RESUMEN
The potential value of hypervascularity detected with power Doppler ultrasonography (PDU) within equine superficial digital flexor tendon (SDFT) as a prognostic factor of SDFT injury is not clear. The purpose of this study was to test the hypothesis that hypervascularity within SDFT is one of the risk factors for subsequent severe SDFT injury and to evaluate the prognostic value. A prospective cohort study of 97 Thoroughbred racehorses without any clinical signs of SDFT injury was conducted. Six variables of age, body weight, sex, the cross-sectional area of SDFT, PDU signal within SDFT and experience of steeplechase were assessed for the possibility of risk factors of subsequent SDFT injury in follow-up period of 1 year. Multivariable logistic regression analyses were used for assessment of the odds ratios (ORs) and 95â¯% confidence intervals (CIs) of SDFT injury. Multivariable logistic regression analysis revealed that the PDU signal within SDFT was a risk factor for the development of SDFT injury in follow-up period (P = 0.017). The adjusted OR of SDFT injury was significantly higher in PDU positive group than in PDU negative group (OR 3.17, 95â¯% CIs 1.20-8.35). Although further studies are required, these results would be useful for early detection and/or prevention of development for clinical severe SDFT injury.
RESUMEN
A retrospective cohort study was conducted to investigate risk factors for the failure of Thoroughbred racehorses to return to racing after an injury of the superficial digital flexor tendon (SDFT). Successful return was defined as the completion of five or more races after SDFT injury. The official Japan Racing Association (JRA) medical records of racehorses with a core-type SDFT injury were reviewed for clinical variables related to the characteristics of the horse and the severity of SDFT injuries at the time of diagnosis. Data on racing outcomes were obtained from the official JRA racing database. Risk factors were screened using univariable logistic regression and subsequent multivariable model building. Forty-nine of 346 (14.2%) horses successfully returned to racing after SDFT injuries. Multivariable model building revealed that an increase in the total number of injured zones (defined as the total number of zones in which the injured hypoechoic area was observed at the time of ultrasonographic diagnosis of SDFT injury) was associated with an increased risk of failure to return to racing after SDFT injury. Horse characteristics, such as age, body mass and sex, were not associated with a successful return to racing. In the rehabilitation of cases with larger (longer) lesions, more effective and careful medical management may be needed for an improvement in the athletic outcomes.
Asunto(s)
Enfermedades de los Caballos/fisiopatología , Carrera , Traumatismos de los Tendones/veterinaria , Animales , Composición Corporal , Estudios de Cohortes , Femenino , Caballos , Masculino , Estudios Retrospectivos , Factores de Riesgo , Traumatismos de los Tendones/diagnóstico por imagen , Traumatismos de los Tendones/fisiopatología , Tendones , Ultrasonografía/veterinariaRESUMEN
REASONS FOR PERFORMING STUDY: There is limited information on clinical use of the new injectable anaesthetic agent alfaxalone in Thoroughbred horses. OBJECTIVES: To compare anaesthetic induction and recovery characteristics and cardiopulmonary responses between alfaxalone, ketamine and thiopental in Thoroughbred horses premedicated with medetomidine and midazolam. STUDY DESIGN: Randomised blinded experimental cross-over study. METHODS: Six Thoroughbred horses were anaesthetised 3 times with alfaxalone 1 mg/kg bwt, ketamine 2.5 mg/kg bwt or thiopental 4 mg/kg bwt after premedication with medetomidine 6 µg/kg bwt and midazolam 20 µg/kg bwt. Qualities of anaesthetic induction and recovery were scored on a scale of 1 (poor) to 5 (excellent). Induction time and recovery time were recorded. Cardiopulmonary values (heart rate, respiratory rate, arterial blood pressures, and arterial blood gases) were recorded throughout anaesthesia. Data were analysed with nonparametric methods. RESULTS: The anaesthetic induction (P = 0.2) and recovery (P = 0.1) quality scores (median, range) were not different amongst protocols and were 4.0, 3-5; 5.0, 4-5; 4.5, 3-5; and 4.5, 3-5; 3.5, 2-5; 4.0, 2-5 for alfaxalone, ketamine and thiopental, respectively. Induction time for ketamine (67, 53-89 s) was significantly longer than that for alfaxalone (49, 40-51 s, P = 0.01) and thiopental (48, 43-50 s, P = 0.01). Time to standing for alfaxalone (44, 40-63 min, P = 0.01) and thiopental (39, 30-58 min, P = 0.01) was significantly longer than that for ketamine (25, 18-26 min). Cardiovascular values were maintained within the clinically acceptable level throughout anaesthesia. Respiratory rate significantly decreased during anaesthesia for all 3 drugs; however, spontaneous breathing did not disappear, and PaCO2 values were maintained at approximately 50 mmHg. CONCLUSIONS: All 3 drugs showed similar effects in relation to anaesthetic induction and recovery qualities and cardiopulmonary responses. However, alfaxalone and thiopental prolonged recovery time compared with ketamine.
