Search PubMed⌕ Search

Biomedical subjects

S Harvey

Publications and source records attributed to S Harvey.

At least 55 records · Page 3Linked to original sources
PubMed1999

Two functional domains of the epsilon subunit of DNA polymerase III.

The epsilon subunit is the 3'-->5' proofreading exonuclease that associates with the alpha and theta subunits in the E. coli DNA polymerase III. Two fragments of the epsilon protein were prepared, and binding of these epsilon fragments with alpha and theta was investigated using gel filtration chromatography and exonuclease stimulation assays. The N-terminal fragment of epsilon, containing amino acids 2-186 (epsilon186), is a relatively protease-resistant core domain of the exonuclease. The purified recombinant epsilon186 protein catalyzes the cleavage of 3' terminal nucleotides, demonstrating that the exonuclease domain of epsilon is present in the N-terminal region of the protein. The absence of the C-terminal 57 amino acids of epsilon in the epsilon186 protein reduces the binding affinity of epsilon186 for alpha by at least 400-fold relative to the binding affinity of epsilon for alpha. In addition, stimulation of the epsilon186 exonuclease by alpha using a partial duplex DNA is about 50-fold lower than stimulation of the epsilon exonuclease by alpha. These results indicate that the C-terminal region of epsilon is required in the epsilonalpha association. To directly demonstrate that the C-terminal region of epsilon contains the alpha-association domain fusion protein, constructs containing the maltose-binding protein (MBP) and fragments of the C-terminal region of epsilon were prepared. Gel filtration analysis demonstrates that the alpha-association domain of epsilon is contained within the C-terminal 40 amino acids of epsilon. Also, the epsilon186 protein forms a tight complex with theta, demonstrating that the association of theta with epsilon is localized to the N-terminal region of epsilon. Association of epsilon186 and theta is further supported by the stimulation of the epsilon186 exonuclease in the presence of theta. These data support the concept that epsilon contains a catalytic domain located within the N-terminal region and an alpha-association domain located within the C-terminal region of the protein.

ATP-Binding Cassette Transporters↗
PubMed1999

A preliminary CD and NMR study of the Escherichia coli DNA polymerase III theta subunit.

The theta subunit of DNA polymerase III, the main replicative polymerase of Escherichia coli, has been examined by circular dichroism and by NMR spectroscopy. The polymerase core consists of three subunits: alpha, epsilon, and theta, with alpha possessing the polymerase activity, epsilon functioning as a proofreading exonuclease, and theta, a small subunit of 8.9 kD, of undetermined function. The theta subunit has been expressed in E. coli, and a CD analysis of theta indicates the presence of a significant amount of secondary structure: approximately 52% alpha helix, 9% beta sheet, 21% turns, and 18% random coil. However, at higher concentrations, theta yields a poorly-resolved 1D proton NMR spectrum in which both the amide protons and the methyl protons show poor chemical shift dispersion. Subsequent 1H-15N HSQC analysis of uniformly-15N-labeled theta supports the conclusion that approximately half of the protein is reasonably well-structured. Another quarter of the protein, probably including some of the N-terminal region, is highly mobile, exhibiting a chemical shift pattern indicative of random coil structure. The remaining amide resonances exhibit significant broadening, indicative of intermolecular and/or intramolecular exchange processes. Improved chemical shift dispersion and greater uniformity of resonance intensities in the 1H-15N HSQC spectra resulted when [U-15N]-theta was examined in the presence of epsilon186--the N-terminal domain of the epsilon-subunit. Further work is currently in progress to define the solution structure of theta and the theta-epsilon186 complex.

Circular Dichroism↗
PubMed1999

Exonucleases and the incorporation of aranucleotides into DNA.

The polymerization of nucleotide analogs into DNA is a common strategy used to inhibit DNA synthesis in rapidly dividing tumor cells and viruses. The mammalian DNA polymerases catalyze the insertion of the arabinofuranosyl analogs of dNTPs (aranucleotides) into DNA efficiently, but elongate from the 3' aranucleotides poorly. Slow elongation provides an opportunity for exonucleases to remove aranucleotides. The exonuclease activity associated with DNA polymerase delta removes araCMP from 3' termini with the same efficiency that it removes a paired 3' deoxycytosine suggesting that the proofreading exonucleases associated with DNA polymerases might remove aranucleotides inefficiently. A separate 30 kDa exonuclease has been purified from mammalian cells that removes araCMP from 3' termini. The activity of this enzyme in the cell could remove aranucleotides from 3' termini of DNA and decrease the efficacy of the analogs. Inhibition analysis of the purified exonuclease shows that this enzyme is inhibited by thioinosine monophosphate (TIMP) with a Ki = 17 microM. When high TIMP levels are generated in HL-60 cells, incorporation of araC in DNA is increased about 16-fold relative to total DNA synthesis. This increased araC in DNA is likely a result of exonuclease inhibition in the cell. Thus, exonucleases in cells might play an important role in removing aranucleotides inserted by DNA polymerases.

