Regulation of neurotrophin receptor expression by retinoic acid in mouse sympathetic neuroblasts

We have studied the effect of retinoic acid on the expression of the neurotrophin receptors trkA, trkC, and p75 by neuroblasts and neurons at different axial levels along the embryonic mouse paravertebral sympathetic chain. In dissociated cultures of sympathetic neuroblasts, retinoic acid inhibited the developmental increase in trkA mRNA expression and the developmental decrease in trkC mRNA expression that normally occurs in these cells but did not affect p75 mRNA expression. At higher concentrations, retinoic acid also increased the proliferation of sympathetic neuroblasts. After sympathetic neuroblasts became postmitotic, retinoic acid no longer affected receptor expression. Studies with retinoic acid receptor agonists and antagonists indicated that the effects of retinoic acid on neurotrophin receptor expression were mediated mainly by alpha retinoic acid receptors, not beta or gamma receptors. The observation that alpha-antagonists increased trkA mRNA expression in intact sympathetic ganglion explants suggests that endogenous retinoic acid is a physiological regulator of trkA receptor expression.     

Differential expression of transcripts encoding retinoid binding proteins and retinoic acid receptors during placentation of the mouse

An isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of human growth hormone (hGH) directly in osmotic shock fluids is described. This methodology allows an initial rapid evaluation of the quality and quantity of hGH being secreted in the bacterial periplasmic space right after, or even during fermentation. Considering that RP-HPLC does not identify size isomers, these were determined via a parallel run of the same osmotic shock fluid on high-performance size-exclusion chromatography, coupled with radioimmunoassay, of the eluted fractions. The methodology provides a complete picture, within 24 h from the beginning of the fermentation process, of the recombinant protein being produced with respect to its activity, identity, yield, and hGH-related contaminants. These latter include sulfoxide and desamido derivatives, dimer and high-molecular-mass forms.     

Molecular cloning of a zinc finger gene eZNF from a human inner ear cDNA library, and in situ expression pattern of its mouse homologue in mouse inner ear

A mathematical model of posttetanic processes launched by rhythmic stimulation of the excitatory and inhibitory inputs to the dendritic spine of a pyramidal neuron in hippocampal field CA3 was used to study conditions for modifying the efficiency of the inhibitory input. The level of dephosphorylation of GABAa and GABAb receptors, which determines the GABA sensitivity of these receptors, was shown to depend on the Ca(2+)-dependent ratio of active protein kinases and protein phosphatases; the level of dephosphorylation decreased monotonically as the intracellular Ca2+ increased. Posttetanic increases and decreases in the Ca2+ concentration, as compared with the level achieved during the previous stimulation, led to increases or decreases respectively in the number of dephosphorylated GABA receptors and to induction of long-term potentiation and depression, respectively, in the efficiency of inhibitory transmission. The extent of the modification effect depended on the ratio of the quantities of inhibitory and excitatory mediators in the synaptic cleft. At very low or very high GABA concentrations, modification of inhibitory transmission was insignificant.     

Correlation between loss of middle ear bones and altered goosecoid gene expression in the branchial region following retinoic acid treatment of mouse embryos in vivo

Various methods are now available to identify the molecular partners of the component of a signal transduction pathway. Some interactions, however, can be technically difficult to detect because they depend upon transient tyrosine phosphorylation. Here, we present a simple affinity chromatography approach based on synthetic phosphopeptides to purify potential partners of phosphotyrosine-containing proteins. With this approach, we confirm the previously characterized interaction between Grb2 and the EGF receptor, and we identify novel partners of the IGF-1 receptor and of the JAK proteins. Methenyltetrahydrofolate synthetase (MTHFS) was identified as a potential mediator of IGF-1R dependent transformation. P85alpha, the regulatory subunit of PI3 kinase, was identified as one of four proteins recruited by a phosphopeptide mimicking a motif conserved in all JAK family members.     

