RESUMEN
Plant range expansion is occurring at a rapid pace, largely in response to human-induced climate warming. Although the movement of plants along latitudinal and altitudinal gradients is well-documented, effects on belowground microbial communities remain largely unknown. Furthermore, for range expansion, not all plant species are equal: in a new range, the relatedness between range-expanding plant species and native flora can influence plant-microorganism interactions. Here we use a latitudinal gradient spanning 3,000 km across Europe to examine bacterial and fungal communities in the rhizosphere and surrounding soils of range-expanding plant species. We selected range-expanding plants with and without congeneric native species in the new range and, as a control, the congeneric native species, totalling 382 plant individuals collected across Europe. In general, the status of a plant as a range-expanding plant was a weak predictor of the composition of bacterial and fungal communities. However, microbial communities of range-expanding plant species became more similar to each other further from their original range. Range-expanding plants that were unrelated to the native community also experienced a decrease in the ratio of plant pathogens to symbionts, giving weak support to the enemy release hypothesis. Even at a continental scale, the effects of plant range expansion on the belowground microbiome are detectable, although changes to specific taxa remain difficult to decipher.
Asunto(s)
Microbiota , Plantas/microbiología , Rizosfera , Bacterias/genética , Bacterias/aislamiento & purificación , Cambio Climático , ADN Bacteriano/análisis , ADN de Hongos/análisis , Europa (Continente) , Hongos/genética , Hongos/aislamiento & purificación , Microbiología del SueloRESUMEN
The T235 allele of the angiotensinogen gene (AGT) has been associated with hypertension. Blood pressure increases faster over time in black children than in white children, and in adults hypertension is more prevalent in blacks. We sought evidence for a role for angiotensinogen to contribute to racial differences in blood pressure in a study of 148 white and 62 black normotensive children (mean age, 14.8 yr). The frequency of the T235 allele was 0.81 in blacks and 0.42 in whites (chi 2 = 77.3, P = 0.0001). The mean angiotensinogen level was 19% higher in blacks than in whites (P = 0.0001 for males, P = 0.004 for females). Genotype was positively related to serum angiotensinogen in white children (P = 0.0001 for males, P = 0.004 for females), but a similar relationship was absent in blacks where the frequency of M235 may have been too low to discern an association. Longitudinal blood pressure (measured twice yearly) adjusted for body mass index showed a marginally significant relationship to the angiotensinogen level (P = 0.07). An independent relationship of serum angiotensinogen with body mass index (P = 0.0001) and race (P = 0.0003) was also observed. In summary, T235 was more frequent, and the level of angiotensinogen was higher in blacks than in whites. Such a racial difference in the renin-angiotensin system may contribute to the disparity in blood pressure levels of white and black young people.
Asunto(s)
Angiotensinógeno/genética , Población Negra/genética , Variación Genética , Población Blanca/genética , Adolescente , Adulto , Aldosterona/sangre , Alelos , Angiotensinógeno/sangre , Secuencia de Bases , Determinación de la Presión Sanguínea , Niño , Femenino , Frecuencia de los Genes , Humanos , Hipertensión/etiología , Indiana , Estudios Longitudinales , Masculino , Datos de Secuencia Molecular , Renina/sangreRESUMEN
The initial steps involved in mediating the transduction of PTH signal via its G protein-coupled receptors are well understood and occur through the activation of cAMP and phospholipase C pathways. However, the cellular and molecular mechanisms for subsequent receptor desensitization are less well understood. Recently, a new family of GTPase activating proteins known as regulators of G protein signaling (RGS), has been implicated in desensitization of several G protein-coupled ligand-induced processes. At present, it is not known whether any of the RGS proteins play a role in PTH signaling. Using the differential display method, we screened for genes that are selectively expressed after a single s.c. injection of human PTH (1-38) (8 microg/100 g) in osteoblast-enriched femoral metaphyseal spongiosa of young male rats (3-4 weeks old). We found and cloned one full-length complementary DNA that encodes a 211-amino acid RGS protein and shares 97% sequence identity with mouse and human RGS2. Based on sequence similarity, we have designated this clone as rat RGS2. Northern blot analysis confirmed that the expression of RGS2 messenger RNA (mRNA) is rapidly and transiently increased by human PTH (1-38) in both metaphyseal (4-to 5-fold) and diaphyseal (2- to 3-fold) bone, as well as in cultured osteoblast cultures (2- to 37-fold). In vitro, forskolin and dibutyryl cAMP similarly elevated RGS2 mRNA. In vivo, PTH analog (1-31) [which stimulates intracellular cAMP accumulation, PTHrP (1-34), and prostaglandin E2] induced RGS2 mRNA expression; whereas PTH analogs (3-34) and (7-34), which do not stimulate cAMP production, had no effect on expression. In tissue distribution analysis, RGS2 is widely expressed and was detected in all tissues examined (heart, spleen, liver, skeletal muscle, kidney, and testis), with significant expression in two nonclassical PTH-sensitive tissues: the brain, and the heart. After PTH injection, RGS2 mRNA expression was induced in rat bone but not in any of the other tissues examined. These findings demonstrate that RGS2 is regulated by PTH, prostaglandin E2, and PTHrP and that regulation by PTH in bone occurs via the cAMP pathway. Additionally, these results suggest the exciting possibility that increased RGS2 expression in osteoblasts may be one of the early events influencing PTH signaling.
