Draft Genome Sequence of a Hypermucoviscous Extended-Spectrum-ß-Lactamase-Producing Klebsiella quasipneumoniae subsp. similipneumoniae Clinical Isolate.

A clinical isolate of extended-spectrum-ß-lactamase-producing Klebsiella quasipneumoniae subsp. similipneumoniae 06-219 with hypermucoviscosity phenotypes obtained from a urine culture of an adult patient was used for whole-genome sequencing. Here, we report the draft genome sequences of this strain, consisting of 53 contigs with an ~5.6-Mb genome size and an average G+C content of 57.36%. The annotation revealed 6,622 coding DNA sequences and 77 tRNA genes.

2D 1H and 13C NMR conformational studies of thienopyridines and carboline biarylic compounds.

In this paper, we report on conformational studies of biarylic compounds, as prepared through the well-known aza-Wittig methodology. The conformational studies were mainly realized by bidimensional (2D) nuclear magnetic resonance spectroscopy (NMR) and NOESY experiments. The conformational behavior showed that these biarylic compounds display an orthogonal symmetry and adopt a characteristic arrangement around the pivotal bond. Molecular modeling calculations were performed to support structure conformations.

[Laparoscopic vesical autoaugmentation: an animal model in rabbits (Oryctolagus cuniculus)]. / Autoaumento vesical por laparoscopia. Modelo experimental en conejos (Oryctolagus cuniculus).

INTRODUCTION: Small contracted bladder remains a challenge in clinical urological practice and treatment options are not always effective. Urothelium sparing surgical techniques represent a reasonable therapeutical option. We aimed to evaluate the feasibility of a rabbit model (Oryctolagus cuniculus) in the teaching and training setting for laparoscopic vesical autoaugmentation. METHODS: Transperitoneal laparoscopic bladder autoaugmentation was performed in five New Zealand male rabbits (Oryctolagus cuniculus). A three port technique was used for all cases. Circumferential detrusorectomy was performed with vascular pedicles sparing, subsequently submucosal detailed blunt dissection allowed the creation of a bladder diverticulum which was verified intraoperatively. Eye-ball cystometry was performed preoperatively and postoperatively to verify bladder capacity variations. Postoperatively cystometry was done immediately after the procedure and 7 and 15 days postoperatively. Statistical analysis was performed with T-student model. A p-value < 0.05 was considered of significance for the analysis. Surgical procedure and perioperative animal care was minutely precised according to the Guidelines of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. RESULTS: Five New Zealand male rabbits (Oryctolagus cuniculus) were surgically treated as described. Median operative time was 68.8 minutes (range 44 -120). Median preoperatory bladder capacity for the series was 25.8 cc (7-52). Median postoperative bladder capacity was diminished in 33% immediately after the operation. Then, bladder capacity augmented 72% and 52% for 7th and 15th postoperative days, respectively. Percentage variation in bladder capacity is statistically significant. Two operative complications (mucosal tear) were reported. A postoperatory abscess was observed in one animal. CONCLUSION: The rabbit (Oryctolagus cuniculus) model for the laparoscopic bladder autoaugmentation is feasible for teaching, training and research. Bigger series and longer follow-up should be addressed.

Autoaumento vesical por laparoscopia. Modelo experimental en conejos (oryctolagus cuniculus) / Laparoscopic vesical autoaugmentation: ana animal model in rabbits (Oryctolagus cuniculus)

Introducción: La vejiga pequeña y contraída representa un cuadro clínico de difícil terapéutica y no siempre efectiva. Se presenta la cirugía preservadora de urotelio como una opción que permite el aumento de la capacidad vesical. Nuestro objetivo fue evaluar la factibilidad del conejo (Oryctolagus cuniculus) como modelo experimental de enseñanza, entrenamiento y aplicación de técnica para el autoaumento vesical por laparoscopia. Material y Métodos: Cinco conejos (Oryctolagus cuniculus), machos, New Zealand, fueron intervenidos laparoscópicamente por técnica transperitoneal de tres puertos, realizándose autoaumento vesical mediante detrusorectomía. Se realizó cistometría simple preoperatoria y postoperatoria inmediata, mediata y tardía, para verificar modificaciones de capacidad vesical. Resultados: 5 conejos machos fueron operados en un tiempo promedio de 68,8 minutos (rango 44-120). Se realizó medición de la capacidad vesical preoperatoria con promedio de 25.8cc (7-52) disminuyendo la postoperatoria inmediata en un 33%. Se verificó aumento de capacidad vesical postoperatoria de 72% y 52% para el séptimo y decimoquinto 0día, respectivamente. Estos porcentajes fueron estadísticamente significativos con 95% de confianza. Se presentaron 2lesiones vesicales como complicación transoperatoria y un absceso como complicación postoperatoria. Conclusiones: El modelo animal en conejos para la técnica de autoaumento vesical por laparoscopia es un modelo factible para enseñanza, entrenamiento y aplicación (AU)