Animals↗
PubMed1999

Absence of the alpha6(IV) chain of collagen type IV in Alport syndrome is related to a failure at the protein assembly level and does not result in diffuse leiomyomatosis.

X-linked Alport syndrome is a progressive nephropathy associated with mutations in the COL4A5 gene. The kidney usually lacks the alpha3-alpha6 chains of collagen type IV, although each is coded by a separate gene. The molecular basis for this loss remains unclear. In canine X-linked hereditary nephritis, a model for X-linked Alport syndrome, a COL4A5 mutation results in reduced mRNA levels for the alpha3, alpha4, and alpha5 chains in the kidney, implying a mechanism coordinating the production of these 3 chains. To examine whether production of alpha6 chain is under the same control, we studied smooth muscle cells from this animal model. We determined the canine COL4A5 and COL4A6 genes are separated by 435 bp, with two first exons for COL4A6 separated by 978 bp. These two regions are >/= 78% identical to the human sequences that have promoter activity. Despite this potential basis for coordinated transcription of the COL4A5 and COL4A6 genes, the alpha6 mRNA level remained normal in affected male dog smooth muscle while the alpha5 mRNA level was markedly reduced. However, both alpha5 and alpha6 chains were absent at the protein level. Our results suggest that production of the alpha6 chain is under a control mechanism separate from that coordinating the alpha3-alpha5 chains and that the lack of the alpha6 chain in Alport syndrome is related to a failure at the protein assembly level, raising the possibility that the alpha5 and alpha6 chains are present in the same network. The lack of the alpha6 chain does not obviously result in disease, in particular leiomyomatosis, as is seen in Alport patients with deletions involving the COL4A5 and COL4A6 genes.

Animals↗
PubMed1999

A unique urinary constituent, 6-hydroxy-6-methyl-3-heptanone, is a pheromone that accelerates puberty in female mice.

BACKGROUND: Olfactorily mediated puberty acceleration in female mice (measured by an increase in uterine weight) has been observed since the 1960s without the active chemosignal being structurally identified. There are many controversies in the literature as to whether this male-originated pheromone is a volatile substance. We investigated the chemical nature of the urinary fractions that are responsible for the characteristic uterine weight increases. RESULTS: The active pheromone was identified as 5,5-dimethyl-2-ethyltetrahydrofuran-2-ol and/or its open-chain tautomer (6-hydroxy-6-methyl-3-heptanone). A series of cyclic vinyl ethers were isolated from chromatographically active fractions of the urine. Because these compounds did not accelerate puberty, we postulated that these ethers were degradation products of a lactol (5,5-dimethyl-2-ethyltetrahydrofuran-2-ol). The lactol was then detected directly in the mouse urine extract using a silylation agent. Synthetic 6-hydroxy-6-methyl-3-heptanone had strong biological activity, whereas its close structural analogs did not. CONCLUSIONS: The male house mouse excretes into its urine a large quantity of a volatile substance that has a unique lactol/hydroxyketone structure. This substance is capable of binding to the less volatile urinary constituents, such as proteins or peptides, and is active in puberty-acceleration bioassays. The controversies regarding the volatility of the puberty-accelerating pheromones can now be explained by considering a complex of volatile lactol/hydroxyketone and urinary proteins.

Animals↗
PubMed1999

Strategic planning for research use in nursing practice.

