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C A Smith

Publications and source records attributed to C A Smith.

At least 289 records · Page 16Linked to original sources
PubMed1994

Molecular and biological characterization of human 4-1BB and its ligand.

4-1BB was originally described as a cDNA expressed by activated murine T cells and subsequently demonstrated to encode a member of the tumor necrosis factor receptor family of integral membrane proteins. Recently, we identified and cloned a murine ligand for 4-1BB (mu4-1BB-L) and demonstrated it to be a member of an emerging family of ligands with structural homology to tumor necrosis factor. To characterize further the role of 4-1BB in the immune response we undertook to clone the human homologue of 4-1BB-L. However, attempts to isolate a cDNA encoding the human 4-1BB-L by cross-hybridization with the murine cDNA were unsuccessful. Therefore we first utilized cross-species hybridization to isolate a cDNA encoding human 4-1BB (hu4-1BB). A fusion protein consisting of the extracellular portion of hu4-1BB coupled to the Fc region of human immunoglobulin G1 (hu4-1BB.Fc) was then used to identify and clone a gene for human 4-1BB-L from an activated CD4+ T cell clone using a direct expression cloning strategy. Human 4-1BB-L shows 36% amino acid identity with its murine counterpart and maps to chromosome 19p13.3. Scatchard analysis demonstrated high-affinity binding of hu4-1BB.Fc to either native or recombinant human 4-1BB-L. Both monoclonal antibody to hu4-1BB and cells transfected with hu4-1BB-L induced a strong proliferative response in mitogen co-stimulated primary T cells. In contrast, ligation of 4-1BB on T cell clones enhanced activation-induced cell death when triggered by engagement of the TCR/CD3 complex.

Amino Acid Sequence↗
PubMed1994

Steroidogenic enzyme activity and ovarian differentiation in the saltwater crocodile, Crocodylus porosus.

It has been hypothesized that steroid hormone production is involved in sexual differentiation of the gonads in reptiles with temperature-dependent sex determination. We have therefore examined steroidogenic enzymes and ovarian differentiation in Crocodylus porosus embryos incubated at 30 degrees, a temperature producing 100% female hatchlings. delta 5-3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) activity was detected only in the adrenal gland before, during, and after the period of ovary differentiation. The same pattern was observed during testis differentiation in embryos incubated at 32 degrees (predominantly male-producing). At no stage was 3 beta-HSD activity detected in the gonads. The tritiated water assay was used to measure aromatase enzyme activity in the gonad-adrenal-mesonephric complex (GAM) during development at 30 degrees C. Aromatase activity in the GAM increased during the period of ovary differentiation. In female C. porosus hatchlings, 85% of the aromatase activity within the GAM was derived from the ovary, 10% from the adrenal, and 5% from the regressing mesonephros. The aromatase inhibitors aminoglutethimide and 4-hydroxyandrostenedione significantly reduced aromatase activity in female hatchling GAMs. In some stage 25 embryos incubated at 32.5 degrees, aromatase activity was low in testes and high in ovaries. These observations show that urogenital tissues of C. porosus are steroidogenically active during gonadal differentiation and that increased aromatase enzyme activity accompanies ovary development at 30 degrees. Steroid synthesis in the GAM of C. porosus embryos may involve interaction between the adrenal glands and the gonads.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Cowpox virus contains two copies of an early gene encoding a soluble secreted form of the type II TNF receptor.

The inverted terminal repeats of the DNA of cowpox virus (Brighton Red strain) contain the crmB gene, an additional member of a family of viral genes that modify cytokine responses to infection. The crmB gene is transcribed from an early promoter. The primary product is a 355-amino-acid protein containing a signal peptide sequence and three potential N-linked glycosylation sites. The mature gene product is a secreted soluble protein that has an apparent molecular mass of 48 kDa. TNF alpha and TNF beta bind to this protein in a competitive manner, consistent with the sequence of its N-terminal 176 amino acids, which closely resembles the ligand-binding domains of the type II (75-kDa) human TNF receptor. The sequence of the C-terminal 161 amino acids of the CrmB protein is unlike that of human TNF receptors, but overall, the CrmB protein is similar to the T2 proteins of the leporipoxviruses (48% identity) and the predicted product of the G4R/G2R open reading frame of variola virus (85% identity), suggesting that not only the TNF-binding domains but also the C-terminal regions contribute to the functions of these viral proteins. These results show that orthopoxiviruses such as cowpox virus encode secreted forms of TNF receptors that can contribute to the modification of TNF-mediated antiviral processes.

