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Biomedical subjects

J B Smith

Publications and source records attributed to J B Smith.

At least 19 recordsLinked to original sources
PubMed1992

Rates of carbamylation of specific lysyl residues in bovine alpha-crystallins.

Previous investigations indicate that some forms of cataract may be due to the reactions of isocyanate with lens proteins. The present investigation was directed toward identifying the products of these reactions and determining rate constants for their formation. Bovine alpha-crystallins were incubated with isocyanate and separated into alpha A- and alpha B-crystallins by reversed-phase HPLC (high-performance liquid chromatography). Products of the reaction of isocyanate with alpha-crystallins were analyzed by mass spectrometry and isoelectric focusing. Proteolytic digests of carbamylated alpha A were analyzed by HPLC and fast atom bombardment mass spectrometry to determine the extent of reaction of each of the 7 lysyl residues present in alpha A. These results demonstrate that incubation of alpha-crystallins in 0.1 M KNCO leads to partial carbamylation of all 7 lysines of alpha A-crystallin. The extent of modification after 24 h of incubation varied from 7% at Lys 88 to 61% at Lys 11. Rate constants for the reaction of specific lysyl residues with isocyanate ranged from 5 to 54 x 10(-2) M-1 h-1. The distribution of reaction products, as determined by isoelectric focusing, indicates that the physiologically relevant initial stages of carbamylation of the 7 lysyl residues of alpha A proceed in a noncooperative manner.

Amino Acid Sequence↗
PubMed1992

Cytosolic calcium as a second messenger for collagen-induced platelet responses.

We showed previously that direct platelet activation by collagen involves an increase in the platelet cytosolic free Ca2+ concentration ([Ca2+]i) but that this increase is not required for the adhesion of platelets to collagen. We now report that collagen-induced arachidonic acid liberation, myosin phosphorylation and 5-hydroxytryptamine secretion are dependent on increases in [Ca2+]i, as they were markedly inhibited in platelets loaded with the acetoxymethyl ester of the Ca2+ chelator BAPTA but not in cells loaded with the acetoxymethyl ester of the non-chelating diazo-3. BAPTA also partially inhibited the rate of collagen-induced phosphatidic acid (PtdA) formation but had little effect on increases in phosphorylation of pleckstrin (47 kDa protein; P47). From these results we infer that collagen-induced increases in [Ca2+]i are required for dense granule secretion and arachidonic acid liberation, but are not necessary for stimulation of the protein kinase C pathway.

Affinity Labels↗
PubMed1992

A sequence element necessary for self-cleavage of the antigenomic hepatitis delta RNA in 20 M formamide.

The genomic and antigenomic RNAs of hepatitis delta virus are capable of self-cleavage and show no significant sequence similarities to other known self-cleaving RNAs. We have derived an antigenomic delta RNA which cleaves to completion in 15 s in 9 mM magnesium at 37 degrees C and is capable of efficient self-cleavage in concentrations of formamide as high as 20 M. Cleavage in high concentrations of denaturant is dependent upon the presence of a polypurine sequence element, GGAGA, located between 81 and 85 nucleotides downstream of the cleavage site. Mutation of the initial G81G82 to C81C82, or removal of the sequence element, results in a loss of the ability to cleave in high formamide concentrations. Changing the final U-2C-1 of a pyrimidine-rich region, UCUUC, just upstream of the cleavage site, to G-2G-1 severely affects the self-cleavage, but introducing the two mutations, GG to CC and UC to GG, into the same molecule, restoring potential base pairing, partially restores the formamide stability. Relocating the GGAGA sequence upstream of the cleavage site also results in partial restoration of the formamide cleavage. Although the GGAGA sequence is important for self-cleavage under denaturing conditions, it does not appear to be necessary for HDV RNA cleavage in normal buffer conditions.

Base Sequence↗
PubMed1992

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.

Amino Acid Sequence↗
PubMed1992

Evidence that adhesion of electrically permeabilized platelets to collagen is mediated by guanine nucleotide regulatory proteins.

Adhesion of electrically permeabilized platelets to collagen was found to be essentially independent of free Ca2+ concentration in the medium. Addition of stable GTP analogues increased the proportion of adhering cells about 5-fold. This effect was inhibited by guanosine 5'-[beta-thio]diphosphate, cytochalasin D or monoclonal antibodies to glycoprotein Ia. In contrast, the protein kinase C inhibitor staurosporine had only a small effect on the GTP-analogue-enhanced adhesion of the permeabilized cells to collagen. These results suggest that a guanine nucleotide regulatory (G)-protein is directly linked to the collagen receptor and is involved in the actin-dependent recruitment of additional collagen receptors.

Alkaloids↗
PubMed1992

Cyclic AMP does not inhibit collagen-induced platelet signal transduction.