BACKGROUND/OBJECTIVE: To prepare for a culture change to integrate research utilization into daily nursing practice, the authors conducted a descriptive survey of all registered nurses (RNs) in an integrated healthcare delivery system. The purposes of this study were to assess RNs' knowledge, attitudes, and practices (KAP) of nursing research activities, assess factors that support a research environment, and determine facilitating and challenging factors related to conducting regional nursing research. METHODS: A 33-item survey based on the Iowa Model for Evidence-Based Practice was developed, validated, and determined to be reliable by the authors. Site coordinators organized and managed the orientation, administration, and collection of data from the 2,736 registered nurses who worked in 6 hospitals, 65 affiliated clinics, and 3 business units. Narrative notes taken by study investigators were analyzed for themes to determine challenging and facilitating factors for conducting regional research. RESULTS: Education and job title significantly predicted knowledge and ability to perform research activities but was not related to willingness to engage in research activities. Several environmental factors were associated with knowledge of, willingness to engage in, and ability to perform research utilization activities. Challenging and facilitating factors to conducting regional research were identified. CONCLUSIONS/IMPLICATIONS: Our research environment is changing to value research as shown in the philosophy, conceptual framework, and bylaws for the professional nursing staff. Novice-to-expert research utilization expectations are included in the promotional model for nursing. All RN job descriptions and the annual performance tool were revised to include responsibilities related to research activities. The Iowa Model for Evidence-Based Practice was adopted as the method for creating practice validation and change. Train-the-trainer educational and experiential sessions are being designed for nurse leaders; all new RN employees complete a self-assessment tool of research utilization knowledge and the nursing division strategic goals incorporate research utilization expectations. The elements of this plan may be useful for nurse executives. Healthcare systems are restructuring throughout the world and within the United States. These changes are occurring to better meet the evolving healthcare needs of the population through cost-effective approaches. Within the United States, emerging organized healthcare systems require research related to patient care outcomes and the health systems that can best address them.

Adult↗
PubMed1999

Foci of tuberculosis transmission in central Los Angeles.

To identify sites of tuberculosis transmission and to determine the contribution of HIV-infected patients to tuberculosis morbidity in an urban area, we prospectively evaluated 249 patients with culture-proven tuberculosis in central Los Angeles. Restriction fragment length polymorphism (RFLP) analysis was performed on Mycobacterium tuberculosis isolates to identify patients infected with the same strain. Using RFLP and clinical and epidemiologic data, we identified the most likely source case and site of transmission for 79 patients. Homelessness, birth in the United States and Native American ethnicity were independent predictors of being a source case, but HIV infection was not. Three homeless shelters were sites of tuberculosis transmission for 55 (70%) of the 79 patients. HIV-infected patients constituted 27% (66/249) of the study population, but only 17% (13/79) of patients were infected by an HIV-infected source case. We conclude that transmission of tuberculosis in central Los Angeles was highly focal, and that the major transmission sites were three homeless shelters. HIV- infected tuberculosis patients did not play a major role in spread of tuberculosis. Tuberculosis control measures targeted at specific homeless shelters can reduce tuberculosis morbidity in urban areas where homelessness is common and the incidence of tuberculosis is high.

AIDS-Related Opportunistic Infections↗
PubMed1999

A missense mutation in the GHR gene of Cornell sex-linked dwarf chickens does not abolish serum GH binding.

Sex-linked dwarfism (SLD) in chickens is characterized by impaired growth despite normal or supranormal plasma growth hormone (GH) levels. This resistance to GH action is thought to be due to mutations of the GH receptor (GHR) gene that reduce or prevent GH binding to target sites. The genetic lesion causing GH resistance in Cornell SLD chickens is, however, not known. Previous studies have shown that hepatic GH-binding activity is abnormally low in these birds, yet the GHR gene is transcribed into a transcript of appropriate size and abundance. Point mutations or defects in translation could therefore account for the impaired GHR activity in this strain. These possibilities were addressed in the present study. A missense mutation resulting in the substitution of serine for the conserved phenylalanine was identified in the region of the GHR cDNA encoding the extracellular domain. Translation of this mutant transcript was indicated by the presence of GHR/GH-binding protein (GHBP)-immunoreactive proteins in liver (55, 70 and 100 kDa) and serum (70 kDa) of normal (K) and SLD birds. Radiolabelled GH did not, however, bind to the hepatic membranes of most SLD chickens. Serum GH-binding activity, in contrast, was readily detectable, although at significantly lower levels than in normal birds. The missense mutation in the SLD GHR gene may thus affect targeting of GHRs to hepatic plasma membranes.

Animals↗
PubMed1999

Growth hormone resistance: clinical states and animal models.

