mariner family transposons are widespread among eukaryotic organisms. These transposons are apparently horizontally transmitted among diverse eukaryotes and can also transpose in vitro in the absence ...of added cofactors. Here we show that transposons derived from the mariner element Himar1 can efficiently transpose in bacteria in vivo. We have developed simple transposition systems by using minitransposons, made up of short inverted repeats flanking antibiotic resistance markers. These elements can efficiently transpose after expression of transposase from an appropriate bacterial promoter. We found that transposition of mariner-based elements in Escherichia coli produces diverse insertion mutations in either a targeted plasmid or a chromosomal gene. With Himar1-derived transposons we were able to isolate phage-resistant mutants of both E. coli and Mycobacterium smegmatis. mariner-based transposons will provide valuable tools for mutagenesis and genetic manipulation of bacteria that currently lack well developed genetic systems.
Mariner-family transposable elements are active in a wide variety of organisms and are becoming increasingly important genetic tools in species lacking sophisticated genetics. The Himar1 element, ...isolated from the horn fly, Haematobia irritans, is active in Escherichia coli when expressed appropriately. We used this fact to devise a genetic screen for hyperactive mutants of Himar1 transposase that enhance overall transposition from approximately equal to 4- to 50-fold as measured in an E. coli assay. Purified mutant transposases retain their hyperactivity, although to a lesser degree, in an in vitro transposition assay. Mutants like those described herein should enable sophisticated analysis of the biochemistry of mariner transposition and should improve the use of these elements as genetic tools, both in vivo and in vitro.
Although the complete DNA sequences of several microbial genomes are now available, nearly 40% of the putative genes lack identifiable functions. Comprehensive screens and selections for identifying ...functional classes of genes are needed to convert sequence data into meaningful biological information. One particularly significant group of bacterial genes consists of those that are essential for growth or viability. Here, we describe a simple system for performing transposon mutagenesis on naturally transformable organisms along with a technique to rapidly identify essential or conditionally essential DNA segments. We show the general utility of this approach by applying it to two human pathogens, Haemophilus influenzae and Streptococcus pneumoniae, in which we detected known essential genes and assigned essentiality to several ORFs of unknown function.
Background: High levels of cadmium exposure are known to cause emphysema in occupationally exposed workers, but little has been reported to date on the association between chronic environmental ...cadmium exposure and pulmonary function. Objective: In this study we examined the association between pulmonary function and cadmium body burden in a subcohort of the Normative Aging Study, a community-based study of aging. Methods: We examined 96 men who had cadmium measured in single 24-hr urinary specimens collected in 1994-1995 and who had one to three tests of pulmonary function between 1994 and 2002 (a total of 222 observations). We used mixed-effect models to predict pulmonary function based on individual 24-hr urinary cadmium output, adjusted for age, height, time elapsed from the baseline, and smoking status. We assessed effect modification by smoking status. Results: Among all subjects, a single log-unit increase in baseline urinary cadmium was inversely associated with forced expiratory volume in 1 sec (FEV₁) percent predicted β = -7.56%; 95% confidence interval (CI) -13.59% to -1.53%; forced vital capacity (FVC) percent predicted (β = -2.70%; 95% CI -7.39% to 1.99%), and FEV₁/FVC ratio (β = -4.13%; 95% CI -7.61% to -0.66%). In models including an interaction between urinary cadmium and smoking status, there was a graded, statistically significant reduction in FEV₁/FVC ratio across smoking status in association with urinary cadmium. Conclusions: This study suggests that chronic cadmium exposure is associated with reduced pulmonary function, and cigarette smoking modifies this association. These results should be interpreted with caution because the sample size is small, and further studies are needed to confirm our findings.
Objectives. We sought to establish the diagnostic accuracy of transesophageal echocardiography (TEE) during cardiopulmonary resuscitation.
Background. Because of its bedside diagnostic capabilities, ...excellent cardiac images and lack of interference with resuscitation efforts, TEE is ideally suited to determine the cause of a circulatory arrest that is not due to severe arrhythmia. However, the diagnostic accuracy of TEE during resuscitation is unknown.
Methods. TEE was performed in patients with prolonged circulatory arrest. The TEE diagnoses were compared with diagnoses from autopsy, surgery and clinical follow-up.
Results. Of the 48 study patients (29 male, 19 female, mean age ± SD 61 ± 20 years), 28 had an in-hospital cardiac arrest and 20 an out-of-hospital onset of arrest. Forty-four patients eventually died; four survived to discharge. The diagnoses made with TEE were cardiac tamponade (n = 6), myocardial infarction (n = 21), pulmonary embolism (n = 6), ruptured aorta (n = 1), aortic dissection (n = 4), papillary muscle rupture (n = 1), other diagnosis (n = 2) and absence of structural cardiac abnormalities (n = 7). A definite diagnosis from a reference standard was available in 31 patients. The TEE diagnosis was confirmed in 27 of the 31—by postmortem examination (n = 19), operation (n = 2), angiography (n = 2) or clinical course (n = 4). In the other four patients the TEE diagnosis proved incorrect by postmortem examination. The sensitivity, specificity and positive predictive value of TEE were 93%, 50% and 87%, respectively. In 15 patients (31%), major therapeutic decisions were based on TEE findings.