Asunto(s)
Comunicación Autocrina/fisiología , Huesos/fisiología , Hormona Paratiroidea/fisiología , Proteínas RGS/biosíntesis , Secuencia de Aminoácidos , Animales , Northern Blotting , Huesos/metabolismo , Células Cultivadas , ADN Complementario/biosíntesis , ADN Complementario/genética , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Osteoblastos/metabolismo , Poli A/aislamiento & purificación , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
PTH stimulates bone formation in animals and humans, and the expressions of a number of genes have been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon we used differential display RT-PCR and screened for genes that are selectively expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single s.c. injection of human PTH-(1-38) (8 microg/100 g). We show that one of the messenger RNAs that is up-regulated in bone is ADAMTS-1, a new member of the ADAM (A disintegrin and metalloprotease) gene family containing thrombospondin type I motifs. ADAMTS-1 consists of multiple domains common to ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. However, unlike other ADAMs, ADAMTS-1 does not possess a transmembrane or cytoplasmic domain and is a secreted protein. Northern blot analysis confirmed that ADAMTS-1 was up-regulated in both metaphyseal (14- to 35-fold) and diaphyseal (4.2-fold) bone 1 h after PTH-(1-38) injection and returned to control levels by 24 h. We also analyzed the regulation of ADAMTS-1 in response to various PTH/PTH-related peptide (PTHrP) analogs and found that PTH-(1-31) and PTHrP-(1-34), which activate the protein kinase A (PKA) pathway, induce ADAMTS-1 expression 1 h after injection, whereas PTH-(3-34) and PTH-(7-34), which do not activate the PKA pathway, did not regulate expression. To investigate the effect of other osteotropic agents, we analyzed ADAMTS-1 expression after a single dose of PGE2 (6 mg/kg) and found that it was up-regulated 1 h after injection and returned to control levels by 6 h. In vitro ADAMTS-1 is expressed in primary osteoblasts and osteoblastic cell lines, but was not detectable in osteoclasts generated from macrophage colony-stimulating factor/receptor activator of NF-kappaB ligand/transforming growth factor-beta1-treated bone marrow cells. Treatment of UMR 106 osteosarcoma cells with PTH, PGE2, forskolin, or (Bu)2cAMP increased ADAMTS-1 expression 7-, 4-, 5-, and 5-fold, respectively. Also, in vitro treatment with 1alpha,25-dihydroxyvitamin D3 increased ADAMTS-1 expression 3-fold. Tissue distribution analysis showed that ADAMTS-1 is expressed at high levels in many tissues, including the heart, lung, liver, skeletal muscle, and kidney. Taken together, these results demonstrate that ADAMTS-1 is specifically up-regulated in bone and osteoblasts by the osteotropic agents PTH, PTHrP, and PGE2 possibly via the cAMP/PKA pathway. We speculate that the rapid and transient increase in ADAMTS-1 expression may contribute to some of the effects of PTH on bone turnover.
