Allele-Specific PCR Method Based on pncA and oxyR Sequences for Distinguishing Mycobacterium bovis from Mycobacterium tuberculosis: Intraspecific M. bovis pncA Sequence Polymorphism

An allele-specific amplification method based on two genetic polymorphisms to differentiate Mycobacterium tuberculosis from Mycobacterium bovis was tested. Based on the differences found at position 169 in the pncA genes from M. tuberculosis and M. bovis, a PCR system which was able to differentiate most of the 237 M. tuberculosis complex isolates tested in one of the two species was developed. All 121 M. tuberculosis strains showed the expected base (cytosine) at position 169. Most of the M. bovis isolates had a guanine at the cited position. Nevertheless, 18 of the 116 M. bovis isolates, all of them goat isolates, showed the pncA polymorphism specific to M. tuberculosis. These results suggest that goat M. bovis may be the nicotinamidase-missing link at the origin of the M. tuberculosis species. Based on the polymorphism found at position 285 in the oxyR gene, the same system was used to differentiate M. tuberculosis from M. bovis. In this case, DNAs from all 121 M. tuberculosis isolates had the expected base (guanine) at this position. In addition, all 116 M. bovis isolates, including those from goats, showed the identical polymorphism (adenine). The oxyR allele-specific amplification method can differentiate M. bovis from M. tuberculosis, is rapid (results can be obtained in less than 3 h), and is easy to perform.     

Androgen receptor independent cardiovascular action of the antiandrogen flutamide

We have previously shown that flutamide (specific antagonist of the androgen receptor) has antihypertensive effects. In the present study we examined the mechanisms of flutamide action in the vasculature. The vascular effects of flutamide were assayed in aortae isolated from male or female Sprague-Dawley rats and from rats or mice lacking a functional androgen receptor ( tfm, testicular feminization mutation). The effect of flutamide on coronary flow was tested in isolated hearts. In addition, male hypertensive rats with tfm mutation were treated with flutamide, and blood pressure was monitored. Flutamide induced a relaxation of rat aortae from all the strains used (maximum relaxation at 10 microM: 51.3+/-5.2% of phenylephrine contraction) and increased the coronary flow. The aortic relaxation to flutamide was abolished by endothelium removal, or by inhibition of nitric oxide synthase, guanylyl cyclase, and tyrosine kinase but not by calmodulin inhibition. Flutamide treatment attenuated the development of hypertension in mouse renin transgenic rats with the tfm mutation. Flutamide produces direct vasodilation by inducing release of NO from the endothelium and causes subsequent activation of soluble guanylyl cyclase in an active androgen receptor independent manner. This response may contribute to the observed antihypertensive actions of flutamide.     

Haplotype analysis of the BRCA2 9254delATCAT recurrent mutation in breast/ovarian cancer families from Spain

A frame-shift 9254del5 mutation was independently identified in 12 families, eleven of them with Spanish ancestors, in a BRCA2 screening performed in 841 breast and/or ovarian cancer families and in 339 women with breast cancer diagnosed before the age of 40 at different centers in France and Spain. We sought to analyze in detail the haplotype and founder effects of the 9254del5 and to estimate the time of origin of the mutation. Eight polymorphic microsatellite markers and two BRCA2 polymorphisms were used for the haplotype analyses. The markers were located flanking the BRCA2 gene spanning a region of 6.1 cM. Our results suggest that these families shared a common ancestry with BRCA2 9254del5, which is a founder mutation originating in the Northeast Spanish, with an estimated age of 92 (95% CI 56-141) generations.     

Evaluation of E-Test for determination of antimicrobial MICs for Pseudomonas aeruginosa isolates from cystic fibrosis patients.