Compartment-selective sensitivity of cardiovascular morphogenesis to combinations of retinoic acid receptor gene mutations

Several aspects of normal cardiovascular development require signaling by the vitamin A metabolite retinoic acid. We have previously established germ-line mutations in mice in the genes that encode the RAR alpha 1, RAR beta, and RXR alpha retinoic acid receptors as a means of studying the function of these receptors in vivo. Although mutation of RXR alpha results in fetal ventricular defects, the RAR alpha 1 and RAR beta mutations are apparently nonphenotypic in the heart and elsewhere. In this study, we have established and analyzed combinations of these receptor gene mutations. Malformations of the ventricular chamber (chamber hypoplasia and muscular ventricular septal defects), conotruncus (double-outlet right ventricle, transposition, and membranous ventricular septal defects), aortic sac (persistent truncus arteriosus and aorticopulmonary window), and aortic arch-derived arteries were recovered in various combinations of the RAR alpha 1, RAR beta, and RXR alpha gene mutations. Depending on the combination of receptor mutations, selective defects were obtained in specific cardiovascular compartments, suggestive of differential expression or function of each receptor within domains of the developing heart.     

Antisense retinoic acid receptor gamma-1 oligonucleotide enhances chondrogenesis of mouse limb mesenchymal cells in vitro

Retinoic acid receptor (RAR) gamma gene is expressed in the precartilaginous cells during chondrogenesis in mouse embryos, but the role of the gene products is still unclear. To examine the role during chondrogenesis, we isolated mesenchymal cells from the limb bud of mouse embryos and exposed them to antisense RAR gamma-1 oligodeoxynucleotide in micromass culture. The antisense oligodeoxynucleotide inhibited RAR gamma-1 protein expression and enhanced chondrogenesis in the exposed cells. These results suggest that the complex of RAR gamma-1 protein and its ligand RA acts as a suppressor of the chondrogenesis in the limb development.     

Distribution of Na,K-ATPase is normal in the inner ear of a mouse with a null mutation of the glucocorticoid receptor

This study was performed in order to test the hypothesis that the glucocorticoid hormone stimulates the formation of Na,K-ATPase in the inner ear of the mouse. An immunohistochemical study with respect to the presence and distribution of glucocorticoid receptors (GR) and Na,K-ATPase in the vestibular and cochlear regions of the inner ear was performed on a C57BL mouse with a null mutation of the glucocorticoid receptor (GR mutant mouse). The wild type C57BL mouse and the CBA mouse served as normal controls. As expected, the homozygous GR mutant mouse showed no specific staining for GR in the inner ear. The heterozygous GR mutant mouse showed faint staining of GR in the spiral limbus, the spiral ganglion, the organ of Corti and the utricle. This staining was markedly less than in the wild type C57BL mouse. Antibody labelling of Na,K-ATPase in the inner ear showed no significant difference between the homozygous and the heterozygous GR mutant mouse as compared to the control wild type C57BL mouse or the CBA mouse. Although earlier studies have shown a positive correlation between levels of glucocorticoid hormone in serum and the concentration of Na,K-ATPase in the inner ear, the hypothesis that glucocorticoid hormones alone stimulate the formation of Na,K-ATPase in the inner ear could not be confirmed by this study. Thus other regulating substances must be considered.     

Alterations in the distributions of the basement membrane components after secondary inner ear immune reaction in guinea pigs

Alterations in the distributions of type IV collagen (C-IV), laminin (La), and fibronectin (Fib), which are important components of the basement membrane, in the inner ear following secondary endolymphatic sac immune response were studied immunohistologically using control animals for comparison. Endolymphatic hydrops developed following direct secondary keyhole limpet hemocyanin (KLH) challenge to the endolymphatic sac in systematically pre-sensitized animals. In the endolymphatic sacs of control animals, C-IV and La were localized continuously just under epithelial cells whereas Fib was present intermittently in subepithelial connective tissue. In the endolymphatic sac, following secondary KLH challenge, linear subepithelial localizations of C-IV and La were interrupted, thinner and more loosely aggregated with numerous inflammatory cellular infiltrates on days 2-4. Following these changes, endolymphatic hydrops in the cochlea developed and peaked on days 4-7. On days 1-7, Fib was strongly but sporadically localized in subepithelial cells. These results suggest that C-IV and La may play important roles in the regulation of endolymph whereas Fib may be related to the restoration of injured endolymphatic sac tissue in animals exposed to a secondary challenge.     

