951 resultados para Soy protein isolate


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The studies presented in this thesis aimed to a better understanding of the molecular biology of Sweet potato chlorotic stunt virus (SPCSV, Crinivirus, Closteroviridae) and its role in the development of synergistic viral diseases. The emphasis was on the severe sweet potato virus disease (SPVD) that results from a synergistic interaction of SPCSV and Sweet potato feathery mottle virus (SPFMV, Potyvirus, Potyviridae). SPVD is the most important disease affecting sweetpotato. It is manifested as a significant increase in symptom severity and SPFMV titres. This is accompanied by a dramatic sweetpotato yield reduction. SPCSV titres remain little affected in the diseased plants. Viral synergistic interactions have been associated with the suppression of an adaptive general defence mechanism discovered in plants and known as RNA silencing. In the studies of this thesis two novel proteins (RNase3 and p22) identified in the genome of a Ugandan SPCSV isolate were shown to be involved in suppression of RNA silencing. RNase3 displayed a dsRNA-specific endonuclease activity that enhanced the RNA-silencing suppression activity of p22. Comparative analyses of criniviral genomes revealed variability in the gene content at the 3´end of the genomic RNA1. Molecular analyses of different isolates of SPCSV indicated a marked intraspecific heterogeneity in this region where the p22 and RNase3 genes are located. Isolates of the East African strain of SPCSV from Tanzania and Peru and an isolate from Israel were missing a 767-nt fragment that included the p22 gene. However, regardless of the absence of p22, all SPCSV isolates acted synergistically with SPFMV in co-infected sweetpotato, enhanced SPFMV titres and caused SPVD. These results showed that p22 is dispensable for development of SPVD. The role of RNase3 in SPVD was then studied by generating transgenic plants expressing the RNase3 protein. These plants had increased titres of SPFMV (ca. 600-fold higher in comparison with nontransgenic plants) 2-3 weeks after graft inoculation and displayed the characteristic SPVD symptoms. RNA silencing suppression (RSS) activity of RNase3 was detected in agroinfiltrated leaves of Nicotiana bethamiana. In vitro studies showed that RNase3 was able to cleave small interferring RNAs (siRNA) to products of ~14-nt. The data thus identified RNase3 as a suppressor of RNA silencing able to cleave siRNAs. RNase3 expression alone was sufficient for breaking down resistance to SPFMV in sweetpotato and for the development of SPVD. Similar RNase III-like genes exist in animal viruses which points out a novel and possibly more general mechanism of RSS by viruses. A reproducible method of sweetpotato transformation was used to target RNA silencing against the SPCSV polymerase region (RdRp) with an intron-spliced hairpin construct. Hence, engineered resistance to SPCSV was obtained. Ten out of 20 transgenic events challenged with SPCSV alone showed significantly reduced virus titres. This was however not sufficient to prevent SPVD upon coinfection with SPFMV. Immunity to SPCSV seems to be required to control SPVD and targeting of different SPCSV regions need to be assessed in further studies. Based on the identified key role of RNase3 in SPVD the possibility to design constructs that target this gene might prove more efficient in future studies.

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-Essential hypertensives display enhanced signal transduction through pertussis toxin-sensitive G proteins. The T allele of a C825T variant in exon 10 of the G protein beta3 subunit gene (GNB3) induces formation of a splice variant (Gbeta3-s) with enhanced activity. The T allele of GNB3 was shown recently to be associated with hypertension in unselected German patients (frequency=0.31 versus 0.25 in control). To confirm and extend this finding in a different setting, we performed an association study in Australian white hypertensives. This involved an extensively examined cohort of 110 hypertensives, each of whom were the offspring of 2 hypertensive parents, and 189 normotensives whose parents were both normotensive beyond age 50 years. Genotyping was performed by polymerase chain reaction and digestion with BseDI, which either cut (C allele) or did not cut (T allele) the 268-bp polymerase chain reaction product. T allele frequency in the hypertensive group was 0.43 compared with 0.25 in the normotensive group (chi2=22; P=0.00002; odds ratio=2.3; 95% CI=1.7 to 3.3). The T allele tracked with higher pretreatment blood pressure: diastolic=105+/-7, 109+/-16, and 128+/-28 mm Hg (mean+/-SD) for CC, CT, and TT, respectively (P=0.001 by 1-way ANOVA). Blood pressures were higher in female hypertensives with a T allele (P=0.006 for systolic and 0.0003 for diastolic by ANOVA) than they were in male hypertensives. In conclusion, the present study of a group with strong family history supports a role for a genetically determined, physiologically active splice variant of the G protein beta3 subunit gene in the causation of essential hypertension.

