Although 24R,25(OH)2D3 has been implicated in bone development, its biological role and mechanism of action remain controversial. In search for evidence of a receptor, nuclear and cytosol extracts were isolated from mandibles and calvaria of E17.5 mice. Competition and saturation analysis identified a saturable, specific and high affinity (Kd=1.1nM) 24R,25(OH) 2D3 binding-protein. The results of these and sucrose sedimentation studies indicate that this protein is not vitamin D receptor (VDR) or vitamin D binding protein (DBP). Tissue specificity experiments suggest that this putative receptor is also present in liver but not brain. / pBDGal4-hRXRalpha bait was used to screen neonate and embryonal mandible/calvaria cDNA libraries using the yeast two-hybrid system. PCR screening was also performed using primers from the zinc-finger region of the VDR. To date no positive clones have been identified. Isolation of this putative receptor will provide valuable insight into the mechanism of this metabolite's role in bone development.

We report that the chitinase-like protein, human cartilage glycoprotein 39 (HC-gp39) is a growth and survival factor. It promotes connective tissue cell survival and growth, and counteracts catabolic processes. Dose-dependent growth stimulation was observed when human synoviocytes and fibroblasts were exposed to HC-gp39. Both the ERK1/2 MAP kinase and the PI3 kinase pathways were activated by HC-gp39. Thus HC-gp39 elicits a signaling cascade leading to increased connective tissue cell proliferation, suggesting that HC-gp39 may play a major role in the pathological conditions leading to tissue fibrosis. In addition to acting as a mitogen, HC-gp39 also reduces stress-induced apoptosis in human connective tissue cells. HC-gp39 significantly inhibited H2 O2 activation of SAPK/JNK and p38 in chondrocytes. A reduction of caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage and apoptotic cell number was also seen. These results suggest that HC-gp39 plays an important role in the survival of connective tissue cells. The protective effect is further illustrated by the ability of HC-gp39 to reduce cellular responses to inflammatory cytokines. IL-1 or TNF-alpha stimulated phosphorylation of SAPK/JNK and p38 were greatly reduced by HC-gp39. Signalling through the NF-kappab pathway was unaffected. These actions resulted in a significant reduction of MMP1, MMP3, MMP13 and IL-8 production, suggesting that HC-gp39 can participate in the catabolic aspects of tissue remodelling as a repair or protecting factor counteracting the catabolic processes. Although HC-gp39 affected the response to IL-1 and TNF-alpha, these cytokines had no effect on the high expression level of HC-gp39 in chondrocytes cultures. However, inhibition of the NF-B signalling pathway, which has been implicated as a major effectory mechanism for TNF-alpha, significantly decreased HC-gp39 protein and mRNA levels in a concentration and time dependent manner. Increased NF-kappaB / Therefore, this work demonstrated that expression of HC-gp39 at inflammation sites is part of the reaction chain in the pathogenesis of joint degeneration and/or inflammation. The activity of the elements regulating the expression of HC-gp39 suggests that it could serve as a negative feedback regulator for the inflammatory cytokines.

Cardiac action potential (AP) properties are not uniform because of functional specialization in different regions. The thesis of these studies was that important aspects of regional cardiac electrical function in health and disease are determined by heterogeneous ionic current expression. / The inward rectifier current, IK1, responsible for the resting membrane potential (RMP) and late repolarization, is greater in ventricle than atrium. Atrial-ventricular IKI differences result in important electrophysiological differences between atrium and ventricle. To gain insight into the mechanisms of this IK1 variation, we compared the expression and distribution of inward rectifier subunits Kir2.1/2.3, believed to underlie IK1, in both regions. Ventricles had greater Kir2.1 and lower Kir2.3 expression than atrium. The subcellular distributions of these subunits also differed between the two regions. Our findings suggest that, in addition to the intensity of subunit expression, cellular subunit location affects local channel function. / To further examine the effects of channel subunit expression and distribution on regional electrophysiology, we studied the pulmonary vein (PV) myocardial sleeve. PVs are important in atrial fibrillation (AF) initiation and maintenance and exhibit unique current profiles compared with adjacent atrial cardiomyocytes. Our findings demonstrate a greater PV expression of ERG and KvLQT1---the channel subunits that underlie the rapid and slow components of the delayed rectifier current, IK, respectively. KvLQT1 was diffusely distributed in atrial cardiomyocytes, in contrast to the transverse tubular (TT) and intercalated disk (ID) PV distribution. Kir2.3 abundance was similar in both regions, but differently distributed with a TT and ID LA distribution and ID localization in PVs. Observed expression and distribution differences between atrial and PV cardiomyocytes may account for the smaller Vmax, less negative RMP and shorter AP duration in PVs, which contribute to their arrhythmic diathesis. / The PVs are thought to act as fibrillatory foci in congestive heart failure (CHF). We compared channel subunit expression in PV cardiomyocytes from (rapid ventricular pacing induced) CHF and control hearts. CHF decreased KvLQT1 expression, increased expression of ERG and the sodium calcium exchange subunit, NCX, and altered the subcellular KvLQT1 distribution. (Abstract shortened by UMI.)

