Formation and Clearance of Norepinephrine Glycol Metabolites in Mouse Brain

To determine the degree of conversion of 3,4-dihydroxyphenylethyleneglycol (DHPG) to 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and the amount of DHPG eliminated unchanged from the brain, we have examined the kinetics of formation and disappearance of mouse brain MHPG and DHPG following clorgyline (10 mg/kg, i.p.) and/or tropolone (75 mg/kg, i.p.) treatment. During the first 10 min after tropolone, brain DHPG levels accumulated linearly at a rate of 1,300 pmol/g/h, whereas MHPG disappeared exponentially at a rate of 411 pmol/g/h. Following clorgyline administration, brain DHPG declined exponentially at a rate of 1,240 pmol/g/h. In contrast, the elimination of MHPG became a first-order process only when catechol-O-methyltransferase (COMT) was also inhibited in addition to monoamine oxidase. Thus, combined clorgyline and tropolone treatment resulted in an exponential decline of MHPG levels at a rate of 524 pmol/g/h, whereas DHPG levels were slightly but significantly elevated compared to control values. When the animals were treated with pargyline (75 mg/kg, i.p.) in combination with clorgyline and tropolone, brain DHPG and MHPG disappeared at rates of 40 and 660 pmol/g/h, respectively. The above observations suggest that mouse brain DHPG is cleared primarily through O-methylation with minimal direct elimination from brain. Assuming the disposition and clearance of norepinephrine metabolites are similar in mouse and human brain, peripherally measured DHPG in humans is likely derived principally from extracerebral sources and reflects peripheral sympathetic function.     

Inhibition of 17,20(17-hydroxyprogesterone)-lyase by progesterone

¹⁴C-17-Hydroxyprogesterone was incubated with 7000 × g × 20 min supernatants of rat testis homogenates in the presence of various concentrations of ³H-progesterone, both under conditions where metabolism would take place and where it would be prevented. When metabolism was prevented, the ratio of progesterone to 17-hydroxyprogesterone in the microsomal fraction was 3 times that which was added to the incubation medium.Progesterone competitively inhibited 17,20-lyase action on added 17-hydroxyprogesterone but not on 17-hydroxyprogesterone formed from the added progesterone. The rate of formation of 17-hydroxyprogesterone from progesterone, however, was inhibited by added 17-hydroxyprogesterone. The results indicate that there is no free exchange of an intermediate between progesterone and androstenedione with the soluble fraction, either inside or outside the microsomal vesicle. The limited exchange with 17-hydroxyprogesterone in solution probably represents exchange with an enzyme-bound intermediate.     

Design and in silico modeling of Indoloquinoxaline incorporated keratin nanoparticles for modulation of glucose metabolism in 3T3-L1 adipocytes

The following study was done to assess the glucose utilizing efficiency of Indoloquinoxaline derivative incorporated keratin nanoparticles (NPs) in 3T3-L1 adipocytes. Indoloquinoxaline derivative had wide range of biological activities including antidiabetic activity. In this view, Indoloquinoxaline moiety containing N, N-dimethyl (3-fluoro-6H-indolo [3,2-b] quinoxalin-6-yl) methanamine compound was designed and synthesized, and further it is incorporated into keratin nanoparticles. The formulated NPs, drug entrapment efficiency, releasing capacity, stability, and physicochemical properties were characterized by various spectral analyzer and obtained results of characterizations were confirmed the properties of NPs. The analysis of mechanism underlying the glucose utilization of NPs was examined through molecular docking with identified target, and observed in silico study reports shown strong interaction of NPs in the binding pockets of AMPK and PTP1B. Based on the in silico screening, the formulated NPs was performed for in vitro cellular viability and glucose uptake studies on 3T3-L1 adipocytes. Interestingly, 40 μg of NPs displayed 78.2 ± 2.76% cellular viability, and no cell death was observed at lower concentrations. Further, the concentration dependent glucose utilization was observed at different concentrations of NPs in 3T3-L1 adipocytes. The results of NPs (40 μg) on glucose utilization have revealed eminent result 58.56 ± 4.54% compared to that of Metformin (10 μM) and Insulin (10 μM). The identified results clearly indicated that Indoloquinoxaline derivative incorporated keratin NPs significantly increased glucose utilization efficiency and protect the cells against the insulin resistance.     

Dynamic large branching hash tree based secure and efficient dynamic auditing protocol for cloud environment

Cluster Computing - Cloud computing model offers various platforms services and provides a scalable, on-demand service at any time-anywhere manner. However, in the outsourcing strategy, users no...     

Development of SCAR markers for the DNA-based detection of the Asian long-horned beetle,Anoplophora glabripennis (Motschulsky)

DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence characterized amplified regions (SCARs) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after screening 230 random primers in a PCR-based assay system. Three pairs of nested 22-mer oligonucleotide primers were designed on the basis of the sequence of this fragment and were used to perform diagnostic PCR. The first pair of primers (SCAR1) amplified a single 745-bp fragment of ALB DNA, but this did not differentiate ALB from other species. The other two pairs of SCAR primers (SCAR2 and SCAR3) amplified bands of 1,237- and 2,720-bp, respectively, that were capable of differentiating ALB from other closely related non-native and native Cerambycids, such as A. chinensis (Forster), A. malasiaca (Thomson), A. nobilis (Ganglbauer), Monochamus scutellatus (Say), Plectrodera scalator (Fab), Saperda tridentata (Olivier), and Graphisurus fasciatus (Degeer). The latter two SCAR markers could be amplified using DNA extracted from body parts of ALB such as the wing, the leg, and the antennae as well as tissues from all the developmental stages including the egg, larva, pupa, and adult. These markers were also capable of identifying ALB using the DNA extracted from frass. Our results demonstrate that the SCAR markers we have identified can be used for unambiguously identifying ALB from other closely related Cerambycids using a simple PCR procedure.     

