Molecular Characterization of Plasmid-Mediated Oxytetracycline Resistance in Aeromonas salmonicida

Using broth conjugation, we found that 19 of 29 (66%) oxytetracycline (OT)-resistant isolates of Aeromonas salmonicida transferred the OT resistance phenotype to Escherichia coli. The OT resistance phenotype was encoded by high-molecular-weight R-plasmids that were capable of transferring OT resistance to both environmental and clinical isolates of Aeromonas spp. The molecular basis for antibiotic resistance in OT-resistant isolates of A. salmonicida was determined. The OT resistance determinant from one plasmid (pASOT) of A. salmonicida was cloned and used in Southern blotting and hybridization experiments as a probe. The determinant was identified on a 5.4-kb EcoRI fragment on R-plasmids from the 19 OT-resistant isolates of A. salmonicida. Hybridization with plasmids encoding the five classes (classes A to E) of OT resistance determinants demonstrated that the OT resistance plasmids of the 19 A. salmonicida isolates carried the class A resistance determinant. Analysis of data generated from restriction enzyme digests showed that the OT resistance plasmids were not identical; three profiles were characterized, two of which showed a high degree of homology.Aeromonas salmonicida is the causative agent of furunculosis, an economically important disease of salmonids (5). Control of this disease in aquaculture may be by prophylaxis (i.e., vaccination) (11) or by chemotherapy with a wide variety of antimicrobial compounds (5). Initially when sulfonamides were administered as food additives, they were successful (12). Subsequently, the usefulness of oxytetracycline (OT) was reported (29), and this antibiotic is still used extensively for control of furunculosis (5, 28). However, continued and widespread use of antibiotics has led to the development of resistant strains (3, 8, 15, 23). Moreover, plasmids encoding antibiotic resistance (R-plasmids) have been isolated from A. salmonicida (2, 15, 26, 27). A second generation of 4-quinolones–fluoroquinolones, notably enrofloxacin and sarofloxacin, effectively inhibits the pathogen and offers promise for the future since plasmid-encoded resistance to these compounds has not been described (7, 14, 19). However, mutational resistance to this class of compounds can develop in A. salmonicida (8, 21, 22, 32).As noted previously (28), it is difficult to make any definite conclusions about the impact of OT usage in aquaculture because of the methodological differences described in the literature. Transferable R-plasmids encoding resistance to tetracycline in A. salmonicida were described in 1971 (2, 31); subsequently, studies indicated that the frequency of OT-resistant strains of A. salmonicida was increasing. In a survey of 444 A. salmonicida isolates collected from Scottish salmon farms during 1988 to 1991, 53% of the isolates were resistant to OT (23). Using a random subsample of these isolates, researchers determined that 27% contained R-plasmids which could be transferred to Escherichia coli K-12 by conjugation (15), although no information concerning the molecular basis for the resistance was provided. Whereas there is no doubt that the results of such studies have value, it is necessary to clarify the situation with regard to the spread of R-plasmids encoding OT resistance within A. salmonicida populations. Only through precise molecular characterization of the genes encoding OT resistance and the plasmids that carry these resistance determinants will it become clear if aquaculture is facing a real threat from the use of antibiotics. To address this issue, a collection of OT-resistant isolates was used in experiments that examined the molecular basis for OT resistance and the potential environmental impact of the R-plasmids of these isolates.     

Ca<Superscript>2+</Superscript> signalling in cardiovascular disease: the role of the plasma membrane calcium pumps

The plasma membrane calcium ATPases (PMCA) are a family of genes which extrude Ca²⁺ from the cell and are involved in the maintenance of intracellular free calcium levels and/or with Ca²⁺ signalling, depending on the cell type. In the cardiovascular system, Ca²⁺ is not only essential for contraction and relaxation but also has a vital role as a second messenger in signal transduction pathways. A complex array of mechanisms regulate intracellular free calcium levels in the heart and vasculature and a failure in these systems to maintain normal Ca²⁺ homeostasis has been linked to both heart failure and hypertension. This article focuses on the functions of PMCA, in particular isoform 4 (PMCA4), in the heart and vasculature and the reported links between PMCAs and contractile function, cardiac hypertrophy, cardiac rhythm and sudden cardiac death, and blood pressure control and hypertension. It is becoming clear that this family of calcium extrusion pumps have essential roles in both cardiovascular health and disease.     

Adaptation for horizontal transfer in a homing endonuclease

Selfish genes of no function other than self-propagation are susceptible to degeneration if they become fixed in a population, and regular transfer to new species may be the only means for their long-term persistence. To test this idea we surveyed 24 species of yeast for VDE, a nuclear, intein-associated homing endonuclease gene (HEG) originally discovered in Saccharomyces cerevisiae. Phylogenetic analyses show that horizontal transmission has been a regular occurrence in its evolutionary history. Moreover, VDE appears to be specifically adapted for horizontal transmission. Its 31-bp recognition sequence is an unusually well-conserved region in an unusually well-conserved gene. In addition, the nine nucleotide sites most critical for homing are also unusually well conserved. Such adaptation for horizontal transmission presumably arose as a consequence of selection, both among HEGs at different locations in the genome and among variants at the same location. The frequency of horizontal transmission must therefore be a key feature constraining the distribution and abundance of these genes.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Characterizing proteolytic cleavage site activity using bio-basis function neural networks

MOTIVATION: In protein chemistry, proteomics and biopharmaceutical development, there is a desire to know not only where a protein is cleaved by a protease, but also the susceptibility of its cleavage sites. The current tools for proteolytic cleavage prediction have often relied purely on regular expressions, or involve models that do not represent biological data well. RESULTS: A novel methodology for characterizing proteolytic cleavage site activities has been developed, which incorporates two fundamental features: activity class prediction and the use of an amino acid similarity matrix for (non-parametric) neural learning. The first solved the problem of predicting proteolytic efficiency. The second significantly improved the robustness in prediction and reduced the time complexity for learning. This study shows that activity class prediction is successful when applying this methodology to the prediction and characterization of Trypsin cleavage sites and the prediction of HIV protease cleavage sites. AVAILABILITY: Requests for software and data should be made respectively to Dr Zheng Rong Yang and Miss Rebecca Thomson.     

