Oligodeoxynucleotide IMT504 induces a marked recovery in a streptozotocin-induced model of diabetes in rats: correlation with an early increase in the expression of nestin and neurogenin 3 progenitor cell markers.

AIMS/HYPOTHESIS: IMT504 is an oligonucleotide that promotes tissue repair in bone injury and neuropathic pain models by stimulating progenitor cells. Here we evaluated the effect of IMT504 on the recovery of islet function in a streptozotocin (STZ)-induced model of diabetes in the rat. METHODS: Male Sprague-Dawley rats were injected with STZ (60 mg/kg, i.p., day 1) or citrate buffer (Control). Animals with glycaemia between 11 and 20 mmol/l on day 4 were injected with IMT504 (4 mg/animal in saline, s.c., STZ-IMT504) or with saline (STZ-Saline) for 10 days. Glycaemia and water and food intake were recorded for 33 days. Intraperitoneal glucose tolerance tests (IPGTTs) were performed on day 30. On day 35, overnight-fasted animals were killed and blood samples and pancreases collected for hormonal and histological studies. A second group of STZ-IMT504 rats was killed, together with Control and STZ-Saline rats, after two consecutive days of blood glucose decreases after the beginning of IMT504 treatment. Pancreases were collected and proliferating cell nuclear antigen (PCNA), nestin and neurogenin 3 (NGN3) detected by immunohistochemistry. RESULTS: IMT504 greatly improved blood glucose and food and water intakes in STZ-IMT504 rats by day 8, as well as IPGTTs on day 30. Significant increases in islet number and beta cell content were observed in STZ-IMT504 rats (day 33). Furthermore, after two to five IMT504 injections, blood glucose decreased, and an increase in pancreatic nestin (mainly in endothelial cells), PCNA and NGN3 production (in islets) was observed in STZ-IMT504 rats. CONCLUSIONS/INTERPRETATION: IMT504 induced a marked recovery of STZ-induced diabetes that correlated with early production of progenitor cell markers, such as nestin and NGN3.

Inducción de fenotipo presentador de antígenos y apoptosis en células de leucemia linfática crónica B incubadas con oligodeoxinucleótidos sintéticos con motivos PyNTTTTGT (IMT504) / Phenotype´s induction presenter of antigens and apoptosis in cells of incubated lymphatic leukemia chronic B with oligodeoxynucleotides synthetics with PyNTTTTGT(IMT504) reasons

Introducción: El sistema inmune no es capaz de reconocer los cambios en células transformadas por mutaciones en la leucemia linfática crónica de estirpe B (LLC-B). Su incubación con linfocitos autólogos no induce citotoxicidad. Puede deberse a la ausencia de linfocitos T citotóxicos o a la incapacidad de las células de LLC en expresar moléculas coestimulatorias (CD80, CD86), a pesar de tener expresión de HLA clase I y II. El ADN bacteriano y los ODNs que contienen motivos PyNTTrrGT, CpG y otros motivos, pueden activar a los monocitos, las células dendríticas y los linfocitos B. Aparte, algunos ODNs tienen efectos directos sobre las células LLC-B. Material y métodos: se incubaron células leucémicas provenientes de 20 pts. con LLC-B con 3 diferentes ODNs: a) IMT504, con motivos PyNTTrrGT b) 2006 con motivos CpG y c) ODN inactivo de control. Previo y posterior a la incubación se efectuaron determinaciones de CD80, CD86, CD40, MHC clase I y II, CD5, CD19 y CD20 por citometría de flujo. Además se determinó (por medio de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la expresión de CD80, CD86 y CD40 en las células incubadas con IMT504 y 2006. Además las células LLC-B expresaron significativamente mayor cantidad de Anexina v. La morfología de las células en cultivo a largo plazo mostró las características de apoptosis. Los estudios efectuados con el ODN de control no mostraron ninguna de las características descritas. Conclusión: La incubación de ODN con motivos PyNTTTTGT (IMT504) y con motivos CpG (2006) induce en las células de LLC-B un fenotipo considerado de células presentadoras de antígenos, y además apoptosis. Perspectiva: En otros estudios preclínicos y clínicos los ODNs han demostrado muy baja toxicidad, lo que permitiria efectuar estudios de Fase I/II en LLC-B

