Leukocyte migration inhibition studies with Epstein-Barr virus (EBV) determined nuclear antigen (EBNA) in relation to the EBV-carrier status of the donor

Cell extracts from EBV-genome-carrying cell lines inhibit the migration of leukocytes from EBV-positive but not seronegative healthy donors. In the present study extracts from EBV-negative lines and their own in vitro EBV-converted sublines were used to induce migration inhibition with leukocytes from seronegative and seropositive individuals. A clear difference was found between the extracts from EBV-negative and positive cell lines. Significant migration inhibition could be obtained with antigen(s) associated with the virus nonproducer state. Since EBNA is known to be expressed by all nonproducer EBV-genome-carrying cells, we have compared the effect of partially purified EBNA and correspondingly prepared mock-EBNA on the leukocyte migration. Purified EBNA inhibited the leukocyte migration of EBV seropositives, whereas mock-EBNA had no such effect.     

A novel neuroprotective agent with antioxidant and nitric oxide synthase inhibitory action

N(alpha)-vanillyl-N(omega)-nitroarginine (N - 1) that combines the active functions of natural antioxidant and nitric oxide synthase inhibitor was developed for its neuroprotective properties. N - 1 exhibited protective effects against hydrogen peroxide-induced cell damage and the inhibitory effect on nitric oxide 'NO' production induced by calcium ionophore in NG 108-15 cells. N - 1 inhibited the constitutive NOS isolated from rat cerebellar in a greater extent than constitutive NOS from human endothelial cells. Low binding energy (-10.2 kcal/mol) obtained from docking N - 1 to nNOS supported the additional mode of action of N - 1 as an nNOS inhibitor. The in vivo neuroprotective effect on kainic acid-induced nitric oxide production and neuronal cell death in rat brain was investigated via microdialysis. Rats were injected intra-peritonially with N - 1 at 75 micromol/kg before kainic acid injection (10 mg/kg). The significant suppression effect on kainic acid-induced NO and significant increase in surviving cells were observed in the hippocampus at 40 min after the induction.     

Identification of phosphorylation sites in glycine N-methyltransferase from rat liver

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.     

Acetylation of N-terminal valine of glycine N-methyltransferase affects enzyme inhibition by folate

Native liver glycine N-methyltransferase (GNMT) is N-acetylated while the recombinant enzyme is not. We show here that acetylation of the N-terminal valine affects several kinetic parameters of the enzyme. Glycine N-methyltransferase is a regulatory enzyme mediating the availability of methyl groups by virtue of being inhibited by folate. N-acetylation does not affect the overall structure of the protein and does not affect basal enzyme activity of GNMT. Binding of both the mono- and pentaglutamate forms of 5-methyltetrahydrofolate is the same for the acetylated and non-acetylated forms of the enzyme, however the pentaglutamate form is bound more tightly than the monoglutamate form in both cases. Although binding of the folates is similar for the acetylated and non-acetylated forms of the enzyme, inhibition of enzyme activity differs significantly. The native, N-acetylated form of the enzyme shows 50% inhibition at 1.3 microM concentration of the pentaglutamate while the recombinant non-acetylated form shows 50% inhibition at 590 microM. In addition, the binding of folate results in cooperativity of the substrate S-adenosylmethionine (AdoMet), with a Hill coefficient of 1.5 for 5-methyltetrahydrofolate pentaglutamate.     

Growth, delayed fluorescence and pigment composition of four Prorocentrum minimum strains growing at two salinities

Prorocentrum minimum is a potentially harmful and widely distributed marine dinoflagellate. Several P. minimum strains have already been studied, showing phylogenetical relations of strains isolated from the same geographical regions. Similarity among the strains was further examined on the basis of their physiology. Pigment composition and concentration, as well as delayed fluorescence (DF) decay kinetics and intensity, were measured in four P. minimum strains isolated from the Baltic and Adriatic Seas. The strains were grown at two salinities characteristic of the Baltic (8 PSU) and North Adriatic Seas (32 PSU). Strain differences in DF decay kinetics and growth did not always follow their genetic relations. While two strains showed similarities to the previously described strains from the Baltic and Adriatic Seas in DF parameters, the other two strains seemed to be specific. The differences among strains isolated from the same sea could stem from adaptations to conditions in the specific habitats. Cluster analysis based on the ratio of individual carotenoid pigments concentrations to the chlorophyll a concentration or to total carotenoids were not conclusive in showing relations among the strains. Among the measured pigments, only peridinin concentration depended on salinity in all st.     

