RESUMEN
The gene Tousled of Arabidopsis Thaliana encodes a protein kinase which, when mutated, results in abnormal flower development. From a library of mRNAs that are translationally upregulated by overexpression of the translation initiation factor 4E, we identified a mammalian Tousled Like kinase (TLK1B). The human TLK1B mRNA contains a 5'UTR 1088-nt-long with two upstream AUG codons, and was found to be very inhibitory for translation. The TLK1B protein localizes almost exclusively to the nuclei. TLK1B overexpression in mammalian cells rendered them more resistant to ionizing radiation (IR). Purified TLK1B phosphorylated histone H3 at S(10) with high specificity both in a mix of core histones and in isolated chromatin, suggesting that histone H3 is a physiological substrate for TLK1B. Moreover, overexpression of TLK1B in transfected cells resulted in a higher degree of H3 phosphorylation. Expression of TLK1B in a yeast strain that harbors a temperature-sensitive mutation of the major H3 kinase, Ipl1, complemented the growth defect; restored normal levels of histone H3 phosphorylation; and increased their resistance to IR. Phosphorylation of H3 has been linked to the activation of the immediate-early genes upon mitogenic stimulation, and to chromatin condensation during mitotic/meiotic events. A possible role for TLK1B in radioprotection is discussed.
Asunto(s)
Histonas/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Tolerancia a Radiación/genética , Regiones no Traducidas 5' , Animales , Células CHO , Cricetinae , Regulación de la Expresión Génica , Biblioteca de Genes , Prueba de Complementación Genética , Humanos , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Homología de SecuenciaRESUMEN
Current evidence points to the existence of multiple processes for bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an alpha-gustducin (Galpha(gust))-mediated stimulation of phosphodiesterase in bitter taste transduction. Additionally, a taste-enriched G protein gamma-subunit, Ggamma(13), colocalizes with Galpha(gust) and mediates the denatonium-stimulated production of inositol 1,4,5-trisphosphate (IP(3)). Using quench-flow techniques, we show here that the bitter stimuli, denatonium and strychnine, induce rapid (50-100 ms) and transient reductions in cAMP and cGMP and increases in IP(3) in murine taste tissue. This decrease of cyclic nucleotides is inhibited by Galpha(gust) antibodies, whereas the increase in IP(3) is not affected by antibodies to Galpha(gust). IP(3) production is inhibited by antibodies specific to phospholipase C-beta(2) (PLC-beta(2)), a PLC isoform known to be activated by Gbetagamma-subunits. Antibodies to PLC-beta(3) or to PLC-beta(4) were without effect. These data suggest a transduction mechanism for bitter taste involving the rapid and transient metabolism of dual second messenger systems, both mediated through a taste cell G protein, likely composed of Galpha(gust)/beta/gamma(13), with both systems being simultaneously activated in the same bitter-sensitive taste receptor cell.
Asunto(s)
Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/metabolismo , Transducción de Señal/fisiología , Gusto/fisiología , Transducina/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Glicinérgicos/farmacología , Isoenzimas/inmunología , Ratones , Ratones Endogámicos , Fosfolipasa C beta , Compuestos de Amonio Cuaternario/farmacología , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/efectos de los fármacos , Estricnina/farmacología , Papilas Gustativas/enzimología , Transducina/inmunología , Fosfolipasas de Tipo C/inmunologíaRESUMEN
OBJECTIVES: To determine the cellular localization in male and female axillary tissue for apocrine secretion odor-binding proteins 1 (ASOB1) and 2 (ASOB2) and the electrophoretic pattern of female apocrine proteins and to begin characterization of the ASOB1 protein. DESIGN: Immunohistochemical techniques were used with biopsy samples from axillary tissue of male and female subjects. Immunological techniques and microsequencing were used to characterize several of the proteins in male and female apocrine secretions. SETTING: A university medical center. PARTICIPANTS: Healthy male and female volunteers who donated apocrine secretions and/or axillary tissue. RESULTS: Specific immunoreactivity was localized only to the apocrine glands in both sexes. Furthermore, only preabsorption with a mixed apocrine secretion sample eliminated all immunoreactivity. The electrophoretic pattern of proteins in female apocrine secretions is similar to that in male secretions. Western blotting of the separated proteins from female samples using serum samples containing antibodies to ASOB1 and ASOB2 yielded identical results to those found with separated proteins from male samples. Partial sequence data obtained from the N-terminus of ASOB1 suggested that it shares homology with the alpha-chain of apolipoprotein J (Apo J). Apocrine secretion odor-binding protein 1 is not immunologically similar to ApoJ, but 2 other apocrine secretion proteins are. CONCLUSIONS: Male and female subjects appear to have the same glycoprotein carriers for (E)-3-methyl-2-hexenoic acid localized to the apocrine glands. The N-terminal sequence for ASOB1 may be homologous to Apo J, but it is not immunologically similar to it. However, 2 other proteins in the apocrine secretion appear to be the monomer and dimer forms of Apo J.
