RESUMEN
Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The activated wild-type enzyme irreversibly denatures at 60 °C with a pseudo-first-order kinetics and a half-life time (t1/2) of 2 min. To increase the thermoresistance of MTG for higher temperature applications, we generated 31 variants based on previous results obtained by random mutagenesis, DNA shuffling and saturation mutagenesis. The best variant TG16 with a specific combination of five of seven substitutions (S2P, S23Y, S24 N, H289Y, K294L) shows a 19-fold increased half-life at 60 °C (t1/2 = 38 min). As measured by differential scanning fluorimetry, the transition point of thermal unfolding was increased by 7.9 °C. Also for the thermoresistant variants, it was shown that inactivation process follows a pseudo-first-order reaction which is accompanied by irreversible aggregation and intramolecular self-crosslinking of the enzyme. Although the mutations are mostly located on the surface of the enzyme, kinetic constants determined with the standard substrate CBZ-Gln-Gly-OH revealed a decrease in KM from 8.6 mM (± 0.1) to 3.5 mM (± 0.1) for the recombinant wild-type MTG and TG16, respectively. The improved performance of TG16 at higher temperatures is exemplary demonstrated with the crosslinking of the substrate protein ß-casein at 60 °C. Using molecular dynamics simulations, it was shown that the increased thermoresistance is caused by a higher backbone rigidity as well as increased hydrophobic interactions and newly formed hydrogen bridges.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Streptomyces/enzimología , Transglutaminasas/química , Transglutaminasas/metabolismo , Proteínas Bacterianas/genética , Clonación Molecular , Estabilidad de Enzimas , Calor , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/química , Streptomyces/genética , Especificidad por Sustrato , Transglutaminasas/genéticaRESUMEN
We report the identification and characterization of two genes from Drosophila melanogaster that encode novel ionotropic glutamate receptor proteins, named DGluR-IB and DNMDAR-II, and that are located on chromosome 3L, region 67AB, and the X chromosome, position 2B, respectively. The DGluR-IB full-length cDNA was isolated from Drosophila embryonic and head libraries. The encoded protein of 1,095 amino acids displays high sequence identity (73%) to DGluR-IA. The DNMDAR-II gene was identified by sequence-homology searches in databases. The deduced protein shows moderate sequence identity (29-31%) to the mouse NMDAR2A-D receptor subunits. Whole-mount in situ hybridization on embryos revealed DGluR-IB and DNMDAR-II transcripts in the CNS. Immunofluorescence analysis of the adult fly brain indicates that the DGluR-IB protein is expressed in neurons implicated in the regulation of the circadian clock.
Asunto(s)
Sistema Nervioso Central/metabolismo , Drosophila melanogaster/genética , Receptores de Glutamato/genética , Animales , ADN Complementario/genética , Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Immunoblotting , Datos de Secuencia Molecular , Receptores de Glutamato/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
In a randomized trial in 102 preterm newborns with respiratory distress syndrome (RDS) it has been shown that early Ambroxol treatment (30 mg kg(-1) over the first 5 days) significantly reduces the incidence of RDS-associated complications [bronchopulmonary dysplasia (BPD), intraventricular haemorrhage, post-natal acquired pneumonia]. The aim of the present analysis was to investigate the effect of Ambroxol treatment on lung function in newborns who developed BPD. Respiratory function testing (RFT) was performed immediately after extubation and at day 28. Tidal volume (VT) and respiratory frequency (f) were measured during tidal breathing using the deadspace free flow-through technique. The lung mechanic parameter VT/maxPes was determined by measuring the maximal oesophageal pressure changes, maxPes, with a catheter tip pressure transducer. In the placebo group 36/50 infants were extubated within the first 28 days of life and 13/36 (36%) developed BPD. In the Ambroxol group 44/52 were extubated and 9/44 (20%) developed BPD. After extubation, RFT showed (i) no statistically significant difference in the ventilatory parameters of either treatment group, (ii) improved (P<0.05) lung mechanics (VT/maxPes) in Ambroxol group compared to controls (94+/-27 ml kPa(-1) vs. 8.1+/-2.6 ml kPa(-1)) and (iii) no statistically significant difference in lung function between infants with and without BPD. At day 28 we found (i) no effect of early Ambroxol treatment on lung functions, (ii) significantly (P < 0.05) higher f (58.5+/-11.7 min(-1) vs. 49.7+/-10.1 min(-1)) and significantly (P<0.01) lower V(T) (9.6+/-1.9 ml vs. 12.3+/-2.7 ml) and V(T)/maxPes (8.9+/-2.6 ml kPa(-1)] vs. 12.0+/-2.9 ml kPa(-1)) in infants with BPD compared to infants without and (iii) these differences are not influenced by early Ambroxol treatment. If the process of BPD development is induced, early Ambroxol treatment has no influence on impaired lung function at day 28.
