Haemophilus influenzae UvrA: overexpression, purification, and in cell complementation

UvrA protein is a major component of ABC endonuclease complex involved in nucleotide excision repair (NER) mechanism. Although NER system is best characterized in Escherichia coli, not much information is available in Haemophilus influenzae. However, based on amino acid homology, uvrA ORF has been identified on H. influenzae genome [gene identification No. HI0249, Science 269 (1995) 496]. H. influenzae Rd uvrA ORF was cloned and overexpressed in E. coli. The expressed UvrA protein was purified using a two-step column chromatography protocol to a single band of expected molecular weight (104 kDa) and characterized for its ATPase and DNA binding activity. In addition, when H. influenzae uvrA was introduced in E. coli uvrA mutant strain AB1886, its UV resistance was restored to near wild type level.     

Relative Abundance and Plasmodium Infection Rates of Malaria Vectors in and around Jabalpur,a Malaria Endemic Region in Madhya Pradesh State,Central India

BackgroundThis study was undertaken in two Primary Health Centers (PHCs) of malaria endemic district Jabalpur in Madhya Pradesh (Central India).MethodsIn this study we had investigated the relative frequencies of the different anopheline species collected within the study areas by using indoor resting catches, CDC light trap and human landing methods. Sibling species of malaria vectors were identified by cytogenetic and molecular techniques. The role of each vector and its sibling species in the transmission of the different Plasmodium species was ascertained by using sporozoite ELISA.ResultsA total of 52,857 specimens comprising of 17 anopheline species were collected by three different methods (39,964 by indoor resting collections, 1059 by human landing and 11,834 by CDC light trap). Anopheles culicifacies was most predominant species in all collections (55, 71 and 32% in indoor resting, human landing and light trap collections respectively) followed by An. subpictus and An. annularis. All five sibling species of An. culicifacies viz. species A, B, C, D and E were found while only species T and S of An. fluviatilis were collected. The overall sporozoite rate in An. culicifacies and An. fluviatilis were 0.42% (0.25% for P. falciparum and 0.17% for P. vivax) and 0.90% (0.45% for P. falciparum and 0.45% for P. vivax) respectively. An. culicifacies and An. fluviatilis were found harbouring both P. vivax variants VK-210 and VK-247, and P. falciparum. An. culicifacies sibling species C and D were incriminated as vectors during most part of the year while sibling species T of An. fluviatilis was identified as potential vector in monsoon and post monsoon season.ConclusionsAn. culicifacies species C (59%) was the most abundant species followed by An. culicifacies D (24%), B (8.7%), E (6.7%) and A (1.5%). Among An. fluviatilis sibling species, species T was common (99%) and only few specimens of S were found. Our study provides crucial information on the prevalence of An. culicifacies and An. fluviatilis sibling species and their potential in malaria transmission which will assist in developing strategic control measures against these vectors.     

Altered Activation of Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein PACT

PACT is a stress-modulated activator of the interferon-induced double-stranded RNA-activated protein kinase (PKR). Stress-induced phosphorylation of PACT is essential for PACT''s association with PKR leading to PKR activation. PKR activation leads to phosphorylation of translation initiation factor eIF2α inhibition of protein synthesis and apoptosis. A recessively inherited form of early-onset dystonia DYT16 has been recently identified to arise due to a homozygous missense mutation P222L in PACT. To examine if the mutant P222L protein alters the stress-response pathway, we examined the ability of mutant P222L to interact with and activate PKR. Our results indicate that the substitution mutant P222L activates PKR more robustly and for longer duration albeit with slower kinetics in response to the endoplasmic reticulum stress. In addition, the affinity of PACT-PACT and PACT-PKR interactions is enhanced in dystonia patient lymphoblasts, thereby leading to intensified PKR activation and enhanced cellular death. P222L mutation also changes the affinity of PACT-TRBP interaction after cellular stress, thereby offering a mechanism for the delayed PKR activation in response to stress. Our results demonstrate the impact of a dystonia-causing substitution mutation on stress-induced cellular apoptosis.     

Comparative susceptibility of three important malaria vectors Anopheles stephensi, Anopheles fluviatilis, and Anopheles sundaicus to Plasmodium vivax

The 3 laboratory-colonized malaria vectors, i.e., Anopheles stephensi, An. sundaicus, and An. fluviatilis, were studied for their comparative susceptibility to Plasmodium vivax sporogony. There was no significant difference in oocyst and sporozoite recruitment by these 3 species, whereas the geometric mean (GM) of the oocyst number per midgut was significantly lower in An. fluviatilis as compared with that in the other 2 species. There was no difference in the GM of oocyst between An. stephensi and An. sundaicus. Adaptability to laboratory conditions and susceptibility to plasmodial infection suggest that An. fluviatilis and An. sundaicus can also be used as a vector model for vector-parasite interaction studies.     

