The phase resetting effects of stimulating the superior laryngeal nerve at different phases of the respiratory cycle in cats were measured in terms of the latency of onset of the cycle following stimulation. Fixed-delay stimulation was also used; for certain combinations of delay, stimulus intensity, and cycles between stimuli, it resulted in (1) a variable, rather than consistent, response, and (2) a transient increase in cycle duration during and after stimulation. Phase resetting and fixed-delay stimulation of a simple three-phase model for neural rhythm generation produce responses that are qualitatively similar to those obtained experimentally. / We consider the dynamical properties of a class of theoretical models of neural networks that have the same mathematical formulation as the above three-phase model, but consist of a larger number of randomly connected elements. A simple transformation of these models shows correspondence with previous neural network models and enables a theoretical analysis of steady states and cycles. Complex aperiodic dynamics are found in networks consisting of 6 or more elements.

The pro-opiomelanocortin gene encodes protein precursor expressed primarily by the anterior pituitary corticotrophs and intermediate lobe melanotrophs. Pituitary expression of the POMC gene provides a paradigm for studying both tissue-specific and hormonally-regulated gene expression. The POMC promoter is composed of at least 13 different regulatory elements binding many different classes of transcription factors. There are several E-box sequences present in the POMC promoter, including elements DE-2C, CE-1A, and CE-1B. E-box elements bind members of the helix-loop-helix family of transcription factors. These factors exhibit either ubiquitous or cell-restricted expression patterns. The DE-2C element binds cell-specific HLH factors termed CUTE (Corticotroph Upstream Transcription Element) binding factors. / This study examines the role of the ubiquitous HLH transcription factors on POMC expression. Northern blot analysis revealed that all of the ubiquitous HLH factors, Pan 1, Pan 2, HEB, and ITF-2 are expressed in POMC-expressing AtT-20 cells, as is the neurally-restricted factor Mash-1. Overexpression of these factors revealed that the ubiquitous factors act on the CE-1B E-box, but not the DE-2C nor CE-1A elements. This finding was confirmed with gel retardation analysis; CE-1B binds overexpressed ITF-2, whereas DE-2C does not. Finally, the activity of the DE-2C element cannot be reconstituted in a non-POMC expressing cell line with the ubiquitous HLH factors alone. Collectively these experiments define a new transcriptional element contributing to POMC gene expression, CE-1B. The ubiquitously expressed members of the HLH family activate POMC transcription through this element; however, these factors alone cannot account for the activity of the CUTE factors.

The cellular activity in bone and cartilage tissue is controlled by substances present in the osseous environment. Using representative cell lines, modulation of osteoblastic and chondrocytic phenotypic markers such as hormone-responsiveness, enzymatic activities, and matrix production by osteotropic factors was examined in vitro. In UMR 106 cells, a rat osteoblast-derived osteosarcoma cell line, a differential distribution of EGF and PTH receptors over proliferating and quiescent cells, respectively, was observed. EGF treatment resulted in expansion of a less differentiated population characterized by decreased PTH- and CT-stimulated adenylate cyclase and binding capacity, and increased CGRP-responsiveness. Using immunocytolysis, a mixed osseous cell population (OBCK6) was derived from fetal rat calvarial cells. Subsequent dilution cloning yielded two cell lines, the CFK1 line with osteoblastic characteristics and the CFK2 line with chondrocytic properties. Although only the OBCK6 cells deposited a mineralized matrix in vitro, organic matrix components were produced by the other two lines. EGF stimulated proliferation in both CFK1 and CFK2 cells. Retinoic acid decreased cell proliferation, PTH-responsiveness, and alkaline phosphatase activity, caused cell shape change and population reorganization, and increased EGF receptors in CFK1 cells. Induction of cartilage specific matrix components (type II collagen, proteoglycan core protein, link protein, and thrombospondin) was observed in CFK2 cells maintained in culture without passaging and could be regulated by EGF, PTH, dexamethasone, and retinoic acid. Thus, peptidergic and steroidal factors have been employed to modulate functional activity and cell cycle kinetics, and have resulted in the characterization of discrete stages of differentiation of osteoblastic and chondrocytic populations in vitro.

Parathyroid glands are responsible for maintaining normal extracellular calcium concentrations through their release of PTH. Calcium and 1,25-(OH)$ rm sb2D sb3$ have been demonstrated to be potent regulators of PTH and CgA synthesis and release. Primary cultures of quiescent bovine parathyroid cells proliferate in response to high concentrations of serum. Next, I examined the role of c-myc in the proliferation of the PT-r cell line, which was cloned from rat parathyroid cells. In order to study the role of c-myc in PT-r cell proliferation more precisely, I used antisense RNA technology to inhibit c-myc mRNA. / In summary, my studies have shown that parathyroid cells respond to selected growth factors. This proliferative response involves increased expression of c-myc, c-fos, c-jun and PTHrP. 1,25-(OH)$ rm sb2D sb3$ inhibits the expression or c-myc, and cell proliferation is inhited. The differentiated parathyroid cell expresses high levels of CgA and PTH. However, during proliferation these high levels are not sustained.

Neurons on layer IV of the primate visual cortex undego changes in connections before and after birth leading to the formation of ocular dominance and orientational selective stripes. We model these changes by an intra-cortical interaction which can in turn be represented by a one-component (Ising-like) model for ocular dominance and a two-component (XY-like) model orientational selectivity. We study these systems numerically by Monte Carlo simulation and by solving Langevin equations. Both models give evidence of stripe, hexagonal, and paramagnetic phases. We discuss the relationship of these phases and of defect formation to experimental results from physiology.