Asunto(s)
Ambroxol/uso terapéutico , Displasia Broncopulmonar/prevención & control , Expectorantes/uso terapéutico , Displasia Broncopulmonar/fisiopatología , Método Doble Ciego , Femenino , Humanos , Recién Nacido , Recien Nacido Prematuro , Masculino , Trastornos Respiratorios/tratamiento farmacológico , Trastornos Respiratorios/fisiopatología , Respiración Artificial/métodos , Pruebas de Función RespiratoriaRESUMEN
UNLABELLED: Macrophages concentrate urokinase-type plasminogen activator (uPA) at the cell surface by expressing urokinase receptors (uPAR) in order to focus the pericellular space plasminogen-dependent proteolysis important in matrix remodeling and cell movement. This study examines the uPAR levels of tumor-associated macrophages (TAM) of invasive breast carcinomas, of TAMs from ductal carcinoma in situ (DCIS) and of macrophages derived from normal (non-tumor) breast tissue. TAMs from invasive breast carcinomas (n = 30), from DCIS (n = 12), and macrophages from normal breast tissue (n = 30) were cultured and immunocytochemically phenotyped by using a panel of antibodies. Urokinase receptor levels were determined by Western blot analysis and in cell-free supernatants by enzyme-linked immunosorbent assay. Urokinase receptor cell surface fluorescence intensity was determined by FACS and by confocal laser scan microscopy. Urokinase-receptor mRNA was detected by in situ hybridization. TAMs of invasive breast carcinomas and of DCIS possess significantly elevated uPAR levels compared with macrophages derived from normal breast tissue. CONCLUSIONS: activated macrophages with elevated uPAR levels belong to inflammatory areas in close vicinity of infiltrating and non-infiltrating (DCIS) tumor cells. Blood monocytes that possess elevated uPAR-levels may be selectively recruited from the bloodstream to inflammatory sites close to carcinoma cells, and/or breast cancer and precursor lesions may induce elevated uPAR-levels in TAMs by paracrine interactions.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma Intraductal no Infiltrante/metabolismo , Macrófagos/metabolismo , Receptores de Superficie Celular/biosíntesis , Antígenos CD18/biosíntesis , Femenino , Humanos , Antígeno de Macrófago-1/biosíntesis , Receptores de Superficie Celular/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Células Tumorales CultivadasRESUMEN
Several studies have demonstrated that ambroxol stimulates surfactant synthesis and has antioxidative and antiinflammatory effects. We investigated the effect of ambroxol on lung function in newborns with respiratory distress syndrome (RDS) weighing <1,500 g. In all, 102 newborns were enrolled (52 received ambroxol and 50 placebo). After extubation, lung function tests were performed weekly using a face mask for ventilatory measurements and a catheter tip pressure transducer (diameter 1.7 mm) for esophageal pressure measurements (Pes) The flow-through technique was used to eliminate apparatus dead space and to allow long-term measurements during quiet sleep. Percentile curves of pulmonary function parameters from healthy newborns were used for comparison. During the first 28 days, 42 newborns were extubated in the ambroxol group and 36 in the placebo group. The ventilatory parameters of both treatment groups were in the normal range and there were no significant differences between the two groups at any time. After extubation, the ratio of tidal volume to maximal esophageal pressure changes (V(T)/P(es,max)) was below the 10th percentile in the ambroxol and placebo-treated groups. In the ambroxol group the 10th percentile was reached on day 10, whereas in the placebo group the 10th percentile was reached significantly later (P < 0.05) on day 23. Modeling of power expenditures was used to identify the optimal breathing pattern so that small differences in ventilatory parameters between the two groups could be analyzed. We conclude that early ambroxol treatment has only a modest effect on lung function in newborns with established RDS. The sensitivity of tidal breathing parameters is not sufficient to detect these small changes in lung mechanics, but small improvements could be demonstrated in lung mechanics 10 days after extubation in the ambroxol-treated group.
