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Abstracts from VI International Symposium on Avian EndocrinologyMarch 31 - April 5, 1996 Chateau Lake Louise, Alberta Continued Testosterone Regulation of Body Compositionin Male Dark-Eyed Juncos: Keep the Muscle, Lose the FatP. Deviche Institute of Arctic Biology, University of Alaska Fairbanks,
Fairbanks, AK 99775-7000, USA Previous studies found that the food intake, metabolic rate, and body mass and composition of small Passerine species change seasonally. In such species, plasma androgen concentrations also vary seasonally, but to what extent these hormonal variations are causally related to feeding and metabolism is not fully understood. Further, most studies on the role of androgens in small bird body composition have only examined effects of these steroids on lipids and not on other body components. To study these questions, male dark-eyed juncos (Junco hyemalis) held on short days in the fall received either testosterone-filled (T; n=24) Silastic capsules to induce physiologically high plasma levels of the steroid or empty (C; n=24) capsules. Food consumption, body mass, and fat score were monitored for all males for the following 30 days. Birds were then sacrificed, and carcasses were analyzed for their composition in total lipids, water, and proteins. During the treatment period, T and C birds ate similar amounts of food. At the end of the study, T males had a lower body mass than C males. T birds also had smaller lipid reserves and to a small extent less body water than C birds, but the two groups did not differ with respect to their body protein content. Thus, T treatment-induced body mass loss was not a consequence of decreased food intake, and this loss was component-specific, being accounted for mostly by decreased lipid reserves. Based on previous studies, it appears that the main mechanism underlying the effects of T administration on body mass and fat reserves may consist of steroid-enhanced active (namely, behavioral) metabolic expenditure. (Supported by NSF Award BNS-9121258) Effects of P450arom Inhibitor on mRNA Expression of P450 C17 and P450arom in the Gonad of the Chicken Embryo: in situ Hybridization AnalysisH. Nishikimi, K. Yoshida, N. Saito, K. Shimada Laboratory of Animal Physiology, School of Agricultural
Sciences, Nagoya University, Chikusa, Nagoya 464-01, Japan Sexual differentiation is influenced by sex steroid hormones. Since cytochrome P450aromatase (P450arom) is a key enzyme converting androgen to estrogen, it is of significance to study mRNA expression of cytochrome P45017a hydroxylase (P450c17) and P450arom in normal chicken embryos and to examine effects of P450arom inhibition (AI) on their expression. This study reports that P450arom mRNA was first detected at day 6.5 of incubation only in genetic females, whereas P450c17 mRNA was first detected at day 5 of incubation in both sexes. AI injection into eggs at day 3 of incubation markedly reduced the expression of both mRNAs in the gonad of the female embryo at day 8 of incubation. Since AI treatment caused 50% of the females to undergo sexreversal when examined at day 18, expression of P450arom gene with its resultant estrogen production may play a role in sexual differentiation and ovarian development in genetically female chickens. Avian Thyroidal Superoxide Radical and Superoxide Dismutase System: Role and Thyrotrophic Regulation with Reference to Iodine MetabolismP. Prakash, P. Kumar G1, M. Laloraya1, M.S. Parihar2 Govt. College Chachaura-Binaganj, Guna, (M.P.) India; 1The
Population Council, Centre for Biomedical Research 1230 York
Avenue, New York, 10021, USA; 2School of Studies in Zoology,
Biochemistry Division, Vikram University, Ujjain (M.P.), 456010,
India Spin trap studies, using EPR spectroscopy, showed that the
thyroid gland of the Indian rock pigeon Columba livia
intermedia generates superoxide anion (O2·) radicals and
its enzymic dismutation by superoxide dismutase (SOD) serves as
an alternative hydrogen peroxide (H2O2) generating system to
drive the H2O2 dependent and peroxidase mediated organic iodine
biosynthesis. The O2· radical also mediates oxidative
activation of thyroidal iodine to its free radical form, 1·.
