Molecular docking analysis of CDK-1 inhibitors from Chrysophyllum cainito leaves

It is of interest to document the molecular docking analysis of Cyclin-dependent kinase 1 (CDK-1) inhibitors from Chrysophyllum cainito leaves towards the treatment of tumors using the known structure of PDB ID: 5HQ0. Data shows that molecules such as 8- (Dimethylamino)-7-(3-(4-ethylphenoxy)-2d, ethyl 6-oxo-5-propylheptanoate, 2,3-dihydro-3, 5-dihydroxy-6-methyl-4h-pyran-4-one, 1,2,3-benzenetriol and 1,4-benzenediol 2,5-bis (1,1-dimethylethyl) identified in methanolic extract of C. cainito have binding features with CDK1 for further consideration.     

Hydrogen peroxide induces hyphal differentiation in Candida albicans

In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H₂O₂ in Candida albicans. This finding is confirmed by the changing intracellular concentration of H₂O₂. In order to induce the same level of differentiation, low concentrations of exogenous H₂O₂ are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.     

P4-ATPase requirement for AP-1/clathrin function in protein transport from the trans-Golgi network and early endosomes

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Delta cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.     

Eucalyptus microsatellites mined in silico: survey and evaluation

Eucalyptus is an important short rotation pulpy woody plant, grown widely in the tropics. Recently, many genomic programmes are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. These sequences can be utilized for analysis of simple sequence repeats (SSRs) and single nucleotide polymorphism (SNPs) available in the transcribed genes. In this study, in silico analysis of 15,285 sequences representing partial and full-length mRNA from Eucalyptus species for their use in developing SSRs or microsatellites were carried out. A total of 875 EST-SSRs were identified from 772 SSR containing ESTs. Motif size of 6 for dinucleotide and 5 for trinucleotide, tetranucleotide, and pentanucleotides were considered in locating the microsatellites. The average frequency of identified SSRs was 12.9%. The dinucleotide repeats were the most abundant among the dinucleotide, trinucleotide and tetranucleotide motifs and accounted for 50.9% of the Eucalyptus genome. Primer designing analysis showed that 571 sequences with SSRs had sufficient flanking regions for polymerase chain reaction (PCR) primer synthesis. Evaluation of the usefulness of the SSRs showed that EST-derived SSRs can generate polymorphic markers as all the primers showed allelic diversity among the 16 provenances of E. tereticornis.     

Activation of the murine interleukin-12 p40 promoter by functional interactions between NFAT and ICSBP

Interleukin (IL)-12 is a heterodimeric cytokine that is critical for the development of a T-helper-1 immune response and immunity against intracellular pathogens. The IL-12 p40 gene product, expressed specifically in macrophages and dendritic cells, heterodimerizes with p35 to form bioactive IL-12, and heterodimerizes with p19 to comprise the cytokine IL-23. Regulation of the murine IL-12 p40 promoter is complex. Multiple cis-acting elements have been characterized that are involved in activation by bacterial products. However, molecular mechanisms through which interferon (IFN)-gamma and bacterial products synergistically activate IL-12 p40 gene expression are less clear. In this study, a composite NFAT/ICSBP binding site at -68 to -54 is identified that is functionally important for p40 promoter activation by lipopolysaccharide (LPS) and LPS plus IFN-gamma. DNA binding of NFAT and ICSBP is demonstrated on the endogenous promoter by chromatin immunoprecipitation. NFAT is required for ICSBP binding to this region. Overexpression of NFAT and ICSBP synergistically activates the p40 promoter. A dominant negative NFAT molecule attenuates LPS- and IFN-gamma-activated endogenous IL-12 p40 mRNA expression. A physical association between NFAT and ICSBP in the absence of DNA is detected by co-immunoprecipitation of endogenous proteins. Three NFAT domains are required for ICSBP interaction. Finally, in LPS- and IFN-gamma-activated RAW-264.7 cells, the association between NFAT and ICSBP is abrogated by IL-10 priming.     

