Purification of recombinant human phosphodiesterase 7A expressed in Dictyostelium discoideum

Phosphodiesterase plays an important role in regulating inflammatory pathways and T cell function. The development of phosphodiesterase 7 inhibitor may give better efficacy profile over phosphodiesterase 4 inhibitors. However, the recombinant phosphodiesterase 7 is required in large quantity for high-throughput screening of new drugs by in vitro enzymatic assays. In the present study, recombinant human PDE7A1 was expressed in Dictyostelium discoideum under the control of constitutively active actin-15 promoter. The cytosolic localization of the expressed protein was confirmed by immunofluorescence studies. Upto 2 mg of recombinant protein was purified using His-Tag affinity column chromatography followed by ion-exchange Resource Q column purification. The recombinant protein expressed in D. discoideum followed Michaelis–Menten kinetics similar to the protein expressed in mammalian system and showed no major changes in affinity to substrate or inhibitors. Thus, our study clearly demonstrates a robust expression system for successful bulk production of pharmacologically active isoform of human PDE7A1 required for high-throughput assays.     

Leishmania donovani pteridine reductase 1: biochemical properties and structure-modeling studies

Pteridine reductase 1 (PTR1, EC 1.5.1.33) is a NADPH dependent short-chain reductase (SDR) responsible for the salvage of pterins in the protozoan parasite Leishmania. This enzyme acts as a metabolic bypass for drugs targeting dihydrofolate reductase, therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Based on homology model drawn on recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecular modeling and docking studies with two compounds of dihydrofolate reductase specificity showing promising antileishmanial activity in vitro. Both the inhibitors appeared to fit well in the active pocket revealing the tight binding of the carboxylic acid ethyl ester group of pyridine moiety to pteridine reductase and identify the important interactions necessary to assist the structure based development of novel pteridine reductase inhibitors.     

Induction,resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio vulnificus in artificial sea water

Vibrio vulnificus, an important food-borne pathogen, is known to enter viable but nonculturable (VBNC) state under low temperature and low nutrition stress conditions. Present study examined the time required for induction of VBNC state and temperature which induces resuscitation of V. vulnificus YJ016. The change in cell morphology and gene expression during VBNC state and in resuscitated cells was also examined. V. vulnificus incubated in artificial sea water at 4 °C entered VBNC state after considerably extended time (70 days). An increase in temperature by 6 °C from the VBNC induction temperature (4 °C) resulted in resuscitation of VBNC cells; however, maximum resuscitation was observed when VBNC cells were held at 23 °C for 24 h. VBNC cells changed their morphology from comma shape to coccoid shape. Two rounds of induction of VBNC and resuscitation were possible with V. vulnificus cells; however, there was progressive reduction in number of resuscitated cells and after 190 days cells failed to resuscitate. Significant up-regulation of genes related to membrane proteins [porinH (10.4-fold), ompU (2.9-fold)], regulatory proteins [envZ (5.6-fold), toxR (4.5-fold), toxS (4.8-fold)], oxidative stress related protein katG (2.3-fold), cell division/maintenance proteins [ftsZ (4.3), mreB (6.5-fold)] and resuscitating promoter factor yeaZ (fourfold) was observed during resuscitation with respect to VBNC state indicating that these genes play a role during resuscitation. Gene expression data presented here would enhance our understanding of resuscitation of V. vulnificus from VBNC state. The results also highlight the importance of maintenance of low temperature during storage of seafood.     

Cross‐compartment proteostasis regulation during redox imbalance induced ER stress

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle‐specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ‐based LC–MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross‐talk between ER‐ and mitochondrial proteostasis exemplified by an Ire1p‐dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross‐talk during stress.     

Determination of strain energy function for arterial elastin: Experiments using histology and mechanical tests

The long-range reversible deformation of vertebrate arteries is primarily mediated by elastin networks that endure several million deformation cycles without appreciable fatigue. To determine how elastin contributes to the composite arterial properties, we studied the three-dimensional microstructure and biomechanics of isolated elastin. We initially estimated the sensitivity of these studies by comparing two elastin isolation protocols, autoclaving and alkali-extraction, and measured their effect on isolated elastin using uniaxial tests and histology. These studies show that autoclaved tissues have a trend for higher modulus (900.79+/-678.02 kPa) than alkali-extracted samples (417.74+/-162.23 kPa)albeit with higher collagen-proteoglycan impurities, and (2) greater optical density (78.6+/-9.1%) than alkali-extracted groups (46.2+/-5.9%), suggesting that autoclaving is superior to alkali-extraction for biomechanical tests on elastin. Using these data we show that an isotopic Mooney-Rivlin model cannot adequately represent arterial elastin. The neo-Hookean model, with coefficient 162.57 (+/-115.44) kPa for autoclaved and 76.94 (+/-27.76) kPa for alkali-extracted samples, fits the uniaxial data better. Autoclaved elastins also show linear stress-strain response and equal stiffness in circumferential and axial directions suggesting equal number of layers in these directions and that elastin may help distribute tensile stresses during vessel inflation. Histology of autoclaved and control porcine arteries reveals axial elastin fibers in intimal and adventitial layers but circumferential medial fibers. We propose an orthotropic material symmetry for arterial elastin with two orthogonally oriented and symmetrically placed mechanically equivalent fibers. An exact form of the constitutive equation will be obtained in a future study.     