Asunto(s)
Glándulas Apocrinas/metabolismo , Chaperonas Moleculares , Odorantes/análisis , Precursores de Proteínas/análisis , Receptores Odorantes/análisis , Adulto , Secuencia de Aminoácidos , Anticuerpos/sangre , Glándulas Apocrinas/química , Glándulas Apocrinas/inmunología , Axila , Western Blotting , Clusterina , Femenino , Glicoproteínas/química , Glicoproteínas/inmunología , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Precursores de Proteínas/química , Receptores Odorantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores SexualesRESUMEN
Although the precise mechanisms of the conversion of predentin to dentin are not well understood, several lines of evidence implicate the noncollagenous proteins (NCPs) as important regulators of dentin biomineralization. Here we compared the in vivo temporospatial expression patterns of two dentin NCP genes, dentin matrix protein 1 (Dmp1), and dentin sialophosphoprotein (DSPP) in developing molars. Reverse transcription-polymerase chain reaction was performed on embryonic day 13 to 1-day-old first molars using Dmp1- and DSPP-specific primer sets. Dmp1 transcripts appeared at the late bud stage, while DSPP mRNA was seen at the cap stage. Expression of both genes was sustained throughout odontogenesis. In situ hybridization analysis revealed interesting differences in the expression patterns of these genes. While Dmp1 and DSPP showed coexpression in young odontoblasts before the start of mineralization, the expression of these genes was notably distinct at later stages. Dmp1 expression decreased in secretory odontoblasts after the appearance of mineral, while high levels of DSPP were sustained in odontoblasts. In early secretory ameloblasts, DSPP expression was transient and down-regulated with the appearance ofdentin matrix. Interestingly, Dmp1 expression became evident in ameloblasts during the maturative phase of amelogenesis. In contrast to Dspp expression that was tooth-specific, Dmp1 was expressed by osteoblasts throughout ossification in the skeleton. Probes directed to the "DSP" and "DPP" regions of the DSPP gene showed identical patterns of mRNA expression. These data show that the developmental expression patterns of Dmp1 and DSPP are distinct, implying that these molecules serve different biological functions in vivo.
Asunto(s)
Fosfoproteínas/genética , Animales , Proteínas de la Matriz Extracelular , Expresión Génica/genética , Expresión Génica/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Hibridación in Situ , Ratones , Minerales/metabolismo , Odontogénesis/genética , Odontogénesis/fisiología , Osteogénesis/genética , Osteogénesis/fisiología , Reacción en Cadena de la Polimerasa , Alveolos Pulmonares/química , Alveolos Pulmonares/embriología , Alveolos Pulmonares/metabolismo , ARN/análisis , ARN/genética , Sondas ARN , Factores de TiempoRESUMEN
Certain eukaryotic cells can sense changes in their extracellular Ca2+ concentration through molecular structures termed Ca(2+)-sensing receptors (CaRs). We have shown recently that in the bone-resorbing osteoclast, a unique cell surface-expressed ryanodine receptor (RyR), functions as the CaR. The present study demonstrates that the sensitivity of this receptor is modulated by physiological femtomolar concentrations of the bone-conserving hormone, calcitonin. Calcitonin was found to inhibit cytosolic Ca2+ responses to both Ca2+ and Ni2+. The latter inhibition was mimicked by amylin (10(-12) M), calcitonin gene-related peptide (10(-12) M), cholera toxin (5 micrograms/l) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) (2.5 x 10(-4) or 5 x 10(-4) M) and was reversed by the protein kinase A phosphorylation inhibitor, IP-20. Finally, using a quench flow module, we showed that cellular cAMP levels rise to a peak within 25 ms of calcitonin application; this is consistent with the peptide's rapid effect on CaR activation. We conclude, therefore, that cAMP plays a critical role in the control of CaR function by calcitonin.
Asunto(s)
Calcitonina/farmacología , Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Espacio Extracelular/metabolismo , Osteoclastos/metabolismo , Animales , Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Proteínas de Unión al Calcio/antagonistas & inhibidores , AMP Cíclico/metabolismo , Citosol/metabolismo , Níquel/farmacología , Concentración Osmolar , Ratas , Ratas WistarRESUMEN
The tasting of bitter compounds may have evolved as a protective mechanism against ingestion of potentially harmful substances. We have identified second messengers involved in bitter taste and show here for the first time that they are rapid and transient. Using a quench-flow system, we have studied bitter taste signal transduction in a pair of mouse strains that differ in their ability to taste the bitter stimulus sucrose octaacetate (SOA); however, both strains taste the bitter agent denatonium. In both strains of mice, denatonium (10 mM) induced a transient and rapid increase in levels of the second messenger inositol 1,4,5-trisphosphate (IP3) with a maximal production near 75-100 ms after stimulation. In contrast, SOA (100 microM) brought about a similar increase in IP3 only in SOA-taster mice. The response to SOA was potentiated in the presence of GTP (1 microM). The GTP-enhanced SOA-response supports a G protein-mediated response for this bitter compound. The rapid kinetics, transient nature, and specificity of the bitter taste stimulus-induced IP3 formation are consistent with the role of IP3 as a second messenger in the chemoelectrical transduction of bitter taste.