Amino Acid Sequence↗
PubMed1994

Three-dimensional structure of lactoferrin in various functional states.

The three-dimensional structures of various forms of lactoferrin, determined by high resolution crystallographic studies, have been compared in order to determine the relationship between structure and biological function. These comparisons include human apo and diferric lactoferrins, metal and anion substituted lactoferrins, the N-terminal half molecule of human lactoferrin, and bovine diferric lactoferrin. The structures themselves define the nature and location of the iron binding sites and allow anti-bacterial and putative receptor-binding regions to be mapped on to the molecular surface. The structural comparisons show that small internal adjustments can allow the accommodation of different metals and anions without altering the overall molecular structure, whereas large-scale conformational changes are associated with metal binding and release, and smaller, but significant, movements accompany species variations. The results also focus on differences in flexibility between the two lobes, and on the importance of interactions in the inter-lobe region in modulating iron release from the N-lobe and in possibly enabling binding at one site to be signalled to the other.

Animals↗
PubMed1994

Synergism and substitution in the lactoferrins.

The anion binding properties of human lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, highlight differences between the two sites, and in the anion binding behaviour when different metals are bound. Carbonate, oxalate and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf although it does for the bovine analogue. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+. The anion sites as determined from the crystal structures of Fe2(CO3)2Lf, Fe2(C2O4)2Lf, Cu2(CO3)2Lf, and Cu2(CO3)(C2O4)Lf have been compared. Both the carbonate and oxalate ions bind in bidentate fashion to the metal, except that the carbonate ion in the N-lobe site of dicupric lactoferrin is monodentate. The hybrid copper lactoferrin complex shows that the oxalate ion binds preferentially in the C-lobe site in a bidentate mode. A series of complexes containing the synergistic anion O,N-chelates with increasing substitution on the N atom (glycinate, iminodiacetate and nitrilotriacetate) have been prepared with iron bovine lactoferrin for comparison with the O,O-chelate oxalate. Overall these observations lead to a generalised model for synergistic anion binding by transferrins and allow comparisons to be made with nonsynergistic anions such as citrate and succinate.

Animals↗
PubMed1994

The mouse Fas-ligand gene is mutated in gld mice and is part of a TNF family gene cluster.

The gene for the mouse Fas ligand was cloned and its chromosomal position determined. Fasl was tightly linked to gld (no crossovers in 567 meiotic events) on mouse chromosome 1 and closely linked with a novel member of the same TNF family of ligands, the Ox40 ligand (Ox40l, 1 crossover in 567 meiotic events). Southern blot analysis did not reveal any difference between the Fasl gene from gld and wild-type mice and levels of Fasl mRNA transcripts were similar in PMA and ionomycin induced wild-type and coisogenic gld T cells. Sequence analysis of the gld gene indicated a single amino acid change (Phe Leu) in the COOH terminal portion of this type II transmembrane protein, and COS cells transfected with Fasl cDNA from gld mice failed to induce apoptosis of Fas-expressing target cells. Thus, the data demonstrate that the gld phenotype is the result of a point mutation in the Fasl gene and that Fasl is part of a complex of ligands structurally related to TNF mapping within a small region of mouse chromosome 1.

Amino Acid Sequence↗
PubMed1994

Metabolic polymorphisms in carcinogen metabolising enzymes and cancer susceptibility.