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The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmuller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1,2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine. 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensis Culberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2,4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suis Davaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis. (C) 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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The effects of tyrosinase, laccase and transglutaminase (TG) were studied in different meat protein systems. The study was focused on the effects of the enzymes on the gel formation properties of myofibrils, and on the textural and water-holding properties of the heated meat systems. The cross-linking efficiency of a novel Trichoderma reesei tyrosinase was compared to that of the commercial Agaricus bisporus tyrosinase. Trichoderma tyrosinase was found to be superior compared to the Agaricus enzyme in its protein cross-linking efficiency and in the incorporation of a small molecule into a complex proteinaceous substrate. Tyrosinase, laccase and TG all polymerised myofibrillar proteins, but laccase was also found to cause protein fragmentation. A positive connection between covalent cross-link and gel formation was observed with tyrosinase and TG. Laccase was able to increase the gel formation only slightly. With an excessive laccase dosage the gel formation declined due to protein fragmentation. Tyrosinase, laccase and TG had different effects on the texture and water-holding of the heated chicken breast meat homogenates. Tyrosinase improved the firmness of the homogenate gels free of phosphate and with a low amount of meat. TG improved the firmness of all studied homogenates. Laccase weakened the gel firmness of the low-meat, low-salt and low-salt/phosphate homogenates and maintained the firmness on the control level in the homogenate free of phosphate. Tyrosinase was the only enzyme capable of reducing the weight loss in the homogenates containing a low amount of meat and a low amount of NaCl. TG was the only enzyme that could positively affect the firmness of the homogenate gel containing both low NaCl and phosphate amounts. In pilot scale the test products were made of coarsely ground chicken breast fillet with a moderate amount of salt. Increasining the amount of meat, salt and TG contents favoured the development of firmness of the test products. The evaporation loss decreased slightly along with increasing TG and NaCl amounts in the experimental conditions used, indicating a positive interaction between these two factors. In this work it was shown that tyrosinase, laccase and TG affected the same myofibrillar proteins, i.e. myosin and troponin T. However, these enzymes had distinguishable effects on the gel formation of a myofibril system as well as on the textural and water-holding properties of the finely ground meat homogenates, reflecting distinctions at least in the reaction mechanisms and target amino acid availability in the protein substrates for these enzymes.

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Fruit flies require protein for reproductive development and actively feed upon protein sources in the field. Liquid protein baits mixed with insecticide are used routinely to manage pest fruit flies, such as Bactrocera tryoni (Froggatt). However, there are still some gaps in the underpinning science required to improve the efficacy of bait spray technology. The spatial and temporal foraging behaviour of B. tryoni in response to protein was investigated in the field. A series of linked trials using either wild flies in the open field or laboratory-reared flies in field cages and a netted orchard were undertaken using nectarines and guavas. Key questions investigated were the fly's response to protein relative to: height of protein within the canopy, fruiting status of the tree, time of day, season and size of the experimental arena. Canopy height had a significant response on B. tryoni foraging, with more flies foraging on protein in the mid to upper canopy. Fruiting status also had a significant effect on foraging, with most flies responding to protein when applied to fruiting hosts. B. tryoni demonstrated a repeatable diurnal response pattern to protein, with the peak response being between 12:0016:00 h. Season showed significant but unpredictable effects on fruit fly response to protein in the subtropical environment where the work was undertaken. Relative humidity, but not temperature or rainfall, was positively correlated with protein response. The number of B. tryoni responding to protein decreased dramatically as the spatial scale increased from field cage through to the open field. Based on these results, it is recommend that, to be most effective, protein bait sprays should be applied to the mid to upper canopies of fruiting hosts. Overall, the results show that the protein used, an industry standard, has very low attractancy to B. tryoni and that further work is urgently needed to develop more volatile protein baits.

