Induction of periosteal callus formation by bone morphogenetic protein-2 employing adenovirus-mediated gene delivery.

Although the chondrogenic response of periosteum is well established in healing fractures, the mechanisms mediating the proliferation and differentiation of periosteal chondroprogenitor cells are poorly understood. In the present study we demonstrate that bone morphogenetic protein-2 (BMP-2), introduced by adenovirus-mediated gene transfer, alone is capable of inducing callus formation at the site of periosteal injection. Both immunohistochemistry and Northern analysis demonstrated activation of type II collagen production between days 4 and 7 after the injection, followed by activation of type X collagen expression. The activation of chondrogenesis was associated with increased expression of L-Sox5 and Sox9, suggesting that the BMP-2 effect is mediated via Sox proteins. This capacity of adenovirus-mediated overproduction of BMP-2 to induce chondrogenesis (and subsequent endochondral ossification) should be useful for tissue engineering of cartilage and bone.     

Epimerization of 3-hydroxyacyl-CoA esters in rat liver. Involvement of two 2-enoyl-CoA hydratases

Interconversion of D- and L-isomers of 3-hydroxy-decanoyl-CoA was catalyzed by rat liver homogenate. Cation exchange chromatography followed by ammonium sulfate precipitation and PBE-94 chromatofocusing column was used to separate the peroxisomal bifunctional protein, the classic 2-enoyl-CoA hydratase (crotonase), and a novel 2-enoyl-CoA hydratase. Epimerization activity was lost during the last purification step. None of the above proteins was capable of catalyzing the epimerization by itself, but reconstitution was achieved by recombining crotonase and the novel 2-enoyl-CoA hydratase. Since hydration by the latter enzyme follows a different stereochemical course from that with crotonase, these two hydratases are distinguished as 2-enoyl-CoA hydratase 1 (crotonase) and 2-enoyl-CoA hydratase 2 (the novel hydratase). The data strongly suggested that epimerization in the rat liver proceeds via dehydration-hydration catalyzed by the two different hydratases. The intermediate of this two step mechanism appears to be trans-2-enoyl-CoA.     

Bare laser‐synthesized Au‐based nanoparticles as nondisturbing surface‐enhanced Raman scattering probes for bacteria identification

The ability of noble metal‐based nanoparticles (NPs) (Au, Ag) to drastically enhance Raman scattering from molecules placed near metal surface, termed as surface‐enhanced Raman scattering (SERS), is widely used for identification of trace amounts of biological materials in biomedical, food safety and security applications. However, conventional NPs synthesized by colloidal chemistry are typically contaminated by nonbiocompatible by‐products (surfactants, anions), which can have negative impacts on many live objects under examination (cells, bacteria) and thus decrease the precision of bioidentification. In this article, we explore novel ultrapure laser‐synthesized Au‐based nanomaterials, including Au NPs and AuSi hybrid nanostructures, as mobile SERS probes in tasks of bacteria detection. We show that these Au‐based nanomaterials can efficiently enhance Raman signals from model R6G molecules, while the enhancement factor depends on the content of Au in NP composition. Profiting from the observed enhancement and purity of laser‐synthesized nanomaterials, we demonstrate successful identification of 2 types of bacteria (Listeria innocua and Escherichia coli). The obtained results promise less disturbing studies of biological systems based on good biocompatibility of contamination‐free laser‐synthesized nanomaterials.

     

Modification of the Monomer Composition of Polyhydroxyalkanoate Synthesized in Saccharomyces cerevisiae Expressing Variants of the β-Oxidation-Associated Multifunctional Enzyme

Expression by Saccharomyces cerevisiae of a polyhydroxyalkanoate (PHA) synthase modified at the carboxy end by the addition of a peroxisome targeting signal derived from the last 34 amino acids of the Brassica napus isocitrate lyase (ICL) and containing the terminal tripeptide Ser-Arg-Met resulted in the synthesis of PHA. The ability of the terminal peptide Ser-Arg-Met and of the 34-amino-acid peptide from the B. napus ICL to target foreign proteins to the peroxisome of S. cerevisiae was demonstrated with green fluorescent protein fusions. PHA synthesis was found to be dependent on the presence of both the enzymes generating the β-oxidation intermediate 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-[CoA]) and the peroxin-encoding PEX5 gene, demonstrating the requirement for a functional peroxisome and a β-oxidation cycle for PHA synthesis. Using a variant of the S. cerevisiae β-oxidation multifunctional enzyme with a mutation inactivating the B domain of the R-3-hydroxyacyl-CoA dehydrogenase, it was possible to modify the PHA monomer composition through an increase in the proportion of the short-chain monomers of five and six carbons.     

Alternatives to the isomerase-dependent pathway for the beta-oxidation of oleic acid are dispensable in Saccharomyces cerevisiae. Identification of YOR180c/DCI1 encoding peroxisomal delta(3,5)-delta(2,4)-dienoyl-CoA isomerase.

