RESUMEN
An HPLC method was developed to determine the various carotenoids in Taiwanese mango (Mangifera indica L.). Initially, the peel and seed of mangoes were removed, the pulps were cut into pieces, freeze-dried, ground into powder, extracted and subjected to HPLC analysis. A mobile phase of methanol-isopropanol (99:1, v/v) (A) and methylene chloride (100%) (B) with the following gradient elution was developed: 100% A and 0% B in the beginning, maintained for 15 min, decreased to 70% A in 45 min, maintained for 15 min and returned to 100% A in 65 min. A total of 25 carotenoids were resolved within 53 min by using a C-30 column with flow rate at 1 mL/min and detection at 450 nm. alpha-Carotene was used as an internal standard to quantify all the carotenoids. All-trans-beta-carotene was present in largest amount (29.34 microg/g), followed by cis isomers of beta-carotene (9.86 microg/g), violaxanthin and its cis isomers (6.40 microg/g), neochrome (5.03 microg/g), luteoxanthin (3.6 microg/g), neoxanthin and its cis isomers (1.88 microg/g), zeaxanthin (1.16 microg/g) and 9- or 9'-cis-lutein (0.78 microg/g).
Asunto(s)
Carotenoides/análisis , Cromatografía Líquida de Alta Presión/métodos , Mangifera/química , Sensibilidad y EspecificidadRESUMEN
Oseltamivir carboxylate is a potent and specific inhibitor of influenza A and B neuraminidase (NA). Oseltamivir phosphate, the ethyl ester prodrug of oseltamivir carboxylate, is the first orally active NA inhibitor available for the prophylaxis and treatment of influenza A and B. It offers an improvement over amantadine and rimantadine which are active only against influenza A and rapidly generate resistant virus. The emergence of virus resistant to oseltamivir carboxylate in the treatment of naturally acquired influenza infection is low (about 1%). The types of NA mutation to arise are sub-type specific and largely predicted from in vitro drug selection studies. A substitution of the conserved histidine at position 274 for tyrosine in the NA active site has been selected via site directed mutagenesis, serial passage in culture under drug pressure in H1N1 and during the treatment of experimental H1N1 infection in man. Virus carrying H274Y NA enzyme selected in vivo has reduced sensitivity to oseltamivir carboxylate. The replicative ability in cell culture was reduced up to 3 logs, as was infectivity in animal models of influenza virus infection. Additionally, pathogenicity of the mutant virus is significantly compromised in ferret, compared to the corresponding wild type virus. Virus carrying a H274Y mutation is unlikely to be of clinical consequence in man.
Asunto(s)
Acetamidas/farmacología , Antivirales/farmacología , Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A/efectos de los fármacos , Mutación/efectos de los fármacos , Neuraminidasa/genética , Acetamidas/química , Acetamidas/uso terapéutico , Sustitución de Aminoácidos , Animales , Antivirales/química , Antivirales/uso terapéutico , Peso Corporal , Línea Celular , Modelos Animales de Enfermedad , Farmacorresistencia Viral/genética , Hurones , Fiebre/etiología , Humanos , Técnicas In Vitro , Inflamación/etiología , Virus de la Influenza A/enzimología , Virus de la Influenza A/genética , Virus de la Influenza A/patogenicidad , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/metabolismo , Oseltamivir , Análisis de Secuencia de ADN , Replicación ViralRESUMEN
The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.
Asunto(s)
Proteínas Fúngicas/fisiología , Cinetocoros/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/fisiología , Poliploidía , Sitios de Unión , Proteínas Fúngicas/genética , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias , Unión Proteica , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiaeRESUMEN
Mutations in the LIS1 gene cause gross histological disorganization of the developing human brain, resulting in a brain surface that is almost smooth. Here we show that LIS1 protein co-immunoprecipitates with cytoplasmic dynein and dynactin, and localizes to the cell cortex and to mitotic kinetochores, which are known sites for binding of cytoplasmic dynein. Overexpression of LIS1 in cultured mammalian cells interferes with mitotic progression and leads to spindle misorientation. Injection of anti-LIS1 antibody interferes with attachment of chromosomes to the metaphase plate, and leads to chromosome loss. We conclude that LIS1 participates in a subset of dynein functions, and may regulate the division of neuronal progenitor cells in the developing brain.