Inducción de fenotipo presentador de antígenos y apoptosis en células de leucemia linfática crónica B incubadas con oligodeoxinucleótidos sintéticos con motivos PyNTTTTGT (IMT504) / Phenotype s induction presenter of antigens and apoptosis in cells of incubated lymphatic leukemia chronic B with oligodeoxynucleotides synthetics with PyNTTTTGT(IMT504) reasons

Introducción: El sistema inmune no es capaz de reconocer los cambios en células transformadas por mutaciones en la leucemia linfática crónica de estirpe B (LLC-B). Su incubación con linfocitos autólogos no induce citotoxicidad. Puede deberse a la ausencia de linfocitos T citotóxicos o a la incapacidad de las células de LLC en expresar moléculas coestimulatorias (CD80, CD86), a pesar de tener expresión de HLA clase I y II. El ADN bacteriano y los ODNs que contienen motivos PyNTTrrGT, CpG y otros motivos, pueden activar a los monocitos, las células dendríticas y los linfocitos B. Aparte, algunos ODNs tienen efectos directos sobre las células LLC-B. Material y métodos: se incubaron células leucémicas provenientes de 20 pts. con LLC-B con 3 diferentes ODNs: a) IMT504, con motivos PyNTTrrGT b) 2006 con motivos CpG y c) ODN inactivo de control. Previo y posterior a la incubación se efectuaron determinaciones de CD80, CD86, CD40, MHC clase I y II, CD5, CD19 y CD20 por citometría de flujo. Además se determinó (por medio de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la detección de fosfatidilserina por la unión ala Anexina V) la capacidad de inducir la apotosis de las células transformadas, y se estudió la morfología de las células en cultivo. Resultados: Se observó el aumento de la expresión de CD80, CD86 y CD40 en las células incubadas con IMT504 y 2006. Además las células LLC-B expresaron significativamente mayor cantidad de Anexina v. La morfología de las células en cultivo a largo plazo mostró las características de apoptosis. Los estudios efectuados con el ODN de control no mostraron ninguna de las características descritas. Conclusión: La incubación de ODN con motivos PyNTTTTGT (IMT504) y con motivos CpG (2006) induce en las células de LLC-B un fenotipo considerado de células presentadoras de antígenos, y además apoptosis. Perspectiva: En otros estudios preclínicos y clínicos los ODNs han demostrado muy baja toxicidad, lo que permitiria efectuar estudios de Fase I/II en LLC-B(AU)

Birth of the domains Bacteria, Archaea and Eucarya and of major taxa within them: a hypothesis.

A hypothesis to explain how the birth of the Bacteria, Archaea and Eucarya domains and of major taxa within them took place is presented. It is proposed that the birth of each domain was an independent event consisting in the genetic isolation of a particular cell from a very diverse pool of "primitive cells". Cells within this pool have a dynamic pattern of cell fusion followed by mostly illegitimate DNA recombination. It is postulated that genetic isolation was achieved: a) by evolution of the peptidoglycan layer in Bacteria, b) by evolution of a glycoproteic cell wall in Archaea, and c) by evolution of the nuclear membrane in Eucarya. It is also postulated that, within each domain, branching was a consequence of sporadic events of fusion between two cells of different phylogenetic lineages, followed by mostly illegitimate DNA recombination and cell wall regeneration. The two fusing cells may have belonged to the same domain, to different domains or even one may have belonged to one of the domains and the other to the pool of "primitive cells". In this last case, new complex phenotypes, previously absent from all the domains, were suddenly introduced in one of them (e.g.: photosynthesis in Bacteria, methanogenesis in Archaea). A corollary of this theory is that genes should have a phylogenetic tree with defined nodes while organisms are characterized by discontinuities instead of nodes.

