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No description available.

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.

Horizontal step-ramp stimuli were used to examine gaze, eye, and head movement dynamics during head-unrestrained pursuit with and without imposed retinal velocity errors (RVE; i.e. open- and closed-loop, respectively) in two rhesus monkeys. In the closed-loop experiment , pursuit was elicited by step-ramp stimuli with a constant velocity of 20--80 deg/s. Each monkey used a combination of eye and head motion to initially fixate and then pursue the target. Additionally, we found that initial eye and head acceleration increased as a function of target velocity. In the open-loop experiment, step-ramp stimuli (40 deg/s) were presented and ~125 ms after pursuit onset, a constant RVE was imposed for a duration of 300 ms. In each monkey, when RVE = 0 deg/s, gaze, eye, and head velocity trajectories were maintained at their current or at a damped velocity. Moreover, the head as well as the eyes mediated the observed increase and decrease in gaze velocity when RVE was +10 and -10 deg/s, respectively. Based on our findings we conclude that the pursuit system uses visual and non-visual signals to drive coordinated eye-head pursuit.

Na+/H+ exchanger (NHE) is a major contributor in controlling intracellular pH. The activity of this protein is allosterically modified by intracellular H+. Histidine residues of the NHE that face the cytoplasm may be involved in determining the intracellular pH set point, with their state of protonation influencing the rate of Na +/H+ exchange. To test this hypothesis, histidine residues in the ubiquitously expressed NHE isoform (NHE1) that are relatively conserved amongst members of the NHE gene family were substituted by site-directed mutagenesis and the mutants were stably transfected into mammalian cells that are deficient in endogenous Na+/H + exchange activity. The pHi sensitivity of each mutant was evaluated by measuring the rate of 22Na + influx as a function of the intracellular H+ concentration. Mutation of the histidines located at the putative cytoplasmic face of the N-terminal transmembraneous domain of NHE1 did not show any significant effect on the pHi sensitivity of the protein. By contrast, substitution of histidines located in the C-terminal cytoplasmic tail activated the exchanger by increasing its sensitivity to H+. These mutants were no longer activated in response to protein kinase C, when compared to wild type. Taken together, these data support the hypothesis that some of the relatively conserved histidine residues in the C-terminal cytoplasmic tail of NHE1 may be involved in determining the pHi "threshold" or "set point" of the transporter.

Acylation stimulating protein (ASP or C3a desArg) is a complement derived product produced by adipocytes and is able to alter their metabolism, stimulating both triglyceride synthesis and glucose transport. This stimulatory activity has been shown to be due to ASP as both plasma derived protein and recombinant protein stimulate glucose transport and triglyceride synthesis. Furthermore, we demonstrated that ASP is functionally distinct from C3a. A two site model for ligand interaction with the receptor is proposed with the carboxy-terminal involved in receptor binding and the disulphide core in stimulating triglyceride synthesis. Functionality of ASP in vivo supported the hypothesis that ASP is involved in plasma triglyceride and glucose clearance postprandially after a fat load. The following data were obtained: (1) Administration of ASP and ASP functional knockouts displayed increased and delayed triglyceride clearance respectively. (2) Administered ASP decreased plasma glucose levels, which was independent from its effects on plasma triglycerides. (3) In ASP functional knockout mice gender differences of greater postprandial lipemia in males and more pronounced reductions of adipose tissue in females were observed. In conclusion the function of ASP was determined as well as structural regions involved. Furthermore, the in vivo physiology of ASP has been determined, with an effect on postprandial metabolism, regulation of adiposity and gender dependent penetrance of the ASP functional knockout phenotype.

This dissertation examines the ovarian development and function in genetically modified mice that lack FSH receptor (FSH-R) signaling. We propose that a complete or partial loss of FSH-R causes ovarian insufficiency resulting in estrogen deficiency and premature aging in female mice. / Targeted disruption of FSH-R caused a gene dose related endocrine and gametogenic abnormality in female mice. The resulting FOllitropin R&barbelow;eceptor K&barbelow;nockO&barbelow;ut (FORKO) mutants were acyclic and infertile due to ovulatory defects, even with very high levels of FSH. Lack of FSH-R signaling in females caused a severe ovarian underdevelopment, producing estrogen deficiency. As a consequence, the null mutants developed obesity and skeletal abnormalities. The expression of nuclear estrogen receptor(s) alpha and beta genes and the corresponding proteins in the ovary and uterus of FORKO mice were maintained intact, as estrogen administration induced uterine growth and decreased accumulation of the adipose tissue. By 12 months of age, 92% of FORKO animals developed ovarian neoplasms of sex cord-stromal type similar to pathology observed in women. Our results showed, for the first time, that the loss of the FSH-R signaling mechanisms predisposes the ovary to molecular and structural changes causing tumor formation. / In contrast to acyclic and infertile FORKO (-/-) females, a phenotype of FORKO mice with a partial (+/-) disruption of the receptor gene exhibits irregular cyclicity and reduced fertility, undergoing early reproductive senescence. Our findings also demonstrate that the loss of a single allele of the FSH receptor gene causes a premature exhaustion of gonadal reserves accompanied by age-related changes in the hypothalamic-pituitary axis. / The study concludes that the FSH receptor signaling offers a protective mechanism, which gradually weakens upon reproductive senescence (menopause in women); therefore this knockout constitutes a unique and promising animal model for studying the physiology and molecular mechanisms of gonadal receptors and hormones.