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Impatiens necrotic spot tospovirus (INSV) is a significant pathogen of ornamentals. The tripartite negative- and ambi-sense RNA genome encodes six proteins that are involved in cytoplasmic replication, movement, assembly, insect transmission and defence. To gain insight into the associations of these viral proteins, we determined their intracellular localization and interactions in living plant cells. Nucleotide sequences encoding the nucleoprotein N, non-structural proteins NSs and NSm, and glycoproteins Gn and Gc of a Kentucky isolate of INSV were amplified by RTPCR, cloned, sequenced and transiently expressed as fusions with autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana. All proteins accumulated at the cell periphery and co-localized with an endoplasmic reticulum marker. The Gc protein fusion also localized to the nucleus. N and NSm protein self-interactions and an NSm-N interaction were observed by using bimolecular fluorescence complementation. A tospovirus NSm homotypic interaction had not been reported previously.

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The situation normally encountered in the high-resolution refinement of protein structures is one in which the inaccurate positions of P out of a total of N atoms are known whereas those of the remaining atoms are unknown. Fourier maps with coefficients (FN -- F'P) × exp (i[alpha]'P) and (mFN -- nF'P) exp (i[alpha]'P), where FN is the observed structure factor and F'P and [alpha]'P are the magnitude and the phase angle of the calculated structure factor corresponding to the inaccurate atomic positions, are often used to correct the positions of the P atoms and to determine those of the Q unknown atoms. A general theoretical approach is presented to elucidate the effect of errors in the positions of the known atoms on the corrected positions of the known atoms and the positions of the unknown atoms derived from such maps. The theory also leads to the optimal choice of parameters used in the different syntheses. When the errors in the positions of the input atoms are systematic, their effects are not taken care of automatically by the syntheses.

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The water soluble carbodiimide mediated condensation of dipeptides of the general form Gly-X was carried out in the presence of mono- and poly-nucleotides. The observed yield of the tetrapeptide was found to be higher for peptide-nucleotide system of higher interaction specificity following mainly the anticodon-amino acid relationship (Basu, H.S. & Podder, S.K., 1981, Ind. J. Biochem. Biophys.,19, 251-253). The yield of the condensation product of L-peptide was more because of its higher interaction specificity. The extent of the racemization during the condensation of Gly-L-Phe, Gly-L-Tyr and Gly-D-Phe was found to be dependent on the specificity of the interaction -the higher the specificity, the lesser the racemization. The product formed was shown to have a catalytic effect on the condensation reaction. These data thus provide a mechanism showing how the specific interaction between amino acids/dipeptides and nucleic acids could lead to the formation of the lsquoprimitiversquo translation machinery.

