HOX proteins are homeodomain-containing transcription factors essential for embryonic patterning. Despite amino acid differences, all HOX homeodomains recognize highly similar sites on DNA. One mechanism by which HOX proteins achieve specificity is through interaction with cofactors of the PBX and MEIS/PREP1 families. Higher order complexes between HOX, PBX and MEIS/PREP1 proteins form in vivo and are essential for target recognition and transcriptional regulation. Another level of control of HOX function is the nuclear availability of its cofactors. This thesis addresses the regulation of the nuclear availability of the PBX protein by MEIS/PREP1 family members. We identified two nuclear localization signals (NLS) in the PBX homeodomain and showed that the NLS are masked in the absence of MEIS/PREP1. Upon a conformational change in PBX induced by MEIS/PREP1 binding, the NLS are exposed and a receptor-mediated active transport of PBX into the nucleus is allowed. This thesis also investigates the mechanisms of transcriptional regulation by the HOX·PBX complexes. We show that HOX·PBX complexes repress transcription and are switched to transcriptional activators in response to cell signaling. We demonstrate that PBX mediates the repression function by recruiting histone deacetylases (HDACs) to HOX target promoters. Inhibition of HDAC activity or stimulation of protein kinase A (PKA) signaling converts the HOX·PBX complex into a net activator of transcription. The activation function is mediated by the HOX protein through its recruitment of CREB-binding protein (CBP), a coactivator with histone acetyl-transferase (HAT) activity. We propose a model whereby HOX·PBX transcriptional activity is determined by cell signaling, and is mediated by the local modification of chromatin structure in the promoter of downstream targets.

The objective of this thesis was to test the hypothesis that the decreased effects of PGE$ sb2$ and PGF$ sb{2 alpha}$ on cerebral metabolism and vasculature in the newborn might result from a deficiency of brain EP and FP receptors, which may be downregulated by the relatively high brain levels of PGE$ sb2$ and PGF$ sb{2 alpha}.$ / This study revealed that the densities of EP and FP receptors and receptor-coupled second messengers in brain synaptosomes and microvessels were much lower in the newborn than in the adult pigs, also the relative distribution of EP receptor subtypes in brain synaptosomes and microvessels differed. For example, in brain synaptosomes, only EP$ sb3$ subtype was present in the newborn, and both EP$ sb2$ and EP$ sb3$ subtypes existed in the adult; EP$ sb1$ subtype was not found in the brain synaptosomes. In contrast, in brain microvessels, more than 80% of EP receptors were of EP$ sb1$ subtype with small amount of EP$ sb3$ subtype. No EP$ sb2$ subtype was detected in the brain microvessels. / In order to determine whether high levels of prostaglandin in the newborn downregulate EP and FP receptors, newborn pigs were treated with cyclooxygenase inhibitors, ibuprofen or indomethacin. The inhibition of prostaglandin synthesis in the newborn pigs significantly increased EP and FP receptor densities as well as receptor-coupled IP$ sb3$ and cAMP production in brain synaptosomes and microvessels of the newborn to levels found in the adult; this effect could be prevented by co-treatment of membranes with stable PG analogs. Vasoconstrictor effects of PGE$ sb2$ and PGF$ sb{2 alpha}$ on cerebral microvessels of the newborn were also increased. / These findings suggest that the relatively low EP and FP receptor densities in the newborn brain are caused by the high levels of prostaglandins and that these receptors and their functions can be upregulated by reducing prostagladin levels.

The mature nervous system is composed largely of two cell types, glial cells and postmitotic neurons. All postmitotic neurons of the mature nervous system derive from proliferating neural precursor cells. To generate a neuron, a precursor must cease dividing and express a number of genes that are characteristic of the neuronal phenotype. How these changes in cell behaviour and phenotype are brought about in mammals is still poorly understood. / This thesis describes experiments that were designed to explore cell intrinsic mechanisms regulating the generation of neurons from neural precursor cells. Specifically, the regulatory region of the rat Talpha1 alpha-tubulin gene, which encodes an isoform of alpha-tubulin expressed in neurons throughout the nervous system immediately following cell cycle exit, was analyzed to identify DNA sequences directing early neuronal gene expression. / A novel 10-nucleotide regulatory sequence, named the neuronal restriction element (NRE), has been identified. In the context of the Talpha1 gene, the NRE inhibits precocious expression in neural precursor cells. Interestingly, the NRE is conserved in the alpha-1 alpha-tubulin gene and is found in a number of neural genes expressed widely and early in development. As such, the NRE may affect the onset time of a battery of neuronal genes and modulate the timing of neuronal differentiation. In vitro , the NRE binds Su(H), a highly conserved transcription factor involved in the repression of neuronal differentiation. / A second novel regulatory element has been identified, the forebrain response element (FRE), which acts to enhance gene expression specifically in the neocortex. The FRE overlaps the NRE and also contains a conserved 30-nucleotide sequence constituting a putative homeodomain recognition sequence. We speculate that the FRE consists of two subelements that act synergistically to promote gene expression in newborn and mature neocortical neurons.

