Free and microencapsulated hepatocytes in an alginate-polylysine-alginate artificial membrane (APA), were implanted intraperitoneally into the Gunn rat, the animal model for Crigler-Najjar syndrome, to reduce serum bilirubin level. Hepatocytes from guinea pigs, Wistar and Sprague-Dawley rats whether free or microencapsulated were equally effective in lowering serum bilirubin levels in the Gunn rat. Buffalo rat hepatocytes however, were immunorejected unless microencapsulated. Decrease in serum bilirubin was concomitant with the appearance of conjugated bilirubin in the bile of Gunn rats as demonstrated by HPLC analysis. Microcapsules containing guinea pig hepatocytes showed less fibrosis than microcapsules containing hepatocytes from different strains of rats and empty microcapsules. In the Gunn rat there is significant accumulation of bilirubin in various tissues which affects the net removal of bilirubin upon implantation of the encapsulated hepatocytes. This deposition increases with time and it is organ-dependent. The kinetic data of UDP-glucuronosyltransferase (UDPGT) indicated that it is a multisubunit enzyme in which there is cooperative binding of the substrate to the subunits. The binding of bilirubin showed positive cooperativity while the binding of UDPGA exhibited kinetics with mixed cooperativity. Encapsulated hepatocytes when incubated with bilirubin and UDP-glucuronic acid can form bilirubin conjugates. This was shown by HPLC analysis.The comparison of UDPGT activity between liver homogenate, intact hepatocytes and encapsulated hepatocytes showed that there is mass transfer resistance of the APA membrane.

In the rat, catecholamine conjugates, particularly dopamine conjugates, are widely distributed in cerebrospinal fluid, sympathetically innervated tissues, plasma, erythrocytes and urine. Sulfoconjugated dopamine, norepinephrine and epinephrine are detected in small quantities, whereas glucuronoconjugation seems to occur only in dopamine, and is by far the predominant form of dopamine conjugation. On the other hand, in man, sulfoconjugation is normally the sole form of catecholamine conjugation. Glucuronoconjugated dopamine constitutes the bulk not only of dopamine, but also of total catecholamines in rat cerebrospinal fluid, plasma and urine, which sharply contrasts with the composition of catecholamines in peripheral tissues where the bulk of total catecholamines is free norepinephrine (in sympathetically innervated tissues) or epinephrine (in adrenals). Sulfo- and glucuronoconjugated catecholamines in rat cerebrospinal fluid are central in origin, whereas the adrenal medulla and peripheral postganglionic sympathetic neurons are important sources of peripheral conjugated catecholamines. In response to stress, rat plasma free catecholamine surges occur with concomitant substantial falls in sulfoconjugated dopamine, norepinephrine and epinephrine as well as glucuronoconjugated dopamine. Under stress, a large portion of rat plasma conjugated dopamine, mainly glucuronoconjugated dopamine is not only deconjugated to free dopamine, but probably also converted to free norepinephrine and epinephrine. Adrenalectomy abolishes these normal stress-induced responses to plasma conjugated catecholamines in the rat. These findings led to the following conclusions. (1) Glucuronoconjugation is an important pathway in the metabolism of central and peripheral dopamine in the rat. (2) In the metabolism of catecholamines, conjugation is qualitatively distinct from O-methylation and deamination in that conjugation represents an intermediate and reversible process, whereas O-methylatio

The functions of somatostatin, norepinephrine and epinephrine in regulation of rhythmic growth hormone secretion were evaluated in male Sprague-Dawley rats. Three experimental strategies were used: (1) the chronic cannulation model to assess the effects of stress, lateral hypothalamic stimulation, monosodium glutamate, and pharmacologic agents on growth hormone dynamics, (2) biochemical mapping of central somatostatinergic pathways involved in growth hormone regulation, and (3) an in vitro perifusion system to study somatostatin release. / It is shown that exictatory and inhibitory neural inputs must be intact to maintain normal rhythmic growth hormone secretion. Growth hormone rises are generated by hypothalamic neurons that liberate a growth hormone releasing factor. These neurons are activated by adrenergic (and probably noradrenergic) inputs. The periventricular and amygdalofugal somatostatinergic systems control ebbs in plasma growth hormone and stress- or lateral hypothalamic stimulation-induced growth hormone suppression. Catecholaminergic regulation of somatostatin release is not defined clearly.

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Decorin is required for the normal fibrillogenesis and spatial arrangement of collagen. As collagen is important in determining the elastic behaviour of the lung, we hypothesized that lung tissue mechanics would be altered in decorin deficient (Dcn-/-) mice. Complex impedance, pressure-volume curves, and length-stress curves of lung parenchyma were measured in C57BL/6 mice, 6 Dcn-/- and 6 wildtype ( Dcn+/+), both in vivo and in vitro. Immunohistochemistry and Western blotting were performed to identify decorin and biglycan in the lung tissues. In vivo, airway resistance was decreased and lung compliance was increased in Dcn-/- mice. In vitro, length-stress curves showed increased compliance in the Dcn-/- mice. Immunohistochemistry showed decorin staining in the airway and vessel walls of Dcn+/+ but not Dcn-/- mice; Western blots showed that biglycan levels were not different in the Dcn-/- mice. These data support a critical role for decorin in the formation of the lung collagen network. Lack of decorin alters lung tissue mechanical behaviour. Additionally, the data from Dcn+/+ mice were compared to those from other species, and is consistent with the evidence in the literature that mouse lungs differ structurally from other species. Finally, differences observed in vivo vs. in vitro suggest that measurements made in the strip more accurately reflect lung tissue properties.

