During lactation, control of the activity of the hypothalamic-pituitary-adrenal (HPA) axis is adjusted in that tonically elevated glucocorticoid secretion is observed concurrently with blunted ACTH secretion following exposure to various stressors. Although a decline in the level of CRF mRNA has been reported in neurons of the paraventricular nucleus (PVN) known to control ACTH secretion, the mechanisms underlying stress hyporesponsiveness during lactation are still largely unknown, In these studies we tested the hypothesis that pituitary responsiveness to secretagogues was altered and that central noradrenergic (NA) inputs to hypothalamic CRF neurons were modified during lactation in the rat. First, at the level of the pituitary the involvement of corticotrope sensitivity changes was examined in mid-lactating and virgin females. Results demonstrate that both virgin and lactating females show a significant ACTH response to a high stress-level dose of CRF but the magnitude of the response was greater in virgin compared to lactating females. Second, to understand the importance of brainstem NA afferents in mediating the stress response during lactation we performed 6OH-DA lesions over the PVN. In virgin females, 6OH-DA lesions caused a significant reduction in the ACTH and corticosterone (B) responses to 5 min swim stress, but lesioning did not affect stress-induced ACTH levels in lactating females. Finally, participation of central NA changes to some aspects of behavior was characterized during lactation. The acoustic startle response (ASR) was examined in groups of cycling and lactating female rats in early-, mid-, or late-lactation. / Overall results suggest that brainstem NA inputs to the PVN act to facilitate ACTH stress response in virgin, but not lactating females. The absence of this facilitation is partially due to a reduced endogenous secretion (as demonstrated under basal conditions) and to a series of modifications in alpha-adrenoreceptor number, synthesis, and function that cause a decreased responsiveness to NA in parvicellular CRF neurons. (Abstract shortened by UMI.)

Traditionally, the vestibulo-ocular reflex (VOR) has been considered a stereotyped ocular counterrotation response to head movement that stabilizes a visual image on the retinae. However, during natural head movements, the appropriate magnitudes and directions of compensatory ocular deviations depend on viewing context. Moment-to-moment adjustments in VOR performance are required as gaze is redirected towards different viewing locations. / This thesis presents an investigation of viewing-context-dependent VOR performance through the development of a physiologically and anatomically based bilateral model structure. Previous theoretical studies of visual-vestibular interactions during head-centered rotation are extended by simulating both ocular responses and those of individual premotor brainstem neuron types in an integrated binocular controller for slow eye movements. Central sensitivities to vestibular canal signals are modulated as a function of instantaneous binocular fixation state to simulate appropriate viewing-location-dependent changes in monocular rotational VOR performance and distinct premotor cell behaviors. / A new hypothesis for the central dynamic processing of sensory otolith signals in the translational VOR is presented. Previous proposals suggested that the unique dynamic characteristics of otolith and canal afferent signals imply additional central processing in the translational as compared to the rotational VOR pathways. The strategy presented here demonstrates that projecting canal and otolith signals onto a shared premotor circuit at unique sites is sufficient to reproduce observed ocular and central behaviors without introducing additional central filters. By implementing this simple strategy in the bilateral model structure the ability to achieve appropriate compensatory responses for different translation directions and viewing locations in the horizontal plane is demonstrated. / Finally, the model is extended to incorporate brainstem-cerebellar interactions. Current conclusions surrounding potential central sites for plasticity underlying long-term VOR gain adaptation are evaluated. The work makes new suggestions for vestibulo-ocular system organization and proposes directions for experimental work in addressing the following general themes: (1) Sensory convergence onto a shared premotor controller; (2) The role of a bilateral topology in motor pattern selection and binocular coordination; (3) The role of central connectivity in the appearance of distinct premotor cell types; (4) The ability to localize central sites for modifications underlying viewing-location-dependent and long-term adaptive changes in reflex performance.

Caulobacter crescentus divides asymmetrically to produce two different progeny, a swarmer progeny that is replication incompetent, and a stalked cell progeny that is replication competent. Upon cell division, a cell cycle regulator protein (CtrA) was identified only in the swarmer cells, and additional circumstantial evidence links this protein to being a repressor of chromosome replication. For example, this response regulator protein binds to five specific sites in the replication origin (Cori) designated [a-e]. We carefully studied the binding characteristics of both phosphorylated (CtrA~P) and unphosphorylated forms of the protein to the five binding sites [a-e] in the replication origin (Chapter 2) and upstream of the ctrA gene (Chapter 3). We showed that phosphorylation significantly enhances binding affinity in the replication origin (Chapter 2) but not upstream of ctrA (Chapter 3). In addition a "pseudo-active" protein form (CtrA D51E) that resembles the phosphorylated form in vivo did not improve the binding characteristics (Chapter 3). These results suggest that enhanced binding on phosphorylation is not the only signal achieved on phosphorylation. In fact, CtrA half-site mutation binding studies shows that phosphorylation stimulates protein/protein interaction and cooperative binding between sites [a] and [b] in the replication origin (Chapter 2). We show that CtrA binding site [b] is the major contributor in the cooperative CtrA binding between [a] and [b] (Chapter 4). We demonstrate that cooperative binding of CtrA~P to sites [a] and [b] repress transcription from a strong promoter (Ps), which in turn blocks plasmid replication. In addition, mutating site [b] to block CtrA binding to [a] and [b] has a deleterious effect on chromosome replication (Chapter 4). / This cooperative CtrA binding at [a] and [b] is independent from the upstream binding sites [c-e] (Chapter 2). CtrA∼P binding in the origin is altered in the presence of the histone-like protein (IHF) that also binds and overlaps CtrA binding site [c] (Chapter 5). In-fact, IHF binds and overlaps binding site [c] (Chapter 5). We propose a replication model in the stalked cell were IHF binding hinders active CtrA binding in the replication origin and regulates cooperative transcription that coincides with replication initiation.

