Aspartic proteinase from wheat seeds: isolation,properties and action on gliadin

Wheat endosperm was shown to contain an aspartic proteinase capable of hydrolyzing the wheat storage protein, gliadin, in vitro. The enzyme was purified to homogeneity by affinity chromatography on bacilliquin-silochrome, diethylaminoethyl-Toyopearl ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide gel electrophoresis. The sedimentation constant of the enzyme was 3.4 S and the relative molecular mass (M_r), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 58000 dalton (Da). The purified enzyme was completely inhibited by pepstain whereas other enzyme inhibitors did not affect its activity. The enzyme was found to hydrolyze mainly - and -gliadins with M_r's of 67000–95000 Da, with maximal activity at pH 4.5. The data make it possible to suggest that the enzyme has an endogenous function by initiating proteolysis of storage proteins in germinating wheat seeds.Abbreviations BSA bovine serum albumin - Da dalton - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Differential expression of wheat aspartic proteinases, WAP1 and WAP2, in germinating and maturing seeds

Two aspartic proteinase (AP) cDNA clones, WAP1 and WAP2, were obtained from wheat seeds. Proteins encoded by these clones shared 61% amino acid sequence identity. RNA blotting analysis showed that WAP1 and WAP2 were expressed in both germinating and maturing seeds. The level of WAP2 mRNA expression was clearly weaker than that of WAP1 in all tissues of seeds during germination and maturation. APs purified from germinating seeds were enzymatically active and digested the wheat storage protein, gluten. To elucidate the physiological functions of WAP1 and WAP2 in seeds, we investigated the localisation of WAP1 and WAP2 by in situ hybridisation. In germinating seeds investigated 24h after imbibition, both WAP1 and WAP2 were expressed in embryos, especially in radicles and shoots, scutellum, and the aleurone layer. In maturing seeds, WAP1 was expressed in the whole embryo, with slightly stronger expression in radicles and shoots. WAP1 was also expressed in the aleurone layer 3 weeks after flowering. Strong signals of WAP1 mRNA were detected in the whole embryo and aleurone layer 6 weeks after flowering. On the other hand, WAP2 was scarcely detected in seeds 3 weeks after flowering, and thereafter weak signals began to appear in the whole embryo. WAP1 and WAP2 were expressed widely in germinating and maturing seeds. Such diversity in site- and stage-specific expression of the two enzymes suggests their differential functions in wheat seeds.     

A fluorimetric method for the determination of pepsin activity

An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.     

Aspergillus flavus infection and aflatoxin contamination in sorghum seeds and their biological management

In order to establish the current scenario of aflatoxigenic fungal infection and aflatoxin contamination in sorghum seeds across India, 58 seed samples were collected from different agro-climatic regions. Among these, 67.2% samples were infected with Aspergillus spp. and 28% were found contaminated with aflatoxins ranging from 0.0 to 130?μg?kg^?1. Greenhouse studies revealed no correlation between incidence of Aspergillus flavus and aflatoxin content, and its effect on seed quality parameters. Among the 37 A. flavus strains isolated, six were non-aflatoxigenic when analysed through cultural, TLC and ic-ELISA. Seed treatment with biocontrol agents (antagonistic Rhizobacteria and Trichoderma) suppressed the growth of A. flavus under laboratory and significantly enhanced seed quality variables under greenhouse conditions to a various extent. Field trials with selected biocontrol agents showed that talcum powder formulations of Pseudomonas putida Has-1/c, Bacillus spp. 3/a, Trichoderma asperellum M5 and T. asperellum T2 improved seedling emergence, % nutrient accumulation in plants, increased plant biomass and 1000 seed weight. Seeds harvested from treated plants showed significant increase in seed quality variables under laboratory and greenhouse conditions in comparison with control, but there was no significant difference in A. flavus infection and aflatoxin was completely absent in all treatments.     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol

The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca²⁺-requiring proteinase required 5–10 µM Ca²⁺ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25–2.0 µM) in the absence or presence of Ca²⁺ (5.0 µM) added. The effect of regucalcin, however, was the greater in the absence of Ca²⁺ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca²⁺ (5.0 µM). In the absence of Ca²⁺, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 µg/ml), and heavy metals (25 µM cadmium or 25 µM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca²⁺-independent neutral cysteinyl-proteinase.     

