RESUMEN
Novel influenza viruses of the H7N9 subtype have infected 33 and killed nine people in China as of 10 April 2013. Their haemagglutinin (HA) and neuraminidase genes probably originated from Eurasian avian influenza viruses; the remaining genes are closely related to avian H9N2 influenza viruses. Several characteristic amino acid changes in HA and the PB2 RNA polymerase subunit probably facilitate binding to human-type receptors and efficient replication in mammals, respectively, highlighting the pandemic potential of the novel viruses.
Asunto(s)
Subtipo H9N2 del Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/diagnóstico , Infecciones por Orthomyxoviridae/transmisión , Animales , Aves , Femenino , Humanos , Subtipo H9N2 del Virus de la Influenza A/patogenicidad , Gripe Humana/diagnóstico , MasculinoRESUMEN
An allyl di-glycol carbonate (ADC) sheet which has been utilised as a neutron detector for personal dosimetry has recently been studied for its application as a device for radiation exposure control for astronauts in space, where protons are the dominant radiation. It is known that the fabrication process, modified by adding some kind of antioxidant to improve the sensitivity of ADC to high energy protons, causes a substantial increase in false tracks, which disturb the automatic counting of proton tracks using the auto-image analyser. This made clear the difficulty of fabricating ADC sheets which have sufficient sensitivity to high energy protons, while maintaining a good surface. In this study, we have tried to modify the fabrication process to improve the sensitivity to high energy protons without causing a deterioration of the surface condition of ADC sheets. We have successfully created fairly good products.
Asunto(s)
Carbonatos/química , Carbonatos/efectos de la radiación , Membranas Artificiales , Protones , Protección Radiológica/instrumentación , Dosimetría Termoluminiscente/instrumentación , Relación Dosis-Respuesta en la Radiación , Diseño de Equipo , Análisis de Falla de Equipo , Ensayo de Materiales , Dosis de Radiación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Propiedades de Superficie , Dosimetría Termoluminiscente/métodosRESUMEN
N-(omega-Hydroxyalkyl)glycolamidobisphosphoric esters (P-O-CH2-CO-NH-(CH2)n -O-P), which are analogues of the aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) substrate fructose 1,6-bisphosphate, were synthesized and used for probing its active site. These phosphate compounds competitively inhibited aldolase activity. The Ki value was lowest when the maximum distance between the phosphorus atoms of the bisphosphate was brought close to that of fructose 1,6-bisphosphate. The inhibitor constants, Ki, were compared to those of alkanediol monoglycolate bisphosphoric esters and alkanediol bisphosphate compounds, which were reported previously by Ogata et al. The values of Ki for the bisphosphate compounds containing an amide group, the amide bisphosphate compounds, were smaller than those for the bisphosphate compounds containing an ester group, the ester bisphosphate compounds, and those for alkanediol bisphosphates were the largest for the same distance between phosphorus atoms in these bisphosphates. The difference spectra of aldolase caused by binding of a saturating concentration of N-(omega-hydroxypropyl)glycolamidobisphosphoric ester resembled that of butanediol monoglycolate bisphosphoric ester. However, the effects of the amide bisphosphate compounds on the absorption spectrum of aldolase were smaller than those of the ester bisphosphate compounds for the same distance between phosphorus atoms in these bisphosphate compounds. These results suggest that the synthesized phosphate compounds bind to aldolase at the active site and the -CO-NH- group of the compounds might be held more tightly than the -CO-O- group by hydrogen bonds, presumably with the amino acid residues in the active site, such as Lys-146 or -229 and Asp-33 or Glu-187. On the other hand, the -CO-O- group might be more effective in changing the environment of the Trp-147 residue in the active site of this enzyme.
