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.     

Evaluating the effectiveness of therapies for urinary incontinence

Subjective criteria, behavioral/emotional factors, and quality-of-life assessments are necessary components in evaluating incontinence therapies. Behavioral regimens often yield a response that lasts beyond the end of therapy and, when combined with pharmacologic therapy, are likely to extend duration of response. In addition to objective efficacy criteria, a number of instruments evaluate quality-of-life issues. Shortened forms of these will likely overcome limitations on patient compliance and completion. New instruments continue to be developed, but until one or more are universally accepted, variations in reported outcomes can be expected.     

Interventions for detrusor overactivity: the case for multimodal therapy

Viable therapeutic alternatives for the management of overactive bladder (OAB) have recently evolved that provide satisfactory symptomatic control for the majority of patients. However, the presupposition that interventions exist as stand-alone entities is not representative of experience in unique populations with the therapeutic benefit of combination therapy, using components drawn from behavioral, physiotherapeutic, neuromodulatory, and, if necessary, surgical alternatives. Even in populations relatively refractory to therapy, the use of multimodal therapy yields additive benefits for patients with OAB symptoms. Herein is detailed the evidence supporting the concept that multimodal therapy provides optimal benefit to patients suffering from this symptom complex.     

Enantiomeric discrimination of Ru-substrates by cytochrome P450cam

Molecules with photosensitizers attached to substrates (Wilker et al., Angew. Chem. Int. Ed. 38 (1999) 90-92) or cofactors (Hamachi et al., J. Am. Chem. Soc. 121 (1999) 5500-5506) can rapidly deliver redox equivalents to buried active sites. The structure of cytochrome P450cam (P450) co-crystallized with a prototypal sensitizer-substrate, [Ru-C9-Ad]Cl2, has been determined (Dmochowski et al., Proc. Natl. Acad. Sci. USA 96 (1999) 12987-12990); and, in separate UV-vis absorption and time-resolved luminescence experiments, the binding of the lambda and delta enantiomers of Ru-C9-Ad to P450 has been measured. The results, KD(delta/lambda) approximately 2, indicate that the bipyridyl ligands of the lambda isomer interact more favorably with hydrophobic residues at the entrance to the substrate channel. We conclude that enantiospecific interactions may be exploited in the design of enzyme-metallosubstrate conjugates.     

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.     

Additional contribution of emerging risk factors to the prediction of the risk of type 2 diabetes: evidence from the Western New York Study

Objective: To examine whether several biomarkers of endothelial function and inflammation improve prediction of type 2 diabetes over 5.9 years of follow‐up, independent of traditional risk factors. Methods and Procedures: A total of 1,455 participants from the Western New York Study, free of type 2 diabetes at baseline, were selected. Incident type 2 diabetes was defined as fasting glucose exceeding 125 mg/dl or on antidiabetic medication at the follow‐up visit. Sixty‐one people who met the case definition (8/1,000 person years) were identified and individually matched with up to three controls on gender, race, year of study enrollment, and baseline fasting glucose (<110 or 110–125 mg/dl). Biomarkers were measured from frozen baseline samples. Results: In conditional logistic regression analyses accounting for traditional risk factors (age, family history of diabetes, smoking, drinking status, and BMI), E‐selectin was positively related (3rd vs. 1st tertile: odds ratio 2.77, 95% confidence interval (CI) 1.13–6.79, P for linear trend = 0.023) and serum albumin was inversely related (3rd vs. 1st tertile: odds ratio 0.36, 95% CI 0.14–0.93, P for linear trend = 0.032) to type 2 diabetes incidence. The addition of E‐selectin, serum albumin, and leukocyte count to a basic risk factor model including only traditional risk factors significantly increased the area under the receiver operating characteristic curve (AUC) (from 0.646 to 0.726, P value = 0.04). Discussion: These results support the role of endothelial dysfunction and subclinical inflammation as important mechanisms in the etiopathogenesis of type 2 diabetes; moreover, they indicate that novel biomarkers may improve the prediction of type 2 diabetes beyond the use of traditional risk factors alone.