This study investigates the interaction between increased gain in the visual feedback loop and motor control of the periphery. Subjects were asked to maintain a constant finger position while utilizing magnified visual feedback. The accuracy of each trial was quantified by taking the standard deviation (trial-error) of the finger position. Trials performed under magnification have lower trial-errors than trials without magnification. The change in trial-error between trials with and without magnification proves greater than the difference between trials at any two magnifications. In contrast, the differences between individual subjects is often greater than the difference between performances at individual magnifications. At higher magnifications performance seems to be limited by the tremor. Trial-error is approximately 2 times the tremor-intensity. When applied to microsurgery these results are in accord with earlier research including results suggesting that the level of magnification used in microsurgery is not the most significant factor in achieving good results, and that tremor is the limiting factor in microsurgical tasks.

Our laboratory identified pyridoxal isonicotinoyl hydrazone (PIH) as an orally effective Fe chelator (BBA 586:278 1979). More recent in vitro and in vivo studies with PIH analogs have demonstrated that halogenated pyridoxal benzoyl hydrazones are more effective iron chelators than PIH. In this study, 33 analogues were synthesized and assessed. The compounds were derived from 3 parent aldehydes: pyridoxal (100 series), salicylaldehyde (200 series) and 2-hydroxy-1-naphthylaldehyde (300 series) in combination with the ortho-, meta -, and para-isomers of halogenated benzhydrazides: chloro (08), fluoro (09), bromo (10), and iodo (10.1). Fe chelators were evaluated on the basis of their ability to mobilize Fe59 in vitro from (i) murine reticulocytes with a high level of non-heme Fe59 and (ii) Fe59 loaded K562 cells. Following incubation of Fe59 loaded cells in the presence of chelators, we also determined the intracellular level of chelator-bound Fe59 that remains in solution following ethanol precipitation of cellular proteins. (Abstract shortened by UMI.)

There is good reason to believe that the known human cysteine protease repertoire is currently incomplete. Recently, two peptide sequences (i.e. CTLA-2alpha and CTLA-2beta) have been identified in the mouse T-lymphocyte. These peptides have been shown to be similar to the propeptide of human cathepsin L. In this project, computer based searches for human homologues to these mouse peptide sequences has been made. One entry from the TIGR express sequence tag database has been identified. As part of this project, the tag was isolated from the human Jurkat cell mRNA, a cell line compatible with the source of RNA used to identify the original sequence tag. / Several novel members of the papain proteases superfamilly have been discovered and characterized in the last couple of years. Of particular note is cathepsin K, which is primarily an osteoclast component that has been shown to be the main mediator of organic matrix degradation during bone resorption. Since many of these proteases demonstrate relevant extracellular matrix degradation in connective tissue, they are of significant interest in the study of joint health and diseases. In this project, qualitative cathepsins mRNA expression analyses in representative human joint-related cells have been assessed by RT-PCR. (Abstract shortened by UMI.)

Although nitric oxide (NO) from inducible nitric oxide synthase (NOSII) is proposed to be the major factor in the vascular abnormalities of sepsis in rats, its role in higher order species is not well established. This thesis thus, addresses the role of NO in septic pigs. / I first hypothesized that induction of NOSII in pigs is the cause of sepsis-induced hypotension as occurs in rodents. To test this, I treated pigs with lipopolysaccharide (LPS) to simulate sepsis. In contrast to what is observed in rats and mice, plasma nitrite did not change during the 4 hours of the LPS infusion and there was no increase in calcium (Ca)-independent nitric oxide synthase (NOS) activity in lung tissue from endotoxemic pigs compared to control animals. Furthermore, there was only minimal induction of NOSII mRNA. / My second hypothesis was that NOS (NOSIII) is not down regulated in septic pigs, and NO from NOSIII, could contribute to peroxynitrite formation. In support of this, western blot analysis of samples from aorta and vena cava showed no down regulation of NOSIII. There also was an increase in Ca ++-dependent NOS activity of aorta and vena cava after 2-hour of endotoxemia, which indicates increased constitutive NOS activity. Moreover, there was an increase in NOSI in the vena cava. LPS produced a leftward shift in the dose-response curves of SVR and RVR in response to the NOS inhibitor, L-NAME (NG -nitro-L-arginine-methyl-ester). / Peroxynitrite is formed from NO and superoxide (O2 -). Since NO was not increased in endotoxemic pigs, I next hypothesized that O2- is increased in endotoxemic pigs and this reacts with NO from constitutive NOS to form peroxynitrite. To test this hypothesis, I first studied rats to confirm that O2 - is increased in arterial vessels of endotoxic rats. There was a small increase in basal O2- in endotoxic rats. Stimulation of NAD(P)H oxidase by giving the substrate, NADH, did not result in different O2- production in control and endotoxic rats. However, inhibition of NOS increased O2 - in endotoxic rats. / I demonstrated by immunohistochemistry and western blot analysis, that the components of NAD(P)H oxidase, gp91phox, p22phox , and p47phox are present in skeletal muscle and it has similar behavior to other non-phagocytic NADPH oxidases. The addition of L-NAME to NADH-treated diaphragm strips of endotoxic rats, increased O 2- production in endotoxic rats indicating that O2- production counters the increased NO formation as we observed in rat aorta. These observations indicate that O2 - generation from NADPH oxidase in skeletal muscles can play a role in the pathophysiology of sepsis. (Abstract shortened by UMI.)

