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Biomedical subjects

B Moffat

Publications and source records attributed to B Moffat.

At least 19 recordsLinked to original sources
PubMed2005

Enhanced surveillance for childhood hepatitis B virus infection in Canada, 1999-2003.

Since hepatitis B virus (HBV) infection can have serious sequelae, especially if infection occurs during childhood, there is a continuing need to examine its epidemiology so as to inform control measures. We analyzed trends in disease incidence and patterns of hepatitis B virus (HBV) transmission in both Canadian-born and non-Canadian-born children from 1999 to 2003, through the Enhanced Hepatitis Strain Surveillance System. Amongst Canadian-born children, the incidence of newly identified HBV infection per 100,000 declined significantly during the study period from 1.4 in 1999, to 0.5 in 2003 (RR, 0.75 per year; 95% CI, 0.60-0.95). Amongst non-Canadian-born children, the incidence of HBV infection per 100,000 ranged from 9.4 to 16.3, during the study period (linear trend test, p=0.69). Poisson regression analysis revealed that non-Canadian-born children were more likely to have HBV infection (RR, 12.3; 95% CI, 7.6 to 19.8), than Canadian-born children. HBV infection was found to be more common among children emigrating from high endemic area, than among Canadian-born children. Current Canadian immunization policy should take into consideration the protection of all children against HBV infection, including those coming from countries where mass hepatitis B vaccination programs have still not been launched.

Journal Article↗
PubMed2001

Vascular endothelial cell growth factor-driven endothelial tube formation is mediated by vascular endothelial cell growth factor receptor-2, a kinase insert domain-containing receptor.

Vascular endothelial cell growth factor (VEGF) binds to 2 related receptor tyrosine kinases, known as kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt-1). The KDR has been shown to mediate VEGF-stimulated endothelial cell mitogenesis, migration, and permeability. The Flt-1 receptor has been suggested to mediate VEGF-stimulated endothelial branching morphogenesis, a process whereby endothelial cells, in the presence of a 3D milieu composed of extracellular matrix components and a mixture of growth factors, undergo a morphological transition into a tubular network with many lumina. In the present study, we have used 2 independent endothelial cell tube formation models and highly selective VEGF mutants for the KDR and Flt-1 receptors. We demonstrate that KDR, not Flt-1, stimulation is responsible for the induction of endothelial tubulogenesis. In addition, we demonstrate a modulatory role for Flt-1 in VEGF-mediated tube formation. We also report that VEGF-driven endothelial tube formation is inhibited by selective inhibitors of mitogen-activated protein kinase activation and p38 protein kinase.

Blood Vessels↗
PubMed2000

Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants.

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.

Capillary Permeability↗
PubMed2000

Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.

Animals↗
PubMed1999

Protection and regeneration of nigral dopaminergic neurons by neurturin or GDNF in a partial lesion model of Parkinson's disease after administration into the striatum or the lateral ventricle.

Both glial cell line-derived neurotrophic factor (GDNF) and its recently discovered congener, neurturin (NTN), have been shown to exert neuroprotective effects on lesioned nigral dopamine (DA) neurons when administered at the level of the substantia nigra. In the present study, we have explored the relative in vivo potency of these two neurotrophic factors using two alternative routes of administration, into the striatum or the lateral ventricle, which may be more relevant in a clinical setting. In rats subjected to an intrastriatal (IS) 6-hydroxydopamine (6-OHDA) lesion, GDNF and NTN were injected every third day for 3 weeks starting on the day after the 6-OHDA injection. GDNF provided almost complete (90-92%) protection of the lesioned nigral DA neurons after both IS and intracerebroventricular (ICV) administration. NTN, by contrast, was only partially effective after IS injection (72% sparing) and totally ineffective after ICV injection. Although the trophic factor injections protected the nigral neurons from lesion-induced cell death, the level of expression of the phenotypic marker, tyrosine hydroxylase (TH), was markedly reduced in the rescued cell bodies. The extent of 6-OHDA-induced DA denervation in the striatum was unaffected by both types of treatment; consistent with this observation, the high rate of amphetamine-induced turning seen in the lesioned control animals was unaltered by either GDNF or NTN treatment. In the GDNF-treated animals, and to a lesser extent also after IS NTN treatment, prominent axonal sprouting was observed within the globus pallidus, at the level where the lesioned nigrostriatal axons are known to end at the time of onset of the neurotrophic factor treatment. The results show that GDNF is highly effective as a neuroprotective and axon growth-stimulating agent in the IS 6-OHDA lesion model after both IS and ICV administration. The lower efficacy of NTN after IS, and particularly ICV, administration may be explained by the poor solubility and diffusion properties at neutral pH.

