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Biomedical subjects

P Hudson

Publications and source records attributed to P Hudson.

At least 73 records · Page 4Linked to original sources
PubMed1987

Porcine relaxin. Gene structure and expression.

A porcine genomic cosmid library was constructed to study the structure and regulation of the porcine relaxin gene. Two overlapping cosmids containing relaxin-specific sequences were isolated, and a 9-kilobase BamHI fragment containing the porcine relaxin gene was sequenced. The sequence derived from these cosmids was used to characterize the relaxin gene transcription unit utilized in the pregnant ovary. Analysis of chromosomal and cosmid DNA has shown that porcine relaxin is encoded by a single copy gene, comprising two exons separated by a 5.5-kilobase intron. The gene contains elements representing two classes of repeated sequences homologous with the human Alu and Kpn families. The transcriptional initiation site of this gene was located by primed extension and direct sequencing of relaxin-specific cDNA. Furthermore, the promoter elements were mapped and sequenced, and transcriptional activity was demonstrated. The promoter contained sequences with homology to regions conserved within retroviral long terminal repeats and within the adenovirus E4 promoter.

Animals↗
PubMed1987

Synthesis of a specific oligodeoxyribonucleotide probe and its use for studying proenkephalin A gene expression.

A 44-mer, complementary to the coding region of bovine adrenal medullary proenkephalin A mRNA (ProEnkA mRNA) has been synthesized as a specific probe for this mRNA. Northern-blot hybridization analysis identified a single band in an RNA extract from bovine, ovine and porcine adrenal medullae. The molecular size of these hybridized bands (1400-1600 nucleotides) was in excellent agreement with that reported previously using cloned cDNA probes. In situ hybridization on bovine and ovine adrenal sections revealed that ProEnkA mRNA was localized selectively in cells at the outer margin of the medulla, a region rich in adrenaline-containing cells. This study both confirms and extends previous findings with cloned cDNA probes on the presence of high concentrations of ProEnkA mRNA in cells at the periphery of the adrenal medulla. In addition, it demonstrates the usefulness of synthetic oligodeoxyribonucleotides as specific and sensitive probes for the study of proenkephalin A gene expression.

Adrenal Medulla↗
PubMed1986

Sertoli cell production of müllerian inhibiting substance in vitro.

Sertoli cell cultures were monitored for müllerian inhibiting substance with a solid phase radioimmunoassay. Sertoli cells from newborn calf testes were placed in defined media free of serum in monolayer culture after treatment with trypsin-collagenase followed by gravity separation. Immunoreactive müllerian inhibiting substance was detected in the culture media of Sertoli cells but not National Institutes of Health 3T3 cells. To verify that the Sertoli cells were intact, cyclic adenosine monophosphate levels were determined after follicle-stimulating hormone stimulation. Cyclic adenosine monophosphate levels were elevated in Sertoli cells but not National Institutes of Health 3T3 cells. The newborn calf Sertoli cell culture provides a useful system in which to study factors affecting müllerian inhibiting substance production and release, and documents this substance as another reliable marker for the Sertoli cell. Müllerian inhibiting substance levels also could be measured in media beneath testicular fragments in organ culture, and were increased and prolonged in comparison to müllerian inhibiting substance release from Sertoli cell monolayer cultures. Modulation of müllerian inhibiting substance release from monolayer and organ cultures then was attempted. Neither gonadotropin nor steroid additions affected the release of müllerian inhibiting substance during 24 to 72 hours in organ or tissue culture. We are using the müllerian inhibiting substance radioimmunoassay to monitor attempts to immortalize a Sertoli cell line capable of continuous müllerian inhibiting substance production using viral deoxyribonucleic acid transfection techniques.

Animals↗
PubMed1986

Synthesis of transthyretin (pre-albumin) mRNA in choroid plexus epithelial cells, localized by in situ hybridization in rat brain.

The sites of synthesis of transthyretin in the brain were investigated using in situ hybridization with [35S]-labeled recombinant cDNA probes specific for transthyretin mRNA. Autoradiography of hybridized coronal sections of rat brain revealed specific cellular localization of transthyretin mRNA in choroid plexus epithelial cells of the lateral, third, and fourth ventricles. Transferrin mRNA was also investigated and, in contrast to transthyretin mRNA, was localized mainly in the lateral ventricles. Our results indicate that substantial synthesis of transthyretin and transferrin mRNA may occur in the choroid plexus.

