Structural basis for the platelet-collagen interaction: the smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproline triplets

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.     

Simultaneous determination of the accuracy and precision of closed-circuit cardiac output rebreathing techniques.

Foreign and soluble gas rebreathing methods are attractive for determining cardiac output (Q(c)) because they incur less risk than traditional invasive methods such as direct Fick and thermodilution. We compared simultaneously obtained Q(c) measurements during rest and exercise to assess the accuracy and precision of several rebreathing methods. Q(c) measurements were obtained during rest (supine and standing) and stationary cycling (submaximal and maximal) in 13 men and 1 woman (age: 24 +/- 7 yr; height: 178 +/- 5 cm; weight: 78 +/- 13 kg; Vo(2max): 45.1 +/- 9.4 ml.kg(-1).min(-1); mean +/- SD) using one-N(2)O, four-C(2)H(2), one-CO(2) (single-step) rebreathing technique, and two criterion methods (direct Fick and thermodilution). CO(2) rebreathing overestimated Q(c) compared with the criterion methods (supine: 8.1 +/- 2.0 vs. 6.4 +/- 1.6 and 7.2 +/- 1.2 l/min, respectively; maximal exercise: 27.0 +/- 6.0 vs. 24.0 +/- 3.9 and 23.3 +/- 3.8 l/min). C(2)H(2) and N(2)O rebreathing techniques tended to underestimate Q(c) (range: 6.6-7.3 l/min for supine rest; range: 16.0-19.1 l/min for maximal exercise). Bartlett's test indicated variance heterogeneity among the methods (P < 0.05), where CO(2) rebreathing consistently demonstrated larger variance. At rest, most means from the noninvasive techniques were +/-10% of direct Fick and thermodilution. During exercise, all methods fell outside the +/-10% range, except for CO(2) rebreathing. Thus the CO(2) rebreathing method was accurate over a wider range (rest through maximal exercise), but was less precise. We conclude that foreign gas rebreathing can provide reasonable Q(c) estimates with fewer repeat trials during resting conditions. During exercise, these methods remain precise but tend to underestimate Q(c). Single-step CO(2) rebreathing may be successfully employed over a wider range but with more measurements needed to overcome the larger variability.     

Genetic analysis of the Linnaean Ulva lactuca (Ulvales,Chlorophyta) holotype and related type specimens reveals name misapplications,unexpected origins,and new synonymies

Current usage of the name Ulva lactuca, the generitype of Ulva, remains uncertain. Genetic analyses were performed on the U. lactuca Linnaean holotype, the U. fasciata epitype, the U. fenestrata holotype, the U. lobata lectotype, and the U. stipitata lectotype. The U. lactuca holotype is nearly identical in rbcL sequence to the epitype of U. fasciata, a warm temperate to tropical species, rather than the cold temperate species to which the name U. lactuca has generally been applied. We hypothesize that the holotype specimen of U. lactuca came from the Indo‐Pacific rather than northern Europe. Our analyses indicate that U. fasciata and U. lobata are heterotypic synonyms of U. lactuca. Ulva fenestrata is the earliest name for northern hemisphere, cold temperate Atlantic and Pacific species, with U. stipitata a junior synonym. DNA sequencing of type specimens provides an unequivocal method for applying names to Ulva species.     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

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.