Androgens drive divergent responses to salt stress in male versus female rat kidneys

Dahl-Iwai (DI) salt-sensitive rats were studied using microarrays to identify sex-specific differences in the kidney, both basal differences and differences in responses to a high-salt diet. In DI rat kidneys, gene expression profiles demonstrated inflammatory and fibrotic responses selectively in females. Gonadectomy of DI rats abrogated sex differences in gene expression. Gonadectomized female and gonadectomized male DI rats both responded to high salt with the same spectrum of gene expression changes as intact female DI rats. Androgens dominated the sex-selective responses to salt. Several androgen-responsive genes with roles potentiating the differential responses to salt were identified, including increased male expression of angiotensin-vasopressin receptor and prolactin receptor, decreased 5 alpha-reductase, and mixed increases and decreases in expression of Cyp4a genes that can produce eicosanoid hormones. These sex differences potentiate sodium retention by males and increase kidney function during gestation in females.     

Chromium is not an essential trace element for mammals: effects of a “low-chromium�?diet

Chromium was proposed to be an essential trace element over 50?years ago and has been accepted as an essential element for over 30?years. However, the studies on which chromium’s status are based are methodologically flawed. Whether chromium is an essential element has been examined for the first time in carefully controlled metal-free conditions using a series of purified diets containing various chromium contents. Male Zucker lean rats were housed in specially designed metal-free cages for 6?months and fed the AIN-93G diet with no added chromium in the mineral mix component of the diet, the standard AIN-93G diet, the standard AIN-93G diet supplemented with 200?μg?Cr/kg, or the standard AIN-93G diet supplemented with 1,000?μg?Cr/kg. The chromium content of the diet had no effect on body mass or food intake. Similarly, the chromium content of the diet had no effect on glucose levels in glucose tolerance or insulin tolerance tests. However, a distinct trend toward lower insulin levels under the curve after a glucose challenge was observed with increasing chromium content in the diet; rats on the supplemented AIN-93G diets had significantly lower areas (P?<?0.05) than rats on the low-chromium diet. The studies reveal that a diet with as little chromium as reasonably possible had no effect on body composition, glucose metabolism, or insulin sensitivity compared with a chromium-“sufficient??diet. Together with the results of other recent studies, these results clearly indicate that chromium can no longer be considered an essential element.     

Conditional Survival in de novo Metastatic Urothelial Carcinoma

Background

Second-line therapy is frequently utilized for metastatic urothelial carcinoma, but there are limited data to guide this approach. While an assessment of overall survival based on registry data may not capture the impact of second- and third-line therapies on clinical outcome, this may be reflected in relative conditional survival (RCS).

Methods

Patients with stage IV urothelial carcinoma diagnosed from 1990–2010 were identified from the Surveillance, Epidemiology and End Results (SEER) dataset. The association of clinicopathologic variables with disease specific survival (DSS) was explored through univariate and multivariate analyses. DSS in subgroups divided by time period (1990–2000 v 2001–2010) was compared using the Kaplan-Meier method and log-rank test. One-year RCS at annual landmarks up to 5 years was compared in subgroups divided by time period.

Results

Of 261,987 patients diagnosed with urothelial carcinoma from 1990–2010, 3,110 patients met criteria for the current analysis. Characteristics of patients diagnosed between 1990 and 2000 (n = 810) and 2001 to 2010 (n = 2,300) were similar and there was no significant difference in DSS between the two groups. On multivariate analysis, older age (age ≥ 80) was associated with shorter DSS (HR 1.79, 95%CI 1.48–2.15), but no association was found between time period of diagnosis and outcome. One-year RCS improved substantially through successive annual landmarks up to 5 years, but no differences were seen in subgroups divided by time of diagnosis.

Conclusions

No difference in RCS was observed amongst patients with stage IV urothelial carcinoma diagnosed from 1990–2000 and 2001–2010. A lack of difference in RCS (more so than cumulative DSS) may reflect a lack of progress in salvage therapies for the disease.     

Characterization of a caveolin‐1 mutation associated with both pulmonary arterial hypertension and congenital generalized lipodystrophy

Congenital generalized lipodystrophy (CGL) and pulmonary arterial hypertension (PAH) have recently been associated with mutations in the caveolin‐1 ( CAV1 ) gene, which encodes the primary structural protein of caveolae. However, little is currently known about how these CAV1 mutations impact caveolae formation or contribute to the development of disease. Here, we identify a heterozygous F160X CAV1 mutation predicted to generate a C‐terminally truncated mutant protein in a patient with both PAH and CGL using whole exome sequencing, and characterize the properties of CAV1 , caveolae‐associated proteins and caveolae in skin fibroblasts isolated from the patient. We show that morphologically defined caveolae are present in patient fibroblasts and that they function in mechanoprotection. However, they exhibited several notable defects, including enhanced accessibility of the C‐terminus of wild‐type CAV1 in caveolae, reduced colocalization of cavin‐1 with CAV1 and decreased stability of both 8S and 70S oligomeric CAV1 complexes that are necessary for caveolae formation. These results were verified independently in reconstituted CAV1 ^?/? mouse embryonic fibroblasts. These findings identify defects in caveolae that may serve as contributing factors to the development of PAH and CGL and broaden our knowledge of CAV1 mutations associated with human disease.