Human alveolar T lymphocyte responses to Mycobacterium tuberculosis antigens: role for CD4+ and CD8+ cytotoxic T cells and relative resistance of alveolar macrophages to lysis.

T cell-mediated cytotoxicity against Mycobacterium tuberculosis (MTB)-infected macrophages may be a major mechanism of specific host defense, but little is known about such activities in the lung. Thus, the capacity of alveolar lymphocyte MTB-specific cell lines (AL) and alveolar macrophages (AM) from tuberculin skin test-positive healthy subjects to serve as CTL and target cells, respectively, in response to MTB (H37Ra) or purified protein derivative (PPD) was investigated. Mycobacterial Ag-pulsed AM were targets of blood CTL activity at E:T ratios of > or = 30:1 (51Cr release assay), but were significantly more resistant to cytotoxicity than autologous blood monocytes. PPD- plus IL-2-expanded AL and blood lymphocytes were cytotoxic for autologous mycobacterium-stimulated monocytes at E:T ratios of > or = 10:1. The CTL activity of lymphocytes expanded with PPD was predominantly class II MHC restricted, whereas the CTL activity of lymphocytes expanded with PPD plus IL-2 was both class I and class II MHC restricted. Both CD4+ and CD8+ T cells were enriched in BL and AL expanded with PPD and IL-2, and both subsets had mycobacterium-specific CTL activity. Such novel cytotoxic responses by CD4+ and CD8+ T cells may be a major mechanism of defense against MTB at the site of disease activity.

Photoinduced DNA and Protein Cleavage Activity of Ferrocene-Conjugated Ternary Copper(II) Complexes

Ferrocene-conjugated ternary copper(II) complexes [Cu(L)(B)](ClO4)(2), where L is FcCH(2)N(CH2Py)(2) (Fc = (eta(5)-C5H4)Fe-II(eta(5)-C5H5)) and B is a phenanthroline base, viz., 2,2'-bipyridine (bpy, 1), 1, 10-phenanthroline (phen, 2), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 3), and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4), have been synthesized and characterized by various spectroscopic and analytical techniques. The bpy complex 1, as its hexafluorophosphate salt, has been structurally characterized by X-ray crystallography. The molecular structure shows the copper(II) center having an essentially square-pyramidal coordination geometry in which L with a pendant ferrocenyl (Fc) moiety and bpy show respective tridentate and bidentate modes of binding to the metal center. The complexes are redox active, showing a reversible cyclic voltammetric response of the Fc(+)-Fc couple near 0.5 V vs SCE and a quasi-reversible Cu(II)-Cu(I) couple near 0.0 V. Complexes 2-4 show binding affinity to calf thymus (CT) DNA, giving binding constant (K-b) values in the range of 4.2 x 10(4) to 2.5 x 10(5) M-1. Thermal denaturation and viscometric titration data suggest groove binding and/or a partial intercalative mode of binding of the complexes to CT DNA. The complexes show good binding propensity to the bovine serum albumin (BSA) protein, giving K-BSA values of similar to 10(4) M-1 for the bpy and phen complexes and similar to 10(5) M-1 for the dpq and dppz complexes. Complexes 2-4 exhibit efficient chemical nuclease activity in the presence of 3-mercapto-propionic acid (MPA) as a reducing agent or hydrogen peroxide (H2O2) as an oxidizing agent. Mechanistic studies reveal formation of hydroxyl radicals as the reactive species. The dpq and dppz complexes are active in cleaving supercoiled (SC) pUC19 DNA on photoexposure to visible light of different wavelengths including red light using an argon-krypton mixed gas ion laser. Mechanistic investigations using various inhibitors reveal the fort-nation of hydroxyl radicals in the DNA photocleavage reactions. The dppz complex 4, which shows efficient photoioduced BSA cleavage activity, is a potent multifunctional model nuclease and protease in the chemistry of photodynamic therapy (PDT) of cancer.

Photoinduced DNA and Protein Cleavage Activity of Ferrocene-Appended L-Methionine Reduced Schiff Base Copper(II) Complexes of Phenanthroline Bases

Ferrocene-appended ternary copper(H) complexes of phenanthroline bases having CuN3OS coordination with an axial Cu-S bond derived from L-methionine reduced Schiff base shows red light induced oxidative DNA cleavage activity following a hydroxyl radical pathway. The dipyridophenazine complex, in addition, displays photoinduced oxidative cleavage of bovine serum albumin protein in UV-A light.