Conclusions. TEE can reliably establish the cause of a circulatory arrest during cardiopulmonary resuscitation.
Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different ...electrophysiological properties and have different distributions in cardiac tissues with disparate conduction properties. We characterized the expression, phosphorylation, turnover, and subcellular distribution of these connexins in primary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and Cx40 mRNA were specifically detected in RNA blots. Immunofluorescent staining with antibodies specific for Cx43 and Cx45 revealed punctate labeling at appositional membranes, but no immunoreactive Cx40 was detected. Double-label immunofluorescence confocal microscopy of cultured myocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were both identified by immunoprecipitation from 35Smethionine-labeled cultures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipitated from cultures metabolically labeled with 32Porthophosphate. Phosphoamino acid analysis demonstrated that Cx45 was modified on serine residues, and Cx43 was phosphorylated on serine and threonine residues. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results implicate multiple mechanisms of gap junction regulation that may differ for different connexins.
Male passerine bird song is a classic example of a sexually selected display. Theory predicts that such displays should be costly, but there are few empirical data on the potential costs of bird ...song. We measured the rate of oxygen consumption by male pied flycatchers, Ficedula hypoleuca, singing inside a respirometry chamber. Song is known to be important in mate choice by pied flycatchers, so we predicted that if passerine song is energetically costly, we should be able to measure the costs in this species. The metabolic rate of singing pied flycatchers (N = 3) was 0.62 ± 0.11 W, which was equivalent to 2.7 ± 0.5 × basal metabolic rate. Metabolism during singing did not differ significantly from that during standing. A power analysis of this test showed that the metabolic rate of singing birds was less than 1.12 ± 0.04 times that of standing birds. Comparing the maximum cost of song production with daily energy expenditure (DEE) during nestling rearing, each hour of singing rather than standing would increase DEE by 0.4%. Thus, song production appears to be energetically cheap in the pied flycatcher in relation to the overall daily energy budget. However, because birds cannot sing and eat at the same time, there could be an energetic constraint to song duration owing to the need to spend sufficient time foraging.
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Linopirdine (DuP 996) has been shown to enhance depolarization‐induced release of several neurotransmitters in the CNS through a mechanism which may involve K+ channel blockade. The ...electrophysiological effects of linopirdine were therefore investigated directly, by use of conventional voltage recording and single electrode voltage‐clamp.
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Linopirdine (10 μm) reduced spike frequency adaptation (SFA) in rat hippocampal CA1 pyramidal neurones in vitro. The reduction of SFA comprised an increase in number of spikes and a reduction in inter‐spike intervals after the first, but with no effect on time to first spike. Linopirdine also caused a voltage‐dependent depolarization of resting membrane potential (RMP).
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M‐current (IM), a current known to underlie SFA and to set RMP, was blocked by linopirdine in a reversible, concentration‐dependent manner (IC50 = 8.5 μm). This block was not reversed by atropine (10 μm).
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Linopirdine did not affect IQ, the slow after‐hyperpolarization following a spike train, or spike duration.
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Linopirdine may represent a novel class of K+ blocker with relative selectivity for the M‐current. This block of IM is consistent with the suggestion from a previous study that linopirdine may affect a tetraethylammonium‐sensitive channel, and it could be speculated that IM blockade may be involved with the enhancement of neurotransmitter release by linopirdine.
We have previously demonstrated that epidermal growth factor induced a rapid, transient decrease in gap junctional communication and increase in serine phosphorylation on the connexin-43 gap junction ...protein in T51B rat liver epithelial cells. The kinase(s) responsible for phosphorylation and specific serine targets in connexin-43 have not been identified. There are three consensus mitogen-activated protein (MAP) kinase serine phosphorylation sequences in the carboxyl-terminal tail of connexin-43 and purified MAP kinase phosphorylated connexin-43 in vitro on tryptic peptides that comigrated with a subset of peptides from connexin-43 phosphorylated in vivo in cells treated with epidermal growth factor. These data suggested that MAP kinase may phosphorylate connexin-43 directly in vivo. We have utilized a glutathione S-transferase fusion protein containing the cytoplasmic tail of connexin-43 to characterize MAP kinase phosphorylation. Site-directed mutagenesis, phosphotryptic peptide analysis, and peptide sequencing have confirmed that MAP kinase can phosphorylate connexin-43 at Ser255, Ser279, and Ser282, which correspond to the consensus sites recognized earlier. Characterization of MAP kinase-mediated phosphorylation of connexin-43 has defined potential targets for phosphorylation in vivo following activation of the epidermal growth factor receptor and has provided the basis for studies of the effects of phosphorylation, at specific molecular sites, on the regulation of gap junctional communication.
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