Asunto(s)
Huesos/enzimología , Desintegrinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Metaloendopeptidasas/genética , Hormona Paratiroidea/farmacología , Proteínas ADAM , Proteína ADAMTS1 , Animales , Calcitriol/farmacología , Bovinos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dinoprostona/farmacología , Desintegrinas/metabolismo , Activación Enzimática/efectos de los fármacos , Fémur , Humanos , Cinética , Masculino , Metaloendopeptidasas/metabolismo , Especificidad de Órganos , Osteoblastos/enzimología , Osteoclastos/enzimología , Osteosarcoma/enzimología , Proteína Relacionada con la Hormona Paratiroidea , Fragmentos de Péptidos/farmacología , Proteínas/farmacología , ARN Mensajero/análisis , Ratas , Ratas Endogámicas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Aldosterone production, estimated from urinary excretion of aldosterone and the plasma aldosterone level, was found in a previous cross-sectional study to be lower in black children than white children. The present study examined aldosterone excretion longitudinally to determine whether the aldosterone excretion rate changed with time and if the racial difference in aldosterone excretion persisted. Urine samples were collected every 6 months for up to 5.5 yr in 351 white and 170 black children for measurements of aldosterone, sodium (Na+), and potassium (K+) excretion. Results were expressed per mumol urinary creatinine. Mean values for excretion rates for the total longitudinal period were determined. Na+ excretion was not significantly different in the two groups, whereas K+ excretion was 18% lower in blacks than whites (P = 0.0001). Body weight and urinary Na+ and K+ excretion were significantly related to aldosterone excretion. After adjusting for these variables, the aldosterone excretion rate was 35% lower in blacks than whites (P = 0.0001), a racial difference that did not change with age. Aldosterone excretion rates showed no longitudinal trend to either increase or decrease. The physiological relevance of the lower aldosterone excretion rate in black children remains unknown.
Asunto(s)
Aldosterona/metabolismo , Adolescente , Población Negra , Niño , Preescolar , Femenino , Humanos , Estudios Longitudinales , Masculino , Sodio/metabolismo , Población BlancaRESUMEN
An insertion (I)/deletion (D) polymorphism of the angiotensin I-converting enzyme (ACE) gene that has been associated with certain cardiovascular disorders accounts for nearly half the variation in serum ACE level in white subjects. Whether a similar association of serum ACE with the I/D polymorphism occurs in other racial groups is not known. We studied the I/D polymorphism of ACE in relation to serum ACE activity in 141 white and 62 black healthy, unrelated children and adolescents (mean age, 14.7 years). The mean level of ACE activity in whites homozygous for the D allele was higher than in heterozygotes (P = .002) and in homozygotes for the I allele (P = .0001), consistent with an earlier study. In blacks, on the other hand, no significant difference in serum ACE activity between genotypes was observed. An additional finding was a significantly positive relationship between serum ACE activity and diastolic pressure (P = .009). In children and adolescents, serum ACE activity is related to the ACE gene I/D polymorphism in whites but not in blacks. The results indicate a potentially important ethnic variation in genetic regulation of serum ACE activity and the relationship of the I/D polymorphism to cardiovascular disease.
Asunto(s)
Angiotensina I/metabolismo , Población Negra/genética , Peptidil-Dipeptidasa A/sangre , Peptidil-Dipeptidasa A/genética , Polimorfismo Genético , Población Blanca/genética , Adolescente , Alelos , Presión Sanguínea/genética , Estudios de Cohortes , Femenino , Genotipo , Humanos , MasculinoRESUMEN
A variant of the angiotensinogen gene (AGT) that encodes for threonine at codon 235 (T235) has been associated with a higher serum angiotensinogen concentration and with hypertension in white subjects. The frequency of T235 is about two times higher in blacks than whites, suggesting that AGT may contribute to the susceptibility to hypertension in blacks more than it does in whites. However, an association of T235 with angiotensinogen level or blood pressure has not been observed in blacks, possibly because the high prevalence of T235 makes it insufficiently informative as a marker. For this reason, we undertook to further differentiate the T235 carrier state by constructing haplotypes with alleles in the 5' upstream region of AGT. One such haplotype, -1074t;T235, showed a significant association with angiotensinogen level in a cohort of black and white children and adolescents (76 blacks, mean age = 12.3 +/- 2.0 [SD] years; 139 whites, mean age = 12.4 +/- 1.8 years). With a linear regression model, the level of serum angiotensinogen was significantly related to body mass index (P = .0017) and the haplotype (P = .0001). Within specific race groups, the haplotype was significantly related to serum angiotensinogen in both the blacks (P = .0277) and whites (P = .0001). The mean level of angiotensinogen was higher in the blacks carrying a single copy of the haplotype than in those without the haplotype (1472.2 +/- 68.4 versus 1274.9 +/- 46.7 nmol angiotensin I/L), a difference that was marginally significant (P = .0609). In the whites, the level of angiotensinogen was also higher in carriers of a single copy than in those with no copy (1527.9 +/- 71.2 versus 1099.2 +/- 20.1 nmol angiotensin I/L) (P = .0003). Serum angiotensinogen level did not increase with two copies of the haplotype, but in each racial group, there were only four individuals who were homozygous. The haplotype showed a marginally significant relation (P = .0757) to the mean of longitudinally determined diastolic pressures adjusted for body mass index, race, sex, and age. In summary, using a haplotype to differentiate further the T235 carrier state, we observed an association of genotype with serum angiotensinogen level and blood pressure in blacks and whites. The findings suggest that AGT may play an important role in blood pressure regulation in both racial groups.
