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Biomedical subjects

J S Garvey

Publications and source records attributed to J S Garvey.

At least 19 recordsLinked to original sources
PubMed1996

Immunolocalization of 17 and 21.5 kDa MBP isoforms in compact myelin and radial component.

Our previous biochemical analyses revealed that the levels of the minor MBP isoforms 21.5 and 17 kDa are elevated relative to the 14 and 18.5 kDa MBP isoforms in the fraction of isolated myelin of murine CNS that is enriched in interlamellar junctions (or radial component). To substantiate the localization of 21.5 and 17 kDa MBP in the myelin sheath, we used immunoelectron microscopy on thin-sections of mouse optic nerve. Two different polyclonal antibodies were used to distinguish 21.5 and 17 kDa MBP from 14 and 18.5 kDa MBP: Ab-MBP21.5, which was raised against a synthetic peptide corresponding to the exon II amino acid sequence 61-83 of mouse 21.5 kDa MBP (LKQSRSPLPSHARSRPGLCHMYK), and Ab-MBP14, which is immunoreactive to all four isoforms of mouse MBP. Our SDS-PAGE/immunoblotting demonstrated that Ab-MBP21.5, unlike Ab-MBP14, recognized only the 21.5 and 17 kDa MBP isoforms from isolated mouse CNS myelin. Immunolabelling of tissue sections indicated that Ab-MBP14 bound tenfold more to junction-free compact myelin than to radial component, whereas Ab-MBP21.5 bound about equally to the two regions of the myelin sheath. In addition, within the junction-free compact myelin, both antibodies bound nearly three fold more to the major dense line than to the intraperiod line.

Amino Acid Sequence↗
PubMed1996

Amino acid composition, immunoreactivity, sequence analysis, and function of bovine hippocampal metallothionein isoforms.

The high concentration of zinc in the hippocampal mossy fiber axon boutons is localized in the vesicles and is mobilized by exocytosis of the zinc-laden vesicles. Because "free" zinc in excess is a neurotoxic substance inhibiting an extensive number of sulfhydryl-containing enzymes and receptor sites, we hypothesized that low-molecular-weight zinc binding proteins must exist in the hippocampus to regulate the steady-state concentration of zinc. In this communication, we report that the bovine hippocampus synthesizes metallothionein (MT) isoforms that are similar, but not identical, to those of the rat brain MT isoforms and cross-react poorly with antibodies formed against the hepatic MT isoforms, suggesting that the immunologically dominant regions of hippocampal MT (residues 1-29) are not conserved. A comparative sequence analysis of bovine hippocampal MTs and bovine hepatic MT isoforms I and II revealed a 90% sequence identity, being mostly different in residues 1-29. The results of these studies suggest that the hippocampal MT isoforms, which are synthesized on a continuous basis, may play a role in regulating the transport, accumulation, and compartmentation of zinc in the hippocampus.

Amino Acid Sequence↗
PubMed1994

Serum zinc and copper concentrations in relation to spontaneous abortion in cows: implications for human fetal loss.

The purpose of this study was to investigate the effect of the status of dietary zinc and serum zinc and copper concentrations on the risk of fetal loss in 570 cows. three herds received no supplements (herds 1, 3, 4), while cows in herd 2 received supplements of either 7 g zinc week-1 (n = 118), as zinc methionine, or a control diet containing methionine (n = 128). Serum zinc, copper and metallothionein concentrations were determined once a month throughout gestation. Logistic regression and survival analysis were used to examine for associations between risk of fetal loss and serum zinc, copper, copper:zinc, or metallothionein concentrations, supplement level, and maternal age at conception. The risk of fetal loss increased when both serum zinc decreased and copper concentrations increased (P < 0.0001; relative risk = 10.28, 95% confidence intervals = 4.69, 22.5). The attributable risk, for a decline in the zinc concentration by 10 mumol l-1 and an increase in the copper concentration by 5 mumol l-1 was 90.27%. Methionine-supplemented cows had a higher risk of fetal loss compared with zinc-methionine-supplemented cows (one-tailed P = 0.0375; relative risk = 2.98). Cows in herds 1, 3 and 4 had a higher risk for abortion than did zinc-methionine-supplemented cows in herd 2 (relative risk = 26.27, 95% confidence intervals = 2.31, 299.38; relative risk = 40.87, 95% confidence intervals = 3.50, 458.43; relative risk = 41.53, 95% confidence intervals = 3.77, 457.02, respectively). Our results suggest that inflammation and zinc nutriture may play an important role in fetal loss in dairy cows.