Molecular genetic analysis is providing us with enormous advances in understanding the pathogenesis of human diseases such as cancer. The study of familial disease and the subsequent mapping and identification of the mutations which contribute to disease susceptibility, is not only providing insights into the factors involved in the pathogenesis of the disease but also identifying new targets for therapy. It is now clear that human tumours result from a complex sequence of mutation events. Each individual step makes the mutated cell more independent of its normal growth regulatory processes, eventually resulting in the formation of a metastatic tumour. There are a multitude of biochemical changes that these mutations confer, which provide preneoplastic cells with a selection advantage. In addition to an increased rate of cell division, such changes may make the cells resistant to cytotoxic insult or to programmed cell death. They can also confer an increased ability to survive independent of a normal hormonal environment. It is now clear that all these types of change may contribute to tumour cell progression.

Cytochrome P-450 Enzyme System↗
PubMed1994

Effects of REM sleep on the ventilatory response to airway occlusion in the dog.

We determined the effects of sleep state on the ventilatory response following transient airway occlusion and on the response to vagal blockade in the unanesthetized sleeping dog. Three tracheotomized dogs underwent repeated occlusions (159 trials) during rapid eye movement (REM) and nonrapid eye movement (NREM) sleep. In all sleep states we found significant but variable transient hyperventilation following release of occlusion. In NREM sleep, a significant central apnea [expiratory time (TE) prolonged 2-10 times control] followed the hyperpneic response, so long as the increase in tidal volume (VT) during the hyperpnea exceeded three times control VT, that is, a volume-dependent apneic threshold. In REM sleep with maintained levels of eye movement density, hyperventilation commonly followed release of obstruction but only very rarely did VT exceed the volume threshold, and central apnea was rare. Cervical vagal blockade was used to show that significant inhibitory pulmonary stretch receptor reflexes were present in both NREM and REM sleep, although the strength of the reflex was diminished in REM. We postulate that the phasic events of REM sleep inhibit the increase in VT in response to the chemical stimuli accumulated during airway occlusion and also interfere with the prolongation of TE in response to lung stretch and/or transient hypocapnia. The result is that central apnea occurs only very rarely in REM sleep.

Airway Obstruction↗
PubMed1994

Debrisoquine and mephenytoin oxidation in Sinhalese: a population study.

The frequency distributions of the 0-8 h urinary metabolic ratios of debrisoquine and mephenytoin were measured in 111 healthy, unrelated Sinhalese resident in Sri Lanka. Blood samples were taken from 77 of these subjects for CYP2D6 genotyping. Bimodality in the distribution of the log10 debrisoquine/4-hydroxydebrisoquine ratio was not evident from visual inspection and by kernel density analysis. The results of genotyping indicated that 82% of the population were either homozygous for the wild-type CYP2D6 gene or heterozygous for the wild type allele and the whole gene deletion. Eighteen per cent of the Sinhalese population were heterozygous for the CYP2D6B mutation and the wild-type allele. All of these genotypes give rise to the extensive metaboliser phenotype in white Caucasians. No CYP2D6A mutations were identified and no individuals who were homozygous for the mutant alleles were detected, which is in accord with an absence of phenotypic poor metabolisers of debrisoquine. The mutant CYP2D6 allele frequency in Sinhalese (9%) is only half that observed in white Caucasians. The S/R-mephenytoin ratio ranged from 0.09 to 2.27 (median 0.38). By visual inspection and kernel density analysis the distribution of the S/R-mephenytoin ratio was bimodal and, using a value of 0.9 for the antimode, 16 (14%) subjects were poor metabolisers. In conclusion, the prevalence of the poor metaboliser phenotype in Sinhalese appears much lower for debrisoquine and higher for mephenytoin than in white Caucasians. These findings are similar to those observed in Indians living in Bombay and in Oriental populations.

Adult↗
PubMed1994

Heterogeneous expression and polymorphic genotype of glutathione S-transferases in human lung.