Asunto(s)
Dineínas/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Mitosis/fisiología , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Células COS , División Celular , Línea Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Perros , Complejo Dinactina , Dineínas/metabolismo , Expresión Génica , Humanos , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Pruebas de Precipitina/métodos , Fracciones SubcelularesRESUMEN
The formation of heterocyclic amines (HAs) in fried fish fiber during processing and storage was studied. Fried fish fiber was prepared by boiling of raw fish, followed by eviscerating, pressing, chopping, and then the fish meat was subjected to frying, during which the various additives such as sugar, soybean sauce, and edible oil were added. The various HAs in fried fish fiber were analyzed by high-performance liquid chromatography with photodiode-array detection. Only four HAs, Norharman, Harman, 2-amino-9H-pyrido[2,3-b]indole, and 2-amino-3-methyl-9H-pyrido[2,3-b]indole were detected in fried fish fiber. The amount of HAs increased with increasing frying temperature. Amino acids might play a more important role for HA formation than reducing sugar during processing of fried fish fiber. During storage, the HAs increased with increasing storage temperature when the fried fish fiber was packed in an aluminum foil bag. However, the relationship between storage temperature and HAs formation was not consistent when the fried fish fiber was packed in a plastic bag.
Asunto(s)
Aminas/química , Productos Pesqueros/efectos adversos , Manipulación de Alimentos , Conservación de Alimentos , Compuestos Heterocíclicos/química , Mutágenos/química , Aminas/análisis , Animales , Cromatografía Líquida de Alta Presión , Culinaria , Peces , Embalaje de Alimentos , Compuestos Heterocíclicos/análisis , Calor , Mutágenos/análisis , Oxidación-ReducciónRESUMEN
Lissencephaly is a brain developmental disorder characterized by disorganization of the cortical regions resulting from defects in neuronal migration. Recent evidence has implicated the human LIS-1 gene in Miller-Dieker lissencephaly and isolated lissencephaly sequence. LIS-1 is homologous to the fungal genes NudF and PAC1, which are involved in cytoplasmic dynein mediated nuclear transport, but it is also almost identical to a subunit of PAF acetylhydrolase, an enzyme which inactivates the lipid mediator platelet activating factor. Recent evidence from our laboratory has revealed that cytoplasmic dynein coimmunoprecipitates with LIS-1 in bovine brain cytosol, supporting a role in the dynein pathway in vertebrates. Overexpression of LIS-1 interferes with cell division, with noteworthy effects on chromosome attachment to the mitotic spindle and on the interaction of astral microtubules with the cell cortex. Other aspects of dynein function, such as the organization of the Golgi apparatus, are not affected. Together, these results suggest a role for LIS-1 in cytoplasmic dynein functions involving microtubule plus-ends. Furthermore, they suggest that mutations in LIS-1 may produce a lissencephalic phenotype either by interfering with the movement of neuronal nuclei within extending processes, or by interference with the division cycle of neuronal progenitor cells in the ventricular and subventricular zones of the developing nervous system.
Asunto(s)
Encéfalo/anomalías , Encéfalo/metabolismo , Citoplasma/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Encéfalo/citología , Complejo Dinactina , Dineínas/química , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Fenotipo , Factor de Activación Plaquetaria/metabolismo , Unión ProteicaRESUMEN
The analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by soaking and drying treatments were studied. The various carotenoids in the flowers of daylily were analyzed using a reversed-phase C(30) HPLC column and a mobile phase of methanol/methylene chloride/2-propanol (89:1:10, v/v/v) with methanol/methylene chloride (45:55, v/v) as sample solvent. Twenty-one pigments were resolved, of which 14 carotenoids were identified, including neoxanthin, violaxanthin, violeoxanthin, lutein-5,6-epoxide, lutein, zeaxanthin, beta-cryptoxanthin, all-trans-beta-carotene, and their cis isomers, based on spectral characteristics and Q ratios. Prior to hot-air-drying (50 degrees C) or freeze-drying, some of the daylily flowers were subjected to soaking in a sodium sulfite solution (1%) for 4 h. Under either the hot-air- or the freeze-drying treatment, the amounts of most carotenoids were higher in the soaked daylily flowers than in those that were not soaked. With hot-air-drying, the amount of cis carotenoids showed a higher yield in soaked samples than in nonsoaked samples. However, with freeze-drying, only a minor change of each carotenoid was observed for both soaked and nonsoaked samples. Also, air-drying resulted in a higher loss of carotenoids than freeze-drying.
