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Biomedical subjects

R H Smith

Publications and source records attributed to R H Smith.

At least 55 records · Page 3Linked to original sources
PubMed1995

Nuclear localization and transcriptional activation activities of truncated versions of the immediate-early gene product of equine herpesvirus 1.

The equine herpesvirus 1 (EHV-1) immediate-early (IE) gene product encodes a nuclear regulatory protein capable of negatively autoregulating its own promoter, transactivating representative EHV-1 early promoters, and acting in a concerted fashion with accessory EHV-1 regulatory factors to transactivate EHV-1 late promoters. To identify IE amino acid sequences involved in nuclear localization and to examine the contribution of C-terminal portions of the IE polypeptide to transactivation, vectors that express various carboxyterminally truncated IE polypeptides were constructed. It is demonstrated that amino acids 963 through 970 of the 1,487-amino-acid IE protein are required for efficient localization of the truncated IE polypeptides to the nuclei of transfected cells. In addition, it is demonstrated that the first 970 amino acids of the IE gene product are sufficient to transactivate the EHV-1 thymidine kinase promoter to significant levels (i.e., approximately 40% of the level of wild-type activation).

Amino Acid Sequence↗
PubMed1994

The equine herpesvirus type 1 immediate-early gene product contains an acidic transcriptional activation domain.

The equine herpesvirus type 1 (EHV-1) immediate-early (IE) gene product, an ICP4 homolog, is the major regulatory protein encoded by EHV-1 during cytolytic infection. The IE gene product has been demonstrated to induce reporter gene expression directed by both homologous and heterologous viral promoters, including the EHV-1 thymidine kinase (tk) promoter, the herpes simplex virus type 1 (HSV-1) tk and ICP4 promoters, and the simian virus 40 early promoter. In this report, the transcriptional activation domain of the EHV-1 IE gene product was mapped to within an acidic, 87-amino-acid region (amino acids 3 to 89) at the amino-terminus of the IE molecule. It is demonstrated that the IE transcriptional activation domain, when fused to the DNA-binding domain of the yeast transcriptional activator GAL4, can activate gene expression in cell lines derived from at least two different species. Moreover, it is shown that the EHV-1 IR2 gene product (Harty and O'Callaghan, J. Virol. 65, 3829-3838, 1991), a truncated form of the IE polypeptide lacking IE amino acid residues 1-322 (and, therefore lacks the deduced transcriptional activation domain), fails to transactivate the EHV-1 tk promoter, but retains the ability to down-regulate the EHV-1 IE promoter. Fusion of the acidic transcriptional activation domain of the HSV-1 virion protein VP16 to the transactivation-deficient IR2 gene product restored the ability of this truncated IE polypeptide to transactivate the EHV-1 tk promoter. These findings suggest a role for the IR2 protein as a trans-repressor of EHV-1 gene expression.

Amino Acid Sequence↗
PubMed1994

Identification and initial characterization of the IR6 protein of equine herpesvirus 1.

The IR6 gene of equine herpesvirus 1 (EHV-1) is a novel gene that maps within each inverted repeat (IR), encodes a potential protein of 272 amino acids, and is expressed as a 1.2-kb RNA whose synthesis begins at very early times (1.5 h) after infection and continues throughout the infection cycle (C. A. Breeden, R. R. Yalamanchili, C.F. Colle, and D.J. O'Callaghan, Virology 191:649-660,1992). To identify the IR6 protein and ascertain its properties, we generated an IR6-specific polyclonal antiserum to a TrpE/IR6 fusion protein containing 129 amino acids (residues 134 to 262) of the IR6 protein. This antiserum immunoprecipitated a 33-kDa protein generated by in vitro translation of mRNA transcribed from a pGEM construct (IR6/pGEM-3Z) that contains the entire IR6 open reading frame. The anti-IR6 antibody also recognized an infected-cell protein of approximately 33 kDa that was expressed as early as 1 to 2 h postinfection and was synthesized throughout the infection cycle. A variety of biochemical analyses including radiolabeling the IR6 protein with oligosaccharide precursors, translation of IR6 mRNA in the presence of canine pancreatic microsomes, radiolabeling the IR6 protein in the presence of tunicamycin, and pulse-chase labeling experiments indicated that the two potential sites for N-linked glycosylation were not used and that the IR6 protein does not enter the secretory pathway. To address the possibility that the unique IR6 gene encodes a novel regulatory protein, we transiently transfected an IR6 expression construct into L-M fibroblasts alone or with an immediate-early gene expression construct along with a representative EHV-1 immediate-early, early, or late promoter-chloramphenicol acetyltransferase reporter construct. The results indicated that the IR6 protein does not affect the expression of these representative promoter constructs. Interestingly, the IR6 protein was shown to be phosphorylated and to associate with purified EHV-1 virions and nucleocapsids. Lastly, immunofluorescence and laser-scanning confocal microscopic analyses revealed that the IR6 protein is distributed throughout the cytoplasm at early times postinfection and that by 4 to 6 h it appears as "dash-shaped" structures that localize to the perinuclear region. At late times after infection (8 to 12 h), these structures assemble around the nucleus, and three-dimensional image analyses reveal that the IR6 protein forms a crown-like structure that surrounds the nucleus as a perinuclear network.

