dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.     

Expression of carbonic anhydrases II, IV, VII, VIII and XII in rat brain after kainic acid induced status epilepticus

Carbonic anhydrases (CAs) are important enzymes in the central nervous system (CNS), where they participate in regulating cerebrospinal fluid (CSF) secretion, blood-brain barrier and glial cell function. Using RT-PCR we found CA XII mRNA in rat and mouse brain. Cloning of rat CA XII revealed 94% homology with the mouse CA XII. To map the putative functional roles of different CAs, we studied the expression and localization of CA II, CA IV, CA VII, CA-related protein (CA-RP) VIII and CA XII mRNAs in rat brain after kainic acid induced epileptic seizures using Northern blot analysis and in situ hybridization. The expression of CA IV, CA VII and CA-RP VIII was somewhat similar: they were expressed in the cortex, hippocampus and midbrain structures and their expression did not change after the kainic acid treatment. The expression of CA II was concentrated in the white matter structures, which is in line with the preferential expression of CA II in the oligodendrocytes. High levels of CA II mRNA were also detected in the choroid plexus. Surprisingly, CA II was induced 3-12 h after seizures in the vulnerable CA1 region. CA XII was expressed in dentate granule cells, cortex and choroid plexus. Kainic acid stimulated CA XII expression throughout the cortical layer I. The observed hippocampal induction of CA II may indicate a pro-apoptotic and/or epileptogenic role of CA II after prolonged seizures. The physiological significance of the observed cortical induction of CA XII remains obscure. Cytosolic CA II is known to participate in CSF secretion, and the high expression of CA XII in the choroid plexus suggests an analogous role for this membrane-bound isozyme.     

Factors affecting outdoor exposure in winter: population-based study

The extent of outdoor exposure during winter and factors affecting it were examined in a cross-sectional population study in Finland. Men and women aged 25–74 years from the National FINRISK 2002 sub-study (n=6,591) were queried about their average weekly occupational, leisure-time and total cold exposure during the past winter. The effects of gender, age, area of residence, occupation, ambient temperature, self-rated health, physical activity and education on cold exposure were analysed. The self-reported median total cold exposure time was 7 h/week (8 h men, 6 h women),<1 h/week (2 h men, 0 h women) at work, 4 h/week (5 h men, 4 h women) during leisure time and 1 h/week (1 h men, 1.5 h women) while commuting to work. Factors associated with increased occupational cold exposure among men were: being employed in agriculture, forestry and industry/mining/construction or related occupations, being less educated and being aged 55–64 years. Factors associated with increased leisure-time cold exposure among men were: employment in industry/mining/construction or related occupations, being a pensioner or unemployed, reporting at least average health, being physically active and having college or vocational education. Among women, being a housewife, pensioner or unemployed and engaged in physical activity increased leisure-time cold exposure, and young women were more exposed than older ones. Self-rated health was positively associated with leisure time cold exposure in men and only to a minor extent in women. In conclusion, the subjects reported spending 4% of their total time under cold exposure, most of it (71%) during leisure time. Both occupational and leisure-time cold exposure is greater among men than women.     

The molecular basis of filamin binding to integrins and competition with talin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.     

The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro.

Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxylysyl and hydroxylysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys(144) and aspartates in positions 187-191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins.     

Involvement of a class III peroxidase and the mitochondrial protein TSPO in oxidative burst upon treatment of moss plants with a fungal elicitor

Production of apoplastic reactive oxygen species (ROS), or oxidative burst, is among the first responses of plants upon recognition of microorganisms. It requires peroxidase or NADPH oxidase (NOX) activity and factors maintaining cellular redox homeostasis. Here, PpTSPO1 involved in mitochondrial tetrapyrrole transport and abiotic (salt) stress tolerance was tested for its role in biotic stress in Physcomitrella patens, a nonvascular plant (moss). The fungal elicitor chitin caused an immediate oxidative burst in wild-type P. patens but not in the previously described ΔPrx34 mutants lacking the chitin-responsive secreted class III peroxidase (Prx34). Oxidative burst in P. patens was associated with induction of the oxidative stress-related genes AOX, LOX7, and NOX, and also PpTSPO1. The available ΔPpTSPO1 knockout mutants overexpressed AOX and LOX7 constitutively, produced 2.6-fold more ROS than wild-type P. patens, and exhibited increased sensitivity to a fungal necrotrophic pathogen and a saprophyte. These results indicate that Prx34, which is pivotal for antifungal resistance, catalyzes ROS production in P. patens, while PpTSPO1 controls redox homeostasis. The capacity of TSPO to bind harmful free heme and porphyrins and scavenge them through autophagy, as shown in Arabidopsis under abiotic stress, seems important to maintenance of the homeostasis required for efficient pathogen defense.     

