Escherichia coli Mutants Permissive for T4 Bacteriophage with Deletion in e Gene (Phage Lysozyme)

Escherichia coli mutants have been isolated that are permissive for the infection by T4 phage with deletion in the cistron for the phage lysozyme, the e gene. Some, but not all, of these mutants are simultaneously permissive for the infection by T4 phage defective in the t gene, the product of which has also been implicated in the release of progeny phages. Most of these mutants shared the following properties: temperature sensitivity in growth and cell division, increased sensitivity towards a number of unrelated antibiotics and colicins, and increased sensitivity towards anionic detergents (sodium dodecyl sulfate and sodium deoxycholate). The possible biochemical basis for these phenotypes is discussed.     

Effect of Naturally Occurring Xanthines on Bacteria: I. Antimicrobial Action and Potentiating Effect on Antibiotic Spectra

The effect of xanthines on various microorganisms was studied. The antibacterial effect was not high; most of the test organisms could easily withstand a concentration of 2,500 μg/ml. Caffeine was more antibacterial than theophylline, and the latter more than theobromine. Caffeine citrate exhibited greater inhibitory effect than did pure caffeine. The effect was both bacteriostatic and bactericidal against susceptible organisms. The susceptibility of organisms to xanthines differed greatly even in related species. The morphology of Aerobacter aerogenes and A. cloacae was affected under the influence of caffeine; filamentation of cells followed sublethal doses. Potentiation was seen with antibiotics and caffeine; resistant strains were killed with a lower dose of drug in the presence of caffeine. This potentiating effect was pronounced with the tetracyclines; with streptomycin, the effect was the contrary.     

TRAF6 promotes TGFβ-induced invasion and cell-cycle regulation via Lys63-linked polyubiquitination of Lys178 in TGFβ type I receptor

Transforming growth factor β (TGFβ) can act either as a tumor promoter or a tumor suppressor in a context-dependent manner. High levels of TGFβ are found in prostate cancer tissues and correlate with poor patient prognosis. We recently identified a novel TGFβ-regulated signaling cascade in which TGFβ type I receptor (TβRI) is activated by the E3 ligase TNF-receptor-associated factor 6 (TRAF6) via the Lys63-linked polyubiquitination of TβRI. TRAF6 also contributes to activation of TNF-α-converting enzyme and presenilin-1, resulting in the proteolytic cleavage of TβRI and releasing the intracellular domain of TβRI, which is translocated to the nucleus to promote tumor invasiveness. In this report, we provide evidence that Lys178 of TβRI is polyubiquitinated by TRAF6. Moreover, our data suggest that TRAF6-mediated Lys63-linked ubiquitination of the TβRI intracellular domain is a prerequisite for TGFβ regulation of mRNA for cyclin D1 (CCND1), expression, as well as for the regulation of other genes controlling the cell cycle, differentiation, and invasiveness of prostate cancer cells.     

Novel morpholine ketone analogs as potent histamine H3 receptor inverse agonists with wake activity

Structure-activity relationship on a novel ketone class of H(3)R antagonists/inverse agonists is disclosed. Compound 4 showed excellent target potency, selectivity and brain penetration. Evaluation of antagonist 4 in the rat EEG/EMG model demonstrated robust wake activity thereby establishing preclinical proof of concept.     

Computational evidence to inhibition of human acetyl cholinesterase by withanolide a for Alzheimer treatment

Alzheimer's disease (AD), a neurodegenerative disorder, is the most common cause of dementia. So far only five drugs have been approved by US FDA that temporarily slow worsening of symptoms for about six to twelve months. The limited number of therapeutic options for AD drives the exploration of new drugs. Enhancement of the central cholinergic function by the inhibition of acetylcholinesterase is a prominent clinically effective approach for the treatment of AD. Recently withanolide A, a secondary metabolite from the ayurvedic plant Withania somnifera has shown substantial neuro-protective ability. The present study is an attempt to elucidate the cholinesterase inhibition potential of withanolide A along with the associated binding mechanism. Our docking simulation results predict high binding affinity of the ligand to the receptor. Further, long de novo simulations for 10_ns suggest that ligand interaction with the residues Thr78, Trp81, Ser120 and His442 of human acetylcholinesterase, all of which fall under one or other of the active sites/subsites, could be critical for its inhibitory activity. The study provides evidence for consideration of withanolide A as a valuable small ligand molecule in treatment and prevention of AD associated pathology. The present information could be of high value for computational screening of AD drugs with low toxicity to normal cells. Accurate knowledge of the 3D structure of human acetylcholinesterase would further enhance the potential of such analysis in understanding the molecular interaction basis between ligand and receptor.     

