Molecular cloning and characterization of Brugia malayi hexokinase

5' EST from filarial gene database has been subjected to 3' rapid amplification of cDNA ends (RACE), semi-nested PCR and PCR to obtain full-length cDNA of Brugia malayi. Full-length hexokinase gene was obtained from cDNA using gene specific primers. The elicited PCR product was cloned, sequenced and expressed as an active enzyme in Escherichia coli. Sequence analysis of B. malayi hexokinase (BmHk) revealed 59% identity with nematode Caenorhabditis elegans but low similarity with all other available hexokinases including human. BmHk, an apparent tetramer with subunit molecular mass of 72 kDa, was able to phosphorylate glucose, fructose, mannose, maltose and galactose. The Km values for glucose, fructose and ATP were found to be 0.035+/-0.005, 75+/-0.3 and 1.09+/-0.5 mM respectively. BmHk was strongly inhibited by ADP, glucosamine, N-acetyl glucosamine and mannoheptulose. The recombinant enzyme was found to be activated by glucose-6-phosphate. ADP exhibited noncompetitive inhibition with the substrate glucose (Ki=0.55 mM) while, mixed type of inhibition was observed with inorganic pyrophosphate (PPi) when ATP was used as substrate (Ki=9.92 microM). The enzyme activity is highly dependent on maintenance of free sulfhydryl groups. CD analysis indicated that BmHk is composed of 37% alpha-helices and 26% beta-sheets. The observed differences in kinetic properties of BmHk as compared to host enzyme may facilitate designing of specific inhibitors against BmHk.     

Pharmacological inhibition of inducible nitric oxide synthase attenuates the development of seizures in mice

The present study has been designed to pharmacologically expound the significance of inducible nitric oxide synthase in the pathophysiological progression of seizures using mouse models of chemically induced kindled epilepsy and status epilepticus induced spontaneous recurrent seizures. Pentylenetetrazole (40 mg kg⁻¹) (PTZ) administration every second day for a period of 15 days was used to elicit kindled seizure activity in mice. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Pilocarpine (100 mg kg⁻¹) was injected every 20 min until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of quantitative assessment of the progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus. Sub-acute PTZ administration induced the development of severe form of kindled seizures in mice. Further, pharmacological status epilepticus elicited a progressive evolution of spontaneous recurrent seizures in the animals. However, treatment of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide synthase, markedly and dose dependently suppressed the development of both PTZ induced kindled seizures as well as pilocarpine induced spontaneous recurrent seizures. Therefore inducible nitric oxide synthase may be implicated in the development of seizures.     

Evaluation of the toxicity of different phytoextracts of Ocimum basilicum against Anopheles stephensi and Culex quinquefasciatus

The larvicidal effect of the crude carbon tetrachloride, methanol and petroleum ether leaf extracts of a widely grown medicinal plant, Ocimum basilicum, against Anopheles stephensi and Culex quinquefasciatus was evaluated. Petroleum ether extract was found to be the most effective against the larvae of both mosquitoes, with LC₅₀ values of 8.29, 4.57; 87.68, 47.25 ppm and LC₉₀ values of 10.06, 6.06; 129.32, 65.58 ppm against A. stephensi and C. quinquefasciatus being observed after 24 and 48 h of treatment, respectively. The efficacy of petroleum ether was followed by that of the carbon tetrachloride and methanol extracts, which had LC₅₀ values of 268.61, 143.85; 446.61, 384.84 ppm and LC₉₀ values of 641.23, 507.80; 923.60, 887.00 ppm against A. stephensi after 24 and 48 h, respectively, and LC₅₀ values of 24.14, 17.02; 63.48, 53.77 ppm and LC₉₀ values of 295.38, 204.23; 689.71, 388.87 ppm against C. quinquefasciatus after 24 and 48 h of treatment, respectively. These extracts are highly toxic against mosquito larvae from a range of species; therefore, they may be useful for the management of mosquito larvae to control vector borne diseases.     

Cellulase production by six <Emphasis Type="Italic">Trichoderma</Emphasis> spp. fermented on medicinal plant processings

Capabilities of cellulase production, using delignified bioprocessings of medicinal and aromatic plants, viz. citronella (Cymbopogon winterianus) and Artemisia annua (known as marc of Artemisia) and garden waste (chiefly containing Cynodon dactylon), by the six species of Trichoderma were comparatively evaluated. Among the members of Trichoderma studied, T. citrinoviride was found to be the most efficient producer of cellulases along with a high level of β- glucosidase (produced 102.4 IU g⁻¹ on marc of Artemisia; 101.33 IU g⁻¹ on garden waste; 81.86 IU g⁻¹ on distillation waste of citronella and 94.77 IU g⁻¹ on pure cellulose). Although T. virens was noticed to be the minimal enzyme producer fungus, it interestingly could not produce complete cellulase enzyme complex on any test waste or pure cellulose, except on marc of Artemisia, where it produced all three enzymes of the complex. Immediate reduction in pH was also noticed during fermentation in the case of pure polymer (cellulose) by all tested fungi, while it was delayed with delignified agrowastes. The pH profile varied with the substrate used as well as with individual species of Trichoderma. On the other hand, no alteration in pH with any species of Trichoderma was noticed when grown on marc of A. annua, which might be due to the buffering capacity of this marc.     

