Perkinsus marinus Extracellular Protease Modulates Survival of Vibrio vulnificus in Eastern Oyster (Crassostrea virginica) Hemocytes

The in vitro effects of the Perkinsus marinus serine protease on the intracellular survival of Vibrio vulnificus in oyster hemocytes were examined by using a time-course gentamicin internalization assay. Results showed that protease-treated hemocytes were initially slower to internalize V. vulnificus than untreated hemocytes. After 1 h, the elimination of V. vulnificus by treated hemocytes was significantly suppressed compared with hemocytes infected with invasive and noninvasive controls. Our data suggest that the serine protease produced by P. marinus suppresses the vibriocidal activity of oyster hemocytes to effectively eliminate V. vulnificus, potentially leading to conditions favoring higher numbers of vibrios in oyster tissues.     

Stochastic simulations on the reliability of action potential propagation in thin axons

It is generally assumed that axons use action potentials (APs) to transmit information fast and reliably to synapses. Yet, the reliability of transmission along fibers below 0.5 μm diameter, such as cortical and cerebellar axons, is unknown. Using detailed models of rodent cortical and squid axons and stochastic simulations, we show how conduction along such thin axons is affected by the probabilistic nature of voltage-gated ion channels (channel noise). We identify four distinct effects that corrupt propagating spike trains in thin axons: spikes were added, deleted, jittered, or split into groups depending upon the temporal pattern of spikes. Additional APs may appear spontaneously; however, APs in general seldom fail (<1%). Spike timing is jittered on the order of milliseconds over distances of millimeters, as conduction velocity fluctuates in two ways. First, variability in the number of Na channels opening in the early rising phase of the AP cause propagation speed to fluctuate gradually. Second, a novel mode of AP propagation (stochastic microsaltatory conduction), where the AP leaps ahead toward spontaneously formed clusters of open Na channels, produces random discrete jumps in spike time reliability. The combined effect of these two mechanisms depends on the pattern of spikes. Our results show that axonal variability is a general problem and should be taken into account when considering both neural coding and the reliability of synaptic transmission in densely connected cortical networks, where small synapses are typically innervated by thin axons. In contrast we find that thicker axons above 0.5 μm diameter are reliable.     

In vitro examination of anti-parasitic,anti-Alzheimer,insecticidal and cytotoxic potential of Ajuga bracteosa Wallich leaves extracts

This research study is mainly focused to evaluate the anti-parasitic, insecticidal, cytotoxic and anti-alzheimer potential of various leaf extracts of Ajuga bracteosa Wallich ex Bentham. 04 different extracts were prepared using solvent of different polarity to determine the best candidate for potent bioactivity i.e. n-hexane (NH), Ethyl acetate (EA), Ethanol (EL) and Chloroform (CH). Concentrations of each extracts were made specified for all activities. All extracts were exploited for broad range of biomedical applications including leishmaniasis, in vitro anti-Alzheimer, insecticidal and cytotoxic studies. Our results showed that A. bracteosa n-hexane extract was highly active against Leishmania Tropica with significant inhibition of 58 ± 1.61 for promastigote and 63 ± 2.29 for amastigote at 1000 μg/mL. Furthermore, promising anti-alzheimer activity acetylcholinesterase (AChE) 46 ± 0.83 and butrylcholineterase (BChE) 49 ± 1.17 was noted for n-hexane. The insecticidal potential of these extracts were test against five different insects (Rhyzopertha dominica, Trogoderma granarium, Tribolium castaneum, Sitophilus oryze, and Callosobruchus analis). The higest mortality rate of insecticidal activity was recorded by n-hexane followed by Ethyl acetate whereas ethanol extract was found to be less effective against all the test species. Significant cytotoxic potential of each plant sample against Artemia salina thus aware us for further detailed research to find out novel drugs. Based on our results we believe that Ajuga bracteosa could be used to develop as a potential botanical insecticide against different insect and pests, such as aphids as well as an excellent source for the compound isolation as anti-tumor agent.     

Illegitimate recombination: An efficient method for random mutagenesis in Mycobacterium avium subsp. hominissuis

ABSTRACT: BACKGROUND: The genus Mycobacterium (M.) comprises highly pathogenic bacteria such as M. tuberculosis as well as environmental opportunistic bacteria called non-tuberculous mycobacteria (NTM). While the incidence of tuberculosis is declining in the developed world, infection rates by NTM are increasing. NTM are ubiquitous and have been isolated from soil, natural water sources, tap water, biofilms, aerosols, dust and sawdust. Lung infections as well as lymphadenitis are most often caused by M. avium subsp. hominissuis (MAH), which is considered to be among the clinically most important NTM. Only few virulence genes from M. avium have been defined among other things due to difficulties in generating M. avium mutants. More efforts in developing new methods for mutagenesis of M. avium and identification of virulence-associated genes are therefore needed. RESULTS: We developed a random mutagenesis method based on illegitimate recombination and integration of a Hygromycin-resistance marker. Screening for mutations possibly affecting virulence was performed by monitoring of pH resistance, colony morphology, cytokine induction in infected macrophages and intracellular persistence. Out of 50 randomly chosen Hygromycin-resistant colonies, four revealed to be affected in virulence-related traits. The mutated genes were MAV_4334 (nitroreductase family protein), MAV_5106 (phosphoenolpyruvate carboxykinase), MAV_1778 (GTP-binding protein LepA) and MAV_3128 (lysyl-tRNA synthetase LysS). CONCLUSIONS: We established a random mutagenesis method for MAH that can be easily carried out and combined it with a set of phenotypic screening methods for the identification of virulence-associated mutants. By this method, four new MAH genes were identified that may be involved in virulence.     

