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Biomedical subjects

J B Smith

Publications and source records attributed to J B Smith.

At least 73 records · Page 4Linked to original sources
PubMed1997

In vivo modification of the C-terminal lysine of human lens alphaB-crystallin.

Both the structural and chaperone-like properties of lens alpha-crystallins have been implicated in maintaining lens transparency. Modifications of lens alpha-crystallins may lead to formation of cataract by affecting the close-packing of the crystallins or by reducing the chaperone-like activity of the alpha-crystallins. A previously unreported modified alphaB-crystallin, whose molecular weight is 72 u greater than unmodified alphaB-crystallin, has been isolated from human lenses by size exclusion chromatography, reversed phase HPLC and ion exchange HPLC. Approximately one nanomole of this modified alphaB-crystallin was obtained from each of five human eye lenses. Molecular weight determinations of peptides produced by digestion with trypsin or endoproteinase Asp-N showed that the modification is in the C-terminal region of alphaB-crystallin. The fragmentation pattern of peptides from the C-terminal region, analysed by tandem mass spectrometry, located the modification of the epsilon-amino group of the C-terminal lysine. The elemental composition of this modification, determined from its exact mass, is C3H4O2. Because this modification decreases the net charge of alphaB-crystallin by one unit, and because the C-terminus has been implicated in the chaperone activity attributed to alphaB-crystallin, this modification at Lys 175 may have a significant role in cataractogenesis.

Adult↗
PubMed1997

Eosinophil cationic protein in tracheal aspirates of preterm infants with bronchopulmonary dysplasia.

Eosinophil cationic protein was elevated during the first week of life in tracheal aspirates from 11 preterm infants in whom bronchopulmonary dysplasia subsequently developed compared with 8 preterm and 8 term infants without bronchopulmonary dysplasia. Eosinophil cationic protein levels increased progressively with continued intubation in the infants with bronchopulmonary dysplasia but remained low in a comparison group of term infants. We suggest that eosinophils participate in the inflammatory process in bronchopulmonary dysplasia and may contribute to lung injury.

Bronchopulmonary Dysplasia↗
PubMed1997

Identification of inflammatory mediators by screening for glucocorticoid-attenuated response genes.

We describe an approach for identifying novel inflammatory mediators, based on screening for immediate early/primary response genes whose induction by an inflammatory stimulus is attenuated by glucocorticoids. This procedure can be applied to a wide range of cell types and tissues, using a variety of inducers. In an initial test of this idea, we identified cDNAs for 12 LPS-induced, glucocorticoid-attenuated response genes (GARGs) by differential hybridization screening of a lambda phage cDNA library from murine 3T3 fibroblasts. Seven of the GARGs were known genes, including the chemokines JE/MCP-1, fic/MARC/MCP-3, and crg2/IP-10. One of the novel cDNAs was a new C-X-C chemokine that we designated LIX, for LPS-induced C-X-C chemokine. Because the 12 GARG cDNAs were identified in a single screening of only 15,000 phage, and four were found as single isolates, these results suggest that there are many GARGs not yet described. Furthermore, six of the seven known GARGs encode proteins that modulate intercellular communication. These results support our hypothesis that GARGs predominantly encode products that function in paracrine cell communication. Here we provide an overview of the GARG strategy and the differential hybridization procedures used in our initial screening. A variety of other methods for identifying differentially expressed genes may be used in future searches for novel GARGs.

3T3 Cells↗
PubMed1997

Phenotypic analysis of adhesion molecules in first-trimester decidual tissue from chorion villus samples.

PROBLEM: First-trimester decidua contain CD56bright/CD16neg lymphocytes that phenotypically resemble a subset of peripheral blood natural killer (NK) cells. Factor controlling their localization to decidua are unknown, but they may relate to trophoblast invasion, endometrial decidualization, and adhesion molecule expression. METHOD OF STUDY: Ninety-one chorion villous samples (CVS) were screened for the presence of decidua, and selected specimens were analyzed for the expression of adhesion molecule pairs using a panel of monoclonal antibodies. RESULTS: Lymphoid cells in CVS-derived decidua expressed CD56, PECAM, intracellular adhesion molecule-1 (ICAM-1), and the integrins LFA-1, alpha E beta 7, and alpha 4 beta 1, and co-receptors for these molecules were expressed by decidual stromal cells, invasive trophoblasts, and venule endothelium. Some endothelium expressed a cuboidal phenotype. CONCLUSIONS: The expression of complimentary pairs of adhesion molecules by maternal lymphoid cells and by either extravillous cytotrophoblast or decidual stromal cells supports the hypothesis that these cells interact within decidua. Also, both LFA-1 and alpha 4 beta 1 may contribute to decidual localization of CD56bright NK cells.

CD56 Antigen↗
PubMed1997

Identification of hydrogen peroxide oxidation sites of alpha A- and alpha B-crystallins.