The aim of the studies underlying this thesis was to characterize the muscarinic and adrenergic receptors expressed in rat brain oligodendrocytes' (OLs); determine if ligand binding alters second messenger levels classically associated with these families of receptors, such as inositol phosphates (InsP), intracellular calcium ( (Ca$ rm sp{2+} rbrack sb{i}),$ and cyclic AMP (cAMP); and the role of neurotransmitters, acetylcholine (Ach) or norepinephrine (NE) on oligodendrocyte growth. / CCH (carbachol), a stable Ach analog, caused a concentration and time dependent increase in the accumulation of InsPs and the mobilization of (Ca$ rm sp{2+} rbrack sb{i},$ which was inhibited by atropine, a specific muscarinic antagonist, and was negatively regulated by acute activation of protein kinase C by the phorbol ester TPA. CCH also negatively regulated the $ beta$-adrenergic-stimulated increase in cAMP levels. Using subtype m1 and m2 specific muscarinic receptor oligonucleotide primers RT-PCR confirmed the presence of, at least, these two muscarinic receptor subtypes. CCH caused a time and concentration-dependent increase in c-fos proto-oncogene mRNA levels as determined by Northern blot analysis. The CCH-stimulated c-fos increase was mediated through a non-phorbol ester sensitive PKC isozyme, and was dependent upon intra and extracellular calcium. Moreover, CCH stimulated DNA synthesis in OLPs, as measured by both ($ sp3$H) -thymidine and BrdU incorporation. / Lastly, the NE-stimulated signal transduction pathway was characterized in developing OLPs. Using selective agonists and antagonists, we determined that NE increased the formation of InsPs through $ alpha sb1$ adrenoceptors. We further subclassified the $ alpha sb1$ receptor to the $ rm alpha sb{1A}$ subtype using more selective reagents; WB4101, a selective antagonist for $ rm alpha sb{1A}$ receptors blocked the response to NE, while chloroethylclonidine, an $ rm alpha sb{1B}$ antagonist had no effect. Furthermore, Pertussis toxin, a bacterial toxin that ADP-ribosylates and inactivates certain G-proteins, EGTA, a calcium chelator, or CdCl$ sb2,$ an inorganic calcium channel blocker, all significantly blocked the NE-stimulated InsP formation. Together these results suggest that OLPs express $ alpha sb1$-adrenoceptors characteristic of the $ rm alpha sb{1A}$ subtype. / In toto, these studies demonstrate that developing OLs express functional muscariaic and adrenergic receptors, and suggest that Ach may function as a trophic factor. These results help to define a mechanism whereby neurons, and OLs may use neurotransmitters to communicate both during development and in the mature central nervous system.

During embryogenesis, B cell precursors that have undergone productive Ig V(D)J rearrangement are selected to expand in oligoclonal follicles of the bursa of Fabricius. Because Ig V(D)J recombination in chickens results in minimal diversity, diversity being generated instead by gene conversion in the follicles of the bursa of Fabricius, B cell precursors express a limited range of Ig specificities prior to colonization of the bursa. It has been proposed that recognition of endogenous ligands by this 'pre-diversified' B cell receptor is critical to the progression of normal B cell development. To test this hypothesis, we constructed a truncated IgM receptor (Tmu) lacking the V and Cmu1 domain, which does not associate with Ig L chain proteins nor does it require IgL chains for surface expression, and used a retroviral gene transfer system to introduce it in developing chick embryos. In these embryos, Tmu+ B cell precursors productively colonized bursal follicles as efficiently as B cell precursors expressing endogenous sIg. Furthermore, we detected low but significant levels of IgL VJ rearrangements in Tmu + bursal cells. The analysis of these VJ junctions revealed no selection for in-frame products as these cells are maintained by the Tmu receptor. Interestingly, we showed that the rearranged VL segments derived from Tmu+ bursal cells underwent gene conversion indistinguishably from rearranged VL segments derived from bursal cells expressing endogenous sIg. Taken together, we have ruled out a role for V(D)J encoded determinants in the normal development of B cells in avian embryos. Sequence analysis of 80 IgL VJ segments derived from Tmu+ bursal allowed the unique opportunity to assess the efficiency of gene conversion in vivo, in the absence of selection. Using this system, we demonstrated that >97% of gene conversion events maintain the sequence in-frame. Following hatching, the bursa undergoes morphological changes initiated by the migration of bursal cells ba

