RESUMEN
OBJECTIVE: We aimed at investigating changes in the expression and physiological function of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in interstitial cells of Cajal (ICC) in diabetic state. MATERIALS AND METHODS: Twenty adult female Sprague-Dawley (SD) rats were randomly assigned to control and Zucker diabetic fatty (ZDF) group. The protein and mRNA expression of HCN isoforms and C-kit in the rat bladders were detected using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The bladder contraction was evaluated using a bladder smooth muscle strip test. Whole cell patch-clamp techniques were used to detect the activity of HCN channels. Immunofluorescent staining was used to the positive expression of HCN and C-kit in ICC. RESULTS: cAMP, as HCN channel-specific stimulant, could increase the frequency and amplitude of spontaneous contractions in both group, while cAMP inducing contraction of ZDF rats, was still significantly lower compared with the control group. Acute bladder ICCs were isolated by collagenase digestion. Classic Ih current pattern was recorded on ICCs while Ih current amplitude of ICCs from ZDF diabetic rats was significantly lower than the control group. The expression and mRNA of HCN1-4 isoforms in ZDF diabetic rats were both significantly lower compared with the control group. Meanwhile, the number of c-kit positive cells in ZDF diabetic rats showed no significant differences compared with controls. The morphological structure of ICC in the bladder of ZDF rats was relatively loose and the number of their cell process was apparently decreased. CONCLUSIONS: The structure of ICCs in ZDF rats was relatively loose, their connection to each other was also diminished. The expression of HCN was down-regulated and its response to cAMP was also decreased. HCN channels in bladder ICCs might regulate detrusor contraction. Changes in HCN expression and activity in bladder ICCs might be one of the most important mechanisms of diabetic cystopathy.
Asunto(s)
Complicaciones de la Diabetes/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Células Intersticiales de Cajal/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Enfermedades de la Vejiga Urinaria/metabolismo , Vejiga Urinaria/metabolismo , Urodinámica , Animales , Complicaciones de la Diabetes/patología , Complicaciones de la Diabetes/fisiopatología , Femenino , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Células Intersticiales de Cajal/patología , Músculo Liso/patología , Músculo Liso/fisiopatología , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Ratas Sprague-Dawley , Ratas Zucker , Transducción de Señal , Vejiga Urinaria/patología , Vejiga Urinaria/fisiopatología , Enfermedades de la Vejiga Urinaria/patología , Enfermedades de la Vejiga Urinaria/fisiopatologíaRESUMEN
Microsatellite markers were isolated using dual-suppression-PCR for the endangered species Excentrodendron hsienmu (Tiliaceae) to evaluate the genetic diversity and population structure of this species. A total of 11 polymorphic microsatellite loci were characterized in E. hsienmu. The number of alleles per locus ranged from 2 to 9, with an average of 5.27. The expected heterozygosity value ranged from 0.053 to 0.780, with an average of 0.568 and the observed heterozygosity value ranged from 0 to 0.595, with an average of 0.268. The polymorphic information content value ranged from 0.051 to 0.740, with an average of 0.521. These newly designed markers will be of great potential significance and profound influence in future research related to the genetic diversity, population structure, and patterns of gene flow of this species, which will contribute to the implementation of conservation and management strategies for this species.
Asunto(s)
Repeticiones de Microsatélite , Tiliaceae/genética , Alelos , ADN de Plantas/genética , Especies en Peligro de Extinción , Sitios Genéticos , Variación Genética , Heterocigoto , Polimorfismo GenéticoRESUMEN
OBJECTIVE: S100A2, a Ca(2+) -binding protein with two EF-hands, is a tumor suppressor in oral cancer. Helix III flanking the C-terminal EF-hand is implicated to participate in the interaction of S100A2 and its target(s). The aim of this study was to examine if the coding sequence polymorphism S100A2_185G>A, leading to the peptide 62 substitution of asparagine (AAC, A allele) for serine (AGC, G allele) in helix III, had modulation effects on S100A-mediated tumor suppression. SUBJECTS AND METHODS: We sequenced the coding sequence of S100A2 gene in normal oral keratinocytes (NOKs), dysplastic oral keratinocytes (DOKs), eight oral cancer lines, and 54 pairwise oral cancer specimens. We also compared the in vitro anti-tumor effect of wildtype (G allele) and variant (A allele) S100A2 expression using cell proliferation, migration, invasion, and colony formation assays. RESULTS: With the exception of CAL27 and SCC-15 cancer lines being heterozygotes of A and G alleles, the remaining oral cells were homozygotic in G alleles. No alterations of anti-growth, anti-migration, anti-invasion, and anti-colony formation were observed between variant and wildtype cells. Moreover, no minor S100A2_185A allele was detected in 54-pairwise clinical specimens. CONCLUSION: The coding sequence polymorphism S100A2_185G>A had no regulatory role in S100A2-mediated tumor suppression in oral cancer.