Two types of aspartic proteinases from buckwheat seed--gene structure and expression analysis

Two types of aspartic proteinase (AP) genes have been isolated from the cDNA library of developing buckwheat seeds. Analysis of their sequences showed that one of these, FeAP9, resembled the structure and shared high homology with the so-called typical plant APs characterized by the presence of a plant-specific insert (PSI), an element unique among APs. The other cDNA, FeAPL1, encoded an AP-like protein lacking that domain. Different expression profiles were observed for FeAP9 and FeAPL1. FeAPL1 mRNAs were restricted to the seeds only, whereas FeAP9 mRNAs were also present in the other plant tissues - leaves, roots, and flowers. Higher levels of FeAP9 were observed in senescent leaves compared with green leaves. The differential expression pattern of these two unique APs raises the interesting possibility that these proteinases have unique substrate specificity and may have different roles in plant development and other physiological processes.     

Biorefinery of sweet sorghum stem

Sweet sorghum has been considered as a viable energy crop for alcohol fuel production. This review discloses a novel approach for the biorefining of sweet sorghum stem to produce multiple valuable products, such as ethanol, butanol and wood plastic composites. Sweet sorghum stem has a high concentration of soluble sugars in its juice, which can be fermented to produce ethanol by Saccharomyces cerevisiae. In order to obtain high ethanol yield and fermentation rates, concentrated juice with an initial total sugar concentration of 300gL(-1) was fermented. The maximum ethanol concentration after 54h reached 140gL(-1) with a yield of 0.49g ethanol per g consumed sugar, which is 97% of the theoretical value. Sweet sorghum bagasse, obtained from juice squeezing, was pretreated by acetic acid to hydrolyze 80-90% of the contained hemicelluloses. Using this hydrolysate as raw material (total sugar 55gL(-1)), 19.21gL(-1) total solvent (butanol 9.34g, ethanol 2.5g, and acetone 7.36g) was produced by Clostridium acetobutylicum. The residual bagasse after pretreatment was extruded with PLA in a twin-screw extruder to produce a final product having a PLA: fiber ratio of 2:1, a tensile strength of 49.5M and a flexible strength of 65MPa. This product has potential use for applications where truly biodegradable materials are required. This strategy for sustainability is crucial for the industrialization of biofuels from sweet sorghum.     

Effects of yeast proteolytic activity on Oenococcus oeni and malolactic fermentation

Alcoholic fermentation of synthetic must was performed using either Saccharomyces cerevisiae or a mutant Deltapep4, which is deleted for the proteinase A gene. Fermentation with the mutant Deltapep4 resulted in 61% lower levels of free amino acids, and in 62% lower peptide concentrations at the end of alcoholic fermentation than in the control. Qualitative differences in amino acid composition were observed. Changes observed in amino acids in peptides were mainly quantitative. After alcoholic fermentation, each medium was inoculated with Oenococcus oeni. Malolactic fermentation in the medium with the Deltapep4 strain took 10 days longer than the control. This difference may have been due to a difference in the nitrogen composition of the two media. Free amino acids and amino acids in peptides were poorly consumed by O. oeni. Thus, the qualitative aspects of nitrogen composition, which depend in part on yeast metabolism, may be a determinant for the optimal growth of O. oeni in wine.     

Protein modification in malting sorghum

Steeping time, moisture content and germination times were deployed in assessing protein modification in sorghum varieties: ICSV400, SK5912 and KSV8. Grains were steeped for 45 h using 6 h wet and 3 h dry cycles and germinated for 8 days. Moisture contents and their effects on protein modification were monitored at various intervals. Optimum moisture contents of 37–43% and out-of-steep values of 32–35% were recorded. Significant positive correlations existed between moisture content and free alpha amino nitrogen (FAN), total non-protein nitrogen (TNPN) and cold water soluble protein (CWS-P), all key protein modification indicators, during steeping. Maximum values for FAN, TNPN and CWS-P were recorded in both ICSV400 and SK5912 after 40 h of steeping, suggesting a similarity in the physiology of the grains in both varieties while those of KSV8 occurred after 45 h. Variety and steeping time significantly affected moisture content at P < 0.01 and P < 0.001, respectively as well as the development of FAN, TNPN and CWS-P during steeping. Optimum values for the above parameters occurred on day 5 of germination in all the sorghum varieties. Variety and germination time highly significantly (P < 0.001) affected protein modification during germination.     