Asunto(s)
Anestésicos/farmacología , Hipnóticos y Sedantes/farmacología , Premedicación/veterinaria , Anestésicos/administración & dosificación , Animales , Quimioterapia Combinada , Caballos , Hipnóticos y Sedantes/administración & dosificación , Ketamina/administración & dosificación , Ketamina/farmacología , Medetomidina/administración & dosificación , Medetomidina/farmacología , Midazolam/administración & dosificación , Midazolam/farmacología , Pregnanodionas/administración & dosificación , Pregnanodionas/farmacología , Tiopental/administración & dosificación , Tiopental/farmacologíaRESUMEN
Countin, a cell-counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild-type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7-fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild-type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild-type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.
Asunto(s)
Dictyostelium/citología , Proteínas Protozoarias/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Dictyostelium/genética , Datos de Secuencia Molecular , Fenotipo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Homología de Secuencia de AminoácidoRESUMEN
A Staphylococcus warneri strain M, newly isolated from processed seafood (smoked Watasenia scintillans), produced an extracellular protease. The protease, designated to as m-PROM (the mature form of PROM), selectively cleaved the carbonyl side of glutamic acid residues in beta-casein. Sequence of N-terminal 27 amino acids of m-PROM, RANVILPNNDRHQINDTTLGHYAPVTF, was found to be similar to those of other glutamyl endopeptidases, V8 protease (Staphylococcus aureus strain V8) and SPase (S. aureus ATCC 12600). To determine the complete primary structure and precursor of PROM, its gene (proM) was cloned and sequenced. The gene proM was found to encode for a protein of 316 amino acids. The amino acid residues from 64 to 90 completely coincided with the N-terminal 27 amino acids of the m-PROM, suggesting that the N-terminal 63 amino acids region of p-PROM (the precursor form of PROM) might be processed posttranslationally. Moreover, the whole amino acid sequence deduced from the primary structure of proM shows significant similarity to those of other glutamyl endopeptidases, V8 protease and SPase. These results suggested that PROM belongs to the glutamyl endopeptidase class. PROM, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.
Asunto(s)
Serina Endopeptidasas/genética , Staphylococcus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Alineación de Secuencia , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Staphylococcus/enzimología , Especificidad por SustratoRESUMEN
We sequenced a gene encoding AP endonuclease DdAPN in Dictyostelium discoideum. The sequence predicts a protein of 542 amino acids, showing high homology to Escherichia coli Endonuclease IV (Endo IV). There is 45% identity to Endo IV using the C-terminal 282 amino acids of the Dictyostelium protein. The DdAPN conserves nine residues for the metal-binding identified in Endo IV. The truncated DdAPN protein containing these sites partially complemented E. coli RPC501 (xth(-), nfo(-)).
Asunto(s)
Liasas de Carbono-Oxígeno/biosíntesis , Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/metabolismo , Dictyostelium/enzimología , Dictyostelium/genética , Proteínas de Escherichia coli , Escherichia coli/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , Daño del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/farmacología , Mitomicina/farmacología , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Plásmidos/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The aim of the present study was to investigate differences in the methods for preparing a large DNA fragment to be used for making transgenic rats from the standpoint of transgenic production efficiency and integrity of the introduced gene. In yeast artificial chromosome (YAC) transgenesis, three methods for preparing DNA for microinjection were compared: amplification of YAC in yeast (AMP), amplification of YAC in yeast and removal of the amplification element (AMP/RE), and no amplification of the YAC in yeast (AMP-). Production efficiency per microinjected ovum with DNA by the AMP method was four times higher than that by the AMP/RE and AMP-. Based on these results, we favor the AMP method in spite of the thymidine kinase gene-induced male sterility. In bacterial artificial chromosome (BAC) transgenesis, linear DNA fragments for microinjection prepared by three kinds of purification procedures were compared: Not I digestion and CsCl gradient ultra-centrifugation (Prep. 1), CsCl gradient ultra-centrifugation, Not I digestion, gel electrophoresis, and beta-agarase digestion (Prep. 2), and CsCl gradient ultra-centrifugation, Not I digestion, pulse field gel electrophoresis, and beta-agarase digestion (Prep. 3). Although the efficiency of producing transgenic rats was similar with all these three DNA preparations, integration of the intact DNA fragment only occurred with the Prep. 3 procedure. We therefore favor the Prep. 3 method for preparing BAC DNA fragments. These results indicate that the method used to prepare a large DNA fragment such as YAC and BAC DNAs is important in order to produce transgenic rats with an intact transgene.
