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M Smith

Publications and source records attributed to M Smith.

At least 901 records · Page 50Linked to original sources
PubMed1989

Identification of an upstream repressor site controlling the expression of an anaerobic gene (ANB1) in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme. A 299-base pair-long fragment from the 5'-flanking region of the ANB1 gene was found to confer oxygen-mediated negative regulation to an heterologous CYC1-LacZ hybrid gene. Studies with deletions of predefined length in this fragment demonstrated the presence of separate elements that comprise an upstream promoter that is active in the absence or presence of oxygen, and an upstream repressor site (URS) which confers strong repression upon the promoter element when oxygen is present. The promoter element is located 5' to the URS in the ANB1 gene. Mixed oligonucleotide-directed mutagenesis was used to obtain nucleotide substitutions in the URS which partially or completely inactivated this sequence without affecting the promoter activity. The URS region has three short direct repeats which seem to be important for function, as nucleotide substitutions within the repeats and not outside them, inactivated URS function. A model to explain the negative regulation of the ANB1 gene by oxygen and heme is proposed.

Anaerobiosis↗
PubMed1989

Mutation-induced perturbation of the cytochrome c alkaline transition.

The possible influence of residue Phe-82 in the cytochrome c alkaline isomerization has been evaluated by spectrophotometric pH titrations of a family of mutant yeast iso-1-cytochromes c in which the identity of the residue at this position has been varied. The pKa for the exchange of the Met-80 heme iron ligand was determined from pH titrations in which the S----Fe charge-transfer band (695 nm) was monitored and was found to be 8.5 for the wild type, 7.7 for Ser-82, 7.7 for Gly-82, 7.2 for Leu-82, and 7.2 for Ile-82. pH-jump experiments [Davis et al. (1974) J. Biol. Chem. 249, 2624] established that substitutions at position 82 affect the alkaline isomerization by lowering the pKa of the titrating group by as much as 1.4 pK units; for the Ser-82 and Gly-82 variants, there is also a small effect on the Keq for the ligand exchange equilibrium. On the basis of these findings, we conclude that one critical role for Phe-82 in the wild-type protein is stabilization of the native heme binding environment.

Cytochrome c Group↗
PubMed1989

Role of arginine-38 in regulation of the cytochrome c oxidation-reduction equilibrium.

Arg-38 is an internal residue of mitochondrial cytochrome c that is close to heme propionate-7. Previous work comparing the behavior of cytochromes c from several species [Moore, G. R., Harris, D. E., Leitch, F. A., & Pettigrew, G. W. (1984) Biochim. Biophys. Acta 764, 331-342] has suggested that Arg-38 lowers the pKa of this propionate group and thereby accounts for the relative pH independence of the cytochrome c reduction potential from pH 5 to pH 8. The influence of Arg-38 on the oxidation-reduction equilibrium of yeast iso-1-cytochrome c has now been investigated by electrochemical, NMR, and theoretical analysis of six specifically mutated forms of this protein in which Arg has been replaced by Lys, His, Gln, Asn, Leu, or Ala. As the electron-withdrawing character of the residue at position 38 decreases, the reduction potential of the protein also decreases, with the largest decrease (ca. 50 mV) observed for the Ala variant. However, the variation in the reduction potentials of the mutants as a function of pH was similar to that observed for the wild-type protein. The effects of some of these mutations on the pKa values of His-33 and His-39 have been determined by NMR spectroscopy and found to be minimal. Calculations of the electrostatic free energy for the Leu-38 variant predict a decrease in the reduction potential of this mutant that is remarkably close to that observed experimentally. This work establishes that while Arg-38 contributes to the relatively high reduction potential of cytochrome c, this residue does not appear to be the sole functionality responsible for lowering the heme propionate-7 pKa.

Amino Acids↗
PubMed1989

Diagnostic value of the apex beat.

100 patients were examined without knowledge of other findings to assess the value of apex displacement as a sign of cardiomegaly; 25 had a radiographic cardiothoracic ratio greater than 50%. The apex was located in only half of the patients, palpability being influenced by frame size, percentage ideal body weight, and percentage body fat. By comparison with the cardiothoracic ratio, apex displacement beyond the midclavicular line as the diagnostic test for cardiomegaly had a specificity of 76%, a sensitivity of 59%, a positive predictive value of 59.4%, and a negative predictive value of 76.9%. Another clinical sign of cardiomegaly, apex more than 10 cm from the midsternal line, was more sensitive but even less specific.