Asunto(s)
Ambroxol/uso terapéutico , Expectorantes/uso terapéutico , Recien Nacido Prematuro/fisiología , Respiración , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Síndrome de Dificultad Respiratoria del Recién Nacido/fisiopatología , Femenino , Humanos , Recién Nacido , Masculino , Pruebas de Función Respiratoria , Resultado del TratamientoRESUMEN
Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.
Asunto(s)
Cinesinas/metabolismo , Miosinas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas ras/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Células COS , Femenino , Humanos , Cinesinas/química , Cinesinas/genética , Masculino , Ratones , Mutagénesis Sitio-Dirigida , Miosinas/química , Miosinas/genética , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Receptor EphB2 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Especificidad por Sustrato , Distribución TisularRESUMEN
HEK2 belongs to the family of EPH-related receptor tyrosine kinases (RTK) which are involved in axonal pathfinding and the formation of the embryonic body plan. The knowledge about intracellular pathways of signal transduction mediated by EPH-related receptors is still limited. Many of the known key players of cellular signalling contain Src homology 2 (SH2) domains, which recognize phosphotyrosine motifs in RTKs. Thus, we examined the interactions of various SH2-containing molecules like PLC-gamma1, rasGAP, p85 subunit of PI3-kinase, Src, Fyn, Crk, Nck, Grb2 and Shc with HEK2 using in vitro binding assays, immunoprecipitations and yeast Two-Hybrid assays. We found that rasGAP, Crk and Fyn bind in a SH2-dependent manner to autophosphorylated HEK2. rasGAP, which contains two SH2- and one SH3-domain, was shown to associate with its N-terminal SH2-domain to HEK2. Furthermore, we demonstrated that a single amino acid substitution (Y614F) clearly reduces the phosphotyrosine content of HEK2 and abrogates its ability to bind rasGAP, Crk and Fyn indicating that this residue functions as major phosphorylation and multi-docking site. The conservation of this predicted binding site among various EPH-related RTKs provides evidence that Fyn, Crk and rasGAP are key players in signal transduction of at least a subset of these receptors.
