Pyridoxal isonicotinoyl hydrazone (PIH) has been described as an orally effective Fe chelator. It is both membrane permeable and plasma soluble, and has a high affinity for Fe, making it an ideal model on which to base future chelators. Ten novel ligands have been synthesized based on these attributes. Characterization experiments were performed to determine the ligands' selectivity and binding affinity for iron, their lipophilicity as both free and Fe-ligand complexes, and their stoichiometric relationship with iron. Efficacy of the chelators has been determined through their ability to effectively mobilize non-heme 59Fe from pre-labeled cells. Intracellular levels of chelator bound 59Fe were also determined. Concentration-dependence and time-dependence mobilization experiments were performed to determine the minimal concentrations of ligands required to elicit maximal 59Fe release. Toxicity experiments with various ligand concentrations were performed in order to determine the concentration which inhibits at least half of cellular growth as compared with control. (Abstract shortened by UMI.)

Chronic ligation of one pulmonary artery results in pulmonary vascular remodelling and bronchial angiogenesis, known as postobstructive pulmonary vasculopathy (POPV). In previous studies of POPV, we found that responses of pulmonary arteries to 5-HT and of veins to histamine were markedly increased, but the role of putative factors, such as structure, endothelial modulation and alterations in receptors remains unknown. First, we examined the role of these mechanisms in the differential responses of pulmonary arteries and veins of normal guinea pigs to histamine and 5-HT, using a novel lung explant technique. We found that veins contracted more to both agonists than arteries, and that H2 receptors were responsible for the differential contractile responses of pulmonary arteries and veins to histamine, whereas endothelium-derived vasoactive substances (nitric oxide and prostacyclin) were responsible for their differential contractile responses to 5-HT. We also investigated relaxation responses, and found that endothelial-dependent NO-mediated relaxation was greater in pulmonary arteries than veins, and that acetylcholine-induced NO-mediated relaxation was reduced by the simultaneous production of cyclooxygenase-derived vasoconstrictors. / Second, we did experiments in guinea pigs with POPV, and found that the maximal contractions of pulmonary arteries to 5-HT and of veins to histamine were increased compared with controls, and that the augmented responses were not due to endothelial dysfunction nor to structural alterations, but probably to changes in the smooth muscle proper. To ascertain responses to endothelin (ET), and the role of altered receptors, specifically ET receptors, in POPV, we produced the model in rats: we found that contractions to ET-1 and ET-3 were increased and that relaxation to ET-1 was reduced significantly only in the pulmonary arteries with POPV compared with controls; these findings were attributed, using receptor binding studies, to an augmented proportion of ETA over ETB receptors. / We conclude that the differential contractile responses of normal pulmonary arteries and veins to histamine and 5-HT, and the altered vascular responses to these amines and to ETs in POPV, are due primarily to differences in receptors or in endothelial modulation, rather than to disparities in vascular structure.

Ceruloplasmin oxidizes the toxic ferrous (Fe(II)) form of iron to the ferric (Fe(III)) form, and promotes iron loading onto transferrin. It is produced by the liver and secreted into the plasma. My work has shown that a novel membrane-anchored form of ceruloplasmin is the major form of this enzyme in the rat brain. / The membrane-anchored form of ceruloplasmin was identified using a monoclonal antibody (mAb 1A1) that specifically labels the surface of astrocytes. Using mAb 1A1 immunoaffinity chromatography, the antigen was purified from detergent extracts of rat C6 glioma cells. Amino acid microsequencing, and additional experiments, revealed that this antigen was highly similar to ceruloplasmin. Further experiments revealed that this form of ceruloplasmin was directly anchored to the surface of astrocytes and C6 glioma cells by a glycosylphosphatidylinositol (GPI) anchor. / Screening of a C6 glioma cDNA library identified a novel alternatively-spliced transcript that codes for this GPI-anchored form of ceruloplasmin. HEK293T cells transfected with the novel full-length cDNA expressed ceruloplasmin on the cell surface. Treating these transfected cells with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that specifically cleaves GPI anchors, eliminated the cell surface localization of ceruloplasmin, confirming that the novel cDNA codes for the GPI-anchored form of the protein. RNase protections on rat brain and liver RNA revealed that GPI-anchored ceruloplasmin is the major form in the brain, whereas the liver expresses predominantly the secreted form. / To better understand the role of ceruloplasmin in vivo, a ceruloplasmin gene knockout mouse was generated. The knockout mice accumulate large amounts of iron in the liver and have reduced serum iron levels. Older knockout mice (15--17 months) have increased iron accumulation in different parts of the CNS, including the cerebellum, spinal cord, and retina. Increased lipid peroxidation is also observed in some regions of the CNS. These biochemical changes were accompanied by deficits in motor skills. Furthermore, dissociated cell cultures of the cerebellum from knockout mice were more susceptible to free radical injury when treated with hydrogen peroxide. These data suggest that ceruloplasmin plays an important role in iron metabolism in the CNS and provides protection against free radical injury.