Animals↗
PubMed1986

Detection of Müllerian inhibiting substance in biological samples by a solid phase sandwich radioimmunoassay.

Müllerian inhibiting substance (MIS) is a 140,000-dalton glycoprotein responsible for regression of Müllerian ducts in a male embryo. It has recently been demonstrated that MIS inhibits the growth of tumors in vivo and in vitro. In this study, we have constructed a sensitive, solid phase sandwich RIA using monoclonal antibodies raised to bovine MIS. The amount of MIS detected was based on the protein concentration of Green 3, the most purified fraction of MIS available. The assay could detect 20 ng Green 3 or 0.14 pmol in physiological samples. There was no evidence of cross-reactivity of the antibodies raised to bovine MIS with chicken, rat, mouse, or human MIS.

Animals↗
PubMed1986

Accidental firearm fatalities in North Carolina, 1976-80.

All accidental gunshot fatalities in North Carolina were reviewed for the years 1976-80. There were 210 cases, of which 94 were self-inflicted and 116 were inflicted by others. Young white males predominated as victims, 31 per cent under the age of 15. Sixty-five per cent of the accidents occurred in the home and 18 per cent occurred in rural, "hunting" locations. Forty-one per cent of the cases involved shotguns, 39 per cent involved handguns, and 16 per cent, rifles. Sixteen per cent of the accidents involved children playing with guns and 14 per cent involved dropped or mishandled weapons. During the same period, there were 2,553 suicides and 2,509 homicides by firearm. Gunshot fatalities are an important American public health problem.

Accidents↗
PubMed1985

Follow-up of occupational asthma caused by crab and various agents.

Sixty-three subjects with occupational asthma caused by crab (n = 31) and various other agents (n = 32) were studied after cessation of exposure at work for mean +/- SD intervals of 12.3 +/- 5.5 and 24.5 +/- 18.7 months (greater than 6 months in every subject), respectively. Nineteen of the subjects with asthma caused by crab and 30 of the subjects with asthma caused by various agents were still symptomatic of asthma, nine subjects of the latter group requiring bronchodilators (with inhaled beclomethasone in five) regularly. No significant changes in baseline spirometry were observed at the time of follow-up as compared with initial assessment, nine subjects (all in the asthma group caused by various agents) demonstrating significant bronchial obstruction. Improvement in bronchial responsiveness to histamine was significant (p less than 0.01) in the group with asthma caused by crab but not in the other group. Forty-eight of 52 subjects still had significant airway hyperexcitability. Subjects with asthma caused by crab who were asymptomatic and those subjects with asthma caused by various agents who used bronchodilators only if they were needed had worked for shorter intervals after onset of symptoms (p less than 0.01 and p less than 0.05, respectively). It is concluded that subjects with occupational asthma caused by various agents can remain symptomatic of asthma and demonstrate a persistence of bronchial obstruction and hyperexcitability for prolonged intervals after cessation of exposure.

Adult↗
PubMed1985

Epicardial versus parietal pericardial defibrillation.

This study evaluated the energy requirements for porous electrodes implanted on the parietal pericardium versus those for porous electrodes implanted on the epicardial surface of the heart. Defibrillation with a 2.5-cm porous electrode implanted on the parietal pericardium was successful in 42% of all episodes of ventricular fibrillation. The minimal energy requirement ranged from 30-68 J, with an average of 46 J. Epicardial defibrillation was successful in approximately 80% of all episodes. The average maximal energy was 21.3 J. There was histological evidence of subepicardial damage in the parietal defibrillation group. This may be related to the higher energy required to defibrillate in this group. However, in this group frequent, large, external shocks were required to defibrillate the dogs' hearts. Theoretically, there may be some physiological and surgical advantages to an intact pericardium. However, the high energy requirement and the low success rate for defibrillation with a porous electrode on the parietal pericardium negate the feasibility of this route of defibrillation with a permanent implantable system.

Animals↗
PubMed1985

The primary structure of the imported mitochondrial protein, ornithine transcarbamylase from rat liver: mRNA levels during ontogeny.