GH exerts pleiotropic effects on growth and metabolism through the GH receptor. A deficiency in the GH receptor gene is thus associated with GH resistance and dwarfism. Complete GH resistance in humans, or Laron syndrome, has been associated with numerous inherited defects in the GH receptor, including point mutations, complete or partial gene deletions, and splice site alterations. Analysis of the GH receptor genes of these patients has provided considerable insight into structure-function relationships of the GH receptor. However, the relative rarity of this disease and the obvious difficulties involved in human research have prompted a search for an animal model of GH resistance. Numerous models have been proposed, including the sex-linked dwarf chicken, the guinea pig, and the Laron mouse. In this review, the characteristics and etiology of Laron syndrome and these animal models will be discussed. The insight provided by these disorders into the roles and mechanism of action of GH will also be reviewed.

Animals↗
PubMed1999

Use of enhanced green fluorescent protein to optimize and quantitate infection of target cells with recombinant retroviruses.

Recombinant retroviral vectors are useful tools for gene transfer in both gene therapy and research applications. An enhanced form of green fluorescent protein has been incorporated into recombinant retroviruses as a marker to follow infected cells. In this paper, we extended the use of the fluorescent reporter to quantify protein expression using such analytical tools as fluorescent microscopy, flow cytometry and fluorescent plate reader analysis. These tools enabled us to rapidly assess the titer of recombinant retrovirus harvested from packaging cells and to optimize parameters for infection of different cell lines.

3T3 Cells↗
PubMed1998

The CC chemokine I-309 inhibits CCR8-dependent infection by diverse HIV-1 strains.

Using a chemokine receptor model based on known receptor sequences, we identified several members of the seven transmembrane domain G-protein superfamily as potential chemokine receptors. The orphan receptor ChemR1, which has recently been shown to be a receptor for the CC chemokine I-309, scored very high in our model. We have confirmed that I-309, but not a number of other chemokines, can induce a transient Ca2+ flux in cells expressing CCR8. In addition, the human erythroleukemic cell line K562 responded chemotactically in a dose-responsive manner to this chemokine. Since several chemokine receptors have been shown to be required as coreceptors for HIV-1 infection, we asked whether human immunodeficiency virus type 1 (HIV-1) could efficiently utilize CCR8. Here we show that the CCR8 receptor can serve as a coreceptor for diverse T-cell tropic, dual-tropic, and macrophage-tropic HIV-1 strains and that I-309 was a potent inhibitor of HIV-1 envelope-mediated cell-cell fusion and virus infection. Furthermore, we show by flow cytometry and immunohistochemistry that antibodies generated against the CCR8 receptor amino-terminal peptide cross-reacted with U-87 MG cells stably expressing CCR8, THP-1 cells, HL-60 cells, and human monocytes, a target cell for HIV-1 infectivity in vivo.

Amino Acid Sequence↗
PubMed1998

Calcitropic peptides: neural perspectives.

In mammals and higher vertebrates, calcitropic peptides are produced by peripheral endocrine glands: the parathyroid gland (PTH), thyroid or ultimobranchial gland (calcitonin) and the anterior pituitary gland (growth hormone and prolactin). These hormones are, however, also found in the neural tissues of lower vertebrates and invertebrates that lack these endocrine organs, suggesting that neural tissue may be an ancestral site of calcitropic peptide synthesis. Indeed, the demonstration of CNS receptors for these calcitropic peptides and their induction of neurological actions suggest that these hormones arose as neuropeptides. Neural and neuroendocrine roles of some of these calcitropic hormones (calcitonin and parathyroid hormone) and related peptides (calcitonin gene related peptide, stanniocalcin and parathyroid hormone related peptide) are thus the focus of this review.

Animals↗
PubMed1998

Growth hormone: a paracrine growth factor in embryonic development?

Although pituitary growth hormone is obligatory for normal postnatal growth and development, early embryonic and fetal growth is generally considered to be independent of pituitary GH. Indeed, in chickens, somatotrophs and serum GH are not detectable until late in embryogenesis, and neither partial decapitation nor pre-hatch GH administration greatly affects embryonic growth. However, since it is now known that GH can be produced and act in many extra-pituitary tissues, early embryonic growth may be independent of pituitary GH but dependent upon the paracrine actions of extra-pituitary GH. The possibility that growth hormone may be a paracrine growth factor during early development will therefore be considered in this brief review, which is based on the embryogenesis of the domestic fowl.