Novel Biginelli dihydropyrimidines with potential anticancer activity: A parallel synthesis and CoMSIA study

Novel Biginelli dihydropyrimidines of biological interest were prepared using p-toluene sulphonic acid as an efficient catalyst. All the thirty-two synthesised dihydropyrimidines were evaluated for their in vitro antioxidant activity using DPPH method. Only, compounds 28 and 29 exhibited reasonably good antioxidant activity. Furthermore, the synthesised Biginelli compounds were subjected for their in vitro anticancer activity against MCF-7 human breast cancer cells. The title compounds were tested at the concentration of 10 μg. Compounds exhibited activity ranging from weak to moderate and, from moderate to high in terms of percentage cytotoxicity. Among them, compounds 10 and 11 exhibited significant anticancer activity. In order to elucidate the three-dimensional structure–activity relationships (3D QSAR) towards their anticancer activity, we subjected them for comparative molecular similarity indices analysis (CoMSIA). Illustration regarding their synthesis, analysis, antioxidant activity, anticancer activity and 3D QSAR study is described.

Disease burden due to Streptococcus dysgalactiae subsp equisimilis (group G and C streptococcus) is higher than that due to Streptococcus pyogenes among Mumbai school children

Streptococcus pyogenes [group A streptococcus (GAS)], a human pathogen, and Streptococcus dysgalactiae subsp. equisimilis [human group G and C streptococcus (GGS/GCS)] are evolutionarily related, share the same tissue niche in humans, exchange genetic material, share up to half of their virulence-associated genes and cause a similar spectrum of diseases. Yet, GGS/GCS is often considered as a commensal bacterium and its role in streptococcal disease burden is under-recognized. While reports of the recovery of GGS/GCS from normally sterile sites are increasing, studies describing GGS/GCS throat colonization rates relative to GAS in the same population are very few. This study was carried out in India where the burden of streptococcal diseases, including rheumatic fever and rheumatic heart disease, is high. As part of a surveillance study, throat swabs were taken from 1504 children attending 7 municipal schools in Mumbai, India, during 2006-2008. GAS and GGS/GCS were identified on the basis of beta-haemolytic activity, carbohydrate group and PYR test, and were subsequently typed. The GGS/GCS carriage rate (1166/1504, 11%) was eightfold higher than the GAS carriage (22/1504, 1.5%) rate in this population. The 166 GGS/GCS isolates collected represented 21 different emm types (molecular types), and the 22 GAS isolates represented 15 different emm types. Although the rate of pharyngitis associated with GGS/GCS is marginally lower than with GAS, high rates of throat colonization by GGS/GCS underscore its importance in the pathogenesis of pharyngitis.

Growth of and trehalase activity in the thermophilic fungusThermomyces lanuginosus

The thermophilic fungus,Thermomyces lanuginosus, was grown in a glucose-asparagine liquid medium. Optimal mycelial growth occurred at 50°C. The conditions for sporulation were different from those required for vegetative growth. the former being favoured by lower nitrogen level and temperature. Trehalase (α, α-glu coside-l-glucohydrolase, EC 3.2.1.28) was one of the most active glycosidases at 50°C. Non-sporulating mycelium had higher levels of this enzyme than the sporulating mycelium. Trehalase was synthesized constitutively and its activity appears to be controlled by catabolite repression.

Characterization of mitochondrial neutral protease activity and the response of lysosomal enzymes to clofibrate feeding in rat liver

The phosphate-inhibitable neutral protease activity of the heavy mitochondrial fraction of rat liver is of lysosomal origin. The activity is essentially due to the thiol proteinases of the lysosomes. Digitonin treatment of the mitochondrial fraction results in the release of about 85 per cent of the neutral protease activity and the residual activity has an alkaline pH optimum and is not inhibited by phosphate. Clofibrate feeding at 0.5 per cent level in the diet results in enhanced levels of lysosomal enzymes. The increase is however restricted to the lysosome-rich fraction such that the activities associated with the heavy mitochondrial fraction show a significant decrease. It is suggested that clofibrate inhibits engulfment of mitochondria by lysosomes and this results in enhanced mitochondrial protein content.

