Functional adaptation of Nef to the immune milieu of HIV-1 infection in vivo

Nef-mediated down-regulation of MHC class I (MHC-I) molecules on HIV-1-infected cells has been proposed to enhance viral persistence through evasion of host CTLs. This conclusion is based largely on demonstrations that Nef from laboratory HIV-1 strains reduces the susceptibility of infected cells to CTL killing in vitro. However, the function and role of Nef-mediated MHC-I down-regulation in vivo have not been well described. To approach this issue, nef quasispecies from chronically HIV-1-infected individuals were cloned into recombinant reporter viruses and tested for their ability to down-regulate MHC-I molecules from the surface of infected cells. The level of function varied widely between individuals, and although comparison to the immunologic parameters of blood CD4(+) T lymphocyte count and breadth of the HIV-1-specific CTL response showed positive correlations, no significant correlation was found in comparison to plasma viremia. The ability of in vivo-derived Nef to down-regulate MHC-I predicted the resistance of HIV-1 to suppression by CTL. Taken together, these data demonstrate the functionality of Nef to down-regulate MHC-I in vivo during stable chronic infection, and suggest that this function is maintained by the need of HIV-1 to cope with the antiviral CTL response.     

Fabrication of cytochrome c-poly(5-amino-2-napthalenesulfonic acid) electrode by one step procedure and direct electrochemistry of cytochrome c

Herein, we reported for the first time one step procedure for the preparation of cytochrome c (cyt c)-poly (5-amino-2-napthalenesulfonic acid) (PANS) modified glassy carbon electrode by cyclic voltammetrically (CV). Hereafter, we called the above modified electrode as cyt c-PANS electrode. The presence of cyt c on modified electrode was investigated with electrochemical quartz crystal microbalance (EQCM), CV, and superoxide radicals reaction studies. The reaction between cyt c in the modified electrode and superoxide radicals in solution, was exemplified by cyclic voltammetric measurements. Surface morphology of the modified electrode was investigated by using atomic force microscopy (AFM). The modified electrode showed a pair of well defined redox peak in PBS solution, pH 6.7. The modified electrode utilized for electrocatalytic reduction as well as amperometric determination of hydrogen peroxide (H(2)O(2)). The detection limit and linear range for H(2)O(2) were 5 and 50 microM to 7 mM, respectively.     

High pH during trisodium phosphate treatment causes membrane damage and destruction of Salmonella enterica serovar enteritidis

Trisodium phosphate (TSP) is now widely used during the processing of poultry and red meats, but the mechanism whereby it inactivates gram-negative bacteria such Salmonella spp. remains unclear. Thus, Salmonella enterica serovar Enteritidis (ATCC 4931) cells were treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) and compared with (i) cells treated with the same pH as the TSP treatments (pH 10.0, 10.5, and 11.0, respectively) and (ii) cells treated with different concentrations of TSP (1.5, 2.0, and 2.5% [wt/vol]) adjusted to a pH of 7.0 +/- 0.2 (mean +/- standard deviation). Cell viability, loss of membrane integrity, cellular leakage, release of lipopolysaccharides, and cell morphology were accordingly examined and quantified under the above treatment conditions. Exposure of serovar Enteritidis cells to TSP or equivalent alkaline pH resulted in the loss of cell viability and membrane integrity in a TSP concentration- or alkaline pH-dependent manner. In contrast, cells treated with different concentrations of TSP whose pH was adjusted to 7.0 did not show any loss of cell viability or membrane integrity. A 30-min pretreatment with 1.0 mM EDTA significantly enhanced the loss of membrane integrity only when followed by TSP or alkaline pH treatments. Measuring the absorbance at 260 nm, agarose gel electrophoresis, Bradford assay, and Tricine-sodium dodecyl sulfate gel electrophoresis of filtrates of treated cell suspensions revealed considerable release of DNA, proteins, and lipopolysaccharides compared to controls and pH 7.0 TSP treatments. Electron microscopic examination of TSP- or alkaline pH-treated cells showed disfigured cell surface topology and wrinkled appearance and showed evidence of a TSP concentration- and pH-dependent disruption of the cytoplasmic and outer membranes. These results demonstrate that TSP treatment permeabilizes and disrupts the cytoplasmic and outer membranes of serovar Enteritidis cells because of the alkaline pH, which in turn leads to release of intracellular contents and eventual cell death.     