Some important findings of our study are: Superoxide Radical Induced Peroxidative Alteration of Avian Thyroid Cell Membrane Fluidity and Molecular Order of Phospholipid Bilayer Under Melatonin Implantation: a Spintrap and Spin-Label Study by EPR SpectroscopyP. Prakash, P.G. Kumar1, M. Laloraya1, M.S. Parihar2 Govt. College Chachaura-Binaganj, Guna, (M.P.) India; 1The
Population Council, Centre for Biomedical Research 1230 York
Avenue, New York, 10021, USA; 2School of Studies in Zoology,
Biochemistry Division, Vikram University, Ujjain (M.P.), 456010,
India We investigated the effect of subcutaneous implants of
melatonin, on O2· /SOD activity, lipid peroxidation, membrane
fluidity and phospholipid molecular order in the thyroid glands
of Columbia livia intermedia. We used spin trap PBN
(N-t-butyl-alpha-phenylnitrone) for O2· trapping and to probe
biophysical status. 16-doxyl stearate was used as the spinlabel
during EPR spectroscopy. After two weeks of melatonin
implantation:- Angiotensin II Receptors in Avian Adrenocortical Cells: Sorting Out their Signals and FunctionR.V. Carsia1, K. Boesze-Battaglia2, J.F. Kocsis1, P.J. McIlroy3, R.J. Schimmel1, K.I. Tilly4, J.L. Tilly4 Departments of 1Cell Biology and 2Molecular Biology,
UMDNJ-School of Osteopathic Medicine, Stratford, NJ 08084, USA;
3Department of Biology, CCAS-Rutgers University, Camden, NJ
08102, USA; 4Vincent Center for Reproductive Biology, Department
of Obstetrics and Gynecology, Massachussetts General
Hospital/Harvard Medical School, Boston, MA 02114, USA In mammalian adrenocortical cells (ACs), diverse signals elicited by angiotensin II (AII) regulate growth, differentiation and steroidogenesis. How signal information is partitioned for these various functions is unclear. In this presentation, we provide evidence that avian (turkey) ACs possess at least two AII receptor subtypes (distinct gene products). These receptors exhibit subtly different pharmacological and physicochemical properties. In addition, they appear to be coupled to disparate signal transduction cascades. One receptor, that is activated by native [Ile5]/[Val5]AII, is relatively insensitive to extracellular K+ ([K+]ex) and is coupled to increases in intracellular Ca2+ ([Ca2+]i). However, this receptor is not linked to aldosterone production (ALDO) since the AII analog, [Sar1, Ile8]AII, induces ALDO without increasing [Ca2+]i and blocks the AIIinduced rise in [Ca2+]i without altering ALDO. This [Ca2+]ilinked receptor may regulate AC survival and differentiation. By contrast, there appears to be another receptor that exhibits a dependency on [K+]ex and that when activated results in ALDO. There is some evidence suggesting that this molecularly distinct receptor is coupled to a phospholipase A2arachidonic acid pathway. In addition, since [K+]ex does not alter [Ca2+]i in turkey ACs, this novel receptor may have intrinsic depolarization or K+ sensor properties or alternatively, is linked to a distinct K+ sensor (receptor). (Supported by USDA 95372062279) Molecular Biology of Avian LH and FSH ReceptorsD.N. Foster, S. You, M.E. El Halawani Department of Animal Science, University of Minnesota, St.