Asunto(s)
Ésteres/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Glicolatos/metabolismo , Compuestos Organofosforados/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Ésteres/química , Glicolatos/química , Espectroscopía de Resonancia Magnética , Músculos/enzimología , Compuestos Organofosforados/química , Análisis EspectralRESUMEN
Alkyl glycolamido phosphoric esters (P-O-CH2-CO-NH-(CH2)n-CH3) and alkyl monoglycolate phosphoric esters (P-O-CH2-CO-O-(CH2)n-CH3), which are analogs of the aldolase substrate fructose-1-phosphate, were synthesized and use for probing the active site of rabbit muscle aldolase. The inhibition constants (Ki) were affected by the length of the alkyl groups of these compounds and a maximum value of Ki was observed between the number of methylene groups 2 and 4, depending on the type of compound. In the previous investigation, N-(omega-hydroxyalkyl)-glycolamido bisphosphoric esters (P-O-CH2-CO-NH-(CH2)n-O-P) and alkanediol monoglyclolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P) have a minimum Ki value between the number of methylene groups 1 and 4. The difference spectra of aldolase caused by binding of alkyl glycoamido phosphoric esters or alkyl monophosphates resembled that of their analogous bisphosphoric esters, but the intensity of absorbance was smaller than that of the bisphosphoric ester analogs. These results suggest that rabbit muscle aldolase has two binding sites for the phosphate groups on the entrance end of the active site cavity, the singly wound beta-barrel of the parallel alpha/beta class structure. The distance between the phosphate binding site Lys-107 in the beta-sheet structure (c) and Arg-148 in the beta-sheet structure (d) may possibly be expanded or contracted by the forms of the bending structure of the biphosphate compounds. Also, the change of distance between the beta-sheet structure (c) and (d) containing Trp-147, may have an effect on the environment of the tryptophan and cause a change of the absorbance of aldolase especially at 295-299 nm. On the other hand, the synthetic monophosphate compounds bind at only one of the two phosphate binding sites and have very little effect on the absorbance of Trp-147, in a similar manner as orthophosphate. The alkyl groups of monophosphate may be repelled by the ionic amino acid side chains, Asp-33, Lys-146, Glu-187 and/or Lys-229 in the middle of the active site cavity. However, the end of the long alkyl group of some monophosphates may possibly contact the hydrophobic bottom of the active site cavity without effect on the environment of Trp-147.
Asunto(s)
Fructosa-Bifosfato Aldolasa/metabolismo , Fructosafosfatos/metabolismo , Glicolatos/síntesis química , Músculos/enzimología , Organofosfatos/síntesis química , Sitios de Unión , Unión Competitiva , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Glicolatos/farmacología , Organofosfatos/farmacologíaRESUMEN
Ubiquinone, known as a component of the electron transfer system in many organisms, has a different length of the isoprenoid side chain depending on the species, e.g., Escherichia coli, Saccharomyces cerevisiae and humans have 8, 6, and 10 isoprene units in the side chain, respectively. No direct evidence has yet shown what factors define the length of the side chain of ubiquinone. Here we proved that the polyprenyl diphosphate that was available in cells determined the length of the side chain of ubiquinone. E. coli octaprenyl diphosphate synthase (IspB) was expressed with the mitochondrial import signal in S. cerevisiae. Such cells produced ubiquinone-8 in addition to the originally existing ubiquinone-6. When IspB was expressed in a S. cerevisiae COQ1 defective strain. IspB complemented the defect of the growth on the non-fermentable carbon source. Those cells had the activity of octaprenyl diphosphate synthase and produced only ubiquinone-8. These results opened the possibility of producing the type of ubiquinone that we need in S. cerevisiae simply by expressing the corresponding polyprenyl diphosphate synthase.
Asunto(s)
Transferasas Alquil y Aril , Transferasas/metabolismo , Ubiquinona/biosíntesis , Ubiquinona/química , Secuencia de Bases , Southern Blotting , Transporte de Electrón , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Humanos , Mitocondrias/enzimología , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
The human chromosome (HSA)19q region has been shown to correspond to swine chromosome (SSC) 6q11-->q21 by bi-directional chromosomal painting and gene mapping. However, since the precise correspondence has not been determined, 26 genes localized in HSA19q13.3-->q13.4 were assigned to the SSC6 region mainly by radiation hybrid (RH) mapping, and additionally, by somatic cell hybrid panel (SCHP) mapping, and fluorescent in situ hybridization (FISH). Out of the 26 genes, 24 were assigned to a swine RH map with LOD scores greater than 6 (threshold of significance). The most likely order of the 24 genes along SSC6 was calculated by CarthaGene, revealing that the order is essentially the same as that in HSA19q13.3-->q13.4. For AURKC and RPS5 giving LOD scores not greater than 6, SCHP mapping and FISH were additionally performed; SCHP mapping assigned AURKC and RPS5 to SSC6q22-->q23 and SSC6q21, respectively, which is consistent with the observation of FISH. Consequently, all the genes (26 genes) examined in the present study were shown to localize in SSC6q12-->q23, and the order of the genes along the chromosomes was shown to be essentially the same in swine and human, though several intrachromosomal rearrangements were observed between the species.