Aged↗
PubMed1989

The pseudoautosomal boundary in man is defined by an Alu repeat sequence inserted on the Y chromosome.

The Y chromosome, which in man determines the male sex, is composed of two functionally distinct regions. The pseudoautosomal region is shared between the X and Y chromosome and is probably required for the correct segregation of the sex chromosomes during male meiosis. The second region includes the sex-determining gene(s), the presence of which is necessary for the development of testes. The two regions have contrasting genetic properties: the pseudoautosomal region recombines between the X and Y chromosome; the Y-specific region must avoid recombination otherwise the chromosomal basis of sex-determination breaks down. The pseudoautosomal region is bounded at the distal end by the telomere and at the proximal end by X- and Y-specific DNA. We have found that the proximal boundary was formed by the insertion of an Alu sequence on the Y chromosome early in the primate lineage. Proximal to the Alu insertion there is a small region where similarity between the X and Y chromosomes is reduced and which is no longer subject to recombination.

Base Sequence↗
PubMed1989

Radiophosphorus (32P) treatment of bone marrow disorders in dogs: 11 cases (1970-1987).

Between March 1970 and February 1987, radiophosphorus (32P) was used to treat bone marrow disorders in 6 dogs; 4 had polycythemia vera and 2 had essential thrombocythemia. Activities of 32P given initially ranged from 2.4 to 3.3 mCi/m2. Four dogs responded well to 32P treatment, with gradual resolution of high RBC or platelet counts. Two of these dogs died of intercurrent disease unrelated to their bone marrow disorder, before blood counts could be stabilized. Two dogs did not respond to the initial 32P treatment nor to additional treatments with 32P, and had clinical signs and blood counts stabilized by use of phlebotomy or chemotherapeutic agents. We reviewed and analyzed 5 other cases of bone marrow disorders in dogs treated with 32P and included the findings from their records with the records of our 6 dogs in this retrospective analysis. Of the 8 dogs with polycythemia vera treated with 32P, 5 were given a single treatment that controlled clinical signs and blood counts for the remainder of the follow-up period. Of the 3 dogs treated for thrombocytosis with 32P, 2 had blood counts that responded to a single treatment.

Animals↗
PubMed1989

Spironolactone metabolism: steady-state serum levels of the sulfur-containing metabolites.

Metabolism of spironolactone in man is extensive and complex. For many years the dethioacetylated metabolite, canrenone, was assumed to be the major metabolite. However, recent studies using specific high performance liquid chromatography (HPLC) have demonstrated the presence of spironolactone and the sulfur-containing metabolites 7 alpha-thiomethylspirolactone (IV) and 6 beta-hydroxy-7 alpha-thiomethylspirolactone (V), in addition to canrenone, in the serum after a single oral dose of spironolactone. The importance of spironolactone and metabolites IV and V relative to canrenone at steady state remains unknown and was the subject of the present investigation. Twelve healthy males received 100 mg spironolactone, once daily, for 15 days. Repeated blood samples were taken on days 1, 8 and 15 for estimation of spironolactone and its metabolites. Peak serum levels [mean (SD)] of spironolactone, canrenone, and sulfur-containing metabolites IV and V were 72 (45), 155 (43), 359 (106) and 101 (26) ng/ml, respectively on day 1 and 80 (20), 181 (39), 391 (118) and 125 (24) ng/ml, respectively on day 15. The AUC (0-24) values of these compounds on day 15 were 231 (50), 2173 (312), 2804 (777) and 1727 (367) ng.hr/ml, respectively and the post-steady state elimination half-life (t1/2) values were 1.4 (0.5), 16.5 (6.3), 13.8 (6.4), and 15.0 (4.0) hours, respectively. It was concluded that unmetabolized spironolactone is present in the serum and that the sulfur-containing metabolite IV rather than canrenone is the major metabolite in serum following single or repeated doses of spironolactone.

Adolescent↗
PubMed1989

Continuous multi-channel intravascular monitoring of the effects of dopamine and dobutamine on plasma potassium in dogs.

The effects of dopamine and dobutamine on plasma potassium were investigated in dogs using continuous, multi-channel, intravascular ion-selective potassium electrodes situated in the aorta and abdominal and thoracic inferior vena cavae. Doses of 10 and 30 micrograms kg-1 min-1 of each drug were used, and the effects on potassium compared with isoprenaline 0.07 and 0.2 microgram kg-1 min-1. Both the drugs caused a biphasic pattern of potassium change consisting of an initial small rise in the potassium level, followed by a sustained period of hypokalaemia. The changes were greater with the higher dose of each drug compared with the lower dose, but there were no significant differences between the drugs. Comparison of the potassium changes between the three vascular sites studied suggested that the rise in potassium may be a result of release of the ion from the liver, and that the liver may also be the principle site of potassium uptake during the hypokalaemic phase.