Asunto(s)
Adenina , Carcinoma de Células Escamosas/genética , Factores Quimiotácticos/genética , Guanina , Neoplasias de la Boca/genética , Sistemas de Lectura Abierta/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas S100/genética , Adulto , Anciano , Alelos , Sustitución de Aminoácidos/genética , Asparagina/genética , Línea Celular Tumoral , Células Cultivadas , Motivos EF Hand/genética , Exones/genética , Femenino , Genotipo , Secuencias Hélice-Asa-Hélice/genética , Heterocigoto , Humanos , Células KB , Queratinocitos/patología , Masculino , Persona de Mediana Edad , Polimorfismo Genético/genética , Serina/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Increasing evidence suggests that the activity of cysteine proteases, including cathepsin L, is important in cancer cell invasion and metastasis. This study was designed to investigate the clinicopathological and prognostic significance of cathepsin L in human urothelial carcinoma of the bladder (UCB). Standard immunohistochemistry was used to determine the presence of cathepsin L and Ki-67 (a marker of proliferation) in paraffin-embedded specimens of 82 human UCB cases. Cathepsin L protein was localized in the cytoplasm of the malignant UCB cells, and was significantly associated with the pathological tumour stage (invasiveness), tumour grade, survival, local tumour recurrence during follow-up, the occurrence of distant metastasis during follow-up and the presence of Ki-67 protein. Multivariate analysis using the Cox proportional hazards model revealed that cathepsin L immunopositivity and pathological tumour stage (invasiveness) were independent significant prognostic factors for overall survival. This study showed that cathepsin L provides significant prognostic information and that it might be a useful therapeutic target in the future.
Asunto(s)
Catepsina L/metabolismo , Neoplasias de la Vejiga Urinaria/enzimología , Neoplasias de la Vejiga Urinaria/patología , Urotelio/enzimología , Urotelio/patología , Anciano , Anciano de 80 o más Años , Proliferación Celular , Citoplasma/enzimología , Citoplasma/patología , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Antígeno Ki-67/metabolismo , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Modelos de Riesgos Proporcionales , Resultado del TratamientoRESUMEN
The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is a phosphatase that antagonizes the phosphoinositol-3-kinase/AKT signaling pathway and suppresses cell survival as well as cell proliferation. PTEN is the second most frequently mutated gene in human cancer after p53. Germline mutations of PTEN have been found in cancer susceptibility syndromes, such as Cowden syndrome, in which over 80% of patients have mutations of PTEN. Homozygous deletion of Pten causes embryonic lethality, suggesting that PTEN is essential for embryonic development. Mice heterozygous for Pten develop spontaneous tumors in a variety of organs comparable with the spectrum of its mutations in human cancer. The mechanisms of PTEN functions in tumor suppression are currently under intense investigation. Recent studies demonstrate that PTEN plays an essential role in the maintenance of chromosomal stability and that loss of PTEN leads to massive alterations of chromosomes. The tumor suppressor p53 is known as a guardian of the genome that mediates the cellular response to environmental stress, leading to cell cycle arrest or cell death. Through completely different mechanisms, PTEN also protects the genome from instability. Thus, we propose that PTEN is a new guardian of the genome. In this review, we will discuss new discoveries on the role of PTEN in tumor suppression and explore mechanisms by which PTEN maintains genomic stability.