Amino Acid Sequence↗
PubMed1993

An unexpected pathway for the metabolic degradation of 1,3-dialkyl-3-acyltriazenes.

In the presence of NADPH, rat liver microsomes catalyzed the degradation of a series of 1,3-dialkyl-3-acyltriazenes, and the extent of the reaction was correlated with compound lipophilicity. In the case of two methylcarbamoyltriazenes, 1-(2-chloroethyl)-3-benzyl-3- (methylcarbamoyl)triazene (CBzM) and 1-(2-chloroethyl)-3-methyl-3-(methylcarbamoyl)triazene (CMM), microsomal metabolites were isolated. Identification of the CBzM metabolites as 1-(2-chloroethyl)-3-benzyl-3-(hydroxymethylcarbamoyl)triazene and 1-(2-chloroethyl-3-benzyl-3-carbamoyltriazine, and the CMM metabolite as 1-(2-chloroethyl)-3-methyl-3-(hydroxymethylcarbamoyl)triazene indicated that the first metabolic step involves hydroxylation of the methylcarbamoyl substituent. Detailed studies of the metabolism of CBzM indicated that the Km for the reaction was 84 microM, and that metabolism was more efficient if microsomes were prepared from male than from female rats. During prolonged incubation, the metabolites of CBzM were also degraded. The degradation of CBzM and its metabolites was inhibited by SKF-525A and metyrapone, suggesting the involvement of a cytochrome P450 isozyme, and supporting the hypothesis that the process is oxidative rather than hydrolytic in both cases. Metabolic oxidation represents an alternative pathway to chemical or enzymatic hydrolysis for the in vivo decomposition of (methylcarbamoyl)triazenes. This mechanism may ultimately explain the antitumor efficacy and low acute toxicity of selected compounds.

Alkylating Agents↗
PubMed1993

DNA sequence and transcriptional analyses of the region of the equine herpesvirus type 1 Kentucky A strain genome encoding glycoprotein C.

DNA sequence and transcriptional analyses were performed on the region of the equine herpesvirus type 1 (EHV-1) genome (KyA strain) (map units 0.129 to 0.152) encoding open reading frames (ORFs) 15 and 16. ORF16 encodes a homolog of glycoprotein C of HSV-1 (herpes simplex virus type 1), while ORF15 corresponds in position to HSV-1 UL45 but exhibits no significant homology at the amino acid level. Sequence analyses revealed that the EHV-1 gC ORF of 468 amino acids and ORF15 of 227 amino acids mapped at nucleotides (nt) 716 to 2119 and 2397 to 3077, respectively (relative to the 5' end of the BamHI recognition site at map unit 0.152). ORF15 exhibited significant homology (69% identity) at the amino acid level to the EHV-4 gene located 3' of the EHV-4 gC homolog. Sequence analyses identified potential CAAT and TATA boxes for the EHV-1 gC ORF and a TATA box for ORF15. While no consensus polyadenylation signal was detected between ORFs 15 and 16, two polyadenylation signals were detected 3' of ORF15. Northern blot and S1 nuclease analyses were used to map and characterize the gC and ORF15 mRNAs, and metabolic inhibitors were used to identify the kinetic class of these two genes. The results revealed that gC is a gamma-1 gene which encodes a 2.8-kb mRNA, while ORF15 is a gamma-2 gene encoding a 0.9-kb mRNA which is 3' coterminal with the gC transcript. The gC and ORF15 mRNAs were shown by S1 nuclease analyses to initiate approximately 34 and 26 nucleotides downstream of their respective TATA boxes and to have a common termination site 18 to 20 nucleotides downstream of a consensus polyadenylation signal. Comparative sequence analysis revealed that the KyA strain gC protein differs in only three amino acid residues from the gC protein of the EHV-1 Ab4 and T431 strains, and one of the three amino acid differences occurred within a segment of six contiguous amino acids showing high degree of hydrophilicity in the gC molecule. Further comparative sequence analysis revealed that KyA strain genome has a major deletion in the region of ORF17 which lies 5' of gC in the Ab4 strain. This finding that 1038 base pairs (bp) of the 1203-bp ORF17 is deleted indicates that ORF17 is nonessential for EHV-1 replication in cell culture. To examine the regulation of the EHV-1 gC gene, transient transfection assays using CAT (chloramphenicol acetyltransferase) reporter gene constructs of gC were performed.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Induction of T-cell immunity to oligosaccharide antigens immobilized on crystalline bacterial surface layers (S-layers).