Extracellular nucleotide signaling in the inner ear

Extracellular nucleotides, particularly adenosine 5'-triphosphate (ATP), act as signaling molecules in the inner ear. Roles as neurotransmitters, neuromodulators, and as autocrine or paracrine humoral factors are evident. The diversity of the signaling pathways for nucleotides, which include a variety of ATP-gated ion channels (assembled from different subtypes of P2X-receptor subunit) and also different subtypes of G protein-coupled nucleotide receptors (P2Y receptors) supports a major physiological role for ATP in the regulation of hearing and balance. Almost invariably both P2X and P2Y receptor expression is apparent in the complex tissue structures associated with the inner-ear labyrinth. However P2X-receptor expression, commonly associated with fast neurotransmission, is apparent not only with the cochlear and vestibular primary afferent neurons, but also appears to mediate humoral signaling via ATP-gated ion channel localization to the endolymphatic surface of the cochlear sensory epithelium (organ of Corti). This is the site of the sound-transduction process and recent data, including both electrophysiological, imaging, and immunocytochemistry, has shown that the ATP-gated ion channels are colocalized here with the mechano-electrical transduction channels of the cochlear hair cells. In contrast to this direct action of extracellular ATP on the sound-transduction process, an indirect effect is apparent via P2Y-receptor expression, prevalent on the marginal cells of the stria vascularis, a tissue that generates the standing ionic and electrical gradients across the cochlear partition. The site of generation of these gradients, including the dark-cell epithelium of the vestibular labyrinth, may be under autocrine or paracrine regulation mediated by P2Y receptors sensitive to both purines (ATP) and pyrimidines such as UTP. There is also emerging evidence that the nucleoside adenosine, formed as a breakdown product of ATP by the action of ectonucleotidases and acting via P1 receptors, is also physiologically significant in the inner ear. P1-receptor expression (including A1, A2, and A3 subtypes) appear to have roles associated with stress, acting alongside P2Y receptors to enhance cochlear blood flow and to protect against the action of free radicals and to modulate the activity of membrane conductances. Given the positioning of a diverse range of purinergic-signaling pathways within the inner ear, elevations of nucleotides and nucleosides are clearly positioned to affect hearing and balance. Recent data clearly supports endogenous ATP- and adenosine-mediated changes in sensory transduction via a regulation of the electrochemical gradients in the cochlea, alterations in the active and passive mechanical properties of the cells of the sensory epithelium, effects on primary afferent neurons, and control of the blood supply. The field now awaits conclusive evidence linking a physiologically-induced modulation of extracellular nucleotide and nucleoside levels to altered inner ear function.     

Developmental expression of alpha 9 acetylcholine receptor mRNA in the rat cochlea and vestibular inner ear

Expression of alpha9 acetylcholine receptor (AChR) mRNA was studied by in situ hybridization in the rat adult and developing cochlea and vestibular inner ear. Alpha9 AChR mRNA was first observed in cochlear hair cells (HCs) at embryonic day 18 (E18), increased markedly after birth, stayed high until postnatal day 10 (P10), and decreased to substantially lower adult levels by P14. High levels of alpha9 AChR mRNA expression were also noted in the developing nonneuronal structures of the inner sulcus, chondrocytes, and/or osteoblasts in the cochlear capsule and interscalar laminae. Both developing and adult bone marrow cells also expressed intense alpha9 AChR mRNA. In the vestibular system, alpha9 AChR mRNA was first observed in HCs at E16 in all sensory epithelia, increased to its highest levels by P0-P4, then decreased slightly to reach adult levels by P10. The results are consistent with the alpha9 AChR subserving efferent neurotransmission to both cochlear and vestibular HCs. The observation of alpha9 AChR mRNA in cochlear HCs 2 weeks prior to functional onset in the cochlea further suggests that expression of this gene is not related to HC activity. The observation of substantial nonneuronal expression of alpha9 AChR mRNA suggests that this receptor also has functions separate from its role in neurotransmission.     

Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor beta 2 induction

We previously showed the involvement of retinoic acid receptor alpha (RAR alpha) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of RAR alpha. Here, we show that the protein synthesis inhibitor, cycloheximide completely blocks the induction of t-PA by RA, which points to the need of an intermediary protein in t-PA stimulation. This intermediary protein is likely to be RAR beta 2 on the basis of the following findings: (1) the induction of RAR beta by RA exactly precedes that of t-PA; (2) HUVECs with elevated RAR beta mRNA levels show an undelayed t-PA induction on stimulation with RA, and this response can be almost completely inhibited with an RAR antagonist; and (3) an antisense oligodeoxynucleotide against the translation initiation site of RAR beta 2 mRNA greatly reduces the t-PA induction by RA. Thus, induction of t-PA by RA in HUVECs involves a 2-step mechanism requiring induction of RAR beta 2 via RAR alpha, followed by induction of t-PA synthesis via RAR beta 2. Each of these steps is shown to have a different activation profile with RA and 9 cis RA.     