Synthesis and the biological evaluation of arylnaphthalene lignans as anti-hepatitis B virus agents

We have previously shown that helioxanthin can suppress human hepatitis B virus gene expression. A series of helioxanthin analogues were synthesized and evaluated for their anti-hepatitis B virus activity. Modifications at the lactone rings and methylenedioxy unit of helioxanthin can modulate the antiviral activity. Among them, compound 32 is the most effective anti-HBV agent. Compound 32 can suppress the secretion of viral surface antigen and e antigen in HepA2 cells with EC₅₀ values of 0.06 and 0.14 μM, respectively. Compound 32 not only inhibited HBV DNA with wild-type and lamivudine-resistant strain but also suppressed HBV mRNA, core protein and viral promoters. In this study, a full account of the preparation, structure–activity relationships of helioxanthin analogues, and the possible mechanism of anti-HBV activity of this class of compounds are presented. This type of compounds possesses unique mode of action differing from existing therapeutic drugs. They are potentially new anti-HBV agents.     

Rescue and production of vaccine and therapeutic adenovirus vectors expressing inhibitory transgenes

Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels. A key advantage to this system is that repression of transgene expression is mediated by the packaging cell line, thus, expression of regulatory products from the adenovector are not required. This provides a simple, broadly applicable system wherein transgene repression is constitutive during vector rescue and growth and there is no effect on adenovector-mediated expression of gene products in transduced cells. Several high-level expression vectors based on first- and second-generation adenovectors were rescued and produced to high titer that otherwise could not be grown. Yields of adenovectors expressing inhibitory transgene products were increased, and the overgrowth of cultures by adenovectors with nonfunctional expression cassettes was prevented. The gene regulation system is a significant advancement for the development of adenovirus vectors for vaccine and other gene transfer applications.     

Significance of the 19-kDa hemolymph protein HP19 for the development of the rice moth Corcyra cephalonica: morphological and biochemical effects caused by antibody application

The hemolymph protein HP19 of the rice moth, Corcyra cephalonica, mediates the 20-hydroxyecdysone (20E)-dependent acid phosphatase (ACP) activity at a nongenomic level. Affinity-purified polyclonal antibody against HP19 (alphaHP19-IgG) was used in the present study to understand the role of HP19 during the postembryonic development of Corcyra. In the in vitro studies, HP19 action was blocked either by immuno-precipitation using alphaHP19-IgG, prior to its addition to the fat body culture or by the addition of the antibody directly to the culture, along with 20E and hemolymph containing HP19. The alphaHP19-IgG blocked the HP19-mediated 20E-dependent ACP activation. In the in vivo studies, the alphaHP19-IgG was injected into the fully developed last (final/Vth) instar larvae of Corcyra, to complex the HP19 in vivo, in order to block the action of HP19. The injection of alphaHP19-IgG resulted in defective development of larvae, which grew either into non-viable larvae or larval-pupal/pupal-adult intermediates relative to the effect of pre-immune IgG injected controls. The present study shows that HP19 plays an important role in controlling the metamorphosis of Corcyra by regulating the 20E-dependent ACP activity. Coupled with the earlier findings, the ecdysteroid hormone regulates this action at a nongenomic level.     

Identification and characterization of a juvenile hormone (JH) response region in the JH esterase gene from the spruce budworm, Choristoneura fumiferana

Using a differential display of mRNA technique we discovered that the juvenile hormone (JH) esterase gene (Cfjhe) from Choristoneura fumiferana is directly induced by juvenile hormone I (JH I), and the JH I induction is suppressed by 20-hydroxyecdysone (20E). To study the mechanism of action of these two hormones in the regulation of expression of this gene, we cloned the 1270-bp promoter region of the Cfjhe gene and identified a 30-bp region that is located between -604 and -574 and is sufficient to support both JH I induction and 20E suppression. This 30-bp region contains two conserved hormone response element half-sites separated by a 4-nucleotide spacer similar to the direct repeat 4 element and is designated as a putative juvenile hormone response element (JHRE). In CF-203 cells, a luciferase reporter placed under the control of JHRE and a minimal promoter was induced by JH I in a dose- and time-dependent manner. Moreover, 20E suppressed this JH I-induced luciferase activity in a dose- and time-dependent manner. Nuclear proteins isolated from JH I-treated CF-203 cells bound to JHRE and the binding was competed by a 100-fold excess of the cold probe but not by 100-fold excess of double-stranded oligonucleotides of unrelated sequence. JH I induced/modified nuclear proteins prior to their binding to JHRE and 20E suppressed this JH I induction/modification. These results suggest that the 30-bp JHRE identified in the Cfjhe gene promoter is sufficient to support JH induction and 20E suppression of the Cfjhe gene.     

Production of dimethylsulphide during the seasonal anoxia off Goa

Subsurface waters over the western Indian continental shelf experience seasonal anoxia towards the end of the southwest monsoon season. During a 3-day study carried out at the Candolim time series site (off the coast of Goa), dimethylsulphide (DMS) concentrations showed a 40-fold increase to a maximum of 442?nM at 25?m depth compared to the oxygenated surface waters. This extremely high DMS was found to be associated with relatively low chlorophyll a, low phytoplankton cell counts and a high concentration of hydrogen sulphide. However, total dimethylsulphoniopropionate, total dimethylsulphoxide and methanethiol concentrations were quite low and unlikely to account for the DMS build-up through presently known pathways of DMS production. While there are several possible mechanisms for the observed accumulation of DMS, we were unable to pinpoint the exact pathway of DMS production. Future work will involve investigation of the source of DMS through sediment slurry experiments, to explore this interesting link between the carbon and sulphur cycles under anoxic conditions.