Animals↗
PubMed1989

The effects of beta blockade on dopamine and dobutamine induced hypokalaemia in dogs.

The effect of beta 1 and beta 2 blockade on dopamine and dobutamine induced hypokalaemia was investigated in dogs. Arterial plasma potassium was continuously monitored using a potassium-sensitive electrode sited in the abdominal aorta. Dopamine and dobutamine were infused at 15 micrograms kg-1 min-1 into animals pre-treated with atenolol (0.5 mg kg-1) or ICI 118551 (0.5 mg kg-1) and the potassium changes compared to those seen in control animals. The inotrope infusions caused a small and transient rise in plasma potassium (p less than 0.05) followed by a more profound hypokalaemia (p less than 0.05) sustained for the duration of the infusion. The potassium falls in the control animals were similar to those in the beta 1 and beta 2 blockade groups. The results suggest that beta 2 receptor stimulation cannot alone be responsible for dopamine and dobutamine induced hypokalaemia and a multiple receptor activation is postulated.

Animals↗
PubMed1989

Cycloheximide-resistant gene of Epstein-Barr virus in freshly infected B lymphocytes.

The transcription of latent Epstein-Barr viral (EBV) genes, i.e., genes encoding EBNA-1, EBNA-2, EBNA-3A, -3B, -3C, and LMP, were detected in human tonsillar lymphocytes early after infection with EBV. Transcription of the BHLF-1 open reading frame was also detected at this initial phase of immortalization. Cycloheximide treatment inhibited the transcription of all the latent EBV genes but not BHLF-1. These results suggest that BHLF-1 might be considered an immediate-early gene of EBV. Cycloheximide treatment of EBV-infected cells reduced not only the degree of the transcription but also the size of the transcript for EBNA-2. We hypothesize that the immediate-early expression of BHLF-1 may be required for the enhanced transcription of the viral genes in lymphocytes early after infection with EBV.

Antigens, Viral↗
PubMed1989

Expression of EBNA-3 family in fresh B lymphocytes infected with Epstein-Barr virus.

Four transcripts belonging to the EBNA-3 family were detected during immortalization of human tonsillar lymphocytes with the Epstein-Barr virus. These four transcripts were also detected in immortalized B-lymphoblastoid cells (LCL) and the Burkitt's lymphoma cell line, Namalwa. A 5.0-kb transcript hybridized with the EBNA-3A probe; a 8.5-kb transcript hybridized with the EBNA-3B and -3C probes; and a 5.6- and a 3.6-kb transcript hybridized with the EBNA-3C probe. Sequencing and cDNA analysis showed that the EBNA-3C transcripts contained exons of the most rightward BamHI-E open reading frames, BERF-3 and BERF-4. Moreover, sequencing of cDNA clones revealed exons in the 5' portion homologous to regions in EBV BamHI-C, -W, and -U. Hybridization experiments with EBV BamHI-W and -U probes identified the 8.5- and 5.6-kb transcripts but not the 3.6-kb transcript in LCL. Organization of these transcripts from the EBNA-3 family is discussed.

Antigens, Viral↗
PubMed1989

Definitive irradiation for localized non-Hodgkin's lymphoma of breast.

A diagnosis of primary non-Hodgkin's lymphoma (NHL) confined to the breast or breast and ipsilateral lymph nodes was biopsy proven in 19 patients who presented to our institution between 1970 and 1984. Four patients were treated with a modified radical mastectomy, and one with chemotherapy only. The remaining 14 patients comprise the basis of this study. All patients were clinically staged. There were 12 patients with 1A-E disease and two with IIA-E disease. All patients were treated with breast radiation. The axillary nodes were included in seven of the patients and two, in addition, received "mantle" irradiation. The majority of the patients received between 3500-4400 cGy, but four received lower doses. One patient, with Stage IIA-E disease, received 6 months of systemic chemotherapy, followed by 3500 cGy to the breast and axilla. Three of the 14 patients relapsed within the treated area, with a local control rate of 78%. One was salvaged with a mastectomy and remains alive and well 15 years after diagnosis. Seven of the 14 patients relapsed distantly, including two with local failures, but only three have died of disease. An additional two patients have died of other causes without evidence of disease, for a survival of 66% at 48 months (Kaplan-Meier Calculations).