Evaluation of the presence of reduced nicotinamide adenine dinucleotide phosphate in bacterial metabolites used as immunostimulators and its role in nitric oxide induction

Bacterial metabolites that act as immunostimulators have aroused interest because of their therapeutic potential in several immune disorders. These metabolites are complex, heterogeneous, and comprise numerous immune‐boosting biomolecules. To better understand their immune stimulatory properties, characterization of their components is essential. An ether extract of metabolites from nine bacterial species was analyzed for the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) or other fluorophores. This metabolite in combination with bile lipids is a licensed immune stimulatory drug. Excitation of the extract at 340 nm resulted in fluorescence with an emission maximum of around 410 nm, which is fairly specific for NADH and NADPH. Reverse‐phase‐HPLC and electro‐spray ionization‐mass analysis confirmed the presence of NADPH in the bacterial metabolites. Quantification by glutathione reductase assay indicated 11.90 ± 0.01 µM of NADPH in the metabolites. Further characterization of the individual bacterial extracts of the metabolite confirmed the presence of NADPH. Subsequently, studies were performed to evaluate the role/s of NADPH in immune‐stimulatory drugs. NADPH is known to be involved in production of nitric oxide (NO), which has versatile roles in the immune system. The biological function of NADPH in NO induction by RAW 264.7 (mouse macrophage) cells was evaluated and it was found that bacterial NADPH has a significant role in inducing NO and that NADPH from individual bacterial extracts is capable of inducing NO. Investigation on the stability and biological potency of NADPH in bacterial metabolites is important because of NADPH's wide therapeutic applications, most of which are associated with its role in NO induction.     

Visiting the cell biology of Salmonella infection

Salmonella, a Gram-negative facultative intracellular pathogen is capable of infecting vast array of hosts. The striking ability of Salmonella to overcome every hurdle encountered in the host proves that they are true survivors. In the host, Salmonella infects various cell types and needs to survive and replicate by countering the defense mechanism of the specific cell. In this review, we will summarize the recent insights into the cell biology of Salmonella infection. Here, we will focus on the findings that deal with the specific mechanism of various cell types to control Salmonella infection. Further, the survival strategies of the pathogen in response to the host immunity will also be discussed in detail. Better understanding of the mechanisms by which Salmonella evade the host defense system and establish pathogenesis will be critical in disease management.     

IL28B alleles exert an additive dose effect when applied to HCV-HIV coinfected persons undergoing peginterferon and ribavirin therapy

Background

Genetic studies have demonstrated a strong association between single nucleotide polymorphisms (SNPs) at IL28B and response to treatment with peginterferon (PEG) and ribavirin (RBV) in HCV monoinfected persons. We sought to test these associations in a prospective PEG / weight based ribavirin (WBR) treatment trial (ACTG A5178) (National Institution of Health registration number {"type":"clinical-trial","attrs":{"text":"NCT00078403","term_id":"NCT00078403"}}NCT00078403) in persons with HCV-HIV coinfection, and to develop a prediction score.

Methods

We selected subjects enrolled in A5178 who completed at least the first 12 weeks of the trial and had DNA available, and genotyped three SNPs at IL28B (rs12979860, rs12980275, rs8099917). We used multivariate logistic regression analysis to evaluate the association between IL28B SNPs and HCV treatment outcomes and to develop the prediction score.

Results

231 HCV/HIV coinfected subjects were included. We observed a strong association between IL28B genotype and response to therapy among those with genotypes 1 or 4 (odds ratio for complete early virologic responses (cEVR) and sustained virologic response (SVR) was 2.98 [1.7–5.3] and 3.4 [1.7–6.9], respectively, for each additional copy of the C allele of rs12979860). Differences in frequency of the responder allele explained some of the discrepancy in HCV treatment outcomes between blacks and whites. A simple pretreatment prediction score that incorporates the IL28B genotype and baseline HCV viral load has a 93% negative predictive value (NPV) for SVR.

Conclusions

IL28B SNPs have an additive allele dose effect in predicting HCV treatment outcomes in HCV/HIV coinfected persons and can be incorporated into a simple pretreatment prediction score that could minimize the risk of exposure to PEG/RBV therapy for persons with unfavorable scores.     

Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules

The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.     

Rates and pattern of ovule abortion vis-à-vis <Emphasis Type="Italic">in situ</Emphasis> pollen germination in some populations of <Emphasis Type="Italic">Trifolium fragiferum</Emphasis> L.

The present study is based on four populations of Trifolium fragiferum L. of the family Fabaceae growing at four different sites in Jammu region, India. The species, which grows as a common weed in the area of study, follows an annual life cycle of about 3½?month in the subtropical climates of Jammu region. While all of these populations were recorded in full bloom during February and March, they displayed a temporal scatter. Detailed studies revealed these population types to be morphologically similar but distinct in the many aspects studied. An interesting phenomenon noted for the plants of this species was in situ pollen germination, which was recorded in about 28.8% of the flowers studied. The species under investigation also showed an appreciable amount of ovule abortion. The ovule abortion in pistils was found to be non-random, with the peduncular ovule aborting at a higher rate than the stylar one. The rates and patterns of ovule abortion were studied vis-à-vis in situ pollen germination and were compared between different populations. Interesting results were obtained, indicative of the fact that precocious pollen germination does affect the ovule abortion in one way or other.