Tissue factor in the antiphospholipid syndrome.

Antiphospholipid (aPL) antibodies are clinically important acquired risk factors for thrombosis and pregnancy loss and are thought to have a direct prothrombotic effect in vivo. Data suggest that a major mechanism by which aPL antibodies contribute to thrombophilia is the upregulation of tissue factor (TF) (CD142) on blood cells and vascular endothelium. TF is the physiological trigger of normal blood coagulation and thrombosis in many hypercoagulable conditions. This article reviews the physiology of TF, the molecular regulation of TF expression and the effects of aPL antibodies on intravascular TF regulation and expression. Inhibition of TF and the pathways by which aPL antibodies induce TF expression are potentially attractive therapeutic targets in the antiphospholipid syndrome.

[Accumulation of heat-shock proteins 70 in insect fat bodies as evidence of stress in Microsporidia-infected insects]. / Nakoplenie belka teplovogo shoka 70 v zhirovom tele nasekomykh, zarazhennykh mikrosporidiiami, kak svidetel'stvo stressa nasekomykh.

At present a concept prevails that pathological alterations in insect hosts infected with microsporidia, and those associated with hormone imbalance may be explained by the production of juvenile hormone-like (JH) substances by microsporidia. According to another view point, this pathology is a consequence of the host response. We suggested that the microsporidian infection can provoke a stress reaction in insects, which may cause JH secretion by these insects. To confirm this hypothesis, we have analysed major stress protein Hsp70 levels in the infected insects. Using affinity chromatography on ATP-agarose and immunoblotting, we have shown that Hsp70 was accumulated in infected crickets, and that it was the host protein. The consequence of events accompanying the infection in the insects is discussed in relation to the response of hormonal system of the host organism.

A human BRCA2 complex containing a structural DNA binding component influences cell cycle progression.

Germline mutations of the human BRCA2 gene confer susceptibility to breast cancer. Although the function of the BRCA2 protein remains to be determined, murine cells homozygous for BRCA2 inactivation display chromosomal aberrations. We have isolated a 2 MDa BRCA2-containing complex and identified a structural DNA binding component, designated as BRCA2-Associated Factor 35 (BRAF35). BRAF35 contains a nonspecific DNA binding HMG domain and a kinesin-like coiled coil domain. Similar to BRCA2, BRAF35 mRNA expression levels in mouse embryos are highest in proliferating tissues with high mitotic index. Strikingly, nuclear staining revealed a close association of BRAF35/BRCA2 complex with condensed chromatin coincident with histone H3 phosphorylation. Importantly, antibody microinjection experiments suggest a role for BRCA2/BRAF35 complex in modulation of cell cycle progression.

[Role of heat-shock proteins in cardioprotection of patients operated on for ischemic heart disease]. / Rol' belka teplovogo shoka v kardioprotektsii u bol'nykh, operirovannykh po povodu ishemicheskoi bolezni serdtsa.

Major stress protein HSP72 is known to participate in protecting cells and organisms against harmful factors including ischemia, trauma etc. Under study was the level of HSP72 in the myocardium of 32 patients with coronary disease operated in Military-medical academy. HSP72 was detected in probes of the right atria before and after pre-cardiopulmonary bypass in all cases induction of HSP72 was observed in 40% of patients, and directly correlated with the time of cardiopulmonary bypass and standing of the disease. The cardioprotective effect of the elevated pre-operational level of HSP72 was shown to be proportionate to the lower activity of cardiospecific enzymes, creatine phosphokinase (CK-MB). It is suggested that HSP72 is involved in the mechanism of cardioprotection during cardiopulmonary bypass.

Effects of exogenous stress protein 70 on the functional properties of human promonocytes through binding to cell surface and internalization.

The presence of antibodies against the major stress protein, Hsp70, in patients with autoimmune diseases led us to hypothesize that Hsp70 may occur extracellularly, and could exert chaperoning and regulatory effects on various cells. We examined the action of pure Hsp/Hsc70 on the main physiological functions of human promonocytic U-937 cells. The protein was isolated from calf muscle and was shown to be a mixture of inducible Hsp70 (60%) and constitutive Hsc70 (40%) isoforms. It was observed that the addition of the protein up-regulated two major monocyte/macrophage differentiation markers, CD11c and CD23, by 20-35%, while it had no effect on CD14. The experiments performed to investigate the influence of Hsp/Hsc70 on the reaction of U-937 cells to differentiation stimuli demonstrated that the addition of the protein prior to PMA was able to inhibit binding of proper transcription factors to double-symmetry and cAMP-response elements of the c-fos early response gene promoter. Administration of exogenous Hsp/Hsc70 prior to treatment with the tumor necrosis factor-alpha significantly lowered the number of apoptotic and necrotic cells. In no case did the control protein, ovalbumin, taken in the same concentration give a comparable effect on U-937 cells. Since the Hsp/Hsc70 effects occurred within the first hour of co-incubation, and therefore they might be explained by its interaction with the cell surface, we assayed binding of the biotinylated protein to U-937 cells by immunoenzyme assay, flow cytometry and indirect immunofluorescence. Using these three techniques we were able to detect Hsp/Hsc70 bound to cells after a 20 min incubation. According to flow cytometry data, at this time 32% of cells were positively stained with streptavidin-FITC. Immunofluorescence studies demonstrated Hsp/Hsc70 bound to the cell surface after a 20 min incubation followed by induction of patch and cap-like structures. One hour later, the majority of the protein had been internalized by U-937 cells.

