The effect of extracellular recombinant human heat shock protein 70 (Hsp70) on protein pattern observed after endotoxin-induced macrophage activation

The protein pattern of mouse macrophage strain (J774) has been investigated using 2D electrophoresis after combined action of bacterial endotoxins (LPS), heat shock treatment (HS) and administration of recombinant human Hsp70. The investigation demonstrated significant protective effect of HS and recombinant Hsp70 treatment applied before LPS introduction. This effect is apparently realized by means of several signal transduction systems. In the course of the investigation, we have identified eight proteins, which exhibited pronounced changes in their synthesis due to combined treatment. The data accumulated may shed light on molecular mechanisms underlying protective antiseptic action of HS and/or recombinant Hsp70 applied before LPS administration.     

Effect of extracellular recombinant human heat shock protein 70 (HSP70) on protein pattern observed after endotoxin-induced macrophage activation

The protein pattern of murine macrophages cell line J774 has been investigated using 2D electrophoresis after combined action of bacterial endotoxins (LPS), heat shock treatment (HS), and administration of recombinant human Hsp70. The investigation demonstrated significant protective effect of HS and recombinant Hsp70 treatment applied before LPS introduction. This effect is apparently realized by means of several signal transduction systems. In the course of the investigation, we identified eight proteins that exhibited pronounced changes in their synthesis due to combined treatment. The data accumulated may shed light on molecular mechanisms underlying protective antiseptic action of HS and/or recombinant Hsp70 applied before LPS administration.     

Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.Key words: MS-HRM, pyrosequencing, digital PCR, heterogeneous DNA methylation, DAPK1, chronic lymphocytic leukemia     

Recombinant human Hsp70 protects against lipoteichoic acid-induced inflammation manifestations at the cellular and organismal levels

It has been previously reported that pretreatment with exogenous heat shock protein 70 (Hsp70) is able to protect cells and animals from the deleterious effects of bacterial lipopolysaccharide (LPS) produced by Gram-negative bacteria. However, the effects of Hsp70 pretreatment on lipoteichoic acid (LTA) challenge resulted from Gram-positive bacteria infection have not been fully elucidated. In this study, we demonstrated that preconditioning with human recombinant Hsp70 ameliorates various manifestations of systematic inflammation, including reactive oxygen species, TNFα, and CD11b/CD18 adhesion receptor expression induction observed in different myeloid cells after LTA addition. Therefore, exogenous Hsp70 may provide a mechanism for controlling excessive inflammatory responses after macrophage activation. Furthermore, in a rat model of LTA-induced sepsis, we demonstrated that prophylactic administration of exogenous human Hsp70 significantly exacerbated numerous homeostatic and hemodynamic disturbances induced by LTA challenge and partially normalized the coagulation system and multiple biochemical blood parameters, including albumin and bilirubin concentrations, which were severely disturbed after LTA injections. Importantly, prophylactic intravenous injection of Hsp70 before LTA challenge significantly reduced mortality rates. Thus, exogenous mammalian Hsp70 may serve as a powerful cellular defense agent against the deleterious effects of bacterial pathogens, such as LTA and LPS. Taken together, our findings reveal novel functions of this protein and establish exogenous Hsp70 as a promising pharmacological agent for the prophylactic treatment of various types of sepsis.     

Regulation of the apoptosis of neutrophils under the action of lipopolysaccharides

Human neutrophils (polymorphonuclear neutrophils, PMN) constitutively undergo apoptosis, and this process is critical for resolution of inflammation. Neutrophil apoptosis can be modulated by a wide variety of agents including lipopolysaccharides (LPS), granulocyte macrophage colony-stimulating factor (GM-CSF), and TNFα. Recent studies of the molecular mechanisms of apoptosis have shown the involvement of members of the Bcl-2 and IAP protein families as well as caspases in the regulation and execution of the neutrophil apoptosis. Cell surface receptors and protein kinases, particularly mitogen-activated protein kinases, also play critical roles in transduction of LPS signals, which results in the apoptosis or extended survival of neutrophils. This review summarizes the current knowledge of the molecular mechanisms and components of LPS-modulated PMN apoptosis.     

Review of the fauna of the bug families Ceratocombidae,Tingidae, Microphysidae,and Reduviidae (Heteroptera) of the Middle and South Urals,with analysis of the zoogeographie structure of the Tingidae fauna