The aim of this thesis study was to investigate the substance P (SP)-immunoreactive (IR) sensory fiber innervation of CNS target tissues in two experimental models. In the first model, we examined the SP-IR boutons apposed to three functional types of dorsal horn neurons and their morphological interaction with calcitonin gene-related peptide (CGRP) and enkephalin (ENK) in the cat spinal cord, using a combination of intracellular electrophysiological recording and horseradish peroxidase injection with ultrastructural immunocytochemistry. In addition to SP-IR only boutons, we detected boutons co-localizing SP plus CGRP and SP plus ENK immunoreactivities presynaptic to nociceptive neurons. Quantitatively, significantly higher numbers of SP-IR, SP+CGRP-IR and SP+ENK-IR boutons were apposed to nociceptive neurons. Non-nociceptive neurons were rarely innervated by boutons which were SP-IR, SP+CGRP-IR and SP+ENK-IR. In contrast, ENK-IR only boutons innervated non-nociceptive neurons considerably. Boutons co-localizing SP and CGRP were considered as originating from primary sensory afferents. Most nociceptive neurons contained ENK immunoreactivity, but non-nociceptive neurons were never ENK-IR. The interaction of SP and $ gamma$-aminobutyric acid (GABA) in the superficial dorsal horn of the cat and rat spiral cord was also investigated. The co-localization of SP and GABA in axonal terminals was detected for the first time in the superficial layers of the dorsal horn of the cat, but not rat, spinal cord. / In the second model, we used immunocytochemistry to study the SP-IR fiber innervation of the white matter of transgenic mice expressing NGF in myelinating oligodendrocytes driven by a MBP promoter. SP-IR fibers were observed in the white matter of the CNS of both transgenic and control mice from postnatal day 0 to day 2. From day 5 on, however, these SP-IR fibers increased markedly to become ectopic fibers in transgenic mice, but decreased dramatically, and finally disappeared, in control mice. The ectopic SP-IR fibers of transgenic mice persisted throughout adulthood. Capsaicin treatment abolished all ectopic SP-IR fibers, indicating their primary sensory origin. / In conclusion, SP-IR fibers specifically innervated nociceptive neurons and co-localized with CGRP, ENK and GABA in the cat dorsal horn. The finding provides anatomical substrates for roles of SP in nociception and for functional interactions of SP with ENK and GABA. Ectopic SP-IR fibers innervated the white matter of the CNS of transgenic mice where NGF was abnormally-produced.

The hemopoietic microenvironment of the bone marrow is an essential regulator of in vivo hemopoiesis. In addition to supporting the growth of normal blood cells, it also influences the growth of leukemia. This thesis describes the use of a rat model to examine three aspects of the function of the hemopoietic microenvironment. First, using a myeloid leukemia cell line (BNML), we showed that the pattern of growth of these cells differed in the bone marrow and spleen, that their presence was associated with a relocalization of normal hemopoietic stem cells from marrow to spleen, and that factors (yet to be defined) released from spleen cells altered the pattern, possibly to create a more permissive environment. Second, we showed that the "ST3" marker of marrow fibroblasts was associated with the Thy-1 molecule, and either directly or indirectly contributed to the in vitro adhesion reaction between marrow fibroblastoid cells and normal and leukemic myeloid precursors. Third, we showed that ectopic bony ossicles induced by subcutaneous implantation of recombinant human bone morphogenetic protein-2 contained marrow expressing the full range of hemopoiesis, including stem cells with a potential for long-term repopulation (demonstrated using a rat Y-chromosome specific DNA probe that we developed), and contained fibroblastoid cells differentiated to express the ST3 antigen in a manner similar to those from femoral bone marrow. These results provide further evidence for, although not final proof of, the hypothesis that the ST3 antigen participates in the function of the rat hemopoietic microenvironment, and points the way to future experiments on the interactions between stromal elements and normal and leukemic myeloid precursors.