Asunto(s)
Genoma Humano/fisiología , Fosfohidrolasa PTEN/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Núcleo Celular/metabolismo , Redes Reguladoras de Genes/fisiología , Inestabilidad Genómica/genética , Humanos , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/fisiología , Modelos Biológicos , Fosfohidrolasa PTEN/química , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/fisiologíaRESUMEN
The SET- and chromo-domains are recognized as signature motifs for proteins that contribute to epigenetic control of gene expression through effects on the regional organization of chromatin structure. This paper reports the identification of a novel subgroup of SET-domain-containing proteins in tobacco and Arabidopsis, which show highest homologies with the Drosophila position-effect-variegation repressor protein SU(VAR)3-9 and the yeast centromer silencing protein CLR4. The tobacco SET-domain-containing protein (NtSET1) was fused to the green fluorescence protein (GFP) that serves as a visual marker for localization of the recombinant protein in living cells. Whereas control GFP protein alone was uniformly dispersed within the nucleus and cytoplasm, the NtSET1-GFP fusion protein showed a non-uniform localization to multiple nuclear regions in interphase tobacco TBY2 cells. During mitosis, the NtSET1-GFP associated with condensed chromosomes with a non-random distribution. The NtSET1 thus appears to have distinct target regions in the plant chromatin. Overexpression of the NtSET1-GFP in transgenic tobacco inhibited plant growth, implicating the possible involvement of the NtSET1 in transcriptional repression of growth control genes through the formation of higher-order chromatin domains.
Asunto(s)
Arabidopsis/genética , Cromatina/química , Nicotiana/genética , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Proteínas de Schizosaccharomyces pombe , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular , Proteínas de Ciclo Celular/genética , Inhibidores de Crecimiento , N-Metiltransferasa de Histona-Lisina , Metiltransferasas/genética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
The insulin-like growth factors (IGF) are important anabolic hormones in the mammalian fetus; their anabolic actions are potentially modulated by alterations in the IGF-binding proteins (IGFBP). We have previously shown that the nutritional state of the fetus affects both IGF-I and the IGFBP concentrations. The present study was designed to determine the effect of alterations in insulin and IGF-I circulating concentrations on the IGFBPs. Because both insulin and IGF-I elicit decreases in glucose and amino acid concentrations, the concentrations of these substrates were clamped during the hormone infusions. Sixteen ovine fetuses were chronically catheterized at approximately 115 days of gestation, and experimental procedures performed at approximately 130 days of gestation. Insulin, IGF-I or both were infused for an 8-h period. Baseline concentrations of hormones and binding proteins were obtained, and concentrations were also obtained at the end of the infusion. Hepatic IGFBP-1 mRNA expression was also determined. Intravenous infusion of IGF-I significantly increased IGF-I concentrations in plasma in the ovine fetus. Intravenous infusion of insulin inhibited hepatic IGFBP-1 gene expression when amino acids and glucose were clamped. In contrast, intravenous infusion of recombinant human IGF-I (rhIGF-I) enhanced hepatic IGFBP-1 gene expression. Neither insulin nor rhIGF-I treatment had an effect on hepatic IGFBP-3 gene expression. Insulin did not alter plasma IGFBP-1 significantly, but it increased IGFBP-3 in plasma. rhIGF-I increased both IGFBP-1 and IGFBP-3 protein levels in plasma. The responses of IGFBP-1 and IGFBP-3 to increased plasma IGF-I and insulin may serve to protect the fetus from exaggerated anabolic effects and to blunt the hypoglycemic potential of circulating IGFs and insulin.
Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Ovinos/embriología , Ovinos/metabolismo , Análisis de Varianza , Animales , Northern Blotting/métodos , Western Blotting/métodos , Femenino , Sangre Fetal/química , Expresión Génica/efectos de los fármacos , Edad Gestacional , Humanos , Infusiones Intravenosas , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/química , Factor I del Crecimiento Similar a la Insulina/análisis , Hígado/química , Embarazo , ARN Mensajero/análisis , Radioinmunoensayo/métodos , Proteínas Recombinantes/farmacologíaRESUMEN
We have previously shown that the tobacco cyclin B1;1 protein accumulates during the G2 phase of the cell cycle and is subsequently destroyed during mitosis. Here, we investigated the sub-cellular localisation of two different B1-types and one A3-type cyclin during the cell cycle by using confocal imaging and differential interference contrast (DIC) microscopy. The cyclins were visualised as GFP-tagged fusion proteins in living tobacco cells. Both B1-type cyclins were found in the cytoplasm and in the nucleus during G2 but when cells entered into prophase, both cyclins became associated with condensing chromatin and remained on chromosomes until metaphase. As cells exited metaphase, the B1-type cyclins became degraded, as shown by time-lapse images. A stable variant of cyclin B1;1-GFP fusion protein, in which the destruction box had been mutated, maintained its association with the nuclear material at later phases of mitosis such as anaphase and telophase. Furthermore, we demonstrated that cyclin B1;1 protein is stabilised in metaphase-arrested cells after microtubule destabilising drug treatments. In contrast to the B1-type cyclins, the cyclin A3;1 was found exclusively in the nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.
