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.     

High-throughput inference of protein-protein interfaces from unassigned NMR data

SUMMARY: We cast the problem of identifying protein-protein interfaces, using only unassigned NMR spectra, into a geometric clustering problem. Identifying protein-protein interfaces is critical to understanding inter- and intra-cellular communication, and NMR allows the study of protein interaction in solution. However it is often the case that NMR studies of a protein complex are very time-consuming, mainly due to the bottleneck in assigning the chemical shifts, even if the apo structures of the constituent proteins are known. We study whether it is possible, in a high-throughput manner, to identify the interface region of a protein complex using only unassigned chemical shifts and residual dipolar coupling (RDC) data. We introduce a geometric optimization problem where we must cluster the cells in an arrangement on the boundary of a 3-manifold, where the arrangement is induced by a spherical quadratic form [corrected] The arrangement is induced by a spherical quadratic form, which in turn is parameterized by a SO(3)xR2. We show that this formalism derives directly from the physics of RDCs. We present an optimal algorithm for this problem that runs in O(n3 log n) time for an n-residue protein. We then use this clustering algorithm as a subroutine in a practical algorithm for identifying the interface region of a protein complex from unassigned NMR data. We present the results of our algorithm on NMR data for seven proteins from five protein complexes, and show that our approach is useful for high-throughput applications in which we seek to rapidly identify the interface region of a protein complex. AVAILABILITY: Contact authors for source code.     

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.     

A polynomial-time algorithm for de novo protein backbone structure determination from nuclear magnetic resonance data.

We describe an efficient algorithm for protein backbone structure determination from solution Nuclear Magnetic Resonance (NMR) data. A key feature of our algorithm is that it finds the conformation and orientation of secondary structure elements as well as the global fold in polynomial time. This is the first polynomial-time algorithm for de novo high-resolution biomacromolecular structure determination using experimentally recorded data from either NMR spectroscopy or X-ray crystallography. Previous algorithmic formulations of this problem focused on using local distance restraints from NMR (e.g., nuclear Overhauser effect [NOE] restraints) to determine protein structure. This approach has been shown to be NP-hard, essentially due to the local nature of the constraints. In practice, approaches such as molecular dynamics and simulated annealing, which lack both combinatorial precision and guarantees on running time and solution quality, are used routinely for structure determination. We show that residual dipolar coupling (RDC) data, which gives global restraints on the orientation of internuclear bond vectors, can be used in conjunction with very sparse NOE data to obtain a polynomial-time algorithm for structure determination. Furthermore, an implementation of our algorithm has been applied to six different real biological NMR data sets recorded for three proteins. Our algorithm is combinatorially precise, polynomialtime, and uses much less NMR data to produce results that are as good or better than previous approaches in terms of accuracy of the computed structure as well as running time.     

Synthesis and evaluation of novel 2-pyridone derivatives as inhibitors of phosphodiesterase3 (PDE3): A target for heart failure and platelet aggregation

Twenty-six 2-pyridone derivatives (8a-8z), which are structurally analogous to amrinone and milrinone two important cardiotonic drugs, are synthesized and characterized. The synthesis of 2-pyridone derivatives involves addition, followed by cyclization between Baylis-Hillman acetates (7a-7k) and enamino esters or nitriles (3a-3e). Thus synthesized pyridones were subjected to PDE3 inhibitory activity, 14 pyridones were found to be hits out of 26 pyridones synthesized and out of 14 hits, there are 5 pyridones found to be lead compounds having excellent PDE3 inhibitory activity. Further we have carried out computational analysis to understand protein/enzyme and 2-pyridone derivative interactions to identify amino acid residues involved in the vicinity of binding and compared with milrinone drug.     

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.     

Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection

Background

Host genetics has been shown to play a role in porcine reproductive and respiratory syndrome (PRRS), which is the most economically important disease in the swine industry. A region on Sus scrofa chromosome (SSC) 4 has been previously reported to have a strong association with serum viremia and weight gain in pigs experimentally infected with the PRRS virus (PRRSV). The objective here was to identify haplotypes associated with the favorable phenotype, investigate additional genomic regions associated with host response to PRRSV, and to determine the predictive ability of genomic estimated breeding values (GEBV) based on the SSC4 region and based on the rest of the genome. Phenotypic data and 60 K SNP genotypes from eight trials of ~200 pigs from different commercial crosses were used to address these objectives.

Results

Across the eight trials, heritability estimates were 0.44 and 0.29 for viral load (VL, area under the curve of log-transformed serum viremia from 0 to 21 days post infection) and weight gain to 42 days post infection (WG), respectively. Genomic regions associated with VL were identified on chromosomes 4, X, and 1. Genomic regions associated with WG were identified on chromosomes 4, 5, and 7. Apart from the SSC4 region, the regions associated with these two traits each explained less than 3% of the genetic variance. Due to the strong linkage disequilibrium in the SSC4 region, only 19 unique haplotypes were identified across all populations, of which four were associated with the favorable phenotype. Through cross-validation, accuracies of EBV based on the SSC4 region were high (0.55), while the rest of the genome had little predictive ability across populations (0.09).

Conclusions

Traits associated with response to PRRSV infection in growing pigs are largely controlled by genomic regions with relatively small effects, with the exception of SSC4. Accuracies of EBV based on the SSC4 region were high compared to the rest of the genome. These results show that selection for the SSC4 region could potentially reduce the effects of PRRS in growing pigs, ultimately reducing the economic impact of this disease.     

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.