We determined the E-Test and National Committee for Clinical Laboratory Standards standardized agar dilution MICs of ceftazidime, ciprofloxacin, piperacillin, and tobramycin for Pseudomonas aeruginosa during tests of 100 rough and mucoid P. aeruginosa isolates from cystic fibrosis patients. The levels of agreement (+/- 1 log2 dilution) between quantitative E-Test and agar dilution MIC results were 80, 97, 73, and 89% for ceftazidime, ciprofloxacin, piperacillin, and tobramycin, respectively. Comparison of the results after converting the MIC data to qualitative categories (susceptible, intermediate, and resistant) yielded levels of agreement of 84, 96, 88, and 93% for the same agents, respectively. Of the 39 qualitative discrepancies, 36 were minor and 3 were very major. We conclude that use of the E-Test is easier and more practical than use of the agar dilution method for most laboratories and that the E-Test furnishes results which are at least as accurate as those obtained by the agar dilution method. However, the higher cost of the E-Test method would likely discourage most laboratories from selecting it over disk diffusion for routine antimicrobial susceptibility testing of P. aeruginosa isolates from cystic fibrosis patients.     

Diarrhea Associated with Vibrio fluvialis in the United States

We report the isolation in the United States of Vibrio fluvialis from the stools of a patient who had severe watery diarrhea without fever and who subsequently died. V. fluvialis, a known enteric pathogen in other parts of the world, should be suspected in patients with watery diarrhea, especially in coastal areas.     

Shape,F-actin,and surface morphology changes during chemotactic peptide-induced polarity in human neutrophils

Background: The exposure of human neutrophils to uniform concentrations of chemoattractants, such as N-formyl peptides, induces morphological cell polarization. In this study we report the temporal sequence of changes in cell shape, F-actin, and cell surface morphology during cellular polarization induced by N-formylmethionyl-leucyl-phenyl-alanine (fMLP) in human neutrophils in suspension. Methods: Neutrophil shape changes induced by 10^?8 M fMLP were observed with DIC microscopy. Size and Cellular granularity were analyzed by flow cytometry measuring their forward and side scattered light. To visualize F-actin distribution, neutrophils were labeled with the fluorescence probe FITC-phalloidin, and were examined with fluorescence and confocal laser scanning microscopy. Cell surface morphology was assessed with scanning electron microscopy (SEM). Results: The stimulation of round-smooth neutrophils with nanomolar concentrations (10^?8 M) of fMLP in suspension induced a temporal sequence of morphological changes during cell polarization, characterized by 1) increase in size as determined by forward angle scattered light, 2) rapid redistribution of F-actin from a diffuse cytoplasmic localization to the cell periphery, and 3) rapid reorganization of cell surface morphological features, with accumulation of plasma membrane in the front of polar cells. Four cell shapes were identified with SEM after stimulation of round-smooth neutrophils: round-ridged, round-ruffled, nonpolar ruffled, and polar cells. These cell shapes were correlated with a cortical localization, focal aggregates, and multipolar distribution of F-actin. In polar neutrophils, F-actin became concentrated in the front of the cell. Conclusions: These findings show the relation between reorganization of the microfilamentous cytoskeleton and modifications in cell shape and surface features during cell polarization induced after fMLP activation in neutrophils. This approach offers a powerful tool for further analysis of receptor distribution in polarized, motile neutrophils. © 1995 Wiley-Liss, Inc.     

Demonstration of an inwardly rectifying K+ current component modulated by thyrotropin-releasing hormone and caffeine in GH3 rat anterior pituitary cells

 Reduction of an inwardly rectifying K⁺ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical activity increases in GH₃ rat anterior pituitary cells. However, the existence of an inwardly rectifying K⁺ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target in pituitary cells, including GH₃ cells. In this report, an inwardly rectifying component of K⁺ current is indeed demonstrated in perforated-patch voltage-clamped GH₃ cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active. Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current. Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K⁺ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is probably mediated by a HERG-like K⁺ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production. Thus, an inwardly-rectifying K⁺ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH₃ cells. Received: 15 May 1997 / Received after revision and accepted: 24 July 1997