The alpha-crystallins are the most abundant structural proteins of the lens and, because of their chaperone activity, contribute to the solubility of the other crystallins. With aging, the lens crystallins undergo a variety of modifications which correlate with a loss of solubility and the development of cataract. A recent study demonstrating that alpha-crystallins exposed in vitro to FeCl3 and H2O2 exhibit decreased chaperone activity, implicates metal catalyzed oxidations of alpha-crystallins in this loss of solubility. The present study has determined that alpha-crystallins incubated with FeCl3 and H2O2 are modified by the nearly complete oxidation of all methionine residues to methionine sulfoxide, with no other detectable reaction products. The modifications were identified from the molecular weights of peptides formed by enzymatic digestion of the alpha-crystallins and located by tandem mass spectrometric analysis of the fragmentation pattern of the mass spectra of the fragments from peptides with oxidized methionine is loss of 64 Da, which corresponds to loss of CH3SOH from the methionine sulfoxide. These fragments are useful in identifying peptides that include oxidized methionine residues.

Amino Acid Sequence↗
PubMed1997

Bryostatin 1 and phorbol ester down-modulate protein kinase C-alpha and -epsilon via the ubiquitin/proteasome pathway in human fibroblasts.

We evaluated the possibility that distinct proteolytic pathways contribute to the down-regulation of a novel (epsilon) or conventional (alpha) isoform of protein kinase C (PKC) in nonimmortalized human fibroblasts. Inhibitors of calpains and other cysteine proteinases, vesicle trafficking, or lysosomal proteolysis did not affect the down-regulation of PKC-alpha or -epsilon produced by bryostatin 1 (Bryo). Lactacystin (Lacta) and certain terminal aldehyde tripeptides or tetrapeptides, which selectively inhibit the proteasome, preserved substantial PKC-alpha and -epsilon protein from down-regulation by Bryo or phorbol-12-myristate-13-acetate. Lacta preserved active kinase in vivo, as shown by the retention of Bryo-induced autophosphorylated PKC-alpha. Concomitant with down-regulation, Bryo produced PKC-alpha and -epsilon species that were larger than the native proteins (80 and 90 kDa, respectively). Western blot analysis showed that the larger PKC-alpha species were ubiquitinylated. Treatment with Bryo plus Lacta synergistically increased multiubiquitinylated PKC-alpha, as expected if Bryo induces ubiquitinylation of PKC-alpha and Lacta blocks its degradation. Bryo also produced a 76-kDa, nonphosphorylated form of PKC-alpha and an 86-kDa form of PKC-epsilon. Phosphatase inhibitors decreased production of 76- and 86-kDa PKC-alpha and -epsilon by Bryo and preserved 80- and 90-kDa PKC-alpha and -epsilon, respectively. Our results suggest that the down-modulation of PKC-alpha and -epsilon occurs principally via the ubiquitin/ proteasome pathway. Dephosphorylation seems to predispose PKC to ubiquitinylation.

Acetylcysteine↗
PubMed1997

Leptomeningeal carcinomatosis: a sequela of cholangiocarcinoma.

Leptomeningeal carcinomatosis is a rare and deceptive presentation of systemic cancer. We report the treatment of a patient with a Klatskin's tumor by surgical resection. The patient presented with symptoms suggestive of liver failure in the early postoperative period. He rapidly developed progressive, multineuroaxis symptoms and died. Routine gastrointestinal evaluation failed to demonstrate any intraabdominal pathologic process that could be responsible for his decline. A MRI of the brain was also unremarkable. Serial lumbar punctures, however, documented leptomeningeal carcinomatosis consistent with a gastrointestinal primary. The clinical presentation of leptomeningeal carcinomatosis is subtle and may masquerade as another disease state. The hallmark of this lethal process is the finding of progressive neurologic deficits at more than one level of the neuroaxis. Serial cerebrospinal fluid examination is often diagnostic. This is the first known report of leptomeningeal carcinomatosis secondary to primary cholangiocarcinoma.

Bile Duct Neoplasms↗
PubMed1997

An orally active selenium-based antihypertensive agent with restricted CNS permeability.

We report here the first orally active, selenium-based antihypertensive agent, and we demonstrate its restricted CNS permeability using inductively coupled plasma/mass spectroscopy (ICP/MS) and operant behavioral analysis. The biochemistry and pharmacology of selenium are subjects of intense current interest. As a consequence of the redox chemistry of the selenium moiety, phenylaminoalkyl selenides possess the remarkable characteristic of propagating a cycle of turnover-dependent local depletion of reduced ascorbate when processed by the key enzyme of catecholamine metabolism, dopamine-beta-monooxygenase. ICP/MS analysis was used to determine the pharmacokinetic parameters for selenide compounds after i.v. administration to anesthetized rats. Analysis of the data using a two-compartment pharmacokinetic model established very rapid initial clearance and a short beta-elimination half-life from blood. We developed an oxidative procedure for digestion and processing of tissue samples in order to obtain ICP/MS data on the tissue distributions of Se-containing metabolites after the administration of selenide compounds. The results establish that aromatic ring hydroxylation of the selenides results in a marked reduction in brain levels of Se-containing metabolites. The comparative effects of selenide compounds on locomotor activity and operant behavior were then investigated, and the results fully corroborate the ICP/MS analytical results. The novel compound, 4-hydroxy-alpha-methyl-phenyl-2-aminoethyl selenide, exhibits both restricted CNS permeability and oral antihypertensive activity in spontaneously hypertensive rats. This compound is the first orally active selenium-based antihypertensive agent ever reported, and it possesses properties that are highly desirable in pharmacological agents being developed for treatment of chronic diseases such as hypertension.