Asunto(s)
Carotenoides/análisis , Manipulación de Alimentos/métodos , Magnoliopsida/química , Aire , Liofilización , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.
Asunto(s)
Acetamidas/farmacología , Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Virus de la Influenza A/enzimología , Neuraminidasa/antagonistas & inhibidores , Animales , Línea Celular , Embrión de Pollo , Perros , Farmacorresistencia Microbiana , Femenino , Hemaglutininas/biosíntesis , Hemaglutininas/genética , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/patogenicidad , Riñón/citología , Ratones , Ratones Endogámicos BALB C , Mutación , Neuraminidasa/genética , Oseltamivir , Ensayo de Placa Viral , Virulencia , Replicación Viral/efectos de los fármacosRESUMEN
Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L. V. Kochian [1998] Plant Physiol 117: 19-27).
Asunto(s)
Aluminio/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Ácido Cítrico/metabolismo , Malatos/metabolismo , Mutación/genética , Raíces de Plantas/metabolismo , Aluminio/metabolismo , Arabidopsis/crecimiento & desarrollo , Resistencia a Medicamentos/genética , Glucanos/metabolismo , Lantano/farmacología , Raíces de Plantas/genéticaRESUMEN
We have recently described GS 4071, a carbocyclic transition-state analog inhibitor of the influenza virus neuraminidase, which has potent inhibitory activity comparable to that of 4-guanidino-Neu5Ac2en (GG167; zanamivir) when tested against influenza A virus replication and neuraminidase activity in vitro. We now report that GS 4071 is active against several strains of influenza A and B viruses in vitro and that oral GS 4104, an ethyl ester prodrug which is converted to GS 4071 in vivo, is active in the mouse and ferret models of influenza virus infection. Oral administration of 10 mg of GS 4104 per kg of body weight per day caused a 100-fold reduction in lung homogenate viral titers and enhanced survival in mice infected with influenza A or B viruses. In ferrets, a 25-mg/kg dose of GS 4104 given twice daily reduced peak viral titers in nasal washings and eliminated constitutional responses to influenza virus infection including fever, increased nasal signs (sneezing, nasal discharge, mouth breathing), and decreased activity. Consistent with our demonstration that the parent compound is highly specific for influenza virus neuraminidases, no significant drug-related toxicity was observed after the administration of oral dosages of GS 4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats. These results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.
Asunto(s)
Acetamidas/farmacología , Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Neuraminidasa/antagonistas & inhibidores , Infecciones por Orthomyxoviridae/prevención & control , Profármacos/farmacología , Acetamidas/administración & dosificación , Acetamidas/metabolismo , Administración Oral , Aminas/administración & dosificación , Aminas/metabolismo , Aminas/farmacología , Animales , Antivirales/administración & dosificación , Antivirales/metabolismo , Modelos Animales de Enfermedad , Femenino , Hurones , Guanidinas , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza B/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Neuraminidasa/efectos de los fármacos , Oseltamivir , Profármacos/administración & dosificación , Profármacos/metabolismo , Piranos , Ratas , Ratas Sprague-Dawley , Ácidos Siálicos/administración & dosificación , Ácidos Siálicos/metabolismo , Ácidos Siálicos/farmacología , Replicación Viral/efectos de los fármacos , ZanamivirAsunto(s)
Aciclovir/farmacología , Antivirales/farmacología , Citosina/análogos & derivados , Resistencia a Múltiples Medicamentos , Foscarnet/farmacología , Herpes Simple/tratamiento farmacológico , Herpesvirus Humano 1/efectos de los fármacos , Organofosfonatos , Compuestos Organofosforados/farmacología , Adulto , Cidofovir , Citosina/farmacología , Farmacorresistencia Microbiana , Herpes Simple/patología , Herpes Simple/fisiopatología , Humanos , Inyecciones Intravenosas , Leucemia Mieloide Aguda/complicaciones , MasculinoRESUMEN
The molecular mechanisms that link cell-cycle controls to the mitotic apparatus are poorly understood. A component of the Saccharomyces cerevisiae spindle, Ase1, was observed to undergo cell cycle-specific degradation mediated by the cyclosome, or anaphase promoting complex (APC). Ase1 was degraded when cells exited from mitosis and entered G1. Inappropriate expression of stable Ase1 during G1 produced a spindle defect that is sensed by the spindle assembly checkpoint. In addition, loss of ASE1 function destabilized telophase spindles, and expression of a nondegradable Ase1 mutant delayed spindle disassembly. APC-mediated proteolysis therefore appears to regulate both spindle assembly and disassembly.
