RESUMEN
We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-envelope and -liposome (HSP65 + IL-12/HVJ). An IL-12 expression vector (IL-12DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine on the basis of C.F.U of number of TB, survival, an induction of the CD8 positive CTL activity and improvement of the histopathological tuberculosis lesions. This vaccine also provided therapeutic efficacy against multi-drug resistant TB (MDR-TB) and extremely drug resistant TB (XDR-TB) (prolongation of survival time and the decrease in the number of TB in the lung) in murine models. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. All monkeys in the control group (saline) died within 8 months, while 50% of monkeys in the HSP65+hIL-12/HVJ group survived more than 14 months post-infection (the termination period of the experiment). Furthermore, the BCG priming and HSP65 + IL-12/HVJ vaccine (booster) by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys from BCG Tokyo alone group were alive (33% survival). Furthermore, this vaccine exerted therapeutic efficacy (100% survival) and augmentation of immune responses in the TB-infected monkeys. These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis including XDR-TB and MDR-TB for human therapeutic clinical trials.
RESUMEN
BACKGROUND: Serum amyloid A (SAA) and C-reactive protein (CRP) have been suggested to be involved in the process of coronary heart disease (CHD) and to be potential markers and/or predictors of CHD. Remnant-like lipoprotein particles (RLPs), which are regarded as atherogenic remnant lipoprotein, are reported to be increased in type 2 diabetic patients. We assessed the association of CHD with SAA, CRP and RLP-cholesterol in type 2 diabetic patients. METHODS: One hundred and twenty-six diabetic patients without CHD and 41 patients with CHD were recruited from our hospital. Plasma SAA was measured by the latex agglutination nephelometric immunoassay. Plasma high-sensitivity CRP was measured by a latex immunoturbidity method. Plasma RLP-cholesterol was measured by an immunoabsorption enzyme method. RESULTS: The mean standard deviation values of RLP-cholesterol in patients with and without CHD were 0.22 (0.26) mmol/L and 0.15 (0.10) mmol/L, respectively (P <0.05). Median (interquartile ranges) for SAA in patients with and without CHD were 7.4 (4.2-11.2) mg/L and 3.9 (2.2-5.9) mg/L, respectively (P <0.001). Median (interquartile ranges) for CRP in patients with and without CHD was 1.14 (0.45-2.08) mg/L and 0.43 (0.19-1.25) mg/L, respectively (P <0.001). For all patients, the Spearman rank correlation statistics for RLP-cholesterol compared with SAA and with CRP were 0.213 (P <0.05) and 0.301 (P <0.01), respectively. CONCLUSION: These data suggest that SAA, CRP and RLP-cholesterol are increased in type 2 diabetic patients with CHD, and that the inflammatory proteins correlate with remnant lipoprotein.
Asunto(s)
Proteína C-Reactiva/análisis , Colesterol/sangre , Enfermedad Coronaria/sangre , Diabetes Mellitus Tipo 2/sangre , Lipoproteínas/sangre , Proteína Amiloide A Sérica/análisis , Triglicéridos/sangre , Anciano , Biomarcadores , Enfermedad Coronaria/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Femenino , Humanos , Pruebas de Fijación de Látex , Masculino , Persona de Mediana Edad , Nefelometría y TurbidimetríaRESUMEN
The bone morphogenetic protein (BMP) family, comprising multifunctional peptide growth factors, regulates many developmental processes in a variety of tissues. We examined the spatiotemporal expression of BMP5 by in situ hybridization in chick embryonic hearts from stages 5 to 33. The BMP5 gene was first expressed in the endoderm underlying the precardiac mesoderm at stages 5 to 8. Thereafter, BMP5 expression was restricted to the myocardium of the atrioventricular (AV) canal and outflow tract (OT) regions, where the valvuloseptal endocardial cushion tissue is induced. These results suggest that BMP5 may play important roles not only in myocardial differentiation, but also in the formation and maintenance of endocardial cushion tissue.