The release of vasopressin and oxytocin from the nerve terminals of magnocellular neurosecretory cells (MNCs) is optimised by increases in firing frequency and the adoption of a phasic pattern of firing in the soma. The temporal summation of post-spike depolarising afterpotentials (DAPs) of consecutive action potentials has been proposed to contribute to the generation of phasic firing. There has not been, however, any experimental evidence to support this hypothesis. In this study, a direct blocker of the DAP is introduced as being Cs +. Using this blocker, it is shown that the DAP plays an important role in the generation of phasic firing. Furthermore, these experiments reveal that external Cs+ causes depolarisation of MNCs when the membrane potential is held between action potential threshold and near -80 mV. External Cs+, however, is also known as a classical blocker of the hyperpolarisation activated inward current (IH). If I H is present in rat MNCs, the blockade of the IH by external Cs+ should lead to hyperpolarisation rather than the observed depolarisation. Using a recently introduced blocker of IH, ZD 7288, I show that IH is indeed expressed in rat MNCs, and that it also plays an important role in excitability and phasic firing of these cells. Finally, the ionic basis for the depolarising effects of external Cs + in rat MNCs is investigated. It is concluded in that extracellular Cs+ blocks both IH and a leakage K+ current that contributes significantly to the resting potential.

Atrial natriuretic factor (ANF) and angiotensin II (ANG-II) have multiple biological actions, most of them related, directly or indirectly, to blood pressure regulation and fluid homeostasis. The present work was undertaken to study the regulation of renal glomerular ANF receptors by renal sympathetic nerves and ANG-II, to characterize ANF and ANG-II receptor subtypes in rat renal preglomerular vessels, and to define the precise localization of these receptors in the rat mesentery. Using radioligand binding techniques, we demonstrated that renal denervation up-regulated glomerular ANF receptors, and ANG-II blunted this effect, independently of modifications in plasma ANF levels. Receptor characterization studies showed that rat renal preglomerular vessels and glomeruli do not express detectable amounts of ANP-B receptors, and that they have a different proportion of ANP-A and ANP-C receptors. In glomeruli, 85% of receptors are ANP-C and 15% are ANP-A. In preglomerular vessels, ANP-A receptors represented 60% whereas ANP-C accounted for 40%. The large majority of ANG-II receptors in rat renal preglomerular vessels corresponds to the AT$ sb1$ subtype. In the rat mesentery, saturation binding experiments demonstrated that all ANF and most ANG-II receptors (90%) were localized not in the arteries, but in the surrounding perivascular adipose tissue. Most ANF receptors were ANP-C, and a minor fraction were ANP-A and ANP-B. Competition studies with selective ANG-II antagonists revealed that only the AT$ sb1$ receptor subtype is expressed in mesenteric adipose tissue. We expect our studies may contribute to the understanding of the physiological significance of tissue-specific distribution of receptors for ANF and ANG-II in intrarenal structures and within the entire vascular tree. Further studies are required to comprehend the challenging role of ANF and ANG-II in adipose tissue.

Two rabbit and three guinea pig corticostatic (anti-adrenocorticotrophic) peptides were isolated from bone marrow cells and identified. The third guinea pig peptide proved to be a novel 13-member anti-parallel dimer. Removal of the two C-terminal arginines from rabbit corticostatin 1 lowered the biologic activity but removal of the two N-terminal arginines from the guinea pig peptides was without effect. Immunocytochemical localization of rabbit corticostatin 1 in the rabbit indicated that it was localized in immune tissues such as spleen and bone marrow but also in non-immune tissues such as lung, placenta, adrenal, anterior pituitary and various parts of the brain, Rabbit corticostatin 1 was measured in maternal and fetal tissues and in blood at 24, 27 and 30 days of pregnancy in the rabbit and marked changes were noted with increasing gestation, Rabbit corticostatin 1 did not inhibit the action of angiotensin II or Atrial Natriuretic Peptide but it did inhibit $ alpha$-Melanotrophic Stimulating Hormone binding to specific zona glomerulosa receptors.

No description available.

Anemia is an almost invariable manifestation of chronic renal failure (CRF) and it often contributes substantially to the morbidity of the condition. In its uncomplicated form, this anemia is due primarily to reduced production of erythropoietin (EPO) by the diseased kidney. The present study was carried out in order to determine the relative role in the anemia secondary to CRF of EPO and insulin-like growth factor-1 (IGF-1), a recently recognized important regulatory factor of erythropoiesis in the normal physiological state. / A mouse model of CRF was employed in this investigation. Six weeks after the surgical induction of renal failure, the mice were characterized in terms of biochemical and hematological parameters which included the response to a 3-week treatment with recombinant human EPO (r-HuEPO). Additionally the kidneys, liver and bone marrow were harvested for the determination of the mRNA expression of EPO and IGF-1 as assessed by the reverse transcription polymerase chain reaction followed by Southern blotting. Normal mice and mice rendered anemic by phlebotomy were included in all experiments. (Abstract shortened by UMI.)