosomes are segregated precisely towards the spindle poles. Although many cellular processes involving motility require ATP-fuelled force generation by motor proteins, most models of the chromosome movement invoke the release of energy stored at strained (owing to GTP hydrolysis) plus ends of microtubules. This energy is converted into chromosome movement through passive couplers, whereas the role of molecular motors is limited to the regulation of microtubule dynamics. Here we report, that the microtubule-depolymerizing activity of MCAK (mitotic centromere-associated kinesin), the founding member of the kinesin-13 family, is accompanied by the generation of significant tension-remarkably, at both microtubule ends. An MCAK-decorated bead strongly attaches to the microtubule side, but readily slides along it in either direction under weak external loads and tightly captures and disassembles both microtubule ends. We show that the depolymerization force increases with the number of interacting MCAK molecules and is ～1?pN per motor. These results provide a simple model for the generation of driving force and the regulation of chromosome segregation by the activity of MCAK at both kinetochores and spindle poles through a 'side-sliding, end-catching' mechanism.     

Transmission of force and displacement within the myosin molecule

Myosin is a repetitive impeller of actin, using its catalysis of ATP hydrolysis to derive repeatedly the required free energy decrements. In each impulsion, changes at the myosin active site are transmitted through a series of structural elements to the myosin propeller (lever arm), almost 5 nm away. While the nature of transmission through most elements is evident, that through the so-called converter is not. To investigate how the converter changes linear displacement into rotation, we tested (one at a time) the effect of two Phe residue mutations (at 721 and 775) in the converter on the overall function of a heavy meromyosin (or subfragment 1) system, after first showing by observing kinetic behaviors that neither mutation affects other elements in the transmission. Using three tests (direct movement of the lever arm, activity in a motility assay with actin filaments, and direct force measurement of lever arm function), we found that these mutations affected only movements of the converter and the lever arm. From interpreting our observations in terms of the structure of the converter, we deduce that the linear-rotational transformation in the converter is mediated by a little machine (two Phe residues linked to a Gly) within a machine.     

Serine and threonine phosphorylation of the low density lipoprotein receptor-related protein by protein kinase Calpha regulates endocytosis and association with adaptor molecules

The low density lipoprotein receptor-related protein (LRP) is a large receptor that participates in endocytosis, signaling pathways, and phagocytosis of necrotic cells. Mechanisms that direct LRP to function in these distinct pathways likely involve its association with distinct cytoplasmic adaptor proteins. We tested the hypothesis that the association of various adaptor proteins with the LRP cytoplasmic domain is modulated by its phosphorylation state. Phosphoamino acid analysis of metabolically labeled LRP revealed that this receptor is phosphorylated at serine, threonine, and tyrosine residues within its cytoplasmic domain, whereas inhibitor studies identified protein kinase Calpha (PKCalpha) as a kinase capable of phosphorylating LRP. Mutational analysis identified critical threonine and serine residues within the LRP cytoplasmic domain that are necessary for phosphorylation mediated by PKCalpha. Mutating these threonine and serine residues to alanines generated a receptor that was not phosphorylated and that was internalized more rapidly than wild-type LRP, revealing that phosphorylation reduces the association of LRP with adaptor molecules of the endocytic machinery. In contrast, serine and threonine phosphorylation was necessary for the interaction of LRP with Shc, an adaptor protein that participates in signaling events. Furthermore, serine and threonine phosphorylation increased the interaction of LRP with other adaptor proteins such as Dab-1 and CED-6/GULP. These results indicate that phosphorylation of LRP by PKCalpha modulates the endocytic and signaling function of LRP by modifying its association with adaptor proteins.     

Salinity-stress-induced changes in the resistance of embryos of the White Sea herringClupea pallasi marisalbi to freshwater

The short-term effects of extremely low salinity (3‰) on the resistance of White Sea herring to freshwater were studied. An increasing resistance of the embryos against a sudden drop in salinity, and a decreasing resistance against gradual changes in salinity (to 0‰) was revealed.     

