RESUMEN

The purpose of this study was to assess sedation, emesis and cardiovascular effects of dexmedetomidine alone or combined with acepromazine in healthy cats. Fourteen male cats aged 0.9 ± 0.5 years and weighing 3.7 ± 0.7 kg were randomly assigned to one of two experimental groups: GD, dexmedetomidine 5 µg/kg; and GDA, dexmedetomidine 5 µg/kg with acepromazine 0.03 mg/kg, all intramuscularly. Measurements were recorded at baseline, at 20 minutes and then at 10-minute intervals following sedation and included heart rate (HR), respiratory rate (FR), systolic arterial pressure (SAP), rectal temperature (RT), number of episodes of emesis and sedation score (0-4). Data were compared using ANOVA for repeated measures followed by Sídák and Dunnet test. Sedation scores were compared between groups at T20 using Mann-Whitney test. Significance was considered when P <0.05. At T20, HR was significantly lower in GDA (99 ± 14 beats/min) compared with GD (133 ± 19 beats/min) and SAP was significantly lower in both groups compared with baseline (126 ± 14 vs. 148 ± 26 and 111 ± 13 vs. 144 ± 17 mmHg in GD and GDA, respectively). Duration of sedation was similar between groups, although sedation scores differed significantly at T20, with 1 (0-4) in GD and 4 (4-4) in GDA. More episodes of emesis were recorded in GD compared with GDA. The combination of dexmedetomidine and acepromazine produced more profound sedation with faster onset and lower incidence of emesis compared with dexmedetomidine alone in healthy cats.

Asunto(s)

Anestesia , Dexmedetomidina , Gatos , Masculino , Animales , Hipnóticos y Sedantes/farmacología , Acepromazina/farmacología , Dexmedetomidina/farmacología , Anestesia/veterinaria , Vómitos/veterinaria

RESUMEN

The purpose of this study was to investigate whether intravenous crotalphine produces significant sedation, as well as physiological changes, in healthy standing horses. Six mares, aged 8 years and weighing 415kg underwent three different treatments in a crossover design: TA (acepromazine: 50μg.kg-1), TC (crotalphine: 0.01μg.kg-1) and TX (xylazine: 1000μg.kg-1), intravenously. At various time points over 60 minutes, physiologic variables were recorded: heart rate, respiratory rate, and rectal temperature. The head height from the ground (HHG) was evaluated in centimeters. Data were analyzed using ANOVA followed by Dunnett’s test or Friedman followed by Dunn’s test, under 5% significance. Heart rate decreased significantly at M5 and M10 compared with Mb in TX (28±7, 26±6 and 40±8 beats/minute-1, respectively; p=0.0004). Respiratory rate and rectal temperature did not differ among groups or time points. The HHG significantly decreased in all groups compared with Mb at various time points (p<0.0001). In conclusion, crotalphine did not produce reliable and durable sedation in healthy standing mares and did not influence cardiorespiratory variables in a clinically relevant manner.

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O objetivo deste estudo foi investigar se a administração de crotalfina intravenosa produz sedação significativa e alterações fisiológicas em equinos saudáveis. Seis éguas, idade média de oito anos e peso médio de 415kg, foram submetidas a três tratamentos distintos: TA (acepromazina: 50μg/kg), TC (crotalfina: 0,01μg/kg) e TX xilazina: 1000μg/kg), por via intravenosa. Em vários momentos, ao de longo de 60 minutos, as variáveis fisiológicas registradas foram frequência cardíaca, frequência respiratória e temperatura retal. A altura de cabeça ao solo (ACS) foi avaliada em centímetros. Os dados foram analisados pela ANOVA, seguida pelo teste de Dunnett ou de Friedman e, depois, pelo teste de Dunn, sob 5% de significância. A frequência cardíaca diminuiu significativamente em M5 e M10 em comparação com Mb em TX (28±7, 26±6 e 40±8 bpm, respectivamente; P=0,0004). A frequência respiratória e a temperatura retal não diferiram entre os grupos ou os pontos de tempo. O HHG diminuiu significativamente em todos os grupos em comparação com Mb em vários momentos (P <0,0001). Em conclusão, a crotalfina não produziu sedação confiável e durável em éguas saudáveis e não influenciou as variáveis cardiorrespiratórias de maneira clinicamente relevante.

