The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Synthesis, structural properties and insulin-enhancing potential of bis(quercetinato)oxovanadium(IV) conjugate

An oxovanadium complex of quercetin (2), exhibits highly potent insulin-enhancing activity in streptozotocin-induced diabetic mice. It also mimics mitogenic potential of insulin as evaluated by [H(3)]thymidine uptake assay making an effective, orally active insulin-enhancing agent for the treatment of both type 1 and type 2 diabetes without any noticeable toxic effects.     

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller)

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.     

Leptin downregulates ethanol-induced secretion of proinflammatory cytokines and growth factor

Inflammatory mediators, including cytokines and growth factors are associated with the pathology of chronic liver diseases. The aim of our present work was to study the effect of exogenous leptin and/or ethanol on the secretion of TNF-alpha, IL-6 and TGF-beta1 both in vivo and in vitro. Forty eight hours after the exposure to ethanol (500 mM) significantly elevated the secretion of TNF-alpha, IL-6 and TGF-beta1 in the cell-free culture supernatant (HepG2 and mouse HCC cell lines), which were decreased on leptin (31.2 nM) treatment. Similarly, leptin administration to ethanol (6.32 g kg(-1) body weight) fed mice for 45 days significantly lowered the concentration of these cytokines in the circulation; however, leptin alone (230 microg kg(-1) body weight i.p. administered every alternate day for the last 15 days) had no such significant effect on cytokine secretion both in vivo and in vitro conditions. We conclude that leptin is involved in the protective mechanisms that allow an organism to cope with the potentially autoaggressive effects of its immune system in alcoholic liver disease.     

Chimeric T helper-B cell peptides induce protective response against Japanese encephalitis virus in mice

Virus neutralizing MAb binding and T helper cell stimulating peptide epitopes from structural and non-structural proteins of Japanese encephalitis virus were delineated. It was observed that priming by T helper peptides potentiated neutralizing antibody response against JE virus. Immunization with chimeric T helper - B cell peptides could thus protect mice from lethal challenge with JE virus.     

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.     

Antibacterial activity against β- lactamase producing Methicillin and Ampicillin-resistants Staphylococcus aureus: fractional Inhibitory Concentration Index (FICI) determination

Background

The present study reports the antibacterial capacity of alkaloid compounds in combination with Methicillin and Ampicillin-resistants bacteria isolated from clinical samples. The resistance of different bacteria strains to the current antibacterial agents, their toxicity and the cost of the treatment have led to the development of natural products against the bacteria resistant infections when applied in combination with conventional antimicrobial drugs.

Method

The antibacterial assays in this study were performed by using inhibition zone diameters, MIC, MBC methods, the time-kill assay and the Fractional Inhibitory Concentration Index (FICI) determination. On the whole, fifteen Gram-positive bacterial strains (MRSA/ARSA) were used. Negative control was prepared using discs impregnated with 10 % DMSO in water and commercially available Methicillin and Ampicillin from Alkom Laboratories LTD were used as positive reference standards for all bacterial strains.

Results

We noticed that the highest activities were founded with the combination of alkaloid compounds and conventional antibiotics against all bacteria strains. Then, results showed that after 7 h exposition there was no viable microorganism in the initial inoculums.

Conclusion

The results of this study showed that alkaloid compounds in combination with conventional antibiotics (Methicillin, Ampicillin) exhibited antimicrobial effects against microorganisms tested. These results validate the ethno-botanical use of Cienfuegosia digitata Cav. (Malvaceae) in Burkina Faso. Moreover, this study demonstrates the potential of this herbaceous as a source of antibacterial agent that could be effectively used for future health care purposes.     

Promise(s) of Mesenchymal Stem Cells as an In Vitro Model System to Depict Pre-Diabetic/Diabetic Milieu in WNIN/GR-Ob Mutant Rats

Background

Development of model systems have helped to a large extent, in bridging gap to understand the mechanism(s) of disease including diabetes. Interestingly, WNIN/GR-Ob rats (Mutants), established at National Centre for Laboratory Animals (NCLAS) of National Institute of Nutrition (NIN), form a suitable model system to study obesity with Type 2 diabetes (T2D) demonstrating several secondary complications (cataract, cardiovascular complications, infertility, nephropathy etc). The present study has been carried out to explore the potent application(s) of multipotent stem cells such as bone marrow mesenchymal stem cells (BM-MSCs), to portray features of pre-diabetic/T2D vis-à-vis featuring obesity, with impaired glucose tolerance (IGT), hyperinsulinemia (HI) and insulin resistance (IR) seen with Mutant rats akin to human situation.

Methodology/Principal Findings

Primary cultures of BM-MSCs (third passage) from Mutants, its lean littermate (Lean) and parental control (Control) were characterized for: proliferation markers, disease memory to mark obesity/T2D/HI/IR which included phased gene expression studies for adipogenic/pancreatic lineages, inflammatory markers and differentiation ability to form mature adipocytes/Insulin-like cellular aggregates (ILCAs). The data showed that BM-MSCs from Mutant demonstrated a state of disease memory, depicted by an upregulated expression of inflammatory markers (IL-6, TNFα); increased stem cell recruitment (Oct-4, Sox-2) and proliferation rates (CD90+/CD29+, PDA, ‘S’ phase of cell cycle by FACS and BrdU incorporation); accelerated preadipocyte induction (Dact-1, PPARγ2) with a quantitative increase in mature adipocyte formation (Leptin); ILCAs, which were non-responsive to high glucose did confer the Obese/T2D memory in Mutants. Further, these observations were in compliance with the anthropometric data.

Conclusions

Given the ease of accessibility and availability of MSCs, the present study form the basis to report for the first time, application of BM-MSCs as a feasible in vitro model system to portray the disease memory of pre-clinical/T2D with IR - a major metabolic disorder of global concern.