Citations

ABSTRACT Six clinical isolates of the nonpigmented, rapidly growing species Mycobacterium mageritense were recovered from sputum, bronchial wash, blood, sinus drainage, and two surgical wound infections from separate patients in Texas, New York, Louisiana, and Florida. The isolates matched the ATCC type strain by PCR restriction enzyme analysis of the 65-kDa hsp gene sequence of Telenti, high-performance liquid chromatography, biochemical reactions, and partial 16S rRNA gene sequencing. These are the first isolates of this species to be described in the United States and the first isolates to be associated with clinical disease. Susceptibility testing of all known isolates of the species revealed all isolates to be susceptible or intermediate to amikacin, cefoxitin, imipenem, and the fluoroquinolones and sulfonamides but resistant to clarithromycin. Because of their phenotypic and clinical similarity to isolates of the Mycobacterium fortuitum third biovariant complex (sorbitol positive), isolates of M. mageritense are likely to go undetected unless selected carbohydrate utilization or molecular identification methods are used.

ABSTRACT Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis , but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We developed a rapid-cycle real-time PCR detection assay for B. anthracis that utilizes the LightCycler instrument (LightCycler Bacillus anthracis kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen ( pagA ) was detected in 29 of 29 virulent B. anthracis strains, and the gene encoding the capsular protein B ( capB ) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only pagA or pagB genes, could be detected and differentiated from virulent strains. The assays were specific for B. anthracis : the results were negative for 57 bacterial strains representing a broad range of organisms, including Bacillus species other than anthracis ( n = 31) and other non- Bacillus species ( n = 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per μl of sample. The LightCycler Bacillus anthracis assay appears to be a suitable method for rapid identification of cultured isolates of B. anthracis . Additional clinical studies are required to determine the usefulness of this test for the rapid identification of B. anthracis directly from human specimens.

ABSTRACT Isoniazid is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis . Isoniazid is a prodrug requiring oxidative activation by the catalase-peroxidase hemoprotein, KatG. Resistance to isoniazid can be obtained by point mutations in the katG gene, with one of the most common being a threonine-for-serine substitution at position 315 (S315T). The S315T mutation is found in more than 50% of isoniazid-resistant clinical isolates and results in an ≈200-fold increase in the MIC of isoniazid compared to that for M. tuberculosis H37Rv. In the present study we investigated the hypothesis that superoxide plays a role in KatG-mediated isoniazid activation. Plumbagin and clofazimine, compounds capable of generating superoxide anion, resulted in a lower MIC of isoniazid for M. tuberculosis H37Rv and a strain carrying the S315T mutation. These agents did not cause as great of an increase in isoniazid susceptibility in the mutant strain when the susceptibilities were assessed by using the inhibitory concentration that causes a 50% decrease in growth. These results provide evidence that superoxide can play a role in isoniazid activation. Since clofazimine alone has antitubercular activity, the observation of synergism between clofazimine and isoniazid raises the interesting possibility of using both drugs in combination to treat M. tuberculosis infections.

Background— Outcomes in patients with mild to moderate renal function (RF) abnormalities presenting with acute coronary syndromes (ACS) are not well defined. Methods and Results— A convenience sample of 4 ACS trial databases including all enrolled patients was assessed to determine 30- and 180-day outcomes. The 4 trials were Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO) IIb, GUSTO-III, Platelet Glycoprotein IIb/IIIa in Unstable Angina: Receptor Suppression Using Integrilin Therapy (PURSUIT), and Platelet IIb/IIIa Antagonism for the Reduction of Acute coronary syndrome events in a Global Organization Network (PARAGON-A). Patients were stratified into ST-segment elevation (STE) and non–ST-segment elevation (NSE) groups and by the presence or absence of abnormal RF (creatinine clearance <70 mL/min). In the STE group, 7670 of 18 621 patients (41%) had abnormal RF. In the NSE group, 8152 of 19 304 (42%) had abnormal RF. Patients with abnormal RF were older, more often female, and more likely to have adverse baseline characteristics. They had higher mortality and higher mortality/nonfatal myocardial infarction (MI) at both 30 and 180 days, regardless of ST-segment status. Creatinine clearance was independently associated with risk of mortality (hazard ratio 0.79 in the STE group and 0.81 in the NSE group) and with risk of mortality/MI (hazard ratio 0.93) in the NSE group at 180 days. Conclusions— Patients presenting with ACS frequently have abnormal RF. Abnormal RF is a marker of adverse baseline clinical characteristics and is independently associated with increased risk of death and death/MI.