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Proteínas Morfogenéticas Óseas/genética , Endocardio/embriología , Tabiques Cardíacos/embriología , Válvulas Cardíacas/embriología , Corazón/embriología , Animales , Proteína Morfogenética Ósea 5 , Proteínas Morfogenéticas Óseas/biosíntesis , Embrión de Pollo , Cartilla de ADN/química , Desarrollo Embrionario y Fetal , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Miocardio/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismoRESUMEN
We have identified chick frizzled (Fz)-10, encoding a Wnt receptor, and examined the expression pattern during embryogenesis. Fz-10 is expressed in the region posterior to the Hensen's node at stage 6. Fz-10 expression is detected in the dorsal domain of the neural tube and the central nervous system of the developing embryo. In the developing limb, Fz-10 expression starts at stage 18 in the posterior-dorsal region of the distal mesenchyme, and gradually expands to the anterior-distal region. Fz-10 is also expressed in the feather bud and branchial arch. Implantation of Sonic hedgehog (Shh)-expressing cells into the anterior margin of the limb bud resulted in the induction of Fz-10 expression in anterior-dorsal mesenchyme.
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Proteínas Aviares , Sistema Nervioso Central/embriología , Transactivadores , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , ADN Complementario , Receptores Frizzled , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Esbozos de los Miembros/embriología , Datos de Secuencia Molecular , Proteínas/genética , Proteínas/metabolismo , Receptores de Superficie Celular/genéticaRESUMEN
The dorsal ectoderm of the limb bud is known to regulate anterior-posterior patterning as well as dorsal-ventral patterning during vertebrate limb morphogenesis. Wnt-7a, expressed in the dorsal ectoderm, encodes a key molecule implicated in these events. In the present study, chicken frizzled-10 (Fz-10) encoding a Wnt receptor was used to study mechanisms of Wnt-7a signaling during chick limb patterning, because its expression is restricted to the posterior-distal region of the dorsal limb bud. Fz-10 transcripts colocalize with Sonic hedgehog (Shh) in the dorsal side of stages 18-23 chick limb buds. It was demonstrated that Fz-10 interacts with Wnt-7a to induce synergistically the expression of Wnt-responsive genes, such as Siamois and Xnr3, in Xenopus animal cap assays. In the chick limb bud, Fz-10 expression is regulated by Shh and a signal from the dorsal ectoderm, presumably Wnt-7a, but not by signals from the apical ectodermal ridge. These results suggest that Fz-10 acts as a receptor for Wnt-7a and has a positive effect on Shh expression in the chick limb bud.
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Proteínas Aviares , Extremidades/embriología , Proteínas Proto-Oncogénicas/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Transactivadores , Animales , Tipificación del Cuerpo , Embrión de Pollo , Clonación Molecular , Receptores Frizzled , Proteínas Hedgehog , Morfogénesis , Proteínas/genética , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Homología de Secuencia de Aminoácido , Proteínas Wnt , Xenopus/embriologíaRESUMEN
Members of the frizzled (Fz) family are involved in Wnt signaling during embryogenesis in the vertebrate. We identified chicken cognates of Fz-2, Fz-3, Fz-4, Fz-6 and Fz-8, and examined spatial and temporal expression patterns in the chick embryos by whole-mount in situ hybridization. Fz-4 is intensely expressed in the apical ectodermal ridge and distal mesenchyme of the limb bud at stages 20 to 27. The transcripts are confined to the posterior-distal end of the digit-forming region at stages 25 to 27. Fz-2 is weakly expressed in the proximal limb mesenchyme at stages 25 to 27, while Fz-3 and Fz-6 expressions are uniform in the limb bud at these stages. Fz-2 is also expressed in the dermatomyotome. No expression signal for Fz-8 is detectable in the embryo at stages 20 to 27. The differential expression patterns of the Fz family, together with spatially restricted expression of the Wnt family members in the developing limb, suggest distinct but overlapping functions to transduce Wnt signal implicated in cellular interaction during embryogenesis.