Ornithine transcarbamylase, one of the enzymes of the urea cycle in ureotelic organisms, is synthesized in the cytoplasm of hepatocytes as a precursor larger than the mature form found in the mitochondrial matrix. We deduced the amino acid sequence of the precursor of ornithine transcarbamylase from rat liver from the nucleotide sequence of overlapping cDNA clones spanning the complete coding region, 3' untranslated region, and most of the 5' untranslated region of the mRNA. The mature enzyme consists of 322 amino acids and is derived from the larger precursor by proteolytic removal of 32 amino acids from the amino-terminus. The amino-terminal extension contains eight basic and no acidic residues. This highly basic character appears to be a feature of presequences on cytoplasmically synthesized mitochondrial proteins. Comparison of the amino acid sequence determined for the enzyme from rat with that from human liver (Horwich et al., 1984) shows that there is a high degree of homology between the sequences of the mature protein (93%) and relatively less homology between the sequences of the amino-terminal extension (72%). The ornithine transcarbamylase from rat liver also shows a considerable degree of amino acid homology (44%) with the enzyme from Escherichia coli (Van Vliet et al., 1984) and leads to suggestions about residues involved in substrate binding and catalysis. An analysis of levels of RNA in fetal and neonatal liver shows that ornithine transcarbamylase mRNA levels increase from about 40% of adult levels at day 14 of gestation to a peak at day 20 of gestation, and, after a drop around the time of birth, rises to adult levels during the second week after birth.

Age Factors↗
PubMed1985

Biosynthesis of calcitonin by human lung cancer cells.

The ectopic secretion of calcitonin (CT) by a wide variety of nonthyroidal human tumors has been studied by CT RIA, but little information is available concerning the biosynthesis of CT in these tumors. In the present study, a human lung cancer cell line (BEN), secreting high mol wt forms of CT was investigated to characterize the CT gene products synthesized. When conditioned medium from BEN cells was chromatographed through a Bio-Gel P-30 column, larger species of immunoreactive CT were detected with mol wt of approximately 8,000 and 18,000. Little, if any, CT of 3,500 mol wt was detected. To examine CT gene products produced in BEN cells, poly A+ RNA was isolated from BEN cells and subjected to cell-free translation assays and DNA/RNA hybridization assays. In the wheat germ cell-free translation assay, a single BEN cell product which migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent mol wt of 17,000 could be specifically immunoprecipitated with CT antisera. A similar sized CT-related translation product is produced in wheat germ assays programmed by mRNA prepared from human medullary thyroid carcinomas. In DNA/RNA hybridization assays, a single BEN cell mRNA species of 1,000 base pairs, identical in size to human thyroidal CT mRNA, hybridized to a radiolabeled CT cDNA probe. Hybridization of the CT cDNA probe with BEN cell mRNA was confirmed by RNA dot blot hybridization and cytoplasmic RNA blotting procedures. These results indicate that larger mol wt forms of CT secreted by BEN cells are derived from a translation product and a mRNA which are of similar, if not identical, size as CT gene products produced in human thyroidal tissues. The inability of lung tumor cells to process the CT precursor to calcitonin of 3,500 mol wt may reflect a lack of specific prohormone processing enzymes in these tumor cells or may be due to structural polymorphism in the CT precursor expressed in the lung cells.

Calcitonin↗
PubMed1984

A highly basic N-terminal extension of the mitochondrial matrix enzyme ornithine transcarbamylase from rat liver.

We have deduced the amino acid sequence of the N-terminal leader peptide of the mitochondrial enzyme ornithine transcarbamylase from a cDNA clone obtained from a rat liver cDNA library. The sequence is remarkable in being highly basic, having 4 arginine, 3 lysine and 1 histidine with no acidic residues in a total of 32 residues. The leader sequence has no extensive hydrophobic stretches, has 72% homology with the leader peptide of human ornithine transcarbamylase [1], and in terms of its basic character resembles the N-terminal extensions on a number of fungal mitochondrial [2-5] and pea chloroplast [6] proteins. Thus the basic nature of these leader peptides may constitute the signal for mitochondrial import.

Amino Acid Sequence↗
PubMed1984

Selection of a cDNA clone which contains the complete coding sequence for the mature form of ornithine transcarbamylase from rat liver: expression of the cloned protein in Escherichia coli. Molecular cloning of rat ornithine transcarbamylase.