RESUMEN

This study aimed to compare the effects of different coinduction agents on the duration and dose of propofol in healthy cats. Six cats aged 4.8 ± 1.0 years and weighing 4.4 ± 1.1 kg participated in 4 treatment groups of propofol combined with: saline or control group (TC); ketamine 2 mg/kg (Tket); fentanyl 1 µg/kg (Tfen); or midazolam 0.3 mg/kg (Tmid). Twenty minutes following premedication with dexmedetomidine at 10 µg/kg, induction followed the same protocol in all groups, starting with a propofol bolus of 1 mg/kg over 1 minute followed by an adjuvant, then propofol again at 1 mg/kg/minute for orotracheal intubation. Variables recorded were (in minutes): time of extubation, time to return of palpebral reflex, eye recentralization, recovery of consciousness, quadrupedal position and total propofol dose used (mg/kg). A comparison between the 4 groups was performed by analysis of variance followed by Dunnett test under 5% significance. There was no significant difference in any of the times evaluated during anesthetic recovery between the groups. The propofol dose used to allow orotracheal intubation was significantly lower in all groups compared to TC (P < .05). Ketamine, midazolam, and fentanyl are indicated as suitable choices for coinduction with propofol in cats.

Asunto(s)

Dexmedetomidina , Ketamina , Propofol , Anestésicos Intravenosos/farmacología , Animales , Gatos , Dexmedetomidina/farmacología , Ketamina/farmacología , Midazolam/farmacología , Propofol/farmacología

RESUMEN

The purpose of this study was to assess the efficacy of a blind technique for sciatic and femoral nerve block in rabbit cadavers by evaluating the spread of 1% methylene blue at two different volumes. Nine recently euthanized rabbits weighing 2.5(0.3kg were used. The sciatic (SN) and femoral (FN) nerves of each limb were randomly assigned for injection with 1% methylene blue at 0.2mL/kg (G0.2) or 0.3mL/kg (G0.3). Nerves were dissected and measured for depth and extension of staining (cm). Mean comparisons were performed using paired t test. The relation between volume and nerve staining ( 2cm was assessed using chi-square test. The mean depth of SN was 1.9±0.2 and 1.6±0.3cm and staining 1.9±1.4 and 2.0±1.2cm, respectively in G0.2 and G0.3. No relation was found between depth and dye spread and there was no association between nerve staining ( 2.0cm and volume of solution. The FN failed to be stained in all subjects. In conclusion, SN injection can be successfully performed without guidance in rabbits. The lower volume (0.2mL/kg) is recommended to avoid systemic toxicity.(AU)

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O objetivo deste estudo foi avaliar a eficácia de uma técnica para bloqueio às cegas dos nervos isquiático e femoral em cadáveres de coelhos, por meio da avaliação da dispersão de azul de metileno 1% em dois volumes distintos. Nove coelhos recém-eutanasiados, com peso 2,5(0,3kg, foram utilizados. Os nervos isquiático (NI) e femoral (NF) de cada membro foram aleatoriamente designados para injeção com azul de metileno 1% a 0,2mL/kg (G0,2) ou 0,3mL/kg (G0,3). Em seguida, foram dissecados e mensurados em relação à sua profundidade e extensão corada (cm). As médias foram comparadas por meio de teste t pareado. A relação entre volume e extensão corada ( 2cm foi avaliada utilizando-se teste de qui-quadrado. A profundidade média do NI foi 1,9±0,2 e 1,6±0,3cm, e a extensão corada 1,9±1,4 e 2,0±1,2cm, respectivamente, no G0,2 e no G0,3. Não houve relação entre a profundidade e a extensão corada ou entre a extensão corada ( 2,0cm e o volume de solução. Não foi observada coloração do NF em nenhum cadáver. Concluiu-se que a injeção do NI pode ser realizada com sucesso sem auxílio de tecnologias em coelhos. O menor volume (0,2mL/kg) é recomendado para evitar toxicidade sistêmica.(AU)