Effects of maternal corticosterone and stress on behavioral and hormonal indices of formalin pain in male and female offspring of different ages

In previous studies, we showed for the first time that prenatal stress in rats produces long-term alterations of formalin-induced pain behavior that are dependent on age and sex, and we demonstrated an important role of the serotonergic system in mechanisms of prenatal stress (Butkevich, I.P. and Vershinina, E.A., 2001; Butkevich, I.P. and Vershinina, E.A., 2003; Butkevich, I.P., Mikhailenko, V.A., Vershinina, E.A., Khozhai, L.I., Grigorev, I.P., Otellin, V.A., 2005; Butkevich, I.P., Mikhailenko, V.A., Khozhai, L.I., Otellin, V.A., 2006). In the present study, we focus on the influence of the maternal corticosterone milieu and its role in the effects of stress during pregnancy on formalin-induced pain and the corticosterone response to it in male and female offspring of different ages. For this purpose, we used adrenalectomy (AD) in female rats 3-4 weeks before mating (as distinct from AD typically performed at the beginning of pregnancy). Since AD is considered a reliable method to treat hypercortisolism, researches on the effects of long-term AD in dams on the systems responsible for adaptive behavior in offspring are important (such studies are not described in the literature). The results demonstrate that the differences in the corticosterone response to injection of formalin and saline are obvious in 90-day-old (adult) female offspring but masked in 25-day-old ones. AD promoted the corticosterone response to formalin-induced pain but not to injection of saline in prenatally non-stressed female offspring of both ages. Prenatal stress canceled the differences in corticosterone response to injection of formalin and saline in 25-day-old offspring of AD dams and in adult offspring of sham-operated (SH) dams but caused similar differences in adult offspring of AD dams. Sex differences were found in basal corticosterone levels in AD prenatally stressed rats of both age groups, with a higher level in females, and in the corticosterone response to formalin-induced pain in the adult rats of all groups investigated, with higher corticosterone levels in females. In regard to pain behavior, AD induced significant changes in flexing + shaking in prenatally non-stressed adult offspring and canceled the differences in this behavior between non-stressed and stressed 25-day-old offspring. There were sex differences in pain behavior of the adult rats: greater flexing + shaking in AD non-stressed males but in SH non-stressed females; greater licking in prenatally-stressed AD and SH females. These results indicate that the long-term influences of maternal corticosterone on formalin-induced pain and the corticosterone response to it are determined by the sex and age of the offspring and suggest that other mechanisms, including serotonergic ones revealed in our previous studies, are involved in the effects of prenatal stress on inflammatory pain behavior.     

Regulation of Platelet-derived Growth Factor Receptor Function by Integrin-associated Cell Surface Transglutaminase

