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Biomedical subjects

R M Wright

Publications and source records attributed to R M Wright.

At least 19 recordsLinked to original sources
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↗
PubMed1989

Magnetic resonance imaging of spinal injury.

Magnetic resonance imaging (MRI) was performed on 30 patients following spinal injury (SI). Spin-echo sequences and surface coils were used for all patients. Plain radiographs, high-resolution computed tomography (CT), and MRI were compared for the delineation of bone, disc, and ligament injury, measurement of sagittal spinal canal diameter and subluxation, epidural hematoma, and spinal cord structure. Myelography or intrathecal contrast-enhanced CT were not performed on any of these patients. Magnetic resonance imaging accurately delineated intraspinal pathology in two of four patients with acute penetrating SI, and was normal in the other two patients. In 16 patients with acute nonpenetrating SI, MRI was superior to CT for visualizing injuries to discs, ligaments, and the spinal cord, while CT was superior to MRI in characterizing bony injury. Computed tomography and MRI provided similar measurements of subluxation in six of six patients and of sagittal spinal canal diameter in three of four patients. In ten patients with chronic SI, MRI demonstrated post-traumatic cysts, myelomalacia, spinal cord edema, and the presence or absence of spinal cord compression. In patients with acute penetrating SI and chronic SI, MRI provided comprehensive clinical information. In patients with acute nonpenetrating SI, the information obtained by MRI complemented the data given by plain radiographs and CT, allowing clinical decisions to be made without the need of invasive imaging modalities.

Adolescent↗
PubMed1989

Bilateral MR imaging with switched mutually coupled receiver coils.

The authors describe a system of bilateral mutually coupled receiver coils and its initial clinical use in magnetic resonance imaging of the knees and temporomandibular joints. Switched mutually coupled coils allow bilateral simultaneous imaging of paired structures without a penalty in signal-to-noise ratio because the coils can be alternated with each section acquisition. With this coil system, there is no restriction on placement or design of coils to prevent interaction. When combined with three-dimensional imaging techniques, switched coils allow simultaneous bilateral acquisition of multiple, contiguous sections through paired anatomic structures in a reasonable amount of imaging time.

Adult↗
PubMed1988

Differential regulation of the two genes encoding Saccharomyces cerevisiae cytochrome c oxidase subunit V by heme and the HAP2 and REO1 genes.

In Saccharomyces cerevisiae, the COX5a and COX5b genes encode two forms of cytochrome c oxidase subunit V, Va and Vb. We report here that heme increases COX5a expression and decreases COX5b expression and that the HAP2 and REO1 genes are involved in positive regulation of COX5a and negative regulation of COX5b, respectively. Heme regulation of COX5a and COX5b may dictate which subunit V isoform is available for assembly into cytochrome c oxidase under conditions of high- and low-oxygen tension.

Blotting, Northern↗
PubMed1988

Neonatal cerebral venous thrombosis.

Five cases of neonatal cerebral venous thrombosis (NCVT) diagnosed by magnetic resonance imaging (MRI) are presented in this report. MRI was specific for the anatomic diagnosis, demonstrating involvement of the superior sagittal sinus in 3 infants or deep venous system in the remaining 2. Four cases were associated with perinatal hypoxia or cranial trauma. Three of these children show significant developmental delays. The increased use of MRI may add significant information on the association of cerebral venous thrombosis with the common systemic illnesses of newborns.

Child, Preschool↗
PubMed1986

Characterization of COX9, the nuclear gene encoding the yeast mitochondrial protein cytochrome c oxidase subunit VIIa. Subunit VIIa lacks a leader peptide and is an essential component of the holoenzyme.

The gene COX9 for subunit VIIa of cytochrome c oxidase from Saccharomyces cerevisiae has been cloned with the aid of an oligonucleotide probe. From the nucleotide sequence of COX9, we deduce that subunit VIIa is derived from a precursor that is 59 amino acids in length (Mr = 6963). This precursor is longer than mature subunit VIIa by one amino acid at its NH2 terminus and four amino acids at its COOH terminus. COX9 exists as a single copy in the haploid genome of S. cerevisiae and produces one major transcript. When the genomic copy of COX9 is removed, cells lack a functional cytochrome c oxidase holoenzyme. From the predicted secondary structure of subunit VIIa, previous data concerning its relationship to the lipid bilayer of the inner membrane and the location of its hydrophobic domains (Power, S.D., Lochrie, M.A., and Poyton, R.O. (1986) J. Biol. Chem. 261, 9206-9209) and the finding that it is essential for the holoenzyme, we propose a model for subunit VIIa which suggests that this small integral protein plays a role in holoenzyme assembly or stability.

Amino Acid Sequence↗
PubMed1986

Clinical utility of magnetic resonance imaging in pediatric neurosurgical patients.

The clinical utility of magnetic resonance imaging in pediatric neurosurgical patients was reviewed in a series of 126 scans. MRI was determined to be superior or equivalent to other imaging techniques in the evaluation of intracranial hypertension, developmental abnormalities, or acute conditions involving the spinal cord. In 74% of the scans performed because of these clinical problems, adequate data were obtained for both specific anatomic diagnosis and surgical planning. The scan proved equivalent to CT in the imaging of structural abnormalities associated with seizure disorders or degenerative conditions. In acute head injuries and generalized infections of the central nervous system, MRI offered no advantage in diagnostic specificity, and had significant logistic limitations.

Acute Disease↗