The present study was designed to further characterize the role of insulin and glucagon in the regulation of glucose fluxes during two hours of mild to moderate intensity exercise in postabsorptive healthy male subjects. Endogenous insulin and glucagon were suppressed by somatostatin (SRIF) infusion and the pancreatic hormones were then replaced singly or in combination to match the hormonal concentrations observed during similar exercise in control subjects. Glucose kinetics were determined using stable isotopes of glucose. The exercise protocol was sufficient to induce a 26% decrease in insulin and a 20% increase in glucagon. In all conditions, there were a 1.5- and 2-fold increase in glucose utilization and in glucose metabolic clearance rate, respectively when compared to the resting levels. Exercise alone was able to maintain the increase in glucose metabolic clearance rate despite a deficiency in insulin. Hepatic glucose production and gluconeogenesis increased to values representing 228 and 144% of the resting level, respectively. In the absence of glucagon, these increases during exercise were totally abolished. From our observations, it is concluded that during mild to moderate intensity exercise insulin was not essential for glucose uptake indicating that muscle contractions per se can stimulate glucose uptake by muscles and the increase in hepatic glucose production and gluconeogenesis are essentially dependent on the increase in glucagon. Insulin remains important, however, for maximal glucose uptake by contracting muscles and for preventing glucose overproduction and possibly hyperglycemia.

Quadriceps, muscle endurance, fatigue and recovery were evaluated in elite and novice rowers in order to elucidate the mechanisms underlying the development and recovery from fatigue following several bouts of exercise. Baseline evaluations recorded the maximum voluntary contraction (MVC) using a force transducer, muscle activation via the Interpolated Twitch Technique (ITT) and the compound muscle action potential (m-wave) from the electromyographic (EMG) signal. Contractile properties of the twitch, which included the peak tension (Pt), time to peak tension (TPT) and half-relaxation time (RT 1/2), were also recorded. The rowers then completed a 2500 meter simulated rowing race and the muscle was re-evaluated. Two successive standard fatigue protocols (SFP) followed with subsequent evaluations taking place after each SFP. The successive bouts of exercise caused the MVC to drop considerably. Results revealed that excitation-contraction (E-C) uncoupling was the major factor behind the development of fatigue in both groups due to the differential effects of fatigue and recovery on Pt versus MVC. Activation levels dropped minimally and m-wave amplitudes were unchanged from pre-fatigue levels. Elite rowers' quadriceps muscles demonstrated superior endurance during both SFPs, but to a lesser extent following the second SFP. These results suggest that possible metabolic differences in the environment of the muscle may lead to increased resistance to local muscle fatigue in elite rowers. This research study contributes to the current understanding of neuromuscular fatigue and adds to the physiological profile of athletes who participate in the sport of rowing.

Neurogenic impotence is a clinical situation commonly resulting from major pelvic surgery. A detailed description of the neural pathways mediating erection has been lacking and this has been implicated as a major cause for the high incidence of post-operative impotence. Nitric oxide (NO) is a gaseous molecule believed to be an important mediator of erection. The synthesis of nitric oxide is regulated by the enzyme nitric oxide synthase. / In the first part of this work, we studied the neuroanatomical pathways responsible for erection in the male lower urogenital tract. Using a histochemical staining technique able to localize a co-factor of nitric oxide synthase, NADPH, we traced the course of the cavernous nerve and its microscopic branch fibers from the pelvic plexus distally until the penile crura. Studies in a rat model and in man demonstrated multiple small nitric oxide releasing nerve fibers located on the anterior and lateral surfaces of the prostatic capsule. The physiologic importance of these fibers was shown in a rat model. Transection of these fibers resulted in a significant loss of intracavernosal pressure rise following cavernous nerve stimulation. / The nitric oxide synthase enzyme is found in three isoforms: neuronal, endothelial, and inducible forms. The neuronal form is believed to be important in the neural regulation of erection as has been demonstrated in histological studies. Using a specific inhibitor of the neuronal form of nitric oxide synthase, 7-nitroindazole, we evaluated and showed the importance of the neuronal form in regulating erection in an animal model.

