Functional characterization of fingers subdomain-specific monoclonal antibodies inhibiting the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), encoded by nonstructural protein 5B (NS5B), is absolutely essential for the viral replication. Here we describe the development, characterization, and functional properties of the panel of monoclonal antibodies (mAbs) and specifically describe the mechanism of action of two mAbs inhibiting the NS5B RdRp activity. These mAbs recognize and bind to distinct linear epitopes in the fingers subdomain of NS5B. The mAb 8B2 binds the N-terminal epitope of the NS5B and inhibits both primer-dependent and de novo RNA synthesis. mAb 8B2 selectively inhibits elongation of RNA chains and enhances the RNA template binding by NS5B. In contrast, mAb 7G8 binds the epitope that contains motif G conserved in viral RdRps and inhibits only primer-dependent RNA synthesis by specifically targeting the initiation of RNA synthesis, while not interfering with the binding of template RNA by NS5B. To reveal the importance of the residues of mAb 7G8 epitope for the initiation of RNA synthesis, we performed site-directed mutagenesis and extensively characterized the functionality of the HCV RdRp motif G. Comparison of the mutation effects in both in vitro primer-dependent RdRp assay and cellular transient replication assay suggested that mAb 7G8 epitope amino acid residues are involved in the interaction of template-primer or template with HCV RdRp. The data presented here allowed us to describe the functionality of the epitopes of mAbs 8B2 and 7G8 in the HCV RdRp activity and suggest that the epitopes recognized by these mAbs may be useful targets for antiviral drugs.     

Voles on small islands: effects of food limitation and alien predation

Ecosystems of three trophic levels may be bottom-up (by food-plant availability) and/or top-down (by predators) limited. Top-down control might be of greater consequence when the predation impact comes from an alien predator. We conducted a replicated two-factor experiment with field voles (Microtus agrestis) during 2004-2005 on small islands of the outer archipelago of the Baltic Sea, south-west Finland, manipulating both predation impact by introduced American mink (Mustela vison) and winter food supply. In autumn 2004, we live-trapped voles on five islands from which mink had been consistently removed, and on four islands where mink were present, and provided half of these islands with 1.8 kg oats per vole. Body mass of female voles increased as a response to supplementary food, whereas both food supplementation and mink removal increased the body mass of male voles in subsequent spring. During winter, there was a positive effect of supplementary food, but in the subsequent summer, possible positive long-term impacts of food supplementation on field voles were not detected. Mink removal appeared not to affect density estimates of field voles during the winter and summer immediately after food addition. Trapping data from 2004 to 2005 and 2007 suggested, however, that in two out of three summers densities of voles were significantly higher in the absence than in the presence of mink. We conclude that vole populations on small islands in the archipelago of the Baltic Sea are mainly bottom-up limited during winter (outside the growing season of food plants), when food availability is low, and limited by mink predation during summer which slows population growth during the reproductive season of voles.     

C and N storage in living trees within Finland since 1950s

Living biomass contains 45 to 60% carbon and 0.05 to 3% nitrogen, in dry weight. Like throughout Europe, the amount of living biomass in Finnish forests has increased on average over the last decades, largely because of changes in forest management. The storage of organic C and N in biomass has also increased.Changes in biomass vary between regions. Data were analysed on changes in the last 30–40 years in C and N storage in living trees in Finland, subdivided into 20 regions. Tree biomass increased in 17 regions, and decreased in 3 regions. The storage rate varied between -170 and +480 kg C ha^-1 a^-1, and between –0.5 and +1.2 kg N ha^-1 a^-1.Nitrogen accumulation in trees was less than 15% of atmospheric N deposition in all regions. Although the eventual increase of the nitrogen concentration in tree tissues was omitted, it is not possible that living biomass has been the major sink for atmospheric N deposition to forests. A hypothesis is presented that the main sink is litter layer and organic soil. Carbon can also be accumulating in soils essentially faster than hitherto estimated in analyses of carbon budgets of European forests.Died on September 2, 1994.     

Habitat Effects on the Breeding Performance of Three Forest-Dwelling Hawks

Habitat loss causes population declines, but the mechanisms are rarely known. In the European Boreal Zone, loss of old forest due to intensive forestry is suspected to cause declines in forest-dwelling raptors by reducing their breeding performance. We studied the boreal breeding habitat and habitat-associated breeding performance of the northern goshawk (Accipiter gentilis), common buzzard (Buteo buteo) and European honey buzzard (Pernis apivorus). We combined long-term Finnish bird-of-prey data with multi-source national forest inventory data at various distances (100–4000 m) around the hawk nests. We found that breeding success of the goshawk was best explained by the habitat within a 2000-m radius around the nests; breeding was more successful with increasing proportions of old spruce forest and water, and decreasing proportions of young thinning forest. None of the habitat variables affected significantly the breeding success of the common buzzard or the honey buzzard, or the brood size of any of the species. The amount of old spruce forest decreased both around goshawk and common buzzard nests and throughout southern Finland in 1992–2010. In contrast, the area of young forest increased in southern Finland but not around hawk nests. We emphasize the importance of studying habitats at several spatial and temporal scales to determine the relevant species-specific scale and to detect environmental changes. Further effort is needed to reconcile the socioeconomic and ecological functions of forests and habitat requirements of old forest specialists.     

Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus

Abstract The putative ribosome binding sites preceding 32 of Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H genes were compared. A highly conserved consensus sequence for the ribosome binding sites of LL-H genes was inferred, GAAAGGAG. This study included the characterization of the last nucleotides of the 3'-end of the 16S rRNA molecule from L. delbrueckii subsp. lactis and its comparison to the ribosome binding site consensus sequence.     

The Effects of 2-5A on Protein Synthesis in Wheat Germ Extracts and Tobacco Protoplasts

Abstract

Nonphosphorylated 2-5A inhibited translation and caused RNA degradation in wheat germ extract, whereas 3-5A had no effect. Protein synthesis inhibition by 2-5A was observed in tobacco protoplasts. 70 kD 2-5A-binding protein was found in potato leaf extracts by chemical crosslinking.     

Testis development in Pieris brassicae parasitized by Apanteles glomeratus

Testis development in Pieris brassicae (L.) parasitized as 1-day-old larvae by Apanteles glomeratus (L.) is considerably retarded from the second-instar onward. Testicular volume is only one sixth and testicular wall thickness only one quarter that of the normal testes at the end of the fifth larval instar. Nevertheless, bundles of sperm are formed in parasitized larvae. After interrupted oviposition, leading to the lowest possible level of parasitism, the effects are of the same magnitude as in more heavily parasitized hosts at comparable age. No significant correlation between number of parasitoids and retardation inflicted is found. The possible source of the effect of parasitism is discussed.

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Zusammenfassung Bei den unparasitierten Wirten läuft die gesamte Hodenentwicklung während der Larvenzeit ab. Bei parasitierten Wirten ist der Entwicklungszeitplan identisch, aber das Hodenvolumen bleibt in Larvenstadium auf einem Sechstel und die Hodenwand (Peritonealhülle und Follikelwand) auf einem Viertel in der normalen Werte. Die allmählich hervortretenden Veränderungen beginnen bereits beim zweiten Larvenstadium des Wirtes. Trotz der Veränderungen entwickeln sich auch in den parasitierten Hoden zeitplangemäss Spermienbündel, deren Fertilität jedoch nach ihrem histologischen Aussehen fragwürdig erscheint. Durch eine Unterbrechung der Eiablage des Parasitenweibchens nach 3-sekundiger Ablage konnte ein schwacher Parasitismus hervorgerufen werden, bei dem die Anzahl der Parasitenlarven pro Wirt von 1–8 variierte. Die bei diesen Wirten beobachteten Veränderungen der Hoden waren gleicher Art und gleich gross wie bei stark parasitiertem Material. Eine gute Korrelation zwischen der Zahl der Parasitlarven pro Wirt und der Intensität der pathologischen Veränderungen konnte nicht festgestellt werden. Mögliche Gründe der vom Parasitismus verursachten Veränderungen werden diskutiert. Dafür kommen vor allem die Parasitenlarven ersten und zweiten Stadiums in Frage, aber der eventuelle Anteil des eierlegenden Parasitenweibchens wird auch berücksichtigt.

     

Joint Russian—Finnish study of radioactive contamination in the NW part of Lake Ladoga

In August 1992 a joint Russian-Finnish expedition was arranged to the NW part of Lake Ladoga to study radioactive contamination in the region. Special attention was paid to the area surrounding the Heinämaa Islands, where the wreck of the former mine carrier ship Kit had been lying about 30 years before it was moved to Novaya Zemlya in 1991. During this period the wreck had been used as a store for radioactive waste containing principally ⁹⁰Sr, ¹³⁷Cs and ^239,24OPu. Lake water, bottom sediment and some biological samples were collected for strontium, plutonium and gammaspectrometric analyses. In all the samples the radioactivity concentrations were very low, indicating radioactive contamination of about the same level as caused by global fallout in the 1960's and the Chernobyl fallout in the area. Only in two water samples taken close to the former site of the wreck slightly elevated ^239,240Pu concentrations were detected. The great water volume of Lake Ladoga and effective water exchange at the wreck site may explain the very low levels of radioactive wastes detected in the aquatic environment.     

Modelling non-uniform soil water uptake by a single plant root

The assumption of uniform water flow to the root or uniform water potential at the root surface was shown by Hainsworth and Aylmore (1986, 1989) to be erroneous. The present paper demonstrates how the non-uniform uptake of water by a single root can be modeled. Differential equations are numerically solved to describe simultaneous water movement in the plant and in the soil. In the plant, boundary conditions are the water potentials at the root surface (Ψ_s) and in the xylem at the root base (Ψ_b). A set of difference equations describe the flow of water radially through the cortex to the xylem and in the xylem axially upwards to the base. For calculating the water flow in the soil and the values of Ψ_s, i.e. the boundary conditions for flow in the root, the finite element method (FEM) is used, the boundary conditions being the flux of water into the plant root and the zero flow across the wall, bottom and surface of a hypothetical soil cylinder surrounding the root. ei]Section editor: B E Clothier