Asunto(s)

Animales , Conejos , Nervios Periféricos , Nervio Ciático , Azul de Metileno/administración & dosificación , Bloqueo Nervioso/métodos

RESUMEN

This study aimed to investigate the effects of a single bolus and continuous rate infusion (CRI) of 1% propofol on cholesterol and triglyceride levels of healthy bitches undergoing elective ovariohysterectomy. 10 healthy bitches undergoing elective ovariohysterectomy had blood samples obtained at baseline (TB), 15 minutes following premedication with acepromazine and morphine (TPM), after an intravenous bolus of propofol (induction to anesthesia, TIND) and following 90 minutes of CRI of propofol started at 0.4 mg kg-1 min-1 and adjusted according to individual requirements (TCRI). Data were initially tested for normality using the Shapiro-Wilk test, and comparisons were performed using Friedman followed by Dunn post-hoc test. Serum cholesterol levels significantly decreased at TIND and TCRI (median [min-max] 201 mg dL-1 [111-234 mg dL-1], and 215 mg dL-1 [111-239 mg dL-1]), respectively, compared with TB (232 [128-245 mg dL-1]) and TPM (206 [115-255 mg dL-1]). No differences were found between TIND and TCRI. Triglyceride levels increased significantly at TIND (120 [67-231 mg dL-1]) and TCRI (229 [73-549 mg dL-1]) compared with TPM (36 [51-29 mg dL-1]), and TCRI compared with TB. In conclusion, 1% propofol lipid emulsion significantly increases serum triglycerides and causes lipemia in healthy dogs at a single bolus or CRI.

Asunto(s)

Anestesia , Propofol , Anestesia/veterinaria , Animales , Colesterol , Perros , Femenino , Histerectomía/veterinaria , Propofol/farmacología , Triglicéridos

RESUMEN

Lidocaine is a versatile drug that not only provides local anesthesia, but also reduces anesthetic requirements of other agents and has antiarrhythmic, pro-kinetic, anti-inflammatory, antiendotoxemic and antioxidant effects. As it is a drug commonly used in critically ill patients, its safety from the cardiovascular system should be ensured. The aim of this study was to determine the effects of a continuous rate infusion (CRI) of lidocaine on left ventricular systolic and diastolic function of healthy rabbits sedated with midazolam by use of transthoracic echocardiography. Ten New Zealand healthy rabbits were sedated with intramuscular midazolam (1 mg/kg) and enrolled in two experimental treatments (control or lidocaine). The control treatment (CT) comprised an intravenous bolus of 0.9% sodium chloride (0.05 mL/kg) followed by CRI at 5 mL/h, whereas the lidocaine treatment (LT) comprised a bolus of 2% lidocaine without epinephrine at 1 mg/kg followed by CRI at 50 µg/kg/minute. Echocardiographic and hemodynamic variables were studied. Variables were recorded at baseline (TB) and 20, 40 and 60 minutes following start of CRI (T20, T40 and T60, respectively). No differences were found between treatments. The results of this study demonstrate that a continuous rate infusion of lidocaine at 50 µg/kg/minute does not impair echocardiographic indices of left ventricular systolic and diastolic function of healthy rabbits sedated with midazolam.

