Studies on nutritional and oxygen requirements for production of L-asparaginase by Enterobacter aerogenes

The carbon and nitrogen sources most suitable for L-asparaginase production by Enterobacter aerogenes were selected and their concentrations optimized in shake-flask cultures. Sodium citrate (1.0%) and diammonium hydrogen phosphate (0.16%) proved to be the best sources of carbon and nitrogen, respectively. Nitrogen catabolite repression of enzyme formation was absent in this bacterium. Cultivation in a reactor showed that the dissolved oxygen level is the limiting factor for L-asparaginase production by E. aerogenes. Glucose was found to be a repressor of enzyme synthesis. Asparagine was absent intracellularly when the L-asparaginase level was high. An increase in the extracellular alanine level when the dissolved oxygen remained low indicated a shift from aerobic to fermentative metabolism. Received: 20 July 1999 / Accepted: 2 October 1999     

NMR detection of side chain–side chain hydrogen bonding interactions in 13C/15N-labeled proteins

We describe the direct observation of side chain–side chain hydrogen bonding interactions in proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the guanidinium nitrogen ¹⁵N of arginine 71, which serves as the hydrogen donor, and the acceptor carboxylate carbon ¹³CO₂ of aspartate 100 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _{N CO2} coupling by employing a novel HNCO-type experiment, soft CPD-HNCO. The ^3h J _{N CO2} coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains. The identification of trans-hydrogen bond J-couplings between protein side chains should provide useful markers for monitoring hydrogen bonding interactions that contribute to the stability of protein folds, to alignments within enzyme active sites and to recognition events at macromolecular interfaces.     

Zebrafish wnt4b expression in the floor plate is altered in sonic hedgehog and gli-2 mutants

The floor plate of the neural tube serves an important function as a source of signals that pattern cell fates in the nervous system as well as directing proper axon pathfinding. We have cloned a novel zebrafish wnt family member, wnt4b, which is expressed exclusively in the floor plate. To place wnt4b in the context of known regulators of midline development, its expression was analyzed in the zebrafish mutants cyclops (cyc), floating head (flh), you-too (yot), and sonic you (syu). wnt4b expression in the medial and lateral floor plate are shown to be regulated independently: medial floor plate expression occurs in the absence of a notochord, while lateral floor plate expression requires a functional notochord, sonic hedgehog and gli-2.     

Influence of heme post-translational modification and distal ligation on the backbone dynamics of a monomeric hemoglobin

The cyanobacterium Synechococcus sp. PCC 7002 uses a hemoglobin of the truncated lineage (GlbN) in the detoxification of reactive species generated in the assimilation of nitrate. In view of a sensing or enzymatic role, several states of GlbN are of interest with respect to its structure-activity relationship. Nuclear magnetic resonance spectroscopy was applied to compare the structure and backbone dynamics of six GlbN forms differing in their oxidation state [Fe(II) or Fe(III)], distal ligand to the iron (histidine, carbon monoxide, or cyanide), or heme post-translational modification (b heme or covalently attached heme). Structural properties were assessed with pseudocontact shift calculations. (15)N relaxation data were analyzed by reduced spectral density mapping (picosecond to nanosecond motions) and by inspection of elevated R(2) values (microsecond to millisecond motions). On the picosecond to nanosecond time scale, GlbN exhibited little flexibility and was unresponsive to the differences among the various forms. Regions of slightly higher mobility were the CE turn, the EF loop, and the H-H' kink. In contrast, fluctuations on the microsecond to millisecond time scale depended on the form. Cyanide binding to the ferric state did not enhance motions, whereas reduction to the ferrous bis-histidine state resulted in elevated R(2) values for several amides. This response was attributed, at least in part, to a weakening of the distal histidine coordination. Carbon monoxide binding quenched some of these fluctuations. The results emphasized the role of the distal ligand in dictating backbone flexibility and illustrated the multiple ways in which motions are controlled by the hemoglobin fold.     

Strain transfer through the aortic valve

The complex structural organization of the aortic valve (AV) extracellular matrix (ECM) enables large and highly nonlinear tissue level deformations. The collagen and elastin (elastic) fibers within the ECM form an interconnected fibrous network (FN) and are known to be the main load-bearing elements of the AV matrix. The role of the FN in enabling deformation has been investigated and documented. However, there is little data on the correlation between tissue level and FN-level strains. Investigating this correlation will help establish the mode of strain transfer (affine or nonaffine) through the AV tissue as a key feature in microstructural modeling and will also help characterize the local FN deformation across the AV sample in response to applied tissue level strains. In this study, the correlation between applied strains at tissue level, macrostrains across the tissue surface, and local FN strains were investigated. Results showed that the FN strain distribution across AV samples was inhomogeneous and nonuniform, as well as anisotropic. There was no direct transfer of the deformation applied at tissue level to the fibrous network. Loading modes induced in the FN are different than those applied at the tissue as a result of different local strains in the valve layers. This nonuniformity of local strains induced internal shearing within the FN of the AV, possibly exposing the aortic valve interstitial cells (AVICs) to shear strains and stresses.     

