RESUMEN
The pipeline for new antibacterials is bleak despite the fact that infectious diseases account for a quarter of all worldwide deaths due to disease. Bacteria are ideal organisms for a systems biology approach to understanding pathogenesis by combined use of genomic technologies and computer algorithms. This approach can be applied to identify control points in molecular networks, which could be targets for novel drugs.
Asunto(s)
Antibacterianos/química , Antibacterianos/uso terapéutico , Diseño de Fármacos , Biología de Sistemas , Alcaloides/química , Alcaloides/uso terapéutico , Animales , Infecciones Bacterianas/tratamiento farmacológico , Benzodioxoles/química , Benzodioxoles/uso terapéutico , Genoma Bacteriano/genética , Humanos , Piperidinas/química , Piperidinas/uso terapéutico , Alcamidas Poliinsaturadas/química , Alcamidas Poliinsaturadas/uso terapéuticoRESUMEN
Protein microarrays are a relatively new technology, which will dramatically impact the pharmaceutical industry. The critical need for more rapid identification of novel drug targets, and for obtaining high-quality information early in the target validation process is a major driver for the industry. High-throughput protein analytical techniques are critical for obtaining biological information beyond that which transcript analysis can provide, given that proteins are the "worker bees" in cells. The vast complexity of proteins when compared to DNA and RNA in terms of sheer number, and structural and biochemical diversity requires a higher degree of sophistication in both assay design and data analysis. High-throughput microarray technology platforms allow for simultaneous, multi-parametric analysis of complex protein mixtures. Protein microarrays have tremendous potential as a tool for the study of protein-protein, enzyme-substrate, and antibody-antigen interactions among others. They can also be used for biomarkers and drug target identification via comparative proteomic analysis of healthy and disease tissues. More recently, cellular microarrays that enable identification of cell-surface receptors and other cell-surface proteins allowing rapid screening of cell-specific, novel drug targets, are being developed. This review will focus on the technical issues and potential applications of protein microarrays in pharmaceutical discovery.
Asunto(s)
Análisis por Matrices de Proteínas/métodos , Tecnología Farmacéutica/métodos , Drogas en Investigación/metabolismo , Unión Proteica , Proteínas/metabolismoRESUMEN
High throughput sequencing of a mouse keratinocyte library was used to identify an expressed sequence tag with homology to the epidermal growth factor (EGF) family of growth factors. We have named the protein encoded by this expressed sequence tag Epigen, for epithelial mitogen. Epigen encodes a protein of 152 amino acids that contains features characteristic of the EGF superfamily. Two hydrophobic regions, corresponding to a putative signal sequence and transmembrane domain, flank a core of amino acids encompassing six cysteine residues and two putative N-linked glycosylation sites. Epigen shows 24-37% identity to members of the EGF superfamily including EGF, transforming growth factor alpha, and Epiregulin. Northern blotting of several adult mouse tissues indicated that Epigen was present in testis, heart, and liver. Recombinant Epigen was synthesized in Escherichia coli and refolded, and its biological activity was compared with that of EGF and transforming growth factor alpha in several assays. In epithelial cells, Epigen stimulated the phosphorylation of c-erbB-1 and mitogen-activated protein kinases and also activated a reporter gene containing enhancer sequences present in the c-fos promoter. Epigen also stimulated the proliferation of HaCaT cells, and this proliferation was blocked by an antibody to the extracellular domain of the receptor tyrosine kinase c-erbB-1. Thus, Epigen is the newest member of the EGF superfamily and, with its ability to promote the growth of epithelial cells, may constitute a novel molecular target for wound-healing therapy.
Asunto(s)
Factor de Crecimiento Epidérmico/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Factor de Crecimiento Epidérmico/metabolismo , Epigen , Escherichia coli , Queratinocitos , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
A novel alpha-chemokine, designated KS1, was identified from an EST database of a murine immature keratinocyte cDNA library. The EST has 94% similarity to a recently cloned human gene, BRAK, that has no demonstrated function. Northern analysis of mouse and human genes showed detectable mRNA in brain, intestine, muscle and kidney. Tumour panel blots showed that BRAK was down-regulated in cervical adenocarcinoma and uterine leiomyoma, but was up-regulated in breast invasive ductal carcinoma. KS1 bound specifically to B cells and macrophages, as well as two B cell lines, CESS and A20, and a monocyte line, THP-1. KS1 showed no binding to naive or activated T cells. In addition, KS1 stimulated the chemotaxis of CESS and THP-1 cells but not T cells. The s.c. injection of KS1 creates a mixed inflammatory response in Nude and C3H/HeJ mice. The above data indicates that KS1 and its human homologue represents a novel non-ELR alpha-chemokine that may have important roles in trafficking of B cells and monocytes. We propose the name B cell- and monocyte-activating chemokine (BMAC) for this molecule to reflect the described biological functions.