Abortion, Spontaneous↗
PubMed1993

Organ distributions of liposome-loaded rat platelets.

Past in vitro functional assays have demonstrated that platelet function is not inhibited by liposome uptake. In the present study, the organ distributions of control and liposome-loaded Sprague-Dawley rat platelets were examined to determine whether liposome uptake enhances RES uptake. Platelets were isolated using STRactan density gradient centrifugation, incubated with small unilamellar liposomes in vitro for 1 hour, labeled with 51Cr and injected into a cohort group of rats. One hour post-injection the spleen, liver, lungs, blood, kidneys and bladder contents were removed and the percentages of the recovered dose localized per total organ (%RD) were determined. The RES index, defined as %RDliver + %RDspleen, were 24.8 +/- 4.5 and 20.5 +/- 5.0 for the control platelets and liposome-loaded platelets, respectively. These results indicate that liposome uptake does not enhance RES uptake.

Animals↗
PubMed1993

Cyclic AMP has a differentiative effect on an immortalized oligodendrocyte cell line.

We investigated the effects of increasing the concentration of intracellular cyclic adenosine monophosphate (cAMP) on genes associated with oligodendrocyte differentiation in an immortalized glial cell line, 6E12, derived from the spinal cord of an MBP-SV40 large T-antigen transgenic mouse. Raising intracellular levels of cAMP induced expression of oligodendrocyte differentiation antigens recognized by O4 and anti-galactocerebroside antibodies, up-regulated expression of the proteolipid protein (PLP) gene, and down-regulated glial fibrillary acidic protein (GFAP) expression. There was no treatment effect on myelin-associated glycoprotein (MAG) expression. These phenotypic changes are consistent with oligodendrocyte differentiation. Treatment of 6E12 cells with dibutyryl cyclic AMP (DBC) down-regulated myelin basic protein (MBP) gene expression, perhaps, because it also up-regulated expression of a putative MBP repressor SCIP/Tst-1. Moreover, the 6E12 cells expressed high levels of MBP mRNA but no MBP translation products were detected in the presence or absence of DBC. This immortalized glial cell line is proposed as a CNS model for cAMP-modulated myelin gene expression and for post-transcriptional regulation of MBP.

Animals↗
PubMed1993

Transgenic mouse model for neurocristopathy: Schwannomas and facial bone tumors.

We have characterized a strain of double transgenic mice with simian virus 40 large tumor antigen and prokaryotic lacZ under the control of the myelin basic protein promoter that develops spindle-cell sarcomas and osteogenic sarcomas at 5-7 months of age. Although poorly differentiated, the spindle-cell sarcomas were characterized as malignant Schwannomas based on their neural association, the presence of basal lamina, and expression of Schwann cell-specific genes. The osteogenic sarcomas were often multiple and appeared predominantly in the facial bones, less frequently in the ribs and vertebral column, and only rarely in the appendicular skeleton. Benign osteoblastic lesions were often observed adjacent to these sarcomas. Both the osteoblastic cells in the facial skeleton and Schwann cells are regarded as neural crest derivatives. The biological properties and anatomical location of these tumors suggest that they may share a common origin from the neural crest or its derivatives. R.P. Bolande [Hum. Pathol. (1974) 5, 409-429] introduced the term neurocristopathy as a unifying concept to describe such lesions arising from the neural crest or its derivatives. Cell lines established from both bone and Schwann cell tumors arising in these transgenic mice express simian virus 40 large tumor antigen mRNA as well as functional large tumor antigen. Such cell lines are potentially valuable in the search for markers that identify mammalian neural crest derivatives.