The adhesion of platelets to collagen and their activation is the primary event in haemostasis. Following adhesion, platelet aggregation mediated by ADP, thromboxane A2 and thrombin leads to the formation of a platelet plug. It is known that platelet activation by each of these agonists involves an increase in the cytosolic free Ca2+ concentration, and this has been thought to be controlled by cyclic AMP. However, we report here that while signal transduction induced by ADP plus a thromboxane mimetic (U46619), or by thrombin, is inhibited by stimulators of adenylate cyclase such as a prostaglandin I2 (PGI2) analogue (Iloprost), PGD2 and forskolin, elevation of cyclic AMP does not inhibit either platelet adhesion to collagen or the associated Ca2+ mobilization, phosphatidic acid formation or 5-hydroxytryptamine secretion. Furthermore, collagen did not lower elevated levels of cyclic AMP in platelets measured in the presence of both a thromboxane antagonist and an ADP-removing system. The present results are discussed in the context of previous findings.

Adenosine Diphosphate↗
PubMed1992

Localization of major histocompatibility complex class I and II mRNA in human first-trimester chorionic villi by in situ hybridization.

Maternal immune recognition of pregnancy occurs despite the nonexpression of classical major histocompatibility complex (MHC) antigenic determinants by chorionic villous trophoblast, which comprise the major surface area where maternal blood contacts fetal-derived cells. cDNA-mRNA in situ hybridization was used to probe expression of transcripts corresponding to nonpolymorphic MHC determinants in first-trimester chorionic villus samples. The HLA-B7 probe hybridization signals were localized to syncytiotrophoblast and to cells of the mesenchyme but not to villous cytotrophoblast. HLA-G mRNA was found only in syncytiotrophoblast. A DR beta clone hybridized to both villous cytotrophoblast and syncytiotrophoblast. The results suggest that expression of trophoblast class I and class II determinants early in gestation (10 wk) may be regulated by posttranscriptional events. This also suggests the potential for maternal antifetal alloimmune responses.

Chorionic Villi↗
PubMed1992

Identification of the posttranslational modifications of bovine lens alpha B-crystallins by mass spectrometry.

A combination of mass spectrometric techniques has been used to investigate the amino acid sequence and post-translational modifications of alpha B-crystallin isolated from bovine lenses by gel filtration chromatography and reversed-phase high performance liquid chromatography. Chromatographic fractions were analyzed by electrospray ionization mass spectrometry to determine the homogeneity and molecular weights of proteins in the fractions. The alpha B-crystallin primary gene product, its mono- and diphosphorylated forms, its N- and C-terminal truncated forms, as well as other lens proteins unrelated to the alpha B-crystallins were identified by their molecular weights. Detailed information about the sites of phosphorylation, as well as evidence supporting reassignment of Asn to Asp at position 80, was obtained by analyzing proteolytic digests of these proteins by fast atom bombardment mass spectrometry. Results of this investigation indicate that alpha B-crystallin is phosphorylated in vivo at Ser 45, Ser 59, and either Ser 19 or 21. From the specificity of phosphorylation of alpha-crystallins, it appears that there may be two different kinases responsible for their phosphorylation.

Amino Acid Sequence↗
PubMed1992

Reversible desensitization of fibroblasts to cadmium receptor stimuli: evidence that growth in high zinc represses a xenobiotic receptor.

The xenobiotic Cd2+ triggers the production of inositol trisphosphate and releases stored Ca2+ in certain cell types, apparently by binding to a zinc site in the external domain of an "orphan" receptor (no known endogenous stimulus). Cd2+ and bradykinin evoke similar spikes in cytosolic free Ca2+. Growth in high Zn2+ (100-200 microM) abolished the free Ca2+ spike evoked by Cd2+ without affecting the spike produced by bradykinin. Growth in high Zn2+ almost abolished Cd(2+)-evoked production of [3H]inositol mono-, bis-, and trisphosphate. Bradykinin-evoked [3H]inositol phosphate production was not affected by growth in high Zn2+. Growth in high Zn2+ nearly prevented the stimulation of 45Ca2+ efflux by Cd2+ without affecting the stimulation of 45Ca2+ efflux by bradykinin or histamine. Removing Zn2+ from the culture medium and incubating the cells for several hours fully restored responsiveness to Cd2+. Cycloheximide, actinomycin D, or tunicamycin prevented the restoration of Cd2+ responsiveness, indicating that resensitization requires macromolecular synthesis. Growth in high Zn2+ reversibly abolished Ca2+ mobilization evoked by two additional stimuli: a decrease in extracellular pH or Na+ concentration. These findings support the hypothesis that the three stimuli (Cd2+ or a decrease in external pH or Na+ concentration) activate the same orphan receptor. Growth in high Zn2+ apparently desensitizes the cells to the Cd2+ receptor stimuli by repressing receptor synthesis.