Birth of the domains Bacteria, Archaea and Eucarya and of major taxa within them: a hypothesis

A hypothesis to explain how the birth of the Bacteria, Archaea and Eucarya domains and of major taxa within them took place is presented. It is proposed that the birth of each domain was an independent event consisting in the genetic isolation of a particular cell from a very diverse pool of [quot ]primitive cells[quot ]. Cells within this pool have a dynamic pattern of cell fusion followed by mostly illegitimate DNA recombination. It is postulated that genetic isolation was achieved: a) by evolution of the peptidoglycan layer in Bacteria, b) by evolution of a glycoproteic cell wall in Archaea, and c) by evolution of the nuclear membrane in Eucarya. It is also postulated that, within each domain, branching was a consequence of sporadic events of fusion between two cells of different phylogenetic lineages, followed by mostly illegitimate DNA recombination and cell wall regeneration. The two fusing cells may have belonged to the same domain, to different domains or even one may have belonged to one of the domains and the other to the pool of [quot ]primitive cells[quot ]. In this last case, new complex phenotypes, previously absent from all the domains, were suddenly introduced in one of them (e.g.: photosynthesis in Bacteria, methanogenesis in Archaea). A corollary of this theory is that genes should have a phylogenetic tree with defined nodes while organisms are characterized by discontinuities instead of nodes.

Birth of the domains Bacteria, Archaea and Eucarya and of major taxa within them: a hypothesis.

A hypothesis to explain how the birth of the Bacteria, Archaea and Eucarya domains and of major taxa within them took place is presented. It is proposed that the birth of each domain was an independent event consisting in the genetic isolation of a particular cell from a very diverse pool of [quot ]primitive cells[quot ]. Cells within this pool have a dynamic pattern of cell fusion followed by mostly illegitimate DNA recombination. It is postulated that genetic isolation was achieved: a) by evolution of the peptidoglycan layer in Bacteria, b) by evolution of a glycoproteic cell wall in Archaea, and c) by evolution of the nuclear membrane in Eucarya. It is also postulated that, within each domain, branching was a consequence of sporadic events of fusion between two cells of different phylogenetic lineages, followed by mostly illegitimate DNA recombination and cell wall regeneration. The two fusing cells may have belonged to the same domain, to different domains or even one may have belonged to one of the domains and the other to the pool of [quot ]primitive cells[quot ]. In this last case, new complex phenotypes, previously absent from all the domains, were suddenly introduced in one of them (e.g.: photosynthesis in Bacteria, methanogenesis in Archaea). A corollary of this theory is that genes should have a phylogenetic tree with defined nodes while organisms are characterized by discontinuities instead of nodes.

Aeromonas salmonicida subsp. pectinolytica subsp. nov., a new pectinase-positive subspecies isolated from a heavily polluted river.

Aeromonas strains which phenotypically and genetically belong to the Aeromonas salmonicida species but that according to their phenotypic properties constitute a new subspecies have been isolated from the water of a heavily polluted river, the Matanza river, situated near the central district of Buenos Aires city. These strains were ascribed to the A. salmonicida species by using 65 biochemical tests and by DNA-DNA hybridization. They produce acid from -sorbitol, an unusual biochemical property found in a few members of the A. salmonicida species. They also utilize urocanic acid and do not ferment L-rhamnose or utilize LD-lactate, and are elastase- and gluconate-negative. The DNA relatedness was over 70%, the current limit accepted for the phylogenetic definition of a species, to the described A. salmonicida subspecies and nearly 100% within the new group of Aeromonas strains. Phenotypic differentiation from other A. salmonicida subspecies was readily achieved using the following characteristics: growth at 37 degrees C, melanin production, indole and Voges-Proskauer assays, growth on KCN broth, mannitol and sucrose fermentation and gas from glucose. A remarkable property of the strains of the new group was their ability to degrade polypectate, an unusual feature among Aeromonas species in general. The complete 16S rRNA gene of one strain of the new group was sequenced. Comparison with rDNA sequences of Aeromonas members available in databases revealed a close relationship between this strain and strains belonging to A. salmonicida subsp. salmonicida, masoucida and achromogenes, in agreement with the biochemical data. Since the new A. salmonicida strains constitute a tight genomic group that can be identified by phenotypic properties it was concluded that they represent a new subspecies for which the name Aeromonas salmonicida subsp. pectinolytica is proposed. The type strain of A. salmonicida subsp. pectinolytica is 34melT (= DSM 12609T).