Generation of a Manganese Specific Restriction Endonuclease with Nicking Activity

A typical feature of type II restriction endonucleases (REases) is their obligate sequence specificity and requirement for Mg2+ during catalysis. R.KpnI is an exception. Unlike most other type II REases, the active site of this enzyme can accommodate Mg2+, Mn2+, Ca2+, or Zn2+ and cleave DNA. The enzyme belongs to the HNH superfamily of nucleases and is characterized by the presence of a beta beta alpha-Me finger motif. Residues D148, H149, and Q175 together form the HNH active site and are essential for Mg2+ binding and catalysis. The unique ability of the enzyme to cleave DNA in the presence of different metal ions is exploited to generate mutants that are specific to one particular metal ion. We describe the generation of a Mn2+-dependent sequence specific endonuclease, defective in DNA cleavage with Mg2+ and other divalent metal ions. In the engineered mutant, only Mn2+ is selectively bound at the active site, imparting Mn2+-mediated cleavage. The mutant is impaired in concerted double-stranded DNA cleavage, leading to accumulation of nicked intermediates. The nicking activity of the mutant enzyme is further enhanced by altered reaction conditions. The active site fluidity of R Eases allowing flexible accommodation of catalytic cofactors thus forms a basis for engineering selective metal ion-dependent REase additionally possessing nicking activity.

A Rapid Bioassay for Measuring Inhibin Activity

A sensitive and rapid bioassay of inhibin is described. Swiss albino female mice, 27 days old, weighing 25-30 g, were primed with 20 IU hCG, given in 2 equal doses, administered at 0900 h and 1800 h. An increment of 200% in uterine weight could be observed when the animals were autopsied the next day at 1000 h. The validity of the endpoint chosen, as a function dependent on FSH, was shown by the fact that neutralization of endogenous FSH by FSH antiserum led to an inhibition of uterine weight increase. Further injection of the inhibin material caused an actual reduction in the heightened levels of FSH (ng/ml) brought about by hCG: Control, 207 ± 13.6; hCG, 365 ± 28.8 and hCG inhibin, below the detection level of radioimmunoassay. Inhibin preparation from ovine testicular extract when tested at 3 dose levels showed a dose dependent and significant suppression in uterine weight increase. The assay was statistically validε = 2.5; slope = 23.9; λ = 0.104 and intra- and interassay variation = 8.3% and 11.7%, respectively. Since the mouse uterine weight assay is specific, has greater sensitivity and is uniformly reproducible, it is suggested that this assay procedure is ideally suited to assess the activity of different inhibin test preparations as well as to follow the purification of inhibin activity from crude material.

Regulation of respiratory activity of brown adipose tissue mitochondria through changes in cytochromes.

Exposure of cold-acclimatized rats to heat (37 degrees C) for a short period decreased brown adipose tissue (BAT) mitochondrial substrate-dependent oxygen uptake and H2O2 generation. Both the concentration and substrate-dependent rate of cytochrome b reduction decreased as early as 3 h of heat exposure. These results identify cytochrome b as the locus of regulation of electron transport in BAT mitochondria under conditions of heat stress.

Synthesis, characterization and antioxidant activity of angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) catalyzes the conversion of angiotensin I (Ang I) to angiotensin II (Ang II). ACE also cleaves the terminal dipeptide of vasodilating hormone bradykinin (a nonapeptide) to inactivate this hormone. Therefore, inhibition of ACE is generally used as one of the methods for the treatment of hypertension. `Oxidative stress' is another disease state caused by an imbalance in the production of oxidants and antioxidants. A number of studies suggest that hypertension and oxidative stress are interdependent. Therefore, ACE inhibitors having antioxidant property are considered beneficial for the treatment of hypertension. As selenium compounds are known to exhibit better antioxidant behavior than their sulfur analogues, we have synthesized a number of selenium analogues of captopril, an ACE inhibitor used as an antihypertensive drug. The selenium analogues of captopril not only inhibit ACE activity but also effectively scavenge peroxynitrite, a strong oxidant found in vivo.