Introduction: Small contracted bladder remains a challenge in clinical urological practice and treatment options are not always effective. Urothelium sparing surgical techniques represent a reasonable therapeutical option. We aimed to evaluate the feasibility of a rabbit model (Oryctolagus cuniculus) in the teaching and training setting for laparoscopicvesical autoaugmentation. Methods: Transperitoneal laparoscopic bladder autoaugmentation was performed in five New Zealand male rabbits (Oryctolagus cuniculus). A three port technique was used for all cases. Circumferential detrusorectomy was performed with vascular pedicles sparing, subsequently submucosal detailed blunt dissection allowed the creation of a bladder diverticulum which was verified intraoperatively. Eye-ball cystometry was performed preoperatively and postoperatively to verify bladder capacity variations. Postoperatively cystometry was done immediately after the procedure and 7 and 15days postoperatively. Statistical analysis was performed with T-student model. A p-value < 0.05 was considered of significance for the analysis. Surgical procedure and perioperative animal care was minutely precised according to the Guidelines of the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. Results: Five New Zealand male rabbits (Oryctolagus cuniculus) were surgically treated as described. Median operative time was 68.8 minutes (range 44 -120). Median preoperatory bladder capacity for the series was 25.8 cc (7-52). Median postoperative bladder capacity was diminished in 33% immediately after the operation. Then, bladder capacity augmented 72%and 52% for 7th and 15th postoperative days, respectively. Percentage variation in bladder capacity is statistically significant. Two operative complications (mucosal tear) were reported. A postoperatory abscess was observed in one animal. Conclusion: The rabbit (Oryctolagus cuniculus) model for the laparoscopic bladder autoaugmentation is feasible for teaching, training and research. Bigger series and longer follow-up should be addressed (AU)

(1)H, (13)C NMR and X-ray crystallographic studies of highly polyhalogenated derivatives of costunolide lactone.

The costunolide lactone, a sesquiterpene compound isolated from Zaluzania triiloba species, reacted with several dihalocarbene sources produced by trihaloform-NaOH under successive phase transfer reactions yielding mono-, bis- and tris-dihalocyclopropane adducts. The structures, as well as the configurational assignments of the different derivatives, were established by (1)H and (13)C NMR spectroscopy and assisted by X-ray crystallographic and molecular modelling studies. The specific shielding of protons in the neighbourhood of different halogens on the cyclopropane moieties was correlated to the pseudocontact interactions.

1H,13C 2D NMR and X-ray studies of the products of the reaction between dibenzylidencyclohexanone and 6-amino-1,3-dimethyl uracil.

The reaction of the 6-amino-1,3-dimethyl uracil with the dibenzylidencyclohexanone (1), provided three adducts whose structures result from nucleophilic attack yielding the monoadduct 3 and two isomeric bisadducts (4 and 5) in moderate yields (50-60%). The structures obtained in this study were elucidated with 2D high resolution NMR experiments, variable temperature NMR and X-ray crystallographic studies. In compound 3, the tricyclic skeleton is essentially planar and the cyclohexane ring addopts an envelope conformation. The structures 4 and 5 correspond to two isomeric spiro compounds.

2D 1H and 13C NMR evidences of the [2 + 2] autodimerization of 2-benzyl-5-benzylidene cyclopentanone yielding two different diphenyl, dispiro cyclobutane derivatives.

