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Biomedical subjects

D L Smith

Publications and source records attributed to D L 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

Detection of benzo[a]pyrene sulfate and glucuronide conjugates in cell culture medium by directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry.

An improved method is described for detecting glucuronide and sulfate conjugates of benzo[a]pyrene in medium from cell cultures treated with benzo[a]pyrene. This method is based on a microbore high-performance liquid chromatograph directly coupled to a high-resolution continuous-flow fast atom bombardment mass spectrometer. Sulfate and glucuronide conjugates, as well as some structural isomers of glucuronide conjugates, were fully separated by the reversed-phase microbore high-performance liquid chromatography conditions used in this study. Since the method does not rely on the use of radiolabeled materials, it may be used to detect conjugates of a wide variety of hydrocarbons. The high sensitivity and selectivity of the method were demonstrated by detecting conjugates in the media of cell cultures derived from mice, hamsters and humans.

Animals↗
PubMed1992

A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer.

DNA recognition sequences for dimeric proteins typically contain two types of information. The first is the DNA sequence of each half-site, and the second is the arrangement of these half-sites. We show that dimers of the yeast homeodomain protein alpha 2, although able to read the first type of information, lack the ability to assess the second type. Rather, alpha 2 dimers bind with equal affinity to artificial operators in which the two half-sites are arrayed as inverted repeats, as direct repeats, or as everted (inside-out) repeats. We show that a second protein-MCM1-sets the exact spacing and orientation of the homeodomains in the alpha 2 dimer so that they accommodate only the geometry of the naturally occurring operators. These experiments show directly how the target specificity of a homeodomain protein is raised by an auxiliary protein, allowing it to distinguish the biologically correct operators from closely related sequences in the cell.

Base Sequence↗
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

Microwave thermal balloon angioplasty in the normal rabbit.

The feasibility of utilizing microwave energy for thermal angioplasty was evaluated in 28 normal New Zealand white rabbits. Angioplasty catheters were inserted via bilateral femoral arteriotomy and balloon angioplasty was performed on the external iliac arteries. There were three variables in the microwave angioplasty procedure: peak temperature, energy duration, and peak balloon pressure. At 1 week after the angioplasty, animals were put to death and the iliac arteries were removed after perfusion fixation. Histologic analysis was performed. Injuries to the medial and intimal proliferation were evaluated using an index based on the circumferential extent and depth of abnormality. There was no relationship noted between intimal proliferation or medial injury and energy duration of peak balloon pressure. There was a direct relationship between peak temperature and medial injury. Furthermore, there was an inverse relationship between medial injury and intimal proliferation. The axial extent of change induced by microwave energy was 1.6 cm. Microwave thermal energy delivery through a balloon angioplasty catheter is feasible and can be performed conveniently.

Angioplasty, Balloon↗
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

5-HT3 receptors are not involved in the modulation of the K(+)-evoked release of [3H]5-HT from spinal cord synaptosomes of rat.

The ability of 5-HT3 receptor agonists to modulate the resting efflux or K(+)-evoked release of [3H]5-HT from superfused synaptosomes from the spinal cord of the rat was investigated. Phenylbiguanide did not alter the resting efflux of [3H]5-HIAA or [3H]5-HT or modify the K(+)-evoked release of [3H]5-HT. 2-Methyl-5-HT (10 microM) caused an increase in resting efflux of [3H]5-HIAA, an effect that was blocked by the inhibitor of the uptake of 5-HT fluoxetine. No effect on K(+)-evoked release of tritium was observed. Bufotenine (100-1000 nM) increased the resting efflux of [3H]5-HT and [3H]5-HIAA. These effects were not antagonized by the 5-HT3 antagonist ICS 205-930 but were antagonized by fluoxetine. The drug ICS 205-930 (1 microM) did not alter resting efflux or block the ability of serotonin (30 and 100 nM) to decrease K(+)-evoked release of tritium. Quipazine, a potent antagonist of peripheral 5-HT3 receptors (subnanomolar concentrations), was also unable to alter resting or K(+)-evoked release of [3H]5-HT. It did, however, attenuate the inhibitory effect 5-HT on K(+)-evoked release. The concentrations required were in the micromolar range, consistent with the ability of the drug to antagonize the 5-HT1B autoreceptor. These results support the idea that 5-HT3 receptors do not act as nerve terminal autoreceptors in the spinal cord of the rat.

Animals↗
PubMed1992

Distribution of toxigenic Escherichia coli serotypes in the intestines of infants.

