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J B Smith

Publications and source records attributed to J B Smith.

At least 55 records · Page 3Linked to original sources
PubMed1998

The murine neutrophil-chemoattractant chemokines LIX, KC, and MIP-2 have distinct induction kinetics, tissue distributions, and tissue-specific sensitivities to glucocorticoid regulation in endotoxemia.

Lipopolysaccharide-induced CXC chemokine (LIX) is a novel murine neutrophil-chemoattractant CXC chemokine cloned as a glucocorticoid-attenuated response gene. We investigated LIX message expression in an acute endotoxemia model. LIX message peaks later than KC or macrophage inflammatory protein-2 (MIP-2) and remains elevated longer in almost all tissues. Induced LIX message expression in heart is 5- to 6-fold greater than in lung and spleen, and 20-fold greater than in liver. In contrast, KC expression is equal in heart, lung, and liver, whereas MIP-2 expression is strongest in the lung. Glucocorticoid regulation of these genes also differs. Endotoxemia-induced LIX message expression in the lung is markedly enhanced in adrenalectomized mice and strongly attenuated by dexamethasone, whereas lung KC and MIP-2 expression are unaffected by glucocorticoids. It is surprising to note that endotoxemia-induced brain expression of LIX (but not KC or MIP-2) is increased by dexamethasone. These observations suggest that LIX may have biological roles distinct from KC and MIP-2.

Acute Disease↗
PubMed1998

In vivo acetylation identified at lysine 70 of human lens alphaA-crystallin.

Posttranslational modification of protein lysyl residues that change the net charge of the molecule may alter the protein conformation. Such modifications are of particular significance among lens proteins, because conformational changes are associated with the development of cataract. A previously unidentified acetylated form of alphaA-crystallin has been isolated from the water-soluble portion of human lenses. The alphaA-crystallins were fractionated by anion exchange HPLC into seven peaks, each containing more than one form of alphaA-crystallin. The previously reported deamidated and phosphorylated forms were identified by their molecular masses, determined by electrospray ionization mass spectrometry. In addition to these modifications, approximately 5% of alphaA-crystallin had a modification that decreased the charge by one and increased the molecular mass by 42 u. This modification, identified as acetylation, was located uniquely at Lys 70. Like any modification that alters the surface charge, acetylation may affect protein conformation and intermolecular interactions, thereby altering the solubility or chaperone properties of alphaA-crystallin. Acetylation of lysine 70 is potentially significant since it is located in a region that has been implicated in the chaperone activity of alphaA-crystallin.

Acetylation↗
PubMed1998

Age-related changes in human lens crystallins identified by two-dimensional electrophoresis and mass spectrometry.

The purpose of this study was to identify the major protein components in adult human lenses and to analyse the specific age-related changes in these proteins using two-dimensional electrophoresis, Edman sequencing, and in conjunction with the data in the accompanying manuscript, mass spectrometry. The majority of changes in the two-dimensional electrophoretic pattern of lens proteins occurred prior to 17 years of age, and included a decrease in proteins migrating to the original positions of beta B1, beta B3, beta A3, gamma C and gamma D, and the appearance of many new species with apparent molecular weights on two-dimensional electrophoretic gels similar to beta B2 and gamma S, but having more acidic pIs. These proteins were identified as deamidated forms of beta B1 and beta A3/A1 missing portions of their N-terminal extensions. With the exception of alpha B, deamidation was detected in all crystallin species. These data indicated that a major fraction of the water-soluble protein of the adult human lens is composed of truncated beta B1 and beta A3/A1 crystallins, and that nearly all human crystallins, including the, beta-crystallins, are susceptible to deamidation. The results also provided the most detailed map to date of the identities of protein species on two-dimensional electrophoresis gels of adult human lenses.

Adolescent↗
PubMed1998

Age-related changes in human lens crystallins identified by HPLC and mass spectrometry.

Analysis of water-soluble crystallins from human lenses, ages 32 week fetal to 55 years has led to identification of the major modifications of the proteins comprising the lens. These modifications were identified by the masses of the proteins determined by electrospray ionization mass spectrometry after the proteins were separated by gel filtration and reversed phase high performance liquid chromatography. Examination of all the proteins isolated from the water soluble portion demonstrated that the major age-related modifications causing significant alteration in the molecular weights of the lens crystallins include truncation of the N-termini of beta B1, beta A3 and beta A1, and partial phosphorylation and C-terminal degradation of alpha-crystallins. N-terminal degradation of beta B1, beta A3 and beta A1 was evident in human lenses less than one year old, and the proportion of these truncated proteins became greater with age. Phosphorylation of alpha A- and alpha B-crystallins increased from the fetal to the 3 year old lens, but did not change with further aging. Minor components indicating truncation of the C-termini of alpha-crystallins were found in older lenses. In contrast to beta B1, beta A3 and beta A1, the masses of the major species of alpha A, alpha B, beta B2, beta A4, gamma S, gamma C, and gamma D did not change with aging. This suggested that the major modifications to these crystallins are limited to deamidation and possibly intra-molecular disulfide bonds. These data, in conjunction with the data in the accompanying manuscript, established deamidation as a common modification, since deamidation, which causes only a one dalton change in mass, is the only modification that is consistent with the absence of a detectable change in molecular weight and the observed increased acidity demonstrated in the two-dimensional gels of the accompanying paper. Other age related changes included a decrease in beta B3 (M(r) 24224), a major component of the fetal lens, which was not detected in lenses older than 3 years, and increases in the ratios of alpha B:alpha A and gamma S:gamma C.