Asunto(s)
Linfocitos B/inmunología , Quimiocinas CXC/genética , Monocitos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , Quimiocinas CXC/análisis , Quimiocinas CXC/metabolismo , Quimiocinas CXC/farmacología , Quimiotaxis/efectos de los fármacos , Citometría de Flujo , Biblioteca de Genes , Humanos , Queratinocitos/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Desnudos , Datos de Secuencia Molecular , Quistes Odontogénicos/inmunología , ARN Mensajero/análisis , Piel/efectos de los fármacos , Piel/inmunologíaRESUMEN
In the synthesis of inorganic polyphosphate (polyP) from ATP by polyphosphate kinase (PPK; EC 2.7.4.1) of Escherichia coli, an N-P-linked phosphoenzyme was previously identified as the intermediate. The phosphate is presumed to be linked to N3 of the histidine residue because of its chemical stabilities and its resemblance to other enzymes known to contain N3-phosphohistidine. Tryptic digests of [32P]PPK contain a predominant 32P-labeled peptide that includes His-441. Of the 16 histidine residues in PPK of E. coli, 4 are conserved among several bacterial species. Mutagenesis of these 4 histidines shows that two (His-430 and His-598) are unaffected in function when mutated to glutamine, whereas two others (His-441 and His-460) mutated to glutamine or alanine fail to be phosphorylated, show no enzymatic activities, and fail to support polyP accumulation in cells bearing these mutant enzymes.
Asunto(s)
Escherichia coli/enzimología , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Secuencia de Aminoácidos , Activación Enzimática/genética , Histidina/genética , Histidina/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Whereas exopolyphosphatases have been purified from yeast and a variety of bacteria, this is the first report characterizing endopolyphosphatases that act on long chain inorganic polyphosphate (polyP). The activity from Saccharomyces cerevisiae, localized in vacuoles, has been purified to homogeneity from a strain that possesses vacuolar proteases. The endopolyphosphatase is a dimer of 35-kDa subunits. Distributive action on polyP750 produces shorter chains to a limit of about polyP60, as well as the more abundant release of polyP3; the Km for polyP750 is 185 nM. Endopolyphosphatases have been identified in a wide variety of sources, except for most eubacteria tested. The activity has been partially purified from rat and bovine brain where its abundance is about 10 times higher than in other tissues but less than 1/10 that of yeast; the limit product of digestion of the partially purified brain enzyme is polyP3.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Polifosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácido Anhídrido Hidrolasas/aislamiento & purificación , Animales , Ratas , Ratas Endogámicas F344RESUMEN
Inorganic polyphosphate (polyP), a linear polymer of hundreds of orthophosphate (Pi) residues linked by high-energy, phosphoanhydride bonds, has been identified and measured in a variety of mammalian cell lines and tissues by unambiguous enzyme methods. Subpicomole amounts of polyP (0.5 pmol/100 micrograms of protein) were determined by its conversion to ATP by Escherichia coli polyphosphate kinase and, alternatively, to Pi by Saccharomyces cerevisiae exopolyphosphatase. Levels of 25 to 120 microM (in terms of Pi residues), in chains 50 to 800 residues long, were found in rodent tissues (brain, heart, kidneys, liver, and lungs) and in subcellular fractions (nuclei, mitochondria, plasma membranes, and microsomes). PolyP in brain was predominantly near 800 residues and found at similar levels pre- and postnatally. Conversion of Pi into polyP by cell lines of fibroblasts, T-cells, kidney, and adrenal cells attained levels in excess of 10 pmol per mg of cell protein per h. Synthesis of polyP from Pi in the medium bypasses intracellular Pi and ATP pools suggesting the direct involvement of membrane component(s). In confluent PC12 (adrenal pheochromocytoma) cells, polyP turnover was virtually complete in an hour, whereas in fibroblasts there was little turnover in four hours. The ubiquity of polyP and variations in its size, location, and metabolism are indicative of a multiplicity of functions for this polymer in mammalian systems.