Chromium(III)-induced apoptosis of lymphocytes: death decision by ROS and Src-family tyrosine kinases

Apoptosis is an active process induced by a variety of physiological and external stimuli, in which elimination of damaged cells are effected through a genetically controlled process. In this study, we have examined the mechanism of chromium(III) [Cr(III)]-induced cytotoxicity with respect to its relationship to oxidative stress. Morphology, flow cytometry, and DNA fragmentation studies show that tris-(1,10-phenanthroline)chromium(III) [Cr(III)-phen], tris-(2,2′-bipyridyl)chromium(III) [Cr(III)-bpy], trans-diaqua[1,2-bis(salicylideneamino)ethanechromium(III)] [Cr(III)-salen], and trans-diaqua[1,3-bis(salicylideneamino)propanechromium(III)] [Cr(III)-salprn] induced apoptosis of lymphocytes. Pentaammineaquachromium(III) [Cr(III)-hpa] does not induce apoptosis. Apoptosis induced by these complexes involves the generation of reactive oxygen species (ROS) as seen by increased fluorescence of dichloroflourescein (DCF) observed through flow cytometry. Pretreatment of lymphocytes with antioxidants completely abrogate apoptosis. Cr(III) treatment also increased the expression and activation of Src-family tyrosine kinases viz. p56lck, p59fyn, and p53/56lyn, as seen by immunoblotting and immune complex kinase assay. PP2, a selective Src-family tyrosine kinase inhibitor, abolishes apoptosis, indicating that Src-family tyrosine kinases are directly involved in eliciting apoptosis. Interestingly, a one-to-one correlation between the expression of Src-family tyrosine kinases and ROS is observed, since antioxidants pretreatment inhibits the expression and the activation of these kinases. These results further indicate that Cr(III)-induced apoptosis is mediated through production of ROS, which in turn activates the Src-family tyrosine kinases. The increased activation of Src-family tyrosine kinases may be a mechanism involved in apoptosis of lymphocytes elicited by various other physiological stimuli that exploit ROS as a second messenger.     

Mechanism of nicotinic acid transport in human liver cells: experiments with HepG2 cells and primary hepatocytes

This study reports on the functional expression of a specific, high-affinity carrier-mediated mechanism for the transport of niacin (nicotinic acid) in human liver cells. Both human-derived liver HepG2 cells and human primary hepatocytes were used as models in these investigations. The initial rate of transport of nicotinic acid into HepG2 cells was found to be acidic pH, temperature, and energy dependent; it was, however, Na(+) independent in nature. Evidence for the existence of a carrier-mediated system that is specific for [(3)H]nicotinic acid transport was found and included the following: 1) saturability as a function of concentration with an apparent K(m) of 0.73 +/- 0.16 microM and V(max) of 25.02 +/- 1.45 pmol.mg protein(-1).3 min(-1), 2) cis-inhibition by unlabeled nicotinic acid and nicotinamide but not by unrelated organic anions (lactate, acetate, butyrate, succinate, citrate, and valproate), and 3) trans-stimulation of [(3)H]nicotinic acid efflux by unlabeled nicotinic acid. Transport of the vitamin into human primary hepatocytes occurs similarly via an acidic pH-dependent and specific carrier-mediated process. Inhibitors of the Ca(2+)-calmodulin-mediated pathway (but not modulators of the PKC-, PKA-, and protein tyrosine kinase-mediated pathways) inhibited nicotinic acid transport into both HepG2 cells and human primary hepatocytes. Maintenance of HepG2 cells (for 48 h) in growth medium oversupplemented with nicotinic acid (or nicotinamide) did not affect the subsequent transport of [(3)H]nicotinic acid into HepG2 cells. These results show, for the first time, the existence of a specific and regulated membrane carrier-mediated system for nicotinic acid transport in human liver cells.     