Little is known about the factors stimulating placental progesterone (P4) production at the time of the luteo-placental shift (6-8 weeks post-conception). To explore the regulatory mechanism, the effects of various steroids and peptides on the production of P4 by placental explants were studied. / In early placental explant culture P4 production was stimulated by 19-nortestosterone (19-NT), androstenedione (A-dione), 5$ alpha$-androstane-3$ alpha,$17$ beta$ diol (3$ alpha$-diol) and 5$ alpha$-androstane-3$ beta,$17$ beta$ diol (3$ beta$-diol). Of all the compounds tested, 19-NT had maximal effect. At term, P4 production was stimulated only by 3$ beta$-diol. 19-NT and A-dione were poorly aromatized in early placental explants compared to another androgen (Androst-5-ene-3$ beta,$17$ beta$ diol). / In accord with the above observations, placental levels of 19-NT and A-dione were higher in early gestation while the diols were higher in late gestation. / 19-NT stimulated P4 production in early placenta by effects on the conversion of P4 both from 25-hydroxycholesterol and from pregnenolone. The stimulatory influences of A-dione and 3$ alpha$-diol were mediated by increasing the P450scc activity. The specific increase of the conversion of P4 from pregnenolone accounted for the P4 stimulation observed by 3$ beta$- diol treatment of culture. / Cyloheximide (CH) treatment abolished the stimulatory influences of the aforementioned steroids on P4 production except for the initial phase of P4 stimulation by 19-NT, suggesting that all but the latter are dependent on protein synthesis. / P4 production was also stimulated and prolonged to 30 days in the presence of human maternal serum (HMS); a good correlation (r = 0.74, P $<$ 0.05) was seen between the histological appearance of the explants and P4 production. The stimulatory activity of HMS was heat labile, non-dialyzable and non-extractable into an organic solvent, suggesting that it is protein in nature. / In conclusion, this study suggests that 19-NT and A-dione are important for placental P4 production at the time of the luteo-placental shift. For in vitro study of placental hormonal regulation, HMS is a better nutrient supplement than fetal bovine serum.

This thesis focuses on the effects of adenosine release on neuronal activity in the CA1 region of rat hippocampal slices, especially during energy deprivation, and related events. The overall aim was to find out how brain function is suppressed when metabolism is greatly reduced by the lack of oxygen or glucose, or by cyanide applications, and therefore to provide some insights in the cellular mechanisms underlying these depressant effects. Experiments were carried out on hippocampal slices kept submerged and constantly superfused with oxygenated saline at 33-34$ sp circ$C. Energy deprivation was produced by either removal of oxygen or glucose, or applying cyanide. Extra-/intracellular and whole-cell patch recordings (both in current- and voltage-clamp mode) were used to assess synaptic function and postsynaptic properties. / A most interesting phenomenon is the reversible suppression of neuronal function that occurs in the very early phase of energy deprivation. Adenosine receptor antagonists reversibly reduce this suppression. In contrast, neither glibenclamide, a blocker of ATP-sensitive K$ sp+$ channels, nor a nitric oxide synthase inhibitor prevents the suppression of neuronal activity induced by energy deprivation. The depressant effect acts selectively on excitatory synapses, since in the presence of excitatory receptor antagonists, anoxia causes only a small reduction of monosynaptic inhibitory responses. Also adenosine antagonists, but not the K$ rm sb{ATP}$ channel blocker, reversibly attenuate anoxia- and cyanide-induced post-synaptic hyperpolarizations. / Furthermore, under normoxic conditions, ongoing adenosine release exerts an inhibitory tone on excitatory synapses but not on inhibitory synapses. The effects of ongoing adenosine release are mainly on synapses, since after blockade of transmitter actions, ongoing adenosine release has no detectable effect on membrane conductance. / The evidence presented in this thesis shows that increased adenosine release induced by energy deprivation is a major cause of the reversible loss of synaptic transmission and fall in membrane resistance, and therefore indicates a mechanism by which adenosine release contributes to the reversal depression of neuronal activity seen during energy deprivation.