Asunto(s)
Animales Modificados Genéticamente/genética , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Levadura/genética , Clonación Molecular/métodos , Escherichia coli/genética , Saccharomyces cerevisiae/genética , Animales , Cruzamiento , ADN Bacteriano , ADN de Hongos , Electroforesis en Gel de Campo Pulsado , Femenino , Masculino , Microinyecciones , Reacción en Cadena de la Polimerasa , Embarazo , RatasRESUMEN
The putative repressor protein Cng (10kDa on an SDS gel) for the lytic pathway of Lactobacillus plantarum phage φg1e was purified using the Escherichia coli Pt7 system, and its DNA-binding ability for the seven operator-like sequences, the GATAC-boxes (Gb1 to Gb7), was investigated in vitro. In gel-shift assays, Cng selectively bound to the DNA fragments containing the GATAC-box(es). In addition, DNase I footprinting analysis with supercoiled DNA demonstrated that Cng can specifically cover about a 25bp region centered around each of the GATAC-boxes, although two boxes, Gb4 and Gb6, were only partially protected. Moreover, protein crosslinking experiments using glutaraldehyde suggested that Cng most likely functions as a dimer. On the other hand, the binding ability of Cpg for the GATAC-boxes in supercoiled DNA was also examined under the same conditions as in Cng; unlike Cng, Cpg covered Gb4 and Gb6 completely sufficiently as well as the other five boxes. Thus, the present and previous [Kakikawa et al., Gene 215 (1998) 371-379; 242 (2000) 155-166] results indicate a possibility that the two proteins Cng and Cpg selectively bind to the GATAC-boxes that act as operators, and can decide between the lytic or lysogenic pathways through repression of the promoter activity of P(R) as well as P(L).
Asunto(s)
Bacteriófagos/genética , Proteínas de Unión al ADN/metabolismo , Lactobacillus/virología , Proteínas Represoras/aislamiento & purificación , Proteínas no Estructurales Virales/aislamiento & purificación , Secuencia de Bases , Sitios de Unión/genética , Reactivos de Enlaces Cruzados , Huella de ADN , ADN Viral/genética , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I , Dimerización , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS-PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.
Asunto(s)
Acetiltransferasas , Aciltransferasas/aislamiento & purificación , Escherichia coli/enzimología , Acetilcoenzima A/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Cinética , Plásmidos , ortoaminobenzoatos/metabolismoRESUMEN
The structural and functional features of the approximately 530 bp P(L)/Gb5-Gb6-cpg-Gb7 region (P(L) overlaps Gb5) for the lysogenic pathway of L. plantarum phage (phi)gle were investigated using the cat gene of E. coli plasmid pKK232-8 as a reporter. In E. coli XL1-Blue, a recombinant plasmid pKPL2 (cat under P(L)/Gb5-Gb6) exhibited distinct CAT activity, whereas the activity of pKPLCP1 (cat under P(L)/Gb5-Gb6-cpg) was only marginal. When pKPL2 was coexistent with a compatible derivative of plasmid pACYC177 carrying P(L)/Gb5-Gb6-cpg, the CAT activity was declined to the level of pKPLCP1. On the other hand, the cpg-encoded protein Cpg was overproduced in E. coli under P(T7). The molecular mass of the purified Cpg (14.5 kDa on a SDS gel) corresponded well with that (15.1 kDa) predicted from the DNA sequence. Gel-shift and footprinting assays demonstrated that Cpg selectively binds to about 25 bp bases centered around the GATAC-box (from 1 to 7). Moreover, protein crosslinking experiments using glutaraldehyde showed that Cpg most likely functions as a dimeric form. Thus, the present results indicate that Cpg probably represses P(L) through binding to the operator GATAC-box(es), and the P(L)/cpg region might participate in the lysogenic pathway.