Purification and characterization of a second type of neutral ceramidase from rat brain: a second more hydrophobic form of rat brain ceramidase

Ceramidases (CDase) are enzymes that catalyze the hydrolysis of N-acyl linkage of ceramide (Cer) to generate sphingosine and free fatty acids. In this study we report the purification and characterization of a novel second type of neutral ceramidase from rat brain (RBCDase II). Triton X-100 protein extract from rat brain membrane was purified sequentially using Q-Sepharose, HiLoad16/60 Superdex 200pg, heparin-Sepharose, phenyl-Sepharose HP, and Mono Q columns. After Mono Q, the specific activity of the enzyme increased by ~15,000-fold over that of the rat brain homogenate. This enzyme has pH optima of 7.5, and it has a larger apparent molecular weight (110kDa) than the previously purified (90kDa) and characterized neutral rat brain CDase (RBCDase I). De-glycosylation experiments show that the differences in molecular mass of RBCDase I and II on SDS-PAGE are not due to the heterogeneity with N-glycan. RBCDase II is partially stimulated by Ca(2+) and is inhibited by pyrimidine mono nucleotides such as TMP and UMP. This finding is significant as it demonstrates for the first time an effect by nucleotides on a CDase activity. The enzyme was also inhibited by both oxidized and reduced GSH. The effects of metal ions were examined, and we found that the enzyme is very sensitive to Hg(2+) and Fe(3+), while it is not affected by Mn(2+). EDTA was somewhat inhibitory at a 20mM concentration.     

Synthesis and biological evaluation of benzoisothiazole derivatives possessing N,N-dimethylformimidamide group as 5-HT₆ receptor antagonists

A series of novel N,N-dimethyl-N'-(5-(Ar-sulfonamido) benzo[d]isothiazol-3-yl)formimidamides was designed and synthesized as 5-HT(6) ligands. Here N,N-dimethyl formimidamides was used as a replacement for an aminoethyl moiety. In vitro functional assays demonstrated compounds 9b and 9i significantly inhibited the 5-HT-induced Ca(2+) increases (9b; IC(50)=0.36 μM and 9i; IC(50)=0.44 μM), indicating that 9b and 9i were potent 5-HT(6) receptor antagonists. Compounds 9i also showed good selectivity on the 5-HT(6) over 5-HT(4) and 5-HT(7) receptors.     

Growth stimulatory effect of Ochrobactrum intermedium and Bacillus cereus on Vigna radiata plants

AIMS: This study assessed the plant growth-promoting ability of the bacterial strains Ochrobactrum intermedium (isolate CrT-1) and Bacillus cereus (isolate S-6). METHODS AND RESULTS: Two chromium resistant bacterial strains isolated from chromium-contaminated wastewater and soils were identified as O. intermedium CrT-1 and B. cereus S-6. These strains were inoculated on seeds of mungbean Vigna radiata var NM-92, which were germinated and grown under chromate salts (300 microg ml(-1) of CrCl(3)or K(2)CrO(4)). The data show that Cr(VI) was more toxic because of its better availability to plants roots when compared with Cr(III). The major part of Cr(VI) supplied to the seedlings was reduced to Cr(III) in the rhizosphere by the bacterial strains, thus lowering the toxicity of chromium to seedlings. CONCLUSIONS: Strains have significant Cr(VI) resistance and reduction potential and have ability to enhance mungbean plant growth under chromium stress. SIGNIFICANCE AND IMPACT OF THE STUDY: These strains could be utilized for the growth of economically important cash crops as well as for the bioremediation of chromium-polluted soils.     

Development and validation of a dried blood spot—LC–APCI–MS assay for estimation of canrenone in paediatric samples

A selective and sensitive liquid chromatography (LC)–atmospheric pressure chemical ionisation (APCI)–mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 μl of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17α-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC–APCI–MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25–1000 ng/ml (r > 0.994). Accuracy (% RE) and precision (% CV) values for within and between day were <20% at the lower limit of quantification (LLQC) and <15% at all other concentrations tested. The LLOQ of the method was validated at 25 ng/ml. Clinical validation of the method was achieved by employing the validated method for analysis of 160 DBS samples from 37 neonatal and paediatric patients.