Breakdown of CTL tolerance to self HLA-B*2705 induced by exposure to Chlamydia trachomatis

There is a strong association between seronegative arthritis and HLA B27, but it is still unresolved whether the contribution of B27 to disease pathogenesis is solely as a restriction element for an arthritogenic peptide, or whether B27 itself serves as an autoantigen. This study uses transgenic rats to address the question as to whether exposure to an arthritogenic pathogen can alter tolerance to B27. Unlike their nontransgenic counterparts, B27-transgenic rats are tolerant of B27 immunization using either B27(+) splenocytes or plasmid DNA and do not develop anti-B27 CTL. However, if splenocytes from such immunized animals are exposed to Chlamydia in vitro, CTL are generated that lyse B27(+) targets. No killing was seen with targets transfected with control B7, B14, B40, or B44. This phenomenon was not observed with immunization by nontransgenic splenocytes, or HLA-A2 DNA alone. Using targets expressing mutated B27, we show that the epitope for autoreactive CTL recognition of B27 involves the Lys(70) amino acid residue in the alpha(1) domain of the MHC class I molecule. The generation of CTL with specificity for B27 under these conditions demonstrates that tolerance to B27 can be subverted by CHLAMYDIA: This indicates a dynamic interrelationship between the pathogen and B27, which may have important implications for B27-related spondyloarthropathies triggered by intracellular bacteria.     

Excitatory action of lead on rat sympathetic preganglionic neurons in vitro and in vivo

Lead exposure elicited an increase in blood pressure and was considered to be a cardiovascular risk factor. The involvements of sympathetic nervous system and circulating catecholamines have been implicated in lead-induced hypertension. This study examined the effects of PbCl(2) on sympathetic preganglionic neurons (SPNs) in vitro and in vivo. In vitro electrophysiological study showed that superfusion of a low concentration (5 microM) of PbCl(2), which had no effects on membrane potential and spontaneous discharge rate, enhanced excitatory postsynaptic potentials (EPSPs) in some of the SPNs examined but inhibited inhibitory postsynaptic potentials (IPSPs) in other SPNs tested. A higher concentration (50 microM) of PbCl(2) inhibited both EPSPs and IPSPs in all SPNs examined. In vivo study showed that intrathecal injection of PbCl(2) (10 and 100 nmol) via an implanted cannula to the T7-T9 segments of urethane-anesthetized rats increased both the heart rate and mean arterial pressure. The pressor and tachycardic responses of intrathecal PbCl(2) (100 nmol) were attenuated by pretreatment with intravenous administration of hexamethonium (10 mg/kg) or intrathecal AP-5 (DL-2-amino-5-phosphonovaleric acid, 100 nmol), but were not significantly antagonized by prior intrathecal administration of CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, 100 nmol). Taken together, these results demonstrated that lead may exert a stimulatory effect on SPNs, which may result from the enhancement of EPSPs and inhibition of IPSPs by low concentrations of lead.     

Testing water-mediated DNA recognition by the Hin recombinase

The Hin recombinase specifically recognizes its DNA-binding site by means of both major and minor groove interactions. A previous X-ray structure, together with new structures of the Hin DNA-binding domain bound to a recombination half-site that were solved as part of the present study, have revealed that two ordered water molecules are present within the major groove interface. In this report, we test the importance of these waters directly by X-ray crystal structure analysis of complexes with four mutant DNA sequences. These structures, combined with their Hin-binding properties, provide strong support for the critical importance of one of the intermediate waters. A lesser but demonstrable role is ascribed to the second water molecule. The mutant structures also illustrate the prominent roles of thymine methyls both in stabilizing intermediate waters and in interfering with water or amino acid side chain interactions with DNA.     

Nucleotide and amino acid sequences of oriT-traM-traJ-traY-traA-traL regions and mobilization of virulence plasmids of Salmonella enterica serovars enteritidis,gallinarum-pullorum,and typhimurium

The virulence plasmid of Salmonella enterica serovar Gallinarum-Pullorum (pSPV) but not those of Salmonella enterica serovars Enteritidis (pSEV) and Typhimurium (pSTV) can be readily mobilized by an F or F-like conjugative plasmid. To investigate the reason for the difference, the oriT-traM-traJ-traY-traA-traL regions of the three salmonella virulence plasmids (pSVs) were cloned and their nucleotide and deduced amino acid sequences were examined. The cloned fragments were generally mobilized more readily than the corresponding full-length pSVs, but the recombinant plasmid containing the oriT of pSPV was, as expected, more readily mobilized, with up to 100-fold higher frequency than the recombinant plasmids containing the oriT of the other two pSVs. The nucleotide sequences of the oriT-traM-traJ-traY-traA-traL region of pSEV and pSTV were almost identical (only 4 bp differences), but differed from that of pSPV. Major nucleotide sequence variations were found in traJ, traY, and the Tra protein binding sites sby and sbm. sby of pSPV showed higher similarity than that of pSEV or pSTV to that of the F plasmid. The reverse was true for sbm: similarity was higher with pSEV and pSTV than with pSPV. In the deduced amino acid sequences of the five Tra proteins, major differences were found in TraY: pSEV's TraY was 75 amino acids, pSTV's was 106 amino acids, and pSPV's was 133 amino acids; and there were duplicate consensus betaalphaalpha fragments in the TraY of pSPV and F plasmid, whereas there was only a single betaalphaalpha fragment in that of pSEV and pSTV.     

