In vitro antioxidant activity of banana (Musa spp. ABB cv. Pisang Awak)

The methanolic extract of Musa ABB cv Pisang Awak was investigated for the polyphenolic contents and antioxidant activity. The total phenol and flavonoid contents of the fruit extract were found to be 120 mg gallic acid equivalents (GAE) and 440 mg quercetin equivalents (QE)/100 g of sample dry weight, respectively. The antioxidant activity of the Pisang Awak methanol extract (PAME) (20-500 microg/ml) was determined using 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, reducing capacity, 2-2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS) radical cation decolourization and hydroxyl radical scavenging capacity (OH*). The EC50 values of DPPH, ABTS and OH* activities of the PAME and butylated hydroxy toluene (BHT) were found to be 65 and 9 microg/ml, 29 and 6 microg/ml, 36 and 42 microg/ml respectively. The reducing capacity increased with increasing concentration (31.5-1000 mg/ml) of the fruit extract and the activity was comparable with the standard BHT. The high performance thin layer chromatography (HPTLC) analysis of the extract revealed the presence of polyphenols. The strong and positive correlations were obtained between total phenol/flavonoid contents (R2 = 0.693-1.0) and free radical scavenging ability was attributed to the polyphenols as the major antioxidants.     

A Novel Rhabdovirus Associated with Acute Hemorrhagic Fever in Central Africa

Deep sequencing was used to discover a novel rhabdovirus (Bas-Congo virus, or BASV) associated with a 2009 outbreak of 3 human cases of acute hemorrhagic fever in Mangala village, Democratic Republic of Congo (DRC), Africa. The cases, presenting over a 3-week period, were characterized by abrupt disease onset, high fever, mucosal hemorrhage, and, in two patients, death within 3 days. BASV was detected in an acute serum sample from the lone survivor at a concentration of 1.09×10⁶ RNA copies/mL, and 98.2% of the genome was subsequently de novo assembled from ∼140 million sequence reads. Phylogenetic analysis revealed that BASV is highly divergent and shares less than 34% amino acid identity with any other rhabdovirus. High convalescent neutralizing antibody titers of >1∶1000 were detected in the survivor and an asymptomatic nurse directly caring for him, both of whom were health care workers, suggesting the potential for human-to-human transmission of BASV. The natural animal reservoir host or arthropod vector and precise mode of transmission for the virus remain unclear. BASV is an emerging human pathogen associated with acute hemorrhagic fever in Africa.     

Characterization of Mycobacterium tuberculosis diaminopimelic acid epimerase: paired cysteine residues are crucial for racemization

Recently, the overproduction of Mycobacterium tuberculosis diaminopimelic acid (DAP) epimerase MtDapF in Escherichia coli using a novel codon alteration cloning strategy and the characterization of the purified enzyme was reported. In the present study, the effect of sulphydryl alkylating agents on the in vitro activity of M. tuberculosis DapF was tested. The complete inhibition of the enzyme by 2-nitro-5-thiocyanatobenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid) and 1,2-benzisothiazolidine-3-one at nanomolar concentrations suggested that these sulphydryl alkylating agents modify functionally significant cysteine residues at or near the active site of the epimerase. Consequently, the authors extended the characterization of MtDapF by studying the role of the two strictly conserved cysteine residues. The putative catalytic residues Cys87 and Cys226 of MtDapF were replaced individually with both serine and alanine. Residual epimerase activity was detected for both the serine replacement mutants C87S and C226S in vitro. Kinetic analyses revealed that, despite a decrease in the K(M) value of the C87S mutant for DAP that presumably indicates an increase in nonproductive substrate binding, the catalytic efficiency of both serine substitution mutants was severely compromised. When either C87 or C226 were substituted with alanine, epimerase activity was not detected emphasizing the importance of both of these cysteine residues in catalysis.     

Molecular basis for proline- and arginine-rich peptide inhibition of proteasome

PR39, a naturally occurring and cell-permeable proline- and arginine-rich peptide, blocks the degradation of inhibitor of nuclear factor κB (IκBα), thereby attenuating inflammation. It is a noncompetitive and reversible inhibitor of 20S proteasome. To identify its basis of action, we used solution NMR spectroscopy and mutational analyses of the active fragment, PR11, which identified amino acids required for human 20S proteasome inhibiting activity. We then examined PR11-mediated changes in the expression of nuclear factor κB-dependent genes in situ. The results provide prerequisites for proteasome inhibition by proline- and arginine-rich peptides, providing a powerful new tool to investigate inflammatory processes. These findings offer new leads in developing drugs to treat heart diseases or stroke.     