Asunto(s)
Encéfalo/metabolismo , Fosfatos/metabolismo , Polifosfatos/análisis , Células 3T3 , Envejecimiento/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Química Encefálica , Cromatografía en Capa Delgada , Embrión de Mamíferos , Escherichia coli/enzimología , Humanos , Riñón/química , Hígado/química , Pulmón/química , Ratones , Ratones Endogámicos BALB C , Microquímica , Miocardio/química , Orgánulos/química , Células PC12 , Radioisótopos de Fósforo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Ratas , Ratas Endogámicas F344 , Sensibilidad y Especificidad , Especificidad de la Especie , Fracciones Subcelulares/químicaRESUMEN
Annexin II is a calcium and phospholipid binding protein and a substrate for protein-tyrosine kinases. Recent investigations have revealed involvement of annexin II in DNA synthesis and cell proliferation. Increased levels of annexin II are observed in cancer cells and tissues. To investigate the expression of annexin II in pancreatic adenocarcinoma cells and primary tumors, we measured the levels of annexin II mRNA and protein in normal human pancreas, five established human pancreatic adenocarcinoma cell lines, three primary pancreatic cancers and one metastatic tumor. All five cell lines examined had 5- to 15-fold higher levels of annexin II as compared to normal pancreas. Significant elevations (2- to 8-fold) of annexin II expression were observed in the three primary pancreatic tumors and one metastatic tumor examined. Immunocytochemical analysis indicates that the increased expression of annexin II is limited to proliferating ductular adenocarcinoma, and annexin II expression co-localizes with cells that express PCNA. In normal pancreas, annexin II expression is seen in ductal and ductular cells and no expression is seen in acinar or islet cells. We conclude from these findings that annexin II has a role in cell proliferation and its regulation is altered in pancreatic cancer.
Asunto(s)
Adenocarcinoma/metabolismo , Anexina A2/biosíntesis , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/patología , Anexina A2/genética , Humanos , Inmunohistoquímica , Metástasis de la Neoplasia , Páncreas/metabolismo , Neoplasias Pancreáticas/patología , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
Modification of A. conoides beta-glucosidase by diethylpyrocarbonate caused rapid inactivation of the enzyme. The kinetic analyses showed that the inactivation by diethylpyrocarbonate resulted from the modification of an average of one histidine residue per mole of enzyme. The modified enzyme showed an increase in absorbance at 240 nm. Sulphydryl, lysine and tyrosine residues were not modified by diethylpyrocarbonate treatment. The substrate offered significant protection against diethylpyrocarbonates modification. The results indicate that diethylpyrocarbonate was interacting with the enzyme at or near the active site.
Asunto(s)
Dietil Pirocarbonato/farmacología , Histidina/fisiología , beta-Glucosidasa/efectos de los fármacos , Sitios de Unión , Yodoacetamida/farmacología , Hongos Mitospóricos/enzimología , Nitrofenilgalactósidos/farmacología , Fosfato de Piridoxal/farmacología , beta-Glucosidasa/aislamiento & purificaciónRESUMEN
Primer recognition proteins (PRP) are cofactors for DNA polymerase alpha and may have a role in lagging-strand DNA replication. PRP is composed of two subunits, which we have previously identified as the protein-tyrosine kinase substrate annexin II and phosphoglycerate kinase (PGK). In this study, we have examined the physiological involvement of these proteins in DNA synthesis and cell proliferation. When exponentially growing human HeLa cells are exposed to antisense phosphorothioate oligodeoxynucleotides to annexin II, ongoing DNA synthesis is reduced. The extent of reduction with antisense oligodeoxynucleotide to PGK was much less than with the antisense annexin II oligodeoxynucleotide. Reductions in the labeling and mitotic indices of HeLa cell cultures are seen after exposure to antisense oligodeoxynucleotides. Flow cytometric analyses indicate that progression from S phase to G2 phase of the cycle is retarded by exposure of cells to the antisense oligodeoxynucleotides. Corresponding sense oligodeoxynucleotides have no inhibitory effects on these parameters. The new synthesis of annexin II and PGK is specifically reduced in the presence of antisense oligodeoxynucleotides, indicating that the complex of newly synthesized annexin II and PGK may participate in PRP function. These experiments indicate that annexin II and PGK may have a physiological role in DNA synthesis and cell cycle progression, and represent the first physiological role for annexin II monomer in cells.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Replicación del ADN/efectos de los fármacos , ADN sin Sentido/farmacología , Oligodesoxirribonucleótidos/farmacología , Fosfoglicerato Quinasa/análisis , Anexinas , Secuencia de Bases , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/fisiología , Células HeLa , Humanos , Datos de Secuencia MolecularRESUMEN
Annexins are a family of calcium- and phospholipid-binding proteins related by amino acid sequence homology. Annexins I and II are substrates for protein tyrosine kinases. Recent investigations have revealed a possible involvement of annexins I and II in mitogenic signal transduction and cell proliferation. To investigate further the involvement of annexins in cell proliferation, we measured the levels of annexins I and II and the enzyme 3-phosphoglycerate kinase (PGK) (annexin II and PGK are components of the primer recognition protein complex) in normal Syrian hamster pancreas, three hamster pancreatic ductal carcinoma cell lines, and allografts of the three cell lines into hamster pancreas. All three carcinoma cell lines had 5-8-fold higher levels of annexin II compared to normal pancreas. An inverse relationship was seen between level of annexin II and the doubling time of the cell culture. In intrapancreatic allografts, annexin II levels were 3-6-fold higher than in normal pancreas. Annexin I levels were 2-3-fold higher in the allografts. Significant increases (5-6-fold) in specific activity of PGK were seen in all allografts examined. However, the level of PGK, as measured by immunoblotting, was not significantly altered. Immunohistochemical staining revealed heterogeneity in the reactivity of the antiannexin and anti-PGK antibodies with tumor cells. Strikingly, the reactivity and staining intensity were greater in the proliferating regions of the primary tumors and in the metastatic foci. Mitotic cells were either unstained or very weakly stained. We conclude from these findings that annexin II and PGK, as primer recognition proteins, may have a role in cell proliferation.