Based on the material of the authors’ collections from the South Ural Reserve (Republic of Bashkortostan), Sverdlovsk and Chelyabinsk provinces, the collections of the Zoological Institute of the Russian Academy of Sciences, the Ilmen State Reserve (Chelyabinsk Province), and the Institute of Plant and Animal Ecology of the Ural Branch of the Russian Academy of Sciences (Yekaterinburg), and also the reliable literature data, an annotated list of the true bug fauna of the Middle and South Urals is compiled for the first time. The list includes representatives of the families Ceratocombidae (1 species), Tingidae (45 species of 14 genera), Microphysidae (1 species), and Reduviidae (2 species of 1 genus). The known fauna of the Middle Urals (Perm Territory and Sverdlovsk Province) includes 24 species of Tingidae and 1 species of Microphysidae; that of the South Urals includes 1 species of Ceratocombidae, 41 species of Tingidae, 1 species of Microphysidae, and 2 species of Reduviidae. Six species are recorded from the Urals for the first time: Ceratocombus (Xylonannus) brevipennis Poppius, 1910 (Ceratocombidae), Acalypta gracilis gracilis (Fieber, 1844), Agramma tropidopterum Flor, I860 (Tingidae), Loricula (Myrmedobia) exilis (Fallén, 1807) (Microphysidae), Empicoris culiciformis (De Geer, 1773), and E. vagabundus (Linnaeus, 1758) (Reduviidae). The families Ceratocombidae and Microphysidae were not previously known from this region. The following numbers of species are recorded for the first time for different regions of the Middle and South Urals: for Perm Territory, 2 species of Tingidae; for Sverdlovsk Province, 11 species of Tingidae and 1 of Microphysidae; for Bashkortostan, 1 species of Ceratocombidae, 13 of Tingidae, 1 ofMicrophysidae, and 2 species of Reduviidae; for Chelyabinsk Province, 3 species of Tingidae. The Tingidae fauna of the Middle and South Urals mostly includes species widespread in the latitudinal and longitudinal directions, including 4 Holarctic (8.9%) and 12 Trans-Palaearctic species (26.7%). Ranges of 24 species (53.3%) mainly lie in the “humid” northern part of the Palaearctic (the humid complex of species). Ranges of 21 species (46.7%) mainly lie in the southern part of the Palaearctic, i.e., the Tethyan Region (the arid complex), the Tingidae fauna of the Middle Urals including only 2 species (8.3%) of that complex. Seven species (17.1%) of Tingidae form the arid element in the fauna of Orenburg Province: Kalama henschi (Puton, 1892), Galeatus vitreus Golub, 1974, G. scrophicus Saunders, 1876, Tingis (Tingis) pusilla (Jakovlev, 1873), T. (Tropidocheila) renovata Golub, 1977, T. (Tr.) maculata (Herrich-Schaeffer, 1838), Dictyla subdola (Horvath, 1905). Ranges of 7 species (15.5% of the whole studied fauna of Tingidae) are limited to the Middle and South Urals in the east and northeast. Ranges of 8 other species (17.8%) extend eastwards, beyond the Urals no farther than the south of Western Siberia and Western Kazakhstan. The mountain territory of the Middle and the South Urals obviously serves as a significant orographic and climatic barrier on the way of eastward expansion of some Western- and Central-Palaearctic species of Tingidae.     

A pilot study of rivastigmine in the treatment of delirium after stroke: A safe alternative

Background

Delirium is a common disorder in the early phase of stroke. Given the presumed cholinergic deficiency in delirium, we tested treatment with the acetylcholinesterase inhibitor rivastigmine.

Methods

This pilot study was performed within an epidemiological study. In 527 consecutive stroke patients presence of delirium was assessed during the first week with the confusion assessment method. Severity was scored with the delirium rating scale (DRS). Sixty-two patients developed a delirium in the acute phase of stroke. Only patients with a severe and persistent delirium (defined as a DRS of 12 or more for more than 24 hours) were enrolled in the present study. In total 26 fulfilled these criteria of whom 17 were treated with orally administered rivastigmine with a total dose between 3 and 12 mg a day. Eight patients could not be treated because of dysphagia and one because of early discharge.

Results

No major side effects were recorded. In 16 patients there was a considerable decrease in severity of delirium. The mean DRS declined from 14.8 on day one to 8.5 after therapy and 5.6 after tapering. The mean duration of delirium was 6.7 days (range; 2–17).

Conclusion

Rivastigmine is safe in stroke patients with delirium even after rapid titration. In the majority of patients the delirium improved after treatment. A randomized controlled trial is needed to establish the usefulness of rivastigmine in delirium after stroke.

Trial registration

Nederlands Trial Register NTR1395

     

Apolipoprotein C3 SstI polymorphism and triglyceride levels in Asian Indians

Background  

A close association between Sst I polymorphism in the 3' untranslated region of the apolipoproteinC3 (APOC3 ) gene and levels of plasma triglycerides (TG) had been reported by different investigators. Hypertriglyceridemia(HTG) is a known risk factor for coronary artery disease (CAD) in the context of Asian Indians. We conducted a study on the relationship between APOC3 SstI polymorphism (S1S1, S1S2 and S2S2 genotypes) and plasma TG levels in a group of 139 male healthy volunteers from Northern India.     

Steroid-induced conformational changes of FITC-labelled sarcoplasmic reticulum Ca2+-ATPase

Interactions between transmembrane and cytoplasmic domains of Ca2+-ATPase from sarcoplasmic reticulum (SR) have been studied. To affect the hydrophobic transmembrane domain, we used four amphiphilic steroids - esters of a dibasic acid and 20-oxypregnene. All four steroids contained cholesterol-like nuclei and differed by the structure of side chains. Steroids with carboxyl groups in the side chains inhibited the rates of ATP hydrolysis and Ca2+ transport, whereas a steroid without the carboxyl group did not appreciably affect Ca2+-ATPase function. Fluorimetric titration of FITC-labelled Ca2+-ATPase in SR vesicles by Nd3+ showed that steroids increased the apparent dissociation constant for Nd3+ bound to the hydrolytic site, the potency order of the steroids being the same as for the sterol-induced inhibition of the hydrolytic activity of Ca2+-ATPase. These results suggest structural changes in the active site. Ca2+ transport was inhibited more efficiently by steroids than the hydrolytic activity of the enzyme. This could be partially due to the increase of the membrane passive permeability induced by steroids, which, in turn, reflected the efficiency of the interaction of the steroids with lipid bilayers. The effects of the steroids were largely dependent on their amphiphilicity (the availability of polar groups in regions A and D), the structure of the side chains, and, possibly, on the distance between the molecular polar groups. We suggest that the inhibition of hydrolytic and transport functions of Ca2+-ATPase in the SR membrane is due to the interaction of the steroids with the transmembrane alpha-helical segments.