Biomedical subjects

R M Wright

Publications and source records attributed to R M Wright.

At least 19 recordsLinked to original sources

PubMed1995

Identification of a low specificity, oxygen, heme, and growth phase regulated DNA binding activity in Saccharomyces cerevisiae.

Cytochrome c oxidase from Saccharomyces cerevisiae is subject to intricate control at the level of transcription of the various genes encoding its subunits. Expression of the subunit VI encoding gene, COX6, is glucose repressed, growth phase induced, and dependent on oxygen and heme availability. An upstream activation region for COX6, UAS6, was found to contain a glucose responsive region, a heme dependent region (HDS1), and a binding site for the transcription factor, BAF1. BAF1 was the only factor observed to form a protein complex with UAS6 in vitro. However, we found that binding of BAF1 was unaffected by oxygen or heme regulation. In the present communication, we have identified a DNA binding activity that was growth phase induced and dependent on oxygen and heme availability. This highly regulated activity was detected by its discrete binding to the heme responsive site, HDS1, in UAS6. Nonetheless, it appears to have weak DNA binding specificity.

Base Sequence↗

PubMed1995

Oxygen regulation of the cytochrome c oxidase subunit VI gene, COX6, in Saccharomyces cerevisiae.

Cytochrome c oxidase (CcO) in Saccharomyces cerevisiae is subject to intricate physiological control. Growth phase, carbon source, and oxygen level are three well recognized modulators of CcO expression. We focused on the subunit VI encoding gene, COX6, and detected unexpectedly complex oxygen regulation. We found that COX6 transcription possessed a critical threshold oxygen regulation between 0 and 2%. COX6 transcription was superinduced by elevated oxygen level up to 45%; however, superinduction was lost at 60% oxygen and above. The COX6 upstream activation region, UAS6, contains both glucose and heme responsive regions, and COX6 oxygen regulation was transduced through UAS6 by heme, as has been described for other oxygen regulated genes in yeast. We found that binding of the UAS6-domain 1 protein, BAF1, was unaltered by oxygen regulation. Nor were the alternative BAF1 complexes observed by growth in different glucose conditions formed by growth at different oxygen levels.

Base Sequence↗

PubMed1995

pH and the cAMP-dependent protein kinase mediate growth phase induction of the cytochrome c oxidase subunit VI gene, COX6, in Saccharomyces cerevisiae.

Respiratory adaptation in Saccharomyces cerevisiae is a complex genetic program that ensures ATP synthesis in a glucose-depleted environment. ATP is generated during respiration by the mitochondrial electron transport chain which is induced by respiratory adaptation. We have studied the terminal enzyme in mitochondrial electron transport, cytochrome c oxidase, from S. cerevisiae. Because subunits in this multisubunit enzyme are coordinately regulated, we have focused upon the well characterized subunit VI gene, COX6. In yeast, COX6 transcription is regulated by several factors thought to mediate respiratory adaptation including growth phase induction, oxygen dependence, and glucose repression. In the present study, we found that in addition to these known regulators, COX6 expression also depends upon pH and the cAMP-dependent protein kinase which may comprise additional factors signaling respiratory adaptation.

Cyclic AMP-Dependent Protein Kinases↗

PubMed1995

Analysis of aldehyde oxidase and xanthine dehydrogenase/oxidase as possible candidate genes for autosomal recessive familial amyotrophic lateral sclerosis.

Recently, point mutations in superoxide dismutase 1 (SOD1) have been shown to lead to a subset of autosomal dominantly inherited familial amyotrophic lateral sclerosis (ALS). These findings have led to the hypothesis that defects in oxygen radical metabolism may be involved in the pathogenesis of ALS. Therefore, we decided to analyze other enzymes involved in oxygen radical metabolism for possible involvement in other forms of ALS. We report here analysis of two genes encoding the molybdenum hydroxylases aldehyde oxidase (AO) and xanthine dehydrogenase/oxidase (XDH) for involvement in ALS. Of particular interest, one gene identified as encoding aldehyde oxidase is shown to map to 2q33, a region recently shown to contain a gene responsible for a familial form of ALS with autosomal recessive inheritance (FALS-AR). The AO gene appears to be located within 280,000 bp of simple sequence repeat marker D2S116, which shows no recombination with the FALS-AR locus. The AO gene is highly expressed in glial cells of human spinal cord. In addition, we mapped a gene for XDH to 2p22, a region previously shown to contain a highly homologous but different form of XDH. Neither of these XDH genes appears to be highly expressed in human spinal cord. This evidence suggests that AO may be a candidate gene for FALS-AR.