Asunto(s)
Ciclo Celular , Ciclinas/metabolismo , Proteínas Luminiscentes/metabolismo , Mitosis , Nicotiana/metabolismo , Huso Acromático , Fracciones Subcelulares/metabolismo , Secuencia de Bases , Benzamidas/administración & dosificación , Cartilla de ADN , Proteínas Fluorescentes Verdes , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/genética , Nicotiana/ultraestructuraRESUMEN
Interleukin (IL)-10 is synthesized in the central nervous system (CNS) and acts to limit clinical symptoms of stroke, multiple sclerosis, Alzheimer's disease, meningitis, and the behavioral changes that occur during bacterial infections. Expression of IL-10 is elevated during the course of most major diseases in the CNS and promotes survival of neurons and all glial cells in the brain by blocking the effects of proapoptotic cytokines and by promoting expression of cell survival signals. Stimulation of IL-10 receptors regulates numerous life- or death-signaling pathways--including Jak1/Stat3, PI 3-kinase, MAPK, SOCS, and NF-kappaB--ultimately promoting cell survival by inhibiting both ligand- and mitochondrial-induced apoptotic pathways. IL-10 also limits inflammation in the brain; it does so by three major pathways: (1) reducing synthesis of proinflammatory cytokines, (2) suppressing cytokine receptor expression, and (3) inhibiting receptor activation. Finally, IL-10 induces anergy in brain-infiltrating T cells by inhibiting cell signaling through the costimulatory CD28-CD80/86 pathway. The multiple functions of IL-10 in the brain will create new and intriguing vistas that will promote a better understanding of neurodegenerative diseases. These discoveries could lead to development of innovative approaches for the use of antiinflammatory cytokines in major debilitating diseases of the CNS.
Asunto(s)
Encéfalo/inmunología , Interleucina-10/inmunología , Transducción de Señal/inmunología , Animales , Expresión Génica , Humanos , Inmunidad/inmunología , Interleucina-10/genética , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Receptores de Interleucina-10RESUMEN
OBJECTIVE: To study Cordyceps (artificial fermented Cordyceps sinensis(Berk.) Sacc) powderin the treatment of asthma in the animal models. METHOD: Pulmonary function and airway inflammation in vivo were investigated. RESULT: Cordyceps, 5g.kg-1(ig), significantly inhibited bronchial challenge of ovalbumin-induced change of RL and Cdyn (P < 0.05) and inhibited antigen-induced increase of eosinophils in the BALF of rats (P < 0.05). CONCLUSION: The results suggested cordyceps could be applied for the prevention and cure of asthma.
Asunto(s)
Asma/fisiopatología , Cordyceps , Medicamentos Herbarios Chinos/farmacología , Lepidópteros , Pulmón/fisiopatología , Fitoterapia , Resistencia de las Vías Respiratorias/efectos de los fármacos , Animales , Asma/tratamiento farmacológico , Cordyceps/química , Medicamentos Herbarios Chinos/aislamiento & purificación , Femenino , Cobayas , Lepidópteros/química , Rendimiento Pulmonar/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Stability and absorption of orally administered fluorescein-isothiocyanate labeled insulin (FITC-insulin) in the gastrointestinal (GI) tract were investigated in newborn and 3-day-old pigs. The uptake of FITC-insulin by the intestinal epithelial cells was visualized using confocal laser scanning microscopy. Following oral administration, 3 h later 56 and 88% of orally administered fluorescence was found in the GI tract in newborn and 3-day-old piglets, respectively. Chromatographic analysis revealed that 15-37% of fluorescence recovered from the gastric and proximal intestinal contents was eluted in the void volume of a Sephadex G-25 column. It was also observed that oral administration of FITC-insulin at a dose of 100 nmol/kg body weight led to a significant decrease in blood glucose in newborn pigs (P<0. 05) but not in 3-day-old pigs. Microscopic examination showed that FITC-insulin was taken up via the vesicular transport mechanism throughout the whole small intestine but the ileum appeared to be a preferred site for FITC-insulin transport in newborn pigs. In 3-day-old pigs, the uptake of FITC-insulin occurred only in the distal part of the small intestine. These findings suggest that milk-borne insulin may partially survive in the GI lumen and subsequently act on the gastrointestinal tract in suckling piglets, while GI absorption of milk-borne insulin is limited to newborn pigs.
