Regional and local spatial genetic structure of Siberian primrose populations in Northern Europe

We have investigated the local and regional scale genetic structure of Siberian primrose (Primula nutans) populations in Northern Europe. The genetic diversity and structure of fifteen populations sampled from the Bothnian Bay in Finland, the Barents Sea in Norway and the White Sea in Russia were assessed using eleven microsatellite markers. We investigated the distribution of genetic variation within and between populations, and studied the local genetic structure using spatial autocorrelation analysis. We found very low genetic and allelic diversity in the Bothnian Bay and Barents Sea populations, and only slightly higher in the White Sea population. The level of genetic differentiation between the regions was very high, whereas differentiation between the populations within the regions was moderate. We found no spatial structuring of populations in any region suggesting efficient dispersal on a local scale. Clonal reproduction seemed to have no effect on genetic structure.     

On-line high performance liquid chromatography measurements of extracellular metabolites in an aerobic batch yeast (<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>) culture

We constructed a bioprocess environment enabling automatic sampling from a bioreactor combined with a compact on-line high performance liquid chromatography (HPLC) unit. This setup allowed us to measure extracellular glucose, ethanol, glycerol, and acetate concentrations automatically at 5 min intervals during the cultivation. This environment also provides mechanical measurement of the optical density (OD) of cells and enables us to collect and store (−35°C) samples for further off-line analyses. Among the available devices, the performance of the sampling-analysis unit is by far the best with regard to speed and number of analytes. Both the sampling and analysis phases are easily controlled by software; thus, providing a unique environment to perform various bioprocess activity tasks, whether they would be cell line screening or optimisation of conditions for growth and productivity. Complex research set-ups can be created and continuous automated measurements empower long-term cultivations with a time series. We provide evidence for the applicability of this environment by performing three comparable batch cultivations with Saccharomyces cerevisiae yeast and show that both the on-line sampling and analysis modes produce reliable data for further use in the monitoring and controlling of bioprocesses. On-line data provided new insight into the dynamics of the diauxic shift during aerobic glucose batch cultivation. When cell growth and carbon dioxide production ceased for the first time during the diauxic shift, acetate accumulation and consumption of the remaining glucose below 0.15 g/L continued to occur for 1 h. At the same time, glycerol and ethanol began to be consumed. Samples were also collected during cultivation for later analysis of intracellular metabolites and to collect more valuable information about metabolism.     

High temperature and bacteriophages can indirectly select for bacterial pathogenicity in environmental reservoirs

The coincidental evolution hypothesis predicts that traits connected to bacterial pathogenicity could be indirectly selected outside the host as a correlated response to abiotic environmental conditions or different biotic species interactions. To investigate this, an opportunistic bacterial pathogen, Serratia marcescens, was cultured in the absence and presence of the lytic bacteriophage PPV (Podoviridae) at 25°C and 37°C for four weeks (N = 5). At the end, we measured changes in bacterial phage-resistance and potential virulence traits, and determined the pathogenicity of all bacterial selection lines in the Parasemia plantaginis insect model in vivo. Selection at 37°C increased bacterial motility and pathogenicity but only in the absence of phages. Exposure to phages increased the phage-resistance of bacteria, and this was costly in terms of decreased maximum population size in the absence of phages. However, this small-magnitude growth cost was not greater with bacteria that had evolved in high temperature regime, and no trade-off was found between phage-resistance and growth rate. As a result, phages constrained the evolution of a temperature-mediated increase in bacterial pathogenicity presumably by preferably infecting the highly motile and virulent bacteria. In more general perspective, our results suggest that the traits connected to bacterial pathogenicity could be indirectly selected as a correlated response by abiotic and biotic factors in environmental reservoirs.     

Bud burst in Norway spruce (Picea abies): preliminary evidence for age-specific rest patterns

This study examines the effect of chilling and photoperiod on rest completion and bud burst in Norway spruce [Picea abies (L.) Karst.] using twigs from both 15-year-old (young trees) and 56-year-old (old trees) trees. The material was transferred between September and May from outdoors to experimental forcing conditions with four different photoperiods. The bud burst percentage of the twigs from young trees generally increased in all photoperiods until the end of the year. After that it decreased until vernal equinox (March 20) and then increased steeply towards spring. This new observation of transient rest completion during autumn suggests that young trees have (I) a transient time window during late autumn when ontogenetic development is possible, and (ii) a secondary rest culminating approximately at the time of vernal equinox. In twigs from old trees the transient rest completion was much weaker as the bud burst percentages generally remained under 20 during autumn and winter. At vernal equinox there were no burst buds in twigs from the old trees in any photoperiod and after that the bud burst percentage increased basically in the same manner as in the young trees. The bud burst percentage of the twigs from young trees was generally higher as the photoperiod increased. However, no evidence for absolute long photoperiod requirement of rest completion was observed.     