Aldehyde Oxidase↗

PubMed1995

Mitochondrial respiration scavenges extramitochondrial superoxide anion via a nonenzymatic mechanism.

We determined that mitochondrial respiration reduced cytosolic oxidant stress in vivo and scavenged extramitochondrial superoxide anion (O2-.) in vitro. First, Saccharomyces cerevisiae deficient in both the cytosolic antioxidant cupro-zinc superoxide dismutase (Cu,Zn-SOD) and electron transport (Rho0 state) grew poorly (P < 0.05) in 21% O2 compared with parent yeast and yeast deficient only in electron transport or Cu,Zn-SOD, whereas anaerobic growth was the same (P > 0.05) in all yeast. Second, isolated yeast and mammalian mitochondria scavenged extramitochondrial O2-. generated by xanthine/xanthine oxidase. Yeast mitochondria scavenged 42% more (P < 0.05) extramitochondrial O2-. during pyruvate/malate-induced respiration than in the resting state. Addition of either antimycin (respiratory chain inhibitor) or FCCP (respiratory chain uncoupler) prevented increased O2-. scavenging. Mitochondria isolated from yeast deficient in the mitochondrial manganous superoxide dismutase (Mn-SOD) increased (P < 0.05) O2-. scavenging 56% during respiration. This apparent SOD activity, expressed in units of SOD activity per milligram of mitochondrial protein, was the same (9 +/- 0.6 vs. 10 +/- 1.0; P = 0.43) as the O2-. scavenging of mitochondria with Mn-SOD, suggesting that respiration-dependent mitochondrial O2-. scavenging was nonenzymatic. Finally, isolated rat liver and lung mitochondria also increased (P < 0.05) O2-. scavenging during respiration. We speculate that respiring mitochondria, via the protonmotive pump, present a polarized, proton-rich surface that enhances nonenzymatic dismutation of extramitochondrial O2-. and that this is a previously unrecognized function of mitochondrial respiration with potential physiological ramifications.

Animals↗

PubMed1993

Arachidonic acid stimulates interleukin-6 release from rat peritoneal macrophages in vitro: evidence for a prostacyclin-dependent mechanism.

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (M phi) in vitro. AA (0.5-16 microM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 microM AA generating a peak of IL-6 release (3-5-fold). AA (0.5-16 microM) also induced an increasing release of the AA metabolite thromboxane B2 (TXB2). The AA-induced release of IL-6 occurred within 1-2 h of incubation, whereas TXB2 concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 microM and 40.0 microM) effectively blocked the generation of TXB2, but increased prostacyclin (PGI2) generation and potentiated the release of IL-6. In addition, PGI2, as well as the PGI2 agonists iloprost and cicaprost, stimulated IL-6 release from M phi by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI2 (but not TXA2) is involved in AA-induced IL-6 release from peritoneal M phi.

Animals↗

PubMed1993

Assay for recombinant and native human intraacrosomal antigen SP-10.

PROBLEM: To develop a method to measure a recombinant sperm protein, SP-10, during scale up and purification for a contraceptive vaccine formulation. METHOD: A quantitative assay method for the human intraacrosomal protein SP-10 was developed utilizing the format of indirect capture enzyme-linked immunosorbent assay (ELISA). A SP-10 specific monoclonal antibody mAb, MHS-10, was used as the capture antibody. Two recognition reagents, a rabbit polyclonal anti-SP-10 antisera (pAb) and a biotin-labeled mAb, MHS-10, were used as the recognition antibodies, respectively. A SP-10 recombinant fusion protein consisting of 125 SP-10 amino acids linked to glutathione transferase was used as a working SP-10 standard. The coefficient of variance for the assay system using the rabbit pAb was in the range of 0.099 to 0.157, and for the assay system using the biotinylated mAb MHS-10 was in the range of 0.081 to 0.084. RESULTS: Employing biotinylated MHS-10 as the recognition antibody, it was found that the native SP-10 molecule had more than one MHS-10 epitope. The concentration of SP-10 in a pool of human sperm extracts was found to be approximately 1% of the total proteins, assayed by both of the recognition antibody systems. CONCLUSIONS: The assay system described is useful to monitor the yield of recombinant SP-10 during scale-up production of the SP-10 vaccine.

