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Biomedical subjects

A McMahon

Publications and source records attributed to A McMahon.

At least 37 records · Page 2Linked to original sources
PubMed1997

Dopamine transporter mRNA levels are high in midbrain neurons vulnerable to MPTP.

The neurotoxin MPTP kills only certain midbrain dopaminergic (DA) neurons to produce a model of Parkinson's disease. The dopamine transporter (DAT) is important to MPTP toxicity because to be neurotoxic, an MPTP metabolite must first gain access to the DA neuron via the DAT. Also, MPTP is less toxic to DA neurons that contain the putative neuroprotective calcium-binding protein calbindin-D28k (CB). The present study examined the relative importance of DAT activity and CB for cellular vulnerability to MPTP-induced degeneration in the C57BL/6 mouse. Cells that were vulnerable to MPTP were found to contain high levels of DAT mRNA, whereas cells that were not vulnerable contained low levels. Also, the few substantia nigra cells remaining after a toxic dose of MPTP contained only low levels of DAT mRNA. However, there was not a strong relationship between cellular resistance to MPTP toxicity and cells containing CB. These data provide in vivo evidence for a direct correlation between midbrain cellular vulnerability to MPTP toxicity and the activity of the DAT.

Animals↗
PubMed1997

A simplified squalene epoxidase assay based on an HPLC separation and time-dependent UV/visible determination of squalene.

A novel and highly simplified enzyme assay for squalene epoxidase (EC 1.14.99.7) has been developed. The assay relies on the UV/visible determination of squalene at 195 nm, as it elutes from an octadecylsilane HPLC column. An acetonitrile/water (95.5/0.5, v/v) mixture was found to provide an ideal mobile phase, into which aqueous enzyme reaction mixture aliquots could be injected. Squalene, the natural substrate for squalene epoxidase, may be quantitatively determined within the concentration range 0-30 microM, with a calibration curve exhibiting an r2 (where r2 is the square of the Pearson correlation coefficient r) of 0.995. The HPLC retention time for squalene was significantly longer (> 15 min) than that for any other component required to prepare an enzyme assay reaction mixture, so facilitating its identification and quantification. In this way HPLC was used to follow enzymic squalene consumption within aliquots taken over a 30-min period. Previously reported squalene epoxidase assays rely on the radiolabeling and subsequent monitoring of squalene as it is metabolized by the enzyme. A highly simplified enzyme assay for squalene epoxidase is therefore reported.

Chromatography, High Pressure Liquid↗
PubMed1997

A role for Indian hedgehog in extraembryonic endoderm differentiation in F9 cells and the early mouse embryo.

Hedgehog genes in Drosophila and vertebrates control patterning of a number of different structures during embryogenesis. They code for secreted signaling proteins that are cleaved into an active aminopeptide and a carboxypeptide. The aminopeptide can mediate local and long range events and can act as a morphogen, inducing differentiation of distinct cell types in a concentration-dependent manner. We demonstrate here that the expression of Indian hedgehog mRNA and protein is upregulated dramatically as F9 cells differentiate in response to retinoic acid, into either parietal endoderm or embryoid bodies, containing an outer visceral endoderm layer. The ES cell line D3 forms embryoid bodies in suspension culture without addition of retinoic acid and also upregulates Indian hedgehog expression. RT-PCR analysis of blastocyst outgrowth cultures demonstrates that whereas little or no Indian hedgehog message is present in blastocysts, significant levels appear upon subsequent days of culture, coincident with the emergence of parietal endoderm cells. In situ hybridization analysis for Indian hedgehog mRNA expression demonstrates the presence of elevated levels of message in the outer visceral endoderm cells relative to the core cells in mature embryoid bodies and in the visceral endoderm of Day 6.5 embryos. Whole-mount in situ hybridization analysis of Day 7.5 and 8.5 embryos indicates that Indian hedgehog expression is highest in the visceral yolk sac at this stage. F9 cell lines expressing a full length Indian hedgehog cDNA express a number of characteristics of differentiated cells, in the absence of retinoic acid. Taken together, these data suggest that Indian hedgehog is involved in mediating differentiation of extraembryonic endoderm during early mouse embryogenesis.

