A systematic analysis of cell cycle regulators in yeast reveals that most factors act independently of cell size to control initiation of division

Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.     

Single-nucleotide polymorphism,linkage disequilibrium and geographic structure in the malaria parasite <Emphasis Type="Italic">Plasmodium vivax</Emphasis>: prospects for genome-wide association studies

Background

The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized.

Results

We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance.

Conclusion

These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.See commentary: http://www.biomedcentral.com/1741-7007/8/90

     

Calcium currents in embryonic and neonatal mammalian skeletal muscle

The whole-cell patch-clamp technique was used to study the properties of inward ionic currents found in primary cultures of rat and mouse skeletal myotubes and in freshly dissociated fibers of the flexor digitorum brevis muscle of rats. In each of these cell types, test depolarizations from the holding potential (-80 or -90 mV) elicited three distinct inward currents: a sodium current (INa) and two calcium currents. INa was the dominant inward current: under physiological conditions, the maximum inward INa was estimated to be at least 30-fold larger than either of the calcium currents. The two calcium currents have been termed Ifast and Islow, corresponding to their relative rates of activation. Ifast was activated by test depolarizations to around -40 mV and above, peaked in 10-20 ms, and decayed to baseline in 50-100 ms. Islow was activated by depolarizations to approximately 0 mV and above, peaked in 50-150 ms, and decayed little during a 200-ms test pulse. Ifast was inactivated by brief, moderate depolarizations; for a 1-s change in holding potential, half-inactivation occurred at -55 to -45 mV and complete inactivation occurred at -40 to -30 mV. Similar changes in holding potential had no effect on Islow. Islow was, however, inactivated by brief, strong depolarizations (e.g., 0 mV for 2 s) or maintained, moderate depolarizations (e.g., -40 mV for 60 s). Substitution of barium for calcium had little effect on the magnitude or time course of either Ifast or Islow. The same substitution shifted the activation curve for Islow approximately 10 mV in the hyperpolarizing direction without affecting the activation of Ifast. At low concentrations (50 microM), cadmium preferentially blocked Islow compared with Ifast, while at high concentrations (1 mM), it blocked both Ifast and Islow completely. The dihydropyridine calcium channel antagonist (+)-PN 200-110 (1 microM) caused a nearly complete block of Islow without affecting Ifast. At a holding potential of -80 mV, the half-maximal blocking concentration (K0.5) for the block of Islow by (+)-PN 200-110 was 182 nM. At depolarized holding potentials that inactivated Islow by 35-65%, K0.5 decreased to 5.5 nM.     

BH3‐in‐groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes

Pro‐apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site‐specific disulfide crosslinking, compartment‐specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3‐in‐groove dimer interface on the MOM surface similar to that observed in crystals. However, after the α5 helix was released into the MOM, the remaining interface with α2, α3, and α4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel α9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3‐in‐groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, α9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by α9 insertion into the MOM and localization to the pore rim. Therefore, the BH3‐in‐groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by α9 dimerization at the rim.     

Preparation and assessment of novel coprocessed superdisintegrant consisting of crospovidone and sodium starch glycolate: A technical note

Summary and Conclusion  Coprocessed superdisintegrant consisting of crospovidone and SSG exhibited good flow and compression characteristics. Cefixime trihydrate and ibuprofen tablets containing coprocessed superdisintegrant exhibited quick disintegration and improved drug dissolution. Publshed: February 2, 2007     

The Role of Iex-1 in the Pathogenesis of Venous Neointimal Hyperplasia Associated with Hemodialysis Arteriovenous Fistula