Background— Although aortic valve replacement can be performed at an acceptable risk level in selected patients with left ventricular systolic dysfunction and low-output, low-gradient aortic stenosis, not all patients presenting with these hemodynamics will benefit from the operation. Some patients may have only mild aortic stenosis, despite a small calculated valve area. We report on the clinical utility of diagnostic dobutamine stimulation during cardiac catheterization in these diagnostically challenging patients. Methods and Results— Thirty-two patients with low-output, low-gradient aortic stenosis and an ejection fraction <40% had dobutamine infusion in the catheterization laboratory. On the basis of the results of the dobutamine test, 21 patients underwent aortic valve replacement. All patients with a final aortic valve area ≤1.2 cm 2 at peak dobutamine infusion and a mean gradient of >30 mm Hg were found to have severe calcific aortic stenosis at operation. In the 15 patients in whom contractile reserve was identified during dobutamine challenge (increase in stroke volume >20%), 1 patient died perioperatively (7% mortality) and 12 patients were alive in New York Heart Association class I or II status at follow-up. Conclusions— In patients with left ventricular systolic dysfunction and aortic stenosis with a low output and a low mean gradient, dobutamine challenge may aid in selecting those who would benefit from an aortic valve operation.

Reaction to stress requires feedback adaptation of cellular functions to secure a response without distress, but the molecular order of this process is only partially understood. Here, we report a previously unrecognized regulatory element in the general adaptation syndrome. Kir6.2, the ion-conducting subunit of the metabolically responsive ATP-sensitive potassium (K ATP ) channel, was mandatory for optimal adaptation capacity under stress. Genetic deletion of Kir6.2 disrupted K ATP channel-dependent adjustment of membrane excitability and calcium handling, compromising the enhancement of cardiac performance driven by sympathetic stimulation, a key mediator of the adaptation response. In the absence of Kir6.2, vigorous sympathetic challenge caused arrhythmia and sudden death, preventable by calcium-channel blockade. Thus, this vital function identifies a physiological role for K ATP channels in the heart.

Background— The gene encoding ACE exhibits an insertion/deletion polymorphism resulting in 3 genotypes (DD, ID, and II), which affects serum and tissue ACE activity as well as other vasoactive substances. Pulmonary function is frequently abnormal in patients with congestive heart failure (CHF), the mechanism of which has not been completely characterized. ACE inhibition has been shown to improve diffusion across the alveolar-capillary membrane and to improve exercise capacity and gas exchange in CHF. The aim of the current study was to determine if ACE genotype is associated with altered pulmonary function and exercise intolerance in patients with treated CHF. Methods and Results— Fifty-seven patients (stratified according to ACE genotype as17 DD, 28 ID, 12 II) with ischemic and dilated cardiomyopathy, left ventricular ejection fraction (LVEF) <35%, and <10 pack-years of smoking history were studied. All patients were receiving standard therapy for left ventricular systolic dysfunction. Pulmonary function, LVEF, serum ACE, plasma angiotensin II, atrial natriuretic peptide, and brain natriuretic peptide were measured at baseline. Peak V̇ o 2 and gas exchange measurements were assessed with graded exercise. Resting LVEF was similar among the genotype groups (25% to 28%), and no differences were observed in diastolic function or pulmonary artery pressures ( P >0.05). Mean peak V̇ o 2 and forced vital capacity (% Pred) were significantly reduced ( P <0.05), whereas mean serum ACE activity and plasma angiotensin II concentration were highest in DD homozygotes. Subjects homozygous for the D-allele also demonstrated higher mean ventilatory equivalents for carbon dioxide (V̇ e /V̇ co 2 ) during exercise ( P <0.05). Conclusions— ACE DD genotype is associated with decreased exercise tolerance in CHF, possibly mediated by altered pulmonary function. Pharmacological strategies effecting more complete inhibition of serum and tissue ACE and/or potentiation of bradykinin may improve exercise capacity in patients with CHF and ACE DD genotype.

ABSTRACT Gene therapy in eukaryotes has met many obstacles. Research into the design of suitable nonviral vectors has been slow. To our knowledge, no nonviral vector has been proposed that allows for the possibility of highly efficient, site‐selective integration into the genome of mammalian cells. On the basis of prior studies investigating the components necessary for transposon, retrovirus‐like retrotransposon, and retroviral integration, we propose a nonviral system that would potentially allow for site‐selective, efficient integration into the mammalian genome. Transposons have been developed that can transform a variety of cell lines. For example, the Sleeping Beauty transposon (SB) can transform a wide range of vertebrate cells from fish to human, and it mediates stable integration and long‐term transgene expression in mice. However, the efficiency of transposition varies significantly among cell lines, suggesting the possible involvement of host factors in SB transposition. Here, we propose the use of a chimeric transposase (i.e., transposase‐host DNA binding domain) to bypass the potential requirement of a host DNA‐directing factor (or factors) for efficient, site‐selective integration. We also discuss another potential method of docking the transposonbased vector adjacent to the host DNA, utilizing repetitive sequences for homologous recombination to promote efficient site‐selective integration, as well as other site‐selective nonviral approaches.—Kaminski, J. M., Huber, M. R., Summers, J. B., Ward, M. B. Design of a nonviral vector for site‐selective, efficient integration into the human genome. FASEB J . 16, 1242–1247 (2002)