Acrosome↗

PubMed1993

Lipopolysaccharide induces interleukin-6 release from rat peritoneal macrophages in vitro: evidence for a novel mechanism.

Interleukin-6 (IL-6) is a cytokine involved in the terminal differentiation of B-cells, T-cell activation, and secretion of hepatic acute phase proteins. The production of IL-6 is regulated by many factors, including IL-1 and lipopolysaccharide (LPS). Because IL-6 may be an important contributor to the effects of LPS in inflammation and septic shock, we investigated the ability of LPS to induce IL-6 release from peritoneal macrophages (m phi) in vitro. M phi were isolated from male Long-Evans rats, and cultured in 96-well tissue culture plates at 1 x 10(5) cells/well in serum-free RPMI-1640 medium. Following a 2-hr attachment period, the cells were rinsed twice to remove the nonadherent cells. LPS (0.006-100 ng/ml) stimulated IL-6 release by six- to 12-fold during a 4 hr incubation. In contrast, IL-1 beta (0.006-100 ng/ml) had no effect. Because cyclooxygenase metabolites of arachidonic acid are increased by LPS, we determined the effects of indomethacin (a cyclooxygenase inhibitor) and CGS8515 (a 5-lipoxygenase inhibitor) on LPS-induced IL-6 release. Neither indomethacin (10 microM) nor CGS8515 (2.5 microM) had any effect on basal or LPS-induced IL-6 release. Very low concentrations of LPS (0.01-1,000 pg/ml) stimulated IL-6 by two- to threefold. Pertussis toxin (10 ng/ml), which inactivates Gi protein, had no effect on LPS-induced IL-6 release from mø. Thromboxane B2 (TXB2) concentrations were also elevated with as little as 0.1 pg/ml LPS; however, pertussis toxin inhibited LPS-stimulated TXB2 release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Purification and microsequencing of the intra-acrosomal protein SP-10. Evidence that SP-10 heterogeneity results from endoproteolytic processes.

The human sperm antigen SP-10 has been shown to be a testis-specific, intra-acrosomal protein that is associated with the membranes and matrix of the acrosomal vesicle. Sperm extracts, analyzed on Western blots with a monoclonal antibody to SP-10, have shown heterogeneity of SP-10 peptides ranging from 17.5-34 kDa. Although the entire SP-10 amino acid sequence of 265 amino acids (28.3 kDa) has been deduced from sequencing SP-10 cDNAs, the nature of multiple SP-10 peptide bands is incompletely understood. In this study, we developed a three-step purification method for SP-10 peptides using monoclonal antibody affinity chromatography, reverse-phase HPLC, and preparative gel electrophoresis. Eight SP-10 peptides separated by this protocol and sequenced using Edman degradation showed amino termini that corresponded to regions on the deduced SP-10 amino acid sequence. Peptides with progressively lower apparent mass aligned further toward the carboxy terminus. On the basis of putative cleavage sites on the SP-10 sequence, endoproteases that act at five different peptide bonds are predicted to cleave SP-10: these hydrolyze following arginine (a trypsin-like protease, possibly acrosin), and following serine, proline, glycine, and glutamic acid (previously undescribed intra-acrosomal protease specificities). The present studies 1) provide a purification method for SP-10 peptides; 2) confirm that the SP-10 cDNAs previously sequenced encode authentic SP-10; and 3) yield indirect evidence that endoproteases act to contribute to SP-10 heterogeneity.

Acrosome↗

PubMed1992

Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli.

Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside. Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bglB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1.

Actinomycetales↗

PubMed1992

Children with cerebral venous thrombosis diagnosed with magnetic resonance imaging and magnetic resonance angiography.