Adolescent↗
PubMed1998

Deamidation and disulfide bonding in human lens gamma-crystallins.

Detailed analysis of the three gamma-crystallins present in the water-soluble portion of human lenses, gammaS, gammaD and gammaC, has identified disulfide bonding and deamidation as the major post-translational modifications of these crystallins. Chromatographic and mass spectrometric techniques were used to isolate and identify water-soluble gamma-crystallins from normal lenses, ages 32 week gestation, 0 day old, 4 day old, 19, 31, 45 and 55 year old. The amino acid sequences of the gamma-crystallins were confirmed and/or corrected by mass spectrometric analysis of peptides produced by enzymatic digestion or chemical fragmentation of the isolated crystallins. The molecular weight of peptides were also used to identify, locate and quantify modifications. Each of the gamma-crystallins had two disulfide bonds as well as several deamidated glutamine and asparagine residues. The extent of disulfide bond formation and deamidation appeared to increase with the age of the lens. This examination of normal human lens gamma-crystallins, the first detailed characterization of the gamma-crystallins, will provide a basis for comparison with modifications found in the water-insoluble portion and in cataractous lenses.

Adult↗
PubMed1997

Influence of protein-glutathione mixed disulfide on the chaperone-like function of alpha-crystallin.

In an earlier report we showed that incubation of alpha-crystallin with oxidized glutathione results in significant loss of its chaperone-like activity. In the present study, we determined the effect of protein-glutathione mixed disulfides (PSSG), formed at Cys-131 in bovine alphaA-crystallin, and Cys-131 and Cys-142 in human alphaA-crystallin, on the function of alpha-crystallin as a molecular chaperone. After incubation of calf and young human alphaL-crystallin fractions with oxidized glutathione, levels of PSSG were determined by performic acid oxidation of the mixed disulfides followed by reversed-phase high pressure liquid chromatography separation of phenylisothiocyanate-derivatized glutathione sulfonic acid. Levels of PSSG increased from 0.01 to 0.14 nmol/nmol (20 kDa) in bovine alphaL-crystallin and from 0.022 to 0.25 nmol/nmol in human alphaL-crystallin. The presence of glutathione adducts at Cys-131 and Cys-142 were confirmed by mass spectral analysis. The chaperone-like activity was determined by the heat denaturation assay using betaL-crystallin as the target protein. To examine the reversibility of the effect of mixed disulfides on chaperone activity, studies were done before and after reduction with the glutathione reductase system. Increased levels of PSSG resulted in lower chaperone activities. Treatment with the glutathione reductase system led to 80% reduction in PSSG levels with a concomitant recovery of the chaperone activity. These results suggest that cysteine(s) in the alphaA-crystallin subunit play an important role in the function of alpha-crystallin as a molecular chaperone.

Animals↗
PubMed1997

Stress and developmental regulation of the yeast C-type cyclin Ume3p (Srb11p/Ssn8p).

The ume3-1 allele was identified as a mutation that allowed the aberrant expression of several meiotic genes (e.g. SPO11, SPO13) during mitotic cell division in Saccharomyces cerevisiae. Here we report that UME3 is also required for the full repression of the HSP70 family member SSA1. UME3 encodes a non-essential C-type cyclin (Ume3p) whose levels do not vary through the mitotic cell cycle. However, Ume3p is destroyed during meiosis or when cultures are subjected to heat shock. Ume3p mutants resistant to degradation resulted in a 2-fold reduction in SPO13 mRNA levels during meiosis, indicating that the down-regulation of this cyclin is important for normal meiotic gene expression. Mutational analysis identified two regions (PEST-rich and RXXL) that mediate Ume3p degradation. A third destruction signal lies within the highly conserved cyclin box, a region that mediates cyclin-cyclin-dependent kinase (Cdk) interactions. However, the Cdk activated by Ume3p (Ume5p) is not required for the rapid destruction of this cyclin. Finally, Ume3p destruction was not affected in mutants defective for ubiquitin-dependent proteolysis. These results support a model in which Ume3p, when exposed to heat shock or sporulation conditions, is targeted for destruction to allow the expression of genes necessary for the cell to respond correctly to these environmental cues.

Adenosine Triphosphatases↗
PubMed1997

Cloning and characterization of the human granulocyte chemotactic protein-2 gene.