Amphetamine↗
PubMed1998

Neurturin exerts potent actions on survival and function of midbrain dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) exhibits potent effects on survival and function of midbrain dopaminergic (DA) neurons in a variety of models. Although other growth factors expressed in the vicinity of developing DA neurons have been reported to support survival of DA neurons in vitro, to date none of these factors duplicate the potent and selective actions of GDNF in vivo. We report here that neurturin (NTN), a homolog of GDNF, is expressed in the nigrostriatal system, and that NTN exerts potent effects on survival and function of midbrain DA neurons. Our findings indicate that NTN mRNA is sequentially expressed in the ventral midbrain and striatum during development and that NTN exhibits survival-promoting actions on both developing and mature DA neurons. In vitro, NTN supports survival of embryonic DA neurons, and in vivo, direct injection of NTN into the substantia nigra protects mature DA neurons from cell death induced by 6-OHDA. Furthermore, administration of NTN into the striatum of intact adult animals induces behavioral and biochemical changes associated with functional upregulation of nigral DA neurons. The similarity in potency and efficacy of NTN and GDNF on DA neurons in several paradigms stands in contrast to the differential distribution of the receptor components GDNF Family Receptor alpha1 (GFRalpha1) and GFRalpha2 within the ventral mesencephalon. These results suggest that NTN is an endogenous trophic factor for midbrain DA neurons and point to the possibility that GDNF and NTN may exert redundant trophic influences on nigral DA neurons acting via a receptor complex that includes GFRalpha1.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1998

Persephin, a novel neurotrophic factor related to GDNF and neurturin.

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

Animals↗
PubMed1998

Solution structure of the fourth metal-binding domain from the Menkes copper-transporting ATPase.

Menkes disease is an X-linked disorder in copper transport that results in death during early childhood. The solution structures of both apo and Ag(I)-bound forms of the fourth metal-binding domain (mbd4) from the Menkes copper-transporting ATPase have been solved. The 72-residue mbd4 has a ferredoxin-like beta alpha beta beta alpha beta fold. Structural differences between the two forms are limited to the metal-binding loop, which is disordered in the apo structure but well ordered in the Ag(I)-bound structure. Ag(I) binds in a linear bicoordinate manner to the two Cys residues of the conserved GMTCxxC motif; Cu(I) likely coordinates in a similar manner. Menkes mbd4 is thus the first bicoordinate copper-binding protein to be characterized structurally. Sequence comparisons with other heavy-metal-binding domains reveal a conserved hydrophobic core and metal-binding motif.

Adenosine Triphosphatases↗
PubMed1997

A GPI-linked protein that interacts with Ret to form a candidate neurturin receptor.

Glial-cell-line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two structurally related, potent survival factors for sympathetic, sensory and central nervous system neurons. GDNF mediates its actions through a multicomponent receptor system composed of a ligand-binding glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) and the transmembrane protein tyrosine kinase Ret. In contrast, the mechanism by which the NTN signal is transmitted is not well understood. Here we describe the identification and tissue distribution of a GPI-linked protein (designated NTNR-alpha) that is structurally related to GDNFR-alpha. We further demonstrate that NTNR-alpha binds NTN (K[d] approximately 10 pM) but not GDNF with high affinity; that GDNFR-alpha binds to GDNF but not NTN with high affinity; and that cellular responses to NTN require the presence of NTNR-alpha. Finally, we show that NTN, in the presence of NTNR-alpha, induces tyrosine-phosphorylation of Ret, and that NTN, NTNR-alpha and Ret form a physical complex on the cell surface. These findings identify Ret and NTNR-alpha as signalling and ligand-binding components, respectively, of a receptor for NTN and define a novel family of receptors for neurotrophic and differentiation factors composed of a shared transmembrane protein tyrosine kinase and a ligand-specific GPI-linked protein.

Amino Acid Sequence↗
PubMed1996

Glycerophosphorylethanolamine (GPEA) identified as an hepatocyte growth stimulator in liver extracts.

Extracts from weanling pig liver were found to act synergistically with growth factors such as hepatocyte growth factor and transforming growth factor-alpha to stimulate hepatocyte growth in serum-free cultures. In the absence of added growth factors, the extracts had no activity. The compound responsible for this activity was isolated by passing heat-treated liver extract through anion-exchange and heparin columns followed by gel filtration at neutral and low pH, reversed-phase HPLC, and a final gel filtration column at low pH. The activity was followed throughout the purification by its ability to increase substantially the incorporation of [3H]thymidine into primary rat hepatocytes cultured serum-free in the presence of hepatocyte growth factor. The active compound was identified by NMR and mass spectrometry as glycerophosphorylethanolamine (GPEA), a breakdown product of the phospholipid phosphatidylethanolamine. The ethanolamine portion of the molecule was critical for the observed activity, whereas the glycerol phosphate portion was not necessary. In the absence of added growth factors, neither GPEA nor ethanolamine had any stimulatory effect on the cells. These results demonstrate that hepatocytes grown in culture, and especially those grown in serum-free media, require a supplement of ethanolamine and/or GPEA. In the absence of these compounds, their response to growth stimuli is greatly reduced.

Animals↗
PubMed1996

High-level expression and characterization of a purified 142-residue polypeptide of the prion protein.

