Phylogenentic and enzymatic characterization of psychrophilic and psychrotolerant marine bacteria belong to γ-Proteobacteria group isolated from the sub-Antarctic Beagle Channel, Argentina.

The phylogenetic and physiological characteristics of cultivable-dependent approaches were determined to establish the diversity of marine bacteria associated with the intestines of benthonic organisms and seawater samples from the Argentina's Beagle Channel. A total of 737 isolates were classified as psychrophlic and psychrotolerant culturable marine bacteria. These cold-adapted microorganisms are capable of producing cold-active glycosyl hydrolases, such as ß-glucosidases, celulases, ß-galactosidases, xylanases, chitinases, and proteases. These enzymes could have potential biotechnological applications for use in low-temperature manufacturing processes. According to polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S ribosomal DNA (ARDRA) and DNA gyrase subunit B (gyrB-RFLP), 11 operational taxonomic units (OTU) were identified and clustered in known genera using InfoStat software. The 50 isolates selected were sequenced based on near full sequence analysis of 16S rDNA and gyrB sequences and identified by their nearest neighbors ranging between 96 and 99 % of identities. Phylogenetic analyses using both genes allowed relationships between members of the cultured marine bacteria belonging to the γ-Proteobacteria group (Aeromonas, Halteromonas, Pseudomonas, Pseudoalteromonas, Shewanella, Serratia, Colwellia, Glacielocola, and Psychrobacter) to be evaluated. Our research reveals a high diversity of hydrolytic bacteria, and their products actuality has an industrial use in several bioprocesses at low-temperature manufacturing.

High resolution films for bone regeneration evaluation.

Diagnostic imaging techniques (DIxT) seem to be a useful tool for evaluating bone formation in both human and animal models. There is little evidence on the use of Soft X-Rays (sXR) with high-resolution films for studying the healing process in critical bone size defects (CSD). The aim of this study was to evaluate the ability of soft X-Ray - High Resolution Films (sXR) to distinguish bone regeneration in CSDs. A CSD was created in each of 16 Wistar rat calvariae. The animals were euthanized at 1, 3 and 6 weeks after surgery. The samples were submitted to cXR (conventional X-rays), sXR techniques and histological procedures (HP). Bone formation was observed at CSD edges at all periods of time. At 6 week there was also new bone in the central area. The CSD was not fully regenerated after any period of time. Histometric results were 0.16%; 0.75% and 0.89% new bone formed at weeks 1, 3 and 6 respectively; radiometric results at cXR were 0% in all samples. Evaluation of sXR shows 0.4%; 0.50% and 3.64% bone at weeks 1, 3 and 6. Mean and Standard Deviation were calculated. The data were submitted to statistical analysis using the Pearson product-moment correlation coefficient test. The r value was 0.581. Under these experimental conditions, sXR was found to be a suitable method for detecting new bone formation, based on the positive correlation between sXR and HP during the bone healing process of CSDs in rat calvaria. Furthermore, the sXR technique allowed us to obtain samples with appropriate spatial orientation.

High resolution films for bone regeneration evaluation

Diagnostic imaging techniques (DIxT) seem to be a useful tool for evaluating bone formation in both human and animal models. There is little evidence on the use of Soft X-Rays (sXR) with high-resolution films for studying the healing process in critical bone size defects (CSD). The aim of this study was to evaluate the ability of soft X-Ray - High Resolution Films (sXR) to distinguish bone regeneration in CSDs. A CSD was created in each of 16 Wistar rat calvariae. The animals were euthanized at 1, 3 and 6 weeks after surgery. The samples were submitted to cXR (conventional X-rays), sXR techniques and histological procedures (HP). Bone formation was observed at CSD edges at all periods of time. At 6 week, there was also new bone in the central area. The CSD was not fully regenerated after any period of time. Histometric results were 0.16%; 0.75% and 0.89% new bone formed at weeks 1, 3 and 6 respectively; radiometric results at cXR were 0% in all samples. Evaluation of sXR shows 0.4%; 0.50% and 3.64% bone at weeks 1, 3 and 6. Mean and Standard Deviation were calculated. The data were submitted to statistical analysis using the Pearson product-moment correlation coefficient test. The r value was 0.581. Under these experimental conditions, sXR was found to be a suitable method for detecting new bone formation, based on the positive correlation between sXR and HP during the bone healing process of CSDs in rat calvaria. Furthermore, the sXR technique allowed us to obtain samples with appropriate spatial orientation.