Animals↗
PubMed1997

Implications for nursing of the NHS R&D funding policy.

In this article, the author reviews the recommendations for NHS research and development funding being implemented as a result of the Culyer report (1994). The implications for nursing research are discussed and nurses are urged to make the most of the opportunities now being created.

Humans↗
PubMed1997

Calbindin-D28k in nerve cell nuclei.

Calbindin-D28k is a member of the large EF-hand family of calcium-binding proteins, that is believed to function, in part as a cytosolic calcium buffer. Recent studies have demonstrated that cells containing Calbindin-D28k are protected from degeneration caused by conditions that elevate intracellular calcium concentrations. Since its initial discovery in 1966, Calbindin-D28k has been localized in the cytoplasm of many neuronal populations, but its nuclear localization has been uncertain. Using light and electron microscopic immunohistochemistry, and nuclear fractionation methods, we demonstrate localization of Calbindin-D28k not only in the cytoplasm, but also in the nucleus of rodent midbrain dopaminergic neurons and cerebellar Purkinje cells. The Calbindin-D28k immunoreactive staining intensity in the nucleus was routinely equal or greater than that in the cytoplasm. Since calcium signals are propagated to the nucleus, where they can regulate gene expression, the existence of nuclear Calbindin-D28k has important implications for cellular function.

Animals↗
PubMed1997

Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb.

Anteroposterior polarity in the vertebrate limb is thought to be regulated in response to signals derived from a specialized region of distal posterior mesenchyme, the zone of polarizing activity. Sonic Hedgehog (Shh) is expressed in the zone of polarizing activity and appears to mediate the action of the zone of polarizing activity. Here we have manipulated Shh signal in the limb to assess whether it acts as a long-range signal to directly pattern all the digits. Firstly, we demonstrate that alterations in digit development are dependent upon the dose of Shh applied. DiI-labeling experiments indicate that cells giving rise to the extra digits lie within a 300 microm radius of a Shh bead and that the most posterior digits come from cells that lie very close to the bead. A response to Shh involves a 12-16 hour period in which no irreversible changes in digit pattern occur. Increasing the time of exposure to Shh leads to specification of additional digits, firstly digit 2, then 3, then 4. Cell marking experiments demonstrate that cells giving rise to posterior digits are first specified as anterior digits and later adopt a more posterior character. To monitor the direct range of Shh signalling, we developed sensitive assays for localizing Shh by attaching alkaline phosphatase to Shh and introducing cells expressing these forms into the limb bud. These experiments demonstrate that long-range diffusion across the anteroposterior axis of the limb is possible. However, despite a dramatic difference in their diffusibility in the limb mesenchyme, the two forms of alkaline phosphatase-tagged Shh proteins share similar polarizing activity. Moreover, Shh-N (aminoterminal peptide of Shh)-coated beads and Shh-expressing cells also exhibit similar patterning activity despite a significant difference in the diffusibility of Shh from these two sources. Finally, we demonstrate that when Shh-N is attached to an integral membrane protein, cells transfected with this anchored signal also induce mirror-image pattern duplications in a dose-dependent fashion similar to the zone of polarizing activity itself. These data suggest that it is unlikely that Shh itself signals digit formation at a distance. Beads soaked in Shh-N do not induce Shh in anterior limb mesenchyme ruling out direct propagation of a Shh signal. However, Shh induces dose-dependent expression of Bmp genes in anterior mesenchyme at the start of the promotion phase. Taken together, these results argue that the dose-dependent effects of Shh in the regulation of anteroposterior pattern in the limb may be mediated by some other signal(s). BMPs are plausible candidates.

Animals↗
PubMed1996

Calbindin-D28K in the nucleus of nerve growth factor-treated PC12 cells.

