Efficacy of healthy weight loss program in obesity treatment: Croatian experience

We evaluated the efficiency of a six-month outpatient weight loss treatment program combining healthy diet, fat reduction, psychological counseling, exercise, and orlistat treatment, by measuring body weight and levels of cardiovascular risk factors in 476 subjects with BMI over 30 or 28 with increased blood pressure, cholesterol, and sugar at the baseline and at the end of program. After four weeks of adjustment to a mild low-calorie diet (1600 kcal/day) and counseling, subjects started receiving orlistat (120 mg TID). The mean weight loss after 6 months was 10.9%. Systolic pressure dropped by 6.7%, diastolic by 4.2%, fasting blood glucose by 10.1%, and total cholesterol by 9.8%. Only 9 subjects (7.8%) poorly tolerated the treatment. More men than women were able to maintain the achieved weight loss six months after the program (70.6% vs. 58.3%, respectively). The healthy weight loss program was an efficient approach to obesity treatment.     

Expression and purification of glycine N-methyltransferases in Escherichia coli

Expression and purification of recombinant mouse, rat, and human glycine N-methyltransferases (GNMTs) in pTYB and pET expression vectors was done in order to prepare the proteins for structure studies of the enzymes from different sources. When human and mouse GNMTs were expressed in pTYB vector as a fusion protein with intein and the chitin binding domain, an unusual cleavage of intein was found. This cleavage takes place at two sites near the N-terminus of intein. This resulted in the appearance of an abnormal GNMT protein after on-column cleavage of the fusion protein, which could not be separated from normal GNMT. For this reason expression of mouse, rat, and human GNMTs was done in the pET-17b expression vector, resulting in the expression of soluble protein at about 20-40mg/L of culture. A new procedure for GNMT isolation after expression in the pET vector was developed. This included only two steps, ammonium sulfate precipitation and ion-exchange chromatography, and resulted in preparations containing 95-97% pure protein. All expressed proteins were tetrameric with molecular weights of 130kDa as determined by size-exclusion chromatography. Activity in Tris buffer at pH 9 of mouse, rat, and human GNMTs was found to be 255, 260, and 540U/mg, respectively. This implies that expressed and purified GNMT proteins are biologically active and suitable for biochemical and structural studies.     

A bacterial selection for the directed evolution of pyruvate aldolases

A novel bacterial in vivo selection for pyruvate aldolase activity is described. Pyruvate kinase deficient cells, which lack the ability to biosynthetically generate pyruvate, require supplementation of exogenous pyruvate when grown on ribose. Supplementation with pyruvate concentrations as low as 50 microM rescues cell growth. A known substrate of the KDPG aldolases, 2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (KHPB), also rescues cell growth, consistent with retroaldol cleavage by KDPG aldolase and rescue through pyruvate release. An initial round of selection against 2-keto-4-hydroxyoctonate (KHO), a nonsubstrate for wild-type aldolase, produced three mutants with intriguing alterations in protein sequence. This selection system allows rapid screening of mutant enzyme libraries and facilitates the discovery of enzymes with novel substrate specificities.     

5-methyltetrahydrofolate is bound in intersubunit areas of rat liver folate-binding protein glycine N-methyltransferase

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.     

Delayed fluorescence in algal growth inhibition tests

A series of 72-hour growth inhibition tests with green alga Desmodesmus (Scenedesmus) subspicatus (ISO 8692) has been performed to test the delayed fluorescence (DF) parameters as possible endpoint measurements. Sensitivity to five toxicants with direct and indirect effects on photosynthesis was tested, and the median effective concentration (EC₅₀) values derived from the cell concentration, absorbance and DF were compared. The sensitivity of DF intensity (DFI) was comparable with the two endpoints suggested in ISO 8692 for all five toxicants: potassium dichromate, 3,5-dichlorophenol (3,5-DCP), DCMU, copper and cadmium. In the case of potassium dichromate and copper, DFI was more sensitive than the other endpoints in this study. The analysis of DF relaxation kinetics showed a specific response to the herbicide DCMU. Additionally, a 24-hour test was performed with the same toxicant concentrations (except copper), where DFI was measured 30 minutes, one hour and 24 hours after the exposure. The influence of toxicants on DFI after a 24-hour exposure was comparable with the effects after 72 hours. Only 3,5-DCP influenced DFI after 30 minutes. DF proved to be a simple, reliable and rapid measurement to assess toxicity in algal tests, which can to some extent differentiate among various toxicants.     

Predictive parameters of gestational diabetes mellitus

Gestational diabetes mellitus is a carbohydrate intolerance recognized in pregnancy. The objective of this study was to determine the prevalence of gestational diabetes mellitus (GDM) of all deliveries at the University Hospital Rijeka, Croatia (34 997 deliveries over 10-year period) using 2-hour 75 g oral glucose tolerant test and to evaluate the impact of GDM on neonatal outcomes and mother's health. Gestational diabetes was diagnosed in 55 of 128 pregnant women with suspected glucose intolerance. Logistic regression analysis was used to examine the relationship between fasting plasma glucose, age, family history, body mass index, maternal weight gain, neonatal weight, neonatal head diameter and Apgar score in the gestational diabetes group and in the non-diabetes group. The results indicate that fasting plasma glucose greater than 7.0 mmol/L and maternal overweight are strong predictors for GDM and macrosomia. There was no difference in the mode of delivery, and vitality and metabolic complications among the infants of all analyzed mothers. We concluded that to prevent GDM as well as to reduce the rate of macrosomic infants good glycemic control should be initiated as soon as possible. The 2-hour 75 g OGTT is worth enough to evaluate GDM. Women should be counseled and encouraged to lose weight before or at the beginning of the conception period.     

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens

Applied Microbiology and Biotechnology - Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes....     

Molecular Epidemiology of Crimean-Congo Hemorrhagic Fever Virus in Kosovo

Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic agent that causes severe, life-threatening disease, with a case fatality rate of 10–50%. It is the most widespread tick-borne virus in the world, with cases reported in Africa, Asia and Eastern Europe. CCHFV is a genetically diverse virus. Its genetic diversity is often correlated to its geographical origin. Genetic variability of CCHFV was determined within few endemic areas, however limited data is available for Kosovo. Furthermore, there is little information about the spatiotemporal genetic changes of CCHFV in endemic areas. Kosovo is an important endemic area for CCHFV. Cases were reported each year and the case-fatality rate is significantly higher compared to nearby regions. In this study, we wanted to examine the genetic variability of CCHFV obtained directly from CCHF-confirmed patients, hospitalized in Kosovo from 1991 to 2013. We sequenced partial S segment CCHFV nucleotide sequences from 89 patients. Our results show that several viral variants are present in Kosovo and that the genetic diversity is high in relation to the studied area. We also show that variants are mostly uniformly distributed throughout Kosovo and that limited evolutionary changes have occurred in 22 years. Our results also suggest the presence of a new distinct lineage within the European CCHF phylogenetic clade. Our study provide the largest number of CCHFV nucleotide sequences from patients in 22 year span in one endemic area.