Asunto(s)
Cromosomas Humanos Par 19/genética , Cromosomas de los Mamíferos/genética , Orden Génico/genética , Mapeo de Híbrido por Radiación/métodos , Homología de Secuencia de Ácido Nucleico , Porcinos/genética , Animales , Cartilla de ADN/genética , Genes/genética , Genoma , Genoma Humano , HumanosRESUMEN
A gene conferring resistance to fosmidomycin (Fs) was cloned from the gene pool of a wild-type strain of Escherichia coli. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 406 amino acids (aa) with a molecular weight of 43303. The gene mapped at 10.9 min on the E. coli chromosome and was designated fsr (fosmidomycin resistance). Maxicell analysis revealed that the Fsr protein migrated in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis as a broad band of 35 kDa. A comparison between the aa sequence of Fsr and sequences in a protein database revealed 18% homology to the bacterial drug-export proteins that mediate resistance to tetracycline and chloramphenicol. Hydropathy analysis of the Fsr protein revealed twelve putative transmembrane segments. The degree of FsR of transformants depended on the number of copies of the plasmid that contained fsr. The levels of ubiquinone-8 and undecaprenyl phosphate in cells that harbored a high-copy-number plasmid that included fsr were almost the same as those in the cells without the plasmid. These results suggest that Fsr does not have any direct effect on the biosynthesis of isoprenoid in E. coli, and that the mechanism for FsR involves the efflux of the drug by a process that is facilitated by Fsr.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/genética , Fosfomicina/análogos & derivados , Genes Bacterianos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Fosfomicina/farmacología , Genes Bacterianos/fisiología , Datos de Secuencia Molecular , Homología de SecuenciaRESUMEN
Following rat heterotopic heart allotransplantation, low to lethal doses of the antimetabolites mizoribine (MIZ), RS-61443 (RS), and AZA were given alone or in combination with subtherapeutic doses of FK506 (0.04 mg/kg/day) for 14 days after transplantation. With the median effect analysis of Chou and Kahan for quantitative drug interactions, substantial therapeutic synergism was demonstrated between FK506 and non-toxic doses of MIZ (2.5, 5, and 10 mg/kg/day) or AZA (5, 30, and 45 mg/kg/day), which was particularly evident with the lowest dose MIZ (2.5 mg/kg/day). When FK506 was used in combination with MIZ or AZA but not with RS, the maximum effect (peak median graft survival) was enhanced significantly from 15 days (MIZ alone) to 26 days (P < 0.05), and from 19 days (AZA alone) to 32 days (P < 0.01). In contrast, RS interacted with FK506 no more than additively. Although RS was the most powerful single antimetabolite, the best overall survival was obtained by combining AZA and FK506. The addition of FK506 did not significantly increase the percent mortality and LD50 of the antimetabolites.
Asunto(s)
Trasplante de Corazón , Inmunosupresores/administración & dosificación , Tacrolimus/administración & dosificación , Animales , Azatioprina/administración & dosificación , Azatioprina/toxicidad , Sinergismo Farmacológico , Quimioterapia Combinada , Supervivencia de Injerto/efectos de los fármacos , Inmunosupresores/toxicidad , Dosificación Letal Mediana , Masculino , Ácido Micofenólico/administración & dosificación , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/toxicidad , Ratas , Ratas Endogámicas ACI , Ratas Endogámicas Lew , Ribonucleósidos/administración & dosificación , Ribonucleósidos/toxicidad , Tacrolimus/toxicidad , Trasplante HomólogoRESUMEN
The bidirectional paradigm of tolerance involving reciprocal host vs. graft and graft vs. host reactions was examined after Lewis (LEW)-->Brown Norway (BN) transplantation of different whole organs (liver, intestine, heart, and kidney) or of 2.5 x 10(8) LEW leukocytes obtained from bone marrow, spleen, lymph nodes, and thymus. The experiments were performed without immunosuppression or under 14 daily doses of postoperative tacrolimus, which were continued in weekly doses to 100 days in a "continuous treatment" subgroup, and to 27 days in a short treatment group. Without immunosuppression, all organs and cell suspensions failed to engraft or were acutely rejected. GVHD (usually fatal) was always caused when either the long or short treatment was used for recipients of intestinal grafts and cell suspensions of spleen and lymph nodes. In contrast, both immunosuppressive protocols allowed engraftment of bone marrow cells, liver, heart, and kidney without clinical GVHD, whereas thymus cell suspensions and small doses of whole blood neither engrafted nor caused GVHD. At 100 days, now drug-free for 73 days, the liver, bone marrow, and heart recipients were tolerant in that they accepted all challenge LEW heart and/or liver grafts for 100 more days despite in vitro evidence of donor-specific reactivity (split tolerance). At 200 days, histopathologic studies of the challenge livers were normal no matter what the priming graft. However, the still-beating challenge hearts had a spectrum from normal to severe chronic rejection that defined the tolerogenicity of the original primary grafts: liver best-->bone marrow next-->heart least. Both the GVHD propensity and tolerogenicity in these experiments were closely associated with recipient tissue chimerism 30 and 100 days after the experiments began. The tissue chimerism was invariably multilineage, but the GVHD outcome was associated with T cell over-representation. These observations provide guidelines that should be considered in devising leukocyte augmentation protocols for human whole organ recipients. The results are discussed in relation to the historical tolerance studies of Billingham, Brent, and Medawar; Good; Monaco; and Calne.