Calbindin-D28K (CB) is a calcium-binding protein found within a subset of neurons, which has been proposed to buffer intracellular calcium and to protect cells from calcium-induced neurodegeneration. The rat pheochromocytoma PC12 cell line has been an invaluable tool for studying neuronal development and characterizing the molecular actions of nerve growth factor (NGF). Using Western and RNAse protection analyses and immunocytochemical and cell fractionation techniques, we now report that NGF induces the expression of CB not only in the cytoplasm and neurites, but also within the nuclei of PC12 cells. These data indicate that PC12 cells can serve as a useful model system in which to study the functions of CB and the role of nuclear CB.

Animals↗
PubMed1996

Cloning and characterization of cDNAs for novel proteins with glutamic acid-proline dipeptide tandem repeats.

cDNAs with identical 3' sequences containing a hexanucleotide repeat -(GAGCCG)9- were isolated from rat pheochromocytoma and brain cDNA libraries. The cDNA with the longest open reading frame codes for a protein of 24.6 kDa containing a 16-fold -(Glu-Pro)- dipeptide repeat within a glutamate and proline rich region at its deduced C-terminus. cDNAs with the identical 3' sequence and a divergent 5' sequence were isolated from a rat skeletal muscle cDNA library. The latter are predicted to code for a protein of 15.5 kDa with a C-terminal repetitive domain identical to that in the pheochromocytoma and brain cDNAs. The cDNAs recognize a 1.8 kb mRNA species present in a variety of tissues, being particularly abundant in cardiac and skeletal muscle.

Amino Acid Sequence↗
PubMed1996

Induction of adrenal tyrosine hydroxylase mRNA by single immobilization stress occurs even after splanchnic transection and in the presence of cholinergic antagonists.

Immobilization (IMO) stress elevates plasma catecholamines and increases tyrosine hydroxylase (TH) gene expression in rat adrenals. This study examined the mechanism(s) of IMO-induced changes in adrenal TH mRNA levels. Innervation of the adrenal medulla is predominantly cholinergic and splanchnicotomy as well as nicotinic receptor antagonists prevent the cold-induced rise in TH mRNA levels. In this study, the IMO-induced rise in plasma catecholamines, but not TH mRNA levels, was reduced by the antagonist chlorisondamine. Muscarinic antagonist atropine also did not prevent the IMO stress-elicited rise in TH mRNA. Furthermore, denervation of the adrenals by unilateral splanchnicotomy did not block the IMO-induced rise in TH mRNA but completely prevented the induction of neuropeptide Y mRNA. These results suggest that (1) the large increase in adrenal TH gene expression elicited by a single IMO stress is not regulated via cholinergic receptors or splanchnic innervation, and (2) there is a dissociation between regulatory mechanisms of catecholamine secretion and elevation of TH gene expression in the adrenal medulla of rats during IMO stress.

Adrenal Medulla↗
PubMed1995

The effects of hexachlorobenzene on circulating levels of adrenal steroids in the ovariectomized rat.

Hexachlorobenzene (HCB), is a global pollutant that resists degradation and possesses a propensity to bioaccumulate. However, the effect of HCB on adrenal function remains largely unknown. Thus, circulating levels of adrenal steroids in HCB-exposed (0.0, 1.0, 10.0, or 100.0 mg/kg/day--for 30 days by gavage) adult ovariectomized Sprague-Dawley rats (N = 32) were investigated. A terminal blood sample was collected for HCB residue analysis, and levels of circulating progesterone (P4), corticosterone (CS), and aldosterone (ALD) were quantified. Mean serum CS levels were significantly (P = 0.02) reduced by HCB exposure, starting with the lowest dose group (1.0 mg/kg/day for 30 days), whereas no differences in mean serum P4 and ALD levels were observed. Since it has been argued that the rodent possesses the ability to produce small amounts of cortisol and that levels of this glucocorticoid are altered in pathological states, serum cortisol (C) levels were also measured. Circulating levels of C were significantly lower (P < 0.05) in the highest dose group compared with controls. The presence of C in serum was confirmed by reverse-phase HPLC. These data suggest that even at the lowest dose studied, HCB exposure induces alterations in steroidogenesis of cells of the adrenal cortex inner zone.

Adrenal Cortex↗
PubMed1995

QT dispersion in essential hypertension.