As part of an ongoing study into the pathogenesis of sudden infant death syndrome (SIDS), the distribution of serotypes of toxigenic and non-toxigenic Escherichia coli within the gastrointestinal tract of babies who had died was investigated. Escherichia coli isolates from the mid-ileum, colon and rectum of six SIDS cases and one case which had died suddenly and unexpectedly but had underlying cardiac pathology were "O" serogrouped and examined for verocytoxic activity and production of heat-labile enterotoxin. In addition, the effect of storage of gut specimens and rectal swabs at 4 degrees C on isolation of toxigenic strains was studied in three of the cases. A diversity of serogroups and toxigenicity was a general finding, however, strains found in the proximal gut were also cultured from the rectum, indicating that faecal specimens would be a valid tool in investigating the role of these organisms in SIDS cases compared with healthy controls. Storage for up to 5 days at 4 degrees C had no appreciable effect on isolation rates of toxigenic bacteria.

Animals↗
PubMed1992

Biologically active gamma-lactones and methylketoalkenes from Lindera benzoin.

Brine shrimp lethality-directed fractionation of the 95% EtOH extract of ripe berries from Lindera benzoin led to the isolation of three new C21 alkane-alkene gamma-lactones designated isolinderanolide, isolinderenolide, and linderanolide as well as the known series of C17 and C19 obtusilactones (isoobtusilactone A, obtusilactone A, isoobtusilactone, and obtusilactone) previously isolated from Lindera obtusiloba. The novel (6Z,9Z,12Z)-pentadecatrien-2-one, the known (6Z,9Z)-pentadecadien-2-one, and the known (+)-(Z)-nerolidol were also isolated as bioactive compounds. The structural elucidation and biological activities of these compounds are reported.

Animals↗
PubMed1992

Gigantetronenin and gigantrionenin: novel cytotoxic acetogenins from Goniothalamus giganteus.

Gigantetronenin [1] and gigantrionenin [6], two new monotetrahydrofuran Annonaceous acetogenins each possessing a double bond along the hydrocarbon chain, have been isolated from the bark of Goniothalamus giganteus by the use of brine shrimp lethality for bioactivity-directed fractionation. The structures were elucidated based on spectroscopic and chemical methods. Compounds 1 and 6 both show selective and potent cytotoxicities to human tumor cells in culture as well as toxicity to brine shrimp. A known cytotoxic acetogenin, annomontacin [11], was also isolated from this plant. The biogenetic pathway of the acetogenins from G. giganteus is discussed.

Animals↗
PubMed1992

Epoetin enhances erythropoiesis in normal men undergoing repeated phlebotomies.

Epoetin may enhance autologous blood donation, but efficacy and dose response have not been established. This multicenter, double-blind trial compared intravenous placebo (n = 23) with epoetin beta, 250 U/kg (n = 23), 500 U/kg (n = 19), and 1000 U/kg (n = 22), administered three times weekly for 26 days. Normal men (age, 28 +/- 7 years; mean +/- SD) received phlebotomies up to three times weekly as long as the hemoglobin remained greater than or equal to 12 gm/dl. Subjects treated with epoetin donated 32% more units of blood (p less than 0.05) compared with placebo. A dose response was not observed. Platelet counts increased with epoetin compared with placebo, but platelet function and bleeding time did not change. Prothrombin times increased and partial thromboplastin times decreased with both epoetin and placebo. The supernatant of packed red blood cells collected after multiple phlebotomies and stored 42 days had slightly lower glucose concentrations and pH after therapy with epoetin. Blood pressure did not change with epoetin or placebo. These findings support the efficacy and safety of epoetin for enhancing the erythropoietic response of normal subjects during intensive phlebotomy.

Adolescent↗
PubMed1992

Multiple effects of mutation on expression of alternative cell surface protein genes in Tetrahymena thermophila.

Genes at the SerH locus of the ciliated protist Tetrahymena thermophila specify the major (H) surface protein on cells grown at 20-36 degrees. Alternative proteins L, T, S and I are expressed under different conditions of temperature and culture media. Mutants unable to express SerH genes were examined for expression of these proteins, also called immobilization or i-antigens, at both H and non-H conditions. In all instances, one or more i-antigens were expressed in the absence of H, and, in most instances, expression of i-antigens under non-H conditions was also affected. Examples of the latter include both the continued expression of H-replacement antigens and the inability to express certain other i-antigens. Such multiple effects were observed in mutants with trans-acting (rseA, rseB, rseC, RseD) and cis-acting (H1-1 and H1-2) mutations, but not in mutants in which SerH is affected developmentally (B2092, B2101, B2103, B2107). These interactions suggest that the wild-type genes identified by mutation exert both positive and negative effects in the regulation of i-antigen gene expression.

Animals↗