Decrease in cardiovascular disease mortality in the Czech Republic from 1984 to 1993 and its possible causes

BACKGROUND: The objective of the investigation was to evaluate the ten-year development of the cardiovascular mortality rate in two population groups in the age bracket from 25 to 64 years, i.e. in subjects living in six districts which participated in the international WHO project MONICA and in the population of the whole Czech Republic. METHODS AND RESULTS: Data on the mortality rate in 1984-1993 for the age group from 25-64 years were provided by the Institute of Health Information and Statistics, information on the prevalence of risk factors was obtained in three cross-sectional studies implemented in six districts as part of the MONICA project in 1985, 1988 and 1992. In the mortality rate per 100,000 population in the six districts the following changes were revealed (in parentheses the values for 1984 and 1993 are given): men - a statistically significant declining trend in the from all caused mortality (849.3-742.5; p < 0.001) and cardiovascular mortality (367.2-280.4; p < 0.001) and cerebrovascular mortality (69.7-44.8; p < 0.001). In the mortality from ischaemic heart disease (215.7-170.6; ns) a declining trend was not recorded. In women aged 25-64 years in the six districts there was a statistically significant decline of the mortality from all caused (359.5-322.1; p < 0.001), the cardiovascular mortality (115.6-100.6; p < 0.001) and cerebrovascular mortality (31.1-23.6; p < 0.001). The mortality from ischaemic heart disease did not change (49.2-48.8; ns). In the population of the Czech Republic in men the following were detected: a drop of the from all caused mortality (907.1-784.8; P < 0.001), the cardiovascular mortality (383.5-308.4; p < 0.001) and cerebrovascular mortality (76.5-55.3; p < 0.001). Also in women of the Czech Republic a decline of the mortality from all caused was recorded (390.1-328.5; p < 0.001), the cardiovascular mortality (135.3-103.8; p < 0.001), ischaemic heart disease (58.0-48.6; p < 0.001) and cerebrovascular mortality (43.5-27.4; p < 0.001). In 1990 an increased cardiovascular mortality was recorded in men different from the trend during 1984-1993, statistically significant in the Czech Republic (p < 0.05) and in the six districts (p < 0.05). The reasons of this trend are not clear. The role of health services in the mortality drop is not clear, although available data indicate their improvement. Favourable changes were found in risk factors: during the period from 1985-1992 the prevalence of hypercholesterolaemia declined significantly in men and women, the prevalence of hypertension in women and the prevalence of smoking in men declined in the six districts. From nationwide data ensues that after 1989 significant changes occurred in the diet of the Czech population. The meat consumption declined by 1993 by 13%, the milk and dairy product consumption by 26.8% the butter consumption by 43.6% the consumption of vegetable fats increased by 16%, of vegetables by 8%, tropical fruit by 43.2%. These changes probably had an impact on the cholesterol level and BMI of the Czech population. CONCLUSIONS: In the declining cardiovascular mortality trend during 1984-1993 the following may have participated: improved medical care, dietary changes, improvement of the risk profile and other, in particular socioeconomic factors. With regard to the close temporal association of the investigated changes it may be assumed that this development is at least partly associated with changes of the political and economic position in the Czech Republic after 1989.     

Rapid DNA sequencing of more than 1000 bases per run by capillary electrophoresis using replaceable linear polyacrylamide solutions

The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solutions has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and separation conditions, and enhanced base-calling software. The influences of LPA molecular weight and concentration on separation were investigated. Additionally, the separation buffer, column temperature, and electric field were adjusted to increase the number of resolvable DNA fragments per run while maintaining an enhanced separation speed. Using low concentrations [2% (w/v)] of high molecular weight LPA polymers (> 5.5 x 10(6) Da), elevated column temperature (50 degrees C) and moderately high field (150 V/cm), rapid sequencing analysis for more than 1000 bases on a model ssM13mp18 template was obtained with 96.8% accuracy.     

Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase

Eight polar amino acid residues in the putative substrate-binding region from Thr-360 to Val-379 in human endothelial nitric-oxide synthase (eNOS) (Thr-360, Arg-365, Cys-368, Asp-369, Arg-372, Tyr-373, Glu-377, and Asp-378) were individually mutated. Only two of these residues, Asp-369 and Arg-372, were found to be essential for enzyme activity. A further series of mutants was generated by replacing these two residues with various amino acids and the mutant proteins were expressed in a baculovirus system. Mutant eNOS had a very low L-citrulline formation activity with the exception of D369E and R372K, which retained 27% and 44% of the wild-type enzyme activity, respectively. Unlike the wild-type enzyme, all mutants except D369E, R372K, and R372M had a low spin heme (Soret peak at 416 nm). All the Asp-369 mutants had higher Kd values for L-arginine (1-10 mM) than wild-type eNOS (0.4 microM) and an unstable heme-CO complex, and except for D369E, had a very low (6R)-5,6,7, 8-tetrahydro-L-biopterin (BH4) content. In contrast, each of Arg-372 mutants retained a considerable amount of BH4, had a moderate reduction in L-arginine affinity, and had a more stable heme-CO complex. 1-Phenylimidazole did not bind to wild-type eNOS heme, but bound to all Asp-369 and Arg-372 mutants (Kd ranged from 10 to 65 microM) except R372K. Heme spin-state changes caused by binding of 3, 5-lutidine appeared to depend on both charge and size of the side chains of residues 369 and 372. Furthermore, all Asp-369 and Arg-372 mutants were defective in dimer formation. These results suggest that residues Asp-369 and Arg-372 in eNOS play a critical role in oxygenase domain active-site structure and activity.     

Biochemical and molecular biological characterization of a lipase produced by the fungus Rhizopus delemar

The results of a comprehensive biochemical and molecular biological investigation of the lipase produced by the mycelial fungus Rhizopus delemar are described. This enzyme cleaves and synthesizes primary esters and related bonds, exhibits 1,3-positional selectivity in its actions on glycerides, and is a member of a family of enzymes that have been widely used in applied biocatalysis. Use of glycerol as main carbon source rather than glucose or lipid supported mycelial growth and lipase production. The enzyme was purified to homogeneity and characterized. Pure lipase was crystallized and its three-dimensional structure determined. The enzyme was found to adopt a configuration similar to those of other members of its homologous family. The structural data also indicated that lipases possess greater configurational mobility than had been previously appreciated. A complementary DNA clone was isolated that contained the full length lipase gene. The nucleic acid sequence of this cDNA indicated that it was initially synthesized as a preproenzyme, and allowed determination of the complete predicted amino acid sequence of the lipase, and its comparison to the sequences of related enzymes. Truncated forms of the cloned cDNA were produced that encoded either mature or prepro-lipase. These DNAs were introduced into a tightly regulated E. coli expression system, overcoming the toxicity of the enzyme while also allowing overproduction of lipase. Molecular modelling was employed to guide the rational mutagenesis of the enzyme, identifying sites within the substrate binding region that regulated substrate selectivity. Mutant lipases were generated with altered substrate specificities, creating novel enzymes and beginning the definition of structure-function relationships in the lipolytic enzymes.     

A sub‐Saharan African perspective on mycotoxins in beer – a review

Beer is an alcoholic beverage made from a cereal grain extract and is widely consumed in sub‐Saharan Africa and the world at large. However, beer consumption could expose consumers to mycotoxins. In this review, we appraised the different mycotoxins associated with beer contamination, elucidating their structures and incidence in cereals involved in beer production. The common mycotoxins that are found within the brewing process are reviewed. These include aflatoxin B₁ (AFB₁), fumonisin (FB), ochratoxin A (OTA), zearalenone (ZEA) and deoxynivalenol (DON), which are the prime contaminants in beer produced in sub‐Saharan Africa. Residual levels of <20% of AFB₁, OTA and FB₂ together with the transformation of ZEA (into a less toxic compound β‐zearalenol) can be achieved during the production of beers originating from Europe/America, while >50% of DON and higher ratios of FB₁ can be recovered in finished beer. Adsorption is the major means of mycotoxin removal during beer production. In contrast, traditional African beer processes show no significant efficient removal of mycotoxins. This is because the prevailing environmental conditions during beer production are favourable to mycotoxigenic fungal proliferation. This subsequently leads to relatively high concentration of mycotoxins in freshly processed beer, with a possible increase during the beer shelf‐life owing to the absence of appropriate microbial stabilisation treatments in the finished processed beer. © 2019 The Institute of Brewing & Distilling     

Peptide-Based Identification of Phytophthora Isolates and Phytophthora Detection in Planta