Immunization of Balb/c mice with conjugates of oligosaccharide haptens and crystalline bacterial surface-layer proteins (S-layers) primed the mice for a strong, hapten-specific, delayed-type hypersensitivity (DTH) response. Conjugates of haptens with bovine serum albumin produced only weak DTH responses but, when mixed with aluminium hydroxide, elicited DTH responses comparable to those against S-layer conjugates. Surface-layer conjugates also elicited strong anti-hapten DTH responses when administered by an oral/nasal route. Apparently, the natural assembly of S-layer proteins into large, two-dimensional arrays endows them with intrinsic adjuvant properties.

Adjuvants, Immunologic↗
PubMed1993

Effects of population density and cadmium toxicity on growth and survival of blowflies.

The blowfly Lucilia sericata has been reared on artificial diets containing from 0 to 200 mg Cd kg(-1) at population densities ranging from 8 larvae per 4-g diet to 8 larvae per 32-g diet. Peak larval weight, development period, pupal weight, adult-emergence weight, and fecundity were measured. Larvae of blowflies respond to food deprivation by a reduction in larval weight and subsequent pupal and adult size. Contaminating the food supply with cadmium produced similar additive effects, reduced the growth rate, and increased the age of pupation. The results are of importance in determining the effects of environmental pollutants on population dynamics.

Journal Article↗
PubMed1993

Distinguishing the experiences of envy and jealousy.

This article describes the nature and significance of the distinction between the emotions of envy and jealousy and reports 2 experiments that empirically investigated it. In Experiment 1, Ss recalled a personal experience of either envy or jealousy. In Experiment 2, Ss read 1 of a set of stories in which circumstances producing envy and jealousy were manipulated independently in a factorial design. Both experiments introduced new methodologies to enhance their sensitivity, and both revealed qualitative differences between the 2 emotions. Envy was characterized by feelings of inferiority, longing, resentment, and disapproval of the emotion. Jealousy was characterized by fear of loss, distrust, anxiety, and anger. The practical importance of this distinction, the reasons for its confusion, and general issues regarding the empirical differentiation of emotions are discussed.

Adult↗
PubMed1993

The equine herpesvirus 1 (EHV-1) UL3 gene, an ICP27 homolog, is necessary for full activation of gene expression directed by an EHV-1 late promoter.

We have previously reported that the equine herpesvirus 1 (EHV-1) XbaI G restriction fragment (nucleotides 1436 to 7943 relative to the left terminus of the EHV-1 genome [Kentucky A strain]) is required in combination with the EHV-1 immediate-early (IE) gene to achieve significant activation of two representative EHV-1 late promoter-chloramphenicol acetyltransferase (CAT) recombinants in transient expression assays. In this report, we demonstrate that the XbaI G-encoded UL3 gene (an ICP27 homolog) provides a trans-acting factor which acts (in combination with the EHV-1 IE gene product) to increase reporter gene expression directed by an EHV-1 late promoter-CAT recombinant plasmid. We show that cloned copies of UL3 can successfully substitute for the XbaI G fragment in CAT assays and that stop codon insertion within the UL3 open reading frame inhibits the ability of UL3 to activate reporter gene expression in trans.

