Mobilization, cloning, and sequence determination of a plasmid-encoded polygalacturonase from a phytopathogenic Burkholderia (Pseudomonas) cepacia.

Phytopathogenic strains of Burkholderia cepacia (synonym Pseudomonas cepacia) produce endopolygalacturonase, whereas strains of clinical and soil origin do not. Growth of a phytopathogenic strain (ATCC25416) at elevated temperatures resulted in nonpectolytic derivatives that were either cured of a resident plasmid or contained a plasmid of reduced mass. The resident 200-kb plasmid (pPEC320) in strain ATCC25416 was tagged with Tn5-Mob. The pPEC320::Tn5-Mob (pPEC321) plasmid was mobilized in B. cepacia strains of soil and clinical origin. Transconjugants containing pPEC321 expressed the endopolygalacturonase and showed differential activity on plant tissue. No evidence for self-transfer of pPEC320 or the tagged derivative was observed. A 285-kb cloned fragment from pPEC320 containing the plasmid-borne pehA gene was sequenced and compared to the pehA gene from Erwinia carotovora subsp. carotovora and Ralstonia solanacearum and the polygalacturonase sequence from Lycopersicon esculentum.

Norepinephrine and serotonin release upon impact injury to rat spinal cord.

Microdialysis sampling was used to characterize the release of norepinephrine and serotonin upon impact injury to the rat spinal cord. Increases in extracellular norepinephrine concentrations in response to injury were small and of short duration. In contrast, serotonin concentrations quickly rose 35-90 times following injury and took 30-45 min to return to control levels. Bleeding caused by injury was probably the major source of the increased serotonin levels. Our results allow a role for serotonin in secondary damage upon injury to the spinal cord but suggest that norepinephrine is not a very significant contributor to such damage.

[Post-cesarean necrotizing fasciitis. Bacterial synergism, or mixed infection of the soft tissues?]. / Fascitis necrotizante postcesárea. Sinergismo bacteriano o infección mixta de tejidos blandos?

It has been demonstrated recently that most of the disemminated infections of the dermis, necrotizing fascitis included are due to a mix bacteria infection working sinergistically, demonstrated by clinical studies as well as in experimental animals. Treatment based on wide spectrum antibiotics and extensive debridement of the necrotic tissue performed by a team of several specialists have diminished mortality to 10%. We report a case of necrotizing fasciitis following a cesarean section in the absence of risk factors. We discuss risk factors, classification, etiology, diagnosis and therapy in light of the current knowledge.

The action of two ethyl carbamates on acetylcholinesterase and reproductive organs of Rhipicephalus microplus.

The effects produced by the new synthetic carbamates ethyl-(4-bromophenyl) carbamate and ethyl-(4-chlorophenyl) carbamate on the acetylcholinesterase (AChE) activity, egg structure and reproductive organs of two Rhipicephalus microplus strains were evaluated. Inhibition kinetic parameters showed that the studied carbamates are weak inhibitors and have a low affinity for R. microplus AChE. Histologically, in oocytes from carbamate-treated engorged female ticks, a loss of shape, cytoplasmic vacuoles, decreased chorion deposition, alterations in cytoplasmic granularity and irregular membranes were observed. In oocyte germinal vesicles, a loss of shape, nucleolar fragmentation and membrane alterations with degenerative signs were observed. The ovarian epithelium was vacuolated, flattened, eroded and contained pyknotic nuclei. These alterations were observed from the first day and persisted and increased in severity until day 7 post-treatment. The ovaries from carbamate-treated ticks had fewer stage IV-V oocytes and more stage I-II oocytes. Additionally, eggs produced by the treated ticks had a modified appearance, decreased size, a reduced superficial waxy layer and a loss of viability. The results of this study show that the effects of carbamates on R. microplus were independent of AChE inhibition and show that the morphological alterations in the reproductive organs were due to carbamate actions on the vitellogenesis and viability of the ovarian cells.

Effect of new ethyl and methyl carbamates on Rhipicephalus microplus larvae and adult ticks resistant to conventional ixodicides.

The effects of six new synthetic carbamates on fully engorged females of four Rhipicephalus microplus strains (one reference strain susceptible to conventional ixodicides, two strains multiresistant to ixodicides and one tick field isolate) were compared. In addition, the effect of two other new synthetic carbamates was tested on larvae from the same strains. The first six tested carbamates reduced egg laying and inhibited egg hatching in the four studied strains (P<0.05). Compared with untreated females, the eggs produced by the treated engorged female ticks of all strains had a dark, dry, opaque appearance and were less adherent. The remaining two tested carbamates induced larval mortality in all of the evaluated strains. The three studied R. microplus strains displayed 50% resistance ratios (RR50) of less than 2 when compared to the susceptible reference strain. These results demonstrate that both carbamates with a larvicidal effect and carbamates that inhibit egg laying and embryo development are efficacious against tick strains that are resistant to commercial ixodicides, no cross resistance was observed.

AVPR2 variants and V2 vasopressin receptor function in nephrogenic diabetes insipidus.

BACKGROUND: The AVPR2 gene encodes the type 2 vasopressin receptor, a member of the vasopressin/oxytocin receptor subfamily of G protein-coupled receptors. Disruption of AVPR2 causes X-linked congenital nephrogenic diabetes insipidus (NDI), yet the functional significance of most gene sequence variations found in association with NDI has not been proven. The large number of naturally occurring AVPR2 mutations constitutes a model system for studying the structure-function relationship of G protein-coupled receptors. This analysis can be aided by examining amino acid sequence variation and conservation among evolutionarily disparate members of the subfamily. METHODS: Twenty-five new NDI patients were evaluated by DNA sequencing for mutations in AVPR2. Receptors encoded by eighteen NDI alleles were tested for physiologic signaling activity in response to varying concentrations of arginine vasopressin (AVP) in a sensitive cell culture assay. Seventeen amino acid sequences from the vasopressin/oxytocin receptor subfamily were aligned and conserved residues were identified and correlated with the locations of NDI associated variations. RESULTS: Twenty-four variant alleles were found among the 25 new patients. Thirteen had no prior family history of expressed NDI. All 18 of the NDI-associated AVPR2 alleles tested for function demonstrated diminished response to stimulation with AVP. Twelve failed to respond at all, whereas six signaled only at high AVP concentrations. Evolutionarily conserved residues clustered in the transmembrane domains and in the first and second extracellular loops, and NDI-associated missense mutations appeared mostly in the conserved domains. CONCLUSIONS: Sporadic cases are frequent and they usually represent the X-linked rather than the autosomal form of NDI. Genetic and functional testing can confirm this in individual cases. Mutations in this study affecting ligand binding domains tend to retain partial signaling in vitro, whereas those that introduce a charged residue in a transmembrane domain are inactive. The minimal partial signaling observed in cultured cells is unlikely to correlate with clinically significant urine concentrating ability. Other AVPR2 mutations with milder effects on receptor function probably exist, but may not be expressed clinically as typical NDI.