The ene-ene [2 + 2] cycloaddition of 2-benzyl-5-benzylidenecyclopentanone proceeds smoothly and spontaneously in benzene-d6 or deuteriochloroform solution to give two different diphenyl dispiro [4.1.4.1] dodecan-4,11-diones. Detailed 1H and 13C 2DNMR spectroscopy (COSY, HMQC, HMBC) were performed in order to prove the existence in solution of two cyclobutane derivatives, one a previously described photodimer obtained by the UV irradiation of crystals of 2-benzyl-5-benzylidenecyclopentanone.

RepA negatively autoregulates the transcription of the repABC operon of the Rhizobium etli symbiotic plasmid basic replicon.

The basic replicon of Rhizobium etli CE3, like other members of the repABC plasmid family, is constituted by the repABC operon. RepC is essential for replication, and RepA and RepB play a role in plasmid segregation. It has been shown that deletion derivatives lacking the repAB genes have an increased copy number, indicating that these genes participate in the control of plasmid copy number. RepA is also a trans-incompatibility factor. To understand the regulation of the repABC operon, in this paper: (i) the transcription start site of the repABC operon was determined; (ii) the promoter region was identified by site-directed mutagenesis of the putative -35 and -10 hexameric elements; and (iii) RepA was recognized as a negative regulator of the transcription of the repABC operon.

13C and 1H NMR of 2,6-diaryl-1-hydroxy piperidin-4-one oximes; substituent effects on cis/trans ratio and conformational equilibria.

The reaction of substituted diarylidene acetones with hydroxylamine hydrochloride affords isomeric N-hydroxy diaryl piperidinone oximes as main products. The structures as well as conformational equilibria of these products were established by 1H and 13C NMR spectroscopy and further studied by variable temperature NMR. It was found that the cis/trans ratio of 2,6-substituted piperidine derivatives depended on the position of the substituent on the aromatic ring.

Compilation and analysis of sigma(54)-dependent promoter sequences.

Promoters recognized by the RNA-polymerase with the alternative sigma factor sigma(54) (Esigma54) are unique in having conserved positions around -24 and -12 nucleotides upstream from the transcriptional start site, instead of the typical -35 and -10 boxes. Here we compile 186 -24/-12 promoter sequences reported in the literature and generate an updated and extended consensus sequence. The use of the extended consensus increases the probability of identifying genuine -24/-12 promoters. The effect of several reported mutations at the -24/-12 elements on RNA-polymerase binding and promoter strength is discussed in the light of the updated consensus.

[Serotypes of Salmonella isolated from the Luján River, Argentina]. / Serotipos de Salmonella aislados del Río Luján, Argentina.

In this work, the serotypes of Salmonella genus polluting the waters of Lujan River, situated in the north east of the province of Buenos Aires, Argentina, were studied. A total of 690 samples of water were collected and analysed from February 1988 through December 1989, at three different sites. They were obtained according to the Moore technique and then preenriched in buffered peptone water and enriched in Rappaport-Vassiliadis Soya peptone broth was carried out. The isolations were realised in brilliant green-desoxycholate agar and bismuth sulphite agar and the presumptive colonies to be Salmonella were tested by the standard biochemical identification. Salmonella spp. was isolated in 434 samples (62.9%). The predominant serotype was S. Anatum, followed in a decreasing order by S. Montevideo, S. Newport and S. Bredeney. A large amount of serotypes that are isolated with very low frequency and very rarely in other hidric courses in the country were: S. Westhampton, S. Poona and S. Saintpaul were found.

2D 1H and 13C NMR evidence for stereoselective formation of a new bond C-N, C-S or C-C in the reaction of ivalin acetate with substituted pyrimidines.

Several pyrimidine derivatives of ivalin acetate were synthesized as potential anti HIV agents. High stereoselective Michael addition to ivaline acetate was observed and a new C-C, C-N or C-S bond was formed. 2D NMR 1H and 13C as well as X-ray crystallographic studies were performed on the compounds herein described to establish the structure and stereochemistry.

In vivo genomic footprinting analysis reveals that the complex Bradyrhizobium japonicum fixRnifA promoter region is differently occupied by two distinct RNA polymerase holoenzymes.