Asunto(s)
Enfermedad Injerto contra Huésped/etiología , Tolerancia Inmunológica , Transfusión de Leucocitos , Quimera por Trasplante , Animales , Rechazo de Injerto , Masculino , Trasplante de Órganos , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Trasplante HomólogoRESUMEN
To clarify the characterization and immunologic mechanisms of endometrial extract as a suppressive factor in tissues of the implantation site, the effects of endometrial extract and IgG on mitogen-stimulated cultures of lymphocytes from human peripheral blood were investigated. The inhibitory activity of endometrial extracts was observed to be augmented markedly in the secretory phase of the menstrual cycle as compared to the proliferative phase. Secretory endometrial extract, at a concentration of 0.6 mg protein/ml, caused 50% suppression of PHA-induced lymphocyte blastogenesis (PHA-BL). Column fractionation of endometrial extract on a Sephadex G-200 column showed a profile with three peak fractions and demonstrated that the 2nd peak fraction was mainly responsible for the suppression of PHA-BL. The 2nd peak fraction was shown to contain IgG by the method of immunodiffusion with anti-human IgG. The 2nd peak fraction from which IgG was removed with affinity chromatography caused significant depression of PHA-BL. Furthermore, the Fc fraction of IgG showed marked suppression compared to the F(ab')2 fraction. From these results, we suggest the possibility of an endogenous substance containing IgG as a suppressive factor which is implicated in the suppression of T cell function. The Fc fragment seemed to be the major fraction possessing such suppressive activity.
Asunto(s)
Endometrio/inmunología , Inmunoglobulina G/farmacología , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Femenino , Humanos , Tolerancia Inmunológica , Fragmentos Fab de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/farmacología , Fitohemaglutininas/farmacología , Linfocitos T/inmunologíaRESUMEN
The combined effects of prostaglandins of the E series (PGE) and progesterone (P), both of which are found to increase locally in the secretory endometrium, were studied in mitogen-stimulated cultures of lymphocytes from human peripheral blood. When added separately to lymphocyte cultures, both PGE and P produced a concentration-dependent inhibition of T-cell, but not B-cell, mitogenesis. If added together, these agents caused a much greater inhibition of T-cell mitogenesis, with marked synergy, than that observed with either agent alone. The synergistic inhibition was achieved with endometrial concentrations of PGE and P. The kinetics of PGE- and P-mediated inhibition investigated at various times during lymphocyte activation indicated that both agents affected the early events of T-cell mitogenesis. PGE- and P-inhibition of T-cell mitogenesis were shown to be reversible phenomena by washing the cells treated with these agents. These results suggest that elevated levels of PGE and P in the secretory endometrium, acting synergistically in inhibiting the proliferative response of T cells, may participate in facilitating implantation of histoincompatible fetal tissue in the maternal uterus as non-specific local immunosuppressive factors.