Plantlet regeneration enhances solasodine productivity in hairy root cultures of <Emphasis Type="Italic">Solanum khasianum</Emphasis> Clarke

Summary Shoot regeneration in hairy root cultures of Solanum khasianum Clarke influences root growth, solasodine production. and permeabilization of solasodine into the medium. These parameters are dependent on exogenously supplied auxin and cytokinin: the effect being both concentration-and clone-dependent. Hairy root cultures with no shoot regeneration showed high permeabilization of solasodine into the medium by the sixth week of incubation, suggesting the medium acts as a sink for the solasodine synthesized by the roots. Solasodine in the culture medium was toxic to the transformed roots and caused browning of root tips. In a separate set of experiments, the hairy root cultures showed regeneration of approximately 50–70 mm long shoots after treatment with indole-3-acetic acid and kinetin. These hairy root cultures had inereased levels of solasodine production, compared to cultures without shoot regeneration. The plantlets formed in the hairy root cultures accumulated some of the solasodine, thereby reducing its permcabilization into the medium. Transport of solasodine from root to shoot reduced the toxic effect of solasodine in the root zone and extended the exponential growth phase by 8-10d.     

Decorin antagonizes Met receptor activity and down-regulates {beta}-catenin and Myc levels

A theme emerging during the past few years is that members of the small leucine-rich proteoglycan gene family affect cell growth by interacting with multiple receptor tyrosine kinases (RTKs), mostly by a physical down-regulation of the receptors, thereby depriving tumor cells of pro-survival signals. Decorin binds and down-regulates several RTKs, including Met, the receptor for hepatocyte growth factor. Here we demonstrate that decorin blocks several biological activities mediated by the Met signaling axis, including cell scatter, evasion, and migration. These effects were mediated by a profound down-regulation of noncanonical β-catenin levels. In addition, Myc, a downstream target of β-catenin, was markedly down-regulated by decorin, whereas phosphorylation of Myc at threonine 58 was markedly induced. The latter is known to destabilize Myc and target it for proteasomal degradation. We also discovered that systemic delivery of decorin using three distinct tumor xenograft models caused down-regulation of Met and a concurrent suppression of β-catenin and Myc levels. We found that decorin protein core labeled with the near infrared dye IR800 specifically targeted the tumor cells expressing Met. Even 68-h post-injection, decorin was found to reside within the tumor xenografts with little or no binding to other tissues. Collectively, our results indicate a role for a secreted proteoglycan in suppressing the expression of key oncogenic factors required for tumor progression.     

Artificial production of agarwood oil in <Emphasis Type="Italic">Aquilaria</Emphasis> sp. by fungi: a review

The resinous part called agarwood of genus Aquilaria Lam has been exploited as a source of incense by mankind since time immemorial besides its medicinal properties. In global market purest oud/agarwood oil enjoys an exorbitant price tag owing to its mystical fragrance. This has led to over-exploitation of Aquilaria sp. at worldwide level. All the Aquilaria sp. are protected under CITES since 2002. Agarwood production is considered to have a pathological relation, secreted actively in response to foreign body infection as a means of defense mechanism in Aquilaria sp. In order to fulfill the demand for sustainable production of agarwood, chemical, physical and biological methods of inducing artificial infection for agarwood enhancement/production have been formulated. In addition, artificial inoculation of fungi has been used to induce agarwood in Aquilaria trees. In this review, we have discussed the success and limitations of various methods employed for agarwood stimulation and provided the analysis of investigation conducted by various researchers to ascertain the contributory role of fungi (endophytic–pathogenic–epiphytic) on agar formation and chemical composition of oil from artificially infected agarwood. Based on available literature, Xylaria sp., Lasiodiplodia sp., Colletotrichum sp. and Botryosphaeria sp. are promising fungal isolates which deserved to be further studied and scaled up to a commercial level for production of agarwood oil.     

Isolation of pure,catalytically active human liver monoamine oxidase B: Antibody complex

Monoamine oxidase B was purified from human liver mitochondria using a monoclonal antibody, MAO B-1C2, which recognizes monoamine oxidase B but not A. Triton X-100 extracts of mitochondria were incubated with purified MAO B-1C2 (IgG₁), and the catalytically active enzyme:antibody complex was isolated by affinity chromatography on Protein A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex revealed the presence of four polypeptide bands (monoamine oxidase B, 57,900 dalton; antibody heavy chain, 52,200 dalton; and two light chains, 29,400 and 27,700 dalton), and indicated a 1:1 stoichiometric ratio of enzyme to antibody. This method gave 154-fold purification of the enzyme from mitochondria.     

The dystonia gene THAP1 controls DNA double-strand break repair choice

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