Asunto(s)
Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina/metabolismo , Dominios Homologos src , Sitios de Unión , Proteínas Activadoras de GTPasa , Humanos , Fosforilación , Unión Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-crk , Proteínas Proto-Oncogénicas c-fyn , Receptor EphB3 , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Recent studies have shown that urokinase (uPA) is an independent prognostic marker in breast cancer. uPA plays a key role in the degradation of tumor matrix and promotes tumor progression. Macrophage expression of uPA appears to be important in this context. Our objective in the present study was to provide evidence that tumor growth factor-beta (TGF-beta) released from breast cancer cells markedly up-regulates uPA expression in tumor-associated macrophages (TAMs). TAMs from 32 breast carcinomas were cultured. Blood monocytes from healthy donors and breast cancer patients as well as tissue macrophages from patients with fibrocystic changes of the breast were also examined. After TGF-beta incubation, uPA levels were tested by ELISA, and uPA mRNA levels were determined by Northern blot analysis. TGF-beta receptor and uPA cell surface fluorescence intensities were determined by flow cytometry; TGF-beta receptors were determined by Western blot analysis. Protein kinase-C dependence was also examined, and immunohistochemical stainings for uPA and TGF-beta were performed. We have demonstrated that TGF-beta markedly up-regulates basal uPA expression (mRNA and protein) in TAMs but only modestly increases uPA production in blood monocytes and tissue macrophages. Exposure of macrophages to TGF-beta1 led to a rapid and sustained increase in uPA mRNA levels, which was independent of de novo protein synthesis and completely inhibited by actinomycin D. H7 markedly reduced the ability of TGF-beta to stimulate uPA expression. Likewise, okadaic acid potentiated the ability of TGF-beta to up-regulate macrophage uPA expression. We suggest that TAMs are more responsive to TGF-beta stimulation than are blood monocytes and tissue macrophages because of different TGF-beta receptor densities. TGF-beta stimulates transcription of the uPA gene, increases uPA-mRNA stability, and activates uPA expression via protein kinase-C-dependent mechanisms. The ability of TGF-beta to induce macrophage uPA expression may provide an indirect mechanism by which this growth factor stimulates angiogenesis. It may be, therefore, that TAMs promote tumor progression and tumor angiogenesis.
Asunto(s)
Neoplasias de la Mama/enzimología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Activadores Plasminogénicos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Neoplasias de la Mama/patología , Femenino , Enfermedad Fibroquística de la Mama/enzimología , Enfermedad Fibroquística de la Mama/patología , Humanos , Monocitos/enzimología , Activadores Plasminogénicos/genética , ARN Mensajero/metabolismo , Valores de Referencia , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Nuclear lamins are thought to play an important role in disassembly and reassembly of the nucleus during mitosis. Here, we describe a Drosophila lamin Dm0 mutant resulting from a P element insertion into the first intron of the Dm0 gene. Homozygous mutant animals showed a severe phenotype including retardation in development, reduced viability, sterility, and impaired locomotion. Immunocytochemical and ultrastructural analysis revealed that reduced lamin Dm0 expression caused an enrichment of nuclear pore complexes in cytoplasmic annulate lamellae and in nuclear envelope clusters. In several cells, particularly the densely packed somata of the central nervous system, defective nuclear envelopes were observed in addition. All aspects of the mutant phenotype were rescued upon P element-mediated germline transformation with a lamin Dm0 transgene. These data constitute the first genetic proof that lamins are essential for the structural organization of the cell nucleus.
Asunto(s)
Proteínas de Drosophila , Drosophila/genética , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Animales , Secuencia de Bases , Química Encefálica , Citoplasma/química , Citoplasma/metabolismo , Drosophila/química , Drosophila/metabolismo , Embrión no Mamífero/fisiología , Fertilidad/genética , Técnica del Anticuerpo Fluorescente Indirecta , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes de Insecto/fisiología , Mutación de Línea Germinal/fisiología , Homocigoto , Intrones/genética , Laminas , Locomoción/genética , Microscopía Electrónica , Microtomía , Datos de Secuencia Molecular , Mutagénesis Insercional/fisiología , Membrana Nuclear/ultraestructura , Proteínas Nucleares/inmunología , Fenotipo , Transformación Genética/fisiologíaRESUMEN
Human embryonal kinase 2 (HEK2) is a protein-tyrosine kinase that is a member of the EPH family of receptors. Transcripts for HEK2 have a wide tissue distribution. Recently, a still growing family of ligands, which we have named LERKs, for ligands of the eph-related kinases, has been isolated. In order to analyze functional effects between the LERKs and the HEK2 receptor, we expressed HEK2 cDNA in an interleukin-3-dependent progenitor cell line 32D that grows as single cells in culture. Within the group of LERKs, LERK-2 and -5 were shown to bind to HEK2. Membrane-bound and soluble forms of LERK-2 were demonstrated to signal through HEK2 as judged by receptor phosphorylation. Coincubation of HEK2 and LERK-2 expressing cells induced cell-cell adhesion and formation of cell aggregates. This interaction could be inhibited by preincubation of HEK2 expressing cells with soluble LERK-2. Coexpression of HEK2 and LERK-2 in 32D cells showed reduced kinase activity and autophosphorylation of HEK2 compared with the juxtacrine stimulation, which seems to be due to a reduced sensitivity of the receptor.