Asunto(s)
Activación de Linfocitos/efectos de los fármacos , Progesterona/farmacología , Prostaglandinas E Sintéticas/farmacología , Adulto , Sinergismo Farmacológico , Endometrio/inmunología , Endometrio/metabolismo , Femenino , Feto/inmunología , Humanos , Tolerancia Inmunológica , Inmunosupresores , Técnicas In Vitro , Mitógenos/farmacología , Embarazo , Linfocitos T/inmunologíaRESUMEN
We have investigated alterations in Epstein-Barr virus antigen specific cytotoxic T-lymphocyte (EBV-CTL) activity during human pregnancy. EBV-CTL activity was determined by a modified EBV induced B-cell focus regression assay and was expressed in terms of a regression index (IR50), i.e. the initial cell concentration required to achieve a 50%-incidence of regression in EBV-infected cell culture. Increased values of IR50 indicate the suppression of EBV-CTL activity. In 113 human female T-cell leukemia type-I (HTLV-I) non-carriers, the IR50 values (mean +/- S.E.) in non-pregnant, pregnant (the first trimester, second trimester and third trimester of pregnancy) and puerperal women were 10.6 +/- 1.4, 16.1 +/- 1.1 (20.1 +/- 2.0, 14.8 +/- 2.0, 14.6 +/- 1.6), and 12.1 +/- 1.9 respectively. Among HTLV-I carriers, the IR50 values (mean +/- S.E.) were likewise 34.6 +/- 8.0, 87.4 +/- 5.2 (101.7 +/- 6.3, 88.3 +/- 8.4 and 79.5 +/- 9.2) and 39.2 +/- 7.1 respectively. This data demonstrate: 1) EBV-CTL activity was suppressed during pregnancy (P < 0.05), especially in the first trimester (P = 0.0003). 2). In HTLV-I carriers, this suppression was shown in the first trimester (P = 0.0002), in the second trimester (P = 0.0002) and in the third trimester of pregnancy (P = 0.0014) and 3). One month after delivery, this suppression had returned to the non-pregnant level in both HTLV-I non-carriers and HTLV-I carriers. Pregnancy therefore has a suppressive effect on antigen specific cytotoxic T-lymphocyte activity and this effect is amplified in HTLV-I carriers.
Asunto(s)
Portador Sano/inmunología , Infecciones por HTLV-I/inmunología , Complicaciones Infecciosas del Embarazo/inmunología , Embarazo/inmunología , Linfocitos T Citotóxicos/inmunología , Adulto , Análisis de Varianza , Antígenos CD/análisis , Pruebas Inmunológicas de Citotoxicidad , Femenino , Citometría de Flujo , Anticuerpos Anti-HTLV-I/sangre , Herpesvirus Humano 4/inmunología , Humanos , Inmunidad Celular , Japón , Subgrupos Linfocitarios , Periodo Posparto/inmunologíaRESUMEN
Upon rehydration of lyophilized Escherichia coli cells with phosphate buffer containing [14C]isopentenyl pyrophosphate (IPP), 14C was incorporated into the cells. Radioactivity was found in ubiquinone-8, an unidentified precursor of ubiquinone-8, demethylmenaquinone-8 and phosphate esters of all-trans-octaprenol and cis, trans-polyprenols. On rehydration of the cells with the buffer containing geranyl pyrophosphate or farnesyl pyrophosphate in combination with [14C]IPP, higher radioactivity was incorporated into the above products and some radioactivity was found in free prenols. Fractionation of the 14C-labeled cells by sucrose-density gradient centrifugation before and after recultivation indicated that the size of 14C-labeled cells had changed during the recultivation. This shows that radioactivity of [14C]IPP was incorporated into live cells but not into dead cells. The metabolism of the radioactive products in the recultivated cells was examined. It was found that the unidentified precursor was converted to ubiquinone-8, but demethylmenaquinone-8 was not converted to menaquinone-8. "Lipid intermediates" in peptidoglycan synthesis increased in the logarithmic growth phase and decreased in the stationary phase. In the stationary phase, however, an increase in cis,trans-polyprenyl monophosphates was observed. These observations suggest the operation of the lipid cycle of peptidoglycan synthesis.
