Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Various S=3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alphaH195Q, alphaH195N, and alphaQ191K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N(2) with the alphaH195Q and alphaH195N variants, but not with the alphaQ191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alphaH195Q or alphaH195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alphaQ191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze (57)Fe M?ssbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alphaH195Q and alphaH195N MoFe proteins can bind N(2), but alphaQ195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N(2) reduction.     

Primers with 5' flaps improve real-time PCR

Primers that contain portions noncomplementary to the target region are usually used to add to the PCR product a utility sequence such as a restriction site or a universal probe binding site. We have demonstrated that primers with short 5'AT-rich overhangs increase real-time PCR fluorescent signal. The improvement is particularly significant for difficult to amplify templates, such as highly variable viral sequences or bisulfite-treated DNA.     

Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic bacteria that can cause life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Previous work has shown that Bcc isolates can persist in membrane-bound vacuoles within amoeba and macrophages without bacterial replication, but the detailed mechanism of bacterial persistence is unknown. In this study, we have investigated the survival of the Burkholderia cenocepacia strain J2315 within RAW264.7 murine macrophages. Strain J2315 is a prototypic isolate of the widespread and transmissible ET12 clone. Unlike heat-inactivated bacteria, which reach lysosomes shortly after internalization, vacuoles containing live B. cenocepacia J2315 accumulate the late endosome/lysosome marker LAMP-1 and start fusing with lysosomal compartments only after 6 h post internalization. Using fluorescent fluid-phase probes, we also demonstrated that B. cenocepacia-containing vacuoles continued to interact with newly formed endosomes, and maintained a luminal pH of 6.4 +/- 0.12. In contrast, vacuoles containing heat-inactivated bacteria had an average pH of 4.8 +/- 0.03 and rapidly merged with lysosomes. Additional experiments using concanamycin A, a specific inhibitor of the vacuolar H+-ATPase, revealed that vacuoles containing live bacteria did not exclude the H+-ATPase. This mode of bacterial survival did not require type III secretion, as no differences were found between wild type and a type III secretion mutant strain. Collectively, our results suggest that intracellular B. cenocepacia cause a delay in the maturation of the phagosome, which may contribute to facilitate bacterial escape from the microbicidal activities of the host cell.     

Knockdown of SPARC leads to decreased cell-cell adhesion and lens cataracts during post-gastrula development in Xenopus laevis

SPARC is a multifunctional matricellular glycoprotein with complex, transient tissue distribution during embryonic development. In Xenopus laevis embryos, zygotic activation of SPARC is first detected during late gastrulation, undergoing rapid changes in its spatiotemporal distribution throughout organogenesis. Injections of anti-sense Xenopus SPARC morpholinos (XSMOs) into 2- and 4-cell embryos led to a dose-dependent dissociation of embryos during neurula and tailbud stages of development. Animal cap explants derived from XSMO-injected embryos also dissociated, resulting in the formation of amorphous ciliated microspheres. At low doses of XSMOs, lens cataracts were formed, phenocopying that observed in Sparc-null mice. At XSMOs concentrations that did not result in a loss of axial tissue integrity, adhesion between myotomes at intersomitic borders was compromised with a reduction in SPARC concentration. The combined data suggest a critical requirement for SPARC during post-gastrula development in Xenopus embryos and that SPARC, directly or indirectly, promotes cell?Ccell adhesion in vivo.     

Effect of marriage on duration of chest pain associated with acute myocardial infarction before seeking care

Background:

Coronary artery disease is the most common cause of death in the Western world, and being married decreases the risk of death from cardiovascular causes. We aimed to determine whether marital status was a predictor of the duration of chest pain endured by patients with acute myocardial infarction before they sought care and whether the patient’s sex modified the effect.

Methods:

We conducted a retrospective, population-based cohort analysis of patients with acute myocardial infarction admitted to 96 acute care hospitals in Ontario, Canada, from April 2004 to March 2005. We excluded patients who did not experience chest pain. Using multivariable regression analyses, we assessed marital status in relation to delayed presentation to hospital (more than six hours from onset of pain), both overall and stratified by sex. In patients who reported the exact duration of chest pain, we assessed the effect of marital status on the delay in seeking care.

Results:

Among 4403 eligible patients with acute myocardial infarction, the mean age was 67.3 (standard deviation 13.6) years, and 1486 (33.7%) were women. Almost half (2037 or 46.3%) presented to a hospital within two hours, and 3240 (73.6%) presented within six hours. Overall, 75.3% (2317/3079) of married patients, 67.9% (188/277) of single patients, 68.5% (189/276) of divorced patients and 70.8% (546/771) of widowed patients presented within six hours of the onset of chest pain. Being married was associated with lower odds of delayed presentation (odds ratio [OR] 0.46, 95% confidence interval [CI] 0.30–0.71, p < 0.001) relative to being single. Among men, the OR was 0.35 (95% CI 0.21–0.59, p < 0.001), whereas among women the effect of marital status was not significant (OR 1.36, 95% CI 0.49–3.73, p = 0.55).

Interpretation:

Among men experiencing acute myocardial infarction with chest pain, being married was associated with significantly earlier presentation for care, a benefit that was not observed for married women. Earlier presentation for medical care appears to be one reason for the observed lower risk of cardiovascular death among married men, relative to their single counterparts.Marriage has long been known to offer health benefits^1,2 and is associated with a lower risk of death^3,4 relative to people who are not married. The effect is more pronounced among men than among women.^5,6 However, the specific mechanisms responsible for the lower rate of cardiovascular deaths in married persons⁷ are not known.Effective, time-sensitive therapy for acute myocardial infarction is available,^8,9 and delays in the emergency department and for in-hospital components of care have been substantially reduced over the past few decades.^10,11 In contrast, patients’ delay in seeking care for acute myocardial infarction has shown little improvement over time,^12,13 despite intensive campaigns to raise public awareness.^14,15 Patients’ delay remains by far the largest component of the overall delay between onset of symptoms and receipt of therapy.¹⁶ No study has examined the effect of marital status on patients’ delay, and only a few small studies have examined predictors of this component of delay by sex.^17,18We examined the effect of marital status, a social factor, on the time from onset of chest pain to arrival in an emergency department or hospital, in a population-based cohort of patients with acute myocardial infarction. We hypothesized that being married or in a common-law relationship would be associated with less delay, because we surmised that a spouse would encourage earlier pursuit of medical care, either directly or indirectly (i.e., even if the spouse was not physically present during the symptoms, his or her existence might spur the patient to seek care earlier). We hypothesized that wives would be more likely than husbands to assume the caregiver role and that the beneficial effect of marriage would therefore be stronger among men than among women.