Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein

Type 1 diabetes results from the selective destruction of insulin-producing beta cells in the islets of Langerhans, and autoimmune T cells are thought to be the mediators of this destruction. T cells are also responsible for allorejection once the islets are transplanted into a patient to reduce the negative consequences of a lack of insulin. To better understand these processes, we have developed a transgenic mouse expressing proinsulin II tagged with a live-cell fluorescent reporter protein, Timer. Timer protein is unique because it changes color from green to red in the first 24 h after synthesis. With this marker, insulin synthesis can be carefully monitored through fluorescent changes over time. To complement this new biotechnological research tool, we designed a body window to allow for in vivo imaging over time of the islets transplanted under the kidney capsule. The window device, which is sutured to replace the underlying skin and body wall over the site of islet transplantation, may be used to simultaneously observe beta cells and T cells that have been labeled with a fluorochrome distinguishable from Timer. The imaging of both insulin-producing cells and T cells may be carried out repeatedly for a week or more with no need for repeated surgery, while preserving the life of the studied animal.     

Treatment of Salmonella enterica serovar Enteritidis with a sublethal concentration of trisodium phosphate or alkaline pH induces thermotolerance

The responses of Salmonella enterica serovar Enteritidis to a sublethal dose of trisodium phosphate (TSP) and its equivalent alkaline pH made with NaOH were examined. Pretreatment of S. enterica serovar Enteritidis cells with 1.5% TSP or pH 10.0 solutions resulted in a significant increase in thermotolerance, resistance to 2.5% TSP, resistance to high pH, and sensitivity to acid and H(2)O(2). Protein inhibition studies with chloramphenicol revealed that thermotolerance, unlike resistance to high pH, was dependent on de novo protein synthesis. Two-dimensional polyacrylamide gel electrophoresis (PAGE) of total cellular proteins from untreated control cells resolved as many as 232 proteins, of which 22 and 15% were absent in TSP- or alkaline pH-pretreated cells, respectively. More than 50% of the proteins that were either up- or down-regulated by TSP pretreatment were also up- or down-regulated by alkaline pH pretreatment. Sodium dodecyl sulfate-PAGE analysis of detergent-insoluble outer membrane proteins revealed the up-regulation of at least four proteins. Mass spectrometric analysis showed the up-regulated proteins to include those involved in the transport of small hydrophilic molecules across the cytoplasmic membrane and those that act as chaperones and aid in the export of newly synthesized proteins by keeping them in open conformation. Other up-regulated proteins included common housekeeping proteins like those involved in amino acid biosynthesis, nucleotide metabolism, and aminoacyl-tRNA biosynthesis. In addition to the differential expression of proteins following TSP or alkaline pH treatment, changes in membrane fatty acid composition were also observed. Alkaline pH- or TSP-pretreated cells showed a higher saturated and cyclic to unsaturated fatty acid ratio than did the untreated control cells. These results suggest that the cytoplasmic membrane could play a significant role in the induction of thermotolerance and resistance to other stresses following TSP or alkaline pH treatment.     

Structure of Plasmodium falciparum triose-phosphate isomerase-2-phosphoglycerate complex at 1.1-A resolution

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.     

Distinctive KIR and HLA diversity in a panel of north Indian Hindus

HLA and KIR are diverse and rapidly evolving gene complexes that work together in human immunity mediated by cytolytic lymphocytes. Understanding their complex immunogenetic interaction requires study of both HLA and KIR diversity in the same human population. Here a panel of 72 unrelated north Indian Hindus was analyzed. HLA- A, B, C, DRB1, DQA1, and DQB1 alleles and their frequencies were determined by sequencing or high-resolution typing of genomic DNA; KIR genotypes were determined by gene-specific typing and by allele-level DNA typing for KIR2DL1, 2DL3, 2DL5, 3DL1, and 3DL2. From HLA analysis, the north Indian population is seen to have several characteristics shared either with Caucasian or East Asian populations, consistent with the demographic history of north India, as well as specific features, including several alleles at high frequency that are rare or absent in other populations. A majority of the north Indian KIR gene profiles have not been seen in Caucasian and Asian populations. Most striking is a higher frequency of the B group of KIR haplotypes, resulting in equal frequencies for A and B group haplotypes in north Indians. All 72 members of the north Indian panel have different HLA genotype and different KIR genotype.     

Design of compound libraries based on natural product scaffolds and protein structure similarity clustering (PSSC)

Recent advances in structural biology, bioinformatics and combinatorial chemistry have significantly impacted the discovery of small molecules that modulate protein functions. Natural products which have evolved to bind to proteins may serve as biologically validated starting points for the design of focused libraries that might provide protein ligands with enhanced quality and probability. The combined application of natural product derived scaffolds with a new approach that clusters proteins according to structural similarity of their ligand sensing cores provides a new principle for the design and synthesis of such libraries. This article discusses recent advances in the synthesis of natural product inspired compound collections and the application of protein structure similarity clustering for the development of such libraries.     

