Genbank accession #: AF 135190

Plant Molecular Biology -     

The energy-yielding reactions of Peptococcus prévotii,their behaviour on starvation and the role and regulation of threonine dehydratase

The principal energy-yielding reactions of the strict anaerobe Peptococcus prévotii comprised the fermentation of l-serine and l-threonine via the enzymes threonine dehydratase, thioclastic enzyme, phosphotransacetylase and acetate kinase.Threonine dehydratase was purified 700-fold and shown to require pyridoxal 5-phosphate as co-enzyme, and a reducing agent for optimum activity. The ratio of threonine and serine dehydratase activities was unaltered during purification. The optimum pH was 8.5 to 9.5 and isoleucine did not inhibit.Lineweaver-Burk plots were linear at l-threonine concentrations above 1.35 mM and the K _m for threonine was 2.5 mM and for serine 29 mM. Below this concentration co-operativity occurred which was not nullified by individual adenine nucleotides: Hill plots were biphasic.However, the enzyme was controlled by the adenylate energy charge in a novel manner; only at very low threonine concentrations (<1 mM) was control manifest, when a high energy charge inhibited and a low energy charge stimulated activity.During starvation for 33 hrs in phosphate buffer, pH 6.8, viability fell to zero but, of the enzymes of the energy-generating sequence, only the total units and specific activity of threonine dehydratase decreased (by 35%), which was insufficient to explain the loss of ability to generate ATP.     

The use of dirty bedding for detection of murine pathogens in sentinel mice

Sentinel Swiss (CD-1) mice, housed without filter bonnets, were seronegative for mouse hepatitis virus (MHV) for 8 consecutive months in an experimental colony of CD-1 mice. MHV titers had been detected sporadically in sentinel mice housed in this colony during a 2 year period. In an effort to determine whether MHV was still present in the colony, two methods of exposing sentinel mice to an animal room environment were compared under routine husbandry practices. Eight cages (12 mice per cage; 2 cages per rack) of experimental virus antibody free sentinel mice, housed without filter bonnets, were placed on the bottom shelf of 4 of 12 racks in the room. Twice each week, four cages of sentinel mice received a composite sample of dirty bedding (bedding used previously by mice in the room). The remaining four cages of experimental sentinels received fresh non-used bedding. Sentinel mice were bled at monthly intervals for MHV serology. After 4 months, mice from two cages which received dirty bedding seroconverted to MHV and mice from one cage were positive for Myobia musculi (mites). Three weeks later, all four cages of mice which received dirty bedding were positive for MHV and three were positive for mites. In contrast, only two of four cages of mice which received fresh bedding were positive for MHV and all were negative for mites. These findings indicate the importance of exposing sentinel mice to dirty bedding and that MHV and mites may go undetected for several months in a mouse colony when the incidence levels are low where standard sanitation procedures are used.     

Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids

Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C(3)) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B(12)-derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B(12) is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C(17) fatty acid heptadecanoate, growth on valerate (C(5)) was significantly enhanced through vitamin B(12) supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B(12)-supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B(12) to mycobacterial pathogenesis and suggests that vitamin B(12) availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.     

Genetic variation in TIMP1 but not MMPs predict excess FEV1 decline in two general population-based cohorts

Background

An imbalance in Matrix MetalloProteases (MMPs) and Tissue Inhibitors of MMPs (TIMPs) contributes to Chronic Obstructive Pulmonary Disease (COPD) development. Longitudinal studies investigating Single Nucleotide Polymorphisms (SNPs) in MMPs and TIMPs with respect to COPD development and lung function decline in the general population are lacking.

Methods

We genotyped SNPs in MMP1 (G-1607GG), MMP2 (-1306 C/T), MMP9 (3 tagging SNPs), MMP12 (A-82G and Asn357Ser) and TIMP1 (Phe124Phe and Ile158Ile) in 1390 Caucasians with multiple FEV₁ measurements from a prospective cohort study in the general population. FEV₁ decline was analyzed using linear mixed effect models adjusted for confounders. Analyses of the X-chromosomal TIMP1 gene were stratified according to sex. All significant associations were repeated in an independent general population cohort (n = 1152).

Results

MMP2 -1306 TT genotype carriers had excess FEV₁ decline (-4.0 ml/yr, p = 0.03) compared to wild type carriers. TIMP1 Ile158Ile predicted significant excess FEV₁ decline in both males and females. TIMP1 Phe124Phe predicted significant excess FEV₁ decline in males only, which was replicated (p = 0.10) in the second cohort. The MMP2 and TIMP1 Ile158Ile associations were not replicated. Although power was limited, we did not find associations with COPD development.

Conclusions

We for the first time show that TIMP1 Phe124Phe contributes to excess FEV₁ decline in two independent prospective cohorts, albeit not quite reaching conventional statistical significance in the replication cohort. SNPs in MMPs evidently do not contribute to FEV₁ decline in the general population.     