Bradykinin↗
PubMed1992

Mass spectrometric analysis of the structure of gamma II bovine lens crystallin.

The amino acid sequence of bovine gamma II-crystallin has been verified by a combination of electrospray and fast atom bombardment mass spectrometry. The molecular weight of gamma II, isolated by gel filtration and ion exchange chromatography, was determined to be 20,967 +/- 3 by electrospray mass spectrometry. Another aliquot of gamma II was completely digested by trypsin in a medium of 20% CH3CN and 0.1 M Tris, pH 8.2. The tryptic peptides were separated by reversed phase HPLC and identified by their molecular weights, as determined by fast atom bombardment mass spectrometry (FABMS). The identification of each peptide was confirmed by digesting the peptide further to give new peptides whose molecular weights were also determined by FABMS and related to the proposed amino acid sequences. The data from both types of mass spectrometric analyses were consistent with the sequence previously proposed by Hay et al. (J. Biol. Chem. 1987, 146, 332-338), including threonine at position 119. The FAB mass spectrum of one HPLC fraction suggested that disulfide bonding between Cys 18 and Cys 22 was present in at least half the protein preparation. Whether the Cys 18/Cys 22 disulfide bond was present in native gamma II or was produced during isolation or enzymic digestion could not be determined from these studies. Samples that had been stored for several weeks showed that several of the cysteines had become disulfide bonded. These studies illustrate the power of mass spectrometric techniques to accurately confirm the primary structure of proteins and to identify post-translational modifications.

Amino Acid Sequence↗
PubMed1992

A putative lectin-binding receptor mediates cadmium-evoked calcium release.

Nanomolar concentrations of cadmium (Cd2+) produce an immediate rise in free Ca2+ in human dermal fibroblasts, which is mostly caused by the release of stored Ca2+ via inositol trisphosphate. Here we have used lectins to evaluate the hypothesis that a cell surface glycoprotein mediates the response to Cd2+. A prior incubation with wheat germ agglutinin (WGA) or certain other lectins inhibited calcium release evoked by Cd2+. WGA reversibly inhibited Cd(2+)-evoked calcium release as indicated by measurements of cytosolic free Ca2+ and 45Ca2+ efflux. WGA half-maximally inhibited Ca2+ release at 1.2 x 10(-7) M. The Kd for the binding of fluoresceinylated WGA was 2.8 x 10(-7) M. Chitotriose dissociated fluoresceinylated WGA from the cells and restored cadmium responsiveness. WGA inhibited Cd(2+)-evoked 45Ca2+ efflux similarly at 18 and 37 degrees C. A brief incubation with chitotriose at 18 or 10 degrees C reversed the inhibition by WGA. WGA neither bound 109Cd2+ nor affected 109Cd2+ uptake by the cells. Succinylated WGA, which binds N-acetylglucosamine but not N-acetylneuraminic acid, failed to inhibit Ca2+ release evoked by Cd2+. WGA probably inhibits Ca2+ release produced by Cd2+ by binding to N-acetylneuraminic acid in the external domain of a plasma membrane receptor.

Agglutination Tests↗
PubMed1992

The number of cells used for immunotherapy of repeated spontaneous abortion influences pregnancy outcome.

One hundred and sixty-eight women were immunized on a single occasion with paternal mononuclear cells (MNC) for recurrent spontaneous abortion (RSA) and pregnancy outcomes were analysed with respect to the number of MNC given. The study was done in a prospective sequential fashion using all MNC recoverable from a unit of the spouse's blood and both patients and investigators were blinded as to the number of cells injected. Women receiving low and mid-range doses of MNC (58-305 x 10(6) and 308-567 x 10(6), respectively) had a significantly higher pregnancy success rate (57%) than those receiving the high (568-2677 x 10(6)) dose of MNC (41%). In 77 consecutive patients the diameter of the largest immediate skin flare reaction at the site of subcutaneous injection was recorded. No correlation was found between the skin flare response and the number of MNC injected. Our data suggest that a blinded trial of paternal MNC immunization comparing what appears to be optimum numbers of cells (100-550 million) to a low dose inoculum (e.g., 10 million), again noting the sizes of the skin flare reactions, might answer questions about efficacy and placebo effects of immunotherapy for RSA.

Abortion, Habitual↗
PubMed1992

The site of blister formation in dermatitis herpetiformis is within the lamina lucida.