A plasmid vector for isolation of strong promoters in Escherichia coli.

In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.

Coprothermobacter platensis sp. nov., a new anaerobic proteolytic thermophilic bacterium isolated from an anaerobic mesophilic sludge.

A new anaerobic, proteolytic, moderately thermophilic bacterium, strain 3RT, was isolated from a methanogenic mesophilic reactor treating protein-rich wastewater. The cells were Gram-negative, non-spore-forming, non-motile rods. The DNA base composition was 43 mol% G + C. The optimum pH and temperature for growth were 7.0 and 55 degrees C respectively. The bacterium fermented gelatin, casein, bovine albumin, peptone and yeast extract. Glucose, fructose, sucrose, maltose and starch were poorly fermented. The major fermentation products from glucose were acetate, CO2 and H2 and, from gelatin, propionate was also detected. Growth on glucose was stimulated by thiosulfate, which was reduced to sulfide. Sulfate and nitrate were not reduced. 16S rRNA gene analysis revealed that the isolated bacterial strain was phylogenetically related to Coprothermobacter proteolyticus (96.3% sequence similarity), the only known species within the genus. DNA-DNA hybridization analysis demonstrated a very low level of homology, indicating that the isolated strain and C. proteolyticus were not related at species level. Therefore, it is proposed to classify the described strain in the genus Coprothermobacter as a new species, Coprothermobacter platensis. The type strain of C. platensis is strain 3RT (= DSM 11748T).

Kluyvera bacteriophage Kvp1: a new member of the Podoviridae family phylogenetically related to the coliphage T7.

A DNA containing bacteriophage, Kvp1, was isolated from the water of a very polluted river, the Matanza river, near the central district of Buenos Aires City. This bacteriophage infects bacteria belonging to the Kluyvera cryocrescens species (strain 21 g) isolated from the same river. Kvp1 is a lytic bacteriophage and its propagation characteristics are: burst size 30, latent period 13 min and rise period 10 min. Morphologically, Kvp1 is a small icosahedral bacteriophage, 59.1 nm in diameter, which possesses a short wedge-shaped tail. Its buoyant density in ClCs is 1.517 g/cm3. Kvp1 DNA is linear, double stranded and approximately 40,000 bp in size. The viral particle is composed of at least nine proteins. SDS-PAGE patterns of these proteins and of those produced during the host infection, in addition to its morphological and genomic characteristics, suggested that Kvp1 is similar to the coliphage T7. Molecular cloning, sequencing and computer-assisted analysis of Kvp1 DNA fragments confirmed the relationship to the coliphage. Taking this into account, the partial sequence of the phage RNA polymerase was used to construct phylogenetic relationships between Kvp1 and other related phages. To our knowledge, Kvp1 is the first bacteriophage described which uses as host a member of the Kluyvera bacterial genus.

Binding of nuclear factors to a satellite DNA of retroviral origin with marked differences in copy number among species of the rodent Ctenomys.