Asunto(s)
Anafase , Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Saccharomyces cerevisiae/citología , Huso Acromático/metabolismo , Secuencia de Bases , Fase G1 , Mitosis , Datos de Secuencia Molecular , Morfogénesis , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/metabolismo , Huso Acromático/ultraestructura , TelofaseRESUMEN
The design, synthesis, and in vitro evaluation of the novel carbocycles as transition-state-based inhibitors of influenza neuraminidase (NA) are described. The double bond position in the carbocyclic analogues plays an important role in NA inhibition as demonstrated by the antiviral activity of 8 (IC50 = 6.3 microM) vs 9 (IC50 > 200 microM). Structure-activity studies of a series of carbocyclic analogues 6a-i identified the 3-pentyloxy moiety as an apparent optimal group at the C3 position with an IC50 value of 1 nM for NA inhibition. The X-ray crystallographic structure of 6h bound to NA revealed the presence of a large hydrophobic pocket in the region corresponding to the glycerol subsite of sialic acid. The high antiviral potency observed for 6h appears to be attributed to a highly favorable hydrophobic interaction in this pocket. The practical synthesis of 6 starting from (-)-quinic acid is also described.
Asunto(s)
Acetamidas/síntesis química , Antivirales/síntesis química , Antivirales/farmacología , Ácidos Carboxílicos/síntesis química , Ciclohexanos/síntesis química , Virus de la Influenza A/efectos de los fármacos , Neuraminidasa/antagonistas & inhibidores , Ácidos Siálicos/síntesis química , Acetamidas/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Ácidos Carboxílicos/farmacología , Línea Celular , Cristalografía por Rayos X , Ciclohexanos/farmacología , Ciclohexenos , Modelos Animales de Enfermedad , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Interacciones Hidrofóbicas e Hidrofílicas , Virus de la Influenza A/enzimología , Virus de la Influenza A/crecimiento & desarrollo , Ácido Quínico/química , Ácidos Siálicos/farmacología , Relación Estructura-Actividad , Ensayo de Placa ViralRESUMEN
Al-sensitive (als) mutants of Arabidopsis were isolated and characterized with the aim of defining mechanisms of Al toxicity and resistance. Most als mutants selected on the basis of root growth sensitivity to Al were recessive, and together the mutants constituted eight complementation groups. Also, in most als mutants, Al sensitivity appeared to be specific for Al relative to La (another trivalent cation), except als2, which was more sensitive to La than wild type. The tendency of roots on mutant seedlings to accumulate Al was examined by staining with morin and hematoxylin, dyes used to indicate the presence of Al. A significant increase in morin staining was observed in als5, consistent with its increased sensitivity to Al. Unexpectedly, als7 and als4 showed less morin staining, suggesting that the roots on these mutants accumulate less Al than wild type seedlings after exposure to Al-containing solutions. Roots of wild-type seedlings produce callose in response to AlCl3 concentrations that inhibit root growth. Only als5 accumulated more callose than wild type in response to low levels (25 mu M) of AICI3 However, als4 and als7 did not accumulate callose at this AlCl3 concentration even though root growth was significantly inhibited. The lack of callose accumulation in als4 and als7 suggests that there is not an obligatory relationship between callose deposition and Al-induced inhibition of root growth.