The objective of this study is to demonstrate differences between erythroid and nonerythroid iron metabolism. Erythroid specific gene expression for transferrin receptor (TfR) which is a transporter of transferrin-bound iron, and for ferrochelatase which uses internalized iron to form heme, is found in dimethylsulfoxide-inducible murine erythroleukemia (MEL) cells. During MEL cell differentiation which parallels erythroid differentiation in vivo, TfR mRNA increases resulting from transcriptional activation of the gene and increased mRNA stability while ferrochelatase mRNA level increases with a 2.7 kb mRNA being induced preferentially. Such preferential induction is effected by utilization of an upstream polyadenylation signal. / TfR-deficient Chinese hamster ovary cells were used to study nonerythroid iron metabolism. Our results show these cells need iron for growth and DNA synthesis and acquire that from transferrin and FeSO$ sb4$ in the medium by a TfR-independent mechanism.

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.

This study examines a reflex mechanism involved in mediating the inhibition of nociceptive input at a heterosegmental spinal level. This heterosegmental inhibitory mechanism is activated by noxious stimulation of a peripheral body region and is regulated by afferent fibers, local spinal neuronal circuitry and supraspinal structures. / A conditioning stimulus, consisting of immersion of one paw of the lightly anesthetized rat in water at a noxious temperature for 90 s elicited an inhibition of a spinal nociceptive reflex in response to a noxious thermal test stimulus in the tail flick test. The magnitude of this antinociception varied with the intensity of the noxious conditioning stimulus, as immersion in water at 40$ sp circ$C, an innocuous temperature, did not evoke an increase in tail flick latency. The antinociceptive response to noxious conditioning stimuli persisted for 3 to 6 min. The response was observed with noxious thermal stimulation of either the hindpaw or the forepaw. Adult animals treated neonatally with capsaicin, which inhibits development of small diameter unmyelinated afferent fibers, did not show this response, suggesting that unmyelinated afferent fibers mediated the input of the conditioning stimulus. Furthermore, in rats transected at the thoracic level, the antinociception following noxious stimulation of the forepaw was blocked while that following hindpaw stimulation was attenuated, indicating that the conditioning stimulus activated heterosegmental inhibitory mechanisms controlled in part by supraspinal structures and in part by spinal structures. / Systemic preadministration of substance P (NK-1) receptor antagonists blocked the antinociceptive effect. These results support an earlier hypothesis that substance P is involved in the transmission of nociceptive information in the spinal cord. Systemic as well as intrathecal preadministration of opiate receptor antagonists blocked the response in a dose-dependent fashion. Therefore, this study suggests that both substance P and endogenous opioids are involved in mediating heterosegmental inhibitory mechanisms.

The pathway of receptor-mediated endocytosis in exocrine pancreatic cells of the rat was studied. The pathway was explored using insulin radiolabeled with iodine-125 detectable by the technique of radioautography. / Insulin was seen at the cell membrane soon after administration of the radiolabeled hormone. After 10 and 20 minutes silver grains were found over compartments deep within the cells, at distances greater than 5 half-distances. Grains were also seen to be associated with endosomal vesicles characterized by heavily stained, often fuzzy, membrane. At this time and later times of 45 minutes, grains were observed over zymogen granules. A group of rats was treated with chloroquine to interfere with intracellular vesicle acidification. The results revealed an accumulation of insulin in endosomes and in presecretory and secretory granules. / Degradation of insulin was found to be significantly inhibited by chloroquine in the liver but not in the pancreas. Degradation of insulin in normal rats was found to be slow in the pancreas but fast in the liver. / The results suggest that insulin is internalized to endosomes within which it may be degraded. Internalized insulin was also seen in the trans Golgi network and zymogen granules, especially after chloroquine treatment. This suggests an access route from endocytic to exocytic compartments which may be related to the pathway of sorting of lysosomal proteins. It may also be significant in the transcytosis of insulin to the pancreatic duct.