Asunto(s)
Bacteriófagos/genética , Regulación Viral de la Expresión Génica , Lactobacillus/virología , Secuencia de Aminoácidos , Unión Competitiva , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN/metabolismo , Escherichia coli/genética , Genes Virales/genética , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificación , Proteínas Represoras/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
A homolog of oscillin, the Ca2+ oscillation-inducing factor of the hamster, was identified from the cellular slime mold Dictyostelium discoideum and designated Dd-oscillin. In the developmental stages of D. discoideum, the gene is expressed at the prestalk region which contains a higher concentration of cytosolic Ca2+ than the prespore region. The Dd-oscillin null strain aggregated but did not develop further when the cells were plated on non-nutrient agar at a density of 1.5x10(6) cells/cm2, showing that the Dd-oscillin gene is important for development. Since the null cells carrying the hamster oscillin gene formed fruiting body, the hamster oscillin was the homolog of Dd-oscillin as far as function is concerned. In addition, the null cells formed fruiting body in the presence of 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ: a specific inhibitor of Ca2+-ATPase activity in the endoplasmic reticulum). These results suggest that Dd-oscillin will increase cytosolic Ca2+ in the cells and promote further development.
Asunto(s)
Calcio/metabolismo , Dictyostelium/fisiología , Proteínas/fisiología , Animales , Proteínas de Unión al Calcio , Cricetinae , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Inhibidores Enzimáticos/farmacología , Prueba de Complementación Genética , Hidroquinonas/farmacología , Mutación , Proteínas/genéticaRESUMEN
A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high-expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human alpha-lactalbumin gene express the transgene in a position-independent manner. The 210 kb YAC was thought to have all the elements necessary for position-independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the alpha-lactalbumin gene had position-independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position-dependent expression. Therefore, all the elements necessary for position-independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the alpha-lactalbumin gene. Furthermore, we replaced the human alpha-lactalbumin promoter with the bovine alphaS1-casein promoter in the 210 kb YAC and produced transgenic rats. Position-dependent expression was observed. The elements required for position-independent expression of the bovine alphaS1-casein gene are different from those required for the human alpha-lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity.
Asunto(s)
Cromosomas Artificiales de Levadura/genética , Regulación de la Expresión Génica/genética , Lactalbúmina/genética , Animales , Animales Modificados Genéticamente , Caseínas/genética , Bovinos , Clonación Molecular , Cósmidos , Hormona de Crecimiento Humana/genética , Humanos , Región de Control de Posición/genética , Proteínas de la Leche/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Mensajero/análisis , RatasRESUMEN
Osteoclast differentiation factor (ODF), a ligand for osteoclastogenesis inhibitory factor (OCIF)/ osteoprotegerin (OPG), is a member of the membrane-associated tumor necrosis factor (TNF) family and induces osteoclast-like cell formation in vitro. In the present study, mouse ODF genomic clones were isolated and sequenced to determine their gene structure. The mouse ODF gene is a single copy gene consisting of five exons and spans approximately 40kb of the mouse genome. The first exon encodes the intracellular and transmembrane domains. The extracellular region of ODF containing the TNF homologous domain is encoded by exons 1 through 5. The translation-termination codon and six polyadenylation signal residues are present in exon 5. A major transcription-initiation site is present 143 nucleotides upstream of the initiation-ATG codon. This genomic organization is similar to that of other members of the TNF family, especially the CD40 ligand.
Asunto(s)
Proteínas Portadoras/genética , Glicoproteínas de Membrana/genética , Animales , Secuencia de Bases , Southern Blotting , Diferenciación Celular/genética , Línea Celular , Clonación Molecular , Exones , Intrones , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Osteoclastos/metabolismo , Filogenia , Ligando RANK , ARN Mensajero/genética , Receptor Activador del Factor Nuclear kappa-B , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.
Asunto(s)
Animales Modificados Genéticamente , Cromosomas Artificiales de Levadura , Vectores Genéticos , Hormona de Crecimiento Humana/genética , Lactalbúmina/genética , Transcripción Genética , Animales , Reactores Biológicos , Cartilla de ADN , Femenino , Técnicas Genéticas , Hormona de Crecimiento Humana/análisis , Hormona de Crecimiento Humana/biosíntesis , Humanos , Masculino , Leche/química , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Ratas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesisRESUMEN
GPS-indexed in-situ and car-borne survey of terrestrial gamma-ray dose rates were carried out in Brunei and adjacent areas using two portable NaI(Tl) counters. The mean and population weighted average dose rates for Brunei are 34 and 33 nGy/h, respectively. The car-borne data and the in-situ data when spectral analysed separately, were found to show fractal behaviour with D of 1.7 and 1.8, respectively. A contour map of the dose rates was also produced.