Asunto(s)
Angiotensinógeno/genética , Población Negra/genética , Presión Sanguínea/genética , Hipertensión/genética , Población Blanca/genética , Adolescente , Alelos , Angiotensinógeno/sangre , Niño , Femenino , Humanos , Hipertensión/sangre , MasculinoRESUMEN
Renin and aldosterone secretion is often lower in blacks than in whites, characteristics that resemble a milder form of Liddle syndrome in which a mutation in the amiloride-sensitive epithelial sodium channel (ENaC) of the kidney results in enhanced resorption of sodium. In the present study, we looked for evidence that the intrinsic level of ENaC activity is indeed higher in blacks than in whites. In overnight urine samples collected from young people (249 white and 181 black subjects, mean age 13.4 years), the urinary aldosterone/potassium ratio, which is typically very low in Liddle syndrome, was lower in blacks than in whites: 0.421+/-0.024 (mean+/-SE) versus 0.582+/-0.016 nmol/mmol (P<0.0001). In addition, all but 1 of 5 molecular variants in ENaC were much more common in blacks than in whites. G442V in the beta-subunit, present in 16% of the blacks and in only 1 white, was associated with parameters reflective of a greater Na retention and potentially a higher ENaC activity: a lower plasma aldosterone concentration (P=0.070), a lower urinary aldosterone excretion rate (P=0.052), a higher potassium excretion rate (P=0.048), and a lower urinary aldosterone/potassium ratio (P=0.027). In a second cohort consisting of 126 black and 161 white normotensive subjects and 232 black and 188 white hypertensive subjects, betaG442V did not show a significant association with hypertension (P=0.089). On the other hand, a variant that was twice as common in whites, alphaT663A, was associated with being normotensive both in blacks (P=0.018) and in whites (P=0.034). Expression of either betaG442V or alphaT663A in Xenopus oocytes did not result in a change in basal Na current, consistent with the variants being in linkage disequilibrium with alleles at active loci. In conclusion, several lines of evidence are presented to suggest that ENaC activity is higher in blacks than in whites, which could contribute to racial differences in Na retention and the risk for hypertension.
Asunto(s)
Aldosterona/metabolismo , Hipertensión/genética , Potasio/metabolismo , Canales de Sodio/genética , Canales de Sodio/metabolismo , Adolescente , Aldosterona/sangre , Aldosterona/orina , Población Negra/genética , Presión Sanguínea/genética , Estudios de Cohortes , Células Epiteliales/metabolismo , Exones , Femenino , Humanos , Masculino , Potasio/sangre , Potasio/orina , Renina/metabolismo , Factores de Riesgo , Población Blanca/genéticaRESUMEN
The serotonin 1c (5-HT1C) receptor is found in many brain regions, but is particularly enriched on the epithelial cells of the choroid plexus. A major challenge in neurobiology is to delineate the molecular processes that regulate the specific pattern of neuronal gene expression in the brain. As an initial step towards identifying cis-acting DNA sequences that control the expression of the 5-HT1C receptor, we have isolated the promoter sequence of its gene. Sequence analysis of a 1.8 kb fragment indicated that the 3' end of this fragment overlaps with the 5' untranslated region of the 5-HT1C receptor mRNA, and primer extension using mouse brain poly(A)+ RNA mapped the transcription initiation site within this fragment. There are a number of sequence elements upstream from the transcription initiation site that are homologous to regulatory elements found in other eucaryotic genes. To determine the promoter activity, a plasmid was constructed that contains this fragment as promoter region and the cDNA for the 5-HT1C receptor as the reporter. When injected into the nucleus of Xenopus oocytes, this construct resulted in functional expression of the reporter gene. Primer extension using the RNA extracted from the injected oocytes indicated a single transcription initiation site of the reporter mRNA. These results suggest that the 5-HT1C receptor was functionally expressed under the promoter activity of the 1.8 kb 5' sequence of its gene. This system will be useful for further analysis of the cis-acting elements in the promoter region of the 5-HT1C receptor gene and the trans-acting factors that regulate tissue-specific expression of the receptor.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Iniciación de la Cadena Peptídica Traduccional/genética , Regiones Promotoras Genéticas , Receptores de Serotonina/genética , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Oocitos , XenopusRESUMEN
The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) exerts diverse physiological effects in the central and peripheral nervous systems and in smooth muscle by interacting with pharmacologically distinct membrane receptors. We report here the cDNA cloning of the mouse 5-HT1C receptor and its functional expression in Xenopus oocytes. This receptor possesses the unusual feature of containing eight hydrophobic domains capable of forming membrane-spanning alpha-helices, contrary to the usual '7-helix' paradigm for other membrane receptors that function through coupling to GTP-binding proteins. By hybridization analysis of Chinese hamster x mouse somatic cell hybrid lines, the gene for the receptor, designated Htr1c, has been assigned to the mouse X chromosome.