Non structural protein of avian influenza A (H11N1) virus is a weaker suppressor of immune responses but capable of inducing apoptosis in host cells

ABSTRACT: BACKGROUND: The Non-Structural (NS1) protein of Influenza A viruses is an extensively studied multifunctional protein which is commonly considered as key viral component to fight against host immune responses. Even though there has been a lot of studies on the involvement of NS1 protein in host immune responses there are still ambiguities regarding its role in apoptosis in infected cells. Interactions of NS1 protein with host factors, role of NS1 protein in regulating cellular responses and apoptosis are quiet complicated and further studies are still needed to understand it completely. RESULTS: NS1 genes of influenza A/Chicken/India/WBNIV2664/2008 (H5N1) and A/Aquatic bird/India/NIV-17095/2007(H11N1) were cloned and expressed in Human embryonic kidney (293T) cells. Microarray based approach to study the host cellular responses to NS1 protein of the two influenza A viruses of different pathogenicity showed significant differences in the host gene expression profile. NS1 protein of H5N1 resulted in suppression of IFN-beta mediated innate immune responses in 293T cells, leading to down-regulation of the components of JAK-STAT pathway like STAT1 which further suppressed the expression of pro-inflammatory cytokines like CXCL10 and CCL5. The degree of suppression of host immune genes was found considerable with NS1 protein of H11N1 but was not as prominent as with H5N1-NS1. TUNEL assay analyses were found to be positive in both the NS1 transfected cells indicating both H5N1 as well as H11N1 NS1 proteins were able to induce apoptosis in transfected cells. CONCLUSIONS: We propose that NS1 protein of both H5N1 and H11N1 subtypes of influenza viruses are capable of influencing host immune responses and possess necessary functionality to support apoptosis in host cells. H11N1, a low pathogenic virus without any proven evidence to infect mammals, contains a highly potential NS1 gene which might contribute to greater virus virulence in different gene combinations.     

IL-6 inhibits IFN-γ induced autophagy in Mycobacterium tuberculosis H37Rv infected macrophages

The significance of IL-6 production in tuberculosis is yet to be fully elucidated, although it is known for quite some time that IL-6 interferes with IFN-γ induced signal. In order to know which cellular process induced by IFN-γ is actually counteracted by IL-6, we studied the role of IL-6 on IFN-γ induced autophagy formation in virulent Mycobacterium tuberculosis infection in THP-1 cells, since it is well characterized that induction of autophagy by IFN-γ eliminates intracellular mycobacterium by overcoming the phagosome maturation block imposed by bacilli. We report here that IL-6 inhibits both IFN-γ and starvation induced autophagy in M. tuberculosis H37Rv infected cells. M. tuberculosis H37Rv infection results in time dependent production of IL-6 in THP-1 cells and neutralization of this endogenous IL-6 by anti-IL-6 antibody significantly enhances the IFN-γ mediated killing of the intracellular bacteria. IL-6 time dependently lowers Atg12-Atg5 complex and therefore inhibits autophagosome biogenesis rather than autophagolysosome formation. IL-6 also affects IFN-γ mediated stimulation of mTOR, p-38 and JNK pathways. These results clearly indicate that virulent mycobacteria strategically upregulate IL-6 production to combat innate immunity.     

The endosomal adaptor protein APPL1 impairs the turnover of leading edge adhesions to regulate cell migration

Cell migration is a complex process that requires the integration of signaling events that occur in distinct locations within the cell. Adaptor proteins, which can localize to different subcellular compartments, where they bring together key signaling proteins, are emerging as attractive candidates for controlling spatially coordinated processes. However, their function in regulating cell migration is not well understood. In this study, we demonstrate a novel role for the adaptor protein containing a pleckstrin-homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1) in regulating cell migration. APPL1 impairs migration by hindering the turnover of adhesions at the leading edge of cells. The mechanism by which APPL1 regulates migration and adhesion dynamics is by inhibiting the activity of the serine/threonine kinase Akt at the cell edge and within adhesions. In addition, APPL1 significantly decreases the tyrosine phosphorylation of Akt by the nonreceptor tyrosine kinase Src, which is critical for Akt-mediated cell migration. Thus, our results demonstrate an important new function for APPL1 in regulating cell migration and adhesion turnover through a mechanism that depends on Src and Akt. Moreover, our data further underscore the importance of adaptor proteins in modulating the flow of information through signaling pathways.     