The Bradyrhizobium japonicum fixRnifA operon is transcribed from two promoters: fixRp1, a -24/-12 promoter recognized by the sigma54-holoenzyme form of the RNA polymerase, and fixRp2, a -35/-10 promoter that is transcribed by a second, unidentified, form of RNA polymerase holoenzyme. The fixRp1 promoter is autoregulated during microaerobiosis by NifA, whereas fixRp2 is also activated, but by a different regulatory protein. The main transcription start sites for these promoters are just two nucleotides apart, such that the conserved -12 and -10 regions of fixRp1 and fixRp2, respectively, must overlap each other, whereas the -24 and -35 regions lie one DNA helical turn apart. Using in vivo genomic dimethyl sulfate and KMnO4 footprinting, we showed that the promoter region is differentially protected, depending upon which holoenzyme is bound. Mutagenesis analyses indicated that positions from -12 to -14 are critical for the activity of both promoters, whereas mutations at -10 and -11 affected mainly fixRp2 expression. When the sequence of the putative -35 region of fixRp2 was modified to match the putative consensus, expression from this promoter was increased 3-fold and the reactivity toward KMnO4, but not the transcriptional start site, moved two nucleotides further upstream, indicating that the altered promoter forms a different open complex. Additionally, we detected NifA-dependent methylation protection of two atypical NifA binding sites and protection of guanine -75. The latter residue is located in a region critical for fixRp2 promoter activation. The results present direct physical evidence of the complexity of the organization, regulation, and function of the fixRnifA promoter region.

Overlapping promoters for two different RNA polymerase holoenzymes control Bradyrhizobium japonicum nifA expression.

The Bradyrhizobium japonicum NifA protein, the central regulator for nitrogen fixation gene expression, is encoded in the fixRnifA operon. This operon is activated during free-living anaerobic growth and in the symbiotic root nodule bacteroid state. In addition, it is expressed in aerobic conditions, albeit at a low level. Here, we report that this pattern of expression is due to the presence of two overlapping promoters: fixRp1, which is of the -24/-12 class recognized by the RNA polymerase sigma 54, and fixRp2, which shares homology with the -35 and -10 regions found in other putative B. japonicum housekeeping promoters. Primer extension analyses showed that fixRp1 directed the synthesis of a transcript, P1, that starts 12 nucleotides downstream of the -12 region. In addition to sigma 54, P1 was dependent on NifA and low oxygen tension. Transcripts originating from fixRp2 started at two sites: one coincided with P1, while the most abundant, P2 initiated just two nucleotides further downstream of P1. Expression from fixRp2 was dependent on the upstream -68 promoter region, a region known to bind a putative activator protein, but it was independent of sigma 54 and NifA. This promoter was expressed in aerobic and anaerobic conditions but was not expressed in 30-day-old bacteroids. Mutations in the conserved 12 region for the sigma 54 promoter did not show any transcript, because these mutations also disrupted the overlapping -10 region of the fixRp2 promoter. Conversely, mutations at the -24 region only affected the sigma 54-dependent P1 transcript, having no effect on the expression of P2. In the absence of omega(54), anaerobic expression from the fixRp(2) promoter was enhanced threefold, suggesting that in the wild-type strain, the two RNA polymerase holoenzymes must compete for binding to the same promoter region.

Estudio taxonómico de bacterias heterotrofas aisladas de aguas del Lago Peñuelas, V región Chile / Taxonomic study of heterotrophic bacteria isolated from water of Peñuelas Lake, V region Chile

Se realizó un estudio taxonómico de 109 cepas de bacterias heterótrofas aisladas de las aguas del Lago Peñuelas, localizado en la V Región, Valparaíso, Chile, junto a 8 cepas de referencia. Se efectuaron 107 pruebas fenotípicas que fueron analizadas por taxonomía numérica. Se encontraron 7 fenones utilizando el coeficiente de apareamiento simple de Sokal y Michener (Ssm) y la técnica de agrupamiento UPGMA, a un nivel de semejanza del 70 porciento. Los fenones A (9 cepas) y F(3), fueron asignados al género Bacillus, 41 cepas fueron incluidas en el género Flavobacterium (fenones C y E), el fenón B (11), fue asignado al género Aeromonas y los fenónes D(37) y G(6) fueron incluídos en el género Acinetobacter

Correlation between cyclophosphamide-induced viral susceptibility and depletion of Junin virus-induced suppressor populations.