Asunto(s)
Escherichia coli/metabolismo , Hemiterpenos , Radioisótopos de Carbono , Células Cultivadas , Centrifugación por Gradiente de Densidad , Cromatografía en Capa Delgada , Escherichia coli/crecimiento & desarrollo , Liofilización , Isomerismo , Compuestos Organofosforados/metabolismo , Compuestos Organofosforados/farmacología , Fosfatos de Poliisoprenilo/metabolismo , Fosfatos de Poliisoprenilo/farmacología , SesquiterpenosRESUMEN
Isopentenyl pyrophosphate (IPP) isomerase, farnesyl pyrophosphate (FPP) synthetase, octaprenyl pyrophosphate (OPP) synthetase and undecaprenyl pyrophosphate (UPP) synthetase were partially purified from Escherichia coli by DEAE-Toyopearl chromatography. FPP synthetase catalyzed the condensation of IPP with dimethylallyl pyrophosphate (DPP) as well as with geranyl pyrophosphate (GPP) to yield FPP as final product. OPP synthetase and UPP synthetase catalyzed the condensation of IPP with FPP to yield OPP and cis,trans-polyprenyl pyrophosphates (the C45-, C50, and C55-compound), respectively. Neither DPP nor GPP acted as a priming substrate for either enzyme. These four enzymes required Mg2+ or Mn2+ for their activities. UPP synthetase required also Triton X-100 for its activity. The addition of Triton X-100 enhanced OPP synthetase, but it did not affect IPP isomerase and FPP synthetase. It seems possible that the combination of the four enzymes ensures the in vivo synthesis of long-chain isoprenoids in E. coli.
Asunto(s)
Transferasas Alquil y Aril , Isomerasas de Doble Vínculo Carbono-Carbono , Escherichia coli/enzimología , Fosfatos de Poliisoprenilo/biosíntesis , Catálisis , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada , Dimetilaliltranstransferasa/aislamiento & purificación , Activación Enzimática/efectos de los fármacos , Hemiterpenos , Concentración de Iones de Hidrógeno , Isomerasas/aislamiento & purificación , Cinética , Metales/farmacología , Octoxinol , Polietilenglicoles/farmacología , Especificidad por Sustrato , Transferasas/aislamiento & purificaciónRESUMEN
The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.
Asunto(s)
Transferasas Alquil y Aril , Escherichia coli/genética , Hemiterpenos , Transferasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Codón , Escherichia coli/enzimología , Geraniltranstransferasa , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Compuestos Organofosforados/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Temperatura , Transferasas/biosíntesisRESUMEN
The method for the preparation of zymosterol was improved (13 mg of zymosterol/g dry cells) by the aerobic adaptation of the cells in the presence of 1 mM DL-ethionine. Lanosterol was also found to accumulate (5.0 mg/g dry cells) when the cells were adapted aerobically in the presence of 10(-4) M buthiobate. Pure lanosterol could be obtained by separation of the unsaponifiable lipids on TLC. Pure [14C]lanosterol with a high specific radioactivity (56 Ci/mol) could be prepared by incubation of the desiccated cells with [14C]isopentenyl pyrophosphate, cofactors such as ATP and NADPH-generating system, and buthiobate in phosphate buffer. The method using desiccated cells may also be applicable to the preparation of other radioactive sterol intermediates.
Asunto(s)
Colesterol , Hemiterpenos , Lanosterol/metabolismo , Saccharomyces cerevisiae/metabolismo , Esteroles/metabolismo , Anaerobiosis , Etionina/farmacología , Ácido Mevalónico/metabolismo , Compuestos Organofosforados/metabolismo , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
Adult T-cell leukemia/lymphoma, a lymphocytic leukemia caused by human T-cell lymphoma virus-I (HTLV-I), is prevalent in southwestern Japan. A Japanese woman with adult T-cell leukemia/lymphoma in the third trimester of pregnancy was delivered of an infant by cesarean section. She did not breast-feed the child, who has remained free from HTLV-I infection.
Asunto(s)
Infecciones por Deltaretrovirus , Complicaciones Neoplásicas del Embarazo , Adulto , Cesárea , Infecciones por Deltaretrovirus/epidemiología , Femenino , Humanos , Recién Nacido , Recién Nacido Pequeño para la Edad Gestacional/microbiología , Japón , Masculino , Embarazo , Complicaciones Neoplásicas del Embarazo/epidemiologíaRESUMEN
PURPOSE: Nitric oxide (NO) has recently been shown to be a neurotransmitter in nonadrenergic noncholinergic (NANC) inhibitory nerves in the human gut. To clarify the physiological significance of NO in the human internal anal sphincter (IAS), we investigated enteric nervous responses in normal IAS muscle strips above the dentate line, obtained from patients with rectal cancer. METHODS: Normal IAS muscle strips above the dentate line, obtained from ten patients who underwent rectal amputation for low rectal cancers were used. The subjects consisted of eight men and two women, aged from 46-72 years (mean age, 54.2 years). A mechanographic technique was used to evaluate in-vitro IAS muscle responses to electrical field stimulation (EFS) of adrenergic and cholinergic nerves before and after treatment with various autonomic nerve blockers, N(G)-nitro-L-arginine (L-NNA) and L-arginine. RESULTS: Excitatory nerves were mainly involved in the regulation of enteric nerve responses to EFS in the baseline condition of the study, and NANC inhibitory nerves acted on the normal IAS. L-NNA concentration-dependently inhibited the relaxation in response to EFS in the human IAS, and this inhibitory effect in the IAS was reversed by L-arginine. CONCLUSIONS: These findings suggest that NANC inhibitory nerves play important roles in regulating relaxation of the human IAS, and that NO plays an important role as a neurotransmitter in NANC inhibitory nerves of the human IAS.