RESUMEN

The study aimed to determine the continuous rate infusion of tramadol associated with peri- and postoperative analgesia for orthopedic surgeries in dogs, as well as cardiorespiratory and adverse effects. Thirty dogs aged 4.2±1.2 years and weighing 15.1±0.9kg were enrolled in the study, premedicated intramuscularly with acepromazine (0.04mg kg-1) and tramadol (2mg kg-1); anesthesia was induced with propofol and maintained with isoflurane in oxygen. Three infusion rates were compared, comprising three experimental groups: G2: 2.0mg kg-1 h-1; G2.5: 2.5mg kg-1 h-1; and G3: 3.0mg kg-1 h-1. Surgery was initiated 15 minutes following the start of tramadol infusion. During anesthesia, animals were monitored in predefined time points: immediately after tracheal intubation and start of inhalation anesthesia (T0); surgical incision (TSI); final suture (TFS) and end of tramadol infusion (TEI), which was maintained for at least 120 minutes and prolonged according to the duration of surgery. Postoperative analgesia was evaluated through an interval pain scoring scale and the Melbourne pain scale. The mean time of tramadol infusion was greater than 120 minutes in all groups and no differences were found among them (141±27 minutes in G2, 137±27 minutes in G2.5 and 137±30 minutes in G3). Perioperative analgesia was regarded as short and did not correlate with infusion rates. Tramadol infusion provided adequate analgesia with cardiorespiratory stability Analgesia was not dose-dependent, however, and residual postoperative effects were short-lasting, which warrants proper postoperative analgesia following tramadol infusion. Additional studies are required using higher infusion rates and standardized nociceptive stimulation in order to determine how doses influence tramadol analgesia and whe therthereis a limit to its effect in dogs.(AU)

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Objetivou-se determinar a infusão de taxa contínua de tramadol associada à analgesia peri e pós-operatória para cirurgias ortopédicas em cães, além de efeitos cardiorrespiratórios e adversos. Foram utilizados 30 cães, com idade média de 4,2±1,2 anos e pesos médios de 15,1±0,9kg, pré-medicados por via intramuscular com acepromazina (0,04mg/kg) e tramadol (2mg/kg). A anestesia foi induzida com propofol e mantida com isoflurano em oxigênio. Foram comparadas três taxas de infusão, compreendendo três grupos experimentais: G2: 2,0mg/kg; G2,5: 2,5mg/kg1; e G3: 3,0mg/kg. A cirurgia começou 15 minutos após o início da infusão de tramadol. Durante a anestesia, os animais foram monitorados nos seguintes momentos: imediatamente após a intubação traqueal e o início da anestesia inalatória (T0); incisão cirúrgica (TSI); final de sutura (TFS) e final da infusão de tramadol (TEI), que foi mantida por, pelo menos, 120 minutos e prolongada de acordo com a duração da cirurgia. A analgesia pós-operatória foi avaliada por escalas de pontuação de dor, conforme a escala intervalar de avaliação de dor e a escala de contagem variável de avaliação de dor da Universidade de Melbourne, a cada uma hora. O tempo médio de infusão de tramadol foi maior que 120 minutos em todos os grupos, e não foram encontradas diferenças entre elas (141±27 minutos em G2, 137±27 minutos em G2,5 e 137±30 minutos em G3). A analgesia perioperatória foi adequada na maioria dos indivíduos e a pós-operatória foi considerada curta, não correlacionada àquelas com diferentes taxas de infusão. A infusão de tramadol nas taxas estudadas produziu analgesia adequada com estabilidade cardiorrespiratória. A analgesia não foi dose dependente, no entanto os efeitos residuais pós-operatórios foram considerados curtos, o que determina a necessidade de analgesia adequada após infusão contínua de tramadol. Estudos adicionais que utilizam taxas mais elevadas de infusão de tramadol e estimulação nociceptiva padrão são necessários para determinar em que medida as doses influenciam a analgesia de tramadol e se há um limite nos seus efeitos nos cães.(AU)

Asunto(s)

Animales , Perros , Tramadol/análisis , Perros/cirugía , Anestesia General/estadística & datos numéricos