Asunto(s)
ADN/genética , Ligamiento Genético , Receptores de Serotonina/genética , Cromosoma X , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Biblioteca Genómica , Ratones , Datos de Secuencia Molecular , Oocitos/fisiología , Receptores de Serotonina/química , Agua , XenopusRESUMEN
The sequence of a splice variant of the rat mineralocorticoid receptor (MR) gene is presented. A cDNA clone corresponding to rat MR was isolated from a rat brain cDNA library. Sequence analysis of the region corresponding to the DNA binding domain revealed the presence of a 12 base pair (bp) insertion. Analysis of mRNA from several rat tissues suggests that the variant is less abundant than the wild type in most tissues. The insertion variant is also a product of the human MR gene, the identical splice variant was also observed in human white blood cell mRNA. Unlike other splice variants reported for the MR, this variant alters the encoded protein by the addition of four amino acid residues in the DNA binding domain. The altered protein may influence the affinity of the MR for mineralocorticoid or glucocorticoid response elements.
Asunto(s)
Empalme Alternativo , Encéfalo/metabolismo , Receptores de Mineralocorticoides/biosíntesis , Receptores de Mineralocorticoides/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Biblioteca de Genes , Variación Genética , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , RatasRESUMEN
Isolating a clone from a cDNA or genomic library often involves screening the library by several rounds of plating and filter hybridization. This is not only laborious and time-consuming, but also is prone to artifacts such as false positives commonly encountered in filter hybridization. These problems can be alleviated by using polymerase chain reaction (PCR) in the early rounds of screening prior to conventional filter hybridization. The advantages of PCR screening are threefold: (1) Positive clones are identified by DNA bands of correct sizes in gel, thus avoiding the confusion from false positive spots in filter hybridization; (2) it saves time, especially in initial rounds of screening; and (3) screening of multiple genes can be performed in the same PCR by using appropriate primers for these genes. After the complexity of the phage pool is reduced and the existence of true positives in the pool confirmed, individual clones can be isolated by conventional methods.
RESUMEN
Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC50=20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg) and significantly reduced (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in the JAK2V617F-induced MPN model (TED50=13.7 mg/kg, twice daily). In contrast, LY2784544 showed no effect on erythroid progenitors, reticulocytes or platelets. These data suggest that LY2784544 has potential for development as a targeted agent against JAK2V617F and may have properties that allow suppression of JAK2V617F-induced MPN pathogenesis while minimizing effects on hematopoietic progenitor cells.