Asunto(s)
Proteínas de Unión al Calcio/análisis , Carcinoma/química , Células PC12/química , Neoplasias Pancreáticas/química , Fosfoglicerato Quinasa/análisis , Animales , Anexinas , Cricetinae , Trasplante de Neoplasias , Células Tumorales Cultivadas/químicaRESUMEN
Primer recognition proteins (PRP) are accessory proteins for DNA polymerase alpha in lagging strand DNA replication. We have previously reported that the PRP consist of a complex of two proteins identified as 3-phosphoglycerate kinase (PGK) and the protein-tyrosine kinase substrate, annexin 2 monomer. The physiological role of annexin 2 is not known. Two pools of annexin 2 exist in cells. A majority of annexin 2 is localized with the plasma membrane as a heterotetramer in association with a light chain. Monomer annexin 2 is cytosolic. The identification of annexin 2 monomer as a part of the PRP complex represents one of the physiological roles of this protein in cells. To function as PRP, annexin 2 and PGK would have to be present in the cell nucleus. To investigate whether monomer annexin 2 is indeed associated with nuclear DNA synthesis, we investigated the presence of annexin 2 and PGK in the cell nucleus. In this paper, we demonstrate the presence of annexin 2 and PGK in nuclear extracts. The nuclear fraction of these proteins represents a small subset of the total cellular pools. Immunoelectron-microscopic analyses using anti-PRP antisera demonstrate the distribution of these proteins in HeLa cell nuclei and cytoplasm. Under identical conditions, an anti-cytokeratin monoclonal antibody preferentially labels the plasma membrane without detectable intracellular staining. The distribution of annexin 2 and PGK in both nuclei and cytoplasm is similarly observed in cells from normal tissues such as freshly isolated rat hepatocytes and hamster pancreatic tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas de Unión al Calcio/análisis , Núcleo Celular/química , Citoplasma/química , Proteínas de la Membrana/análisis , Fosfoglicerato Quinasa/análisis , Animales , Anexinas , Ciclo Celular , Membrana Celular/química , Núcleo Celular/enzimología , Transformación Celular Neoplásica , Cricetinae , Citoplasma/enzimología , Replicación del ADN , Células HeLa , Humanos , Hígado/química , Hígado/ultraestructura , Microscopía Inmunoelectrónica , Páncreas/química , Páncreas/ultraestructura , RatasRESUMEN
Two endoglucanases, designated Endo I and Endo II, were purified from the culture filtrates of a nematode trapping fungus, Arthrobotrys oligospora. The purification procedure entailed ammonium sulphate precipitation, gel filtration and preparative PAGE. Both the preparations (Endo I and Endo II) were homogeneous by PAGE, had molecular weights of 24,300 and 44,500 respectively as determined by non-denaturing PAGE, and yielded only cellobiose as the main product of CM-cellulose hydrolysis. The optimum pH and temperature for Endo I were 6.0 and 50 degrees C, and for Endo II, pH 5.6-6.4 and 50 degrees C. The two enzymes differed with respect to their Km (Endo I, 5.04 mg/ml; Endo II, 3.2 mg/ml) and energy of activation values (Endo I, 10.7 kCal; Endo II, 9.5 k Cal). Both enzymes were completely inhibited by 1.25 mH Hg2+ and partially by Pb2+, DTNB and p-HMB while DTT and GSH enhanced their activities.