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Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/metabolismo , Esbozos de los Miembros/fisiología , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Transducción de Señal , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Embrión de Pollo , Receptores Frizzled , Hibridación in Situ , Datos de Secuencia Molecular , Proteínas/genética , Receptores de Neurotransmisores/genética , Proteínas WntRESUMEN
Members of the Wnt family are known to play diverse roles in the organogenesis of vertebrates. The full-coding sequences of chicken Wnt-5a were identified and the role it plays in limb development was examined by comparing its expression pattern with that of two other Wnt members, Wnt-4 and Wnt-11, and by misexpressing it with a retrovirus vector in the limb bud. Wnt-5a expression is detected in the limb-forming region at stage 14, and in the apical ectodermal ridge and distal mesenchyme of the limb bud. The signal was graded along the proximal-distal axis at stages 20-28 and also along the anterior-posterior axis during early stages. It disappeared in the cartilage-forming region after stage 26, and was restricted to the region surrounding the phalanges at stage 34. Wnt-4 and Wnt-11, other members of the Wnt-5a-subclass, were expressed with a distinct spatiotemporal pattern during the later phase. Wnt-4 was expressed in the articular structure and Wnt-11 was expressed in the dorsal and ventral mesenchyme adjacent to the ectoderm. Wnt-5a expression was partially reduced after apical ectodermal ridge removal, whereas Wnt-11 expression was down-regulated by dorsal ectoderm removal. Therefore, expression of these Wnt was differentially regulated by the ectodermal signal. Misexpression of Wnt-5a in the limb bud with the retrovirus resulted in truncation of long bones predominantly in the zeugopod because of retarded chondrogenic differentiation. Distal elements, such as the phalanges and metacarpals, were not significantly reduced in size. These results suggest that Wnt-5a is involved in pattern formation along the proximal-distal axis by regulation of chondrogenic differentiation.
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Tipificación del Cuerpo , Cartílago/embriología , Condrogénesis , Esbozos de los Miembros/embriología , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Animales , Huesos de la Extremidad Superior/embriología , Diferenciación Celular , Embrión de Pollo , Ectodermo , Inducción Embrionaria , Expresión Génica , Regulación de la Expresión Génica , Glicoproteínas/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Alas de Animales/embriología , Proteínas Wnt , Proteína Wnt-5a , Proteína Wnt4RESUMEN
Smads are central mediators of signal transduction for the TGFbeta superfamily. However, the precise functions of Smad-mediated signaling pathways in early development are unclear. Here we demonstrate a requirement for Smad2 signaling in dorsoanterior axis formation during Xenopus development. Using two point mutations of Smad2 previously identified in colorectal carcinomas, we show that Smad2 ushers Smad4 to the nucleus to form a transcriptional activation complex with the nuclear DNA-binding protein FAST-1 and that the mutant proteins interact normally with FAST-1 but fail to recruit Smad4 into the nucleus. This mechanism of inhibition specifically restricts the dominant-negative activity of these mutants to the activin/Vg1 signaling pathway without inhibiting BMPs. Furthermore, expression of these mutants in Xenopus animal caps inhibits but does not abolish activin and Vg1 induction of mesoderm and in the embryo results in a truncated dorsoanterior axis. These studies define a mechanism through which mutations in Smad2 may block TGFbeta-dependent signaling and suggest a critical role for inductive signaling mediated by the Smad2 pathway in Xenopus organizer function.
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Proteínas de Unión al ADN/genética , Inhibinas/genética , Transducción de Señal/genética , Transactivadores/genética , Proteínas de Xenopus , Xenopus/embriología , Receptores de Activinas Tipo I , Activinas , Animales , Células COS , Desarrollo Embrionario , Factores de Transcripción Forkhead , Regulación del Desarrollo de la Expresión Génica/genética , Morfogénesis , Mutación , Factores de Crecimiento Nervioso , Fosforilación , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento/genética , Proteínas Smad , Proteína Smad2 , Proteína Smad4 , Factores de Transcripción/genética , Activación Transcripcional/genética , Transfección , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Bone morphogenetic proteins (BMPs) perform diverse functions in vertebrate development. Here we demonstrate that the heterodimeric BMP-4/7 protein directly induces ventral mesoderm and blood in Xenopus animal caps, and BMP-2/7 heterodimers may function similarly. We also provide indirect evidence that BMP heterodimers function in embryos, using assays with dominant-negative BMP ligands. Homodimeric BMP-2 and BMP-4 proteins do not induce mesoderm, but they ventralize mesoderm induction by activin. In contrast, BMP-7 protein interferes with mesoderm induction by activin, but BMP-7 stimulates ventral mesoderm induction by the heterodimer, BMP-4/7. This novel property of BMP-7 distinguishes it from other BMPs. BMP-7 may therefore function in early embryogenesis to antagonize activin signals and potentiate BMP signals. We propose that BMP heterodimers convey signals for ventral mesoderm induction and patterning in Xenopus development.