[Effect of an extracellular heat shock protein on chromosome variability in Indian muntjak cultured skin fibroblasts]. / Vliianie vnekletochnogo belka teplovogo shoka na khromosomnuiu izmenchivost' v kletochnoi kul'ture fibroblastov kozhi Indiiskogo muntzhaka.

It is known that the essential function of extracellular HSP70 (e-HSP70) to protect cell processes, indirectly associated with the genetic structures. A direct influence of e-HSP70 on chromosomal stability has not been studied. This explains actuality of the suggested investigation. A study was made of the influence of e-HSP70 on chromosomal variability at different phases of the first mitotic cycle in both intact and ciprofloxacin (CF) treated cells of the Indian muntjac skin fibroblasts. E-HSP70 (10 mg/ml) exerts no influence on the level of chromosomal aberrations, typical of the control. After a joint action of e-HSP70 and CF (50 mg/ml) no influence was also exerted on the antibiotic induced genotoxicity effect. CF and e-HSP70 (50 and 100 mg/ml, resp.) acting apart on intact cells during 6 and 24 h induce a significant increase of chromosomal aberrations compared to the control, primarily at the expense of chromatid or chromosomal breaks (depending on the duration of respective effect). CF and e-HSP70 acting apart on intact cells during 6 h with the following cultivation for 18 h in the fresh medium prior to fixation also induce a significant increase of chromosomal aberrations compared to the control, primarily at the expense of both breaks and dicentrics (telomeric associations). The joint action of CF and e-HSP70 on cultivated cells during 6 and 24 h and when CF and e-HSP70 were added respectively on 24 and 6 h prior to fixation, a significant decrease in chromosomal aberrations compared to the control level was induced. A simultaneous addition of CF and e-HSP70 in 6 h with the following cultivation for 18 h in fresh medium prior to fixation exerted no influence on the degree of genotoxicity effect, typical of the separate action of these agents. However a significant decrease in the number of dicentrics occurred. Apparently, e-HSP70 has a protective effect on chromosomal stability mainly at phase 62 of the mitotic cycle. The denaturated e-HSP70 (50, 100 mg/ml) has a genotoxicity effect similar to that of the infact e-HSP70 under above conditions. The joint action of CF and denaturated e-HSP70 (50 mg/ml) during 24 h exerts no influence on the degree of genotoxicity effect, induced by the separate action of these agents or leads to an increase in the number of dicentrics, acting separately or jointly compared to the control. The denaturated e-HSP70 (100 mg/ml) acting jointly with CF for 24 h, increases the degree of genotoxicity effects, induced by separate actions of the agents. Lipopolysacharide (50 mg/ml) exerts no influence on the number of chromosomal aberrations in the control. The sensitivity of individual chromosomes and their regions to agents inducing chromosomal instability is different. The preferential involvement of some chromosomes in dicentric formation was observed. A possible role of dicentrics in adaptation of cells belonging to "markerless" lines to infavourable factors of the environment, and possible mechanisms of protecting effect exerted by e-HSP70 on chromosomes are discussed.

Exogenous heat shock protein hsp70 activates potassium channels in U937 cells.

With the use of patch clamp technique, the effect of exogenous heat shock protein hsp70 on ion channel properties in the plasma membrane of human promonocyte U937 cells has been examined. Cell-attached experiments showed that the addition of 30-100 micrograms/ml hsp70 to the pipette solution resulted in an activation of outward currents through potassium-selective channels of 9 pS unitary conductance. The activity of K(+)-selective channels did not depend on membrane voltage and could be controlled by the intracellular free calcium concentration as revealed in inside-out recordings. K+ channels with similar conductance and kinetic behaviour were found in normal cell-attached patches very rarely. Outside-out experiments showed that the addition of hsp70 to the external solution induced a channel-like stepwise increase of inward current which may provide cation entry from the extracellular medium. The interaction of extracellular hsp70 with the membrane surface of the native cell and of the excised fragment was found to be different. The results suggest that hsp70-induced activation of Ca-dependent K channels in monocyte-macrophage cells may be due to a local increase of free Ca2+ concentration just near the inner membrane side.

The characterization and use of different antibodies against the hsp70 major heat shock protein family for the development of an immunoassay.

The hsp70 family of major stress proteins is composed of several different members exhibiting similar structural and functional properties. In order to obtain an antiserum with wide epitope reactivity, rabbits were immunized with a mixture of native and denatured hsp70 purified from bovine muscle by ATP-affinity chromatography. Screening for antibody specificity was performed by a "sandwich" enzyme linked immunosorbent assay (ELISA). Immunoprecipitation and immunoblotting analyses demonstrated that the polyclonal antiserum obtained by us and a monoclonal antibody raised against a different preparation of antigen recognized the same determinant on the native hsp70 molecule (inducible form). With a different specificity the polyclonal antiserum recognized only the denatured monomers of the other members of the hsp70 family. These results are discussed in relation to the immunological features of the hsp70 molecule and to the development of an immunoassay for the detection of hsp70 in cell and tissue extracts.