G4 motifs affect origin positioning and efficiency in two vertebrate replicators

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome‐wide studies in vertebrates have recently identified a consensus G‐rich motif potentially able to form G‐quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200‐bp cis‐regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.     

Curcumin ameliorated myocardial infarction by inhibition of cardiotoxicity in the rat model

Cardiovascular diseases are the main cause of death globally. Many attempts have been done to ameliorate the pathological changes after the occurrence of myocardial infarction. Curcumin is touted as a polyphenol phytocompound with appropriate cardioprotective properties. In this study, the therapeutic effect of curcumin was investigated on acute myocardial infarction in the model of rats. Rats were classified into four groups; control, isoproterenol hydrochloride (ISO) (100 mg/kbw), curcumin (50 mg/kbw), and curcumin plus ISO treatment groups. After 9-day administration of curcumin, levels of lactate dehydrogenase (LDH), creatine kinase (CK), and cardiac troponin I (cTnI) were determined. Superoxide dismutase (SOD) and malondialdehyde (MDA) contents were measured to investigate the oxidative status in infarct rats received curcumin. By using H & E staining, tissue inflammation was performed. Masson’s trichrome staining was conducted to show cardiac remodeling and collagen deposition. The number of apoptotic cells was determined by using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Data showed the serum decrease of LDH, CK, and cTnI in infarct rats after curcumin intake compared to the rats given (ISO) ( P < 0.05). Curcumin was found to reduce oxidative status by reducing SOD and MDA contents ( P < 0.05). Gross and microscopic examinations revealed that the decrease of infarct area, inflammation response and collagen deposition in rats given ISO plus curcumin ( P < 0.05). We noted the superior effect of curcumin to reduce the number of apoptotic cardiomyocytes after 9 days. Data point the cardioprotective effect of curcumin to diminish the complication of infarction by the reduction of cell necrosis and apoptosis in a rat model of experimental infarction.     

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.     

Application of water super absorbents in waste air biofiltration

Water super absorbents are low cross-linked hydrophilic polymers that absorb water in amounts up to several hundred times their dry weight. In this study, the effect of adding these materials to the bed of a biofilter was investigated. Two equal size biofilters were used for this purpose. One of the biofilters was packed with a mixture of perlite and a commercial polyacrylamide based super absorbent (2.3% dry weight), and the other was packed with perlite to perform as a control. The biofilters were inoculated with a bacterial culture that was able to grow on n-hexane as the sole source of carbon and energy. Both biofilters removed up to 90% of the entering pollutants when using an inlet n-hexane concentration of 1 g/m³, and an air flow rate of 0.3 L/min (mass loading of 18.34 g/m³/h, and empty bed residence time of 3.27 min). The super absorbent had a positive effect on the performance of the biofilter. While the difference in the performance of the biofilters was marginal when frequent moistening was applied, the difference was considerable when moistening was less frequent.     

Rapid clonal analysis of recurrent tuberculosis by direct MIRU-VNTR typing on stored isolates

Background  

The application of molecular tools to the analysis of tuberculosis has revealed examples of clonal complexity, such as exogenous reinfection, coinfection, microevolution or compartmentalization. The detection of clonal heterogeneity by standard genotyping approaches is laborious and often requires expertise. This restricts the rapid availability of Mycobacterium tuberculosis (MTB) genotypes for clinical or therapeutic decision-making. A new PCR-based technique, MIRU-VNTR, has made it possible to genotype MTB in a time frame close to real-time fingerprinting. Our purpose was to evaluate the capacity of this technique to provide clinicians with a rapid discrimination between reactivation and exogenous reinfection and whether MIRU-VNTR makes it possible to obtain data directly from stored MTB isolates from recurrent episodes.     

Optimisation of growth medium for the production of cyclodextrin glucanotransferase from Bacillus stearothermophilus HR1 using response surface methodology

Cyclodextrin glucanotransferase (CGTase) activity was observed when the bacterium was grown in the medium at various initial pH values, containing carbon, nitrogen, phosphorus and mineral salt sources at 50 °C for 24 h in the shake flasks. The optimisation of this growth medium was carried out using response surface methodology. The design contains a total of 32 experimental trials involving 10 star points and 6 replicates at the centre points. The design was employed by selecting sago starch, peptone from casein, K₂HPO₄, CaCl₂ and initial pH as five independent variables in this study. The optimal calculated values of tested variables for maximal production of CGTase were found to be comprised of: sago starch, 16.02 g/l; peptone from casein, 20 g/l; K₂HPO₄, 1.4 g/l; CaCl₂, 0.2 g/l and initial pH, 7.54 with a predicted CGTase activity of 14.20 U/ml. These predicted optimal parameters were tested in the laboratory and the final CGTase activity obtained was very close to the predicted value at 14.80 U/ml.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.