This study was designed as an investigation of the role of changes in muscle force and changes in muscle length on the EMG for the Tibialis Anterior (TA). / Using surface electrodes we examined the EMG for 4 contraction levels at 5 ankle positions over 60$ sp circ$ of ankle rotation. The change in median frequency with muscle length identified a significant shift in the power spectrum to lower frequencies with increasing muscle length. / To further investigate our results we performed three other experiments: First, using X-rays to identify the relative change in distance between two intramuscular wire electrodes we found the change in TA muscle length for this study to be 15% over the 60$ sp circ$ of ankle rotation. Second, to test for synergist contamination we used fine wire electrodes in the Extensor Digitorum Longus and the Peroneus. We found no evidence to support significant contamination. Third, we examined the role of smaller electrodes with a smaller interelectrode distance on our findings. The EMG showed drastic changes with even a slight shift in electrode position most likely due to the large number of innervation zones. / Therefore, the results indicate a shift in the power spectrum with a change in muscle length. In addition, surface EMG results are heavily dependent on the innervation zones and on the electrode geometry, all of which are important considerations in developing the EMG as an accurate diagnostic tool.

Natural killers (NK) are a subpopulation of lymphocytes that are defined by their pattern of cytotoxicity against other normal and neoplastic cells of predominantly hemopoietic origin. Although they have been programmed to recognize a limited spectrum of targets, their activity can be augmented by certain immunoregulatory substances. The molecules that mediate the recognition events have not yet been identified. / I describe here the derivation and characteristics of a variant clone (Clone I) of the human leukemic cell line K562. These cells, selected for decreased binding to peripheral blood lymphocytes, were less sensitive than the parent to lysis by NK in the resting, but not in the augmented state. Although their major plasma membrane proteins appeared identical to those of K562, they contained an additional minor group of fucosylated glycolipids. A later subclone of Clone I, selected for resistance to Concanavalin A, reverted to an NK sensitive pattern and exhibited the parental profile of glycolipids. / The results illustrate in an in vitro model how a leukemic cell can modulate its membrane to escape surveillance by NK cells, and suggest that the glycolipids might be involved (directly or indirectly) in the mechanism.

Uveal melanoma is the most common primary malignant intraocular tumor in adults. The management of uveal melanoma remains a clinical dilemma, which reflects our poor understanding of this life-threatening disease. A major challenge facing researchers investigating this malignancy has been to develop a suitable animal model. The purpose of this work is to characterize, in detail, the processes of tumor development, malignant cell dissemination and metastasis in a 10-week albino rabbit model of uveal melanoma. Intraocular tumors successfully developed, and metastatic disease was present in all animals at the end of the experiment. For the first time using an animal model of uveal melanoma, the presence of circulating malignant cells in the bloodstream was demonstrated. Knowledge gained from this study has led to a better overall understanding of the progression of the disease in this experimental model and may facilitate the development of methods for the prevention, early detection and treatment of metastatic uveal melanoma.

The occurrence of high frequency, gamma EEG activity (30-60 Hz) was bands and in investigated by spectral analysis in relationship to other frequency association with the natural sleep-waking cycle and spontaneous behaviors of the rat. High frequency, gamma waves are present and occur intermittently on the filtered and unfiltered EEG across the sleep-waking cycle. Gamma activity is distinctive from other high frequency bands, in that the amplitude varies systematically in association with (1) state, having the highest amplitude in Waking and Paradoxical Sleep (PS) as compared to Slow Wave Sleep (SWS) and, (2) behavior, having the highest amplitude in attentive and active waking behaviors and 'active' sleep with twitches indicative of PS, as compared to quiet waking and 'quiet' sleep. Coherence in gamma activity also varies as a function of state-behavior, having the highest values in Waking moving and PS. Gamma activity varies positively with theta and inversely with delta activity over the sleep-waking cycle. Gamma activity is thus shown to reflect the degree of behavioral and cortical arousal.