Isolation and characterization of two glycerol-fermenting clostridial strains from a pilot scale anaerobic digester treating high lipid-content slaughterhouse waste

Two obligately anaerobic bacterial strains were isolated from the contents of a pilot scale, anaerobic digester treating slaughterhouse waste with a high protein and lipid content. The isolates, LIP1 and MW8, were characterized as spore-forming, Gram-positive rods, capable of fermenting glycerol. Isolate LIP1 was also observed to be lipolytic and was able to hydrolyse tallow and olive oil. Both isolates grew optimally at 37 degrees C and formed either acetate and formate (LIP1), or acetate and butyrate (MW8), as major glycerol fermentation products. Both isolates produced ethanol as the major reduced fermentation end-product. Neither MW8 nor LIP1 had growth and metabolism inhibited by the addition of stearic acid at concentrations normally considered bactericidal. Analysis of the 16S rRNA gene sequences, in conjunction with the phenotypic data, confirmed that the isolates are members of the genus Clostridium (sensu lato), clustering with species of clostridial clusters I (MW8) and XIVa (LIP1).     

The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana

The irregular xylem 2 (irx2) mutant of Arabidopsis thaliana exhibits a cellulose deficiency in the secondary cell wall, which is brought about by a point mutation in the KORRIGAN (KOR) beta,1-4 endoglucanase (beta,1-4 EGase) gene. Measurement of the total crystalline cellulose in the inflorescence stem indicates that the irx2 mutant contains approximately 30% of the level present in the wild type (WT). Fourier-Transform Infra Red (FTIR) analysis, however, indicates that there is no decrease in cellulose in primary cell walls of the cortical and epidermal cells of the stem. KOR expression is correlated with cellulose synthesis and is highly expressed in cells synthesising a secondary cell wall. Co-precipitation experiments, using either an epitope-tagged form of KOR or IRX3 (AtCesA7), suggest that KOR is not an integral part of the cellulose synthase complex. These data are supported by immunolocalisation of KOR that suggests that KOR does not localise to sites of secondary cell wall deposition in the developing xylem. The defect in irx2 plant is consistent with a role for KOR in the later stages of secondary cell wall formation, suggesting a role in processing of the growing microfibrils or release of the cellulose synthase complex.     

A genetic linkage map of quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis>) based on AFLP,RAPD, and SSR markers

Quinoa (Chenopodium quinoa Willd.) is an important seed crop for human consumption in the Andean region of South America. It is the primary staple in areas too arid or saline for the major cereal crops. The objective of this project was to build the first genetic linkage map of quinoa. Selection of the mapping population was based on a preliminary genetic similarity analysis of four potential mapping parents. Breeding lines Ku-2 and 0654, a Chilean lowland type and a Peruvian Altiplano type, respectively, showed a low similarity coefficient of 0.31 and were selected to form an F₂ mapping population. The genetic map is based on 80 F₂ individuals from this population and consists of 230 amplified length polymorphism (AFLP), 19 simple-sequence repeat (SSR), and six randomly amplified polymorphic DNA markers. The map spans 1,020 cM and contains 35 linkage groups with an average marker density of 4.0 cM per marker. Clustering of AFLP markers was not observed. Additionally, we report the primer sequences and map locations for 19 SSR markers that will be valuable tools for future quinoa genome analysis. This map provides a key starting point for genetic dissection of agronomically important characteristics of quinoa, including seed saponin content, grain yield, maturity, and resistance to disease, frost, and drought. Current efforts are geared towards the generation of more than 200 mapped SSR markers and the development of several recombinant-inbred mapping populations.     

Molecular insight into patterns of colony composition and paternity in the common mole-rat Cryptomys hottentotus hottentotus

We report the discovery of intraspecific variation in both colony composition and patterns of paternity in two populations of the social common mole-rat Cryptomys hottentotus hottentotus. These two populations represent the mesic and arid habitat extremes of the species' broad ecological range in South Africa. Until recently colonies of the common mole-rat were thought to consist of familial groups whereby all colony members were the offspring of a monogamous reproductive pair. The remaining colony members were thought to forego reproduction until both social and ecological conditions favoured dispersal and opportunities for independent outbreeding. Results from genetic assignment tests using microsatellite markers indicate that while colony composition is dominated by familial groups, colonies within both populations included both adult and subadult foreign conspecifics. Analysis of parentage reveals that the social organization of C. h. hottentotus is not that of strict monogamy; paternity of offspring was not assigned consistently to the largest, most dominant male within the colony. Moreover, a number of significantly smaller males were found to sire offspring, suggesting a sneak-mating strategy by subordinate within-colony males. Extra-colony extra-pair paternity (ECP) was also found to characterize C. h. hottentotus colonies, occurring with similar frequencies in both habitats. Both dominant established breeding males and subordinate males were identified as siring young in nonsource colonies. Furthermore, established breeding males were found to sire extra-colony young in the same season as siring young within their source colonies. We discuss the significance of these results within the context of the divergent ecological regimes characterizing the two sites and observe that our results revisit the accuracy of using behavioural and morphological characters, which have structured the basis of our understanding of the behavioural ecology of this species, as indicators of breeding status in mark-recapture studies.     