Asunto(s)
Adhesión Celular , Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Secuencia de Bases , Línea Celular , Membrana Celular/metabolismo , Medios de Cultivo , Activación Enzimática , Efrina-B1 , Humanos , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fosforilación , Unión Proteica , Receptor EphB3RESUMEN
We have identified the nucleotide sequence of the cDNA encoding the human counterpart of the mouse gene Plk (polo-like kinase). The sequence of the human gene, PLK, predicts a serine/threonine kinase of 603 aa. Expression of PLK mRNA appeared to be strongly correlated with the mitotic activity of cells. Resting peripheral lymphocytes did not express the gene at all. When primary T cells were activated by phytohemagglutinin, a high level of PLK transcripts resulted within 2-3 days. In some cases, addition of interleukin 2 to these cells increased the expression of PLK mRNA further. In contrast, primary cultures of human peripheral macrophages, which were not dividing under the culture conditions applied, showed very little or no PLK mRNA. Stimulation of these cells by bacterial lipopolysaccharide, an inducer of several cytokines in macrophages, totally abrogated the expression of PLK mRNA. In line with a function of PLK mRNA expression in mitotically active cells is our finding that six immortalized cell lines examined expressed the gene. In A-431 epidermoid carcinoma cells this expression was down-regulated by serum starvation and enhanced after serum was added again. Tumors of various origin (lung, colon, stomach, smooth muscle, and esophagus as well as non-Hodgkin lymphomas) expressed high levels of PLK transcripts in about 80% of the samples studied, whereas PLK mRNA was absent in surrounding tissue, except for colon. The only normal tissues where PLK mRNA expression was observed were colon and placenta, both known to be mitotically active. No PLK transcripts were found in normal adult lung, brain, heart, liver, kidney, skeletal muscle, and pancreas. In Northern blot experiments with RNA from lymphocytes which were treated with phytohemagglutinin and cycloheximide, PLK transcripts were not detectable, suggesting that PLK is not an early growth-response gene.
Asunto(s)
Proteínas de Drosophila , Proteínas Serina-Treonina Quinasas/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , División Celular , Cicloheximida/farmacología , ADN Complementario/genética , ADN de Neoplasias/genética , Regulación hacia Abajo , Inducción Enzimática , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Datos de Secuencia Molecular , Neoplasias/enzimología , Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
A sensitive isocratic HPLC method for the analysis of AWD 122-14 (1) in plasma and urine was developed. The extraction was processed on-line on a short column. The pharmacokinetics of 1 was studied in rats. The plasma concentration-time course was described by an 1-compartment-model. The pharmacokinetic parameters were estimated. The absorption and elimination of 1 are relatively fast. A single oral dose (D = 1 x 10(-5) mol/kg) was rapidly absorbed (absorption rate constant: kI = 6.85 h-1). The elimination rate constant is kE = 1.59 h-1. The absolute bioavailability of a single oral dose equals 11%. In rats 1 is mainly metabolized. Less than 5% of the dose is excreted in the urine as unchanged drug. The chemical structures of 10 of the 12 isolated metabolites were determined using high-resolution mass spectrometry.