Asunto(s)
Sistema Digestivo/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Insulina/análogos & derivados , Animales , Animales Recién Nacidos , Animales Lactantes , Glucemia/análisis , Fluoresceína-5-Isotiocianato/metabolismo , Insulina/sangre , Insulina/metabolismo , Microscopía Confocal , PorcinosRESUMEN
BACKGROUND: Insulin-like growth factor (IGF)-I is present in the milk of various species. A prerequisite for any biological activity of milk-borne IGF-I in the suckling young is to survive the gastrointestinal luminal digestion. In the present study, the stability of IGF-I was examined in the gastrointestinal lumen in neonatal pigs. METHODS: Iodine-labeled IGF-I was incubated in the gastrointestinal luminal fluids of 3-day-old suckling and 45-day-old weaned pigs at 37 degrees C for 20 minutes. Degradation of the peptide was analyzed by trichloroacetic acid (TCA) precipitation, liquid chromatography, and receptor binding assay. RESULTS: IGF-I remained unchanged in the gastric fluids of suckling and weaned pigs when determined by TCA precipitation. IGF-I degraded 3%, 18%, and 37% in the luminal fluids of the proximal, mid and distal small intestine in suckling piglets compared with 53%, 62%, and 54% in weaned pigs. The results were supported by the chromatography and receptor binding analysis. Porcine colostrum had a capacity to protect IGF-I from gastrointestinal luminal digestion in weaned pigs. CONCLUSION: Milk-borne IGF-I is stable in the gastrointestinal lumen in suckling pigs and may play a role in regulating postnatal development in the suckling young.
Asunto(s)
Animales Recién Nacidos/metabolismo , Animales Lactantes/metabolismo , Sistema Digestivo/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Porcinos/metabolismo , Animales , Líquidos Corporales/metabolismo , Precipitación Química , Cromatografía Liquida , Calostro/fisiología , Digestión , Estabilidad de Medicamentos , Endopeptidasas/metabolismo , Hidrólisis , Radioisótopos de Yodo , Receptor IGF Tipo 1/metabolismo , Ácido Tricloroacético , DesteteRESUMEN
Activation of cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Although cyclin gene expression has been extensively studied in plants, not much is known at the level of the protein stability and function. Here, we demonstrated by using the highly synchronizable tobacco BY2 cell culture, that endogenous cyclin B1 protein undergoes cell cycle-dependent proteolysis and is stabilized when the spindle checkpoint has been activated. Furthermore, we established transgenic tobacco BY2 cell cultures expressing under the control of an inducible promoter, cyclin B1 protein as well as its non-degradable form as fusion proteins with GFP and found that the ectopic expression of these proteins did not dramatically disturb the cell cycle progression. These results indicate that, to a certain extent, cell cycle exit is possible without cyclin B1 proteolysis.