The preparation of a novel complex, ferric bromopyruvate, is described. In solutions from which most of the carbonate has been removed, ferric bromopyruvate can be used both as an iron and pyruvate source for the full iron saturation of apotransferrin. Using ferric bromopyruvate as an iron donor, iron incorporation into human apotransferrin is biphasic; the N-terminal domain is saturated three times faster than its homologous C-terminal iron binding site. Following the reaction of apotransferrin with ferric bromopyruvate, 4 moles of pyruvate per mole of transferrin are covalently bound. Based on the effect of acetylation on pyruvate and iron binding, it is suggested that lysyl residues could be the target of pyruvate bonding. However, the reaction of pyruvate with other positively charged amino acid residues cannot be excluded. The possible sites of pyruvate binding within the N-terminal domain of human serum transferrin are discussed. Covalent attachment of pyruvate to cationic amino acid residues decreased both in vitro and in vivo iron release, preferentially from the N-terminal domain of transferrin. The decreased rate of iron incorporation from iron-pyruvate-transferrin complexes by rabbit reticulocytes caused a lower iron incorporation into heme. It is suggested that an impairment of iron release from transferrin may decrease the rate of heme synthesis in reticulocytes. In vitro studies on the iron removal from iron-pyruvate-transferrin complexes showed that pyrophosphate can remove iron from this complex at an acid pH to a similar extent to the cellular mediated iron release from this complex. Based on this data, a model for the intravesicular iron release from transferrin is proposed.

During the process of endochondral bone formation, Indian Hedgehog (Ihh), a member of the hedgehog (Hh) family of secreted morphogens, and parathyroid hormone-related peptide (PTHrP) play key roles in the regulation of cartilage cell (chondrocyte) differentiation. Previous studies have established that Ihh coordinates chondrocyte differentiation indirectly by activating PTHrP expression, which in turn delays their transition to the terminal hypertrophic state. In the present study, using an in vitro system of cultured CFK-2 chondrocytic cells, we have explored the possibility that Ihh may also influence chondrocytic differentiation directly and investigated how this process is modulated by PTHrP, Engrailed-1 (En-1), and heparan sulfate glycosaminoglycans (HS-GAG). We show that Ihh signaling enhances, rather than inhibits, expression of markers of chondrocytic differentiation and that an inactivating missense mutation mapping to its NH2-terminal domain (N-IhhW160G) abolishes this capacity. Moreover, activation of protein kinase A (PKA) by PTHrP-signaling also perturbs Ihh-mediated differentiation. Indicative of a novel regulatory mechanism, Ihh in turn downregulates PKA activity downstream of the PTH/PTHrP-receptor (PTH1R). En-1, detected in prehypertrophic growth plate chondrocytes in situ, influences chondrocytic differentiation and impedes Hh-signaling in vitro, suggesting a novel role for this protein in cartilage development. Finally, we show that HS-GAGS are required for proper mediation of Hh-signaling and thus also participate in modulating chondrocytic differentiation. Taken together, this study provides experimental evidence that Ihh harbors the capacity to directly induce rather than impede chondrogenic differentiation, and that this function is modulated by the cellular actions of PTHrP, En-1, and HS-GAG.

From our everyday life, we know that our hearts beat with a rhythm which is not perfectly periodic. Even an isolated spontaneously beating cardiac cell, devoid of neural, hormonal, and intracardiac regulatory input, does not beat perfectly regularly. I investigate the hypothesis that the beat-to-beat fluctuations in transmembrane potential of spontaneously beating cardiac cells are due to stochastic gating of the ionic channels in the cell membrane. / Recordings of transmembrane potential from small clusters of spontaneously beating 7-day-old embryonic chick ventricular cells were analyzed to characterize the voltage waveform and the regularity of beating. I constructed a deterministic Hodgkin-Huxley-type ionic model which reproduces spontaneous activity in our experimental recordings, as well as the experimental results of applying various ion channel blockers (D-600, almokalant, and Ba2+). The model consists of six currents: a calcium current (ICa), three potassium currents (IKs, I Kr, IK1), a background current ( Ib), and a seal-leak current (I seal). / The deterministic Hodgkin-Huxley-type model was then reformulated into a stochastic single-channel model. The single-channel model reproduces the irregularity of beating seen experimentally: e.g. the coefficient of variation of interbeat interval was 4.4% vs. 3.9% in the clusters. In the model, IKs is the current giving the major contributions to fluctuations in interbeat interval. / Phase resetting of the spontaneous activity of cardiac pacemaker cells by a brief stimulus pulse was simulated in Hodgkin-Huxley-type models and single-channel models of slow-upstroke (central) and fast-upstroke (peripheral) rabbit sinoatrial node cells. In the Hodgkin-Huxley-type models the phase-resetting response is continuous, but can be extremely delicate in the fast-upstroke model, in that a tiny difference in the stimulus timing can change the stimulus response from a delayed action potential to an advanced one. Therefore, the noise in the fast-upstroke single-channel model can cause a stimulus with fixed amplitude and fixed timing to have widely different effects: sometimes it will induce an action potential but in other cases it will delay an action potential, as seen previously in experiments on cardiac preparations.