Administration, Oral↗
PubMed1996

Sites of glycation of beta B2-crystallin by glucose and fructose.

We determined the sites of glycation of bovine beta B2-crystallin by glucose and fructose. After incubation with glucose or fructose, glycated tryptic peptides were purified by affinity chromatography/reverse-phase HPLC and identified by electrospray ionization mass spectrometry (ESIMS). The results gave evidence of glycation at lysine 10, 75, 100, 107, 120, 139, 167 and 171 by both glucose and fructose, while glycated lysine 119 and 47 or 67 were detected only after fructosylation. We conclude that glucose and fructose have similar glycation specificity.

Amino Acid Sequence↗
PubMed1996

Ubiquitination of protein kinase C-alpha and degradation by the proteasome.

Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-alpha became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.

Acetylcysteine↗
PubMed1996

The glucocorticoid attenuated response genes GARG-16, GARG-39, and GARG-49/IRG2 encode inducible proteins containing multiple tetratricopeptide repeat domains.

In a search for glucocorticoid attenuated response genes (GARGs) induced by lipopolysaccharide (LPS) in murine Swiss 3T3 cells, we cloned 12 GARG cDNAs (J. B. Smith and H. R. Herschman, 1995, J. Biol. Chem. 270, 16756-16765). Analysis of complete cDNA sequences indicates that three of these genes encode members of a highly conserved family of proteins containing multiple tetratricopeptide repeat (TPR) domains. GARG-16 is a homologue of the interferon-induced human IFI-56K gene. GARG-39 is a homologue of the interferon-induced human ISG-54K and hamster CL-54K genes. The predicted GARG-49/IRG2 protein is 60-75 amino acids shorter than other known members of this gene family, and its carboxyl-terminal half is relatively divergent. Homologues of GARG-49/IRG2 in other species have not been reported. The predicted GARG-16 and GARG-39 proteins, and their homologues, contain 10 TPR domains. GARG-49/IRG2 shares the first 6 domains and part of the 7th, but lacks domains 8,9, and 10. Message levels of GARG-16, GARG-39, and GARG-49/IRG2 are increased by LPS stimulation in Swiss 3T3 cells and in peritoneal macrophages. Unlike many primary response genes, these three genes are not induced by serum stimulation in Swiss 3T3 cells. All three are induced in the RAW 264.7 macrophage cell line by LPS, by interferons-alpha/beta, and by interferon-gamma. Despite these similarities, quantitative differences in their responses to different stimuli indicate that GARG-16, GARG-39, and GARG-49/IRG2 are regulated independently. We speculate that the proteins encoded by these LPS- and interferon-inducible genes may participate in multicomponent assemblies via their TPR domains.

3T3 Cells↗
PubMed1996

The hemorrhagin catrocollastatin inhibits collagen-induced platelet aggregation by binding to collagen via its disintegrin-like domain.

Catrocollastatin, a 50 kDa snake venom protein purified from Crotalus atrox, specifically inhibits platelet-collagen adhesion and collagen-induced aggregation. Catrocollastatin is composed of an N-terminal domain, a metalloproteinase domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. The present studies show that catrocollastatin exerts its effect by binding to collagen. Based on the amino acid sequence and homology analysis, a cyclic oligopeptide corresponding to a conservative fragment containing the sequence SECD in the disintegrin-like domain has been synthesized. Like its protein parent, the synthetic peptide inhibits collagen-induced aggregation and possesses the ability to bind to collagen. This is the first snake venom protein with a disintegrin-like structure shown to bind to an integrin ligand matrix molecule instead of an integrin.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1996

The sequence of human betaB1-crystallin cDNA allows mass spectrometric detection of betaB1 protein missing portions of its N-terminal extension.

The sequence of human betaB1-crystallin cDNA encoded a protein of 251 amino acids in length. Mass spectrometric analysis of intact betaB1 from young human lens confirmed the deduced amino acid sequence. Lenses of human donors newborn to 27 years of age also contained partially degraded forms of betaB1 missing 15, 33, 34, 35, 36, 39, 40, and 41 amino acid residues from their N-terminal extensions. The similarity of the cleavage site between residues 15 and 16 in human betaB1 to the cleavage occurring in bovine betaB1 suggested that lenses of both species may contain a similar proteolytic activity. The remaining cleavage sites occurring in human betaB1 did not closely match those occurring in other species, possibly due to the widely divergent amino acid sequence of the N-terminal extension of betaB1 amoung species. Results from animal models suggest that cleavage of the N-terminal extension of betaB1-crystallin could enhance protein insolubilization and cataract in lens. However, the presence of partially degraded betaB1-crystallins in both water-soluble and water-insoluble fractions of lenses of young donors suggested that the rate that proteolyzed betaB1-crystallins become water-insoluble is relatively slow in humans.

Amino Acid Sequence↗