Synthesis and characterization of bioconjugates of S-layer proteins

The self-assembling proteins that form crystalline surface layers (S-layers) on many microbial species have found numerous applications due to their nanostructured nature. To devise a new method to construct surface displays that exploit S-layer self-assembly activity and nanostructural properties, we have constructed polymer bioconjugates of S-layer proteins. The conjugates formed are similar in function to the monomer alkanethiols that form self-assembled monolayers (SAMs) on gold surfaces. However, the self-assembly is driven by the protein "headgroup" that positions polymer-tethered endgroups on a surface. This paper examines the integration of protein purification, conjugation, and surface assembly that has led to the development of this new method for the formation of nanostructured surfaces. Purified S-layer proteins from Lactobacillus brevis were conjugated with small molecule probes and polymers using amine-based reactions. To keep multiple labeling of protein amine groups to acceptable levels, the conjugations were performed at pH 6.5, allowing for limited yields (24-39%) as determined by mass spectrometry and SDS-polyacrylamide gel electrophoresis. As the presence of high levels of unlabeled S-layer proteins is undesired, we have developed a protocol for further purification that employs monomeric avidin affinity chromatography. The surface self-assembly of the polymer bioconjugates onto amine-terminated microspheres was studied using epi-fluorescence, confocal, and scanning electron microscopy. The surfaces obtained exhibited homogeneous distributions of tethered molecules. Also, in cases where the modular assembly of two distinct types of tethered endgroups was accomplished, there was no evidence for phase separation in the surfaces. The modular assembly method will provide a potential route to controlling surface display density as the starting assembly conditions guide displayed endgroup concentrations in mixed molecular monolayers.     

Expression and functional contribution of hTHTR-2 in thiamin absorption in human intestine

The aim of this study was to investigate expression and relative contribution of human thiamin transporter (hTHTR)-2 toward overall carrier-mediated thiamin uptake by human intestinal epithelial cells. Northern blot analysis showed that the message of the hTHTR-2 is expressed along the native human gastrointestinal tract with highest expression being in the proximal part of small intestine. hTHTR-2 protein was found, by Western blot analysis, to be expressed at the brush-border membrane (BBM), but not at the basolateral membrane, of native human enterocytes. This pattern of expression was confirmed in studies using a fusion protein of hTHTR-2 with the enhanced green fluorescent protein (hTHTR2-EGFP) expressed in living Caco-2 cells grown on filter. Pretreating Caco-2 cells (which also express the hTHTR-2 at RNA and protein levels) with hTHTR-2 gene-specific small interfering RNA (siRNA) led to a significant (P < 0.01) and specific inhibition (48%) in carrier-mediated thiamin uptake. Similarly, pretreating Caco-2 cells with siRNA that specifically target hTHTR-1 (which is expressed in Caco-2 cells) also significantly (P < 0.01) and specifically inhibited (by 56%) carrier-mediated thiamin uptake. When Caco-2 cells were pretreated with siRNAs against both hTHTR-2 and hTHTR-1 genes, an almost complete inhibition in carrier-mediated thiamin uptake was observed. These results show that the message of hTHTR-2 is expressed along the human gastrointestinal tract and that expression of its protein in intestinal epithelia is mainly localized to the apical BBM domain. In addition, results show that this transporter plays a significant role in carrier-mediated thiamin uptake in human intestine.     

Conformationally restricted analogs of deoxynegamycin

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC(50)=8.2 microM; 44, IC(50)=6.6 microM; 35e(2), IC(50)=1 microM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16 microg/mL; Klebsiella pneumoniae, MIC=8 microg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED(50)=4.8 mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.     

Caspase-3: Its potential involvement in Cr(III)-induced apoptosis of lymphocytes

In this study, we have examined the role of caspase-3 in apoptosis of lymphocytes induced by the chromium(III) complexes viz. tris-(1,10-phenanthroline)chromium(III) chloride (Cr(III)-phen) and trans-diaqua[1,3-bis(salicylideneamino)propanechromium(III)] perchlorate (Cr(III)-salprn). Evidence for caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage in lymphocytes exposed to Cr(III) complexes is revealed through Western blotting analysis. Blocking the activity of caspase-3 with z-DEVD-fmk, prevents apoptosis as evidenced through [3H]-thymidine incorporation, DNA fragmentation assay and measurement of sub-G1 cells by flow cytometry. Pretreatment of lymphocytes with free radical scavengers completely attenuates the activity of caspase-3 suggesting that reactive oxygen species (ROS) are upstream activators of caspase-3. Preincubation of lymphocytes with PP2, a selective Src-family tyrosine kinase inhibitor, abolishes the activation of caspase-3 indicating that Src-family tyrosine kinases viz. p56lck, p59fyn and p53/56lyn are mediators of caspase-3 activation during Cr(III) exposure. Collectively, our findings support a plausible mechanism in which Cr(III) mediates ROS generation that precedes the up-regulation of p56lck, p59fyn and p53/56lyn which eventually activates caspase-3 to promote apoptotic cell death of lymphocytes. To our knowledge, this is the first report suggesting the importance of Src-family tyrosine kinases for the activation of caspase-3 in metal-induced apoptotic cell death.