The major satellite DNA of the subterranean rodent Ctenomys, named RPCS, contains several consensus sequences characteristic of the U3 region of retroviral long terminal repeats (LTRs), such as a polypurine tract, CCAAT boxes, binding sites for the CCAAT/enhancer-binding protein (C/EBP), a TATA box and putative polyadenylation signals. RPCS presents an enormous variation in abundance between species of the same genus: while C. australis or C. talarum have approximately 3 x 10(6) copies per genome, C. opimus has none. A sequence (RPCS-I) with identity to the SV40-enhancer core element, present in all the repeating units of the satellite is specifically protected in DNase I footprintings. Competitions of band-shift assays with different transcription factor binding sites indicate that binding to RPCS-I is specific and involves CCAAT proteins related to NF-1, but not to C/EBP. By the use of quantitative protein/DNA binding assays we determined that, despite of their conspicuous difference in RPCS copy number, C. talarum and C. opimus have equivalent amounts and identical quality of RPCS-binding proteins. These results are consistent with the observation, by in situ hybridization, that RPCS is clustered in heterochromatic regions, where it might have restricted accessibility to transcription factors in vivo. This is the first report of the binding of transcription factors to a satellite DNA of retroviral origin.

Retroviral-like features in the monomer of the major satellite DNA from the South American rodents of the genus Ctenomys.

It is well known that uninfected mammalian cells contain DNA sequences which are closely related to retroviral genomic segments. However, these sequences seldom (if ever) have been found associated to highly repetitive (satellite) DNA. RPCS is a 348 bp monomer of a major satellite DNA from the South American rodents of the genus Ctenomys. It was found that RPCS contains several elements which are typical of the U3 region of retroviral LTRs. These elements are: a) a polypurine tract; b) two enhancer core sequences; c) two NF1 binding sites; d) two C/EBP binding sites; e) two CCAAT-motifs; f) a TATA box, and g) two putative polyadenylation motifs. Furthermore, the relative positions of these elements are as in the U3 retroviral regions.

Effect of DNA concentration on recombinant plasmid recovery after blunt-end ligation.

We describe conditions for optimal recovery of recombinant plasmids after blunt-end ligation. It was found that one of the most critical parameters of the blunt-end ligation reaction is total DNA concentration (vector plus incoming DNA). This concentration was optimal in the range of 1-5 micrograms/ml of reaction mixture. Concentrations larger than 10 micrograms/ml result in strong inhibition. The optimal molar relationship between incoming DNA and vector was found to be 1 or less. Under these conditions, using dephosphorylated vector, recombinants are generated at a frequency of 10(6) colonies per microgram of insert, provided that transforming efficiency is about 5 x 10(7) colonies per microgram of plasmid DNA.

A staphylococcal plasmid that replicates and expresses ampicillin, gentamicin and amikacin resistance in Escherichia coli.

Plasmid pPG1 from Staphylococcus aureus coding for ampicillin (Apr), gentamicin (Gmr) and amikacin (Akr) resistance was transformed into Escherichia coli. Transformation efficiency was about 2 x 10(3) transformants/micrograms of plasmid DNA. The plasmids present in the E. coli transformants were identical to pPG1 according to their restriction patterns. The copy number of pPG1 was estimated to be at least 20-times less in E. coli than in S. aureus. The minimal inhibitory concentrations (MICs) for Ap and Gm were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus. pPG1 was maintained in the E. coli transformants for at least 80 generations at 37 degrees C without antibiotic selection pressure.

Efficient expression of a Trypanosoma cruzi antigen in Escherichia coli and Staphylococcus aureus and its rapid purification.

A Trypanosoma cruzi antigen gene was closed into a fusion vector based on the IgG binding domain of Staphylococcus aureus protein A. This vector transformed into Escherichia coli or Staphylococcus aureus and produced about 12 mg fusion protein/l culture. In E. coli, the product remained intracellular while in S. aureus it was excreted into the growth medium. The hybrid protein was purified by IgG Sepharose affinity chromatography. The presence of a cleavage site for enterokinase between protein A and the T. cruzi antigen in the fusion protein allowed the efficient release of the unfused antigen by enzymatic treatment. Further affinity chromatography through IgG Sepharose resulted in the production of the T. cruzi antigen free of protein A.