Risk behaviors for getting HIV infection among the Croatian men who have sex with men

This study was conducted with the aim of obtaining the very first information on the sexual risk behavior of Croatian men who have sex with men (MSM). There were 1127 respondents recruited at four venues: three physical meeting places of the Croatian MSM population (disco club, bar and sauna) in Zagreb and one virtual (gay oriented web-site) meeting place of the Croatian MSM population. The overall response rate was only 19%. The rate of condom use during last anal intercourse was 59% and 56% of those who used a lubricant had chosen an incorrect product to use with latex condoms. There was no connection between drug-use and unprotected anal intercourse (UAI). Only a fifth of those who had also had sex with women (MSM/MSW) during last 12 months regularly used protection. The obtained findings will serve for focused and effective prevention activities and a basis for comparison in future research.     

Prosurvival effect of DHCR24/Seladin-1 in acute and chronic responses to oxidative stress

DHCR24/seladin-1, a crucial enzyme in sterol synthesis, is of lower abundance in brain areas affected by Alzheimer's disease. While high levels of DHCR24/seladin-1 exert antiapoptotic function by conferring resistance against oxidative stress, the molecular mechanism for this protective effect is not fully understood. Here we show that DHCR24/seladin-1 expression is up-regulated in an acute response and down-regulated in a chronic response to oxidative stress. High levels of DHCR24/seladin-1 were associated with elevated cholesterol concentrations and a general increase in cholesterol biosynthesis upon oxidative stress exposure in neuroblastoma SH-SY5Y cells. DHCR24/seladin-1 overexpression conferred resistance to oxidative stress in a cholesterol-dependent manner. Mutating the reductase activity within DHCR24/seladin-1 abolished this protective effect. Conversely, DHCR24/seladin-1 levels diminished upon chronic exposure to oxidative stress. Low levels of DHCR24/seladin-1 were associated with reduced p53 levels, independent of DHCR24 activity and cholesterol concentrations. Additionally, ablation of DHCR24/seladin-1 prevented apoptosis of primary neurons in a p53-dependent manner and reduced the response of critical p53 targets due to deficient stabilization of p53 and therefore elevated p53 ubiquitination and degradation. Our findings reveal a dual capacity of DHCR24/seladin-1, which appears to be involved in two mechanistically independent prosurvival effects, exerting an acute response and a chronic response to oxidative stress.     

Contributions to molecular systematics of water scavenger beetles (Hydrophilidae,Coleoptera)

Phylogenetic relationships within Hydrophilidae were examined by analyses of separate and combined nuclear and mitochondrial markers (28S rRNA, 18S rRNA, 16S rRNA, 12S rRNA, COI and COII genes). The preferred (Bayesian) tree topology suggests a sister group relationship between Spercheidae and Hydrophilidae, supporting the ‘hydrophilid lineage’; Epimetopidae are placed on the base of the ‘helophorid branch’, the monophyly of Sphaeridiinae is highly supported, nested deeply within Hydrophilidae closest to Enochrus, making Hydrophilinae and Acidocerini paraphyletic; Hydrobius appears as sister taxon to (Hydrochara + Hydrophilus) without a closer relationship to Acidocerini; the hydrophiloid–histeroid sister group relationship is confirmed. The topology of several taxa remains contradictory, and requires further investigations with a larger taxon sampling and additional molecular markers.     

Glycerol-induced membrane stiffening: the role of viscous fluid adlayers

Lipid interfaces, ranging from cell membranes to thin surfactant layers that stabilize lung alveoli, are integral to living systems. Such interfaces are often subjected to mechanical forces, and because of their membrane-like geometry, they can easily deform by bending into localized folds. In this work, we explore the role of small molecules (i.e., glycerol) on the mechanical stability of model lung surfactant monolayers. We demonstrate that the presence of glycerol increases local monolayer bending stiffness by orders of magnitude. Our x-ray and neutron reflectivity measurements indicate that water is preferentially depleted, or glycerol is preferentially enriched, at the lipid headgroup/solvent interface, and that this glycerol-enriched layer extends beneath the monolayer with an adsorption free energy of −2.5 to −4.6 kJ/mol. The dramatic change in membrane bending stiffness in the presence of the sugar adlayer is understood in terms of two models: 1), lipid antiplasticization by glycerol; and 2), a continuum mechanical model of the viscous adlayer.     

F508del-CFTR increases intracellular Ca(2+) signaling that causes enhanced calcium-dependent Cl(-) conductance in cystic fibrosis

In many cells, increase in intracellular calcium ([Ca(2+)](i)) activates a Ca(2+)-dependent chloride (Cl(-)) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl(-) transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca(2+)](i) increase is Cl(-) dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca(2+). This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca(2+)](i). Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP(3) (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca(2+) signaling.