Bacterial Fibrinolytic Enzyme Coding Sequences from Indonesian Traditional Fermented Foods Isolated Using Metagenomic Approach and Their Expression in Escherichia Coli

Fibrinolytic proteins play an important role in treatment of cardiovascular disease. This study aimed to express coding sequences of new bacterial fibrinolytic protein from Indonesian traditional fermented foods obtained by a PCR-based metagenomic approach and to test their biological activity. New amino acid variations were found in nattokinase (NAT) at several positions i.e. D41N and V192A from dried Tauco, V4F, D41N, and V192A from yellow Oncom but not in Douchy Fibrinolytic Enzyme (DFE) from Terasi. The NAT and DFE recombinant versions were overproduced in Escherichia coli BL21(DE3) using pET16b(+). Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis analysis showed all clones produced mature soluble fibrinolytic proteins of 28 kDa. All recombinant proteins displayed caseinolytic and fibrinolytic activities. In conclusion, metagenomic approach can be applied to obtain and to express coding sequences of new variant bacterial fibrinolytic protein as active soluble mature form in E. coli.     

Folate food intake and red blood cell folate concentrations in women from Recife, Northeast of Brazil

Folate food intake and red blood cell folate concentrations were assess in women from Recife, Northeast Brazil. Following a two stages sampling procedure, a cross-sectional study was carried out involving 360 women, between 15-45 y, attending in nine health care public unit in 2007-2008. Folate intake was evaluated by a Quantitative Food Frequency Questionnaire, and compared with the values of the dietary reference intakes-DRI's. Folate status was evaluated by red blood cell folate concentrations. Geometric mean of folate intake was 627.1 [IC 95% 600.4-655.0] microg/day. The frequency of women at risk for folate intake below the recommendation was 16.0% for adolescents (<330 microg/day) and 6.3% for young adult (<320 microg/ day). The prevalence of women whose consumption exceeds the maximum tolerable intake was 48.0% (> 800 microg/day) and 13.7% (> 1000 microg/day) for adolescents and adults, respectively. The mean of red blood cell folate concentrations was 1797.8 +/- 357.1 nmol/L. Folate rich-food intake did not show any correlation with red blood cell folate concentrations (r = 0.058 and p = 0.274). Higher red blood cell folate concentrations were observed in adult young women (p = 0.004) and among those with income up to two minimum wages (p = 0.042). Folate rich-food intake as well as red blood cell folate concentrations among women from Recife were above the international recommendations.     

Vitamin A deficiency in preschool children of Recife, Northeast of Brazil

The purpose of the study was to evaluate the extent of vitamin A deficiency (VAD) among preschool children in the city of Recife, Northeast Brazil. The sample comprised 344 children of both sexes, 24 to 60 months old, in 18 public day care centres in the city of Recife, in 2007. The nutritional status of vitamin A was assessed by biochemical (serum retinol) and dietetic (vitamin A rich-food consumption) indicators and the pondo-stature status through anthropometric indicators weight-for-age, height-for-age and weight-for-height. The prevalence of hyporetinolemia (< 0.70 micromol/L) was 7.7% (IC 95% 4.88 - 11.81), which characterizes the VAD as a light-type public health problem, according to World Health Organization criteria. On the other hand, 29.6% (IC 95% 24.22 - 35.63) of children had acceptable or marginal levels (0.70 to 1.04 micromol/L) of retinol. Regarding the vitamin A rich-food intake, values below the EAR (Estimated Average Requirement) - 210 microg/ day for children of 1 to 3 years old and 275 microg/day for children of 4 to 8 years old - were 8.1% and 21.3% respectively. The prevalence of anthropometrical deficits (< -2 scores -Z) in preschool children were 2.5% for the indicator weight-for-age, 8.6% for height-for-age and 1.5% for weight-for-height. The research findings point out to the importance of institutionalization for the appropriate nutritional status of children and maintenance of adequate reserves of vitamin A. However, more studies are needed focusing on non-institutionalized preschool, or children living outside the privileged environment of public day care centres.     