A functional collaboration between growth factor receptors such as platelet derived growth factor receptor (PDGFR) and integrins is required for effective signal transduction in response to soluble growth factors. However, the mechanisms of synergistic PDGFR/integrin signaling remain poorly understood. Our previous work showed that cell surface tissue transglutaminase (tTG) induces clustering of integrins and amplifies integrin signaling by acting as an integrin binding adhesion co-receptor for fibronectin. Here we report that in fibroblasts tTG enhances PDGFR-integrin association by interacting with PDGFR and bridging the two receptors on the cell surface. The interaction between tTG and PDGFR reduces cellular levels of the receptor by accelerating its turnover. Moreover, the association of PDGFR with tTG causes receptor clustering, increases PDGF binding, promotes adhesion-mediated and growth factor-induced PDGFR activation, and up-regulates downstream signaling. Importantly, tTG is required for efficient PDGF-dependent proliferation and migration of fibroblasts. These results reveal a previously unrecognized role for cell surface tTG in the regulation of the joint PDGFR/integrin signaling and PDGFR-dependent cell responses.Adhesion of cells to the extracellular matrix (ECM)² regulates a wide range of cellular processes, including cell survival, growth, migration, and differentiation. A central paradigm in the field entails both physical association and functional collaboration between integrins and growth factor receptors (GFRs) in the regulation of cell responses to the ECM and soluble growth factors (1). In particular, the engagement of β1 and αvβ3 integrins with ECM ligands transiently activates platelet-derived growth factor (PDGF) receptor-tyrosine kinase even in the absence of its soluble ligands and promotes and sustains growth factor-initiated signaling by PDGFR (2). Despite a significance of this synergistic signaling, the molecular mechanisms underlying the cross-talk between the two receptor systems remain unknown. A direct or indirect association between these two types of signaling receptors may be enhanced by their co-sequestering in cholesterol-enriched membrane microdomains (3). Because integrins and receptor-tyrosine kinases share many downstream signaling targets, integrin-ECM interaction may also increase availability of signal relay enzymes and adapter proteins to receptor-tyrosine kinases by promoting their recruitment from cytosol to the plasma membrane (4).PDGF is a major survival factor, mitogen, and motogen for mesenchymal cells (5). This ligand-receptor pair is implicated in tumor-associated processes, including autocrine growth stimulation of tumor cells, tumor angiogenesis, and regulation of stromal fibroblasts (6). Atherosclerosis in the vessel wall and restenosis after angioplasty also involve hyperactivation of the PDGF-PDGFR signaling axis in vascular smooth muscle cells (7). Likewise, skin wound healing and liver, lung, and kidney fibrosis depend on PDGF-mediated signaling and cell responses (8). Importantly, ECM composition and cell-matrix interactions modulate cell responsiveness to PDGF (9).Upon binding a dimeric PDGF molecule, PDGFR undergoes dimerization and autophosphorylation of tyrosine residues in trans because of the juxtaposition of cytoplasmic tails of the receptor. Phosphorylation of the conserved tyrosine residue in the kinase domain (Tyr-849 of PDGFRα and Tyr-857 of PDGFRβ) increases catalytic activity of the kinases, whereas autophosphorylation of tyrosine residues outside the kinase domain creates docking sites for signal transduction proteins containing Src homology 2 domains. The latter include various enzymes such as phosphatidylinositol 3-kinase, phospholipase Cγ, the Src family tyrosine kinases, the tyrosine phosphatase Shp-2, and the GTPase activating protein for Ras, RasGAP. Other PDGFR binding partners including Grb2, Grb7, Nck, Shc, and Crk lacking enzymatic activity but serve adapter functions in the downstream signaling pathways (10).Previous studies revealed a transient PDGF-independent tyrosine phosphorylation of PDGFRβ in human fibroblasts during adhesion on fibronectin or collagen type I, whereas similar PDGFRβ activation response was reproduced by application of external strain to quiescent cells (2). Clustering of integrins with fibronectin-coated beads was shown to stimulate PDGFR phosphorylation in fibroblasts (11). Furthermore, fibronectin was found to promote PDGF-mediated signaling in fibroblasts by increasing association of phosphatase Shp-2 with PDGFR and limiting the time that the negative signaling regulator, RasGAP, interacts with the receptor (4). Whereas these results implicate cell-ECM interactions and integrin function in the regulation of PDGFR activity, many details of this functional cross-talk remain unknown.Tissue transglutaminase (tTG) is a multifunctional protein that possesses Ca²⁺-dependent transamidating and GTPase activities (12). On the surface of various cells, all the tTG forms stable non-covalent complexes with β1 and β3 integrins and functionally collaborates with these receptors by acting as a co-receptor for fibronectin (13). This adhesive function of tTG is involved in the assembly of fibronectin matrices and cell migration on fibronectin (14–16). tTG broadly affects integrin signaling by promoting their clustering and increasing activation of focal adhesion kinase and RhoA (13, 17). Thus, we set to examine whether signaling mediated by GFRs, which depends on the integrin function, is altered by tTG.Here we present a novel mechanistic insight into the cross-talk between integrin and PDGFR signaling pathways. We provide evidence that tTG interacts with PDGFR on the cell surface and mediates its physical association with integrins. In turn, the formation of stable integrin-tTG-PDGFR ternary complexes promotes PDGFR activation and downstream signaling, regulates the receptor turnover, and amplifies PDGFR-mediated cellular responses. These studies reveal a novel function of tTG in coupling the adhesion-mediated and growth factor-dependent signaling pathways. They suggest that this tTG activity might be involved in pro-inflammatory function of this protein in normal wound healing and tissue fibrosis (18), vascular remodeling (19), and tumor metastasis (20).     

Interrelation of lymphocyte subpopulations in peripheral blood under cervical papillomavirus infection

T(CD3⁺)-, B(CD19⁺)-lymphocytes and their subsets (CD4⁺, CD8⁺, CD3⁺, DR⁺, CD3⁻ DR⁺) in peripheral blood of patients with CIN I, CIN II, CIN III and cancerin situ associated with HPV infection were evaluated. In peripheral blood of women with CIN II, CIN III and cancerin situ the number of T-lymphocytes which expressed CD3⁺ DR⁺ antigen decreased. In patients with CIN I, CIN III and cancerin situ the level of the CD4⁺ cells decreased; the level of the CD8⁺ cells increased. These patients had a lower CD4/CD8 ratio, the number of B cells being standard. The results may have important implications in the prognosis and immunotherapy of HPV infection. Presented at theInternational Conference on Recent Problems in Microbiology and Immunology, Košice (Slovakia), 13–15 October 1999.