Paul, MN 55108, USA We have used mammalian consensus sequence PCR primers, chicken and turkey genomic DNA, ovarian RNA, and PCR/RT-PCR to amplify both chicken and turkey DNA fragments (592 bp) specific for the transmembrane spanning (TM) region of both LH receptor (LHR) and FSH receptor (FSHR). The chicken LHR fragment was used to probe a chicken ovary cDNA library (a gift from Dr. Pat Johnson, Cornell University) to isolate a 1.8 kb LHR cDNA (which lacks 160 amino acids of the coding region). Based on chicken sequence data we were able to use avian specific primers and RT-PCR to amplify a 1.3 kb turkey LHR fragment that contains 0.8 kb of the TM domain plus 0.5kb of the extracellular domain (EX). Northern blot analysis revealed two transcripts (3.0 and 1.4 kb) that hybridized to stromal RNA when both TM and EX probes were used but only a single 3.0 kb transcript that hybridized to the TM probe, suggesting that a truncated isoform of LHR is present which lacks the transmembrane domain. Such a truncated avian LHR isoform could be potentially important in the regulation of LH binding to its membrane bound receptor and could be a factor in regulating ovulation. We have used similar approaches to clone FSHR cDNAs which are being analyzed. Quantitative RT-PCR demonstrated that steady-state LHR mRNA levels gradually increased during follicular development, both in theca and granulosa layers, and high levels were found in post-ovulatory and regressed follicles. FSHR mRNA levels remained relatively constant during follicular development, with expression in post-ovulatory and regressed follicles. Ca2+ Signalling in Avian Granulosa Cells during Ovarian Follicular DevelopmentB.K. Tsang, J.a. Soboloff, M.G. Wade, a.Sorisky, M. Désilets Reproductive Biology Unit, Departments of Obstetrics and
Gynaecology and Physiology, University of Ottawa and Loeb Medical
Research Institute, Ottawa, ON K1Y 4E9 Canada Although gonadotropins have long been recognized as the primary regulators of ovarian function, current research has emphasized the importance of paracrine and autocrine regulation by intraovarian factors. Neurotransmitters (e.g. Ach) and cytokines (e.g. TNFa) are believed to regulate follicular development, atresia and ovulation. Carbachol (a muscarinic agonist, Cch) induced transient increases in intracellular Ca2+ concentration ([Ca2+]I) in hen granulosa cells throughout follicular development, although the percentage of responding cells, magnitude of [Ca2+]I increase and maximal rate of rise of [Ca2+]I increases significantly between F3 and F1, but not F5,6 and F3, developmental stages. Fast Cch-induced Ca2+ transients, observed primarily in F1 cells, were characterized by a rapid and high peak followed by a secondary elevation superimposed with minor oscillations in [Ca2+]I; slow Cch-induced Ca2+ transients, observed primarily in F3 and F5,6 cells, were characterized by relatively small and slow changes of [Ca2+]I. There was a change in the primary source of Ca2+ from extracellular to intracellular stores during follicular development. The production of inositol trisphosphate was elevated in F1 and F5,6 granulosa cells after Ach or Cch treatment, although to a lesser extent in the less differentiated cells. TNFa, on the other hand, induced a small and delayed Ca2+ channel-mediated Ca2+ transient, with significantly higher percentage of responding cells in the latter cell type. TNFa also reversibly increased the magnitude of the Cch response in cells previously incapable of producing large Cch-induced Ca2+ transients. The cytokine also decreased sphingomyelin levels and increased ceramide concentration, while exogenous sphingomyelinase and sphingosine induced Ca2+ transients in granulosa cells. These studies demonstrate a complex, follicular stage-dependent interaction between TNFa and cholinergic input in the control of the Ca2+ signalling pathway for the regulation of granulosa cell function. (Supported by MRC and NSERC, Canada) Ion Channel Activities Underlying Avian Granulosa Cell Excitability and SecretionP. Morley, G. Mealing, J.F. Whitfield Institute for Biological Sciences, National Research Council
of Canada, Ottawa, ON K1A 0R6 Canada It has been proposed that LH-triggered [Ca2+]i surges stimulate progesterone production in chicken granulosa cells. However, Fura-2 fluorescence measurements of [Ca2+]i have shown that agonists which stimulate Ca2+ release from internal cellular stores and/or Ca2+ entry into granulosa cells do not stimulate progesterone production. It now appears that LH stimulates steroidogenesis through a Ca2+-modulated cAMP-triggered (but not a Ca2+-triggered) mechanism. However, granulosa cells, secrete other non-steroidal products needed for follicular maturation and do so by exocytosis, a Ca2+-dependent process. Indeed, preliminary results shows that extracellular ATP triggers [Ca2+]i oscillations, resulting from the release of Ca2+ from internal stores and Ca2+ influx, rapidly (30 seconds) increasing the release of