Phosphate-buffered saline-based nucleofection of primary endothelial cells

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.     

Genetic Diversity and Differentiation in Hedychium spicatum, a Valuable Medicinal Plant of Indian Himalaya

Hedychium spicatum, a perennial rhizomatous medicinal plant distributed in subtropical and temperate parts, is considered nearly endemic to the Himalayan region.In this study allozyme markers were utilized to assess genetic variations and relationships among 12 distinct populations of this species from the West Himalaya of India. A high level of genetic diversity was found among the populations. Of the 13 loci generated using eight enzymes, 12 (92%) were polymorphic. F-statistics suggested a prevalence of a high heterozygote excess among populations (F(IS) = -0.51). Gene flow estimates and geographic distances between populations did not correlate significantly (r = -0.0258, P < 0.3550). The excess heterozygosity may be attributed to high pollinator mobility and inbreeding coefficient within the subpopulation, relative to the total F(IS) value. High frequencies of several alleles at different loci probably reflect the breeding pattern, as the species propagates clonally as well as through seeds. We also discuss conservation implications for the target species.     

A repertoire of biomarkers helps in detection and assessment of therapeutic response in epithelial ovarian cancer

In epithelial ovarian cancer (EOC), the cancer antigen 125 (CA-125) has been conventionally used to help in diagnosis and assessment of response to treatment. Currently, YKL-40 (Tyrosine–Lysine–Leucine-40) and circulating cell-free DNA are being evaluated for possession of similar ability. In this study, we aimed to assess the ability of a repertoire of potential biomarkers in detecting and assessing therapeutic response, in advanced EOC. Blood levels of CA-125, YKL-40, total cell-free DNA (CFDNA), cell-free nuclear DNA (CFnDNA), and cell-free mitochondrial DNA (CFmDNA) levels were measured in 100 untreated patients of advanced EOC from November 2009 to June 2011, and again on treatment completion from the 20 patients who appeared for follow-up analysis. Significantly, higher proportion of untreated patients had serum CA-125 >3 times upper limit of normal (ULN) (90.0 %; P < 0.0001) and plasma YKL-40 >ULN (77.0 %; P < 0.0001), both of which significantly decreased, Posttherapy. posttherapy, CFDNA (P < 0.0001), and CFnDNA (P < 0.0001) levels significantly decreased as compared to pretreatment levels. Positive and significant correlations existed between pretherapy CFDNA and CFnDNA [Spearman rho (ρ) = 1.000; P < 0.0001], and also with CFmDNA (ρ = 0.301; P = 0.002), separately between CFnDNA and CFmDNA (ρ = 0.303; P = 0.002), as well as between plasma YKL-40 and patient age (ρ = 0.353; (P < 0.0001). On treatment completion, CFDNA and CFnDNA levels showed positive and significant correlation (ρ = 1.000; P < 0.0001). Therefore serum CA-125 and plasma YKL-40 aid detection and assessment of therapeutic response, in advanced EOC. CFDNA and CFnDNA help in estimating extent of therapeutic response in advanced EOC.     

Structural and Functional Insights into the Catalytic Inactivity of the Major Fraction of Buffalo Milk Xanthine Oxidoreductase

Background

Xanthine oxidoreductase (XOR) existing in two interconvertible forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species.

Methods and Findings

Buffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (k _cat/K _m) of 0.11 sec⁻¹ µM⁻¹ of buffalo XO was 8–10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε_{430 nm}) of 46,000 M⁻¹ cm⁻¹ suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum) were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD⁺ entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum.

Conclusion

A major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way.     

Central leptin gene therapy blocks ovariectomy-induced adiposity

Objective: In this study, we tested the hypothesis that insufficiency of leptin restraint in the hypothalamus is responsible for promoting weight gain and adiposity after ovariectomy (ovx). Whether increasing leptin transgene expression can overcome the diminution in leptin restraint was evaluated in ovx rats. Research Methods and Procedures: Enhanced leptin or green fluorescent protein (GFP; control) transgene expression was induced by a single intracerebroventricular injection of recombinant adeno‐associated viral vector encoding either leptin gene (rAAV‐lep) or GFP gene (rAAV‐GFP; control) in acutely and chronically ovx rats. Body weight and food intake responses were monitored weekly. White adipose tissue (WAT) mass and serum levels of WAT‐derived hormones, leptin, and adiponectin were analyzed at termination of the experiments. Results and Discussion: An increase in leptin transgene expression in the hypothalamus initiated soon after ovx blocked hyperphagia and body weight gain and markedly suppressed WAT mass and adipokines, leptin, and adiponectin. Similar suppression of weight gain and adiposity and serum leptin and adiponectin levels after intracerebroventricular rAAV‐lep injection in chronically ovx rats were observed concomitant with unchanged daily food intake. These findings are consistent with the hypothesis that in the absence of ovarian steroids, the existent insufficiency of leptin restraint at the hypothalamic level can be overcome with ectopic leptin expression, thereby reinstating central control on weight and adiposity.