The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was approximately 50%, and from each liter of E. coli culture, approximately 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt.

Amino Acid Sequence↗
PubMed1995

Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult brain.

Glial-cell-line-derived neurotrophic factor (GDNF) promotes survival of embryonic dopaminergic neurons in culture, and its expression pattern suggests a role as a transient target-derived trophic factor for dopaminergic neurons of the substantia nigra. These neurons participate in the control of motor activity, emotional status and cognition, and they degenerate in Parkinson's disease for unknown reasons. To test whether GDNF has a trophic effect on dopaminergic neurons in the adult brain, we used a rat model in which these neurons are induced to degenerate by transecting their axons within the medial forebrain bundle. We report here that axotomy resulted in loss of half the tyrosine hydroxylase-expressing neurons in the substantia nigra. This loss was largely prevented by repeated injections of GDNF adjacent to the substantia nigra. Our findings suggest that GDNF or related molecules may be useful for the treatment of Parkinson's disease.

Animals↗
PubMed1994

Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa.

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.

Anticoagulants↗
PubMed1993

Fibre areas and histochemical fibre types in the quadriceps muscle of paraplegic subjects.

With the advent of functional electrical stimulation (FES) there is the possibility of paraplegic patients regaining some degree of locomotor activity. It is of interest to document the changes in composition histochemistry and size of muscle fibres in such patients both before and after such therapy. We have examined biopsy specimens from quadriceps muscles obtained from 7 male patients, age range 24-47 years, who had been paraplegic for times ranging from 11 months to 9 years and we report the histochemical appearance of the muscle the fibre type composition and the mean fibre areas. In 5 of the 7 subjects there was a marked or complete predominance of fibres which stained as type 2 with the ATPase reaction at pH 9.4. At acid pH these fibres were seen to be predominantly 2B (fast fatigable). The 2 subjects who had been paralysed for the shortest periods had proportions of type 1 fibres which were relatively well preserved. The mean fibre areas of type 2 fibres were below the normal range (2500-7500 microns 2) in every case as were the type 1 fibres in the 4 patients in which these were still present. There was no relationship between the length of time the patient had been paralysed and the mean fibre areas which suggests that atrophy occurs fairly quickly following loss of voluntary control and precedes the loss of type 1 characteristics. Our findings provide an explanation for the rapid onset of fatigue in paraplegic patients taking part in FES programmes since muscles deficient in type 1 fibres will be unable to maintain force for any length of time.

Adult↗
PubMed1992

The disposition of a human relaxin (hRlx-2) in pregnant and nonpregnant rats.

The pharmacokinetics and tissue distribution of a human relaxin were investigated after intravenous (iv) bolus administration to pregnant or nonpregnant rats. Human gene-2 relaxin (hRlx-2) serum concentrations after iv bolus administration were described as the sum of three exponentials. The pharmacokinetics were comparable in pregnant and nonpregnant rats. The serum clearance (CL) was 7.4-10.2 ml/min/kg at doses of 46-93 micrograms/kg and was linear in this range. The half-lives were 1.1-2.0, 15.1-16.4, and 53.7-67.9 min, respectively. The volume of the central compartment (Vc) was 48-79 ml/kg and the volume of distribution at steady state (Vss) was 271-336 ml/kg. Increasing the dose to 463 micrograms/kg increased the dose-corrected area under the serum concentration-time curve and significantly decreased CL and Vss. The distribution of radioactivity in the tissues of pregnant rats was followed after iv bolus dosing with hRlx-2 internally labeled with 35S-cysteine. Comparison of the extent of organ uptake of radiolabel after 35S-hRlx-2 or 35S-cysteine administration suggested that the kidneys were the principal site of uptake; the liver was of secondary importance. In perfusion experiments utilizing livers isolated from pregnant or nonpregnant rats, 36-52% of the dose of hRlx-2 was cleared from the perfusate in 2 hr. These studies showed that the pharmacokinetics of hRlx-2 in rats appeared to be unaffected by pregnancy and suggested that the kidneys and liver both play a role in the elimination of hRlx-2.

Animals↗
PubMed1991

Lung surfactant protein SP-C from human, bovine, and canine sources contains palmityl cysteine thioester linkages.

Lung surfactant is a complex mixture of lipids and proteins that coats the alveoli to reduce surface tension and prevent airspace collapse. One of the principal protein constituents, surfactant protein C (SP-C), has been characterized following isolation from human, canine, and bovine sources. In each species, this highly hydrophobic protein is composed of 33-35 amino acids, the differences being due to NH2-terminal heterogeneity. A COOH-terminal leucine is conserved throughout. The cysteines in each species were found by fast atom bombardment mass spectrometry to be present as thioesters of palmitic acid. Acylation of recombinant SP-C with palmityl coenzyme A, followed by characterization before and after release of the acyl group with 1,4-dithiothreitol, provided corroborating evidence for the native structure.

Acylation↗