S35-methionine labelled products from granulosa cells. It is well established that electrical activity can modulate endocrine cell secretory activity. Patch-clamp studies have demonstrated that chicken granulosa cells are excitable, i.e., they can generate action potentials (APs) either spontaneously or in response to depolarizing current pulses. The APs are due to the interaction of a fast-inactivating inward depolarizing Ca2+ current, a slow Ca2+-dependent inward Cl- current, and a delayed outward hyperpolarizing K+ current. How these APs are triggered physiologically, and their involvement in granulosa cell steroidogenesis or the production and secretion of other non-steroidal products remains to be established. Intracellular Mechanism of GH ActionJ. Burnside Department of Animal and Food Sciences, University of
Delaware, Newark, DE 19717-1303, USA The growth hormone (GH) receptor (GHR) is a member of a large family of membrane receptors that includes receptors for various cytokines (i.e., the interleukins and interferons) and hormones (i.e., erythropoietin and prolactin). The members of this family share both structural features and newly-identified common signal transduction pathways. This signaling pathway involves association with various members of the Janus family of tyrosine kinases (JAK), followed by activation of one or more of a family of latent transcription factors. An important challenge is understanding how a large spectrum of ligands can achieve specificity through activation of relatively small families of intracellular signaling proteins. Our approach to gain a better understanding of the specificity of GH signaling and the mechanism of GH action is to identify GH-regulated genes and the promoter elements that respond to GH. We have used the mRNA differential display technique to compare gene expression in the liver of normal and GHR-deficient sex-linked dwarf chickens. We have identified several genes which are expressed at higher levels in normal chicken liver, compared to dwarf liver, and thus are candidates for GH-regulated genes. We have isolated cDNA and genomic clones of one differentially expressed gene that shows significant sequence similarity to a family of cysteine protease inhibitors. The developmental expression of this gene parallels the plasma GH profile. A portion of the promoter shows GH dependent expression of a reporter construct transfected into primary hepatocytes. Using this approach, we will be able to identify not only genes which are regulated by GH, but the cis-active elements that bind the specific transcription factors involved in GH-specific gene activation. Growth Hormone Receptors in the Avian Immune SystemK. Hull, S. Harvey Department of Physiology, University of Alberta, Edmonton, AB
T6G 2H7, Canada It is well established that the activity and proliferation of lymphoid cells and lymphoid organs are stimulated by growth hormone. These actions on lymphoid cells may be direct or mediated by actions on the epithelial and non-immune tissue cells that regulate immune function. The occurrence and cellular localization of growth hormone receptors in immune tissues has therefore been investigated to determine the target sites of growth hormone action. Growth hormone receptor mRNA was first detected by Northern blotting in the spleen, bursa of Fabricius (bursa) and thymus of domestic fowl. In addition to the 4.4 kb transcript thought to encode the full-length growth hormone receptor, smaller transcripts of 2.8 kb and 1.0 kb were also occasionally observed that may encode growth hormone binding proteins. The abundance of the 4.4 kb transcript in the thymus was directly related to GH status, as it was increased by GH administration and decreased by GH immunoneutralization. Further analysis using the polymerase chain reaction revealed that mRNA sequences encoding the extracellular and intracellular domains of the growth hormone receptor were present in all tissues and highly homologous with hepatic transcripts. Translation of these transcripts also occurs in immune tissues, since immunoreactive growth hormone binding proteins or growth hormone receptors of approximately 56 kDa were detected in hepatic, splenic, thymic and bursal extracts. Immunocytochemistry of these tissues subsequently revealed that macrophages probably contain the bulk of this immunoreactivity, although some thymic medullary epithelial cells (including Hassal_s corpuscles), and splenic ellipsoids and interdigitating cells were also immunoreactive. This immunoreactivity is present in immune tissues of newly-hatched and adult chickens. Importantly, B-lymphocytes were rarely, if ever, immunoreactive, and T-lymphocytes containing growth hormone receptors or binding proteins were not observed. These results suggest that a number of primary (thymus and bursa) and secondary (spleen) lymphoid tissues in the chicken contain growth hormone receptors and are thus target sites for growth hormone action. The distribution of growth hormone receptor/growth hormone binding protein immunoreactivity in these tissues would further suggest that growth hormone plays a major role in macrophage proliferation and/or activity and may indirectly affect lymphocyte maturation and storage via effects on thymic and splenic stromal cells. (Supported by NSERC of Canada) Thyroid Hormones are Involved in Insulin-like Growth Factor (IGF)-I Production by Regulating Growth Hormone Receptor (GHR) in the ChickenA. Tsukada, T. Ohkubo1, K. Sakaguchi2, M. Tanaka2, K. Nakashima2, K. Hayashida, M. Wakita, S. Hoshino Department of Animal Science, Faculty of Bioresources; 1Center