The retina of a newborn rabbit is not fully mature in terms of morphology, chemical composition or capabilities. Taurine is the predominant component of the free amino acid pool of vertebrate retinas. In the rabbit it is present at birth and increases four-fold by maturity reaching levels close to 50 mM in the adult. However, until the present studies its cellular localization was unknown. This study confirmed the postnatal (PN) increase in retinal taurine levels and examined PN changes in the immunocytochemical localization of retinal and optic nerve taurine. I have localized taurine immunoreactivity (taurine-IR) of developing and adult retinas and optic nerves using a highly specific antibody that was developed in our laboratory. / My localization results indicate that the PN quadrupling of taurine content is due not only to photoreceptor cell development but also to the transient and/or stable expression of taurine-IR in other cell types as they differentiate. Particularly striking is the localization in horizontal cells where it is a candidate for a trophic factor in early PN synaptic organization, and in adult retinas as the yet unidentified inhibitory transmitter. The transient localization of taurine-IR in ganglion cell axons within the retina and optic nerve corresponds in timing to the "critical period" for the formation of retinogeniculate connections. (Abstract shortened by UMI.)

The present work was aimed at studying the release of arachidonic acid and formation of lipoxygenase metabolites in rat brain slices maintained in vitro, as well as exploring possible physiological roles for them in the mammalian central nervous system. A particularly active 12-(S)-lipoxygenase activity was found, which could be stimulated by various stimuli including the neurotransmitters norepinephrine and glutamate. Activation of $ alpha$-adrenergic and N-methyl- scD-aspartate (NMDA) receptor subtypes appear responsible for the effect observed in each case. Arachidonic acid on the other hand was found to have profound effects on synaptic transmission, inducing a long-lasting potentiation which appears dependent on the formation of lipoxygenase metabolites. In return, pharmacological conditions which can potentially lead to long-term potentiation (LTP) of synaptic transmission and for most of them activate NMDA receptors also induced arachidonic acid release. As these observations suggest, it is proposed that arachidonic acid and its lipoxygenase metabolites belong to a new group of messengers in the nervous system possibly acting as modulator of synaptic transmission both intra- and transcellularly. This new class of messengers constitutes an essential component of the molecular machinery involved in synaptic plasticity.

Membrane-associated guanylate kinases (MAGUKs) are a family of molecular scaffolds that organize and cluster protein complexes at subcellular specializations. CASK, a multimodular MAGUK containing CamK, PDZ, SH3 and GK domains, is involved in a variety of molecular events including EGF receptor localization and signalling, KIF-17 dependent NMDA receptor containing vesicle transport, presynaptic vesicle exocytosis and basolateral localization of certain transmembrane proteins. CASK also translocates to the nucleus upon interaction with the Tbr-1 transcription factor where it functions as a transcription regulator. We used the CASK PDZ domain in a yeast two-hybrid screen to identify novel members involved in CASK function. TRIP, a TGF-beta Receptor II (TBRII) Interacting Protein that modulates TBRII-dependent gene expression, was identified as a CASK binding protein through its consensus C-terminal PDZ type-II interacting motif. Here we show that CASK, TRIP and TBRII interact in vitro assays, heterologous expression systems and form a tripartite complex in brain.

Due to a 195 fold cardiac overexpression of beta2-Adrenoreceptor (beta2AR), the hearts of transgenic TG4 mice are chronically overstimulated and, indeed, show little stimulatory response to further betaAR agonists. Previous investigations had suggested that signaling from the overexpressed beta 2ARs was dampened in the atria of TG4 mice. Regulators of G-protein Signaling (RGSs) are a family of negative regulators of G-Protein Coupled Receptor (GPCR) signaling that are frequently induced by GPCR stimulation. Using an RT-PCR based strategy, we have identified RGS5 as being a candidate RGS that is up-regulated in the atria of TG4 mice. Northern blot analysis demonstrated that RGS5 levels are 2--3 fold higher in the atria of TG4 mice when compared to their non-transgenic littermates. To further characterize RGS5 expression, we generated an RGS5 specific anti-serum. Results indicate that RGS5 is a housekeeping RGS in the heart and in skeletal muscle but its betaAR mediated induction in the atria suggests that it also has a highly specialized function.

The Na+/H+ exchangers (NHE) mediate the electroneutral exchange of sodium for protons and play integral roles in sodium, acid-base and cell volume homeostasis. Presently, eight isoforms of the NHE (NHE1 to NHE8) have been identified that are targeted to distinct membrane compartments. The focus of this study is to characterize in greater detail the biosynthesis and differential sorting of two closely related organellar NHE isoforms, NHE6 and NHE7. Previous studies have established that NHE7 accumulates in the trans-Golgi network and associated endosomes, whereas the localization of NHE6 remains controversial. In one study, HeLa cells transiently expressing low levels of a green fluorescent protein-tagged construct of NHE6 showed close co-localization with mitochondrion-specific dyes. However, when NHE6 was overexpressed in COS7 cells, significant accumulation was observed throughout the cell in membrane vesicles derived from the endoplasmic reticulum. To further address this discrepancy, NHE6 engineered to contain the influenza virus hemagglutinin (HA) epitope at its C-terminus (NHE6HA), was subcloned into the ecdysone-inducible expression vector pIND and stably transfected into Chinese hamster ovary cells that constitutively express the ecdysone receptor. NHE6HA expression was stimulated by ponasterone A, an ecdysone analogue. The localization of NHE6 was determined biochemically by subcellular fractionation of cell lysates and visually by immunofluorescence confocal microscopy of intact cells using antibodies that recognize the HA-epitope and various organellar specific markers. Similar studies were conducted with an NHE7-inducible mammalian expression system. Our findings indicate that NHE6 is differentially processed through distinct Golgi-dependent and -independent pathways, ultimately accumulating in recycling endosomal vesicles. Little evidence was found to support sorting to mitochondria. Furthermore, we show that NHE6 is synthesiz