Codon↗
PubMed1993

Cocaine and benzoylecgonine in saliva, serum, and urine.

We studied the feasibility of using saliva to detect cocaine and benzoylecgonine. Saliva was collected as an ultrafiltrate directly in the mouth with an osmotic device. We analyzed by immunoassay matched samples of urine, blood, and salivary ultrafiltrate from 69 patients who had used cocaine within 24 h of sample collection. Cocaine concentrations were 4.9 times higher in saliva than in serum; benzoylecgonine concentrations were 2.5 times higher in serum. Seven urine and two serum samples had undetectable concentrations of cocaine, but all 69 saliva samples were positive for the drug. For benzoylecgonine detection, all urine samples were positive and three serum and one saliva sample were negative. We also analyzed 43 samples of saliva by gas chromatography/mass spectrometry: all were positive for benzoylecgonine, whereas 40 were positive for cocaine. We conclude that simultaneous measurement of cocaine and benzoylecgonine in saliva is suitable in screening for recent cocaine use.

Adolescent↗
PubMed1993

Testosterone concentration is increased in whole saliva, but not in ultrafiltrate, after toothbrushing.

The concentration of testosterone in whole saliva is significantly increased (by 9%) after toothbrushing. In ultrafiltrates of saliva collected at the same time as the whole saliva, testosterone concentrations after toothbrushing were unchanged. In 88% of the 162 whole-saliva specimens, but not in the ultrafiltrates, we also measured higher hemoglobin concentrations after toothbrushing. We conclude that the increase of testosterone in whole saliva after toothbrushing can be attributed to a protein-bound fraction. For analytes that are bound to serum proteins, salivary measurements can give spurious results. This problem can be avoided by using as a diagnostic medium an ultrafiltrate of saliva collected directly in the mouth.

Adult↗
PubMed1992

Characterization of the regulatory functions of the equine herpesvirus 1 immediate-early gene product.

Use of the translation-inhibiting drug cycloheximide has indicated that the equine herpesvirus 1 (EHV-1) immediate-early (IE) gene, the sole EHV-1 IE gene, encodes a major viral regulatory protein since IE mRNA translation is a prerequisite for all further viral gene expression (W.L. Gray, R. P. Baumann, A. T. Robertson, G. B. Caughman, D. J. O'Callaghan, and J. Staczek, Virology 158:79-87, 1987). An EHV-1 IE gene expression vector (pSVIE) in combination with chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs was used in transient transfection assays to characterize the regulatory functions of the IE gene product. These experiments demonstrated that (i) the EHV-1 IE gene product is a bifunctional protein capable of both positive and negative modulation of gene expression; (ii) the IE gene product possesses an autoregulatory function which represses the IE promoter; (iii) IE autoregulation is dependent on IE promoter sequences mapping within positions -288 to +73 relative to the transcription initiation site (+1) of the IE gene; (iv) the IE gene product can independently activate the EHV-1 tk promoter (an early promoter) by as much as 60-fold; (v) two EHV-1 beta-gamma (leaky late) promoters, those of IR5 (gene 5 in the inverted repeat) and the glycoprotein D gene, demonstrate a requirement for both the IE gene product as well as a gene product encoded within the EHV-1 XbaI G fragment for significant activation; and (vi) the IE gene product is capable of activating heterologous viral promoters.

Animals↗
PubMed1991

Synthesis and characterization of a Pertussis toxin-biotin conjugate.

We prepared a Pertussis toxin-biotin conjugate and found its biological properties to be similar to those of native Pertussis toxin with respect to the hemagglutination, Chinese hamster ovary cell, and lymphocyte proliferation assays. Direct binding to Chinese hamster ovary and Jurkat cells was observed using fluorescence microscopy. Pertussis toxin-biotin was also found to possess similar glycoconjugate binding specificities as those of 125I-labeled Pertussis toxin.

Animals↗
PubMed1991

A mechanism for maintaining an up-to-date GenBank database via Usenet.

In this paper, we describe an automated system for distributing updates to the GenBank nucleic acid sequence database, using the Usenet news system as the underlying transport mechanism. Our system allows new loci to be distributed as soon as the sequences are available, over existing networks, using existing Usenet software and infrastructure currently available on a wide range of computer systems.

Amino Acid Sequence↗