A cDNA clone corresponding to the mature form of ornithine transcarbamylase (OTCase) was selected from a rat liver cDNA library constructed in bacteriophage lambda gt10. OTCase clones were selected using a synthetic DNA probe of 15 bases corresponding to the 3' end of the OTCase mRNA [Horwich, A. L., Kraus, J.P., Williams, K., Kalousek, F., Königsberg, W. & Rosenberg, L.E. (1983) Proc. Natl Acad. Sci. USA, 80, 4258-4262]. Putative OTCase clones were subcloned into the expression vector, pUC9, and the identity of inserts confirmed by colony immunoassay and by electrophoretic transfer of cloned proteins from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose filters followed by probing with monospecific anti-OTCase antibodies and 125I-labelled protein A. A clone corresponding to the full-length mature form of rat liver OTCase (plus 15 amino acids from Escherichia coli beta-galactosidase) was obtained and the identity of the clone was confirmed by comparison of the 5' sequence with a limited N-terminal amino acid sequence [Lusty, C., Jilka, R. L. & Nietsch, E. H. (1979) J. Biol. Chem. 254, 10030-10036]. A sequence discrepancy between the published sequence (Lusty et al.) and the sequence predicted from the cDNA structure is noted.

Amino Acid Sequence↗
PubMed1984

Relaxin gene expression in human ovaries and the predicted structure of a human preprorelaxin by analysis of cDNA clones.

In earlier studies we identified in a human genomic library a gene (human relaxin gene H1) coding for a relaxin-related peptide. We now have evidence that the human genome possesses an additional relaxin-related gene (designated human relaxin gene H2) which appears to be selectively expressed in the ovary during pregnancy. Nucleotide sequence analysis revealed striking differences in the predicted structures of relaxin encoded by these two genes. Chemical synthesis of biologically active relaxin based on the sequence obtained from ovarian cDNA clones confirmed that the expressed gene (H2) encodes an authentic human relaxin. The expressed gene appears to be transcribed into two different sized mRNAs and preliminary evidence suggests that the mRNA transcripts possess different 3'-untranslated regions. There was no evidence for the expression of human relaxin gene H1 in the ovary and so far it is unclear whether gene H1 is expressed in another tissue or whether it represents a pseudogene. From the sequence data presented here it will now be possible to construct oligonucleotide probes and raise antibodies against synthetic peptides which could then be used to identify sites of relaxin biosynthesis and specifically quantitate the expression from either the H1 or H2 relaxin genes.

Amino Acid Sequence↗
PubMed1984

Two human relaxin genes are on chromosome 9.

We have recently cloned two different human relaxin gene sequences. One of these (H1) was isolated from a human genomic clone bank and the other (H2) from a cDNA library prepared from human pregnant ovarian tissue. Southern gel analysis of the relaxin genes within the genomes of several unrelated individuals showed that all genomes contained both relaxin genes. Hence it is unlikely (p less than 0.001) that the two relaxin gene sequences are alleles. Rather, it is probable that there are two relaxin genes within the human genome. It is likely that relaxin and insulin genes have evolved from a common ancestral gene by gene duplication, since structural similarities between insulin and relaxin are evident at both the peptide and gene level. To investigate the evolutionary relationship between the two human relaxin genes and the insulin gene, we have determined the chromosomal position of the relaxin genes using mouse/human cell hybrids. We found that the human insulin and relaxin genes are on different chromosomes. Both human relaxin genes are located on the short arm region of chromosome 9.

Alleles↗
PubMed1984

Splenic autotransplant and residual partial spleen: prevention of septicemia.

Postsplenectomy septicemia carries an ominous prognosis. Accompanying disseminated intravascular coagulation and adrenal hemorrhage result in a high mortality, despite aggressive treatment by antibiotics. The efficacy of prevention by in situ partial spleen and splenic auto-transplant were evaluated in Sprague-Dawley rats. All totally splenectomized rats died following intravenous challenge of live pneumococcus. Both partial spleens and autotransplants gave substantial protection. The rats which succumbed to pneumococcal sepsis demonstrated massive fibrin thrombi in renal glomeruli and frank adrenal hemorrhage, strikingly similar to clinical observations.

Animals↗