RESUMEN

Deciphering the phylogenetic relationships within the species-rich Millettioid clade has persisted as one of the major challenges in the systematics and evolutionary history of papilionoid legumes (Leguminosae, Papilionoideae). Historically, the predominantly neotropical lianas of subtribe Diocleinae in the Millettioid legumes have been taxonomically tangled together with the largely heterogeneous tribe Phaseoleae. This work presents a comprehensive molecular phylogenetic analysis based on nuclear and chloroplast markers and includes all genera ever referred to Diocleae except for the monospecific Philippine Luzonia, resolving several key generic relationships within the Millettioid legumes. The first of two separate analyses includes 310 matK accessions and strongly supports the reestablishment of tribe Diocleae as a branch of the Millettioid clade. This work sheds greater light on the higher-level phylogeny of Diocleae and allows the recognition of three major lineages: the Canavalia, Dioclea, and Galactia clades. The second set of phylogenetic analyses utilized nuclear (ITS/5.8S and ETS) and plastid (matK and trnT-Y) DNA sequences to reveal (i) the monophyly of Canavalia and Cleobulia; (ii) the monophyly of Bionia with the exclusion of Bionia bella; (iii) the paraphyly of Dioclea with respect to Cleobulia, Cymbosema, and Macropsychanthus; (iv) the paraphyly of Cratylia with respect to the broadly polyphyletic Camptosema; and (v) the polyphyly of Galactia with species scattered widely across the tree.

Asunto(s)

Dioclea/clasificación , Filogenia , Teorema de Bayes , Evolución Biológica , Núcleo Celular/genética , Cloroplastos/genética , ADN de Plantas/análisis , ADN de Plantas/genética , Flores/genética , Humanos , Plastidios/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN

RESUMEN

O presente estudo investigou os benefícios da anestesia por tumescência com lidocaína em cadelas submetidas à mastectomia, visando ao conforto do paciente e à sua recuperação pós-operatória. Foram utilizados sete animais, de peso e raças variadas, que apresentavam neoplasia em região de cadeia mamária e que foram submetidos à cirurgia de mastectomia. Todos os animais receberam o mesmo protocolo anestésico, sendo utilizado como MPA a associação entre acepromazina e morfina, nas doses de 0,04mg/kg e 0,4mg/kg (IM), respectivamente. Após 15 minutos, foi alocado um cateter em veia cefálica e realizou-se a indução com propofol 4mg/kg e midazolam 0,2mg/kg, seguida de manutenção anestésica com isofluorano. Posteriormente à instrumentação, procedeu-se à técnica de anestesia por tumescência com solução gelada composta por ringer lactato, lidocaína 2% sem vasoconstritor e adrenalina, em um volume total de 15mL/kg. Em média, o tempo de duração do procedimento foi de 74±18 minutos. O pico plasmático de lidocaína deu-se entre 30 e 60 minutos após a infiltração da solução. O resgate analgésico foi realizado após sete horas, aproximadamente, da infiltração. Pode-se concluir que a anestesia por tumescência com lidocaína deve ser considerada como constituinte do protocolo anestésico e analgésico de cadelas a serem submetidas à cirurgia de mastectomia, proporcionando estabilidade de parâmetros, segurança e recuperação pós-operatória de boa qualidade.

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The present study investigated the benefits of tumescent anesthesia with lidocaine in dogs undergoing mastectomy, seeking the patients' comfort and their postoperative recovery. Seven animals, with different weight and breed, who had cancer in the region of mammary chain underwent mastectomy surgery. All animals received the same anesthetic protocol being used as the association between acepromazine and morphine doses of 0.04mg.kg-1 and 0.4mg.kg-1 (IM), respectively. After 15 minutes a catheter was placed in the cephalic vein and induction with propofol 4mg.kg-1 and 0.2mg.kg-1 followed by maintenance with isoflurane anesthesia was done. After instrumentation, we proceeded to the tumescent anesthesia technique with ice-cold solution consisting of Ringer's lactate, lidocaine 2% without epinephrine and adrenaline in a total volume of 15mL.kg-1. The average duration of the procedure was 74±18 minutes. The plasmatic peak of lidocaine was between 30 and 60 minutes after infiltration. The rescue analgesic was performed after approximately seven hours of infiltration. It can be concluded that the tumescent anesthesia with lidocaine should be considered as a constituent of anesthetic and analgesic protocol in dogs undergoing mastectomy surgery providing parameter stability, safety and good quality postoperative recovery.