for Molecular Genetics; 2Department of Biochemistry, Faculty of
Medicine, Mie University, Tsu, Mie 514, Japan To investigate the effect of thyroid states on IGF-I production in growing chickens, 5-week-old White Leghorn cockerels were fed 0.1% propylthiouracil (PTU) mixed feed or received sc thyroxine (T) injection (100 µg/kg). The PTU feed was fed for 8 days and T was given daily for 4 days from 5th to 8th day of the treatment. All birds were killed on the 9th to obtain tissues and blood. Concentrations of serum hormones were determined by appropriate radioimmunoassays. The expression of GHRmRNA and IGF-1mRNA in tissues was estimated by Northern blot hybridization and RNase protection assays, respectively. Based on thyroid weight and serum thyroid hormone levels, PTU clearly induced hypothyroidism, which was completely restored by T injection. Concentrations of serum GH were not affected by PTU or T treatments, whereas those of serum IGF-I significantly decreased in PTU-fed group as compared with the control and restored to control levels by T injection. Both GHRmRNA and IGF-I mRNA expressions were significantly reduced with PTU treatment in the liver, and restored by T. In addition, the results of labelled GH binding to the liver membrane were consistent with those of GHRmRNA expression in the liver. These results indicate that thyroid hormones are regulating IGF-I production in the liver by affecting GHR expression. Molecular Cloning, Tissue Distribution and Expression during Various Reproductive States of the Prolactin Receptor in TurkeysJ.R. Zhou, D. Zadworny, D. Guemene1, U. Kuhnlein Department of Animal Science, McGill University, Montreal, PQ
H9X 3V9 Canada; 1Station de Recherches Avicoles, INRA, 37380
Nouzilly, France The cDNA coding for the prolactin receptor (tPRLR) in turkeys was cloned and sequenced (2723 nt) and the ORF predicted an 831 amino acid protein with a structure similar to chicken and pigeon PRLR. The tPRLR mRNA was identified by Northern blot analysis to be a single transcript with a molecular size of about 3 kb. Transcripts were detected in all 26 tissues examined using RT PCR with the highest levels of receptor in the pituitary gland, hypothalamus, crop sac, duodenum and gizzard. The levels of expression of tPRLR in 17 tissues were compared using semi-quantitative RT PCR in non-photostimulated, laying, out-of-lay, incubating, maternal hens and male birds. In most tissues examined, there was no obvious relationship between blood levels of PRL, reproductive states and estimated concentrations of the receptor mRNA. In the pituitary gland and hypothalamus, plasma levels of PRL and levels of tPRLR transcript were inversely correlated. In the hypothalamus, increasing blood levels of PRL were associated with decreasing levels of the receptor transcript, whereas, the opposite was observed in the pituitary gland (P < 0.05). The presence of receptors in the hypothalamus suggests a short-loop feedback mechanism operates in the brain, whereas, receptors on the pituitary reveals a possible autocrine and/or paracrine role of PRL in the regulation of pituitary hormone secretion. These findings support the hypothesis that PRL, itself, may participate in the neuroendocrine control of incubation behaviour through actions on both the hypothalamus and pituitary gland. Distribution of Growth Hormone Receptors in Chicken LiverL. Vleurick, P.P. Van Veldhoven1, E. Decuypere, E.R. Kühn Leuven Poultry Research Group, Naamsestraat 61, B-3000 Leuven,
Belgium; 1Department of Pharmacology, Campus Gasthuisberg,
KULeuven, Belgium In rats, following growth hormone (GH) binding to the plasma membrane (PM) GH receptor (GHR), the GH-GHR complex is internalized and receptors are partly recycled, along with newly synthesized receptors, through the Golgi apparatus to the PM. To restore GHR levels at the cell surface after a plasma GH pulse, both PM and Golgi receptors undergo cycles that are in synchrony with GH pulsatility. To locate GHR on subcellular membranes of chicken liver, Golgi vesicles (GV) and PM fragments were isolated by differential centrifugation and flotation in discontinuous sucrose gradients. Binding of [125I]-labelled GH was measured in all fractions. The total binding assay was unreliable due to severe membrane protein loss during removal of endogenous GH by MgCl2. Binding to unoccupied receptors was proportional