Purine nucleoside phosphorylase synthesis and turnover in human lymphoid cells

We have studied the turnover and synthesis of purine nucleoside phosphorylase by using a polyclonal rabbit antiserum to this protein. The turnover of purine nucleoside phosphorylase was studied in the B lymphoblast cell, WI-L2, by specific immunoprecipitation of [3H]leucine-labeled proteins. The half-lives for total protein and purine nucleoside phosphorylase were 14.5 and 14.1 hr, respectively. For cells cultured in the presence of inosine the half-life of purine nucleoside phosphorylase was reduced to 11.2 hr. The synthesis of purine nucleoside phosphorylase was analyzed during phytohemagglutinin-stimulated T cell transformation by pulse labeling cells with [35S]methionine. Purine nucleoside phosphorylase synthesis increased greater than 10-fold during the first 12 hr of transformation and continued to a maximum of 30-fold. The relative rate of purine nucleoside phosphorylase labeled to total proteins was 0.04% in unstimulated T cells and increased to 0.18% 12 hr after stimulation. These studies identify some preferential synthesis of purine nucleoside phosphorylase during the early stages of T cell transformation.     

Introduction of the alpha subunit mutation associated with the B1 variant of Tay-Sachs disease into the beta subunit produces a beta-hexosaminidase B without catalytic activity

Lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) occurs in two major isozyme forms, hexosaminidase A (alpha beta) and hexosaminidase B (beta beta). Although dimer formation is required for enzymatic activity, both subunits contain active sites which share many common substrates. However, the alpha subunit alone confers on hexosaminidase A the specificity for negatively charged substrates, e.g. GM2 ganglioside. Recently, a point mutation, producing a single amino acid substitution in the alpha subunit (Arg178-His), has been found to be associated with the B1 variant phenotype of Tay-Sachs disease (Ohno, K., and Suzuki, K. (1988) J. Neurochem. 50, 316-318). This variant is characterized by normal levels of hexosaminidase A as measured by a common artificial substrate, but an absence of activity toward alpha subunit-specific substrates. However, because of the presence of an active beta subunit in the mutant hexosaminidase A, it has not been possible to determine whether the affected alpha subunit has undergone a change in substrate specificity or become totally inactive. In order to define the full effect of the B1 mutation we have taken advantage of the common evolutionary origin of the genes coding for the alpha and beta subunits. Since the B1 mutation occurs in a region of extended identity between the two subunits, we have duplicated the Arg178-His mutation in a cDNA coding for the human beta subunit (Arg211-His). By expression of the mutant construct in monkey COS cells we have been able to examine the effect of this mutation on beta subunits which are capable of forming stable, active homodimers, an experiment that could not readily be accomplished with heterodimeric hexosaminidase A. Our data show that beta homodimers containing the Arg211-His substitution are formed and are transported into the lysosome in a manner identical to that of normal pro-hexosaminidase B. However, the mutant homodimers are processed at a slower rate and are less stable in the lysozyme. Their most striking feature was a total lack of normal hexosaminidase B activity. We conclude that while the effect of the Arg178-His substitution is not strictly limited to the active site, the severe B1 phenotype results from a totally inactive alpha-subunit in hexosaminidase A.     

Purine nucleoside phosphorylase (Np) in the mouse: Linkage relationship of Np-2 to esterase-10 (Es-10) and Np-1 on chromosome 14

An improved method for detecting four Np-1 (purine nucleoside phosphorylase) alleles in mouse erythrocytes by cellulose acetate electrophoresis is described. The previous linkage of Np-1 and Es-10 (esterase-10) was confirmed, with a map distance of 13.0±2.6 cM. Np-2 was detected by either specific activity assay or starch gel electrophoresis and shown to be linked to Es-10, 15.9 ± 3.1 cM, on chromosome 14. No recombinants between Np-1 and Np-2 were observed in 52 offspring, indicating either that these loci are either closely associated or that Np-2 represents simply a property of existing allelic products of the Np-1 locus.This research was supported by Medical Research Council of Canada grants to F.G.B. and F.F.S.     

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

Polymer optoelectronic structures for retinal prosthesis

This commentary highlights the effectiveness of optoelectronic properties of polymer semiconductors based on recent results emerging from our laboratory, where these materials are explored as artificial receptors for interfacing with the visual systems. Organic semiconductors based polymer layers in contact with physiological media exhibit interesting photophysical features, which mimic certain natural photoreceptors, including those in the retina. The availability of such optoelectronic materials opens up a gateway to utilize these structures as neuronal interfaces for stimulating retinal ganglion cells. In a recently reported work entitled “A polymer optoelectronic interface provides visual cues to a blind retina,” we utilized a specific configuration of a polymer semiconductor device structure to elicit neuronal activity in a blind retina upon photoexcitation. The elicited neuronal signals were found to have several features that followed the optoelectronic response of the polymer film. More importantly, the polymer-induced retinal response resembled the natural response of the retina to photoexcitation. These observations open up a promising material alternative for artificial retina applications.     

Apoptosis,cytokine and chemokine induction by non-structural 1 (NS1) proteins encoded by different influenza subtypes

Background

Influenza pandemic remains a serious threat to human health. Viruses of avian origin, H5N1, H7N7 and H9N2, have repeatedly crossed the species barrier to infect humans. Recently, a novel strain originated from swine has evolved to a pandemic. This study aims at improving our understanding on the pathogenic mechanism of influenza viruses, in particular the role of non-structural (NS1) protein in inducing pro-inflammatory and apoptotic responses.

Methods

Human lung epithelial cells (NCI-H292) was used as an in-vitro model to study cytokine/chemokine production and apoptosis induced by transfection of NS1 mRNA encoded by seven infleunza subtypes (seasonal and pandemic H1, H2, H3, H5, H7, and H9), respectively.

Results

The results showed that CXCL-10/IP10 was most prominently induced (> 1000 folds) and IL-6 was slightly induced (< 10 folds) by all subtypes. A subtype-dependent pattern was observed for CCL-2/MCP-1, CCL3/MIP-1α, CCL-5/RANTES and CXCL-9/MIG; where induction by H5N1 was much higher than all other subtypes examined. All subtypes induced a similar temporal profile of apoptosis following transfection. The level of apoptosis induced by H5N1 was remarkably higher than all others. The cytokine/chemokine and apoptosis inducing ability of the 2009 pandemic H1N1 was similar to previous seasonal strains.

Conclusions

In conclusion, the NS1 protein encoded by H5N1 carries a remarkably different property as compared to other avian and human subtypes, and is one of the keys to its high pathogenicity. NCI-H292 cells system proves to be a good in-vitro model to delineate the property of NS1 proteins.