Aged↗
PubMed1989

An unusual case of dermatofibrosarcoma protuberans.

A dermatofibrosarcoma protuberans involving the thumb of a nondominant hand is reported and represents the first case reported in this location. Dermatofibrosarcoma protuberans is a soft tissue neoplasm. It is a slow growing, locally aggressive fibrous tumor that occurs with equal frequency on the chest, abdomen, back, and proximal aspect of the extremities. Only two cases involving the hand are reported in the literature.

Adult↗
PubMed1989

Testing for syphilis.

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Emergency Service, Hospital↗
PubMed1989

The p24 leucocyte membrane antigen: modulation associated with lymphocyte activation and differentiation.

Monoclonal antibodies of the CD9 cluster recognize a 24 kD protein (p24) found on platelets and endothelium, and expressed by lymphocytes at restricted stages of maturation and activation. In this study, we explore the possibility that p24 is involved in the response of lymphocytes to signals delivered by lymphokines. p24 is expressed only very weakly by resting B lymphocytes. We found no increase in expression when cells were activated with anti-immunoglobulin together with interleukin-4, or induced to proliferate by low-molecular weight B cell growth factor (LMW-BCGF). Culture of activated B cells with B cell differentiation factor was associated with an increased mean expression of p24. In cells from a patient with chronic lymphocytic leukaemia (CLL), culture with LMW-BCGF up-regulated p24 expression. Resting T cells (p24-negative) were induced to express p24 strongly when activated with antibody against CD3. CD9 antibody did not modulate B or T cell responses to activation stimuli. The results suggest that the p24 molecule is not involved in the primary interaction of cells with lymphokine, but rather may be involved in a secondary reaction, such as ion flux, which follows as a consequence of the action of lymphokines on cells.

Antigens, CD↗
PubMed1989

Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis.

Protein P.69 is localized on the outer membrane of Bordetella pertussis and is one of the virulence factors believed to contribute to the disease state of whooping cough. We demonstrate that protein synthesis of P.69 is under genetic control of the vir locus. Using oligonucleotide probes derived from the protein sequence of a cyanogen bromide fragment, we have cloned the gene for P.69 from B. pertussis CN2992. Analysis of the DNA sequence reveals a G + C-rich gene capable of encoding a protein of 910 amino acids with a Mr of 93,478, suggesting that P.69 is a processed form of a larger precursor. In common with some of the genes in the pertussis toxin operon, the sequence CCTGG was found 5' to the ATG initiation codon. At the 3' end, 29 bases after the TAA stop codon, the sequence GTTTTTCCT was found and may have some function in transcription termination. A full-length clone of the gene for P.69 carried by the cosmid pBPI69 was unable to direct the expression of P.69 protein in an Escherichia coli host. The generation of P.69-fusion products allowed the detection of P.69-specific protein products synthesized in E. coli.

Amino Acid Sequence↗
PubMed1989

Loss of heterozygosity suggests tumor suppressor gene responsible for primary hepatocellular carcinoma.

Primary hepatocellular carcinoma (PHC), epidemiologically associated with chronic hepatitis B virus (HBV) infection, has historically been felt to be caused by the activation or introduction of an oncogene. However, transforming sequences from human PHC have not been reproducibly isolated. In this paper, evidence is presented that suggests PHC may result instead from the loss of an anti-oncogene. Seven of 12 human primary liver tumors tested against a panel of restriction fragment length polymorphisms (RFLPs) demonstrated loss of constitutional heterozygosity for markers on chromosome 4. Tumor and nontumor liver tissue were typed for 11 chromosome 4 RFLPs. In addition, at least one RFLP on nine other chromosomes (1, 2, 6, 7, 9, 11, 13, 14, and 17) was tested for allelic loss. Seven of nine tumors constitutionally heterozygous for chromosome 4q markers showed allele loss in tumor tissue. Six of the seven samples were jointly informative for both 4p and 4q markers. Five of the six demonstrated loss for only 4q RFLPs. In one individual, in which two samples were taken from distant locations within the same tumor, both samples showed loss of the same alleles. Among the other chromosomes informative for allele loss, one tumor showed changes on 13q. No other changes were observed in RFLPs located on the eight other chromosomes tested. These results indicate that an anti-oncogene may be located on 4q and suggest a mechanism for PHC and other cancers seroepidemiologically related to virus infection. Liver cancer caused by chronic HBV infection or other environmental agents may be linked through genetic events responsible for the loss of a tumor suppressor locus (anti-oncogene) located on chromosome 4.

Carcinoma, Hepatocellular↗