Autotaxin and its product lysophosphatidic acid suppress brown adipose differentiation and promote diet-induced obesity in mice

Brown adipose tissue is a thermogenic organ that dissipates stored energy as heat to maintain body temperature. This process may also provide protection from development of diet-induced obesity. We report that the bioactive lipid mediator lysophosphatidic acid (LPA) markedly decreases differentiation of cultured primary brown adipocyte precursors, whereas potent selective inhibitors of the LPA-generating enzyme autotaxin (ATX) promote differentiation. Transgenic mice overexpressing ATX exhibit reduced expression of brown adipose tissue-related genes in peripheral white adipose tissue and accumulate significantly more fat than wild-type controls when fed a high-fat diet. Our results indicate that ATX and its product LPA are physiologically relevant negative regulators of brown fat adipogenesis and are consistent with a model in which a decrease in mature peripheral brown adipose tissue results in increased susceptibility to diet-induced obesity in mice.     

Hierarchical recruitment into nascent ribosomes of assembly factors required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

To better define the roles of assembly factors required for eukaryotic ribosome biogenesis, we have focused on one specific step in maturation of yeast 60 S ribosomal subunits: processing of 27SB pre-ribosomal RNA. At least 14 assembly factors, the ‘B-factor’ proteins, are required for this step. These include most of the major functional classes of assembly factors: RNA-binding proteins, scaffolding protein, DEAD-box ATPases and GTPases. We have investigated the mechanisms by which these factors associate with assembling ribosomes. Our data establish a recruitment model in which assembly of the B-factors into nascent ribosomes ultimately leads to the recruitment of the GTPase Nog2. A more detailed analysis suggests that this occurs in a hierarchical manner via two largely independent recruiting pathways that converge on Nog2. Understanding recruitment has allowed us to better determine the order of association of all assembly factors functioning in one step of ribosome assembly. Furthermore, we have identified a novel subcomplex composed of the B-factors Nop2 and Nip7. Finally, we identified a means by which this step in ribosome biogenesis is regulated in concert with cell growth via the TOR protein kinase pathway. Inhibition of TOR kinase decreases association of Rpf2, Spb4, Nog1 and Nog2 with pre-ribosomes.     

Biomarkers of mild hyperthermia related to flashover training in firefighters

Flashover phenomenon occurs when surfaces exposed to thermal radiation reach the ignition temperature, and the fire rapidly spreads in enclosed area. Flashover training (FOT) performed by firefighters is a simulation of flashover phenomenon under controlled conditions. The study aimed to test thermal and physical strain in male firefighters and instructors attending FOT and its influence on DNA damage, exhaled breath condensate (EBC) pH, and fraction of exhaled nitric oxide (FeNO). DNA damage markers were analyzed in 51 attendees and 7 instructors, and EBC pH and FeNO in 40 respiratory healthy non-smoking subjects (34 attendees and 6 instructors).The average body temperature and pulse increase was 1.1 °C and 30 beats per minute, respectively. A prominent increase in the alkali-labile sites' level has been observed in instructors' peripheral leukocytes compared to first-time attendees (tail length p=0.050, % of DNA in tail p=0.005). FOT was related only to physiological FVC and FEV₁ increase (by 4% and 2.7% on average), and FeNO dropped after the exercise by 2 ppb in comparison with basal values (P=0.034). EBC pH did not change during FOT, but FeNO was inversely correlated to EBC pH after the exercise (Spearman's rho=−0.66, P=0.013). With respect to the thermal and physical strain, FOT is considered to be a safe training procedure for healthy firefighters. The increase rate in primary DNA damage found in the instructors' peripheral leukocytes requires further examination in a larger sample size.     

Distribution of Chlamydia trachomatis Serotypes in Clinical Urogenital Samples from North-Eastern Croatia

The purpose of this study was to determine prevalence of Chlamydia trachomatis (Ct) urogenital infection and its serotype distribution from clinical samples in north-eastern Croatia. During a 3-year period, 2,379 urogenital samples were analyzed by real-time polymerase chain reaction (A group), while 4,846 genital swabs were analyzed by direct fluorescent antibody test (B group). 132 Ct positive specimens were genotyped by omp1 gene sequencing. The prevalence rate of Ct was 3.2 % in A and 1 % in B group. The most prevalent chlamydial genotype was E (44 %), followed by F (33 %), K (11.5 %), G (8 %), J/UW (5.3 %), D-IC (4.4 %), D-B120 (1.8 %), and B/IU, J/IU, Ia/IU (0.9 % each) serotypes. Single-nucleotide polymorphisms (SNPs) of omp1 gene were detected in E, K, and G serotypes. Some of these SNPs (C/T at position 272 and G/A at position 813 in E strain; C/T at position 884 in D strain) might represent novel omp1 variants.     

Oral health awareness in Croatian and Italian urban adolescents

Purpose of this study was to investigate and compare differences in oral health awareness between Croatian and Italian urban adolescents. The sample consisted of primary school last grade students aged between 13 and 15 years, 300 children from Zagreb (Croatia) and 298 children from Bari (Italy). Oral health awareness was evaluated using a self-administered standardized questionnaire. Self-perception of oral health proved to be different between the two groups (p < 0.001). The Croatians reported that their oral health was "excellent" or "very-good" more often than the Italians (68.6% vs. 50.2%). The reasons given for visiting a dentist were different (p < 0.001). The Italians cleaned their teeth more often than the Croatians ("two or more times a day", 83.1% vs. 72.2%, p < 0.003). Wooden toothpicks were preferred by the Croatians (p < 0.001), while floss was preferred by the Italians (p = 0.03). The awareness regarding the use of fluoridated toothpaste was higher in the Italian group (95.6% vs. 72.5%, p < 0.001). The Croatians were consuming sweetened foods more often than the Italians (p < 0.001). Croatian adolescents reported more indicators of a lower level of oral health awareness than the Italians, while on the contrary Croatians had higher esteem of their oral health. Defining national preventive strategies is essential for improving adolescents' attitudes toward oral health in both countries, particularly in Croatia.     

Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-deficient mouse

Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.     

A Simple and Efficient DNA Isolation Method for Salvia officinalis

We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4?M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6?μg DNA g^?1 of dry leaf tissue, and the absorbance ratios 260/280 and 260/230?nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.     

Metabolic control analysis of cellular respiration in situ in intraoperational samples of human breast cancer

The aim of this study was to analyze quantitatively cellular respiration in intraoperational tissue samples taken from human breast cancer (BC) patients. We used oxygraphy and the permeabilized cell techniques in combination with Metabolic Control Analysis (MCA) to measure a corresponding flux control coefficient (FCC). The activity of all components of ATP synthasome, and respiratory chain complexes was found to be significantly increased in human BC cells in situ as compared to the adjacent control tissue. FCC(s) were determined upon direct activation of respiration with exogenously-added ADP and by titrating the complexes with their specific inhibitors to stepwise decrease their activity. MCA showed very high sensitivity of all complexes and carriers studied in human BC cells to inhibition as compared to mitochondria in normal oxidative tissues. The sum of FCC(s) for all ATP synthasome and respiratory chain components was found to be around 4, and the value exceeded significantly that for normal tissue (close to 1). In BC cells, the key sites of the regulation of respiration are Complex IV (FCC?=?0.74), ATP synthase (FCC?=?0.61), and phosphate carrier (FCC?=?0.60); these FCC(s) exceed considerably (~10-fold) those for normal oxidative tissues. In human BC cells, the outer mitochondrial membrane is characterized by an increased permeability towards adenine nucleotides, the mean value of the apparent K(m) for ADP being equal to 114.8?±?13.6?μM. Our data support the two-compartment hypothesis of tumor metabolism, the high sum of FCC(s) showing structural and functional organization of mitochondrial respiratory chain and ATP synthasome as supercomplexes in human BC.     

Associations of HLA DR and DQ molecules with Lyme borreliosis in Latvian patients

ABSTRACT: BACKGROUND: Many autoimmune diseases are associated with variants of HLA genes such as those encoding the MHC complex. This correlation is not absolute, but may help in understanding of the molecular mechanism of disease. The purpose of this study was to determine HLA-DR,-DQ alleles in Latvian patients with Lyme borreliosis and control (healthy) persons. Case patients and control subjects were similar in age, gender and ethnic heritage and differed only as regards the presence of Borrelia burgdorferi infection. The study included 20 patients with clinical stage - erythema migrans and 25 control (healthy) persons. HLA genotyping was performed by PCR with sequence-specific primers. RESULTS: The results show difference in HLA-DRB1 alleles distribution between patients and control subjects. The frequencies of HLA-DRB1 *04 (OR 8.65; p<0.022) and HLA-DRB1 *17 (03) (OR 7.00; p<0.048) were increased in the Lyme disease patients. And the frequency of allele DRB1*13 (OR 0.13; p<0.033) was lower in Borreliosis patients and higher in control group. But, significant differences in frequencies of HLA-DQ alleles we did not detect. CONCLUSIONS: HLA predisposition to Lyme borreliosis appears not to be limited to HLA molecules, but some HLA-DR alleles also have a significant influence, and, may have implications in our understanding of pathogenesis of this disease. In particular, HLA-DRB1*04 and DRB1 *17 (03) may contribute to the Lyme borreliosis development in Latvian population KEYWORDS: Lyme borreliosis, HLA alleles, PCR.     

<Emphasis Type="Italic">In vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis> antioxidant effects of three Veronica species

The aim of the present study was to examine the antioxidant activity of three Veronica species (Plantaginaceae). The antioxidant potential of various extracts obtained from aerial flowering parts was evaluated by DPPH-free (1,1-diphenyl-2-picrylhydrazyl-free) radical scavenging activity and ferric-reducing antioxidant power assays. Considerable antioxidant activity was observed in the plant samples (FRAP values ranged from 0.97 to 4.85 mmol Fe²⁺/g, and DPPH IC₅₀ values from 12.58 to 66.34 μg/ml); however, these levels were lower than the activity of the control compound butylated hydroxytoluene (BHT) (FRAP: 10.58 mmol Fe²⁺/g; DPPH IC₅₀: 9.57 μg/ml). Also, the in vivo antioxidant effects were evaluated in several hepatic antioxidant systems in rats (activities of glutathione peroxidase, glutathione reductase, peroxidase, catalase, xanthine oxidase, glutathione content and level of thiobarbituric acid reactive substances) after treatment with different Veronica extracts, or in combination with carbon tetrachloride (CCl₄). Pretreatment with 100 mg/kg b.w. of Veronica extracts inhibited CCl₄-induced liver injury by decreasing TBA-RS level, increasing GSH content, and bringing the activities of CAT and Px to control levels. The present study suggests that the extracts analyzed could protect the liver cells from CCl₄-induced liver damage by their antioxidative effect on hepatocytes.