Calcium-triggered membrane fusion proceeds independently of specific presynaptic proteins

Complexes of specific presynaptic proteins have been hypothesized to drive or catalyze the membrane fusion steps of exocytosis. Here we use a stage-specific preparation to test the roles of SNAREs, synaptotagmin, and SNARE-binding proteins in the mechanism of Ca2+-triggered membrane fusion. Excess exogenous proteins, sufficient to block SNARE interactions, did not inhibit either the Ca2+ sensitivity, extent, or kinetics of fusion. In contrast, despite a limited effect on SNARE and synaptotagmin densities, treatments with high doses of chymotrypsin markedly inhibited fusion. Conversely, low doses of chymotrypsin had no effect on the Ca2+ sensitivity or extent of fusion but did alter the kinetic profile, indicating a more direct involvement of other proteins in the triggered fusion pathway. SNAREs, synaptotagmin, and their immediate binding partners are critical to exocytosis at a stage other than membrane fusion, although they may still influence the triggered steps.     

Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes

Recombinant mammalian glycoproteins produced by the baculovirus-insect cell expression system usually do not have structurally authentic glycans. One reason for this limitation is the virtual absence in insect cells of certain glycosyltransferases, which are required for the biosynthesis of complex, terminally sialylated glycoproteins by mammalian cells. In this study, we genetically transformed insect cells with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes. This produced a new insect cell line that can express both genes, serve as hosts for baculovirus infection, and produce foreign glycoproteins with terminally sialylated N-glycans.     

Vitamin C transport systems of mammalian cells

Vitamin C is essential for many enzymatic reactions and also acts as a free radical scavenger. Specific non-overlapping transport proteins mediate the transport of the oxidized form of vitamin C, dehydroascorbic acid, and the reduced form, L-ascorbic acid, across biological membranes. Dehydroascorbic acid uptake is via the facilitated-diffusion glucose transporters, GLUT 1, 3 and 4, but under physiological conditions these transporters are unlikely to play a major role in the uptake of vitamin C due to the high concentrations of glucose that will effectively block influx. L-ascorbic acid enters cells via Na+-dependent systems, and two isoforms of these transporters (SVCT1 and SVCT2) have recently been cloned from humans and rats. Transport by both isoforms is stereospecific, with a pH optimum of approximately 7.5 and a Na+:ascorbic acid stoichiometry of 2:1. SVCT2 may exhibit a higher affinity for ascorbic acid than SVCT1 but with a lower maximum velocity. SVCT1 and SVCT2 are predicted to have 12 transmembrane domains, but they share no structural homology with other Na+ co-transporters. Potential sites for phosphorylation by protein kinase C exist on the cytoplasmic surface of both proteins, with an additional protein kinase A site in SVCT1. The two isoforms also differ in their tissue distribution: SVCT1 is present in epithelial tissues, whereas SVCT2 is present in most tissues with the exception of lung and skeletal muscle.     

Beta-(1 --> 4)-galactosyltransferase activity in native and engineered insect cells measured with time-resolved europium fluorescence

To evaluate the ability of insect cells to produce complex-type N-glycans, beta-(1 --> 4)-galactosyltransferase (beta4GalT) activity in several insect cell lines was analyzed. For this purpose, we developed a simple and highly sensitive assay for beta-(1 --> 4)-galactosyltransferase (beta4GalT) activity, which is based on time-resolved fluorometry of europium. Bovine serum albumin (BSA) modified with GlcNAc (GlcNAc(44)-BSA) was used as the acceptor. GlcNAc(44)-BSA was coated on a 96-well microplate, and after incubation with the enzyme sample in the presence of UDP-Gal, Eu-labeled RCA(120) (Ricinus communis aggutin I), was added. RCA(120) binds to the Galbeta(1 --> 4)GlcNAc structure in the product, and the bound Eu-RCA(120) was measured by the fluorescence of europium. When bovine beta4Gal-T-I was used as a standard reference enzyme, a linear relationship between enzyme activity and fluorescent signal was obtained over the range of 0-1000 microUnits (IU). Using this system, we were able to measure a low but significant level of beta4GalT activity in Trichoplusia ni cells ('High Five'). In contrast, no endogenous beta4GalT activity was detected in a Spodoptera frugiperda (Sf-9) cell line. However, Sf-9 cells stably transfected with the bovine beta4GalT-I gene and 'High Five' cells infected with a baculovirus containing the same gene produced activity levels that were comparable to or greater than those found in Chinese hamster ovary cells. We also showed that the beta4GalT activity level observed in the baculovirus-infected T. ni cells under the control of immediate early promoter was highly dependent on the post-infection time, suggesting that galactosylation level may also be variable during the infection period.