Recombinant Endopeptidases IdeS and IdeZ and Their Potential Application.

Endopeptidases IdeS and IdeZ (streptococcal virulence factors that specifically cleave IgG heavy chains) are of particular interest because of their potential use in biotechnology, medicine, and veterinary. Genes encoding these enzymes were cloned and expressed in Escherichia coli heterologous expression system (ideS was cloned from a Streptococcus pyogenes collection strain; ideZ from Streptococcus zooepidemicus was synthesized). The 6His-tag was introduced into the amino acid sequence of each endopeptidase, and IdeS and IdeZ were purified by metal affinity chromatography to an apparent homogeneity (according to polyacrylamide gel electrophoresis). Purified enzymes were active against human and animal IgGs; their specificity toward human IgGs was confirmed by polyacrylamide gel electrophoresis. Recombinant IdeZ was used for immunological analysis of equine strangles infection (diagnostics and determination of the titer of specific antibodies in blood). Hence, IdeZ can be used in veterinary and sanitary microbiology to diagnose infections caused by Streptococcus equi and S. zooepidemicus in addition to its application in medicine and biotechnology.

A Simple Protocol for the Determination of Lysostaphin Enzymatic Activity.

Antibacterial lysins are enzymes that hydrolyze bacterial peptidoglycan, which results in the rapid death of bacterial cells due to osmotic lysis. Lysostaphin is one of the most potent and well-studied lysins active against important nosocomial pathogen Staphylococcus aureus. Similarly to most other lysins, lysostaphin is composed of enzymatic and peptidoglycan-binding domains, and both domains influence its antibacterial activity. It is thus desirable to be able to study the activity of both domains independently. Lysostaphin cleaves pentaglycine cross-bridges within the staphylococcal peptidoglycan. Here, we report the protocol to study the catalytic activity of lysostaphin on the isolated pentaglycine peptide that is based on the chromogenic reaction of peptide amino groups with ninhydrin. Unlike previously reported assays, this protocol does not require in-house chemical synthesis or specialized equipment and can be readily performed in most laboratories. We demonstrate the use of this protocol to study the effect of EDTA treatment on the lysostaphin enzymatic activity. We further used this protocol to determine the catalytic efficiency of lysostaphin on the isolated pentaglycine and compared it to the apparent catalytic efficiency on the whole staphylococcal cells. These results highlight the relative impact of enzymatic and peptidoglycan-binding domains of lysostaphin on its bacteriolytic activity.

Morphine to codeine concentration ratio in blood and urine as a marker of illicit heroin use in forensic autopsy samples.

A morphine to codeine ratio greater than unity (M/C>1) has been suggested as an indicator of heroin use in living individuals. The aim of this study was to examine the morphine to codeine ratio in a large population (N=2438) of forensically examined autopsy cases positive for 6-monoacetylmorphine (6-MAM) and/or morphine in blood and/or urine. Blood and urine concentrations of 6-MAM, morphine and codeine were examined using GC-MS and LC-MS/MS methods. In 6-MAM positive samples, the M/C ratio was greater than unity in 98% (N=917) of the blood samples and 96% (N=665) of the urine samples. Stratification of 6-MAM negative cases by M/C above or below unity revealed similarities in morphine and codeine concentrations in cases where M/C>1 and 6-MAM positive cases. Median blood and urine morphine concentrations were 8-10 times greater than codeine for both groups. Similarly to 6-MAM positive cases, 25-44 year-old men prevailed in the M/C>1 group. In comparison to cases where M/C ≤ 1, the M/C ratio was a hundred times higher in both 6-MAM positive and M/C>1 cases. The range of morphine concentration between the lowest and the highest quintile of codeine in M/C>1 cases was similar to that in 6-MAM positive cases. This range was much higher than for M/C ≤ 1 cases. Moreover, linear regression analyses, adjusted for age and gender, revealed a strong positive association between morphine and codeine in 6-MAM positive and M/C>1 cases. The M/C ratio appeared to be a good marker of heroin use in post-mortem cases. Both blood and urine M/C>1 can be used to separate heroin users from other cases positive for morphine and codeine.

Dietary patterns, food groups, and nutrients as predictors of plasma choline and betaine in middle-aged and elderly men and women.

BACKGROUND: Choline and betaine are linked to phospholipid and one-carbon metabolism. Blood concentrations or dietary intake of these quaternary amines have been related to the risk of chronic diseases, including cardiovascular disease and the metabolic syndrome. OBJECTIVE: We aimed to determine dietary predictors of plasma choline and betaine among middle-aged and elderly subjects recruited from an area without folic acid fortification. DESIGN: This is a population-based study of 5812 men and women aged 47-49 and 71-74 y, within the Hordaland Health Study cohort. Plasma concentrations per increasing quartile of intake of foods, beverages, and nutrients were assessed by multiple linear regression analysis, and dietary patterns were assessed by factor analysis. RESULTS: Plasma choline was predicted by egg consumption (0.16 micromol/L; P < 0.0001) and cholesterol intake (0.16 micromol/L; P < 0.0001), and betaine was predicted by consumption of high-fiber bread (0.65 micromol/L; P < 0.0001); high-fat dairy products (-0.70 micromol/L; P < 0.0001); complex carbohydrates, fiber, folate, and thiamine (0.66-1.44 micromol/L; P

Divergent associations of plasma choline and betaine with components of metabolic syndrome in middle age and elderly men and women.

