Characterization of transformation-deficient mutants of Acinetobacter calcoaceticus

Three Acinetobacter calcoaceticus transformation-deficient mutants, obtained by insertional mutagenesis with the nptll gene, have been characterized physiologically. One mutant (AAC211) was found to be completely transformation deficient, while two others, AAC213 and AAC214, were severely impaired in transformation efficiency (100-1000 times lower than the wild type). The latter applied to both chromosomal as well as plasmid DNA. Analysis of the chromosomal DNA fragments flanking the nptll gene in the mutants showed that mutants AAC213 and AAC214 had an insertion of the nptll gene in the same chromosomal region, but that they were the result of two independent mutational events, whereas the insertion in mutant AAC211 was at a different position. None of the three mutants showed phenotypic or genotypic characteristics typical of a RecA-deficient strain.     

Intramolecular masking of the nuclear location signal and dimerization domain in the precursor for the p50 NF-kappa B subunit.

We show that the non-DNA-binding precursor for the p50 subunit (p110), like NF-kappa B, is subject to control of nuclear uptake. In contrast to p50, p110 was excluded from nuclei and unable to associate detectably with p50 or p65 NF-kappa B subunits. The nuclear location signal in the N-terminal half of p110 was not accessible for monospecific antibodies. Removal of only 191 amino acids from the C-terminus of p110 restored antibody accessibility as well as nuclear uptake. The C-terminal half of p110, which is linked to the p50 portion via a glycine-rich hinge, could also noncovalently bind to p50. This helps to explain why p50, after cleavage of the precursor in intact cells, was still retained in an inactive form in the cytoplasm. Our study describes a novel mechanism of nuclear uptake control by masking of a nuclear location signal through a remote domain within a precursor molecule.     

Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling

Summary Collagens of most connective tissues are subject to continuous remodelling and turnover, a phenomenon which occurs under both physiological and pathological conditions. Degradation of these proteins involves participation of a variety of proteolytic enzymes including members of the following proteinase classes: matrix metalloproteinases (e.g. collagenase, gelatinase and stromelysin), cysteine proteinases (e.g. cathepsin B and L) and serine proteinases (e.g. plasmin and plasminogen activator). Convincing evidence is available indicating a pivotal role for matrix metalloproteinases, in particular collagenase, in the degradation of collagen under conditions of rapid remodelling, e.g. inflammation and involution of the uterus. Under steady state conditions, such as during turnover of soft connective tissues, involvement of collagenase has yet to be demonstrated. Under these circumstances collagen degradation is likely to take place particularly within the lysosomal apparatus after phagocytosis of the fibrils. We propose that this process involves the following steps: (i) recognition of the fibril by membranebound receptors (integrins?), (ii) segregation of the fibril, (iii) partial digestion of the fibril and/or its surrounding noncollagenous proteins by matrix metalloproteinases (possibly gelatinase), and finally (iv) lysosomal digestion by cysteine proteinases, such as cathepsin B and/or L. Modulation of this pathway is carried out under the influence of growth factors and cytokines, including transforming growth factor β and interleukin 1α.     

Molecular evolution and host adaptation of Bordetella spp.: phylogenetic analysis using multilocus enzyme electrophoresis and typing with three insertion sequences.

A total of 188 Bordetella strains were characterized by the electrophoretic mobilities of 15 metabolic enzymes and the distribution and variation in positions and copy numbers of three insertion sequences (IS). The presence or absence of IS elements within certain lineages was congruent with estimates of overall genetic relationships as revealed by multilocus enzyme electrophoresis. Bordetella pertussis and ovine B. parapertussis each formed separate clusters, while human B. parapertussis was most closely related to IS1001-containing B. bronchiseptica isolates. The results of the analysis provide support for the hypothesis that the population structure of Bordetella is predominantly clonal, with relatively little effective horizontal gene flow. Only a few examples of putative recombinational exchange of an IS element were detected. Based on the results of this study, we tried to reconstruct the evolutionary history of different host-adapted lineages.     

Construction and Characterization of a 10-Genome Equivalent Yeast Artificial Chromosome Library for the Laboratory Rat,Rattus norvegicus

Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescencein situhybridization were found to be chimeric, indicating a proportion of about 20–30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species.     

Evolutionary history shapes patterns of mutualistic benefit in Acacia–rhizobial interactions

The ecological and evolutionary factors that drive the emergence and maintenance of variation in mutualistic benefit (i.e., the benefits provided by one partner to another) in mutualistic symbioses are not well understood. In this study, we evaluated the role that host and symbiont phylogeny might play in determining patterns of mutualistic benefit for interactions among nine species of Acacia and 31 strains of nitrogen‐fixing rhizobial bacteria. Using phylogenetic comparative methods we compared patterns of variation in mutualistic benefit (host response to inoculation) to rhizobial phylogenies constructed from housekeeping and symbiosis genes; and a multigene host phylogeny. We found widespread genotype‐by‐genotype variation in patterns of plant growth. A relatively large component of this variation (21–28%) was strongly influenced by the interacting evolutionary histories of both partners, such that phylogenetically similar host species had similar growth responses when inoculated with phylogenetically similar rhizobia. We also found a relatively large nonphylogenetic effect for the average mutualistic benefit provided by rhizobia to plants, such that phylogenetic relatedness did not predict the overall benefit provided by rhizobia across all hosts. We conclude that phylogenetic relatedness should frequently predict patterns of mutualistic benefit in acacia‐rhizobial mutualistic interactions; but that some mutualistic traits also evolve independently of the phylogenies.     