Arteriovenous fistulas (AVFs) used for hemodialysis fail because of venous neointimal hyperplasia (VNH). There are 1,500,000 patients that have end stage renal disease worldwide and the majority requires hemodialysis. In the present study, the role of the intermediate early response gene X-1 (IEX-1), also known as IER-3 in the pathogenesis of VNH was evaluated. In human samples removed from failed AVF, there was a significant increase in IEX-1 expression localized to the adventitia. In Iex-1 ^−/− mice and wild type (WT) controls, chronic kidney disease was induced and an AVF placed 28 days later by connecting the carotid artery to jugular vein. The outflow vein was removed three days following the creation of the AVF and gene expression analysis demonstrated a significant decrease in vascular endothelial growth factor-A (Vegf-A) and monocyte chemoattractant protein-1 (Mcp-1) gene expression in Iex-1 ^−/− mice when compared to WT mice (P<0.05). At 28 days after AVF placement, histomorphometric and immune-histochemical analyses of the outflow vein demonstrated a significant decrease in neointimal hyperplasia with an increase in average lumen vessel area associated with a decrease in fibroblast, myofibroblast, and Ly6C staining. There was a decrease in proliferation (Ki-67) and an increase in the TUNEL staining in Iex-1 KO mice compared to WT. In addition, there was a decrease in Vegf-A, Mcp-1, and matrix metalloproteiniase-9 (Mmp-9) staining. Iex-1 expression was reduced in vivo and in vitro using nanoparticles coated with calcitriol, an inhibitor of Iex-1 that demonstrated that Iex-1 reduction results in decrease in Vegf-A. In aggregate, these results indicate that the absence of IEX-1 gene results in reduced VNH accompanied with a decrease in proliferation, reduced fibroblast, myofibroblast, and Ly6C staining accompanied with increased apoptosis mediated through a reduction in Vegf-A/Mcp-1 axis and Mmp-9. Adventitial delivery of nanoparticles coated with calcitriol reduced Iex-1 and VNH.     

Simultaneous determination of different endogenetic plant growth regulators in common green seaweeds using dispersive liquid–liquid microextraction method

A simple and rapid HPLC-based method was developed for simultaneous determination of major classes of plant growth regulators (PGRs) in Monostroma and different species of Ulva. The plant growth regulators determined included gibberellic acid (GA₃), indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA), salicylic acid and kinetin riboside (KR) and their respective elution time was 2.75, 3.3, 3.91, 4.95, 5.39 and 6.59 min. The parameters optimized for distinct separation of PGRs were mobile phase (60:40 methanol and 0.6% acetic acid in water), column temperature (35 °C) and flow rate (1 ml/min). This method presented an excellent linearity (0.2–100 μg/ml) with limit of detection (LOD) as 0.2 μg/ml for ABA, 0.5 μg/ml for KR and salicylic acid, and 1 μg/ml for IAA, IBA and GA₃. The precision and accuracy of the method was evaluated after inter and intra day analysis in triplicates. The effect of plant matrix was compensated after spiking and the resultant recoveries estimated were in the range of 80–120%. Each PGR thereby detected were further characterized by ESI-MS analysis. The method optimized in this study determined IBA along with IAA for the first time in the seaweed species investigated except Ulva linza where the former was not detected. In all the species studied, ABA level was detected to be the highest while kinetin riboside was the lowest. In comparison to earlier methods of PGR analysis, sample preparation and analysis time were substantially reduced while allowing determination of more classes of PGRs simultaneously.     

Minicells: versatile vectors for targeted drug or si/shRNA cancer therapy

Effective cancer therapy continues to be a daunting challenge due mainly to considerable tumor cell heterogeneity, drug-resistance, and dose-limiting toxicity of therapeutics. Here we review a versatile nano-cellular (minicell) delivery vehicle that can be packaged with therapeutically effective concentrations of chemotherapeutic drugs, siRNAs or shRNAs and can be targeted to tumors via minicell-surface attached bispecific antibodies. A range of minicell-based therapeutics have shown highly effective tumor stabilization/regression in the murine xenograft model and in case studies in canines with late-stage endogenous tumors. Repeat intravenous dosing shows absence of toxicity or immunogenicity in both species. The minicell-based therapeutic has potential applications in personalized cancer medicine.     

Kappaphycus alvarezii as a source of bioethanol

The present study describes production of bio-ethanol from fresh red alga, Kappaphycus alvarezii. It was crushed to expel sap - a biofertilizer - while residual biomass was saccharified at 100 °C in 0.9 N H₂SO₄. The hydrolysate was repeatedly treated with additional granules to achieve desired reducing sugar concentration. The best yields for saccharification, inclusive of sugar loss in residue, were 26.2% and 30.6% (w/w) at laboratory (250 g) and bench (16 kg) scales, respectively. The hydrolysate was neutralized with lime and the filtrate was desalted by electrodialysis. Saccharomyces cerevisiae (NCIM 3523) was used for ethanol production from this non-traditional bio-resource. Fermentation at laboratory and bench scales converted ca. 80% of reducing sugar into ethanol in near quantitative selectivity. A petrol vehicle was successfully run with E10 gasoline made from the seaweed-based ethanol. Co-production of ethanol and bio-fertilizer from this seaweed may emerge as a promising alternative to land-based bio-ethanol.