Sepsis-induced diaphragmatic force loss and failure are associated with an increased exposure to proinflammatory mediators. The septic diaphragm has recently been reported to overexpresse chemokines, including the CC chemokine MCP-1 (monocyte chemoattractant protein-1). This thesis seeks to address the significance of MCP-1 overproduction in diaphragm proinflammatory mediator expression and skeletal muscle contractile function. Neutralization of endogenous MCP-1, produced following administration of LPS, decreased transcription of iNOS, IL-6, IL-1alpha, IL-1beta and MCP-1 in the diaphragm and prevented a decrease in diaphragm force production. Furthermore, exogenous MCP-1 stimulated IL-6 and MCP-1 transcription in primary diaphragm myotubes, and injection of MCP-1 in the healthy EDL muscle led to contractile weakness. Taken together, these results suggest that increased MCP-1 production in the septic diaphragm stimulates proinflammatory mediator production by diaphragm myocytes, contributing to the muscle's contractile dysfunction.

Iron is not a trace element in mammalian physiology. Using textbook values for blood volume (5.5 L), red blood cell (RBC) count (5 million/muL), and a lifespan of 120 days for red blood cells, the equilibrium value for the erythrocyte generation/death rate in the average adult male human is over 2 million/sec. It follows that the amount of iron required for hemoglobin synthesis in one day amounts to about 25 mg. Virtually every atom of that 25 mg is recycled by macrophages of the reticuloendothelial system (RES) that provide iron to the plasma for its subsequent delivery back to the erythron (with a small fraction going to other tissues). In light of the certain toxicity of unprotected iron, both erythroid precursors and RES macrophages perform remarkable tasks in handling such copious amounts of the catalytic metal. In my studies, I have examined specific aspects of iron metabolism in these two tissues. / Iron is taken up by nearly every cell through a mechanism of receptor-mediated endocytosis, whereby the plasma iron binding protein transferrin (Tf) binds to its cognate receptor (TfR) on the cell surface, followed by internalization of the complex into a membrane bound organelle. Subsequent to endocytosis, the endosome is acidified by a v-ATPase proton pump, facilitating the release of iron from Tf. Through an unknown mechanism, iron is targeted to the inner membrane of the mitochondria, where the enzyme that inserts Fe2+ into protoporphyrin IX, ferrochelatase, resides. Although it has been demonstrated that the divalent metal transporter, DMT1, is responsible for the egress of reduced Fe from the vesicle, the immediate fate of the iron atoms after their transport across the vesicular membrane remains unknown. Therefore, we have investigated the uptake of iron in reticulocytes, cells that are taking up large amounts of iron for the synthesis of hemoglobin. Through both biochemical and imaging techniques, we have demonstrated that iron is transferred via a direct interorganellar relation between the endosome and mitochondria. / The "haemoglobin-deficit" (hbd) mouse has an erythroid-specific mutation which is responsible for its microcytic, hypochromic phenotype. Previous studies have shown that these mice have normal dietary iron acquisition and normal to elevated serum iron levels. We therefore investigated the handling of iron in reticulocytes from these animals to determine whether the mutated gene possibly plays a role in the trafficking of transferrin-iron-containing organelles. A systematic examination of the steps in the transferrin pathway revealed that the intracellular trafficking of the protein is compromised in the hbd mice. / The rapid turnover of iron by macrophages of the RES requires heme oxygenase-1 (HO-1), which catalyzes the rate-limiting step in heme degradation. This highly inducible enzyme, besides its major role in erythrocyte iron recycling, has been demonstrated to confer astonishing cytoprotectivity to cells and tissues in which its expression is elevated (either through chemical induction or genetic manipulation). In addition to, reportedly protective, carbon monoxide and biliverdin, the HO-1 catalyzed reaction releases ferrous iron, which itself is a potent pro-oxidant. Also, it is unlikely that there exists a significant amount of free heme in most tissues (i.e., non-erythroid, non-erythrophagocytic), to provide significant amounts of substrate to this enzyme. Hence, it is tempting to speculate that the mechanism of heme oxygenase cytoprotection is removed from its function of heme catabolism. Therefore, we investigated whether increased expression of heme oxygenase will, in and of itself, alter iron metabolism in cultured cells. My experiments show that in the absence of exogenous hemin, elevation of HO-1 protein levels does not have any effect on cellular iron metabolism in cultured cells.