The first objective of this study was to investigate the contralateral and summatory effects of electroacupuncture (EA) stimulation on the flexion reflex (FR) in man. The second objective was to delineate whether the FR (a physiological measure) will vary systematically with subjective pain sensation (a psychological measure) under different stimulation conditions. / In the first study, in 16 subjects, we compared the FR area "values" obtained electromyographically in the biceps femoris under three different stimulation procedures: placebo-TENS (placebo), electroacupuncture to the contralateral leg (EA$ sb{ rm Leg}$ and electroacupuncture to the contralateral arm and leg (EA$ sb{ rm Arm+Leg}$). A statistically significant decrease in the FR area (p $<$ 0.01) was observed in 7, 8 and 9 subjects respectively for placebo, EA$ sb{ rm Leg}$ and EA$ sb{ rm Arm+Leg}$ stimulation. The results demonstrated a significantly greater inhibitory response in the BF FR area with EA$ sb{ rm Leg}$ stimulation than with placebo stimulation (p $<$ 0.01). In the second part to this study we undertook to determine the relationship between the FR and pain sensation. It appeared that electroacupuncture could modulate perceptual output at the cortical level in the same or opposite manner from the spinal motorneuronal level (FR), depending upon whether this reflex motor output (FR) is inhibited or facilitated.

Effects of acute and chronic cocaine on the respiratory pattern, and ventilatory responses to hypoxia and hypercapnia, were studied in awake and anesthetized adult rats. In addition, effects of the drug on vagal reflexes, respiratory mechanics and gas exchange were investigated in anesthetized animals. / Acute cocaine elicited rapid and shallow breathing in awake rats. Ventilatory excitation, but not tachypnea, was present in anesthetized animals. In untreated rats, respiratory rate (f) increased during 10% O$ sb2$ and 5% CO$ sb2$ exposures; f decreased after cocaine, but in absolute terms, it was still higher after than before treatment. In anesthetized animals, augmentation of minute ventilation and f recorded in response to low O$ sb2$ and high CO$ sb2$ were higher with than without acute cocaine. / Chronic cocaine depressed ventilatory responses to both stimuli in awake, but not in the anesthetized chronic rats. Neither acute nor chronic cocaine affected respiratory mechanics and diffusion capacity of the lungs. Finally, strength of the expiratory-promoting reflex increased following acute cocaine. / The data suggest that while acute cocaine acts as ventilatory stimulant triggering behavioral polypnea, it depresses ventilation in chronically treated rats.

No description available.

Previous research has demonstrated that although women may find post-operative pain more intense than men, males are more disturbed than females by low levels of pain that last over time. In these studies, females had a tendency to rate the intensity of pain higher than males, but males had stronger affective responses following the surgical placement of intra-oral implants. However, these findings have not been investigated in an experimental setting. This experiment examined the pain responses of 20 healthy subjects (10 males, 10 females), who were subjected to capsaicin-induced pain on the face and ankle (on separate sessions). During the experiment, all subjects rated their pain intensity, unpleasantness, and anxiety on visual analog scales (VAS). In addition, throughout the experiment, heart rate was monitored every five minutes and mood was assessed once before and after the experiment. Finally, subjects also completed the McGill Pain Questionnaires (MPQ) once at the end of every session. Results revealed that although there were generally no statistically significant sex differences in the pain ratings during the experiment, there was a sex * time interaction with males displaying increasing anxiety scores over time with the capsaicin patch on the face while the anxiety scores of females decreased over time with the capsaicin patch on the face (F = 1.64, P = 0.02). Also, there was a tendency for the relative unpleasantness (unpleasantness/intensity ratio) to be greater for males than females over time on the face (F = 3.43, P = 0.08). Males and females did not differ in both the mean number of words chosen and the pain rating index of the MPQ for all categories. In addition, there were no sex differences for heart rate and mood for both the ankle and face regions throughout the experiment. Taken together, these results replicate previous findings that men may find low levels of pain more disturbing than women.

Huntingtin interacting protein-1 (HIP1) is an endocytic protein that associates with clathrin on coated vesicles, but it also binds directly to actin and microtubules. Close histological examination of the testis of HIP1-/- mice from 7-30 wks of age revealed that HIP1-deficiency was particularly detrimental to spermatids. The testis showed a significant decrease in the diameter of seminiferous tubules, a reduction in the number of late spermatids and the sloughing of germ cells, which were evident as round cells in the epididymal lumen. Major abnormalities in spermatids included structural deformations of heads, bent flagella, the presence of proacrosomic vesicles with the complete or partial absence of an acrosome or its detachment from the nucleus, and retention of the cytoplasm enveloping the spermatid head. Abnormalities with respect to the association of elongating spermatids with ectoplasmic specializations of Sertoli cells were noted as well. Sperm counts and sperm motility parameters were significantly decreased in HIP1-/- mice compared to their wild-type littermates and these differences accounted for reduced fertility levels noted in HIP1-/- mice. / Taken together, differences in sperm counts, morphology and their motility parameters suggest a functional role for HIP1 in relation to actin and microtubules during sperm development in the testis.