Animals↗
PubMed1993

Distinct hypomyelinated phenotypes in MBP-SV40 large T transgenic mice.

To study the effect of SV40 large T-antigen expression in myelin-forming cells of both the central and peripheral nervous system, a series of transgenic mice were generated expressing the SV40 large T-antigen under control of the myelin basic protein (MBP) promoter. Two neurologic phenotypes, designated A and B, appeared among individual transgenic founders and their progeny. The A mice developed a severe action tremor at about 10 days of age that progressed into periods of convulsions and early death by three to four weeks of age. In contrast, the B mice exhibited a progressive hindlimb ataxia and had a more normal lifespan. The A mice displayed hypomyelinating lesions in the central nervous system (CNS), whereas the B mice had lesions in either the peripheral nervous system (PNS) alone or in both the PNS and CNS. Immunohistochemical staining of spinal cord sections of a type A mouse showed a substantial depletion in MBP. Moreover, T-antigen-positive cells appeared predominantly in white matter tracts as randomly distributed single cells. Double labeling immunocytochemistry demonstrated that some of these T-antigen-positive cells were positive for oligodendrocyte differentiation markers MBP and O4. Thus, T-antigen expression appeared to coincide with a terminal stage of oligodendrocyte differentiation.

Animals↗
PubMed1992

Differential effect of adrenalectomy on rat liver metallothionein mRNA levels in basal and stress conditions.

Liver metallothionein (MT) mRNA and serum MT levels of adrenalectomized (ADX) and sham-ADX rats in basal and stress (1, 3 or 6 h of restraint) conditions have been measured. Serum MT levels were overall lower in ADX than in sham-ADX rats. Basal liver MT mRNA levels were increased in ADX rats, suggesting that glucocorticoids have an inhibitory role on the regulation of liver MT synthesis. In contrast, liver MT mRNA levels were increased by stress in sham-ADX but not in ADX rats, suggesting a stimulatory role for glucocorticoids. These results suggest that glucocorticoids have a different role in liver MT regulation depending on the physiological situation.

Adrenalectomy↗
PubMed1991

Glial immunoreactivity for metallothionein in the rat brain.

A series of frozen and vibratome coronal sections of the rat brain were examined by immunocytochemistry for the presence of a cysteine-rich metal binding protein, metallothionein (MT). Astrocytes throughout the brain and brainstem stained positively for MT; neurons and oligodendroglia were unstained. Ependymal cells and tanycyte processes in the hypothalamus were also immunoreactive, along with a narrow zone of immunopositivity along the margins of the area postrema. Gomori-positive astrocytes in the hypothalamus, identifiable by toluidine blue staining, metal-containing cytoplasmic granules, represented a subset of MT-positive astrocytes that may be involved in reactions to blood-borne metal compounds that penetrate into circumventricular organs of the brain.

Animals↗
PubMed1991

Role of extracellular zinc and copper on metallothionein regulation in cultured rat hepatocytes.

Cellular and extracellular metallothionein contents of rat hepatocytes cultured in the presence of albumin (30 mumol/L) with zinc (1, 10, 50 and 100 mumol/L), copper (1, 10 and 50 mumol/L), zinc and copper (1, 10 and 50 mumol/L of each metal) or no metals in the culture medium have been measured by radioimmunoassay. Cellular metallothionein levels increased steadily with culture time regardless of the metal treatment and showed little dependence (only a twofold increase) on extracellular zinc or copper at all metal concentrations and times (up to 3 days) studied. In contrast, the presence of both metals simultaneously in the culture medium strongly increased cellular metallothionein contents, acting synergistically in some cases. Significant extracellular metallothionein was observed when copper or zinc and copper were present in the culture medium, most of which is likely a consequence of cell leakage because no evidence of physiological secretion was observed. Total metallothionein production (cellular and extracellular metallothionein levels) indicated that copper was a better metallothionein inducer than zinc in these experimental conditions. These results indicate that metallothionein regulation in the hepatocyte is different depending on the extracellular metal levels and composition and that attention must be given to metallothionein release from the hepatocyte.