Phytophthora is arguably one of the most damaging genera of plant pathogens. This pathogen is well suited to transmission via the international plant trade, and globalization has been promoting its spread since the 19th century. Early detection is essential for reducing its economic and ecological impact. Here, a shotgun proteomics approach was utilized for Phytophthora analysis. The collection of 37 Phytophthora isolates representing 12 different species was screened for species-specific peptide patterns. Next, Phytophthora proteins were detected in planta, employing model plants Solanum tuberosum and Hordeum vulgare. Although the evolutionarily conserved sequences represented more than 10% of the host proteome and limited the pathogen detection, the comparison between qPCR and protein data highlighted more than 300 protein markers, which correlated positively with the amount of P. infestans DNA. Finally, the analysis of P. palmivora response in barley revealed significant alterations in plant metabolism. These changes included enzymes of cell wall metabolism, ROS production, and proteins involved in trafficking. The observed root-specific attenuation in stress–response mechanisms, including the biosynthesis of jasmonates, ethylene and polyamines, and an accumulation of serotonin, provided the first insight into molecular mechanisms behind this particular biotic interaction.     

DNA sequence analysis of Prinker-modified restriction fragments after collection from capillary electrophoresis with replaceable matrices

This paper demonstrates the procedure of sequencing DNA restriction fragments isolated by a recently developed fraction collector after CE separation. In particular, using pBr 322 plasmid as a model system, a double digest was performed with Eco RI and Pst 1 restriction enzymes to produce two fragments of 749 base pairs (bp) and 3612 bp, both with cohesive ends. Prinkers, specific linkers complementary to the cohesive ends, were then ligated to both fragments (increasing the size by 59 bp each). These Prinker-modified fragments were separated by CE and collected. The success of the collection was demonstrated by reinjection of each isolated fraction with laser-induced fluorescence detection, using ethidium bromide as intercalater. The 808 bp isolated fragment was then polymerase chain reaction-amplified with appropriate primers for the Prinker ends, followed by cycle sequencing. Both strands of the fragment were run on an ABI 373, sequencing 427 bases and 450 bases, respectively, with a read accuracy of 99.3%. This approach with Prinker-modified restriction fragment and automated CE fraction collection can be used as a general procedure for sequencing unknown genomic DNA as well as mutated DNA mixtures.     

Optimization of fermentation conditions for ting production using response surface methodology

This study investigated the effect of fermentation conditions (time and temperature) of sorghum on the composition of ting, using the Doehlert design of response surface methodology (RSM). Fermentation temperature and time were optimized and pH, titratable acidity (TTA), total viable bacteria count (TBC), total lactic acid bacteria count (TLABC), total fungal and yeast count (TFYC), tannin content (TNC), total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities (AA) were determined. Experimental and predicted values obtained were similar, with statistical indices indicating the validity of the models generated (R² between 93.45 and 99.71%, AAD values close to 0, B_f and A_f values close to 1). Numerical multi‐response optimization of parameters suggested optimal fermentation conditions to be 34 °C for 24 hr. Physicochemical characterization of ting samples using scanning electron microscopy (SEM), X‐ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FTIR) showed slight changes in morphology, similarity in diffraction patterns and presence of different functional groups, respectively. Results of this study could provide information for the commercialization of quality ting.

Practical applications

Response surface methodology was used to study the influence of fermentation conditions on the quality of ting and optimal fermentation conditions were obtained at 34 °C for 24 hr. The findings in this study will be useful for ting processors to obtain a product with maximal beneficial composition and traits.     

Endothelial nitric-oxide synthase. Evidence for bidomain structure and successful reconstitution of catalytic activity from two separate domains generated by a baculovirus expression system

A baculovirus system was used to express the oxygenase and reductase domains of human endothelial nitric-oxide synthase (ecNOS) as distinct proteins. The oxygenase domain (residues 1-491) was expressed using a vector containing a His6 tag at the N terminus. The purified oxygenase domain had an apparent molecular mass of approximately 54 kDa, and retained the ability to bind L-arginine and form the ferrous CO complex. The purified reductase domain (residues 492-1244) had an apparent molecular mass of approximately 82 kDa and retained the ability to catalyze NADPH-dependent cytochrome c reduction, which was enhanced 10-fold by the presence of Ca2+/calmodulin. Both purified domains exhibited immunoreactivity to rabbit anti-ecNOS IgG. The NOS activity was successfully reconstituted by mixing the two domains. These results demonstrate for the first time that the two domains of ecNOS are catalytically intact and can be reconstituted in vitro.