Plasma membrane electron transport in Saccharomyces cerevisiae depends on the presence of mitochondrial respiratory subunits

Most investigations into plasma membrane electron transport (PMET) in Saccharomyces cerevisiae have focused on the inducible ferric reductase responsible for iron uptake under iron/copper-limiting conditions. In this paper, we describe a PMET system, distinct from ferric reductase, which reduces the cell-impermeable water-soluble tetrazolium dye, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-1), under normal iron/copper conditions. WST-1/1-methoxy-phenazine methosulphate reduction was unaffected by anoxia and relatively insensitive to diphenyleneiodonium. Dye reduction was increased when intracellular NADH levels were high, which, in S. cerevisiae , required deletion of numerous genes associated with NADH recycling. Genome-wide screening of all viable nuclear gene-deletion mutants of S. cerevisiae revealed that, although mitochondrial electron transport per se was not required, the presence of several nuclear and mitochondrially encoded subunits of respiratory complexes III and IV was mandatory for PMET. This suggests some form of interaction between components of mitochondrial and plasma membrane electron transport. In support of this, mitochondrial tubular networks in S. cerevisiae were shown to be located in close proximity to the plasma membrane using confocal microscopy.     

A comparison of blood pressure measurement over a sleeved arm versus a bare arm

Background

The measurement of blood pressure is a common clinical exam with important health consequences. We sought to determine whether the measurement of blood pressure over a sleeved arm varies from that taken on a bare arm.

Methods

We recruited 376 patients between 18 and 85 years of age from a family medicine clinic between September 2004 and November 2006. They all had their blood pressure recorded using the same automatic oscillometric device, with the cuff placed over their bare arms for the first reading. Each patient was then randomly assigned to either the bare-arm group, for which the second blood pressure reading was also taken on a bare arm, or the sleeved-arm group, for which the second reading was taken with the cuff placed over the patient''s sleeve.

Results

The mean age of the 376 participants was 61.6 years (standard deviation 15.0), 61% of the participants were male, 41% had hypertension and 11.7% had diabetes. We found no clinically important differences between the bare-arm group (n = 180) and the sleeved-arm group (n = 196) in age, sex or body mass index. The mean differences between the first and second readings for patients in the bare-arm group were 4.1 mm Hg (95% confidence interval [CI] 2.8 to 5.5) for systolic blood pressure and 0.1 mm Hg (95% CI –0.7 to 0.9) for diastolic blood pressure. The mean differences between the first and second readings for patients in the sleeved-arm group were 3.4 mm Hg (95% CI 2.1 to 4.7) for systolic blood pressure and 0.4 mm Hg (95% CI –0.4 to 1.3) for diastolic blood pressure. The between-group differences in these values was 0.76 mm Hg (95% CI –1.13 to 2.65) for systolic and –0.31 mm Hg (95% CI –1.48 to 0.86) for diastolic blood pressure; neither of these differences was clinically important or statistically significant.

Interpretation

We found that there was no significant difference in blood pressure recorded over a sleeve or on a bare arm. For practical purposes, the decision to measure blood pressure on a bare arm or over a sleeved arm should be left to the judgment of the health care professional taking the blood pressure.The measurement of blood pressure is one of the most common examinations undertaken in family practice and has important health and management consequences for the patient. Accurate assessment of blood pressure, therefore, is very important. Current guidelines outline standards for obtaining accurate and reproducible blood pressure measurements.¹ These standards include guidelines for the sizes of blood pressure cuffs and the position of the patient''s arm, as well as a recommendation that the patient be seated for 5 minutes before his or her blood pressure is taken. In addition, it has been recommended that blood pressure be measured over the patient''s bare arm.¹ However, the current recommended method to determine blood pressure² has several limitations (e.g., high variability of blood pressure at different times of day or if measured only once, loss of proper technique post-training).Three previous studies have assessed whether blood pressure varies significantly when taken over a sleeved arm compared with a bare arm. Details of the literature review are described in Appendix 1, available online at www.cmaj.ca/cgi/content/full/178/5/585/DC2. In one study, involving 36 patients, Holleman et al³ found no significant differences in systolic or diastolic blood pressure readings taken over the sleeved or bare arm of each patient. However, this study was limited because of its small sample and because blood pressure measurements were taken on both arms simultaneously. A study by Kahan et al,⁴ involving 201 patients, compared blood pressure measurements taken over a sleeved arm, a bare arm and below a rolled-up sleeve. They found that the degree of clothing under the sphygmomanometer cuff did not have a clinically important effect on the reading. Although they found no significant difference in the effect of clothing on blood pressure readings, the study was limited by their design of measuring blood pressure below a rolled-up sleeve. The third study, conducted by Liebl et al,⁵ was published while our study was in progress. Their study, which involved 201 patients, compared blood pressure measurements taken over a sleeved arm and a bare arm with both sphygmomanometric and oscillometric devices. They concluded no significant difference in readings between the sleeved and bare-arm groups. In contrast to previous studies, we sought to determine whether the measurement of blood pressure over a sleeved arm varies from that taken over a bare arm through the use of a control group in which patients'' blood pressure was taken only over a bare arm.     

Site-directed, Ligase-Independent Mutagenesis (SLIM) for highly efficient mutagenesis of plasmids greater than 8kb

Modifying the Site-directed, Ligase-Independent Mutagenesis (SLIM) protocol from a single reaction mode to a two-reaction mode enables highly efficient mutagenesis of plasmid constructs that exceed 8kb. This modified approach reduces the complexity of the PCR step and is optimised for generation of heteroduplexes from long PCR products. The two-reaction mode SLIM has 92% efficiency.