Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N.

The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated M(r) of 95,368, which agrees with the apparent M(r) of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis.     

Safety of the Surrogate Microorganism Enterococcus faecium NRRL B-2354 for Use in Thermal Process Validation

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products.     

Noncontact dipole effects on channel permeation. III. Anomalous proton conductance effects in gramicidin

Proton transport on water wires, of interest for many problems in membrane biology, is analyzed in side-chain analogs of gramicidin A channels. In symmetrical 0.1 N HCl solutions, fluorination of channel Trp(11), Trp-(13), or Trp(15) side chains is found to inhibit proton transport, and replacement of one or more Trps with Phe enhances proton transport, the opposite of the effects on K(+) transport in lecithin bilayers. The current-voltage relations are superlinear, indicating that some membrane field-dependent process is rate limiting. The interfacial dipole effects are usually assumed to affect the rate of cation translocation across the channel. For proton conductance, however, water reorientation after proton translocation is anticipated to be rate limiting. We propose that the findings reported here are most readily interpreted as the result of dipole-dipole interactions between channel waters and polar side chains or lipid headgroups. In particular, if reorientation of the water column begins with the water nearest the channel exit, this hypothesis explains the negative impact of fluorination and the positive impact of headgroup dipole on proton conductance.     

Lifestyle modifications to prevent and control hypertension. 4. Recommendations on physical exercise training. Canadian Hypertension Society,Canadian Coalition for High Blood Pressure Prevention and Control,Laboratory Centre for Disease Control at Health Canada,Heart and Stroke Foundation of Canada

OBJECTIVE: To provide updated, evidence-based recommendations for health care professionals concerning the effects of regular physical activity on the prevention and control of hypertension in otherwise healthy adults. OPTIONS: People may engage in no, sporadic or regular physical activity that may be of low, moderate or vigorous intensity. For sedentary people with hypertension, the options are to undertake or maintain regular physical activity and to avoid or moderate medication use; to use another lifestyle modification technique; to commence or continue antihypertensive medication; or to take no action and remain at increased risk of cardiovascular disease. OUTCOMES: The health outcomes considered were changes in blood pressure and in morbidity and mortality rates. Because of insufficient evidence, no economic outcomes were considered. EVIDENCE: A MEDLINE search was conducted for the period 1966-1997 with the terms exercise, exertion, physical activity, hypertension and blood pressure. Both reports of trials and review articles were obtained. Other relevant evidence was obtained from the reference lists of these articles, from the personal files of the authors and through contacts with experts. The articles were reviewed, classified according to study design and graded according to level of evidence. VALUES: A high value was placed on avoidance of cardiovascular morbidity and premature death caused by untreated hypertension. BENEFITS, HARMS AND COSTS: Physical activity of moderate intensity involving rhythmic movements with the lower limbs for 50-60 minutes, 3 or 4 times per week, reduces blood pressure and appears to be more effective than vigorous exercise. Harm is uncommon and is generally restricted to the musculoskeletal injuries that may occur with any repetitive activity. Injury occurs more often with jogging than with walking, cycling or swimming. The costs include the costs of appropriate shoes, garments and equipment, but these were not specifically measured. RECOMMENDATIONS: (1) People with mild hypertension should engage in 50-60 minutes of moderate rhythmic exercise of the lower limbs, such as brisk walking or cycling, 3 or 4 times per week to reduce blood pressure, (2) Exercise should be prescribed as an adjunctive therapy for people who require pharmacologic therapy for hypertension, especially those who are not receiving beta-blockers. (3) People who do not have hypertension should participate in regular exercise as it will decrease blood pressure and reduce the risk of coronary artery disease, although there is no direct evidence that it will prevent hypertension. VALIDATION: These recommendations agree with those of the World Hypertension League, the American College of Sports Medicine, the report of the US Surgeon General on physical activity and health, and the US National Institutes of Health Consensus Development Panel on Physical Activity and Cardiovascular Health. These guidelines have not been clinically tested. SPONSORS: The Canadian Hypertension Society, the Canadian Coalition for High Blood Pressure Prevention and Control, the Laboratory Centre for Disease Control at Health Canada, and the Heart and Stroke Foundation of Canada.     

The indefinite self-association of lysozyme: consideration of composition-dependent activity coefficients

An improved iterative method for computing association constants from sedimentation equilibrium results obtained with self-interacting protein systems is presented which accounts for the composition-dependence of the activity coefficients of all oligomeric species. The method is based on the calculation of virial coefficients from covolume and charge considerations, the statistical mechanical basis of which is discussed in relation to the DLVO theory. The method is applied to results obtained with lysozyme in diethylbarbiturate buffer of pH 8.0 and ionic strength 0.15 at 15°C. It is shown that these results, encompassing a range of total solute concentration up to 19.7 g/liter are consistent with self-association patterns comprising either a monomer-dimer-trimer system or an isodesmic indefinite self-association of the monomer, the latter being favored. A firmer distinction between these possibilities is sought on the basis of the dependence of the weight-average partition coefficient, determined by frontal gel chromatography, on total solute concentration (up to 56.6 g/liter). This analysis accounts for the composition-dependence of the ratio of the activity coefficients of partitioning monomer in mobile and stationary phases. It is concluded that all results are consistent with an indefinite self-association of lysozyme governed by a single association constant of 4.61 × 10² liter/mole.