Analysis of Variance↗
PubMed1991

Metallothionein in rat lung during postnatal development.

Zn and Cu concentration, content, and distribution in lung were quantitated in neonatal and adult Lewis rats. Total lung Cu and Zn content rose dramatically during postnatal development, paralleling increases in lung and body weight. Cu concentration was 2-fold higher in adult lung than in neonatal lung whereas Zn concentration was unaffected by developmental age. A Cu-containing protein with an approximate molecular weight of 10,000 Da was identified immunologically as metallothionein (MT). At term, native MT concentration in the lung was 4-fold higher than in adult lungs. By day 4 postpartum, MT concentration was reduced by half and reached a level characteristic of the adult by 7 days postpartum. MT was localized in nuclear and cytoplasmic compartments of positive lung cells by immunofluorescent techniques; initially, nuclear MT staining was more intense than cytoplasmic staining.

Animals↗
PubMed1991

Effect of morphine administration on rat liver metallothionein and zinc metabolism.

The putative involvement of opioid receptors in rat liver metallothionein (MT) regulation has been studied by means of morphine administration. Rat liver MT levels were significantly increased by morphine (10 mg kg-1). This increase was blocked by the opiate antagonist naloxone (4 mg kg-1), suggesting that the effect of morphine on liver MT could involve opioid receptors. The effect of morphine appears to be mediated, at least in part, by glucocorticoids and catecholamines, inasmuch as the administration of specific receptor blockers, RU 486 (100 mg kg-1) for glucocorticoids and labetalol (5 mg kg-1) for catecholamines, diminished liver MT increase induced by morphine. These results identify a potential mediation mechanism for regulating liver MT levels. The putative role of endogenous opioids on liver MT response to stress was also studied by means of opioid receptor blockers. The effect of stress on liver MT levels was not altered by blockade of opioid receptors with either naloxone (4 mg kg-1) or naltrexone (4 mg kg-1) or diprenorphine (4 and 20 mg kg-1), suggesting that endogenous opioids are not involved in MT response to stress. Zn metabolism was also altered by morphine, as morphine administration increased liver cytosolic Zn and decreased serum Zn levels. In contrast to those found in liver MT, these changes were not naloxone-sensitive. The results obtained with RU 486 and labetalol suggest that the effect of morphine on Zn metabolism was mediated in part by glucocorticoids and catecholamines.

Animals↗
PubMed1990

Extracellular metallothionein effects on lymphocyte activities.

Metal cation influences on the immune response have been reported in a wide variety of experimental systems. These influences can either result in the augmentation or suppression of immunological activities. In order to investigate possible mechanisms of these influences, we examined the role that a metal cation-induced protein, metallothionein (MT), might play. Our findings suggest that thioneins, either as apoproteins or when complexed as Cd,Zn-MT, Zn-MT, or Cd-MT, are capable of inducing lymphocyte proliferation. This level of induction is substantially reduced when Zn,Cd-MT is added to lymphocyte cultures in the presence of 50 microM 2-mercaptoethanol. Apoprotein, Zn,Cd-MT, Zn-MT and Cd-MT also augment LPS-induced proliferation of splenic lymphocytes. Only the Zn,Cd-MT preparation significantly augmented ConA-induced proliferation. Hg-MT and Cu-MT were inhibitory as additions in either LPS or ConA mitogen proliferation assays, and did not stimulate proliferation when added alone to lymphocyte cultures. The capacity to induce proliferation correlates with the measurable thiol level of the particular thionein. Interestingly, Zn,Cd-MT and apothionein had an equivalent number of accessible thiols. Although Zn, Pb, Hg and Cu lowered the number of these sites, the immunoreactivity of these MTs was not altered substantially except by Pb. These results suggest that some metal influences on lymphocytes might be through a thionein intermediary. Our results also demonstrate that thioneins complexed with certain metal cations are detrimental to the normal cellular activities of lymphocytes. At least in these circumstances, MT does not play a role as a protective agent.