Fatty acids with double bonds at odd-numbered positions such as oleic acid can enter beta-oxidation via a pathway relying solely on the auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase, termed the isomerase-dependent pathway. Two novel alternative pathways have recently been postulated to exist in mammals, and these additionally depend on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (di-isomerase-dependent) or on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (reductase-dependent). We report the identification of the Saccharomyces cerevisiae oleic acid-inducible DCI1 (YOR180c) gene encoding peroxisomal di-isomerase. Enzyme assays conducted on soluble extracts derived from yeast cells overproducing Dci1p using 3,5,8,11,14-eicosapentenoyl-CoA as substrate demonstrated a specific di-isomerase activity of 6 nmol x min(-1) per mg of protein. Similarly enriched extracts from eci1Delta cells lacking peroxisomal 3,2-isomerase additionally contained an intrinsic 3,2-isomerase activity that could generate 3, 5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA but not metabolize trans-3-hexenoyl-CoA. Amplification of this intrinsic activity replaced Eci1p since it restored growth of the eci1Delta strain on petroselinic acid for which di-isomerase is not required whereas Eci1p is. Heterologous expression in yeast of rat di-isomerase resulted in a peroxisomal protein that was enzymatically active but did not re-establish growth of the eci1Delta mutant on oleic acid. A strain devoid of Dci1p grew on oleic acid to wild-type levels, whereas one lacking both Eci1p and Dci1p grew as poorly as the eci1Delta mutant. Hence, we reasoned that yeast di-isomerase does not additionally represent a physiological 3,2-isomerase and that Dci1p and the postulated alternative pathways in which it is entrained are dispensable for degrading oleic acid.     

Phytoplankton community responses to nutrient and iron enrichment under different nitrogen to phosphorus ratios in the northern Baltic Sea

The impact of nutrient enrichment on the phytoplankton community structure, and particularly cyanobacteria, was studied in a 3-week mesocosm experiment conducted in August 2001 in the Archipelago Sea, a part of the northern Baltic Sea. The factorial design experiment included daily additions of nitrogen (N) and phosphorus (P) at two mass ratios, 1N:1P and 7N:1P, respectively, additions of iron (Fe) and a synthetic chelator, ethylenediaminetetraacetic acid (EDTA). The floating enclosures (400 l) were sampled for analyses of phytoplankton biomass and community structure, phytoplankton primary production, chlorophyll a, nutrients, and hepatotoxins. Chlorophyll a concentration, phytoplankton biomass and primary production increased most in the 7N:1P treatment. The increase was mainly due to an abundant growth of chlorophytes (Dictyosphaerium subsolitarium, Kirchneriella spp., Monoraphidium contortum, and Oocystis spp.), pennate diatoms (especially Nitzschia spp.), dinophytes and the chroococcalean cyanobacterium Synechococcus sp. The nutrient enrichments had no effect on the total biomass of N₂-fixing cyanobacteria. Nevertheless, the biomass of Anabaena spp. was highest in the enrichments with a low N/P ratio. Chlorophyll a concentration and total phytoplankton biomass were not affected by Fe or EDTA, but Fe alone had a positive effect on the chlorophyte Kirchneriella sp. The N₂-fixing cyanobacteria Aphanizomenon sp. responded positively to Fe alone and to both Fe and EDTA added together. The hepatotoxin concentration increased during the experiment, but no clear responses to nutrient enrichments were found. Our study showed species-specific responses to nutrient enrichments among the N₂-fixing cyanobacteria. Although the total phytoplankton production was not Fe-limited; the availability of Fe clearly affected the phytoplankton community structure.     

Insights into virus evolution and membrane biogenesis from the structure of the marine lipid-containing bacteriophage PM2

Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane proteins, constitutes a rare visualization of an in vivo membrane. The viral membrane proteins P3 and P6 are organized into a lattice, suggesting a possible assembly pathway to produce the mature virus.     

Effects of nutrients and fish on periphyton and plant biomass across a European latitudinal gradient

Replicated, factorial mesocosm experiments were conducted across Europe to study the effects of nutrient enrichment and fish density on macrophytes and on periphyton chlorophyll a (chl-a) with regard to latitude. Periphyton chl-a densities and plant decline were significantly related to nutrient loading in all countries. Fish effects were significant in a few sites only, mostly because of their contribution to the nutrient pool. A saturation-response type curve in periphyton chl-a with nutrients was found, and northern lakes achieved higher densities than southern lakes. Nutrient concentration and phytoplankton chl-a necessary for a 50% plant reduction followed a latitudinal gradient. Total phosphorus values for 50% plant disappearance were similar from Sweden (0.27 mg L⁻¹) to northern Spain (0.35 mg L⁻¹), but with a sharp increase in southern Spain (0.9 mg L⁻¹). Planktonic chl-a values for 50% plant reduction increased monotonically from Sweden (30 μg L⁻¹) to València (150 μg L⁻¹). Longer plant growing-season, higher light intensities and temperature, and strong water-level fluctuations characteristic of southern latitudes can lead to greater persistence of macrophyte biomass at higher turbidities and nutrient concentration than in northern lakes. Results support the evidence that latitudinal differences in the functioning of shallow lakes should be considered in lake management and conservation policies.     

Tawny owl reproduction and offspring sex ratios under variable food conditions

Tawny owl reproduction and offspring sex ratios have been considered to depend on the abundance of small voles. We studied reproductive performance (laying date, clutch and brood size) during 1995–2003 and offspring sex ratios from 1999 to 2003 in relation to the abundance of small voles and food delivered to the nest in a tawny owl population in southern Finland. Abundance of small voles (field and bank voles) was based on trappings in the field, and estimates of food delivery was based on diet analysis of food remains in the nest boxes. In this population, reproductive output was not related to the abundance of small voles. Analysis of food delivered to the nest showed that the prey weight per offspring varied more than twofold between years and revealed that this difference was mainly related to the proportion of water voles in the diet. Only the number of water voles correlated with laying dates. Offspring sex ratios were weakly male biased (55%) but did not differ from parity. Sex ratios were not related to the abundance of small voles, and we found no evidence that parents delivered more food to nests with proportionally more offspring of the larger (female) sex. Our results underline the notion that populations may differ in their sex allocation pattern, and suggest such differences may be due to diet.     

Genetics for Pseudoalteromonas provides tools to manipulate marine bacterial virus PM2

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (Corticoviridae) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative Pseudoalteromonas species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.