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Proteínas Morfogenéticas Óseas/fisiología , Inducción Embrionaria/efectos de los fármacos , Mesodermo/efectos de los fármacos , Factor de Crecimiento Transformador beta , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Activinas/fisiología , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Proteína Morfogenética Ósea 4 , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/biosíntesis , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/genética , ADN Complementario/genética , Dimerización , Regulación del Desarrollo de la Expresión Génica , Genes Dominantes , Hematopoyesis , Microinyecciones , Datos de Secuencia Molecular , Multimerización de Proteína , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genéticaRESUMEN
Angiotensin (AT) II, the bioactive octapeptide in the renin-angiotensin system that plays a key role in cardiovascular homeostasis, exerts its multiple effects through the different types of AT receptors, AT1a, AT1b, and AT2. Previously, we showed chronic hypotension in angiotensinogen (the precursor of AT)-deficient mice and a dramatic increase in renin mRNA levels in its kidney, but it remains unclear which types of AT receptors regulate the blood pressure and renin gene expression. In order to elucidate the physiological roles of AT1a receptor, we generated mutant mice with a targeted replacement of the AT1a receptor loci by the lacZ gene. In the heterozygous mutant mice, the strong lacZ staining was found in the glomerulus and juxtaglomerular apparatus of the renal cortex, which coincided with that of the signals detected by in situ hybridization. Chronic hypotension was observed in the heterozygous and homozygous mutant mice, with 10 and 22 mm Hg lower systolic blood pressure, respectively, than that of wild-type littermates. Both levels of renin mRNA in the kidney and plasma renin activity were markedly increased only in the homozygous mutant mice. These results demonstrated that an AT1a-mediated signal transduction pathway is, at least in part, involved in the regulation of blood pressure and renin gene expression.
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Angiotensina II/fisiología , Hipotensión/etiología , Receptores de Angiotensina/deficiencia , Renina/sangre , Animales , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Mutantes , Receptores de Angiotensina/genética , Receptores de Angiotensina/fisiología , Renina/genéticaRESUMEN
We have cloned the rat angiotensin II receptor type 2 (AT2) gene, whose physiological function remains unclear. Sequence analysis indicated that exons 1 and 2 exist in the 5'-untranslated region and the initiation codon ATG is located in exon 3. The 1.6-kb genomic fragment at positions -1567 to +26 relative to the putative transcription start site was found to contain a functional promoter region using transient chloramphenicol acetyltransferase assay. This is the first report demonstrating the nucleotide sequence of the promoter region of this gene.
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Receptores de Angiotensina/genética , Animales , Secuencia de Bases , Clonación Molecular , Exones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Receptores de Angiotensina/químicaRESUMEN
A cDNA that encodes a new member of the TGF-beta superfamily most similar to activin beta A, beta B, and recently reported beta C has been isolated from Xenopus laevis. Expression of the gene in early embryos suggested its biological importance in Xenopus embryogenesis. Microinjection of a synthetic mRNA transcribed from the cDNA into ventral blastomeres of early Xenopus embryo led to the formation of secondary body axis. Mesodermal marker genes were induced in isolated animal cap by a similar mRNA injection. These results demonstrate that the gene encodes a new member of activin subfamily whose function is closely related to mesoderm induction.
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Inhibinas/genética , Mesodermo , Activinas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Clonación Molecular , ADN Complementario , Inducción Embrionaria , Regulación del Desarrollo de la Expresión Génica , Inhibinas/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
The effect of plasma adsorption (PA) on bilirubin removal by a porous type anion exchange resin (Medisorba BL, Kuraray) was evaluated in vitro PA and in vivo PA and plasma exchange (PE) in postoperative hyperbilirubinemia patients. Serum and plasma bilirubin were measured by routine biochemical tests, fractionated by high-performance liquid chromatography, and its reduction and rebound were compared. In vitro and in vivo PA indicated the selectivity in bilirubin removal against measured molecules. Changes in bilirubin fractions after PA showed higher adsorbance of conjugated bilirubin (21.4-30.8%) and less adsorbance of delta (85.7%) and unconjugated bilirubin (73.7%). These differences were not observed after PE. But rebound of serum total bilirubin levels after PA was more than that after PE. It is suggested that PA by the resin is selective and has a different metabolic effect on bilirubin during and after PA as compared with PE.