Use of thiazolidinediones and the risk of bladder cancer among people with type 2 diabetes: a meta-analysis

Background:

Patients with type 2 diabetes have a 40% increased risk of bladder cancer. Thiazolidinediones, especially pioglitazone, may increase the risk. We conducted a systematic review and meta-analysis to evaluate the risk of bladder cancer among adults with type 2 diabetes taking thiazolidinediones.

Methods:

We searched key biomedical databases (including MEDLINE, Embase and Scopus) and sources of grey literature from inception through March 2012 for published and unpublished studies, without language restrictions. We included randomized controlled trials (RCTs), cohort studies and case–control studies that reported incident bladder cancer among people with type 2 diabetes who ever (v. never) were exposed to pioglitazone (main outcome), rosiglitazone or any thiazolidinedione.

Results:

Of the 1787 studies identified, we selected 4 RCTs, 5 cohort studies and 1 case–control study. The total number of patients was 2 657 365, of whom 3643 had newly diagnosed bladder cancer, for an overall incidence of 53.1 per 100 000 person-years. The one RCT that reported on pioglitazone use found no significant association with bladder cancer (risk ratio [RR] 2.36, 95% confidence interval [CI] 0.91–6.13). The cohort studies of thiazolidinediones (pooled RR 1.15, 95% CI 1.04–1.26; I² = 0%) and of pioglitazone specifically (pooled RR 1.22, 95% CI 1.07–1.39; I² = 0%) showed significant associations with bladder cancer. No significant association with bladder cancer was observed in the two RCTs that evaluated rosiglitazone use (pooled RR 0.87, 95% CI 0.34–2.23; I² = 0%).

Interpretation:

The limited evidence available supports the hypothesis that thiazolidinediones, particularly pioglitazone, are associated with an increased risk of bladder cancer among adults with type 2 diabetes.People with type 2 diabetes are at increased risk of several types of cancer, including a 40% increased risk of bladder cancer, compared with those without diabetes.^1,2 The strong association with bladder cancer is hypothesized to be a result of hyperinsulinemia, whereby elevated insulin levels in type 2 diabetes stimulate insulin receptors on neoplastic cells, promoting cancer growth and division.^1,3–5 Additional risk factors for bladder cancer include increased age, male sex, smoking, occupational and environmental exposures and urinary tract disease.⁶ Exogenous insulin and other glucose-lowering medications such as sulfonylureas, metformin and thiazolidinediones, may further modify the risk of bladder cancer.¹Data from the placebo-controlled PROactive trial of pioglitazone (PROspective pioglitAzone Clinical Trial in macroVascular Events) suggested a higher incidence of bladder cancer among pioglitazone users than among controls.⁷ Subsequent randomized controlled trials (RCTs) and observational studies have reported conflicting results for pioglitazone, with various studies reporting a significant increase,^8,9 a nonsignificant increase¹⁰ and even a decreased risk¹¹ of bladder cancer.To test the hypothesis that pioglitazone use is associated with an increased risk of bladder cancer, we conducted a systematic review and meta-analysis of RCTs and observational studies reporting bladder cancer among adults with type 2 diabetes taking pioglitazone. To clarify the possibility of a drug-class effect, we also examined data for all thiazolidinediones and for rosiglitazone alone.     

A mathematical model for astrocytes mediated LTP at single hippocampal synapses

Many contemporary studies have shown that astrocytes play a significant role in modulating both short and long form of synaptic plasticity. There are very few experimental models which elucidate the role of astrocyte over Long-term Potentiation (LTP). Recently, Perea and Araque (Science 317:1083-1086, 2007) demonstrated a role of astrocytes in induction of LTP at single hippocampal synapses. They suggested a purely pre-synaptic basis for induction of this N-methyl-D-Aspartate (NMDA) Receptor-independent LTP. Also, the mechanisms underlying this pre-synaptic induction were not investigated. Here, in this article, we propose a mathematical model for astrocyte modulated LTP which successfully imitates the experimental findings of Perea and Araque (Science 317:1083-1086, 2007). Our study suggests the role of retrograde messengers, possibly Nitric Oxide (NO), for this pre-synaptically modulated LTP.