Asunto(s)

Animales , Femenino , Perros , Anestesia Local , Anestesia Local/veterinaria , Mastectomía/veterinaria , Neoplasias/veterinaria , Lidocaína/efectos adversos , Lidocaína/toxicidad

RESUMEN

The tetralin biodegradation genes of Sphingomonas macrogolitabida strain TFA are clustered in two closely linked and divergent operons. To analyze expression of both operons under different growth conditions, transcriptional and translational gene fusions of the first genes of each operon to lacZ have been constructed in plasmids unable to replicate in Sphingomonas and integrated by recombination into the genome of strain TFA. Expression analysis indicated that the transcription of both genes is induced in similar ways by the presence of tetralin. Gene expression in both operons is also subjected to overimposed catabolic repression. Two additional genes named thnR and thnY have been identified downstream of thnCA3A4 genes. ThnR is similar to LysR-type regulators, and mutational analysis indicated that ThnR is strictly required for expression of the thn operons. Unlike other LysR-type regulators, ThnR does not repress its own synthesis. In fact, ThnR activates its own expression, since thnR is cotranscribed with the thnCA3A4 genes. ThnY is similar to the ferredoxin reductase components of dioxygenase systems and shows the fer2 domain, binding a Cys4[2Fe-2S] iron sulfur center, and the FAD-binding domain, common to those reductases. However, it lacks the NAD-binding domain. Intriguingly, ThnY has a regulatory role, since it is also strictly required for expression of the thn operons. Given the similarity of ThnY to reductases and the possibility of its being present in the two redox states, it is tempting to speculate that ThnY is a regulatory component connecting expression of the thn operons to the physiological status of the cell.

Asunto(s)

Regulación Bacteriana de la Expresión Génica , Sphingomonas/genética , Sphingomonas/metabolismo , Tetrahidronaftalenos/metabolismo , Fusión Artificial Génica , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Biodegradación Ambiental , ADN Bacteriano/química , Genes Bacterianos , Genes Reguladores , Genes Reporteros , Datos de Secuencia Molecular , Mutación , Operón , Oxidorreductasas/genética , Oxidorreductasas/fisiología , Filogenia , ARN Bacteriano/análisis , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/genética , Análisis de Secuencia de ADN , Homología de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transcripción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

Two new genes whose products are involved in biodegradation of the organic solvent tetralin were identified. These genes, designated thnE and thnF, are located downstream of the previously identified thnD gene and code for a hydratase and an aldolase, respectively. A sequence comparison of enzymes similar to ThnE showed the significant similarity of hydratases involved in biodegradation pathways to 4-oxalocrotonate decarboxylases and established four separate groups of related enzymes. Consistent with the sequence information, characterization of the reaction catalyzed by ThnE showed that it hydrated a 10-carbon dicarboxylic acid. The only reaction product detected was the enol tautomer, 2,4-dihydroxydec-2-ene-1,10-dioic acid. The aldolase ThnF showed significant similarity to aldolases involved in different catabolic pathways whose substrates are dihydroxylated dicarboxylic acids and which yield pyruvate and a semialdehyde. The reaction products of the aldol cleavage reaction catalyzed by ThnF were identified as pyruvate and the seven-carbon acid pimelic semialdehyde. ThnF and similar aldolases showed conservation of the active site residues identified by the crystal structure of 2-dehydro-3-deoxy-galactarate aldolase, a class II aldolase with a novel reaction mechanism, suggesting that these similar enzymes are class II aldolases. In contrast, ThnF did not show similarity to 4-hydroxy-2-oxovalerate aldolases of other biodegradation pathways, which are significantly larger and apparently are class I aldolases.

Asunto(s)

Aldehído-Liasas/metabolismo , Escherichia coli/enzimología , Hidrolasas/metabolismo , Tetrahidronaftalenos/metabolismo , Aldehído-Liasas/genética , Biodegradación Ambiental , Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano , Hidrolasas/genética , Filogenia , Análisis de Secuencia de ADN , Solventes/química , Solventes/metabolismo , Tetrahidronaftalenos/química

RESUMEN

Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.