BACKGROUND: Because the initial neutrophilic infiltrate in dermatitis herpetiformis is within the dermal papillae, most investigators have assumed the vesicles occur in this same area. This was supported by electron microscopy studies. In 1983 Klein et al. refuted this concept, suggesting that vesicle formation was within the lamina lucida above the lamina densa. Despite this study, current literature continues to state that blister formation is below the lamina densa. OBJECTIVE: Our purpose was to determine the ultrastructural site of blister formation in early and late vesicles of dermatitis herpetiformis. METHODS: We evaluated eight biopsy specimens from four patients by immunomapping with antibodies to bullous pemphigoid antigen, laminin, type IV collagen, and epidermolysis bullosa acquisita antigen. RESULTS: In both early and late vesicles blister formation was found to be above the lamina densa in the lamina lucida. CONCLUSION: These findings are contrary to the commonly held concept that the blister in dermatitis herpetiformis is below the lamina densa and confirm the findings of Klein et al. that the site of blister formation in dermatitis herpetiformis is above the lamina densa within the lamina lucida.

Basement Membrane↗
PubMed1992

Child health outcomes among Central American refugees and immigrants in Belize.

The purpose of this study was to investigate the effect of international migration, including refugee status, upon child health outcomes. Data were drawn from a survey conducted in 1989 in three settlements in Belize, Central America, that have a high proportion of refugees and economic immigrants living side-by-side with the local population. In two of the settlements, the entire population of mothers with children under 6 was interviewed; in the third settlement a two-thirds random sample was interviewed. Health history data were obtained for 255 children of 134 mothers, from whom sociodemographic data were also collected. The majority of children were born to Salvadoran or Guatemalan mothers, but native and naturalized Belizeans in the survey communities were included for comparison purposes. Migration, the exposure variable, was characterized by mother's residency/refugee legal status, nationality, and duration of time in country. Socioeconomic and proximate control variables were included as suggested by the Mosley-Chen framework. Despite normal birthweight averaging 3374 g, a large proportion of children are at the lowest percentiles of the weight-for-age curves (44% below the tenth percentile for the international reference population). A high incidence of diarrheal and respiratory illnesses (30% and 47% of children, respectively, having frequent episodes), and 50% of children with measles vaccination appropriate for age, indicate a population with high potential morbidity. Logistic regression was used to model the effects of migration on weight-for-age and frequency of diarrheal and respiratory tract episodes independent of socioeconomic and proximate factors, as suggested by the Mosley-Chen framework.(ABSTRACT TRUNCATED AT 250 WORDS)

Belize↗
PubMed1992

Predictors for live birth after unexplained spontaneous abortions: correlations between immunologic test results, obstetric histories, and outcome of next pregnancy without treatment.

Our objective was to determine whether results from tests for maternal serum antinuclear antibodies, cytotoxic antibodies to paternal lymphocytes, parental histocompatibility types, and blocking factors for maternal-paternal mixed lymphocyte reactions were predictive of pregnancy outcome without immunologic treatment. Pregnancy outcome data from 95 women with a history of unexplained recurrent spontaneous abortions who underwent immunologic tests at Jefferson Medical College were evaluated with multiple logistic regression analyses. The number of prior spontaneous abortions, history of another relevant diagnosis, parental sharing of one histocompatibility antigen, and maternal age were related to the outcome of the next pregnancy in women given no immunologic treatment (p = 0.05). No significant correlation was found between results from the immunologic tests or other history characteristics evaluated and outcome of the next pregnancy. The immunologic tests evaluated were not clinically useful predictors of pregnancy outcome.

Abortion, Habitual↗
PubMed1992

Deriving a 67-nucleotide trans-cleaving ribozyme from the hepatitis delta virus antigenomic RNA.

RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage, and thus have the potential for serving as ribozymes in a trans-cleaving reaction. Because the catalytic core of such an enzymatic RNA was not evident from phylogenetic data, we took a step-wise approach to identifying the core, reducing the RNA in size, and characterizing various properties for each size class. Thus, a 186-nucleotide antigenomic RNA (termed Ag180) was found to be capable of cleaving well in 20 M formamide (Smith and Dinter-Gottlieb, 1991), and this unusual stability in formamide was lost by reducing the 3' end of the molecule, leaving a 140-nucleotide RNA (Ag 140). Both RNAs showed only intramolecular cleavage at a wide range of concentrations, and a number of conformers could be seen in the Ag140 RNA, some of which were resistant to cleavage at 37 degrees C. Since Ag140 could not cleave in 20 M formamide, the 5' and 3' termini of Ag180 were truncated and produced Ag5-84, which cleaved to 100% at 37 degrees C in less than 0.25 min. Internal deletions of the Stem IV region resulted in Ag5-73, still capable of efficient cleavage, although with a lessened stability in formamide. A trans-cleaving enzyme-substrate pair was finally derived from this RNA, and it consisted of a 67-nucleotide enzyme that cleaved a 13-nucleotide RNA substrate.

Base Sequence↗