Megakaryocytes are polyploid cells which upon maturation release cytoplasmic fragments known as platelets. At this time, we do not have a clear understanding of the mechanisms of control of megakaryocytopoiesis. Evidence supports the existence of a humoral factor, designated thrombopoietin (TSF), regulating megakaryocyte differentiation. Current research involving the mechanism of action of thrombopoietin is hindered greatly by the lack of characterization and isolation of a pure fraction of thrombopoietin. Purification and characterization of this factor are partially dependent upon the development of an adequate assay for thrombopoietin. This study was designed to develop an in vitro assay for thrombopoietin utilizing tritiated thymidine incorporation in whole bone marrow. The sensitivity of this assay is adequate for use in studies of megakaryocyte differentiation, humoral control mechanisms, and purification of thrombopoietin Platelets were harvested from guinea pig blood and injected into rabbits to elicit production of a rabbit-anti-guinea pig platelet antibody (RAG(,p)PS). Upon injection of the antisera into guinea pigs, the animals were rendered acutely thrombocytopenic thus producing increased levels of endogenous thrombopoietin. Thrombopoietin-rich serum was collected for use in the assay For the assay for thrombopoietin, whole bone marrow was harvested from the femora and tibiae of male, Hartly albino guinea pigs. Cells were suspended and washing in calcium and magnesium free Hank's (CMFH) and transferred to Williams's E medium containing ('3)H-thymidine and incubated at 37(DEGREES)C. To the test samples TSF-rich sera was added. Normal guinea pig serum and saline served as controls. After 4 hours of incubation, samples were removed and fixed in 4% paraformaldehyde and prepared for autoradiography. Labeling indices were determined by the enumeration of the number of labeled megakaryocytes/total megakaryocytes Results showed a significant increase of labeled megakaryocytes in the TSF-incubated samples as compared to NGP serum or saline controls. In addition, a 4N megakaryocyte with a band-shaped nucleus and cell diameter of 19-24 micrometers has been shown to respond to TSF stimulus by incorporating tritiated thymidine. An in vitro assay for thrombopoietin utilizing tritiated thymidine incorporation in whole bone marrow has been developed in this investigation / acase@tulane.edu

acase@tulane.edu

acase@tulane.edu

No description available.

Abstract Not Available.

The tools we choose to perform a given task affect not only the result of the task but also how it is conceived. An examination of tools, tasks, and interdisciplinary technology transfer suggests that new ways of thinking, rather than increased efficiency, are the primary means of technological advancement. The history of the relationship of the human body to architecture can be seen as a progression from embodiment to projection to a new paradigm of extension, or prosthesis. These ideas, together with surveys of bridge types and prosthetic technology, lead to the design of a pedestrian bridge linking two existing buildings. The bridge is conceived and designed using the tools of medical prosthetics and orthotics. Human and animal vertebrae suggest structural principles from which a light-weight, articulated bridge form is developed to satisfy the requisite conditions.

The first part of my work evaluates bilateral supramaximal transcutaneous phrenic nerve stimulation as a diagnostic test for respiratory muscle fatigue. I found that twitch transdiaphragmatic pressure (Pdi,T) was inversely and linearly related to lung volume (V$ sb{ rm L}$) both before and after fatigue. Although fatigue caused significant decrease in Pdi,T amplitude at all V$ sb{ rm L}$, the fractional decrease in Pdi,T was greater at high V$ sb{ rm L}$, indicating the importance of V$ sb{ rm L}$ as an independent variable that needs to be controlled whenever Pdi,T is determined. Twitch mouth pressure (Pm,T) was found to be linearly related to twitch esophageal pressure (Pes,T), to Pdi,T, and to V$ sb{ rm L}$. All these relationships were reproducible. Diaphragmatic fatigue resulted in significant decrease in Pm,T proportional to the decrease in Pdi,T for a given V$ sb{ rm L}$ so that Pm,T-Pes,T and Pm,T-Pdi,T relationships were unchanged. Thus the Pm,T-V$ sb{ rm L}$ relationship can be used to assess diaphragmatic fatigue non-invasively. Paired phrenic nerve shocks which were well tolerated by normal subjects can be used to obtain a measure of the pressure-frequency curves of the diaphragm, which were reproducible. In particular, I showed that the pressure ratio of diaphragmatic twitch elicited by the second shock at 10Hz over that at 100Hz (T2$ sb{10/100}$) is a valuable index of low frequency fatigue. / In the second part of my work I studied the effect of respiratory muscle fatigue on ventilatory response to CO$ sb2$ and respiratory muscle recruitment. The data showed that ventilatory response and respiratory muscle recruitment patterns were different in a number of aspects between diaphragmatic fatigue and global inspiratory muscle fatigue. After diaphragmatic fatigue, the only change was an increase in the recruitment of rib cage muscles, which fully compensated for decreased diaphragmatic contractility because all the ventilatory parameters were constant. After global fatigue, both the diaphragm and rib cage muscles contributed less to breathing but expiratory muscles were recruited resulting in a decrease in end-expiratory P$ sb{ rm L}$ and an increased contribution of elastic energy stored within the respiratory system to inspiratory tidal volume generation. In spite of this, rapid shallow breathing developed while minute ventilation remained constant. These data suggest that the ventilatory control system can detect fatigue and has sufficient plasticity to alter inspiratory drive appropriately. The overall ventilation level can thus be maintained.