Asunto(s)
Cardiotónicos/farmacocinética , Morfolinas/farmacocinética , Piridinas/farmacocinética , Administración Oral , Animales , Biotransformación , Cardiotónicos/administración & dosificación , Cardiotónicos/sangre , Cardiotónicos/orina , Cromatografía Líquida de Alta Presión , Humanos , Inyecciones Intravenosas , Masculino , Morfolinas/administración & dosificación , Morfolinas/sangre , Morfolinas/orina , Unión Proteica , Piridinas/administración & dosificación , Piridinas/sangre , Piridinas/orina , Ratas , Ratas Wistar , Espectrofotometría UltravioletaRESUMEN
Lung cancer is the leading cause of death from cancer in Western countries. For improved diagnosis and refined therapeutical approaches it is of major importance to understand by what mechanisms carcinoma of the lung develop. The analysis of primary lung cancer revealed specific chromosomal alterations and allelic losses of the short arm of chromosome 3. Genetic aberrations have been observed in proto-oncogenes such as H-ras, K-ras, C-myc and raf-1 as well as in the tumor suppressor genes Rb and p53. Rearrangement of rlf and elevated expression in certain lung tumors have also been reported. The development of lung cancer also involves the altered activation of genes coding for growth factors such as TGF beta 2 and certain growth factor receptor genes such as c-erbB-2, HEK2 and FGFR-4.
RESUMEN
Using a polymerase chain reaction-mediated approach we have characterized cDNAs from human and mouse origin representing a novel type of receptor protein tyrosine kinase (RTK). The deduced amino acid sequence (855 amino acids) of the longest open reading frame has a unique extracellular region encompassing a factor VIII-like domain, not previously described for RTKs. The most closely related RTKs are members of the neurotrophin receptors (TRK), which showed 47-49% homology with the kinase domain of the new RTK. Therefore, the new gene has been called TKT (Tyrosine-Kinase related to TRK). TKT orthologs from man and mouse were 98% similar. In both species a major transcript of 10 kb was found to be expressed at high levels in heart and lung. Low levels of this mRNA-species were detected in human brain, placenta, liver, skeletal muscle, kidney and in mouse brain and testis. Analysing human/mouse somatic cell hybrids we demonstrated that TKT segregates with human chromosome 1.
Asunto(s)
Mapeo Cromosómico , Factor VIII/química , Factor VIII/genética , Regulación Enzimológica de la Expresión Génica/genética , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Química Encefálica , ADN/análisis , ADN/genética , Factor VIII/análisis , Humanos , Hígado/química , Ratones , Datos de Secuencia Molecular , Músculos/química , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Quinasas Receptoras/análisisRESUMEN
We have previously amplified cDNA subfragments of protein-tyrosine-kinases (PTKs) by using the polymerase chain reaction (PCR) and specific sets of oligonucleotide primers derived from nucleotide sequences of their kinase domain. In this study we have used a more directed approach to identify new members of the EPH/elk-family by PCR of human embryonic cDNA: we utilized oligonucleotide primers specifically designed to a highly conserved N-terminal motif and the kinase region of EPH/elk-PTKs in RNA-PCRs. The 5' and 3' elongation of the primary PCR product was achieved by the RACE (rapid amplification of cDNA ends)-technique. Sequence analysis of 3.8 kb of overlapping PCR products allowed to identify a novel receptor-PTK, HEK 2 (human embryo kinase 2), as an additional member of this family, without the need to screen a cDNA library. This approach should be useful for the rapid isolation of other PTK-genes as well. Analysis of genomic DNA placed HEK 2 on chromosome 3. Northern blot analysis demonstrated the expression of a 4.6 kb HEK 2-mRNA in lung, brain, pancreas, liver, placenta, kidney, skeletal muscle, heart and several human cells. In a protein kinase assay with HEK 2-specific immunoprecipitates from the human epidermoid carcinoma cell line A431, a protein of 130 kDa was found phosphorylated.