RESUMEN
Expression of the gene for tissue factor, the cell-surface initiator of blood coagulation, was examined by stimulating growth-arrested human fibroblasts with serum and measuring changes in the cellular content of tissue factor mRNA, antigen, and activity. Maximum tissue factor mRNA levels were reached within 1 h following serum induction and slowly declined to basal levels from 24 to 48 h after stimulation. The appearance of the tissue factor mRNA was followed by an increase in tissue factor antigen and activity. The parallel rise in antigenically positive protein and procoagulant activity was first observed about 2 h after serum stimulation with a peak at 12 h followed by a slow decline during the next 36 h. The serum-induced synthesis of the tissue factor mRNA was independent of de novo protein synthesis as demonstrated by the increased tissue factor mRNA levels generated in the presence of cycloheximide. The results of this study suggest that the synthesis of tissue factor in human fibroblasts is regulated principally at the level of transcription. In one strain of fibroblasts the activity/antigen ratio, during the period of maximum synthesis, was indistinguishable from that of tissue factor which had been immunoaffinity purified from human brain and reconstituted into phospholipid vesicles. However, during serum starvation the activity/antigen ratio in these cells was significantly reduced. Western blot analysis revealed that in serum-starved cells there was an accumulation of truncated forms of the tissue factor antigen while in the serum-stimulated cells only the full-length antigen was observed.
Asunto(s)
Regulación de la Expresión Génica , ARN Mensajero/biosíntesis , Tromboplastina/genética , Sangre , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/metabolismo , Humanos , Immunoblotting/métodos , Pulmón/citología , Tromboplastina/biosíntesis , Tromboplastina/fisiologíaRESUMEN
Short duration exposure to cellular stresses have been shown to activate p38 mitogen-activated protein kinase (MAPK) in cultured rat ventricular cardiomyocytes and isolated perfused hearts; however, effects of chronic stress on p38 MAPK are not well understood. This study determined whether alterations in the p38 MAPK pathway occurred prior to end-stage human heart failure. The p38 MAPK alpha isoform was detectable in human cardiac tissue. However, carefully controlled analysis of protein and message in this study demonstrated an absence of the p38 MAPK beta -isoform. Low levels of message for the non-SB203580 sensitive p38 MAPK gamma and delta isoforms were also detected in both normal and failing human myocardium. Ischemic and idiopathic end-stage failing human hearts were compared to non-failing hearts for both p38 alpha MAPK protein level and total p38 MAPK activity. Western blotting techniques demonstrated no significant changes in total p38 alpha MAPK content. However, approximately 75% decreases in active/phosphorylated p38 MAPK (P<0.005) were observed in both ischemic and idiopathic failing hearts compared to non-failing hearts. In-gel kinase assays confirmed that activated p38 MAPK, detected by Western blotting, phosphorylated its potential downstream targets. When compared to non-failing hearts, approximately 46% decreases in p38 MAPK phosphorylation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2) were observed in ischemic and idiopathic failing hearts (P=0.03 and P=0.04 respectively). Active p38 MAPK was localized to sarcomeric structures in the cytosol of myocytes by confocal immunofluorescence microscopy. The correlation between decreased MAPKAPK-2 phosphorylation and loss of active p38 MAPK in failing human myocytes suggests that decreases in the activation of p38 MAPK alpha, the predominant cardiac isoform, occur prior to end-stage heart failure.
Asunto(s)
Insuficiencia Cardíaca/enzimología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adulto , Western Blotting , Inhibidores Enzimáticos/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Microscopía Confocal , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/genética , Fosforilación , Isoformas de Proteínas/genética , Piridinas/farmacología , Sarcómeros/inmunología , Sarcómeros/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Hypertrophy is an adaptive response of the heart to myocardial injury or hemodynamic overload that may progress and contribute to cardiac decompensation and eventually to heart failure. The signaling pathways controlling this response in the cardiac myocyte are poorly understood. A data mining effort of a human failed heart cDNA library was undertaken in an effort to identify novel signaling molecules involved in cardiac hypertrophy. This effort identified a novel kinase (MLK7) homologous to the mixed lineage kinase family of proteins. The mixed lineage kinases are mitogen-activated protein kinase kinase kinases (MAPKKKs) which activate stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 kinase pathways. They contain a catalytic domain with homology to both serine/threonine and tyrosine-specific kinases and a dual leucine zipper. MLK7 is identical to leucine zipper and sterile-alpha motif protein kinase (ZAK) through the leucine zipper domain but has a completely divergent COOH-terminus and shares approximately 40% homology with the other MLKs overall. Expression of MLK7 mRNA is most abundant in skeletal muscle and heart, with expression restricted to the cardiac myocyte. The recombinant histidine tagged MLK7 expressed and purified from insect cells exhibited serine/threonine kinase activity in vitro with myelin basic protein as substrate. When expressed in cardiac myocytes, MLK7 activated SAPK/JNK1, and ERK and p38 to a lesser extent. Additionally, MLK7 altered fetal gene expression and increased protein synthesis in cardiac myocytes. These data suggest that MLK7 is a new member of the mixed lineage kinase family that modulates cardiac SAPK/JNK pathway and may play a role in cardiac hypertrophy and progression to heart failure.