Las tecnicas de diagnostico por Imagenes (DxI) han demostrado su utilidad para evaluar formacion osea en situaciones de salud y enfermedad. Son utilizadas tanto en humanos como en modelos animales; aunque la tecnica de rayos X blandos en placas de alta resolucion (rXb) ha sido escasamente aplicada. El objetivo de este trabajo fue evaluar la capacidad tecnica de los rayos X blandos en placas de alta resolucion (rXb) para distinguir la neoformacion osea en defectos oseos criticos (DOC) en calotas de ratas, durante el proceso de regeneracion osea. En 16 ratas Wistar hembras (150 ± 50 g), se realizaron DOC circulares en calota. Los animales fueron eutanasiados a la 1o, 3o y 6o semana post-quirurgica. Las muestras experimentales (MEx) recibieron rayos X convencionales (rXc), rayos X blandos (rXb) y luego fueron procesadas histologicamente (TH). Se realizaron estudios histometricos y radiometricos; utilizando soft Image J (NIH). Los resultados histologicos demostraron presencia de tejido de granulacion en el area del DOC a la 1° semana y se observo tejido fibroso desde la 3° semana. En todos los periodos de tiempo, se observo formacion osea en los bordes del DOC, mientras que a la 6° semana, fue evidente en el area central del mismo. No se evidencio regeneracion osea en ningun periodo estudiado. Los resultados histometricos fueron 0,16%; 0,75% y 0,89% a la 1o, 3o y 6o semana respectivamente. Los resultados radiometricos obtenidos utilizando placas radiograficas convencionales (rXc) fueron de 0% en todos los casos; mientras que en placas de alta resolucion con rayos X blandos (rXb) fueron 0,4%; 0,50% y 3,64% a las 1o, 3o y 6o semanas respectivamente. Se calcularon la media y Desvio Estandar a la 1°, 3° y 6° semana. Ademas se utilizo el coeficiente rho de Pearson, para estimar la correlacion existente entre rXb y TH; obteniendo un valor r de 0,581. En las condiciones experimentales utilizadas, podemos concluir que la tecnica de rXb fue un metodo apropiado para la deteccion de neoformacion osea, ya que demostro una correlacion positiva con la TH, durante los periodos de tiempo estudiados; ademas de facilitar la orientacion de las MEx durante su procesamiento histologico.

Chromate reductase activity in Streptomyces sp. MC1.

Biological transformation of Cr(VI) to Cr(III) by enzymatic reduction may provide a less costly and more environmentally friendly approach to remediation. In a previous report a Cr(VI) resistant actinomycete strain, Streptomyces sp. MC1, was able to reduce Cr(VI) present in a synthetic medium, soil extract and soil samples. This is the first time optimal conditions such as pH, temperature, growth phase and electron donor have been elucidated in vitro for Cr(VI) reduction by a streptomycete. Chromate reductase of Streptomyces sp. MC1 is a constitutive enzyme which was mainly associated with biomass and required NAD(P)H as an electron donor. It was active over a broad temperature (19-39 degrees C) and pH (5-8) range, and optimum conditions were 30 degrees C and pH 7. The enzyme was present in supernatant, pellet and cell free extract. Bioremediation with the enzyme was observed in non-compatible cell reproduction systems, conditions frequently found in contaminated environments.

Bioremediation of chromium(VI) contaminated soil by Streptomyces sp. MC1.

This work provides quantitative information on Cr(VI) reduction in soil samples by an indigenous actinomycete. Streptomyces sp. MC1, previously isolated from sugarcane, has shown ability to reduce Cr(VI) in liquid minimal medium. A reduction of 100 and 75% was obtained at initial Cr(VI) concentrations of 5 and 50 mg l(-1), respectively, after 48 h of incubation. Bioremediation ability of Streptomyces sp. MC1 was assayed in soil extracts and soil samples. Relative growth of Streptomyces sp. MC1 was 77 and 38% when grown in soil extract with 10 and 50 mg l(-1) of Cr(VI), respectively. MC1 was able to reduce 30% of Cr(VI) after 96 h of incubation with 10 mg l(-1) of Cr(VI), and reduction coincided with the exponential growth phase at pH 7 and 30 degrees C.In soil samples, Streptomyces sp. MC1 was able to reduce up to 94% of the Cr(VI) bioavailability (50 mg kg(-1)) after 7 d. These results were compared with non-inoculated soil samples with Cr(VI). Bioremediation activity of Streptomyces sp. MC1 was not inhibited by natural soil microbial flora. Besides, Streptomyces sp. MC1 growth was not inhibited by 50 mg kg(-1) of Cr(VI). In contrast to findings obtained by other authors, our results showed almost complete Cr(VI) removal from soil without any previous treatment, and without addition of any substrate and with a normal soil humidity level. These results confirm the Cr(VI)-contaminated soil bioremediation potential of Streptomyces sp. MC1.