From 1985 to 1991, 13 children were diagnosed at the University of Illinois College of Medicine at Peoria, Saint Francis Medical Center, with cerebral venous thrombosis (CVT) by magnetic resonance imaging scan. Ages ranged from newborn to 5 years. Six children were premature neonates, five were term neonates and two were 5 years old. In the premature neonates, thrombosis was usually associated with other problems. All the term neonates had seizures. In all neonates, thrombosis resolved without any specific treatment. In the two older children, one presented with pseudotumor cerebri and one with coma. These children required neurosurgical intervention. Follow-up magnetic resonance imaging scans were obtained in 9 of 13 children and showed thrombus resolution in each case. Three children were studied in the acute and convalescent stages by magnetic resonance angiography using time-of-flight techniques. Each follow-up magnetic resonance angiogram showed improvement in venous flow consistent with their clinical course and other imaging studies. We conclude that 1) CVT in children encompasses a range of clinical conditions which may or may not require neurosurgical intervention; 2) magnetic resonance imaging is superior to other modalities for the diagnosis of CVT; and 3) magnetic resonance angiography is an alternative means to monitor the evolution of CVT and efficacy of therapeutic intervention.

Cerebral Angiography↗

PubMed1990

The use of magnetic resonance imaging for the diagnosis of fetal intracranial anomalies.

While fetal cranial sonography has been used for the sensitive detection of ventriculomegaly, ancillary imaging techniques may be needed for precise delineation of structural abnormalities. This report outlines the radiologic and clinical results using maternal magnetic resonance imaging (MRI) in ten patients with suspected fetal intracranial anomalies. Imaging was accomplished at 17-39 weeks gestational age, using spin-echo, a multislice technique with intramuscular morphine sulfate for sedation. In four cases, MRI significantly clarified the sonographic diagnosis, while in two cases the scan agreed with the sonographic findings. In one patient, MRI failed to image a lumbar meningomyelocele associated with the Chiari II malformation. In two patients with the Chiari II malformation, both sonography and MRI failed to delineate the anatomic pathology completely. Optimal imaging resolution was achieved in the third trimester. Four patients died in the perinatal period. All the surviving patients required shunting to treat intracranial hypertension: only two patients were meeting cognitive milestones. We conclude that due to the high incidence of multiple anomalies in the fetus with ventriculomegaly, precision in neuroradiological diagnosis is essential. MRI can be a useful adjunct to cranial sonography for the specific delineation of abnormalities of the fetal central nervous system.

Brain↗

PubMed1990

Identification of human acrosomal antigen SP-10 in primates and pigs.

The intra-acrosomal human sperm protein SP-10 was previously designated a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. In the present study, a monoclonal antibody to SP-10 (MHS-10) was employed on Western blots to identify immunoreactive SP-10 in sperm extracts from baboon (Papio cyanocephalus anubis) and two macaques (Macaca mulatta and Macaca fascicularis). In each of these primates, the MHS-10 monoclonal antibody recognized a polymorphic pattern of immunoreactive peptides similar to that in humans. Immunoreactive SP-10 was also demonstrated in pig sperm. Using purified preparations of the previously described intra-acrosomal molecules acrosin and sperminogen in the pig, we observed that the MHS-10 monoclonal antibody did not react with these proteins, indicating SP-10 is distinct from these known acrosomal components. Sperm from several common species including the rabbit, bull, rat, guinea pig and cat did not immunoreact with the MHS-10 monoclonal antibody. By use of a radioactive probe spanning 628 nucleotides of the open reading frame for SP-10 on Northern blots of poly A + RNA obtained from testes of Macaca fascicularis, Papio papio, and Papio cyanocephalus anubis, a 1.35-kb mRNA of identical size to the mRNA from human testes was identified. These results indicate that baboons, macaques, and pigs may be appropriate models for testing an SP-10-based contraceptive vaccine.

Acrosome↗

PubMed1990

Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10.

cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis. Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta. Complete sequencing of these cDNAs, designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein. Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide. Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein. Interestingly, cDNA SP-10-10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10-5, suggesting that alternative splicing generates more than one SP-10 mRNA. The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks. A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum. This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.

Acrosome↗

PubMed1990

Release of two Saccharomyces cerevisiae cytochrome genes, COX6 and CYC1, from glucose repression requires the SNF1 and SSN6 gene products.