University students' knowledge of, and attitudes towards, hiv and AIDS, homosexuality and sexual risk behaviour: a questionnaire survey in two finnish universities

Summary This study describes Finnish university students' knowledge and attitudes towards HIV and AIDS, homosexuality and sexual risk behaviour. Finnish-speaking students were randomly selected from all registered students at two universities in Finland (N=9715, n=950). The data were collected by using a modified version of the State University of New York at Buffalo School of Nursing AIDS Study Questionnaire on sexual risk behaviour developed by Held and Chng. The total response rate was 35% (n=333). The data were analysed using quantitative statistical methods. Normally distributed data were analysed by t-test and one-way ANOVA, with Bonferroni corrections. Non-normally distributed data were analysed using the Mann-Whitney U-test and Kruskal-Wallis test, followed by a post-hoc test. The majority of students were familiar with HIV and AIDS, including its mode of transmission. However, there were still some misconceptions concerning HIV and AIDS. The oldest students and women had a more positive attitude towards people living with HIV and AIDS (PLWHA). Of patients with HIV or AIDS, intravenous drug users were perceived most negatively. Male students had more homophobic attitudes. Students who reported that religion had an important role in their lives had significantly stricter attitudes towards sexual risk behaviour. Students' knowledge correlated positively with general attitudes towards HIV and AIDS. Knowledge about HIV and AIDS will lead to more positive attitudes towards HIV and AIDS as a disease, towards those infected as well as homosexual people. There is a need to focus on preventive health care and sexual health promotion by educating young people and changing their attitudes towards sexual risk behaviour.     

Spontaneous remodeling of HDL particles at acidic pH enhances their capacity to induce cholesterol efflux from human macrophage foam cells

HDL particles may enter atherosclerotic lesions having an acidic intimal fluid. Therefore, we investigated whether acidic pH would affect their structural and functional properties. For this purpose, HDL(2) and HDL(3) subfractions were incubated for various periods of time at different pH values ranging from 5.5 to 7.5, after which their protein and lipid compositions, size, structure, and cholesterol efflux capacity were analyzed. Incubation of either subfraction at acidic pH induced unfolding of apolipoproteins, which was followed by release of lipid-poor apoA-I and ensuing fusion of the HDL particles. The acidic pH-modified HDL particles exhibited an enhanced ability to promote cholesterol efflux from cholesterol-laden primary human macrophages. Importantly, treatment of the acidic pH-modified HDL with the mast cell-derived protease chymase completely depleted the newly generated lipid-poor apoA-I, and prevented the acidic pH-dependent increase in cholesterol efflux. The above-found pH-dependent structural and functional changes were stronger in HDL(3) than in HDL(2). Spontaneous acidic pH-induced remodeling of mature spherical HDL particles increases HDL-induced cholesterol efflux from macrophage foam cells, and therefore may have atheroprotective effects.     

Diversity in prokaryotic glycosylation: an archaeal-derived N-linked glycan contains legionaminic acid

VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV‐1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by β‐elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy. Such analyses showed that the major VP4‐derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5‐N‐formyl‐legionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid‐like sugar, in an archaeal‐derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with N‐acetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC‐ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4‐derived peptides bearing the N‐glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N‐glycosylation apparatus, as determined by LC‐ESI/MS of heterologously expressed VP4. Here, however, the N‐linked pentasaccharide was the same as shown to decorate the S‐layer glycoprotein in this species. Hence, N‐glycosylation of the haloarchaeal viral protein, VP4, is host‐specific. These results thus present additional examples of archaeal N‐glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.     

Conformation study of 2-arylbenzimidazoles as inhibitors of Chlamydia pneumoniae growth

Chlamydia pneumoniae is a worldwide cause of various respiratory track diseases ranging from asymptomatic pharyngeal infection to severe, sometimes fatal pneumonia. We have previously identified 2-arylbenzimidazoles as highly active antichlamydial compounds. In this work the importance of conformational effects on the structure-activity relationship of these compounds was studied. To simplify calculations, properly substituted N-phenylbenzamides, or the corresponding heterocyclic compounds, and 2-arylbenzimidazoles were used as model compounds. They were energy minimized and the energy differences between certain conformations were calculated. The main finding was that the compounds which can more easily adopt a non-planar conformation show higher bioactivity. This finding can be utilized in designing new derivatives or in constructing a pharmacophore model.