Identification and characterization of cysteine proteinases of Trypanosoma evansi

Trypanosoma evansi is a causative agent of `surra', a common haemoprotozoan disease of livestock in India causing high morbidity and mortality in disease endemic areas. The proteinases released by live and dead trypanosomes entail immunosuppression in the infected host, which immensely contribute in disease pathogenesis. Cysteine proteinases are identified in the infectious cycle of trypanosomes such as cruzain from Trypanosoma cruzi, rhodesain or brucipain from Trypanosoma brucei rhodesiense and congopain from Trypanosoma congelense. These enzymes localised in lysosome-like organelles, flagellar pocket and on cell surface, which play a critical role in the life cycle of protozoan parasites, viz. in host invasion, nutrition and alteration of the host immune response. The paper describes the identification of cysteine proteinases of T. evansi lysate, activity profile at different pH optima and inhibition pattern using a specific inhibitor, besides the polypeptide profile of an antigen. Eight proteinases of T. evansi were identified in the molecular weight (MW) ranges of 28-170 kDa using gelatin substrate-polyacrylamide gel electrophoresis (GS-PAGE), and of these proteinases, six were cysteine proteinases, as they were inhibited by L-3-carboxy-2,3-transepoxypropionyl-lecuylamido (4-guanidino)-butane (E-64), a specific inhibitor. These proteolytic enzymes were most reactive in acidic pH between 3.0 and 5.5 in the presence of dithiothreitol and completely inactive at alkaline pH 10.0. Similarly, the GS-PAGE profile of the serum samples of rats infected with T. evansi revealed strong proteolytic activity only at the 28-kDa zone at pH 5.5, while no proteolytic activity was observed in serum samples of uninfected rats. Further, the other zones of clearance, which were evident in T. evansi antigen zymogram, could not be observed in the serum samples of rats infected with T. evansi. The polypeptide pattern of the whole cell lysate antigen revealed 12-15 polypeptide bands ranging from 28 to 81 kDa along with five predominant polypeptides bands (MW of 81, 66, 62, 55 and 45 kDa), which were immunoreactive with hyperimmune serum (HIS) and serum of experimentally infected rabbits with T. evansi infection. The immunoblot recognised antibodies in experimentally infected rabbits and against HIS as well, corresponding to the zone of clearances at lower MW ranges (28-41 kDa), which may be attributed to the potential of these proteinases in the diagnosis of T. evansi infection. Since these thiol-dependent enzymes are most active in acidic pH and considering their inhibition characteristics, these data suggest that they resemble to the mammalian lysosomal cathepsin B and L.

Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of beta-amyloid peptides (A beta) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma A beta and a decrease in brain A beta monomer after 7 d, indicating increased transport of A beta from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the A beta-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain A beta levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain A beta. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain A beta, indicating that increase in liver LRP and sLRP occurring independent of A beta concentration could result in clearance of A beta. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for A beta clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.

Ag@AgI, Core@Shell Structure in Agarose Matrix as Hybrid: Synthesis, Characterization, and Antimicrobial Activity

A novel in situ core@shell structure consisting of nanoparticles of Ag (Ag Nps) and AgI in agarose matrix (Ag@ AgI/agarose) has been synthesized as a hybrid, in order to have an efficient antibacterial agent for repetitive usage with no toxicity. The synthesized core@shell structure is very well characterized by XRD, UV-visible, photoluminescence, and TEM. A detailed antibacterial studies including repetitive cycles are carried out on Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria in saline water, both in dark and on exposure to visible light. The hybrid could be recycled for the antibacterial activity and is nontoxic toward human cervical cancer cells (HeLa cells). The water insoluble Ag@AgI in agarose matrix forms a good coating on quartz, having good mechanical strength. EPR and TEM studies are carried out on the Ag@AgI/agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria.

Discovery of novel glitazones incorporated with phenylalanine and tyrosine: Synthesis, antidiabetic activity and structure-activity relationships

We report a series of new glitazones incorporated with phenylalanine and tyrosine. All the compounds were tested for their in vitro glucose uptake activity using rat-hemidiaphragm, both in presence and absence of insulin. Six of the most active compounds from the in vitro screening were taken forward for their in vivo triglyceride and glucose lowering activity against dexamethazone induced hyperlipidemia and insulin resistance in Wistar rats. The liver samples of rats that received the most active compounds, 23 and 24, in the in vivo studies, were subjected to histopathological examination to assess their short term hepatotoxicity. The investigations on the in vitro glucose uptake, in vivo triglyceride and glucose lowering activity are described here along with the quantitative structure-activity relationships. (C) 2012 Elsevier Inc. All rights reserved.