Asunto(s)
Compuestos de Aluminio/toxicidad , Aluminio/toxicidad , Arabidopsis/genética , Cloruros/toxicidad , Mutación , Aluminio/metabolismo , Cloruro de Aluminio , Compuestos de Aluminio/metabolismo , Arabidopsis/metabolismo , Transporte Biológico , Cloruros/metabolismo , Mapeo Cromosómico , Cruzamientos Genéticos , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Prueba de Complementación Genética , Glucanos/biosíntesis , Lantano/toxicidad , Raíces de Plantas/efectos de los fármacosRESUMEN
The plasma membrane (PM) of higher plants contains numerous proteins; however, due to their low abundance, only a few have been identified and characterized by direct biochemical approaches. The major intrinsic protein (MIP) family is a class of highly hydrophobic integral membrane proteins thought to function as channels that facilitate the passage of water, small solutes, and possibly other moieties through the membrane. A family of PM intrinsic proteins was purified and characterized from PM vesicles derived from storage tissue of Beta vulgaris L. using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate. This PM intrinsic protein-enriched fraction also contains high levels of UDP-glucose:(1,3)-beta-glucan (callose) synthase activity. Dithiothreitol is required to visualize the monomeric species of these highly hydrophobic integral membrane proteins. Sequence analysis of tryptic fragments derived from polypeptides of 31 and 27 kD revealed significant homologies to plant MIPs identified from cloned sequences. These MIPs include clone 7a from pea and RD28 from Arabidopsis, both of which are water-stress proteins, a tomato ripening-associated membrane protein, and PIP 2b, a PM-bound water channel protein from Arabidopsis. MIPs, therefore, represent abundantly occurring components of PMs derived from beet storage tissue.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Plantas/metabolismo , Proteínas de Schizosaccharomyces pombe , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Glucosiltransferasas/aislamiento & purificación , Glucosiltransferasas/metabolismo , Immunoblotting , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Proteínas de Plantas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Escherichia coli is capable of synthesizing the apo-glucose dehydrogenase enzyme (GDH) but not the cofactor pyrroloquinoline quinone (PQQ), which is essential for formation of the holoenzyme. Therefore, in the absence of exogenous PQQ, E. coli does not produce gluconic acid. Evidence is presented to show that the expression of an Erwinia herbicola gene in E. coli HB101(pMCG898) resulted in the production of gluconic acid, which, in turn, implied PQQ biosynthesis. Transposon mutagenesis showed that the essential gene or locus was within a 1.8-kb region of a 4.5-kb insert of the plasmid pMCG898. This 1.8-kb region contained only one apparent open reading frame. In this paper, we present the nucleotide sequence of this open reading frame, a 1,134-bp DNA fragment coding for a protein with an M(r) of 42,160. The deduced sequence of this protein had a high degree of homology with that of gene III (M(r), 43,600) of a PQQ synthase gene complex from Acinetobacter calcoaceticus previously identified by Goosen et al. (J. Bacteriol. 171:447-455, 1989). In minicell analysis, pMCG898 encoded a protein with an M(r) of 41,000. These data indicate that E. coli HB101(pMCG898) produced the GDH-PQQ holoenzyme, which, in turn, catalyzed the oxidation of glucose to gluconic acid in the periplasmic space. As a result of the gluconic acid production, E. coli HB101(pMCG898) showed an enhanced mineral phosphate-solubilizing phenotype due to acid dissolution of the hydroxyapatite substrate.
Asunto(s)
Coenzimas/biosíntesis , Erwinia/genética , Gluconatos/metabolismo , Hidroxiapatitas/metabolismo , Quinolonas/metabolismo , Acinetobacter calcoaceticus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Análisis Mutacional de ADN , Elementos Transponibles de ADN , Durapatita , Erwinia/metabolismo , Escherichia coli/genética , Genes Bacterianos/genética , Glucosa 1-Deshidrogenasa , Glucosa Deshidrogenasas/biosíntesis , Datos de Secuencia Molecular , Complejos Multienzimáticos/biosíntesis , Mutagénesis Insercional , Cofactor PQQ , Homología de Secuencia de Ácido Nucleico , SolubilidadRESUMEN
The Schwanniomyces occidentalis (formerly castellii) ATCC 26077 (CBS 2863) alpha-amylase (AMY 26077) gene was cloned in Saccharomyces cerevisiae and sequenced. An open-reading frame encoding the AMY consists of 1536 base pairs and contains 512 amino-acid residues, which is almost the same in size as the AMY of Sch. occidentalis ATCC 26076 and CCRC 21164. The amino-acid sequence of AMY 26077 differed from that of ATCC 26076 alpha-amylase (AMY 26076) at two residues and from that of CCRC 21164 alpha-amylase (AMY 21164) at three residues. Comparison of the AMY 26077 gene with its homologues from two other strains (Sch. alluvius CBS 1153 and Sch. persoonii CBS 2169) using several restriction enzymes revealed that the AMY 26077 was very similar to AMY CBS 1153 but different from that of CBS 2169.