Extracellular matrix protein 1 interacts with the domain III of fibulin-1C and 1D variants through its central tandem repeat 2

Extracellular matrix protein 1 (ECM1), a widely expressed glycoprotein, has been shown to harbor mutations in lipoid proteinosis (LP), an autosomal recessive disorder characterized by profound alterations in the extracellular matrix of connective tissue. The biological function of ECM1 and its role in the pathomechanisms of LP are unknown. Fibulins comprise a family of extracellular matrix components, and the prototype of this family, fibulin-1, is expressed in various connective tissues and plays a role in developmental and pathologic processes. In this study, we demonstrate that ECM1, and specifically the second tandem repeat domain which is alternatively spliced, interacts with the C-terminal segments of fibulins 1C and 1D splice variants which differ in their C-terminal domain III. The interactions were detected by yeast two-hybrid genetic system and confirmed by co-immunoprecipitations. Kinetics of the binding between ECM1 and fibulin-1D, measured by biosensor assay, revealed a K(d) of 5.71 x 10(-8) M, indicating a strong protein-protein interaction. Since distinct splice variants of ECM1 and fibulin-1 have been shown to be co-expressed in tissues affected in LP, we propose that altered ECM1/fibulin-1 interactions may play a role in the pathogenesis of this disease as well as in a number of processes involving the extracellular matrix of connective tissues.     

The gene family of ABC transporters--novel mutations, new phenotypes

Members of the ABC (ATP-binding cassette) superfamily of genes encode transmembrane proteins that are involved in the transport of a variety of substrates both in and out of the cells, in addition to across intracellular membranes. Recently, mutations in two ABC-transporter genes, ABCC6 and ABCA12, have been demonstrated to underlie phenotypically different diseases affecting the skin (pseudoxanthoma elasticum and harlequin ichthyosis, respectively), attesting to the spectrum of ABC gene mutations in human diseases. These findings have a major impact on the molecular genetics of these devastating disorders in terms of DNA-based prenatal testing and pre-implantation genetic diagnosis.     

Single amino acid substitutions in procollagen VII affect early stages of assembly of anchoring fibrils

Procollagen VII is a homotrimer of 350-kDa pro-alpha1(VII) chains, each consisting of a central collagenous domain flanked by the noncollagenous N-terminal NC1 domain and the C-terminal NC2 domain. After secretion from cells, procollagen VII molecules form anti-parallel dimers with a C-terminal 60-nm overlap. Characteristic alignment of procollagen VII monomers forming a dimer depends on site-specific binding between the NC2 domain and the triple-helical region adjacent to Cys-2634 of the interacting procollagen VII molecules. Formation of the intermolecular disulfide bonds between Cys-2634 and either Cys-2802 or Cys-2804 is promoted by the cleavage of the NC2 domain by procollagen C-proteinase. By employing recombinant procollagen VII variants harboring G2575R, R2622Q, or G2623C substitutions previously disclosed in patients with dystrophic epidermolysis bullosa, we studied how these amino acid substitutions affect intermolecular interactions. Binding assays utilizing an optical biosensor demonstrated that the G2575R substitution increased affinity between mutant molecules. In contrast, homotypic binding between the R2622Q or G2623C molecules was not detected. In addition, kinetics of heterotypic binding of all analyzed mutants to wild type collagen VII were different from those for binding between wild type molecules. Moreover, solid-state binding assays demonstrated that R2622Q and G2623C substitutions prevent formation of stable assemblies of procollagen C-proteinase-processed mutants. These results indicate that single amino acid substitutions in procollagen VII alter its self-assembly and provide a basis for understanding the pathomechanisms leading from mutations in the COL7A1 gene to fragility of the dermal-epidermal junction seen in patients with dystrophic forms of epidermolysis bullosa.     

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.     

A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.     

Nitric oxide synthase in human salivary glands

The distribution of the three nitric oxide synthase (NOS) isoforms was determined immunohistochemically in the human minor and major salivary glands with comparison to that of rat salivary glands. In contrast to rat glands, which contained a dense plexus of neuronal NOS-immunoreactive nerve fibers, only a minority of the nerve fibers in human glands showed neuronal NOS immunoreactivity. Human labial and submandibular glands contained sparse NOS-immunoreactive fibers, while only occasional nerve fibers in the parotid or sublingual glands were stained. Furthermore, in contrast to the animal glands, most duct epithelial cells in all human salivary glands were immunoreactive for neuronal NOS. No specific immunoreactivity for inducible or endothelial NOS were observed in the nerve fibers or duct epithelium. We provide evidence to suggest that the role of nitric oxide in the regulation of salivary gland function is different in human as compared to experimental animals. Nitricergic innervation in human tissue is very sparse and thus nitric oxide is probably of minor importance as a neural regulator of salivary glands. Instead, NOS localized in duct epithelial cells suggests that nitric oxide might directly regulate saliva secretion and it is a putative source of nitrates previously reportedly secreted into the saliva.