Increased QT dispersion is associated with sudden cardiac death in congestive heart failure, hypertrophic cardiomyopathy, and following acute myocardial infarction. Patients with hypertension, in particular those with left ventricular hypertrophy, are also at greater risk of sudden cardiac death. We examined whether QT dispersion, which is easily obtained from a routine ECG, correlates with echo LVH. Sixty-nine untreated patients with essential hypertension had QT dispersion measured from a surface 12-lead electrocardiogram, and two-dimensional echocardiography performed to measure interventricular septal thickness, posterior wall thickness, and left ventricular internal diameter. Office blood pressure was recorded, and in 56 patients, 24 h ambulatory blood pressure monitoring was also done. Multivariate analysis demonstrated significant relationships between QT dispersion and office systolic blood pressure, and left ventricular mass index. Similar findings were obtained when QT dispersion was corrected for heart rate (QTc dispersion). After patients with electrocardiographic left ventricular hypertrophy (n = 5) were excluded from the analysis, the above relationships persisted. Increased QT dispersion is thus found in those essential hypertensives at greatest risk of sudden death. Since this relationship persists even in the absence of electrocardiographic left ventricular hypertrophy, measurement of QT dispersion might be a simple, non-invasive screening procedure to identify those hypertensives at greatest risk of sudden death.

Adolescent↗
PubMed1995

Ultrastructural changes in ovarian follicles of monkeys administered hexachlorobenzene.

OBJECTIVE: To test hexachlorobenzene (HCB), an environmental pollutant, for its potential toxicity to the ovary. DESIGN: Nonhuman primates were orally administered the pollutant at doses between 0.01 and 10 mg of HCB/kg of body weight to test viability of ovarian follicles. At the end of dosing period, the monkeys received a compound that contained follicle-stimulating and luteinizing hormones to stimulate development of follicles that would be examined by electron microscopy. ANIMALS: Twenty, 6- to 13-year-old cynomolgus monkeys were randomly assigned to 5 groups. PROCEDURE: The HCB, in concentrations of 0.01, 0.1, 1.0, and 10.0 mg/kg, was orally administered with glucose in gelatin capsules for 13 weeks. Monkeys receiving capsules containing glucose only served as controls. After the 13th week, monkeys were given a compound that contained follicle-stimulating and luteinizing hormones daily during days 2 to 7 from the start of menses. On day 8 of the menstrual cycle, 5,000 IU of human chorionic gonadotropin was administered, and 35 to 38 hours later, 1 ovary from each monkey was obtained during laparotomy. Approximately 1-mm cubes of tissue from each ovary were harvested, fixed by immersion in buffered 2% glutaraldehyde, and processed for transmission electron microscopy. RESULTS: Ultrastructure of ovarian follicles was altered in the monkeys administered HCB. Lesions were observed in the follicles from monkeys given the lowest concentrations of HCB, and comprised condensed mitochondria in the developing ova and follicular cells that contained nuclei with deep indentations and abnormal accumulation of cytoplasmic lipid droplets. Alterations, such as herniation of the ooplasm, degeneration of the follicular cells, and appearance of abnormal spaces between follicular cells were observed in the follicles from monkeys of the 0.1 to 1.0 mg of HCB/kg dosage groups. The most relevant alterations were seen in the mitochondrion, an organelle that appeared to be most sensitive to the compound. Mitochondria were condensed, with abnormal intracristal spaces in the lower-dosage groups, and were markedly degenerated in the 10 mg/kg group. The effect of HCB were dose-related. CONCLUSION: The HCB is a reproductive system toxicant, and its damaging effects may be a result of augmentation of lipid peroxidation, especially in the primary follicle, which abnormally affects cellular membranes and thus, impairs their permeability.

Administration, Oral↗
PubMed1994

Induction of tyrosine hydroxylase gene expression by a nonneuronal nonpituitary-mediated mechanism in immobilization stress.