Asunto(s)
Canal Anal/efectos de los fármacos , Canal Anal/fisiología , Óxido Nítrico/fisiología , Anciano , Arginina/administración & dosificación , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Sistema Nervioso Entérico/efectos de los fármacos , Sistema Nervioso Entérico/fisiología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Nitroarginina/farmacologíaRESUMEN
AIM: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates the homing of lymphocytes to gut-associated lymphoid tissues (GALT). We performed a semiquantative analysis of MAdCAM-1 expression during small bowel graft rejection. METHODS: Orthotopic small bowel transplantations (SBT) were performed from BN rats to LEW rats. Isografted animals served as controls. Animals were sacrificed on days 3, 4, 5, 6, and 7 after SBT. Cryostat sections were prepared from grafts, including Peyer's patches (PPs). Indirect immunoperoxidase staining was performed using mAbs against MAdCAM-1. The degree of vascular endothelial staining on high endothelial venules (HEV) in the PPs was graded from 1 (low levels) to 5 (high levels), and in the vessels of the lamina propria from 1 (faint), 2 (low at the base of villi), 3 (low to the middle of villi), 4 (high to the middle of villi), to 5 (high to villus tip). RESULTS: MAdCAM-1 expression on HEVs in PPs was down-regulated during rejection. In contrast its expression on endothelial cells of vessels in the lamina propria was up-regulated during rejection. CONCLUSION: Alteration in MAdCAM-1 expression may be associated with the development of SB graft rejection. The vessels at the base of villi, which are associated with lymphocyte recruitment, may become sites of intense immune reactivity during the early phase of small bowel allograft rejection.
Asunto(s)
Rechazo de Injerto/inmunología , Inmunoglobulinas/metabolismo , Intestino Delgado/trasplante , Mucoproteínas/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Animales , Rechazo de Injerto/patología , Inmunoglobulinas/análisis , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Mucosa Intestinal/trasplante , Intestino Delgado/inmunología , Intestino Delgado/patología , Microvellosidades/inmunología , Microvellosidades/patología , Modelos Animales , Mucoproteínas/análisis , Ratas , Ratas Endogámicas Lew , Receptores Mensajeros de Linfocitos/análisisRESUMEN
INTRODUCTION: We investigated the extent of apoptosis in crypt cells and Peyer's patches (PPs) during small bowel allograft rejection in rats to examine whether the Fas/FasL pathway participates in apoptosis within grafts during rejection. MATERIALS AND METHODS: Orthotopic small bowel transplantation with portocaval drainage was performed from Brown Norway to Lewis (LEW) rats. Isografted (LEW --> LEW) and nontransplanted animals served as the controls. Animals were sacrificed on days 3, 5, on 7 after SBT (each n = 5). An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas or Fas-L. RESULTS: The number of ISEL-positive enterocytes in the allografts increased significantly on days 3, 5, and 7. Similarly, in the PPs of the allografts, the number of ISEL-positive mononuclear cells increased significantly on days 3, 5, and 7. On day 7 the number of Fas- and FasL-positive enterocytes were increased significantly in the allografts compared with the nontransplanted controls. Similarly, in the PPs, Fas- and FasL-positive mononuclear cells also increased significantly on day 7 in the allograft. CONCLUSION: Although an increase, number of apoptotic enterocytes and lymphocytes were observed in the early phase, activation of Fas/FasL system occurred during the late phase of small bowel graft rejection. These findings suggest that both rejection-associated and sepsis-induced forms of apoptosis may be associated with small bowel graft rejection.