Regulated conformation of myosin V

We have found that myosin V, an important actin-based vesicle transporter, has a folded conformation that is coupled to inhibition of its enzymatic activity in the absence of cargo and Ca(2+). In the absence of Ca(2+) where the actin-activated MgATPase activity is low, purified brain myosin V sediments in the analytical ultracentrifuge at 14 S as opposed to 11 S in the presence of Ca(2+) where the activity is high. At high ionic strength it sediments at 10 S independent of Ca(2+), and its regulation is poor. These data are consistent with myosin V having a compact, inactive conformation in the absence of Ca(2+) and an extended conformation in the presence of Ca(2+) or high ionic strength. Electron microscopy reveals that in the absence of Ca(2+) the heads and tail are both folded to give a triangular shape, very different from the extended appearance of myosin V at high ionic strength. A recombinant myosin V heavy meromyosin fragment that is missing the distal portion of the tail domain is not regulated by calcium and has only a small change in sedimentation coefficient, which is in the opposite direction to that seen with intact myosin V. Electron microscopy shows that its heads are extended even in the absence of calcium. These data suggest that interaction between the motor and cargo binding domains may be a general mechanism for shutting down motor protein activity and thereby regulating the active movement of vesicles in cells.     

The GAT domains of clathrin-associated GGA proteins have two ubiquitin binding motifs

Ubiquitin (Ub) attachment to membrane proteins can serve as a sorting signal for lysosomal delivery. Recognition of Ub as a sorting signal can occur at the trans-Golgi network and is mediated in part by the clathrin-associated Golgi-localizing, gamma-adaptin ear domain homology, ARF-binding proteins (GGA). GGA proteins bind Ub via a three-helix bundle subdomain in their GAT (GGA and target of Myb1 protein) domain, which is also present in the Ub binding domain of target of Myb1 protein. Ubiquitin binding by yeast Ggas is required to direct sorting of ubiquitinated proteins such as general amino acid permease (Gap1) from the trans-Golgi network to endosomes. Using affinity chromatography and nuclear magnetic resonance spectroscopy, we have found that the human GGA3 GAT domain contains two Ub binding motifs that bind to the same surface of ubiquitin. These motifs are found within different helices within the three-helix GAT subdomain. When functionally analyzed in yeast, each motif was sufficient to mediate trans-Golgi network to endosomal sorting of Gap1, and mutation of both motifs resulted in defective Gap1 sorting without defects in other GGA-dependent processes.     

Anti-inflammatory potential of an ethyl acetate fraction isolated from Justicia gendarussa roots through inhibition of iNOS and COX-2 expression via NF-κB pathway

Justicia gendarussa Burm.f. (J. gendarussa) is a plant used as traditional medicine in different parts of India and China to treat inflammatory disorders like rheumatoid arthritis. But its mechanism of anti-inflammatory action is still unclear. Hence in this context, the objective of our study is to reveal the mechanism of anti-inflammatory activity of J. gendarussa which would form an additional proof to the traditional knowledge of this plant. The anti-inflammatory function and mechanism(s) of action was studied in an ethyl acetate fraction isolated from methanolic extract of J. gendarussa roots (EJG). Anti-inflammatory studies were conducted on rats using partitioned fractions isolated from methanolic extract of J. gendarussa roots. In carrageenan-induced rat paw edema, ethyl acetate fraction brought about 80% and 93% edema inhibition at 3rd and 5th hour at a dose of 50 mg/kg, when compared to other extracts and Voveran. We investigated whether EJG inhibits the release of cycloxygenase (COX), 5-lipoxygenase (5-LOX), interleukin-6 (IL-6) and nuclear factor kappa B (NF-κB) in LPS stimulated human peripheral blood mononuclear cells (hPBMCs). Results shows that EJG dose dependently inhibited LPS-activated COX, 5-LOX, IL-6, and NF-κB in hPBMCs. EJG also reduced LPS induced levels of iNOS and COX-2 mRNA expression in hPBMCs. This study provides an insight into the probable mechanism(s) underlying the anti-inflammatory activity of EJG and therefore, we report the first confirmation of the anti-inflammatory potential of this traditionally employed herbal medicine in vitro.