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The kinetics of estrogen-induced accumulation of riboflavin-carrier protein in the plasma was investigated in immature male rats using a specific and sensitive homologous radio-immunoassay procedure developed for this purpose. Following a single injection of the steroid hormone, plasma riboflavin-carrier protein levels increased markedly after an initial lag period of approximately 24 h, reaching peak levels around 96 h and declining thereafter. A 1.5 fold amplification of the inductive response was evident on secondary stimulation with the hormone. The magnitude of the response was dependent on hormonal dose, whereas the initial lag phase and the time of peak riboflavin-carrier protein induction were unaltered within the range of the steroid doses (0.1–10 mg/ kg body wt.) tested. Simultaneous administration of progesterone did not affect either the kinetics or the maximum level of the protein induced. The hormonal specificity of this induction was further adduced by the effect of administration of antiestrogens viz., En and Zu chlomiphene citrates, which effectively curtailed hormonal induction of the protein. That the induction involvedde novo-protein synthesis was evident from the complete inhibition obtained upon administration of cycloheximide. Passive immunoneutralization of endogenous riboflavin-carrier protein with antiserum to the homologous protein terminated pregnancy in rats confirming the earlier results with antiserum to chicken riboflavin-carrier protein.

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Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.

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Basidiomycetous white-rot fungi are the only organisms that can efficiently decompose all the components of wood. Moreover, white-rot fungi possess the ability to mineralize recalcitrant lignin polymer with their extracellular, oxidative lignin-modifying enzymes (LMEs), i.e. laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP). Within one white-rot fungal species LMEs are typically present as several isozymes encoded by multiple genes. This study focused on two effi cient lignin-degrading white-rot fungal species, Phlebia radiata and Dichomitus squalens. Molecular level knowledge of the LMEs of the Finnish isolate P. radiata FBCC43 (79, ATCC 64658) was complemented with cloning and characterization of a new laccase (Pr-lac2), two new LiP-encoding genes (Pr-lip1, Pr-lip4), and Pr-lip3 gene that has been previously described only at cDNAlevel. Also, two laccase-encoding genes (Ds-lac3, Ds-lac4) of D. squalens were cloned and characterized for the first time. Phylogenetic analysis revealed close evolutionary relationships between the P. radiata LiP isozymes. Distinct protein phylogeny for both P. radiata and D. squalens laccases suggested different physiological functions for the corresponding enzymes. Supplementation of P. radiata liquid culture medium with excess Cu2+ notably increased laccase activity and good fungal growth was achieved in complex medium rich with organic nitrogen. Wood is the natural substrate of lignin-degrading white-rot fungi, supporting production of enzymes and metabolites needed for fungal growth and the breakdown of lignocellulose. In this work, emphasis was on solid-state wood or wood-containing cultures that mimic the natural growth conditions of white-rot fungi. Transcript analyses showed that wood promoted expression of all the presently known LME-encoding genes of P. radiata and laccase-encoding genes of D. squalens. Expression of the studied individual LME-encoding genes of P. radiata and D. squalens was unequal in transcript quantities and apparently time-dependent, thus suggesting the importance of several distinct LMEs within one fungal species. In addition to LMEs, white-rot fungi secrete other compounds that are important in decomposition of wood and lignin. One of these compounds is oxalic acid, which is a common metabolite of wood-rotting fungi. Fungi produce also oxalic-acid degrading enzymes of which the most widespread is oxalate decarboxylase (ODC). However, the role of ODC in fungi is still ambiguous with propositions from regulation of intra and extracellular oxalic acid levels to a function in primary growth and concomitant production of ATP. In this study, intracellular ODC activity was detected in four white-rot fungal species, and D. squalens showed the highest ODC activity upon exposure to oxalic acid. Oxalic acid was the most common organic acid secreted by the ODC-positive white-rot fungi and the only organic acid detected in wood cultures. The ODC-encoding gene Ds-odc was cloned from two strains of D. squalens showing the first characterization of an odc-gene from a white-rot polypore species. Biochemical properties of the D. squalens ODC resembled those described for other basidiomycete ODCs. However, the translated amino acid sequence of Ds-odc has a novel N-terminal primary structure with a repetitive Ala-Ser-rich region of ca 60 amino acid residues in length. Expression of the Ds-odc transcripts suggested a constitutive metabolic role for the corresponding ODC enzyme. According to the results, it is proposed that ODC may have an essential implication for the growth and basic metabolism of wood-decaying fungi.