Asunto(s)
Proteínas Tirosina Quinasas/aislamiento & purificación , Receptores de Superficie Celular/aislamiento & purificación , Secuencia de Aminoácidos , Amiloide/química , Amiloide/genética , Animales , Secuencia de Bases , Southern Blotting , Línea Celular , Secuencia Conservada , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Ratas , Receptor EphA2 , Receptor EphA3 , Receptor EphB3 , Receptores de Superficie Celular/químicaRESUMEN
In order to test the ability of Ambroxol to improve the clinical course of respiratory distress syndrome and to reduce the incidence of complications a multicentre, randomized, placebo-controlled double-blind trial was conducted. Entry was limited to infants with a birth weight below 1500 g. A total of 179 neonates were enrolled, but 31 were later excluded because they had other diseases. Of the remaining 148 babies, 74 received Ambroxol (birth weight 1190 +/- 216 g; gestational age 29.1 +/- 1.9 weeks) and 74 placebo (birth weight 1168 +/- 216 g; gestational age 28.9 +/- 1.9 weeks). In the Ambroxol group 23 (31%) and in the placebo group 27 (37%) infants died during the first 5 months of life. In 28 day-survivors Ambroxol was able to significantly improve the PaO2/FiO2 ratio, mean airway pressure, phospholipid profile of tracheal effluent and pulmonary mechanics of spontaneously breathing infants. In addition, the incidences of bronchopulmonary dysplasia (29% vs 54%), intraventricular haemorrhage (25% vs 44%) and postnatally acquired pneumonia (15% vs 36%) were significantly reduced in the Ambroxol group as compared to the control group. No adverse events attributed to the Ambroxol treatment were reported.
Asunto(s)
Ambroxol/uso terapéutico , Recién Nacido de Bajo Peso , Síndrome de Dificultad Respiratoria del Recién Nacido/tratamiento farmacológico , Método Doble Ciego , Humanos , Recién Nacido , Masculino , Síndrome de Dificultad Respiratoria del Recién Nacido/complicaciones , Síndrome de Dificultad Respiratoria del Recién Nacido/mortalidad , Tasa de Supervivencia , Resultado del TratamientoAsunto(s)
Benzoatos/farmacología , Gránulos Citoplasmáticos/fisiología , Hidrazonas/farmacología , Inhibidores de la Lipooxigenasa , Mastocitos/ultraestructura , Bases de Schiff/farmacología , Animales , Benzoatos/química , Gránulos Citoplasmáticos/efectos de los fármacos , Fabaceae/enzimología , Liberación de Histamina/efectos de los fármacos , Hidrazonas/química , Mastocitos/efectos de los fármacos , Estructura Molecular , Octoxinol , Cavidad Peritoneal/citología , Plantas Medicinales , Polietilenglicoles/farmacología , Protaminas/farmacología , Ratas , Bases de Schiff/químicaRESUMEN
The cyclization of ethyl 2-benzoylthioureidothiophen-3-carboxylates under basic conditions is known to give 2-thioxothieno[2,3-d]pyrimidin-4(1H, 3H)-ones. On the other hand, we have found that ethyl 2-benzoylthioureidothiophen-3-carboxylates 10-16 on treatment with concentrated sulphuric acid or polyphosphoric acid/ethanol undergo cyclization to give the new 2-aminothieno[2,3-d][1,3]thiazin-4-ones 17-23, in some cases 5,6-anellated. Reaction of 10-16, which are readily available from ethyl 2-aminothiophen-3-carboxylates 3-9 and benzoyl isothiocyante affords the title compounds 17-23 in good yields. Mass spectral fragmentation of 17-23 is discussed. A series of 2-aminothieno[2,3-d][1,3]thiazin-4-ones, which contain a free or substituted amino group was evaluated in the rat active cutaneous anaphylaxis test for anti-allergy activity. One compound, 23 had weak activity in the range of theophylline. Only high concentrations of this compound inhibited weakly the histamine release from rat peritoneal mast cells activated by protamine sulfate.