Dissecting the frog inner ear with Gaussian noise. II. Temperature dependence of inner ear function

Wiener kernel analysis was used to characterize the auditory pathway from tympanic membrane to single primary auditory nerve fibers in the European edible frog, Rana esculenta. Nerve fiber signals were recorded in response to white Gaussian noise. By cross-correlating the noise stimulus and the nerve fiber response, we computed (1) the full second-order Wiener kernel, and (2) the diagonals of the zeroth- to fourth-order Wiener kernels. These diagonals are usually referred to as polynomial correlation functions. The measured Wiener kernels were fitted with a 'sandwich' model. A new fitting procedure was used to compute the response characteristics of (1) the first filter, (2) the static nonlinearity, and (3) the second filter, which form the functional components of the model. The first filter is a bandpass filter. In the majority of low frequency fibers, with best excitatory frequency (BEF) < 800 Hz, this filter was tuned to two frequencies. This dual tuning mechanism gives rise to 'off-diagonal' components in the second-order Wiener kernel. The static nonlinearity resembles a rectifier, and is dominated by second-order (quadratic) nonlinearity. As a function of BEF, the shape of the nonlinearity changes systematically. Finally, the last filter in the model was a low pass filter. Across fibers, its cutoff frequency f-3dB ranged from 106 to 434 Hz.     

Cloning and characterization of a mouse brain calcitonin receptor complementary deoxyribonucleic acid and mapping of the calcitonin receptor gene

We have identified and characterized a mouse brain calcitonin receptor (CTR) complementary DNA (cDNA). This cDNA encodes a receptor protein that, after expression, has high affinity binding for salmon calcitonin (Kd approximately, 12.5 nM) and is coupled to adenylate cyclase. The binding affinity of this expressed receptor for salmon calcitonin is lower than that described for the previously cloned porcine renal and human ovarian CTRs, but is similar to that of the recently described rat brain CTR, designated the C1b form of the receptor. Analysis of the deduced structure of the mouse brain CTR reveals that it is highly related to the other CTR cDNAs that belong to a distinct family of G-protein-coupled receptors with seven transmembrane-spanning domains. The major structural feature that distinguishes the mouse cDNA clone from the other CTRs is the presence of a consecutive 111-basepair nucleotide sequence that encodes a 37-amino acid sequence which is predicted to localize to the first extracellular loop between the second and third transmembrane-spanning domains. We have mapped the CTR gene in the mouse to the proximal region of chromosome 6, which is homologous to the 7q region of human chromosome 7; only a single CTR gene was identified. Preliminary analysis of the mouse CTR gene reveals that it is complex, consisting of multiple exons separated by lengthy introns that would allow for splice variants consistent with the existence of multiple CTR isoforms predicted from the CTR cDNA clones. The differential cellular and tissue distribution of these functionally distinct CTR isoforms provides the molecular basis for the previously reported widespread distribution and functional heterogeneity of the CTR.     

Regulation of IRF and STAT gene expression by retinoic acid

Ex vivo production of cytokines as determined by whole blood stimulation and supernatant ELISA is partly determined by heritability. To assess the ability of this system to distinguish between high and low producers the laboratory error and individual variation were investigated. Whole blood samples from healthy volunteers were collected using endotoxin-free tubes and were incubated with 0 to 1000 ng/ml lipopolysaccharide concentrations for 4 and 24 h, and subsequently centrifuged. In the supernatants, TNF-alpha and IL10 were measured by ELISA. Coefficients of variation for the day-to-day variation in the blood sampling, transport and stimulation as well as in the whole blood stimulation per se ranged from 7.5% to 12.3%. The intra-individual variation was 15% (TNF-alpha) and 19% (IL10) in contrast to the inter-individual variation of, on average, 35%. No interchanging of ranks between high and low producers was observed after repeating the whole blood stimulation on distinct days. The whole blood stimulation system is able to distinguish high and low producers of TNF-alpha and IL10.