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Bilirrubina/sangre , Hiperbilirrubinemia/terapia , Complicaciones Posoperatorias/terapia , Adsorción , Resinas de Intercambio Aniónico , Humanos , Hiperbilirrubinemia/etiología , Técnicas In Vitro , Intercambio PlasmáticoRESUMEN
We recently found a mouse unusual per repeat genomic gene showing circadian expression in the suprachiasmatic nucleus (SCN) of rat brain. As an initial step to the better understanding of biological functions of mammalian per repeat family, we isolated a new cDNA clone that encodes for the putative open reading frame of 133 amino acids, designating as mp41, having a per repeat sequence of (ACAGC)32 which lacks one base pair from a mouse unusual per repeat sequence (ACAGGC)n. In situ hybridization showed that the mRNA of mp41 gene expression is detected in the rat pancreas, uterus, ovary, liver, adrenal glands, kidney, intestine, spleen and brain. In brain, daily fluctuations of mp41 mRNA levels were found in the SCN under light and dark cycles--high during the day time and lower during the night time, even in constant darkness for 15 days. After exposing rats to light, mp41 mRNA increased only during the subjective night of the circadian cycle when light also induced the c-fos mRNA expression in the SCN. These results suggest that the transcriptional control of mp41 gene is regulated by light and a circadian clock and indicate that mp41 is a new marker gene for a cycling transcript in the SCN.
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Ritmo Circadiano , Regulación de la Expresión Génica , Luz , Proteínas Nucleares/biosíntesis , Biosíntesis de Proteínas , Proteínas , Secuencias Repetitivas de Ácidos Nucleicos , Núcleo Supraquiasmático/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Femenino , Biblioteca de Genes , Genes fos , Hibridación in Situ , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Especificidad de Órganos , Proteínas Circadianas Period , Proteínas Proto-Oncogénicas c-fos/biosíntesis , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Bazo/metabolismoRESUMEN
We have isolated two cDNAs of 1.7 and 3.0 kb, produced by alternative splicing, that encode a angiotensin II (AII) receptor from a Xenopus laevis heart cDNA library. The two clones had identical coding regions with each other and were found to belong to the G protein-coupled receptor superfamily like the mammalian type 1 AII receptors (AT1); their amino acid sequence was 68.7% homologous with the human AT1 receptor sequence. However, there was a 1.3 kb insertion at the 3'-untranslated region of the longer clone. The insertion contained 9 repeats of an ATTTA motif, suggesting that the two mRNAs undergo distinct post-transcriptional regulation by virtue of a difference in their stability. Although the Xenopus receptor exhibited distinct specificities for AII receptor antagonists compared with mammalian AII receptors, several common characteristics, including the effect of dithiothreitol and guanosine 5'-O-(3-thiotriphosphate), demonstrated that the cloned receptor is a counterpart of the mammalian AT1 receptor. Moreover, the cloned receptor was expressed most abundantly in the Xenopus heart, which is inconsistent with the tissue distribution of mammalian AII receptors. This indicated that the Xenopus heart, unlike that of mammals, plays a major role in the AII-dependent regulation of blood pressure and extracellular fluid volume.
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Empalme Alternativo , Angiotensina II/metabolismo , Receptores de Angiotensina/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Membrana Celular/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Ditiotreitol/farmacología , Expresión Génica , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Cinética , Mamíferos , Datos de Secuencia Molecular , Peso Molecular , Miocardio/metabolismo , Poli A/aislamiento & purificación , Poli A/metabolismo , ARN/aislamiento & purificación , ARN/metabolismo , ARN Mensajero , Receptores de Angiotensina/efectos de los fármacos , Receptores de Angiotensina/metabolismo , Transcripción Genética , Xenopus laevisRESUMEN
Activin exhibits a potent mesoderm inducing activity towards the ectodermal tissue (animal cap) of Xenopus laevis blastulae. Thus in order to investigate the role of activin in morphogenesis of early Xenopus embryos, activation of genes for activin beta A and beta B was examined by the reverse transcription polymerase chain reaction. In vivo, activin beta B mRNA appears to be present in embryonic stage 1 whereas beta A mRNA is undetectable prior to gastrulation. beta B and beta A mRNAs were noted to accumulate after stages 9 and 15 respectively. Activin gene expression in Xenopus animal caps was examined after treatment with various concentrations of activin A. Under these treatment conditions, both activin beta A and beta B mRNAs accumulated in a dose-dependent fashion after 24 h. The same effect was noted for treatment with similar concentrations of activin B. Accumulation of mRNAs was inhibited by the addition of cycloheximide to the culture medium, consistent with the proposition that activin gene expression requires certain protein factors. In total, therefore, these data suggest that an autoinduction mechanism is involved in the regulation of activin mRNA levels in normal Xenopus embryos and that this mechanism may play a pivotal role during early embryonic development.