Vitamin A deficiency in elderly attending the Health Family Programme in Camaragibe, PE, Brazil

Vitamin A deficiency (VAD) is a major nutritional problem in many developing countries. However, the extent of the problem among elderly people has not been well established. The current study aimed at identifying the prevalence of VAD among elderly people attending the Family Health Programme (FHP) in the city of Camaragibe, PE, Northeast Brazil. Following a systematic sampling procedure, a cross-sectional study was carried out involving 315 subjects = 60 years, of both sexes, in 2003. VAD was assessed by serum retinol levels and vitamin A-rich-food intake by a food frequency method. The prevalence of VAD (Serum ret. < 1.05 microMol/L) was 26.1% (95% CI 21.2 - 31.6) and the frequency of animal and vegetal origin vitamin A-rich foods intake = 3x/week was 46.1% (IC95% 40.7 - 52.0) and 63.2% (IC95% 57.5 - 69.5), respectively. Serum retinol levels were not correlated to sex (p = 0.54) and age (p = 0.34) distribution. In the same way, serum retinol was not related to vitamin A rich-food intake (p > 0.05). VAD seems to be very prevalent among elderly people attending the HFP in Camaragibe. Concerted actions to prevent and control VAD are strongly recommended in this ecological context.     

Alimentary, nutritional and health aspects of elderly women in attendance at the Núcleo de Atenção ao Idoso, Recife/2005

Aging has become a worldwide problem. In order to identify food, nutritional and health aspects of elderly women, a across-sectional study was carried out in 2005 with 106 women over 60 years of age cared for at Núcleo de Aten??o ao idoso-NAI at Federal University of Pernambuco. Clinical and social-demographic variables, along with the nutritional status by BMI (OMS and Lipschtz) were assessed, and the frequency of food intake according to food groups. The results disclosed that 38% of the elderly were pre-obese (WHO) and 47.2% were overweight (Lipschitz). As for blood pressure (BP) 69% was found to be hypertensive and 31% normotensive. The BMI mean of hypertensive and normal elderly women was statistically significant (p=0.027). Cereal and bread groups were reported to have been the highest consumption on a daily basis by the elderly women, 89% and 82% respectively. Legumes were present in 53% of the women's meals followed by vegetables with 61% and fruits with 66%. Concerning water intake, 51.9% stated to have ingested one to four glasses of water daily. There was a positive correlation between BMI and systolic (SP) and diastolic pressure (DP) (p<0.001). SP and DP levels showed no correlation with the food intake studied. The results are indicative of an overweight and not conscientious population as regards adequate food intake and point to the necessity of more effective actions in control and/or prevention, particularly for those still young.     

Changes in Non-Deamidated versus Deamidated Epitope Targeting and Disease Prediction during the Antibody Response to Gliadin and Transglutaminase of Infants at Risk for Celiac Disease

Celiac disease (CeD) is a conditional autoimmune disorder with T cell-mediated immune response to gluten coupled with antibody production to gliadin and the self-protein tissue transglutaminase (TG2). TG2 contributes to the CeD pathomechanism by deamidating gliadin, thereby generating more immunogenic peptides. Anti-gliadin antibodies may appear before the autoantibody production. The scope of this study was to dissect these early antibody responses by investigating serum samples collected during the PreventCD prospective double-blind study, where infants with high CeD risk were randomized to 200 mg daily gluten intake or placebo from 4 to 6 months of age, followed by frequent blood testing on regular gluten consumption in both groups. After primary gluten intake, children with or without later CeD produced IgA and IgG antibodies which preferentially recognized non-deamidated gliadin peptides. At CeD development with anti-TG2 seroconversion, there was a significant increase in the antibody reaction toward deamidated gliadin peptides (DGP), with maturation in the binding strength for the PEQPFP gamma-gliadin core peptide. The earliest produced autoantibodies targeted TG2’s celiac epitope 2. Our results reveal a qualitative change in the gliadin-directed humoral immune response at the time when anti-TG2 antibodies appear, but anti-DGP antibodies in the absence of anti-TG2 antibodies are not disease-predictive.     

Biochemical Characterisation of Human Transglutaminase 4

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell–cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.