Asunto(s)
Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Musculares , Miocardio/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Cardiomegalia/enzimología , Cardiomegalia/metabolismo , Células Cultivadas , Clonación Molecular , ADN Complementario , Femenino , Regulación de la Expresión Génica , Biblioteca de Genes , Corazón/fisiología , Humanos , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas/fisiología , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Miocardio/citología , Miocardio/metabolismo , Fosforilación , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Ratas , Proteínas Recombinantes/metabolismo , Distribución TisularRESUMEN
Angiotensinogen (ANG) is the specific substrate of the renin-angiotensin system, a major participant in blood pressure control. We have identified a natural mutation at the -30 amino acid position of the angiotensinogen signal peptide, in which an arginine is replaced by a proline (R-30P). Heterozygous individuals with R-30P showed a tendency to lowered plasma angiotensinogen level (1563 ng of ANG I/ml (range 1129-1941)) compared with normal individuals in the family (1892 ng of ANG I/ml (range 1603-2072)). Human angiotensinogen mRNA has two in-phase translation initiation codons (AUG) starting upstream 39 and 66 nucleotides from the cap site. R-30P occurs in a cluster of basic residues adjacent to the first AUG codon that may affect intracellular sorting of the nascent protein. Pulse-chase experiments in transiently transfected cultured cells revealed that the R-30P mutation was associated with reduced amounts of both intra- and extracellular protein. In a cell-free system, we found that two forms of native angiotensinogen were generated by alternative initiation of translation at either AUG codon. Alteration of either the first or second AUG codons abolished the synthesis of the longer and the shorter form of native angiotensinogen, respectively. Furthermore, the rate of secretion of the shorter form was lower than that of the longer form. By transplanting angiotensinogen signal peptide onto green fluorescence protein, however, we found that both forms of the signal peptide could target green fluorescence protein, normally localized in the cytoplasm, to the secretory pathway. Although the R-30P mutation may not affect intracellular sorting of angiotensinogen in a qualitative manner, it leads to a quantitative reduction in the net secretion of mature angiotensinogen through decreased translocation or increased residence time in the endoplasmic reticulum.
Asunto(s)
Angiotensinógeno/genética , Hipertensión/genética , Mutación Puntual , Polimorfismo Conformacional Retorcido-Simple , Secuencia de Aminoácidos , Angiotensinógeno/química , Animales , Secuencia de Bases , Células COS , Codón/genética , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , TransfecciónRESUMEN
The most prominent structural feature of the G protein-coupled receptor superfamily is their seven hydrophobic domains, which are postulated to form membrane-spanning alpha helices. Some members of the G protein-coupled receptor family, specifically several serotonin (5-HT) receptors, possess eight hydrophobic domains. The importance of this extra hydrophobic domain, located at the N terminus of the receptor, is unknown. This question was addressed by deleting the extra hydrophobic region from the 5-HT2C receptor and comparing its function and topology with those of the wild-type receptor. Immunofluorescence microscopy was used to determine the location of the N terminus of the epitope-tagged wild-type and mutant receptors. The N terminus of both receptors was extracellular, suggesting that the extra hydrophobic domain does not change the topology of this receptor and is unlikely to be a membrane-spanning alpha helix. Radioligand-binding studies in transfected cells and expression studies in Xenopus oocytes demonstrated that seven hydrophobic domains were sufficient for normal function in these assays. Interestingly, the mutant receptor, now containing seven hydrophobic domains, is expressed at higher levels in transfected cells than the wild-type receptor containing eight hydrophobic domains, suggesting that the extra hydrophobic domain does impact the activity of this receptor by regulating its expression.
Asunto(s)
Proteínas de Unión al GTP/química , Receptores de Serotonina/química , Receptores de Serotonina/genética , Secuencia de Aminoácidos , Animales , Unión Competitiva/fisiología , Transporte Biológico/efectos de los fármacos , Cloruros/metabolismo , Ergolinas/farmacología , Técnica del Anticuerpo Fluorescente , Expresión Génica , Ligandos , Datos de Secuencia Molecular , Mutagénesis , Oocitos/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Transfección , Tritio , Xenopus laevisRESUMEN
Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).