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.

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.

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.

Papillomaviruses in human skin warts and their incidence in an Argentine population.

Human papillomavirus genomic types present in human warts of an Argentine population were studied. HPV DNA from single warts was obtained using an alkaline extraction procedure that resulted in a clean DNA preparation, which could be analyzed with several endonucleases. This method was used to isolate and insert the HPV DNAs of two genomic types into the Bam HI site of the pBR322 plasmid. Restriction maps of both HPV DNAs were constructed. According to these maps, one of the genomic variations was identical to HPV1a and the other to HPV2a. The incidence of HPV2 and of HPV1 in different types of skin warts was studied by a dot blot hybridization assay. Twenty-two out of 28 common warts were positive for HPV2 and negative for HPV1; four were positive for HPV1 and negative for HPV2 and two were negative for both. Five out of six plantar warts were positive for HPV1, and one was negative for both. Three out of seven filiform warts were positive for HPV2, three were positive for both probes, and one was negative for both. Southern blot analysis of HPV2 positive samples indicated that 80% were HPV2a and 20% another subtype not yet characterized. All plantar warts contained HPV1a. Msp I/Hpa II restriction analysis confirms previous results indicating that HPV1a DNA is partially methylated, while no evidence of methylation was found for HPV2a 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.

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.

Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane.

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.

Adherence of multiresistant strains of Klebsiella pneumoniae to cerebrospinal fluid shunts: correlation with plasmid content.

A nosocomial multiresistant Klebsiella pneumoniae strain (KMD01) isolated from a patient with an infected ventriculoperitoneal (V-P) shunt was found to contain three plasmids of mol. wts (10(6)) c. 85, 50 and 2.4. A derivative isogenic strain (KMD11) carrying only the plasmids of mol. wts (10(6)) 50 and 2.4 was obtained spontaneously by plating the parent strain. The absence of the plasmid of mol. wt 85 X 10(6) in strain KMD11 correlated with an increased adherence to V-P catheters and glass surfaces, as well as autoagglutination in minimal medium. Bacterial cells containing the whole set of plasmids (strain KMD01) also showed the incorporation into the outer membrane of a new polypeptide (mol. wt, c. 41 X 10(3)), when grown in minimal medium. The presence of this polypeptide correlated with absence of autoagglutination, as shown by strain KMD01 under these cultural conditions. These data suggest that the cell-surface characteristics in K. pneumoniae may be affected by the plasmid content of the strain. Since nosocomial strains of K. pneumoniae usually contain one or more plasmids, and strains easily exchange these extrachromosomal elements, it seems reasonable to speculate that new variants with higher V-P shunt colonisation effectiveness, like the one described in this work, may also evolve in nature.

An outbreak of multiply resistant Pseudomonas aeruginosa in a neonatal unit: plasmid pattern analysis.

An outbreak of infection due to multiply resistant Pseudomonas aeruginosa occurred from March to April 1986 in a neonatal unit. Affected neonates were receiving ventilation support and the mortality rate was high. Plasmid analysis and antibiograms indicated that the outbreak was due to a single strain. A survey of bacteria isolated from respirators, potable water and hands of personnel working in the unit failed to recover the outbreak strain. Lack of sterilization of respirators and overcrowding were considered to be the causes of the outbreak and reinforcement of the importance of aseptic techniques helped in its termination.

Treatment of recurrent common warts with one low dose of intralesional natural human leukocyte interferon alpha.

Nine patients with recurrent and long lasting common warts were treated with intralesional Hu-IFN-alpha. The schedule was a single dose per wart, ranged between 10(5) and 2 x 10(5) IU. Placebo was also administered in 3 of these patients. Complete remission was observed in 7 of the 9 patients. The pattern of warts involution and the possible interferon mechanism of action are discussed. A significant pain relief, produced by interferon injection was observed in the patients with plantar warts and in one patient with subungueal wart.

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.

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.

[Development of molecular probes for the diagnosis of mycobacteria]. / Construcción de sondas moleculares para el diagnóstico de micobacterias.

The presence of a repetitive sequence in Mycobacterium bovis DNA was demonstrated. This sequence was also found in M. tuberculosis DNA but was absent in M. kansasii, M. flavescens, M. fortuitum, M. vaccae, M. leprae, M. phlei, M. smegmatis and M. marinum. This repetitive sequence was found to be polymorphic in M. tuberculosis but not in BCG. Dot hybridization analysis suggested that the repetitive sequence could be useful in the identification of pathogenic Mycobacterium in clinical samples.