Zebrafish caspase-3: molecular cloning, characterization, crystallization and phylogenetic analysis

Apoptosis plays an important role in maintaining the normal function of various tissues and organs in different species. Caspase-3 is a terminal caspases which plays an important role in the execution of apoptosis in all vertebrates. It was cloned from zebra fish embryos and its properties were identified through Western blotting and biological activity. In the cells over-expressing caspase-3, Western blotting with an anti-His-tag antibody confirmed the presence of caspase-3 in the three bands that were proposed to correspond to the precursor form (33 kDa), the mature forms processed at the prodomain alone (29 kDa, large subunit) and small sub unit (13 kD). Fish kidney cells were transiently co-transfected with the beta-galactosidase reporter gene and either vector alone (mock), pZCASP3His (caspase-3) or pZCASP3His mutant (caspase-3 mutant). After 72 h following transfection of fish kidney cells, 35% of cells transfected with the zebra fish caspase-3 construct, pZCASP3His, showed apoptotic morphology when compared with cells transfected with the mock vector or an expression construct (pZCASP3His mutant) encoding the caspase-3 mutant lacking Cys. The fusion proteins were expressed in Escherichia coli, isolated from cell lysates by nickel-affinity column chromatography, and cleaved with thrombin. A thrombin cleavage recognition site was positioned at the fusion junction to release the caspase-3 from the fusion protein. Phylogenetic analysis showed that the cloned zebra fish caspase was a member of the caspase-3 subfamily with approximately 60% identity with caspase-3 from Xenopus, chicken and mammals. We have obtained structural information by X-ray crystallography. Orthorhombic crystals of the caspase-3 that diffracted to 1.8 A were obtained in a mixture of 0.1 M imidazole (pH 6.0) and 0.4 M NaOAc (pH 7.0 -7.5), containing 30% glycerol. The space group is C222 with cell dimensions of a = 36.07 A, b = 38.80 A, c = 135.20 A.     

Ambulatory-Based Standardized Therapy for Multi-Drug Resistant Tuberculosis: Experience from Nepal, 2005–2006

Objective

The aim of this study was to describe treatment outcomes for multi-drug resistant tuberculosis (MDR-TB) outpatients on a standardized regimen in Nepal.

Methodology

Data on pulmonary MDR-TB patients enrolled for treatment in the Green Light Committee-approved National Programme between 15 September 2005 and 15 September 2006 were studied. Standardized regimen was used (8Z-Km-Ofx-Eto-Cs/16Z-Ofx-Eto-Cs) for a maximum of 32 months and follow-up was by smear and culture. Drug susceptibility testing (DST) results were not used to modify the treatment regimen. MDR-TB therapy was delivered in outpatient facilities for the whole course of treatment. Multivariable analysis was used to explain bacteriological cure as a function of sex, age, initial body weight, history of previous treatment and the region of report.

Principal Findings

In the first 12-months, 175 laboratory-confirmed MDR-TB cases (62% males) had outcomes reported. Most cases had failed a Category 2 first-line regimen (87%) or a Category 1 regimen (6%), 2% were previously untreated contacts of MDR-TB cases and 5% were unspecified. Cure was reported among 70% of patients (range 38%–93% by Region), 8% died, 5% failed treatment, and 17% defaulted. Unfavorable outcomes were not correlated to the number of resistant drugs at baseline DST. Cases who died had a lower mean body weight than those surviving (40.3 kg vs 47.2 kg, p<0.05). Default was significantly higher in two regions [Eastern OR = 6.2; 95%CL2.0-18.9; Far West OR = 5.0; 95%CL1.0-24.3]. At logistic regression, cure was inversely associated with body weight <36 kg [Adj.OR = 0.1; 95%CL0.0-0.3; ref. 55–75 kg] and treatment in the Eastern region [Adj.OR = 0.1; 95%CL0.0-0.4; ref. Central region].

Conclusions

The implementation of an ambulatory-based treatment programme for MDR-TB based on a fully standardized regimen can yield high cure rates even in resource-limited settings. The determinants of unfavorable outcome should be investigated thoroughly to maximize likelihood of successful treatment.