Independent prognostic value of coronary artery calcium score and coronary computed tomography angiography in an outpatient cohort of low to intermediate risk chest pain patients

Background

Limited studies report on the additional prognostic value of coronary computed tomography angiography (CCTA) and the coronary artery calcium score (CACS).

Methods

For a median of 637 days, 1551 outpatients with chest pain, without known coronary artery disease (CAD) and low or intermediate pre-test probability of CAD, were followed for major adverse cardiac events (MACE), defined as death, myocardial infarction or late revascularisation. Cox proportional hazard regression was used to evaluate the independent prognostic value of CCTA and CACS.

Results

MACE occurred in 23 patients (1.5?%): death (3, 0.2?%), myocardial infarction (4, 0.3?%) and late revascularisation (16, 1.3?%). Multivariate analysis showed an independent prognostic value of CCTA (p?<?0.001), CACS of 100–400 (p?=?0.035) and CACS of >?400 (p?=?0.021). CCTA showed obstructive CAD in 3.1?% of patients with CACS?=?0. No events occurred in patients with CACS?=?0 without obstructive CAD at CCTA, whereas 2/23 patients (9?%) with CACS?=?0 with obstructive CAD had a MACE.

Conclusions

Our study shows that both CCTA and higher CACS categories have independent prognostic value in chest pain patients with low to intermediate pre-test probability of obstructive CAD, in which CCTA is appropriate. Furthermore a non-negligible amount of patients with CACS?=?0 have obstructive CAD at CCTA. CCTA can be used in these patients to identify those at risk for MACE.

     

The mechanism of potentiation of horseradish peroxidase-catalysed oxidation of NADPH by porphyrins.

Several porphyrins, including HpD (haematoporphyrin derivative), potentiate the oxidation of NADPH by horseradish peroxidase/H2O2. To elucidate the mechanism of potentiation, the following observations are relevant. During peroxidase-catalysed NADPH oxidation, O2-.(superoxide radical) is generated, as judged from superoxide dismutase-inhibitable cytochrome c reduction. This generation of O2-. is suppressed by HpD. Peroxidase-catalysed NADPH oxidation is stimulated by superoxide dismutase and by anaerobic conditions. Under anaerobic conditions HpD has no influence on peroxide-catalysed NADPH oxidation. Previous studies have shown that horseradish peroxidase is inhibited by O2-.. Thus the experimental results indicate that the potentiating effect of HpD can be explained by its ability to inhibit O2-. generation in the horseradish peroxidase/H2O2/NADPH system.     

Cardiac function and coronary flow in chronic endotoxemic pigs

We have reported that myocardial inotropism was depressed in acute and chronic endotoxemia. One possible mechanism for this observation is that endotoxemia reduces myocardial perfusion and indeed, we observed reduced myocardial perfusion in acute endotoxemia. This study tested the hypothesis that reduced inotropism of chronic endotoxemia was accompanied by reduced coronary artery blood flow. Fifteen pigs were equipped with left atrial and ventricular catheters, circumflex coronary and pulmonary artery flow meters, left ventricular pressure transducer, and ultrasonic crystals in the anterior-posterior axis to measure internal short axis diameter by sonomicrometry. The pigs recuperated for 3 days before basal data were collected over the next 3-5 days. After at least 7 postoperative days, an osmotic pump containing Salmonella enteriditis endotoxin was implanted in 12 pigs. Endotoxin was delivered at 10 micrograms/hr/kg for 2 days, at which time the animals were sacrificed. Osmotic pumps containing sterile saline were implanted in 3 pigs. Eight of the 12 endotoxemic pigs survived; 4 died before the morning of the second day. The survivors exhibited elevated heart rate, peak left ventricular systolic pressure, and cardiac output. Inotropism was evaluated by calculating the slope of the end-systolic pressure-diameter relationship (ESPDR) and % diameter-shortening. ESPDR was significantly depressed on the second endotoxemic day, while % diameter-shortening was depressed on both endotoxemic days. Coronary artery blood flow was significantly elevated on both endotoxemic days, while cross-sectional stroke work was unchanged. Therefore, the ratio of coronary blood flow to stroke work increased on both endotoxemic days. Nonsurvivors exhibited reduced heart rate, cardiac output, peak left ventricular systolic pressure, ESPDR, and % diameter-shortening. Neither coronary artery blood flow nor flow-to-work ratios increased in this group. Sham endotoxemic pigs demonstrated no cardiac or hemodynamic changes over 3 days. These results indicate that depressed inotropism during chronic endotoxemia was not caused by reduced coronary blood flow; rather, the myocardium was relatively overperfused.     

A new method for early in utero experiments in rat embryos: endoscopy

The merits of in vitro and in vivo techniques for experiments in rat embryos are discussed in this paper. Time limitation of culture, which is only feasible during 48 hours, up to day 13 post coitum (p.c.) is a major draw-back in the in vitro whole embryo culture. With the in utero operation technique used to date, no controlled experiments can be performed in rat embryos of 15 days p.c. and younger due to the high mortality of the embryos. Therefore a new technique has been developed, in which successful in utero operations can be performed as early as day 12 of gestation. Controlled micro-injection with the help of an endoscope can be given in any desired embryonic organ or structure. This paper describes this technique. Endoscopy in rat embryos of 12 days p.c. onwards has proven to be a new facility for in utero operations.