Skeletal muscle fatigue and insufficient power output were two biological constraints impeding the application of this endogenous energy source for support of the failing ventricle. Our ability to manipulate muscle phenotypic expression by a transforming and continuous low frequency electrical stimulation and the development of synchronizable pulse-train stimulators have overcome these historical obstacles. / This thesis addresses the hypothesis that fatigue-resistant transformed skeletal muscle can support the failing circulation. The work is divided into two major sections involving animal laboratory and clinical human experimentation. / In acute dog models of heart failure we have demonstrated the feasibility of transformed latissimus dorsi muscle assisting the heart in various configurations: (1) to power a potentially implantable pericardiovascular mechanical assist device and (2) in hybrid union for biventricular failure with cardiomyoplasty and mechanical support for the right and left ventricles respectively. / In another series of experiments using coronary sinus drainage as a surrogate marker for epicardial coronary flow we did not witness any compromise of epicardial coronary flow by the overlying muscle graft. / In our clinical series of 5 patients with chronic heart failure, who were all rejected for transplantation and underwent dynamic cardiomyoplasty, our follow-up investigations have revealed insights into appropriate patient selection criteria and the potential underlying pathophysiologic mechanisms of this new surgical technique in the palliation of refractory heart failure.

Female Sexual Dysfunction (FSD) is a significant problem affecting 25%--63% women in the U.S. alone. Although the research community has begun to increase efforts to understand FSD, there is a lack of an animal model in which this condition can be studied. The current research provides data for a new model of sexual arousal in the female Sprague-Dawley rat. The vascular effects in the clitoris and vagina in response to clitoral and pelvic plexus nerve stimulations were studied. Modulation of these effects by infusion of Nitric Oxide, Vasoactive Intestinal Peptide and Calcitonin-Gene Related Peptide agonists and antagonist in the clitoris was also observed. Histological sections of the clitoris were examined for the endogenous presence of these neurotransmitters. These data provide the framework for a reproducible model of sexual arousal in the female rat.

Liver xenotransplantation could eliminate the liver donor shortage, but currently it is not possible because of a lack of understanding of liver xenograft rejection. Hyperacute xenograft rejection is initiated by the binding of preformed naturally occurring xenoreactive antibodies (XNA) to the xenograft endothelium. The XNA bind to the xenograft endothelium, leading to complement-mediated endothelial injury. / Liver xenotransplantation will be initially offered to patients with severe liver failure as a bridge to a human liver transplantation. The hypothesis tested in this thesis is that hyperacute rejection of liver xenografts placed into recipients with liver failure will be diminished because of the complement deficiency that accompanies liver failure. The experiments described in this thesis detail the development of an in vitro pig-to-human liver xenotransplant model incubating cultured pig hepatic endothelial cells (PHEC) and human serum in culture. We showed that either classical or alternative complement pathways could initiate endothelial injury. Next we developed the dog-to-pig liver xenograft model and characterized the lethal coagulopathy that results from hyperacute rejection. The coagulopathy results from the lack of function of platelets as well as their disappearance from the circulation. We then used the galactosamine induced liver injury model in porcine recipients of canine liver xenografts to demonstrate that hyperacute rejection in the setting of liver failure is diminished. We showed that; tissue injury, coagulopathy and platelet defect, and endothelial injury were diminished. Our experiments suggested that the cause of the decreased injury was the lack of complement in the pigs with galactosamine induced liver injury since the XNA levels were no different than in control animals. Our final experiments evaluated serum from patients with liver failure and compared the injury caused by incubation with PHEC. Serum from the liver failure patients had similar levels of XNA when compared with normal subjects, but had less complement activity, and less C3 and C4. Incubation of liver failure serum with PHEC caused much less injury and complement activation than serum from control subjects. The results in this thesis suggest that liver failure will have a significant impact on liver xenograft rejection, helping to diminish hyperacut

Exposure of males to drugs or environmental chemicals can alter reproductive health and affect progeny outcome. The quality of the male germ cell is determined in part by the presence and function of stress response mechanisms. During spermatogenesis, different germ cell types display differential susceptibility to toxicants, suggesting there is variation in the presence and function of stress response mechanisms. Exposure of male rats to cyclophosphamide, a commonly used anticancer and immunosuppressive drug, alters male fertility and progeny outcome in a male-germ cell phase specific manner. The hypothesis of this thesis is that stress response mechanisms are differentially expressed during rat spermatogenesis and that their expression is involved in the response to the alkylating agent cyclophosphamide. The first objective of this thesis was to elucidate the expression of stress response genes at different stages of male germ cell development. Using cDNA arrays, the expression of several gene families was profiled in rat pachytene spermatocytes, round spermatids and elongating spermatids. Stress response genes were found to be differentially expressed during spermatogenesis. The second objective was to determine whether the expression of stress response genes was modified by exposure to cyclophosphamide. Acute and chronic cyclophosphamide treatment altered gene expression in a cell- and treatment-specific manner. Due to the known deleterious effects of cyclophosphamide on meiotic events, the last goal was to assess the ability of pachytene spermatocytes to undergo the meiotic G21/M transition. The delayed meiotic progression induced by acute cyclophosphamide treatment, likely to be a checkpoint response, contrasted with the lack of delay after chronic drug exposure. Together these results show that male germ cells are differentially equipped with mechanisms of stress response. Furthermore, cyclophosphamide alters the expression of stres