Animals↗
PubMed1998

Autoregulation of growth hormone receptor and growth hormone binding protein transcripts in brain and peripheral tissues of the rat.

Growth hormone (GH) differs from other pituitary hormones in that it can affect a wide spectrum of cellular activities in many different tissues. These disparate actions are, however, mediated by a common receptor, suggesting tissue-specific differences in the post-receptor mechanisms and/or tissue sensitivities to GH stimulation may confer specificity. Tissue sensitivity depends upon the abundance of GH receptors (GHRs) and may be modulated by the amplitude and pulsatility of GH secretion. It may also be dependent upon the presence of non-signal transducing GH-binding proteins (GHBPs), which result from the alternate splicing of GHR gene transcripts. Tissue-specific autoregulation of GHRs and GHBPs could, therefore, contribute to differential tissue responsiveness to GH action. The autoregulation of GHR and GHBP gene transcription in novel central (hypothalamus, brainstem, and cortex/neocortex) and peripheral (spleen) tissues was therefore examined in adult, male Sprague-Dawley rats. For comparative purposes, GHR/GHBP gene expression was also examined in the liver, which has traditionally been considered the major GH-target site. Chronic hyposomatotropism, induced by hypophysectomy, exerted tissue-specific effects on the abundance of GHR gene products 10 days post-hypophysectomy. Both GHR and GHBP transcripts were reduced in the hypothalamus of hypophysectomized rats by 20% (P < 0.001), although neither transcript was affected in the liver, spleen, cortex/neocortex or brainstem. In contrast, 2 h after a single bolus GH injection that was designed to simulate a pulsatile increase in circulating GH concentrations, GHR and GHBP mRNA content was significantly increased by 25-30% (P < 0.001) in all brain regions and in the spleen of hypophysectomized or sham-hypophysectomized rats. Production of the two transcripts was differentially regulated, however, as GHBP, but not GHR, transcripts were increased in the liver (P < 0.001), whereas the GHR:GHBP ratio was decreased in the hypothalamus of GH-treated rats (P < 0.001). These results suggest that GHR gene transcription and splicing are acutely autoregulated in a tissue-specific way.

Animals↗
PubMed1998

The transforming power of research on practice: involving practitioners in analysis.

Nurses were invited to participate in the interpretation of transcribed interviews during the data analysis phase of a phenomenological study. They worked with the investigators in learning and using hermeneutic methods to identify and interpret meanings embedded in exemplars. Their expert clinical knowledge of the field of practice added rigor to the analytical process and depth to the understanding achieved. As the analysis progressed, the nurses began to spontaneously describe transformation occurring in their practice. Involving nurses in data analysis has transformative power on practice.

Diffusion of Innovation↗
PubMed1998

Autoregulation of central and peripheral growth hormone receptor mRNA in domestic fowl.

Growth hormone (GH) regulates numerous cellular functions in many different tissues. A common receptor is believed to mediate these tissue-specific effects, suggesting that post-receptor signalling molecules or tissue sensitivity to GH may differ between tissues. Tissue sensitivity depends upon the abundance of GH receptors (GHRs), thus tissue-specific GHR regulation could enable tissue-specific GH actions. The comparative autoregulation of GHR gene transcription in central (whole brain or hypothalami) and peripheral (liver, bursa, spleen and thymus) tissues was therefore examined in domestic fowl. In all tissues, a 4.4 kb GHR gene transcript that encodes the full-length GHR was identified. The abundance of this transcript was inversely related to endogenous GH status; it was lower in males with high circulating concentrations of GH and higher in females with lower basal concentrations of plasma GH. The abundance of this transcript was also rapidly downregulated in response to a bolus systemic injection of recombinant chicken GH, designed to mimic an episodic burst of endogenous GH release. This autoregulatory response was observed within 2 h of GH administration and was of greater magnitude in the brain than in peripheral tissues. Intracerebroventricular injections of GH also downregulated GHR gene expression in the brain, although hepatic GHR transcripts were unaffected 24 h after central administration of GH. In contrast, the induction of hyposomatotropism by passive GH immunoneutralization increased the abundance of the GHR transcript in the thymus, but not in other central (brain) or peripheral (bursa, liver) tissues. GH is not the sole regulator of GHR abundance, however; hypersomatropism induced by hypothyroidism was associated with an increase in GHR mRNA. The expression of the GHR gene in the domestic fowl would thus appear to be autoregulated by GH in a tissue-specific way.

Animals↗