to membrane protein. Only in microsomes, purified PM and GV, specific binding exceeded non-specific binding. The distribution of free GHR and marker enzymes for PM (alkaline phosphatase, alkaline phosphodiesterase) and for GV (galactosyltransferase) were compared. Microsomes (the starting fraction for GV isolation) were enriched in all marker enzymes and in GHR. In the Golgi gradient, GHR peaked in the `light' and `intermediate' Golgi fraction. Galactosyltransferase was concentrated in the `intermediate' and `heavy' fraction and PM markers did not float beyond the `heavy' GV. Purified PM was highly enriched in PM markers, but, to a lesser extent, also in Golgi marker. The concentration of (free) GHR was higher in GV than in PM. Due to Golgi contamination of the PM fraction, the real GHR level in the PM may be even smaller. We can therefore hypothesize that the majority of GHR are located in a Golgi pool from which they are transferred to the PM to resensitize the cell for the next GH pulse. Genetic Evidence for an Interaction between GH and GH-Receptor Genesin White LeghornsX.P. Feng, S. Joseph, S.E. Aggrey, J.S. Gavora1, R.W. Fairfull, D. Zadworny, U. Kuhnlein Department of Animal Science, Macdonald Campus of McGill
University, Ste. Anne de Bellevue, PQ Canada; 1Centre for Food
and Animal Research, Agriculture Canada, Ottawa, ON, Canada Two restriction fragment length polymorphisms (RFLPs), one in the GH and one in the GH-receptor gene, were determined in 360 chickens of an un-selected White Leghorn strain (effective population size >300) and analyzed for associations with egg production traits. Associations were significant (P<0.05) for body weight at 133 days of age, egg weight, feed consumption and feed efficiency for egg mass. A comparison of the different genotypic classes revealed that significant trait-associations of the GH genotype were only observed in the presence of one of the two GH-receptor RFLP alleles. The favourable GH allele was mostly dominant and accounted for a difference of about 10% of the trait values. The result indicates that (1) there are variants of the genes of the GH-axis which affect egg production traits in White Leghorns and (2) the effect of a genetic variation in one gene may depend on the variation in another gene. A Missense Mutation in the Cornell Sex-Linked Dwarf (SLD) Growth Hormone Receptor Gene does not Prevent Plasma GH-BindingK.L. Hull, W.C.J. Janssens, S. Harvey Department of Physiology, University of Alberta, Edmonton, AB
T6G 2H7 Canada Sex-linked dwarfism (SLD) in chickens is characterised by impaired growth despite normal or supranormal plasma growth hormone (GH) levels. This resistance to GH action is primarily thought to be due to mutations of the GH receptor (GHR) gene (major gene deletions, abnormal splicing, and missense mutations) that reduce or prevent GH binding to target sites. The etiology of the GH resistance in the Cornell SLD is, however, unknown. Previous studies have demonstrated that normally bioactive GH but abnormally low levels of hepatic GH-binding activity are present in these birds, and that the GHR gene is transcribed into a transcript of appropriate size and abundance. These transcripts were partially sequenced, and no deletions or point mutations were detected. A point mutation in non-sequenced regions or a defect in translation could, however, result in impaired receptor activity in the Cornell SLD. This possibility was addressed in the present study, in which additional regions of the SLD GHR mRNA were sequenced and GHR-like proteins were identified by Western analysis. A missense mutation was identified in the region of the GHR cDNA encoding the extracellular domain. This T-C transition at position 370 would result in the substitution of serine for the conserved phenylalanine at position 112. This mutant transcript was, however, translated, since hepatic proteins of 55 kDa and 100 kDa were detected in normally-growing (K) and SLD chickens by a polyclonal antibody raised against the extracellular domain of the chicken GHR. This antibody would detect full-length receptors as well as soluble GH-binding proteins (GHBPs), which are homologous to the extracellular domain of the GHR. Small proteins of 45 - 55 kDa were similarly detected in SLD and K serum by Western analysis, and hepatic and serum immunoreactivity was not detected if the antibody was replaced by preimmune serum. This serum immunoreactivity may putatively correspond to functional GHBPs, as proteins capable of binding radiolabelled GH were present in SLD serum. Preliminary results would suggest that a 55 kDa protein present in SLD and K serum (which may correspond to the GHR-like protein detected by Western Analysis) is partially responsible for this GH-binding activity. It is, however, possible that SLD serum contains large amounts of a GH-binding protein that is not homologous to the GHR, and that the GHR-like proteins detected by Western analysis are dysfunctional. (Supported by NSERC of Canada) |