We show here that SNF1 and SSN6 are required for derepression of the glucose-repressible yeast genes COX6 and CYC1, which encode the mitochondrial proteins cytochrome c oxidase subunit VI and iso-1-cytochrome c, respectively. In an snf1 mutant genetic background, the transcription of both COX6 and CYC1 continued to be repressed after cells were shifted into derepressing media. In an ssn6 mutant genetic background, both COX6 and CYC1 were expressed constitutively at high levels in repressing media. SSN6 acted epistatically to SNF1 in the regulation of both cytochrome genes. These findings are similar to previous findings on the effects of SNF1 and SSN6 on SUC2 expression in Saccharomyces cerevisiae and are consistent with a model proposing that SNF1 exerts its effect through SSN6 on COX6 and CYC1.

Blotting, Northern↗

PubMed1989

Detection of bone marrow involvement in patients with cancer.

Current methods for the study of bone marrow to evaluate possible primary or metastatic cancers are reviewed. Bone marrow biopsy, radionuclide scan, computed tomography and magnetic resonance imaging (MRI) are analyzed with regard to their clinical usefulness at the time of diagnosis and during the course of the disease. Bone marrow biopsy is still the examination of choice not only in hematologic malignancies but also for tumors that metastasize into the marrow. Radionuclide scans are indicated for screening for skeletal metastases, except for those from thyroid carcinoma and multiple myeloma. Computed tomography is useful for cortical bone evaluation. MRI shows a high sensitivity in finding occult sites of disease in the marrow but its use has been restricted by high cost and limited availability. However, the future of MRI in bone marrow evaluation seems assured. MRI is already the method of choice for diagnosis of multiple myeloma, when radiography is negative, and for quantitative evaluation of lymphoma when a crucial therapeutic decision (i.e. bone marrow transplantation) must be made. Finally, methods are being developed that will enhance the sensitivity and specificity of MRI studies of bone marrow.

Adult↗

PubMed1989

Transcription of yeast COX6, the gene for cytochrome c oxidase subunit VI, is dependent on heme and on the HAP2 gene.

The COX6 gene encodes subunit VI of cytochrome c oxidase. Previously, this gene and its mRNAs were characterized, and its expression has been shown to be subject to glucose repression/derepression. In this study we have examined the effects of heme and the HAP1 (CYP1) and HAP2 genes on the expression of COX6. By quantitating COX6 RNA levels and assaying beta-galactosidase activity in yeast cells carrying COX6-lacZ fusion genes, we have found that COX6 is regulated positively by heme and HAP2, but is unaffected by HAP1. Through 5' deletion analysis we have also found that the effects of heme and HAP2 on COX6 are mediated by sequences between 135 and 590 base pairs upstream of its initiation codon. These findings identify COX6 as the fourth respiratory protein gene that is known to be regulated positively by heme and HAP2. The other three, CYC1, COX4, and COX5a, encode iso-1-cytochrome c, cytochrome c oxidase subunit IV, and an isolog, Va, of cytochrome c oxidase subunit V, respectively. Thus, it appears that the biogenesis of two interacting proteins, cytochrome c and cytochrome c oxidase, in the mitochondrial respiratory chain, are under the control of common factors.

Blotting, Northern↗

PubMed1989

Organization and expression of the COX6 genetic locus in Saccharomyces cerevisiae: multiple mRNAs with different 3' termini are transcribed from COX6 and regulated differentially.

COX6 and its surrounding genetic locus have been characterized for the yeast Saccharomyces cerevisiae. Flanking genes are found closely spaced upstream and downstream of COX6. The upstream gene and COX6 are transcribed from opposite strands and are separated by no more than 300 bp. COX6 is transcribed into three different size classes of mRNA (1000b, 830b, and 700b) differing in length in their 3' untranslated regions. All three classes of mRNAs are found on polysomes and, hence, are most likely translated. The different COX6 mRNAs vary in abundance during growth in rich media and are affected differentially as cells are shifted into media containing high or low glucose concentrations. The largest mRNA is much more susceptible to glucose repression/derepression than are the two smaller mRNAs, whereas the smallest RNA is preferentially accumulated during growth in rich media. These findings demonstrate that COX6 mRNAs with different 3'-termini are either synthesized differentially or differ in stability and suggest the existence of a complex system regulating COX6 expression.

Amino Acid Sequence↗