We recently described a novel murine CXC chemokine, designated lipopolysaccharide-induced CXC chemokine (LIX). In an ongoing search for new human chemokines related to LIX, we cloned the gene for human granulocyte chemotactic protein-2 (GCP-2) as well as previously described CXC chemokine genes, including epithelial cell-derived neutrophil-activating peptide-78 (ENA-78). Both coding and noncoding portions of the GCP-2 gene have very high nucleotide similarity to ENA-78, except for the occurrence of a long interspersed DNA-1 sequence 5' of the GCP-2 gene. The GCP-2 gene encodes a propeptide of 114 amino acid residues. The predicted 77-residue mature peptide is identical with the GCP-2 protein previously isolated from MG-63 osteosarcoma cells, except for two additional residues at the carboxyl terminus. We confirmed expression of the gene by Northern analysis and by cloning a portion of the cDNA from reverse transcribed MG-63 cell RNA. Despite 85% identity of the first 270 nucleotides 5' of the transcription start sites, GCP-2 and ENA-78 show cell-specific differences in regulation. GCP-2 is induced in MG-63, but not A549 cells by TNF-alpha, IL-1beta, and LPS, while ENA-78 is expressed in both cell types. Analysis of nucleotide sequence relationships does not support the proposal, by others, that LIX is murine GCP-2. LIX is no more closely related to human GCP-2 than to human ENA-78 and is more distant from both human genes than is porcine alveolar macrophage chemotactic factor-II.

Amino Acid Sequence↗
PubMed1997

Size of human lens beta-crystallin aggregates are distinguished by N-terminal truncation of betaB1.

The aggregates formed by the interactions of the human lens beta-crystallins have been particularly difficult to characterize because the beta-crystallins comprise several proteins of similar structure and molecular weight and because their sequences were not known until recently. Previously, it could not be ascertained whether the species of various acidities were different proteins or modifications of the same proteins. The recent determination of the sequences permits calculation of molecular weights and unambiguous identification of the various beta-crystallins and their modified forms by mass spectrometry. In this investigation, the components of the three sizes of beta-crystallin aggregates, beta1 (approximately 150,000), beta2 (approximately 92,000), and beta3 (approximately 46,000), were determined. The principal differences among the different beta-crystallin aggregates was the presence of betaA4 in beta1 and beta2, but not beta3, and the length of the N-terminal extension of betaB1. The size of the beta-crystallin aggregate correlated with the length of the N-terminal extension of betaB1, indicating that the flexible N terminus of betaB1 is critical to the formation of higher molecular weight aggregates of beta-crystallins. Separation of the components by ion exchange under non-denaturing conditions showed that betaB2 occurs as homo-dimers and homo-tetramers as well as contributing to hetero-oligomers. Other beta-crystallins were present only as hetero-oligomers.

Crystallins↗
PubMed1997

Sequence analysis of betaA3, betaB3, and betaA4 crystallins completes the identification of the major proteins in young human lens.

A combination of Edman sequence analysis and mass spectrometry identified the major proteins of the young human lens as alphaA, alphaB, betaA1, betaA3, betaA4, betaB1, betaB2, betaB3, gammaS, gammaC, and gammaD-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human betaA1, betaA3, betaA4, and betaB3-crystallin subunits were predicted by determining cDNA sequences. Mass spectrometric analyses of each intact protein as well as the peptides from trypsin-digested proteins confirmed the predicted amino acid sequences and detected a partially degraded form of betaA3/A1 missing either 22 or 4 amino acid residues from its N-terminal extension. These studies were a prerequisite for future studies to determine how human lens proteins are altered during aging and cataract formation.

Amino Acid Sequence↗
PubMed1997

Treatment of acquired factor VIII inhibitor using intravenous immunoglobulin in two patients with systemic lupus erythematosus.

Acquired inhibitors of factor VIII have rarely been reported in the presence of systemic lupus erythematosus (SLE). Treatment is usually corticosteroids, with or without immunosuppressive agents such as cyclophosphamide or azathioprine. We report here case histories of 2 patients, one with documented SLE, the other with a forme fruste of SLE. Both patients had inadequate responses to corticosteroids and immunosuppressive agents. Both responded to intravenous immunoglobulin, with a decrease in their titers of factor VIII inhibitor and significantly decreased frequency of bleeding episodes despite persistent low-level inhibitor titers. Better control of their SLE symptoms and findings was also observed. Previously reported treatments of acquired factor VIII inhibitors in SLE are discussed.

Adult↗
PubMed1997

Sequence similarities of a subgroup of CXC chemokines related to murine LIX: implications for the interpretation of evolutionary relationships among chemokines.

The murine CXC chemokine LIX has distinctive sequence features that suggest it is a novel chemokine. Among known human chemokines, ENA-78 and GCP-2 are the two most closely related to LIX. We have recently cloned the human GCP-2 gene. Phylogenetic analysis shows that the LIX coding region is more distant from both human GCP-2 and ENA-78 than is porcine AMCF-II, the chemokine with greatest sequence similarity to LIX. Human GCP-2 and ENA-78 have very high nucleotide similarity in non-coding as well as coding sequences, which suggests that these genes are the result of an evolutionarily recent gene duplication event. If this duplication occurred during primate evolution, then non-primate species may have only a single chemokine corresponding to this pair of human genes. This example shows that a one-to-one genetic correspondence does not necessarily exist between all the chemokine genes in two different species. These observations may have important implications for other chemokines that belong to clusters of closely related genes.

Amino Acid Sequence↗