Choline is involved in the synthesis of phospholipids, including blood lipids, and is the immediate precursor of betaine, which serves as a methyl group donor in a reaction converting homocysteine to methionine. Several cardiovascular risk factors are associated with plasma homocysteine, whereas little is known about their relationship to choline and betaine. We examined the relation of plasma choline and betaine to smoking, physical activity, BMI, percent body fat, waist circumference, blood pressure, serum lipids, and glucose in a population-based study of 7074 men and women aged 47-49 and 71-74 y. Overall plasma concentrations (means +/- SD) were 9.9 +/- 2.3 micromol/L for choline and 39.5 +/- 12.5 micromol/L for betaine. Choline and betaine were lower in women than in men and in younger subjects compared with older (P < 0.0001). Multivariate analyses showed that choline was positively associated with serum triglycerides, glucose, BMI, percent body fat, waist circumference (P < 0.0001 for all), and physical activity (P < 0.05) and inversely related to HDL cholesterol (P < 0.05) and smoking (P < 0.0001). Betaine was inversely associated with serum non-HDL cholesterol, triglycerides, BMI, percent body fat, waist circumference, systolic and diastolic blood pressure (P < 0.0001 for all), and smoking (P < 0.05) and positively associated with HDL cholesterol (P < 0.01) and physical activity (P < 0.0001). Thus, an unfavorable cardiovascular risk factor profile was associated with high choline and low betaine concentrations. Choline and betaine were associated in opposite directions with key components of metabolic syndrome, suggesting a disruption of mitochondrial choline dehydrogenase pathway.

Dietary fat and plasma total homocysteine concentrations in 2 adult age groups: the Hordaland Homocysteine Study.

BACKGROUND: The intake of n-3 (formerly called omega-3) fatty acids (FAs) may be inversely associated with plasma total homocysteine (tHcy) concentrations, but the epidemiologic data are sparse. OBJECTIVE: We examined the association between dietary fat and tHcy in a Norwegian population. DESIGN: A cross-sectional, population-based study of 5917 subjects in 2 age groups (47-49 and 71-74 y old) was conducted with the use of food-frequency questionnaires and measurement of plasma tHcy concentrations. RESULTS: The intake of saturated FAs (SFAs) was positively and significantly (P for trend < 0.001) associated with tHcy concentrations; the difference in plasma tHcy concentrations between the highest and lowest quartiles of SFAs was 8.8%. The intake of marine very-long-chain n-3 FAs was inversely associated with tHcy concentrations; the difference in plasma tHcy concentrations between the lowest and the highest quartiles was -5.0% (P for trend < 0.001). Intakes of total and monounsaturated fat also were positively associated with plasma tHcy concentrations (P for trend < 0.001 and < 0.005, respectively), whereas the intake of polyunsaturated fat was positively associated with tHcy concentrations only in the younger subjects (P for trend = 0.03). The associations were weakened by additional adjustment for B vitamin intake but remained significant for SFA intake (P < 0.001). When stratified for total B vitamin intake, the inverse association between tHcy concentrations and very-long-chain n-3 FAs was significant only in the highest quartile of B vitamin intake (P for trend = 0.001), regardless of supplement use. CONCLUSIONS: High intakes of SFAs are associated with high plasma concentrations of tHcy. The inverse association between dietary intakes of very-long-chain n-3 FAs and plasma tHcy concentrations is apparent only at high B vitamin intakes.

Dietary predictors of plasma total homocysteine in the Hordaland Homocysteine Study.

Established dietary predictors of plasma total homocysteine (tHcy) include folate, riboflavin, and vitamins B6 and B12, while information is scarce regarding other dietary components. The aim of this study was to examine the relation between a variety of food groups, food items and nutrients, and plasma tHcy in a large population-based study. The study population included 5812 men and women aged 47-49 and 71-74 years who completed a 169-item FFQ. tHcy was examined across quartiles of dietary components by multiple linear regression analyses adjusting for age, sex, energy intake, various risk factors for elevated tHcy, as well as for dietary and plasma B-vitamins. Among 4578 non-users of vitamin supplements, intake of vegetables, fruits, cereals, eggs, fish and milk, as well as chicken and non-processed meats were inversely associated with tHcy level. The estimated mean difference in tHcy per increasing quartile of intake ranged from - 0.11 (95 % CI - 0.21, - 0.01) micromol/l for milk to - 0.32 (95 % CI - 0.42, - 0.22) micromol/l for vegetables. Positive associations were found for sweets and cakes. Whole-grain bread was significantly inversely related to tHcy only after additional adjustment for dietary and plasma B-vitamins. The nutrients folate, vitamin B6, B12, and riboflavin were inversely related to tHcy. Complex carbohydrates were inversely, and fat positively associated with tHcy, also after adjustment for dietary and plasma B-vitamins. In conclusion, food items rich in B-vitamins and with a low content of fat and sugar were related to lower tHcy levels. Eggs, chicken, non-processed meat, fish and milk were inversely associated with tHcy.