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Regulación de la Expresión Génica , Inhibinas/genética , Activinas , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cartilla de ADN , Humanos , Inhibinas/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Proteínas Recombinantes/genética , Xenopus laevis/embriologíaRESUMEN
We have previously shown that the animal pole tissue from a st.10+ early gastrula Xenopus embryo stimulates the primary differentiation of erythrocytes in the ventral mesoderm in combination culture. To characterize the nature of this stimulation, various sizes and different portions of animal pole tissue were combined with the ventral mesoderm explants. The erythrocyte differentiation in terms of globin expression depended on the size of the animal pole tissue that was combined with the ventral mesoderm. No difference was observed in the potency of stimulation between the ventral and dorsal halves of animal pole tissue. We also found that animal pole tissue from as late as st.7 failed to stimulate both mRNA and protein levels of globin in the explant. Histological studies of the combination explant with st.7 animal pole tissue showed that epidermis, vesicle structure, and blood-cell-like cells developed in the explant, but very few blood cells expressed globin molecules. However, the stimulation of erythroid differentiation was restored if total (20 ng) or poly(A)+ (0.2 ng) RNA from st.10+ animal pole tissue was previously injected at the 2-cell stage and the resulting animal pole tissue at st.7 was combined with st.10+ ventral mesoderm. Erythroid differentiation was also restored by injection with 1 ng of Xenopus bone morphogenetic protein-4 (XBMP-4) RNA. The effect of an extremely small dose of poly(A)+ RNA on erythroid differentiation suggests that in addition to XBMP-4 there exist substances, expressed later than st.7 in the animal pole region, which can stimulate erythrocyte differentiation in the ventral mesoderm.
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Eritropoyesis , Gástrula/fisiología , Mesodermo/fisiología , Xenopus/embriología , Animales , Proteínas Morfogenéticas Óseas , Regulación de la Expresión Génica , Globinas/genética , Proteínas/fisiología , ARN Mensajero/análisisRESUMEN
We have recently reported fluctuations in the expression of the period repeat sequence, pp2.5, during light-dark cycles in the suprachiasmatic nucleus (SCN) of rat. Presently, we performed in situ hybridization which shows that the fluctuation of pp2.5 expression continues during constant darkness conditions in the SCN of rat. The light exposure during subjective night but not subjective day triggered its elevated expression in a time-dependent manner which is parallel to that of c-fos expression. In this review, the cloning and characterization of multiple per repeat sequences from mouse genom and rat brain mRNA were summarized. The abundance of a novel per repeat mRNA (designated as RB15) fluctuates during a light-dark cycle in the SCN. These findings suggest that per repeat sequence may have a role for the mammalian circadian rhythms. The evolutionary relationship between the mammarian per repeat sequence and the Drosophila period gene is also discussed.
Asunto(s)
Ritmo Circadiano/fisiología , Secuencias Repetitivas de Ácidos Nucleicos/fisiología , Núcleo Supraquiasmático/fisiología , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , RatasRESUMEN
The biochemical properties of recombinant amphibian bone morphogenetic protein-4 (BMP-4), the cDNA of which has been cloned recently by screening of a Xenopus cDNA library, was characterized. The protein was expressed by the transfection of Chinese hamster ovary (CHO) cells with the cDNA cloned into expression vectors bearing a cytomegalovirus promoter or a simian virus 40 promoter. Northern-blot analysis showed that the latter vector was more efficient for Xenopus BMP-4 expression. Specific antiserum against Xenopus BMP-4 peptide demonstrated that the protein is synthesized as a large precursor, processed to the mature form and then secreted from the cells as a homodimer. Analysis of the biological activity in the conditioned medium revealed that Xenopus BMP-4 has a potent alkaline phosphatase-inducing activity on mouse osteoblastic cells.