Asunto(s)

Bacteriocinas/genética , Enterococcus faecium/genética , Bacterias Grampositivas/efectos de los fármacos , Secuencia de Aminoácidos , Bacteriocinas/aislamiento & purificación , Bacteriocinas/farmacología , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Enterococcus faecium/aislamiento & purificación , Enterococcus faecium/metabolismo , Microbiología de Alimentos , Genes Bacterianos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Operón , Plásmidos , Mapeo Restrictivo

RESUMEN

A culture medium, named olive juice broth, which resembles the natural environment of Lactobacillus plantarum in the traditional Spanish-style green olive fermentation was obtained from green olives. In this medium, the bacteriocin-producing L. plantarum LPCO10 strain was able to produce bacteriocin throughout the incubation time (15 days). Bacteriocin purification from olive juice broth was achieved by a protocol including ammonium sulphate precipitation of cell-free, L. plantarum LPCO10 culture supernatants, and cation-exchange, hydrophobic-interaction and reversed-phase chromatographies. In a series of mixed cultures in olive juice broth, L. plantarum LPCO10 was able to dominate the bacteriocin-sensitive L. plantarum 128/2 strain, whereas the non-bacteriocin-producing, LPCO10 strain derivative, L. plantarum 55-1 strain did not show such capability. These results indicated that olive juice broth may be a valuable experimental substitute for olive fermentation brine in gaining more knowledge about the role of the bacteriocin-producing L. plantarum strains in the control of the Spanish-style green olive fermentation.

Asunto(s)

Bacteriocinas/biosíntesis , Frutas/microbiología , Lactobacillus/crecimiento & desarrollo , Bacteriocinas/aislamiento & purificación , Cromatografía en Agarosa , Cromatografía por Intercambio Iónico , Recuento de Colonia Microbiana , Medios de Cultivo , Fermentación , Lactobacillus/metabolismo , Mutación , España

RESUMEN

A 4.5-kb region of chromosomal DNA carrying the locus responsible for the production of plantaricin S, a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10 (R. Jiménez-Díaz, J. L. Ruiz-Barba, D. P. Cathcart, H. Holo, I. F. Nes, K. H. Sletten, and P. J. Warner, Appl. Environ. Microbiol. 61:4459-4463, 1995), has been cloned, and the nucleotide sequence has been elucidated. Two genes, designated plsA and plsB and encoding peptides alpha and beta, respectively, of plantaricin S, plus an open reading frame (ORF), ORF2, were found to be organized in an operon. Northern blot analysis showed that these genes are cotranscribed, giving a ca. 0.7-kb mRNA, whose transcription start point was determined by primer extension. Nucleotide sequences of plsA and plsB revealed that both genes are translated as bacteriocin precursors which include N-terminal leader sequences of the double-glycine type. The role of ORF2 is unknown at the moment, although it might be expected to encode an immunity protein of the type described for other bacteriocin operons. In addition, several other potential ORFs have been found, including some which may be responsible for the regulation of bacteriocin production. Two of them, ORF8 and ORF14, show strong homology with histidine protein kinase and response regulator genes, respectively, which have been found to be involved in the regulation of the production of other bacteriocins from lactic acid bacteria. A third ORF, ORF5, shows homology with gene agrB from Staphylococcus aureus, which is involved in the mechanism of regulation of the virulence phenotype in this species. Thus, an agr-like regulatory system for the production of plantaricin S is postulated.