Structural independence of the two EF-hand domains of caltractin

Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli. Ca2+ binding affinities and the Ca2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays. Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca2+-dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain. Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 microm) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals but is activated by the binding of a single ion.     

Saccharomyces cerevisiae flap endonuclease 1 uses flap equilibration to maintain triplet repeat stability

Flap endonuclease 1 (FEN1) is a central component of Okazaki fragment maturation in eukaryotes. Genetic analysis of Saccharomyces cerevisiae FEN1 (RAD27) also reveals its important role in preventing trinucleotide repeat (TNR) expansion. In humans such expansion is associated with neurodegenerative diseases. In vitro, FEN1 can inhibit TNR expansion by employing its endonuclease activity to compete with DNA ligase I. Here we employed two yeast FEN1 nuclease mutants, rad27-G67S and rad27-G240D, to further define the mechanism by which FEN1 prevents TNR expansion. Using a yeast artificial chromosome system that can detect both TNR instability and fragility, we demonstrate that the G240D but not the G67S mutation increases both the expansion and fragility of a CTG tract in vivo. In vitro, the G240D nuclease is proficient in cleaving a fixed nonrepeat double flap; however, it exhibits severely impaired cleavage of both nonrepeat and CTG-containing equilibrating flaps. In contrast, wild-type FEN1 and the G67S mutant exhibit more efficient cleavage on an equilibrating flap than on a fixed CTG flap. The degree of TNR expansion and the amount of chromosome fragility observed in the mutant strains correlate with the severity of defective flap cleavage in vitro. We present a model to explain how flap equilibration and the unique tracking mechanism of FEN1 can collaborate to remove TNR flaps and prevent repeat expansion.     

Update on: Shigella new serogroups/serotypes and their antimicrobial resistance

Shigellosis represents a major burden of disease in developing countries. A low infectious dose allows the disease to be spread effectively. Although shigellosis is mostly a self‐limiting disease, antibiotics are recommended to reduce deaths, disease symptoms and organism‐shedding time. However, in India, antimicrobial resistance among the genus Shigella is more common than among any other enteric bacteria. Notably, new serotypes or subserotypes in Shigella are reported from various parts of the world. Identification of new subserotypes of Shigella spp. is becoming a major issue as these strains are nontypeable by conventional serotyping. The commercially available antisera may not cover all possible epitopes of the O lipopolysaccharide antigen of Shigella serotypes. Therefore, molecular methods which most closely approach the resolution of full serotyping are necessary to identify such strains. In addition, the knowledge of a prevalent serotype in various geographic regions may assist in formulating strategies such as the development of a vaccine to prevent infection especially when the immunity to disease is serotype specific, and to understand the disease burden caused by new Shigella serotypes.     

The Genome Sequence of Lone Star Virus,a Highly Divergent Bunyavirus Found in the Amblyomma americanum Tick

Viruses in the family Bunyaviridae infect a wide range of plant, insect, and animal hosts. Tick-borne bunyaviruses in the Phlebovirus genus, including Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) in China, Heartland virus (HRTV) in the United States, and Bhanja virus in Eurasia and Africa have been associated with acute febrile illness in humans. Here we sought to characterize the growth characteristics and genome of Lone Star virus (LSV), an unclassified bunyavirus originally isolated from the lone star tick Amblyomma americanum. LSV was able to infect both human (HeLa) and monkey (Vero) cells. Cytopathic effects were seen within 72 h in both cell lines; vacuolization was observed in infected Vero, but not HeLa, cells. Viral culture supernatants were examined by unbiased deep sequencing and analysis using an in-house developed rapid computational pipeline for viral discovery, which definitively identified LSV as a phlebovirus. De novo assembly of the full genome revealed that LSV is highly divergent, sharing <61% overall amino acid identity with any other bunyavirus. Despite this sequence diversity, LSV was found by phylogenetic analysis to be part of a well-supported clade that includes members of the Bhanja group viruses, which are most closely related to SFSTV/HRTV. The genome sequencing of LSV is a critical first step in developing diagnostic tools to determine the risk of arbovirus transmission by A. americanum, a tick of growing importance given its expanding geographic range and competence as a disease vector. This study also underscores the power of deep sequencing analysis in rapidly identifying and sequencing the genomes of viruses of potential clinical and public health significance.