Asunto(s)
Saccharomycetales/genética , alfa-Amilasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Genes Fúngicos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Saccharomyces cerevisiae/genética , Saccharomycetales/enzimología , Homología de Secuencia de Ácido Nucleico , Levaduras/enzimología , Levaduras/genética , alfa-Amilasas/químicaRESUMEN
Unidirectional Ca influxes into brain and cerebrospinal fluid (CSF) were measured at different plasma concentrations of ionized Ca ([Ca]i) in pentobarbital-anesthetized rats. Plasma [Ca]i was varied acutely from 0.6 to 3.0 mumol/ml by intravenous infusion of EGTA, NaCl or CaCl2 or by thyroparathyroidectomy. Ca influx was determined from the 15-min uptake of 45Ca after intravenous injection. There were significant regional differences in 45Ca uptake into the CNS, with a approximately 20-fold greater rate into ventricular CSF than into frontal cortex. Autoradiographs of 45Ca uptake demonstrated that uptake into frontal cortex reflects primarily transport across the cerebral capillaries, whereas uptake into ventricular CSF reflects transport across the choroid plexuses. At both sites, Ca influx was a linear function of plasma [Ca]i and extrapolated to zero at [Ca]i = 0. Infusion of EGTA or CaCl2 did not alter the integrity of the blood-brain barrier, as determined by the permeability to [14C]sucrose. These results indicate that Ca influx into the CNS is not regulated by a saturable mechanism that is sensitive to acute changes in plasma [Ca]i. The proportionality between influx and concentration is suggestive of passive diffusional transport. The brain is protected from acute changes in plasma [Ca]i by the low cerebrovascular permeability to Ca, approximately 5 X 10(-8) cm/s.
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Encéfalo/metabolismo , Calcio/metabolismo , Animales , Autorradiografía , Transporte Biológico , Calcio/sangre , Calcio/líquido cefalorraquídeo , Ácido Egtácico/farmacología , Masculino , Glándulas Paratiroides/fisiología , Ratas , Sacarosa/metabolismo , Glándula Tiroides/fisiologíaRESUMEN
The transport of weak bases (pKa values 1.4 through 10.3) across in vitro and in vivo preparations of rat gastric mucosa have been compared. Asymmetric movements were observed in all cases, and the in vitro and in vivo data showed similar profiles of relation between the degrees of asymmetry and the pKa values of transported compounds. These profiles could be described by an equation based on a three-compartment model system, but it was necessary to postulate two such systems in parallel to give a satisfactory fit to the full range of pKa values tested. One system (I) is analogous to that described previously in small intestine. The properties of this system [(Pi/Pni)a = 2.5 x 10(-3), (Pi/Pni)b = 7.0 x 10(-1)] suggested that the intermediate compartment is a subepithelial extracellular compartment in which an alkaline pH is maintained [pHx = 8.5]. The second system (II) is the opposite polarity [(Pi/Pni)a = 1 x 10(0), (Pi/Pni)b = 3 x 10(-1)], and this system may be represented by the acidic lumen of the gastric tube [pHx = 2.0]. The principal differences between the in vivo and in vitro data could be ascribed to poor stirring of the luminal bulk phase in the in vivo situation. It was concluded that the determinants of weak base transport identified in studies with in vitro preparations are pertinent to the absorptive and secretory processes that occur in the intact stomach.
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Electrólitos/metabolismo , Mucosa Gástrica/metabolismo , Animales , Transporte Biológico , Proteínas Sanguíneas/metabolismo , Técnicas In Vitro , Absorción Intestinal , Masculino , Modelos Biológicos , Unión Proteica , Ratas , Ratas EndogámicasRESUMEN
Development of tolerance to phencyclidine (PCP) was assessed in male ICR mice, using motor incoordination as a parameter. The implantation of a PCP (1-3 mg/day/mouse for 1-5 days)-containing osmotic minipump, induced tolerance, as evidenced by a gradual reduction of the duration of motor incoordination. The degree of tolerance exhibited dose and time dependency. Even after the removal of the PCP pump (1 mg/day/mouse for 5 days), the tolerance remained to the same degree for at least 4 days. The hepatic microsomal cytochrome P-450, cytochrome b5 and nicotinamide adenine dinucleotide phosphatase (NADPH)-cytochrome c reductase activities were found to be elevated in tolerant mice (2 mg/day/mouse for 5 days). The half-life of PCP in the brains of tolerant mice was likewise decreased. These data indicate a dispositional tolerance for PCP. It appears that the administration of PCP by the osmotic minipump offers a convenient method for inducing PCP tolerance.