Animals↗
PubMed1990

Liver, brain, and heart metallothionein induction by stress.

To date, stress has been reported to induce metallothionein (MT) synthesis in the liver only. In the present experiment, the effects of food and water deprivation alone or of immobilization stress plus food and water deprivation on liver, brain, and heart MT have been studied in adult male rats. Liver and brain MT levels were increased by immobilization stress as soon as 6 h after the onset of stress. Eighteen hours of immobilization, which is accompanied by food and water deprivation, further increased liver and brain MT levels and significantly increased heart MT content. A specific effect of immobilization was evident in all three tissues, because the effect of food and water deprivation alone was significantly lower than that of immobilization plus starvation. Changes in MT apparently were not related to changes in cytosolic Zn.

Animals↗
PubMed1990

On the metallothionein, glutathione and cysteine relationship in rat liver.

Stress and starvation increased liver metallothionein (MT) and decreased liver glutathione (GSH) levels. Serum cysteine plus cystine levels were increased by stress. The exogenous administration of GSH, while not modifying hepatic GSH content, increased liver MT levels in basal and starved rats but not in stressed rats. Liver and serum cysteine levels were increased by GSH administration, a process partially reverted by the irreversible inhibitor of gamma-glutamyl transpeptidase, alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid. Mouse and rat liver MT levels were also increased by buthionine sulfoximine, an inhibitor of GSH synthesis, indicating that GSH is not a necessary precursor of MT. In addition, the hepatic MT content was increased by the administration of cysteine in a dose-response manner. These results suggest that hepatic MT synthesis is elevated by increased cysteine pools, and that MT, GSH and cysteine levels are somehow inter-related. MT, besides GSH, may be contemplated as a putative intracellular reservoir of cysteine in the liver of adult rats.

Animals↗
PubMed1989

Induction of pulmonary metallothionein following oxygen exposure.

Metallothionein (MT) levels were measured by radioimmunoassay in lungs of animals exposed 0, 3, and 6 days to 85% oxygen. MT levels increased with duration of exposure from 112.0 ng/lung in sham air control animals to 872.6 ng/lung in animals exposed for 6 days to oxygen. Gel chromatographic analysis of lung homogenates from oxygen-exposed animals revealed the presence of a copper- and zinc-binding component with an approximate molecular weight of 12,000 Da. It was heat stable and cross-reacted with anti-MT. The induction of pulmonary Cu/Zn-thionein was accompanied by an acute phase response, characterized by elevated serum Cu and ceruloplasmin levels and depressed serum Zn. Total lung Cu and Zn also increased, perhaps as a consequence of normal repairative processes necessitated by the oxidant injury. Increased adrenal weight and coincident thymic atrophy in oxygen-exposed animals suggested the participation of adrenocorticosteroids in the induction process.

Animals↗
PubMed1989

Preparative isolation of adult human liver metallothionein isoforms.

Preparative human liver metallothionein (MT) isolation is described. MT was saturated with cadmium to follow MT purification spectrophotometrically instead of by metal content and to increase the stability of the protein. A concentrated, MT-rich fraction of the liver cytosol was prepared by selective organic solvent (acetone or acetone/methanol) fractionation. Conventional gel filtration and ion-exchange chromatographies resolved two MT isoforms, MT-I and MT-II. When needed, purification of MT from other low-molecular weight proteins was further increased by gel filtration chromatography at zero ionic strength, i.e., in distilled water. Reversed-phase high performance liquid chromatography of both MT isoforms resolved further peaks sharing MT properties not only from the MT-I but also from the MT-II ion-exchange isoform. The results show that it is feasible to perform a human liver MT isolation from an entire human liver with a reasonable laboratory capability.

Adult↗