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.     

Estimating western corn rootworm (Coleoptera: Chrysomelidae) larval susceptibility to event DAS‐59122‐7 maize

Susceptibility of Diabrotica virgifera virgifera (LeConte) larvae to DAS‐59122‐7 maize was evaluated using a laboratory technique that measures rootworm survival to adulthood on maize seedlings. This method produces direct measures of larval susceptibility using realistic exposure to the same range of insecticidal protein concentrations found in field‐grown DAS‐59122‐7 maize roots. First, second and third instars were reared to adulthood on DAS‐59122‐7 maize seedlings or a non‐transgenic, near‐isoline maize. Data on survival, adult gender ratio, adult weight and median emergence were collected. Overall, larval susceptibility to DAS‐59122‐7 maize was lower than earlier predictions ( Storer et al. 2006 ). Neonate survival on DAS‐59122‐7 maize was approximately 33% of isoline survival after 17 days, and the same 33% recovered and developed to adulthood when the isoline maize was substituted. Survival rate on DAS‐59122‐7 maize increased with instar. The mean survivorship was 0.5%, 26% and 65% when exposure to DAS‐59122‐7 maize began at the first, second and third instars, respectively. Exposure to DAS‐59122‐7 maize led to sub‐lethal effects on adult gender ratio, weight and median emergence. These effects decreased when exposure to DAS‐59122‐7 maize began at later instars. The killing effect of DAS‐59122‐7 maize on rootworm larvae appeared to result from the combined chronic effects and absence of a suitable host as perceived by the larvae. The relevance of these data and the methodology of estimating rootworm susceptibility to plant‐incorporated protectants are discussed in the context of the US Environmental Protection Agency’s functional definition of ‘high dose’ and use of refuge for resistance management ( EPA 1998a ). Based on these results it is evident that DAS‐59122‐7 maize does not meet the functional definitions of high dose as described by EPA (1998a,b) and ILSI (1999) , and the utility of refuge, refuge size and refuge placement for delaying rootworm resistance should be further investigated.     

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.     

Specialized morphology for a generalist diet: evidence for Liem's Paradox in a cichlid fish

The stable isotope ratio and seasonal changes in diet of Alluaud's haplo Astatoreochromis alluaudi, a cichlid fish with massive pharyngeal jaws well known for its ability to process hard‐bodied prey, are described. The diet of A. alluaudi was quantified in Lake Saka, Uganda, over a period of 30 months. Variation in physico‐chemical variables (mean monthly rainfall, water temperature, turbidity and dissolved oxygen), as well as potential competitor density and food abundance, was measured throughout the second half of the study (14 months). Stomach contents and isotope analysis revealed a diet comprised mainly of fishes and insects, with a low contribution of molluscs (0–33%) in any given month. No correlation was detected between diet and either macroinvertebrate abundance or competitor abundance. The running average rainfall was positively related to the percentage of fish consumed per month. Although A. alluaudi exhibits an apparent molluscivorous trophic morphology in Lake Saka, molluscs did not appear to compose a major portion of its diet. Gradients of rainfall seemed to be the most important environmental predictor of diet choice in Lake Saka. These results are discussed with reference to Liem's Paradox that apparently morphologically specialized fishes often function as generalist feeders in the wild.     

Slow response of soil organic matter to the reduction in atmospheric nitrogen deposition in a Norway spruce forest

Global nitrogen (N) deposition rates in terrestrial environments have quadrupled since preindustrial times, causing structural and functional changes of ecosystems. Different emission reduction policies were therefore devised. The aim of our study was to investigate if, and over what timescale, processes of soil organic matter (OM) transformation respond to a decline in atmospheric N deposition. A N‐saturated spruce forest (current N deposition: 34 kg ha^?1 yr^?1; critical N load: 14 kg ha^?1 yr^?1), where N deposition has been reduced to 11.5 kg ha^?1 yr^?1 since 1991, was studied. Besides organic C and organic and inorganic N, noncellulosic carbohydrates, amino sugars and amino acids were determined. A decline in organic N in litter indicated initial effects at plant level. However, there were no changes in biomarkers upon the reduction in N deposition. In addition, inorganic N was not affected by reduced N deposition. The results showed that OM cycling and transformation processes have not responded so far. It was concluded that no direct N deposition effects have occurred due to the large amount of stored organic N, which seems to compensate for the reduction in deposited N. Obviously, the time span of atmospheric N reduction (about 14.5 years) is too short compared with the mean turnover time of litter to cause indirect effects on the composition of organic C and N compounds. It is assumed that ecological processes, such as microbial decomposition or recycling of organic N and C, react slowly, but may start within the next decade with the incorporation of the new litter.