Asunto(s)

Bacteriocinas/genética , Lactobacillus/genética , Secuencia de Aminoácidos , Bacteriocinas/biosíntesis , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Lactobacillus/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Operón , Reacción en Cadena de la Polimerasa , Transcripción Genética

RESUMEN

The N-terminal region of AfsR, a putative pleiotropic regulatory protein for antibiotic production in Streptomyces coelicolor A3(2), is homologous to RedD and Actil-ORF4, pathway-specific regulatory proteins required for the production of the antibiotics undecylprodigiosin (Red) and actinorhodin (Act), respectively. The recent identification of afsS, which lies immediately 3' of afsR and which stimulates antibiotic production when cloned at high copy number, questioned whether afsR was a pleiotropic regulatory gene. In this study we demonstrate that multiple copies of afsR can stimulate both Act and Red production and that, despite its homology, it cannot substitute for the pathway-specific regulatory genes. Moreover, an in-frame deletion that removed most of the afsR coding sequence resulted in loss of Act and Red production, and a marked reduction in the synthesis of the calcium-dependent antibiotic (CDA), but only under some (non-permissive) nutritional conditions. Although additional copies of afsR resulted in elevated levels of the actII-ORF4 and redD transcripts, transcription of the pathway-specific regulatory genes under non-permissive conditions was unaffected by deletion of afsR. While afsR may operate independently of the pathway-specific regulatory proteins to influence antibiotic production, the activity of ActII-ORF4 and of RedD under non-permissive conditions could depend on interaction with, or modification by, AfsR.

Asunto(s)

Antibacterianos/biosíntesis , Proteínas Bacterianas/genética , Proteínas de Unión al ADN , Genes Bacterianos , Streptomyces/genética , Streptomyces/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Antraquinonas/metabolismo , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Amplificación de Genes , Eliminación de Gen , Genes Reguladores , Mutación , Prodigiosina/análogos & derivados , Prodigiosina/biosíntesis , Mapeo Restrictivo

RESUMEN

A cloned DNA fragment from Anabaena sp. strain PCC 7120 that complements an arginine auxotrophic mutant from the same organism was found to include an open reading frame encoding a 427-residue polypeptide that is homologous to N-acetylornithine aminotransferase from Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae. The gene encoding N-acetylornithine aminotransferase in bacteria has been named argD. The expression of Anabaena sp. strain PCC 7120 argD, as well as of argC, was analyzed at the mRNA level. Both genes were transcribed as monocistronic mRNAs, and their expression was not affected by exogenously added arginine. Primer extension analysis identified transcription start points for both genes which were preceded by sequences similar to that of the E. coli RNA polymerase sigma 70 consensus promoter. A second transcription start point for the argD gene that is not preceded by a sigma 70 consensus promoter was detected in dinitrogen-grown cultures.

Asunto(s)

Aldehído Oxidorreductasas , Anabaena/genética , Arginina/biosíntesis , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Transaminasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , Datos de Secuencia Molecular , Nitrógeno/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Factor sigma/genética , Transcripción Genética

RESUMEN

Arginine auxotrophs of the dinitrogen-fixing cyanobacterium Anabaena species strain PCC 7120 were isolated after ultraviolet light mutagenesis and penicillin enrichment. Two of these auxotrophs were complemented by a cosmid gene library of the wild-type strain established in Escherichia coli that was transferred en masse to the mutants by conjugation. The gene complementing one of those mutants was found to complement an E. coli argC mutant. Sequencing analysis of the gene showed that it encodes a 322-residue polypeptide that is homologous to the ArgC protein of E. coli, Bacillus subtilis and Streptomyces clavuligerus and to the C-terminal moiety of the Saccharomyces cerevisiae ARG5,6 gene product, N-acetylglutamate semialdehyde dehydrogenase. A cysteine residue present in a highly conserved domain in the five proteins is probably located in the active site of the enzyme. Conserved among the ArgC proteins, sequences resembling the primary structure of nucleotide-binding domains are also found. Downstream of the Anabaena argC gene seven nearly perfect repeats of a heptanucleotide (consensus sequence:5'-CTAATGA-3') are found.

Asunto(s)

Aldehído Oxidorreductasas/genética , Anabaena/aislamiento & purificación , Arginina/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Secuencia de Aminoácidos , Anabaena/genética , Anabaena/crecimiento & desarrollo , Secuencia de Bases , Clonación Molecular , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Homología de Secuencia