Asunto(s)
Contaminantes Radiactivos del Aire/análisis , Rayos gamma/efectos adversos , Contaminantes Radiactivos del Aire/efectos adversos , Brunei , Humanos , Dosis de Radiación , RadiometríaRESUMEN
Steady-state kinetic parameters for the hydrolysis of cellooligosaccharides by almond beta-glucosidase were evaluated at pH 5.0 and 25 degrees C in relation to the subsite theory (K. Hiromi, Biochem. Biophys. Res. Commun., 40, 1-6, 1970). The value of k0/Km decreased monotonously with increasing degree of polymerization (DP) of the substrates (DP = 2-6). Also, the Km and k0 values for cellotriose were smaller than those for cellobiose. These DP dependencies differ from those of most amylases and glucosidases studied so far, to which the subsite theory has been successfully applied. The subsite parameters could not be consistently obtained, which suggests that one or both of the two basic assumptions of the subsite theory might not be applicable to the hydrolysis of cellooligosaccharides by the enzyme. That is, the intrinsic rate of the hydrolysis may depend on the DP and/or there may be interaction between subsites for binding the glucose residues of a substrate.
RESUMEN
Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S. typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities. All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities. The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes<2,4-DNT<2,2'-dimethyl-5, 5'-dinitroazoxybenzene (2,2'-DM-5, 5'-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)<<4, 4'-dimethyl-3,3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB), and aminonitrotoluenes (2A4AT, 4A2NT)<2,4-DNT<4HA2NT4,4'-dimethyl-3, 3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB)<2HA4NT, respectively. In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2, 6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2'-dimethyl-3, 3'-dinitroazoxybenzene (2,2'-DM-3,3'-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively. These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT.
Asunto(s)
Carcinógenos/toxicidad , Dinitrobencenos/toxicidad , Salmonella typhimurium/genética , Acetiltransferasas/química , Carcinógenos/metabolismo , Dinitrobencenos/metabolismo , Pruebas de Mutagenicidad , Nitrorreductasas/química , Oxidación-Reducción , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/enzimologíaRESUMEN
The Lactobacillus plantarum phage og1e (42
Asunto(s)
Bacteriófagos/genética , Proteínas de Unión al ADN , Genes Virales , Lactobacillus/virología , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas no Estructurales Virales/genética , Proteínas Estructurales Virales/genética , Bacteriófagos/metabolismo , Secuencia de Bases , Sitios de Unión , Cloranfenicol O-Acetiltransferasa/biosíntesis , Clonación Molecular , Escherichia coli/genética , Genotipo , Lactobacillus/genética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/biosíntesis , Proteínas Represoras/metabolismo , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Lactobacillus plantarum phage phi gle has two consecutive cell lysis genes hol-lys (Oki et al., 1996b). In the present study, functional and structural properties of the hol protein (Hol) were characterized in Escherichia coli. Electron microscopic examinations showed that hol under plac in E. coli XL1-Blue injured the inner membrane to yield empty ghost cells with the bulk of the cell wall undisturbed. Northern blot analysis indicated that hol-lys genes under plac were co-transcribed, although the amount of hol transcript was larger than that of lys, ceasing via an apparently rho-independent terminator just downstream of hol. However, deletion and/or fusion experiments suggested that: (1) the N-terminal half of phi gle Hol composed of three putative transmembrane domains may be responsible for interaction with membrane; (2) the N-terminal end (five amino acids) seems nonessential; and (3) the C-terminal half containing charged amino acids appears to be involved in proper hol function. These results suggest that phi gle Hol is a member of the lambdoid holin family, but divergent in several properties from lambda holin.
Asunto(s)
Bacteriófagos/genética , Escherichia coli/virología , Lactobacillus/virología , Proteínas de la Membrana/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Bacteriólisis , Secuencia de Bases , Membrana Celular/ultraestructura , ADN Viral/química , Genes Virales/genética , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , ARN Viral/análisis , Proteínas Recombinantes de Fusión , Mapeo Restrictivo , Proteínas Virales/fisiología , Proteínas Estructurales Virales/genéticaRESUMEN
The complete genome sequence of a Lactobacillus temperate phage phi g1e was established. The double-stranded DNA is composed of 42,259 bp, and encodes for sixty-two possible open reading frames (ORF) as well as several potential regulatory sequences. Based on comparative analysis with other related proteins of the Lactobacillus and Lactococcus phages as well as the Escherichia coli phages (such as lambda), functions were putatively assigned to several phi g1e ORFs: cng and cpg (encoding for repressors), hel (helicase), ntp (NTPase), and several ORFs (e.g., minor capsid proteins). An about 1000-bp DNA region of phi g1e containing cpg and cng was inferred to function as a promoter/repressor system for the phi g1e lysogenic and lytic pathway.