This thesis addresses the expression, regulation, and functional aspects of NOS in normal and developing skeletal muscles, and their role in contractile dysfunction of respiratory muscles associated with septic shock. Normal skeletal muscles of mammalian species express only ecNOS and nNOS to varying degrees. NOS activity in these muscles is mainly Ca2+/calmodulin-dependent and it associates with fast-twitch muscle fibers in rat and mice, but no such correlation exists in other species. Therefore, NOS activity is not the only factor that specifies contractility of skeletal muscles. In developing skeletal muscles, there is a transient increase in NOS Ca2+-dependent activity and the expressions of cNOS isoforms are upregulated. This coincides with skeletal muscle differentiation and maturation. Despite the negative influence of NOS activity on skeletal muscle contractility, little inhibition is observed on force generated by the developing diaphragm. Therefore, NO may regulate other processes than contraction in developing skeletal muscles. The in-vivo induction of iNOS protein and mRNA in skeletal muscles of septic rat is matched by a parallel induction in GTP-cyclohydrolase-I, the rate-limiting enzyme for BH4 biosynthesis. NOS Ca2+ -independent activity increases several fold mainly in the respiratory muscles. In addition, the expressions of cNOS enzymes are upregulated in septic rat muscles. During 24 hrs of endotoxemia of rats, iNOS is induced by 6 hrs, peak by 12 hrs and disappear by 24 hrs after LPS injection. nNOS and ecNOS expression is upregulated by 6 hrs and remained higher than control values after 24 hrs of LPS injection. The regulation of NOS isoforms is matched by an increase in total and Ca2+/calmodulin-independent NOS activity. Furthermore, peroxynitrite was detected in septic respiratory muscles, and nitrated proteins were detected in these muscles 12 hrs after LPS injection. Submaximal force generated by diaphragm strips was significantly inhibit

The present study examines the distribution of the low density lipoprotein receptor related protein-2 (LRP-2) in the reproductive tracts of male and female rats. LRP-2 has been shown to facilitate the endocytosis, and lysosomal degradation of the glycoprotein SGP-2 in cultured cells. SGP-2 has been found in the male reproductive tract, is thought to be involved in sperm maturation. The apical surfaces of the epithelia of the efferent ducts as well as the intermediate zone, proximal caput, corpus, and cauda regions of the epididymis showed pronounced peroxidase staining for LRP-2. Immunogold labeling representing LRP-2 was observed in endocytic vesicles and early endosomes in the single labeling experiment. Labeling was much more significant in the efferent ducts than the epididymis. The double labeling experiment for LRP-2 and SGP-2 showed colocalization in the endocytic vesicles and early endosomes. SGP-2 also labeled the late endosomes and lysosomes. These results suggest that, LRP-2 mediates the endocytosis of SGP-2, and afterward is recycled back to the cell surface. The second aspect of the study examines the distribution of LRP-2 and SGP-2 in the epithelium of the uterus and the oviduct, in relation to the different stages of the estrus cycle. The surface epithelium and the epithelial cells lining the glands of the uterus were for the most part devoid of staining for LRP-2, except in the estrus stage when a strong reaction was seen in the apical region of the cells. The epithelium lining the glands of the uterus showed strong staining, while weak immunoperoxidase staining was observed for SGP-2 in the uterine surface epithelium in every stage of the estrus cycle. The epithelium of the proximal and distal, oviduct showed very little staining if at all for LRP-2 and SGP-2 for all stages of the estrus cycle. (Abstract shortened by UMI.)

Injection of 2mg of estradiol valerate (EV) to cycling female rats causes cell death among the hypothalamic beta-endorphin population that results in increased mu-opioid receptor binding in the hypothalamic MPOA. We suspect that the subsequent opioid suppression of the GnRH system is responsible for the constellation of defects that occur in pituitary LH production and release. / In order to determine the mechanisms by which a defective GnRH pattern affects pituitary LH functions, we quantitated LH-beta and alpha mRNA in EV-injected animals using Northern blot analysis. To determine whether estradiol has direct effects at the pituitary level, we studied estradiol implanted (E2) animals that do not have the hypothalamic lesion. In order to observe possible effects on prolactin and FSH (normal plasma levels) we also quantitated them in EV or E2-treated animals. / Our results indicate that LH-beta, but not alpha or FSH-beta RNA are below control levels in both EV and E2 treated animals. Thus estrogen can modulate LH-beta production at both the hypothalamic (EV) and pituitary (E2) levels. Prolactin was sometimes below that of control animals which is surprising since estradiol is a known stimulator of prolactin production.