Angiotensinergic neurons in sympathetic coeliac ganglia innervating rat and human mesenteric resistance blood vessels

In contrast to the current belief that angiotensin II (Ang II) interacts with the sympathetic nervous system only as a circulating hormone, we document here the existence of endogenous Ang II in the neurons of rat and human sympathetic coeliac ganglia and their angiotensinergic innervation with mesenteric resistance blood vessels. Angiotensinogen - and angiotensin converting enzyme-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia, while renin mRNA was untraceable. Cathepsin D, a protease responsible for cleavage beneath other substrates also angiotensinogen to angiotensin I, was successfully detected in rat coeliac ganglia indicating the possibility of existence of alternative pathways. Angiotensinogen mRNA was also detected by in situ hybridization in the cytoplasm of neurons of rat coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia as well as with mesenteric resistance blood vessels. By using confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally with the mesenteric resistance blood vessels.     

Evaluation of an immunodot test to manage white spot syndrome virus (WSSV) during cultivation of the giant tiger shrimp Penaeus monodon

A monoclonal antibody-based immunodot test was compared to a polymerase chain reaction (PCR) assay for managing white spot syndrome virus (WSSV) on shrimp farms at Kundapur and Kumta situated in Udupi and Uttar Kannada Districts, respectively, of Karnataka on the west coast of India. Of 12 grow-out farms in Kundapur, 6 (F1 to F6) yielded shrimp samples that were negative for WSSV by both immunodot test and 1-step PCR from stocking to successful harvest. Samples from the other 6 farms (F7 to F12) were positive for WSSV by both immunodot test and 1-step PCR at various times post stocking, and their crops failed. In the 2 farms at Kumta (F13, F14), immunodot and 1-step PCR results were both negative, and harvests were successful. In contrast to 1-step PCR results, farms F5, F6, F13, and F14 gave positive results for WSSV by 2-step PCR, and they were successfully harvested at 105 d post stocking. Our results indicate that an inexpensive immunodot assay can be used to replace the more expensive 1-step PCR assay for disease monitoring.     

Effects of the JAK2 inhibitor, AZ960, on Pim/BAD/BCL-xL survival signaling in the human JAK2 V617F cell line SET-2

The Janus-associated kinase 2 (JAK2) V617F mutation is believed to play a critical role in the pathogenesis of polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. We have characterized a novel small molecule JAK2 inhibitor, AZ960, and used it as a tool to investigate the consequences of JAK2 V617F inhibition in the SET-2 cell line. AZ960 inhibits JAK2 kinase with a K(i) of 0.00045 microm in vitro and treatment of TEL-JAK2 driven Ba/F3 cells with AZ960 blocked STAT5 phosphorylation and potently inhibited cell proliferation (GI(50)=0.025 microm). AZ960 demonstrated selectivity for TEL-JAK2-driven STAT5 phosphorylation and cell proliferation when compared with cell lines driven by similar fusions of the other JAK kinase family members. In the SET-2 human megakaryoblastic cell line, heterozygous for the JAK2 V617F allele, inhibition of JAK2 resulted in decreased STAT3/5 phosphorylation and inhibition of cell proliferation (GI(50)=0.033 microm) predominately through the induction of mitochondrial-mediated apoptosis. We provide evidence that JAK2 inhibition induces apoptosis by direct and indirect regulation of the anti-apoptotic protein BCL-xL. Inhibition of JAK2 blocked BCL-XL mRNA expression resulting in a reduction of BCL-xL protein levels. Additionally, inhibition of JAK2 resulted in decreased PIM1 and PIM2 mRNA expression. Decreased PIM1 mRNA corresponded with a decrease in Pim1 protein levels and inhibition of BAD phosphorylation at Ser(112). Finally, small interfering RNA-mediated suppression of BCL-xL resulted in apoptotic cell death similar to the phenotype observed following JAK2 inhibition. These results suggest a model in which JAK2 promotes cell survival by signaling through the Pim/BAD/BCL-xL pathway.     

Novel 3-methylindoline inhibitors of EZH2: Design,synthesis and SAR

EZH2 (enhancer of zeste homologue 2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the methylation of lysine 27 of histone H3 (H3K27). Dysregulation of EZH2 activity is associated with several human cancers and therefore EZH2 inhibition has emerged as a promising therapeutic target. Several small molecule EZH2 inhibitors with different chemotypes have been reported in the literature, many of which use a bicyclic heteroaryl core. Herein, we report the design and synthesis of EZH2 inhibitors containing an indoline core. Partial saturation of an indole to an indoline provided lead compounds with nanomolar activity against EZH2, while also improving solubility and oxidative metabolic stability.     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

Synthesis and antibacterial activity of novel (un)substituted benzotriazolyl oxazolidinone derivatives

A series of novel (un)substituted benzotriazolyl oxazolidinone derivatives has been synthesized and tested for in vitro antibacterial activities by MIC determination against a panel of susceptible and resistant Gram-positive and Gram-negative microorganisms, some of which are resistant to methicillin and vancomycin. Compounds 20, 21, 24, 29 and 30 from this series were found to be equipotent or more potent than linezolid in vitro.     

Bioconversion of 3 beta-acetoxypregna-5,16-diene-20-one to androsta-1,4-diene-3,17-dione by mixed bacterial culture

AIMS: To isolate a bacterium capable of degrading 3 beta-acetoxypregna-5,16-diene-20-one (16-DPA) to androsta-1,4-diene-3,17-dione (ADD) and to decipher the biodegradation pathway. METHODS AND RESULTS: Isolation on mineral salt agar containing 16-DPA as sole carbon source yielded two bacteria identified as Pseudomonas diminuta and Comamonas acidovorons. These bacteria failed to degrade 16-DPA individually in pure cultures but converted 16-DPA to ADD in a mixed culture. The intermediates accumulated during the bioconversion were identified as pregna-4,16-diene-3,20-dione and pregna-1,4,16-triene-3,20-dione. CONCLUSIONS: The degradation pattern of 16-DPA by mixed bacterial culture revealed the reaction sequence as (i) cleavage of C-3 acetyl function, (ii) dehydrogenation at C-1 and C-2 positions and (iii) cleavage of C-17 side-chain. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work opens a new approach towards the production of a female sex hormone precursor and elucidates the biodegradation pathway of 16-DPA by mixed bacterial culture.     

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.     

Formulation and roentgenographic studies of naproxen-pectin-based matrix tablets for colon drug delivery

A study has been carried out to assess the potential use of pectin in combination with two added hydrocolloids, i.e., hydroxy-propyl-methyl cellulose and hydroxyethyl cellulose in varied concentrations and coated with ethyl cellulose and cellulose acetate phthalate. The results of in vitro drug release showed that the matrix tablets prepared with pectin, hydroxy ethyl cellulose (20 percent) when coated with ethyl cellulose and cellulose acetate phthalate were found to be 63.0 percent, 8.4 percent, and 4.5 percent, respectively, in after eight hours during drug release study period. These results were confirmed with the results of roentgenographic studies in nine healthy human volunteers to find the shape and integrity of the dosage form. The X-ray photographs revealed that the enteric-coated tablet was visible only up to 5.5 hours and at the end of eighth hour, the photograph has not shown any presence of tablet indicating the loss of shape and size by the microflora present in the colon region. So, the results of in vitro and roentgenographic studies revealed that pectin, hydroxy ethyl cellulose (20 percent) base coated with ethyl cellulose and cellulose acetate phthalate was found to be a promising carrier for naproxen to colon.