BACKGROUND: Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics by conjugation with glutathione. One of the mu class genes of this superfamily of enzymes, GSTM1, is polymorphic because of a partial gene deletion. This results in a failure to express GSTM1 in approximately 50% of individuals. Several studies have linked GSTM1 null status to an increased risk of lung carcinoma. This study investigated the expression and distribution of GST isoenzymes in human lung, and developed a polymerase chain reaction (PCR) assay which would allow genotyping of archival, paraffin embedded lung tissue. METHODS: Distribution was examined using a panel of polyclonal anti-GST antibodies for immunohistochemistry in normal tissue of 21 tumour-bearing lungs. DNA for PCR was extracted from paraffin blocks and a control group of 350 blood lysates. As a positive control each assay amplified part of GSTM4, a mu class gene which is not polymorphic but which shows strong sequence homology to GSTM1. The presence of GST in bronchoalveolar lavage fluid was sought by Western analysis. RESULTS: Proximal airways contained pi class GST, alpha class GST, and mu class GST with expression concentrated in the brush border. In distal airspaces no alpha GST was expressed but pi GST and mu GST were present in alveolar cells and also alveolar macrophages. Pi class GST was present in bronchoalveolar lavage fluid. The PCR assay enabled genotypic determination using DNA extracted from archival material. Of the control group 56% were null at the GSTM1 locus. CONCLUSIONS: The distribution of GST isoenzymes in the lung is heterogeneous with an apparent decrease in GST in distal lung. Since GSTM1 status has already been associated with susceptibility to disease, the PCR assay developed will allow further studies of the relation between genotype and structural disorders in the lung using archival pathological material.

Base Sequence↗
PubMed1994

Do carotid chemoreceptors inhibit the hyperventilatory response to heavy exercise?

In this paper two types of evidence are presented which question the commonly presumed role of carotid chemoreceptor stimulation as the primary mediator of the hyperventilatory response to heavy exercise. First, carotid-body denervation in ponies increases their hyperventilatory response to heavy exercise. Second, the awake dog and the goat at rest show an immediate and substantial depression of tidal volume and of ventilation when their isolated carotid chemoreceptors are made hypocapnic. Accordingly, it is proposed that during heavy exercise the carotid chemoreceptors are inhibitory to respiratory motor output and that the cause of the hyperventilatory response originates from extrachemoreceptor, locomotor-linked, feed-forward stimuli.

Animals↗
PubMed1994

The antiadrenergic effect of cyclopentyladenosine on myocardial contractility is reduced in vivo in diabetic rats.

The aim of this study was to test the hypothesis that the antiadrenergic action of adenosine is reduced in diabetes. This was determined by evaluating the effect of experimental diabetes mellitus on the in vivo myocardial antiadrenergic action of cyclopentyladenosine, and adenosine A1-receptor agonist. Changes in heart rate and ventricular performance in response to infusion of dobutamine, a beta 1-adrenergic agonist, were determined in the absence and presence of cyclopentyladenosine, in anesthetized, 10- to 12-week male diabetic (60 mg/kg streptozotocin), insulin-treated diabetic and control rats. Intravenous dobutamine (16 micrograms/kg) increased +dP/dtmax and -dP/dtmax in control rats from 7,706 +/- 553 and 5,449 +/- 403 mmHg/s (1 mmHg = 133.3 Pa) to 19,170 +/- 465 and 8,855 +/- 317 mmHg/s, respectively. In diabetic rats dobutamine increased +dP/dtmax and -dP/dtmax from 5,733 +/- 541 and 4,016 +/- 426 to 15,015 +/- 1,521 and 7,039 +/- 809 mmHg/s, respectively. Cyclopentyladenosine significantly attenuated dobutamine-stimulated increases in +dP/dtmax and -dP/dtmax in both control and diabetic rats in a dose-dependent (0.1-3.0 micrograms/kg) manner. Cyclopentyladenosine potency to attenuate dobutamine-enhanced +dP/dtmax was reduced significantly (p < 0.05) in diabetic rats compared with controls (ID50, 1.07 vs. 0.59 micrograms/kg, respectively) with no change in efficacy. The magnitude of cyclopentyladenosine inhibition of dobutamine-enhanced -dP/dtmax was greater in control than diabetic rats (81 vs. 54%, respectively), but ID50 values were not different. Insulin treatment of diabetic rats prevented the observed changes. These data suggest that the antiadrenergic action of adenosine is compromised in diabetes and that this may contribute to the development of diabetic cardiomyopathy.

Adenosine↗