Asunto(s)
Ciclo Celular , Ciclina B/biosíntesis , Nicotiana/metabolismo , Plantas Tóxicas , Células Cultivadas , Clonación Molecular , Ciclina B/genética , Ciclina B/metabolismo , Ciclina B1 , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Fase G2 , Proteínas Fluorescentes Verdes , Leupeptinas/farmacología , Proteínas Luminiscentes , Mitosis , Complejos Multienzimáticos/metabolismo , Plantas Modificadas Genéticamente , Complejo de la Endopetidasa Proteasomal , Proteínas Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/citologíaRESUMEN
Annexins interact in a calcium-dependent manner with membrane phospholipids. Although their exact function is not known, annexins have been proposed to be involved in a variety of cellular processes. To determine whether plant annexins are implicated in cell division, we have isolated cDNAs encoding annexin from TBY2 cells. Based on sequence analysis, these cDNAs fall into two families, differing mainly by deletions or insertions in their 5'- and 3'-untranslated regions. The two annexins Ntp32.1 and Ntp32.2 encoded by these cDNAs are homologous to p32 from bell pepper (Cap32.1): we propose that these Solanaceae annexins constitute a distinct type which we call Sp32 annexins. There are two genes (Ntan.1 and Ntan.2) derived from the separate progenitor species of Nicotiana tabacum and analysis of Southern blots is consistent with the presence of these two genes. We show that Sp32 transcript amounts are developmentally modulated in tobacco plants: RNA levels are highest in growing and dividing tissues. Sp32 annexin gene expression is also regulated in TBY2 cultured cells: transcripts and proteins are detected only in exponentially growing cells. In synchronized TBY2 cells, Sp32 annexin transcripts are expressed at the G2/M transition, in the M phase and at the G1/S transition. These results are the first evidence that the expression of plant annexins is modulated during the cell cycle. The Sp32 annexin proteins accumulate during the cell cycle and peak at the end of mitosis. Immunolocalization shows that the majority of Sp32 annexins is present in intercellular junctions, forming a ring structure under the plasma membrane. Since annexins are known to bind secretory vesicles during exocytosis, their localization at cell junctions suggests that annexins could be involved in cell wall maturation.
Asunto(s)
Anexinas/genética , Nicotiana/química , Proteínas de Plantas , Plantas Tóxicas , Secuencia de Aminoácidos , Anexinas/química , Ciclo Celular/genética , ADN Complementario/química , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Inmunohistoquímica , Datos de Secuencia Molecular , Semillas/químicaRESUMEN
Translocation of the catalytic domain of diphtheria toxin (DT) across the endosomal membrane to the cytoplasm of mammalian cells requires the low-pH-dependent insertion of a hydrophobic helical hairpin (TH8-TH9) that is buried within the T domain of the native protein. Mutations of Pro345, which terminates helix TH8, have been reported to block toxicity for Vero cells. We found that mutant toxins in which Pro345 had been replaced by Cys, Glu, or Gly were profoundly defective at low pH in forming channels in planar phospholipid bilayers and in permeabilizing phospholipid vesicles to entrapped fluorophores. Experiments with isolated T domain containing a polarity-sensitive fluorophore attached to Cys at position 332 suggest that the P345E mutation blocks membrane insertion. None of the Pro345 mutations shifted the pH-dependence of binding in solution of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 7-sulfonate. The results indicate that proline at position 345 is required for the T domain to insert into phospholipid bilayers or to adopt a functional conformation within the bilayer.
Asunto(s)
Membrana Celular/química , Toxina Diftérica/química , Liposomas/química , Animales , Transporte Biológico/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Liposomas/metabolismo , Mutación , Prolina/química , Prolina/genética , Células VeroRESUMEN
Agrobacterium-mediated transformation is the method of choice to engineer desirable genes into plants. Here we describe a protocol for demonstrating T-DNA transfer from Agrobacterium into the economically important graminaceous plant maize. Expression of the T-DNA-located GUS gene was observed with high efficiency on shoots of young maize seedlings after cocultivation with Agrobacterium.