Therapeutic Potential of the Medicinal Plant <i>Tinospora cordifolia</i>–Minireview

For thousands of years, plant based herbal medicines have been utilized by millions of people all over the world. Plant materials or products are used in different folk/traditional medical systems, such as the Chinese, African and Indian medical systems, like Siddha, Ayurveda, Unani, and Homeopathy. Tinospora cordifolia (TC) is a medicinal plant belonging to the family Menispermaceae. It is a big deciduous, climbing shrub growing prevalently in the tropical part of Indian subcontinent regions such as India, Pakistan, Nepal, Bhutan, Bangladesh and Srilanka, and in Myanmar, and China. Guduchi, Giloy, Shindilkodi, and Amritha are all the common names for this plant. Extracts from different parts of this herbal plant have been used to treat many diseases. In Ayurvedic medicine, extract from this plant is used for preparing “rasayanas”, which is known to cure diabetes, skin diseases, allergic conditions, jaundice, cardiovascular diseases, rheumatoid arthritis, poisoning, and microbial infections. T. cordifolia has a many bioactive phytochemicals that have been isolated from its aerial parts and roots. Many bioactive principles have been reported from this plant which belong to various classes like alkaloids, aliphatic compounds, diterpenoid lactones, phenolics, flavonoids, glycosides, sesquiterpenoids, lignans, steroids and polysaccharides. T. cordifolia possesses medicinal properties such as antioxidant, antiallergic, antiinflammatory, antimicrobial, antiviral, antidote, antitumor, antileprotic, antispasmodic, and antidiabetic properties. The present review will provide a comprehensive therapeutic potential of T. cordifolia.     

Interpolyelectrolyte Complexes of Eudragit? EPO with Hypromellose Acetate Succinate and Eudragit? EPO with Hypromellose Phthalate as Potential Carriers for Oral Controlled Drug Delivery

The objective of this study was to compare a novel controlled release tablet formulation based on interpolyelectrolyte complex (PEC). Interpolymer interactions between the countercharged polymers like Eudragit® EPO (polycation) and hypromellose acetate succinate (polyanion) and Eudragit® EPO and hypromellose phthalate (polyanion) were investigated with a view to their use in per oral controlled release drug delivery systems. The formation of inter-macromolecular ionic bonds between cationic polymer and anionic polymer was investigated using Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry. The FT-IR spectra of the tested polymeric matrices are characterized by visible changes in the observed IR region indicating the interaction between chains of two oppositely charged copolymers. The performance of the in situ formed PEC as a matrix for controlled release of drugs was evaluated, using acetaminophen as a model drug. The dissolution data of these matrices were fitted to different dissolution models. It was found that drug release followed zero-order kinetics and was controlled by the superposition of the diffusion and erosion. These profiles could be controlled by conveniently modifying the proportion of the polymer ratio, polymer type, and polymer concentration the in the tablets.KEY WORDS: Eudragit E, hypromellose acetate succinate, hypromellose phthalate polyelectrolyte complexation     

Minimum fluidization velocity and friction factor in a liquid-solid inverse fluidized bed reactor

In the present study the hydrodynamic characteristics (bed expansion and pressure drop) of low-density polyethylene (LDPE) and polypropylene (PP) are studied in a liquid-solid inverse fluidized bed reactor as a function of particle diameter, liquid viscosity and density. The bed expansion and pressure drop data are used to determine the minimum fluidization velocity, U_mf and friction factor, P. It was found that the U_mf increased with an increase in the particle diameter and a decrease in solid density and was independent of initial bed height (solid loading). In addition, the U_mf decreased with an increase in the CMC concentration. The friction factor Reynolds number plot was found similar to that of classical fluidization. Dimensionless equations were proposed for the. prediction of the friction factor and the U_mf.     

SMS: sequence, motif and structure--a database on the structural rigidity of peptide fragments in non-redundant proteins

Structure prediction methods aim to identify the relationship between the amino acid sequence of an unknown protein and information comprised in databases of known protein structures. Towards this end, we created a database by combining the amino acid sequences and the corresponding three-dimensional atomic coordinates for all the 25% non-redundant protein chains available in the Protein Data Bank. It contains information about the peptide fragments that are 5 to 10 residues long. In addition, options are provided for the users to visualize the individual motifs and the superposed fragments in the client machine. Further, useful functionalities areprovided to look for similar sequence motifs in all the sequence databases like PDB, 90% non-redundant protein chains, Genome database, PIR and Swiss-Prot. The database is being updated at regular intervals and the same can be accessed over the World Wide Web interface at the following URL: http://pranag.physics.iisc.ernet.in/sms/.