Characterization of bradykinin receptors in canine cultured corneal epithelial cells: Pharmacological and functional studies

The pharmacological properties of bradykinin (BK) receptors were characterized in canine cultured corneal epithelial cells (CECs) using [(3)H]-BK as a radioligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant of 0.34 +/- 0.07 nM and a maximum receptor density of 179 +/- 23 fmol/mg protein. Neither a B(1) receptor-selective agonist (des-Arg(9)-BK) nor antagonist ([Leu(8), des-Arg(9)]-BK) significantly inhibited [(3)H]-BK binding to CECs, thus excluding the presence of B(1) receptors in canine CECs. The specific binding of [(3)H]-BK to CECs was inhibited by B(2) receptor-selective agonists (BK and kallidin) and antagonists (Hoe 140 and [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK), with a best fit using a one-binding-site model. The order of potency for the inhibition of [(3)H]-BK binding was BK = Hoe 140 > kallidin > [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK. Stimulation of CECs by BK produced a concentration-dependent accumulation of inositol phosphates (IP) and an initial transient peak of intracellular Ca(2+). B(2) receptor-selective antagonist ([D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK) significantly antagonized the BK-induced responses with dissociation constants of 6.0-6.1. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin did not alter the BK-induced IP accumulation. Incubation of CECs in the absence of external Ca(2+) led to a significant attenuation of the IP accumulation induced by BK. These results demonstrate that BK directly stimulates phospholipase C-mediated signal transduction through BK B(2) receptors via a PTX-insensitive G protein in canine CECs. This effect may function as the transducing mechanism for BK-mediated cellular responses.     

Identification of sequence motifs responsible for the adhesive interaction between exon v10-containing CD44 isoforms

Previous studies have demonstrated that CD44 isoforms containing the alternatively spliced exon v10 promote cell-cell adhesion via a mechanism that involves the recognition of chondroitin sulfate side chains presented on the surface of interacting cells in association with other CD44 molecules. Sequence analysis revealed the presence within exon v10 of two motifs that may be relevant to this interaction, a B[X(7)]B motif that may contribute to the recognition and binding of chondroitin sulfate and a serine-glycine motif that may serve as a site of chondroitin sulfate attachment. To determine whether either of these two motifs explain the unique adhesive activity of exon v10-containing CD44 isoforms, each was targeted by site-directed mutagenesis, and the adhesive activity of the resultant mutants was determined using a quantitative cell-cell binding assay. The data obtained demonstrate conclusively that it is the exon v10-encoded B[X(7)]B motif that is solely responsible for the enhanced adhesive activity of exon v10-containing CD44 isoforms.     

Characterization of human Smg5/7a: a protein with similarities to Caenorhabditis elegans SMG5 and SMG7 that functions in the dephosphorylation of Upf1

Nonsense-mediated mRNA decay (NMD) in mammalian cells depends on phosphorylation of Upf1, an RNA-dependent ATPase and 5'-to-3' helicase. Upf1 phosphorylation is mediated by Smg1, a phosphoinositol 3-kinase-related protein kinase. Here, we describe a human protein, which we call hSmg5/7a, that manifests similarity to Caenorhabditis elegans NMD factors CeSMG5 and CeSMG7, as well as two Drosophila melanogaster proteins that are also similar to the C. elegans NMD factors. Results indicate that hSmg5/7a functions in the dephosphorylation of Upf1. Furthermore, hSmg5/7a copurifies with Upf1, Upf2, Upf3X, Smg1, and the catalytic subunit of protein phosphatase 2A. We also demonstrate that Upf2, another factor involved in NMD, is a phosphoprotein. However, hSmg5/7a plays no role in the dephosphorylation of Upf2. These data indicate that hSmg5/7a targets protein phosphatase 2A to Upf1 but not Upf2. Results of Western blotting reveal that hSmg5/7a is mostly cytoplasmic in HEK293T cells.     

Proteomics in biomarker discovery and drug development

Proteomics is a research field aiming to characterize molecular and cellular dynamics in protein expression and function on a global level. The introduction of proteomics has been greatly broadening our view and accelerating our path in various medical researches. The most significant advantage of proteomics is its ability to examine a whole proteome or sub-proteome in a single experiment so that the protein alterations corresponding to a pathological or biochemical condition at a given time can be considered in an integrated way. Proteomic technology has been extensively used to tackle a wide variety of medical subjects including biomarker discovery and drug development. By complement with other new technique advances in genomics and bioinformatics, proteomics has a great potential to make considerable contribution to biomarker identification and to revolutionize drug development process. This article provides a brief overview of the proteomic technologies and their application in biomarker discovery and drug development.