Asunto(s)
ADN Bacteriano/genética , Plantas Modificadas Genéticamente , Transfección/métodos , Zea maysRESUMEN
Although the basic mechanisms which control the progression through the cell cycle appear to be conserved in all higher eukaryotes, the unique features of the plant developmental programme must be somehow reflected in a plant-specific regulation of the factors which control cell division. In the last few years, considerable progress has been achieved in identifying the major components of the cell cycle in plants. The question of how these components direct expression of specific genes at specific stages of the cell cycle, and how they are themselves regulated, constitutes a challenge for the present and the next years. This review summarizes our current knowledge at molecular and biochemical levels of cell cycle-regulated expression in the model system, the synchronized tobacco BY2 cell suspension, and discusses the results in comparison to those obtained by different methods and in other plant systems.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/fisiología , Genes cdc/fisiología , Nicotiana/genética , Fenómenos Fisiológicos de las Plantas , Plantas Tóxicas , Factores de TiempoRESUMEN
Stability and distribution of orally administered epidermal growth factor (EGF) were examined in newborn and 5-day-old pigs. Forty-five minutes after oral administration of iodine-125 labeled EGF, 60 and 50% of the radioactivity administered were recovered from the internal organs in newborn and 5-day-old pigs, respectively. In both age groups, over 95% of the recovered radioactivity was found in the gastrointestinal tract, of which 78-86% was found in the luminal contents with the remaining found in the gastrointestinal wall. Within the gastrointestinal tract, 65-71% of radioactivity was found in the stomach, 27-30% in the proximal and mid small intestine and 3-4% was found in the distal part of the small intestine. There were no significant differences in the overall distribution of orally administered radioactivity between two age groups. Based on liquid chromatography and trichloroacetic acid precipitation, a substantial amount of EGF recovered from the luminal contents (63-86%) and the gastrointestinal wall (42-81%) remained "intact". The receptor binding ability of the EGF recovered from the gastric contents was 96-102% comparable to the native EGF tracer. The receptor binding ability remained high (40-58%) in the proximal small intestinal lumen and it decreased to 15% in the distal small intestinal lumen in newborn pigs. In 5-day-old pigs, EGF recovered from the small intestinal contents had 5 to 24% receptor binding ability when compared with native EGF tracer. The receptor binding ability of the EGF recovered from all other organs was below 5% with an exception of the gastric wall, from which recovered EGF retained 9 to 26% receptor binding ability. These results indicate that most of orally ingested EGF remained in the gastrointestinal tract in neonatal pigs 45 min after oral ingestion, and significant amount of the ingested EGF remained biologically active. It suggests that milk-borne EGF can survive in the gastrointestinal tract and may play a role in regulating gut development in neonatal animals.
Asunto(s)
Animales Recién Nacidos/metabolismo , Factor de Crecimiento Epidérmico/farmacocinética , Administración Oral , Animales , Biotransformación , Cromatografía en Gel , Receptores ErbB/metabolismo , Humanos , Absorción Intestinal , Radioisótopos de Yodo , Proteínas Recombinantes/metabolismo , Porcinos , Distribución TisularRESUMEN
We have previously established a reverse genetic system for studying excision of the transposable element Ds1 in maize plants. Ds1 carried by the genome of maize streak virus (MSV) is introduced into maize plants by agroinfection. Excision of Ds1 from the MSV genome depends on the presence of an active Ac element in the recipient maize plants. With the purpose of exploiting MSV-Ds1 as vector for maize transformation, we studied different genes encoding the transposase (TPase) for their efficiency of activating Ds1 excision. These genes were inserted in the same T-DNA carrying MSV-Ds1 and introduced into maize plants by Agrobacterium-mediated transformation. We showed that the wild-type TPase transcribed by the 2' promoter produced much higher efficiency of Ds1 excision than that transcribed by the Ac promoter. In contrast to what had been observed in tobacco and petunia, the truncated TPase (103-807) lacking the amino-terminal 102 amino acids gave a much more reduced Ds1 excision efficiency than the wild-type TPase when both genes were transcribed by the 2' promoter.
Asunto(s)
Elementos Transponibles de ADN , Geminiviridae/genética , Transposasas/biosíntesis , Zea mays/genética , Zea mays/virología , Eliminación de Gen , Genoma Viral , Cinética , Regiones Promotoras Genéticas , Rhizobium/genética , Transcripción Genética , Transformación Genética , Transposasas/genéticaRESUMEN
Mullerian Inhibiting Substance (MIS) functions to promote regression of the Mullerian duct during male development. Maintaining the sexually dimorphic pattern of MIS expression is essential for proper mammalian reproductive tract development. Here, we show that the intricate spatial and temporal pattern of MIS expression is directed by a remarkably small proximal promoter of only 180 base pairs in length. Expression of the MIS-human growth hormone transgene (MIS/GH) is restricted to Sertoli cells in embryonic testis and to granulosa cells of postnatal ovary, consistent with the known MIS expression pattern. The proximal MIS promoter is therefore sufficient to direct the initiation and the maintenance of MIS gene expression in both sexes. Moreover, in vivo MIS promoter activity requires an intact binding site for the orphan nuclear receptor SF-1. Taken together, these data strongly suggest that SF-1 directly activates MIS in embryonic and postnatal gonads. Consistent with the proposed role of SF-1 in mammalian sex-determination, our study provides physiological evidence that a SF-1 binding site is essential for gene activation of an embryonic testis-specific marker.