Stress stimulates the sympathoadrenal system, causing activation of the catecholamine biosynthetic enzymes. Here we examine the changes of gene expression of tyrosine hydroxylase (TH; EC 1.14.16.2), the initial enzyme of catecholamine biosynthesis, with stress. A single immobilization of rats led to a large transient elevation in TH mRNA and a small elevation in TH immunoreactive protein and activity. Repeated daily immobilizations triggered more sustained changes in TH mRNA levels. After two immobilizations, the levels remained elevated even 3 days later. The rise in TH mRNA was followed by increased immunoreactive protein but only a small elevation in activity. With seven repeated immobilizations, the animals did not appear to adapt and still manifested a further rise in TH mRNA. TH activity was markedly elevated and returned to control levels 7 days after the immobilization. The rise in TH mRNA with a single immobilization occurred even in adrenals of hypophysectomized rats that underwent splanchnic nerve section. Immobilization for 30 min was sufficient to increase TH mRNA. The effect was abolished by the transcriptional inhibitor actinomycin D. Mobility gel-shift assays revealed increased binding of c-Fos and c-Jun to the AP-1 transcription factor site after a single immobilization, and the binding was not further elevated with repeated stress. This study shows that a single immobilization can activate TH gene expression by a nonneuronal nonpituitary-mediated pathway associated with increased binding of AP-1 transcription factors.

Adrenal Glands↗
PubMed1994

Experimental control of axial pattern in the chick blastoderm by local expression of Wnt and activin: the role of HNK-1 positive cells.

Small grafts from transfected mammalian cell lines that secrete activin or express Wnt-1 RNA were made to the marginal zone of entire chick blastoderms in culture. Grafts from appropriate control cell lines produced no effects on development. The activin-secreting grafts, implanted before streak formation, could cause the streak to form opposite their marginal position even when this was 180 degrees distant around the blastoderm from the original presumptive streak site. Alternatively, opposed twin streaks were observed, one at the original presumptive site and one in relation to the graft. Wnt-expressing grafts implanted early could also reposition axis formation, but only to graft sites within approximately 100 degrees of angular distance from the host's presumptive streak origin. No Wnt-induced twinning was observed. Grafts of both experimental cell types intermixed were the most effective in reorientating, twinning, or globally disturbing the axial pattern and led to second axes with the least delay, relative to normal development, in reaching headfold stages. The incidence and distribution of cells positive for the epitope HNK-1 was investigated during early stages of normal and of experimentally twinned development. Only two nonhypoblast regions of HNK-1 expression were consistently observed in normal early development; a sector in the germ wall area opaca, behind the site of streak formation, and then a localised region of intensely, newly expressing cells arising in epiblast and in anteriormost parts of the (epiblast-derived) streak at the half-length streak stage. Both "activin only" and "activin/Wnt" mixed grafts, although not control grafts, became surrounded by new sectors of "germ wall" HNK-1 positivity. Such positivity may therefore mark a cell group with a signaling role (but no anatomical participation) in streak initiation. However, there was no change of the local background incidence of epiblastic HNK-1 positivity in the structure of streaks induced by "activin only" grafts. This indicates that most cells of the streak are specified by relatively local induction, rather than deriving from selective aggregation. Only grafts including the Wnt-expressing cells gave rise to obvious new HNK-1 expression within epiblast-derived cells anteriorly, as does the complete normal streak. This suggests that the Wnt class of response pathway can complement the activin one in producing rostrocaudally complete axial pattern, as has been suggested for amphibian development.

Activins↗
PubMed1994

Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors.

Changes in intermediate filament gene expression occur at key steps in the differentiation of cell types in the mammalian CNS. Neuroepithelial stem cells express the intermediate filament protein nestin and down-regulate it sharply at the transition from proliferating stem cell to postmitotic neuron. Nestin is also expressed in muscle precursors but not in mature muscle cells. We show here that in transgenic mice, independent cell type-specific elements in the first and second introns of the nestin gene consistently direct reporter gene expression to developing muscle and neural precursors, respectively. The second intron contains an enhancer that functions in CNS stem cells, suggesting that there may be a single transcriptional mechanism regulating the CNS stem cell state. This enhancer is much less active in the PNS. The identification of these elements facilitates analysis of mechanisms controlling the switch in gene expression that occurs when muscle and brain precursors terminally differentiate.

Animals↗