In contrast to lymphocytic choriomeningitis virus, another arenavirus, Junin virus (JV), the etiologic agent of Argentine hemorrhagic fever, when inoculated into suckling mice, induces lethal meningoencephalitis characterized by a delayed-type hypersensitivity (DTH)-like immune response. However, the adult BALB/c mouse is resistant to infection and no DTH reaction can be seen. This different viral sensitivity may be related to the development of an antigen non-specific DTH-suppressor cell pathway at work in the adult mouse. When the resistant mice are treated with cyclophosphamide (Cy) (50 mg/kg each dose) given at days -1,+1,+4 (zero: infection day), animals become susceptible and develop DTH reaction in brain that leads to death. We analyze the influence of the timing of Cy administration on the suppressor system developing after infection. It was found that Cy depletes the previously described JV-induced suppressor populations (Tsv) but a new suppressor cell (Tsv*) is disclosed bearing the Thy 1+ Ly1+2- phenotype which is unable to depress DTH in Cy-treated animals. With only two doses of Cy corresponding to days -1 and +1, the target of Tsv* cells is depleted but the third dose is still required to achieve full depletion of Tsv cells which are able to employ the Cy-resistant antigen-specific suppressor cells as targets. Since the Cy treatment is able to deplete the Tsv population together with the target of Tsv* cells, animals became unable to regulate lethal DTH reaction. Thus, a cellular explanation for an empirically established Cy schedule able to abrogate the adult mouse resistance to JV is proposed.

Junin virus-induced non-specific suppressor cells interact with unrelated antigen-specific suppressor cells.

Junin virus (JV) infection of adult (resistant) BALB/c mice induces antigen non-specific delayed-type hypersensitivity (DTH) suppressor T cells, termed Tsv, bearing the Thy-1+, Ly-1+2- phenotype. These cells may be related to survival to infection since DTH reaction is associated with lethal meningoencephalitis. Employing several xenogeneic red blood cell (RBC) sensitization schedules to induce different cell subpopulations, we have attempted to establish the target of JV-induced suppressor cells (Tsv). The target of Tsv cells was actually included in the antigen-specific suppressor cell compartment, as demonstrated for the RBC system. Tsv cells were able to trigger suppressor cells to act without loss of their specificity. The presence of two sets of sheep RBC-induced DTH suppressor cells bearing the Ly-1+2- and Ly-1-2+ phenotypes was disclosed in low (10(6))-dose sensitized mice. Both sets were simultaneously required by Tsv to achieve DTH suppression. In contrast, in high (10(8] SRBC-dose sensitized animals treated with cyclophosphamide (doses of 50 mg/kg), a single Ly-1-2+ suppressor cell was required.

[Incidence of rotavirus in intensive-production poultry farms]. / Incidencia de los rotavirus en establecimientos avícolas de producción intensiva.

Rotavirus are the major etiological agent of acute gastroenteritis in various species of mammals and birds. This study was aimed to explore the frequency of rotavirus in flocks of commercial laying hens and industrial broiler chicken. Feces and caecal content were sampled from 589 birds belonging to 17 farms located in the neighbourhood of Lujan National University. RNA patterns were explored by polyacrylamide gel electrophoresis. Viral particles were found with moderate frequency throughout the period of study: 7.06% in 1987; 7.45% in 1988; 12.57% in 1989. This finding demonstrates the permanence of rotavirus infection in the farm under study and emphasizes the importance of routine rotavirus screening in industrial poultry farms.

Incidencia de los rotavirus en establecimientos avícolas de producción intensiva. / [Incidence of rotavirus in intensive-production poultry farms]

Rotavirus are the major etiological agent of acute gastroenteritis in various species of mammals and birds. This study was aimed to explore the frequency of rotavirus in flocks of commercial laying hens and industrial broiler chicken. Feces and caecal content were sampled from 589 birds belonging to 17 farms located in the neighbourhood of Lujan National University. RNA patterns were explored by polyacrylamide gel electrophoresis. Viral particles were found with moderate frequency throughout the period of study: 7.06

in 1987; 7.45

in 1988; 12.57

in 1989. This finding demonstrates the permanence of rotavirus infection in the farm under study and emphasizes the importance of routine rotavirus screening in industrial poultry farms.