Rat Lefty2/EBAF gene: isolation and expression analysis in the oviduct during the early pregnancy stage.

It has been demonstrated, by RNA Arbitrarily Primed Polymerase Chain Reaction (RAP-PCR), that the endometrial bleeding associated factor (ebaf or lefty2) is expressed in rat oviduct. In this work we isolated and sequenced the full-length lefty2 cDNA from Rattus norvegicus oviducts and described its expression level in this organ during the estrous cycle and early pregnancy stage. The coding deduced sequence (CDS) codifies a 40.91 kDa protein with a highly conserved TGF-beta functional domain. RT-PCR semiquantitative analysis indicated that oviduct cells transcribe lefty2 among different stages of the estrous cycle with the maximum expression at diestrus phase. The highest expression of lefty2 was at the 4(th) day after mating (five folds respect to day one), just when the embryos have completed their transit through the oviduct. The lefty2 expression declined rapidly thereafter and the levels of their transcripts in the oviduct remained low until 7(th) days after mating.

Chromium(VI) resistance and removal by actinomycete strains isolated from sediments.

Forty-one isolated actinomycetes were used to study qualitative and semi-quantitative screening of chromium(VI) resistance. Chromate-removing activity was estimated using the Cr(VI) specific colorimetric reagent 1,5-diphenylcarbazide. Twenty percent of the isolates from El Cadillal (EC) and 14% of isolates from a copper filter plant (CFP) were able to grow at 13 mM of Cr(VI). All isolates from sugar cane (SCP) could grow up to Cr(VI) concentration of 17 mM. EC, CFP and SCP strains were able to remove 24%, 30% and more than 40% of Cr(VI), respectively. The highest and lowest Cr(VI) specific removal values were 75.5 mg g(-1) cell by M3 (CFP), and 1.5 mg g(-1) cell by C35 (EC) strains. Eleven Cr(VI) resistant strains were characterized and identified as species of the genera Streptomyces (10) and Amycolatopsis (1). Differences on actinomycete community composition between contaminated and non-contaminated soil were found. This study showed the potential capacity of actinomycetes as tools for Cr(VI) bioremediation.

RNA fingerprinting using RAP-PCR identifies an EBAF homologue mRNA differentially expressed in rat oviduct

As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100 per cent identical to a rat DNA sequence (Accession No. NW_047400)downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process.

RNA fingerprinting using RAP-PCR identifies an EBAF homologue mRNA differentially expressed in rat oviduct

As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100 per cent identical to a rat DNA sequence (Accession No. NW_047400)downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process. (AU)

RNA fingerprinting using RAP-PCR identifies an EBAF homologue mRNA differentially expressed in rat oviduct.

As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100% identical to a rat DNA sequence (Accession No. NW_047400) downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process.

RNA fingerprinting using RAP-PCR identifies an EBAF homologue mRNA differentially expressed in rat oviduct.

As a step towards the identification of genes preferentially expressed in the oviduct during early rat embryo development, we isolated a cDNA fragment (Pr14) by using RNA arbitrarily primed PCR (RAP-PCR), being its expression restricted to oviduct and uterus; its mRNA is mainly expressed in oviduct during late luteal phase and early pregnancy. This fragment is 100

identical to a rat DNA sequence (Accession No. NW_047400) downstream the terminal exon of a Ratturs norvegicus gene (Locus Link Accession No. LOC289316) similar to ebaf (endometrial bleeding-associated factor), a novel member of the Transforming Growth Factor superfamily. Northern analyses showed that this sequence hybridizes with 2.9 kb and 4.1 kb mRNAs in early pregnant rat oviducts. However, only the 4.1 kb mRNA was detected in the oviduct of non-pregnant rats, showing an increase from proestrus to diestrus. The expression of this oviduct-uterus specific mRNA suggests that the products of this gene may play a role in the oviductal reproductive process.