Regulation of biological activity of laminin-5 by proteolytic processing of gamma2 chain

Laminin-5 (LN5), which regulates both cell adhesion and cell migration, undergoes specific extracellular proteolytic processing at an amino-terminal region of the gamma2 chain as well as at a carboxyl-terminal region of the alpha3 chain. To clarify the biological effect of the gamma2 chain processing, we prepared a human recombinant LN5 with the 150-kDa, non-processed gamma2 chain (GAA-LN5) and natural LN5 with the 105-kDa, processed gamma2 chain (Nat-LN5). Comparison of their biological activities demonstrated that GAA-LN5 had an about five-times higher cell adhesion activity but an about two-times lower cell migration activity than Nat-LN5. This implies that the proteolytic processing of LN5 gamma2 chain converts the LN5 from the cell adhesion type to the cell migration type. It was also found that human gastric carcinoma cells expressing the LN5 with the non-processed gamma2 chain is more adherent but less migratory than the carcinoma cells expressing a mixture of LN5 forms with the processed gamma2 chain and with the unprocessed one. The functional change of LN5 by the proteolytic processing of the gamma2 chain may contribute to elevated cell migration under some pathological conditions such as wound healing and tumor invasion.     

Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets

All parasitic protozoa contain multiple proteases, some of which are attracting attention as drug targets. Aspartic proteases are already the targets of some clinically useful drugs (e.g. chemotherapy of HIV infection) and a variety of factors make these enzymes appealing to those seeking novel antiparasite therapies. This review provides a critical analysis of the current knowledge on Plasmodium aspartic proteases termed plasmepsins, proposes a definitive nomenclature for this group of enzymes, and compares these enzymes with aspartic proteases of humans and other parasitic protozoa. The present status of attempts to obtain specific inhibitors of the parasite enzymes that will be useful as drugs is outlined and suggestions for future research priorities are proposed.     

Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.     

高粱抗蚜基因的RAPD分析

应用RAPD技术BSA法,对高梁抗蚜基因进行分析,筛选了500个随机引物,共扩增出1614条谱带,得到了10个具有稳定多态性标记的引物,分别为OPA－01、OPP－09、OPP－14、OPH－19、OPN－08、OPN－07、OPN－20、OPY－14、OPS－20、OPJ－06。     

Development of a sorghum chain in the Italian Campania Region: From the field to the celiac patient's table

Abstract

Although sorghum has been used for centuries as a food-crop in Africa and India, researchers in the United States and Europe have only relatively recently become interested in the potential of this unique cereal. Much of this interest focuses on the potential use of sorghum in food product development for individuals with allergies to foods containing wheat-based flours. Because it lacks gluten, sorghum is considered safe for people diagnosed with celiac disease, a condition marked by intolerance to gluten. Recent studies have shown that certain sorghum varieties, tan-plant sorghums, can be used to produce high-quality food and beverage products including cookies, waffles, flour, bread, noodles and beer. The intention is to promote the use and marketing of these sorghums in the Italian Campania Region for sorghum flours suitable as food for celiac patients.     

Gene expression profiling of mammary glands of cathepsin E-deficient mice compared with wild-type littermates

Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system and has been implicated in various physiological and pathological processes. Because of physiological substrates of cathepsin E have not yet been identified, however, the physiological significance of this protein still remains speculative. To better understand the physiological significance of cathepsin E in the mammary gland, we investigated the effect of the deficiency of this protein on the gene expression profile of the tissue. Here we used mammary glands derived from multiparous and non-pregnant 11-month-old syngenic wild-type (CatE(+/+)) and cathepsin E-deficient (CatE(-/-)) mice for extraction of total RNA from each tissue and subsequent mRNA amplification, DNA fragmentation, and hybridization with cDNA mixroarray chips. A total of 654 genes were identified as overexpressed (>2-fold) in CatE(-/-) mammary glands compared with CatE(+/+) counterparts. These included genes related to signal transduction, immune responses, growth factor activity, and milk proteins, which occupied a large portion of the gene fragments identified as overexpressed. In contrast, a total of 665 known genes were identified as underexpressed in the mammary gland of CatE(-/-) mice compared with CatE(+/+) counterparts. These included genes related to cytoskeleton, cell differentiation, cell cycle arrest and apoptosis, which occupied the majority of the gene fragments identified as underexpressed. The results thus suggest that cathepsin E in mammary glands plays a crucial role in the regulation of proteins involved in signaling, development, differentiation and proliferation in the mammary gland.     

高粱种质材料幼苗期耐盐碱性评价

采用Hoagland营养液砂培法,以NaCl和Na2CO3组成的混合盐碱对高粱幼苗进行胁迫处理,建立高粱幼苗期耐盐碱评价方法,并评价了66份高粱种质材料的耐盐碱性.结果表明:盐浓度在8.0～12.5 g·L-1时,高粱耐盐碱品种‘TS-185’与盐碱敏感品种‘Tx-622B’在幼苗期的耐盐碱性差异明显,表明进行高粱幼苗期耐盐碱性评价时适宜的盐浓度范围为8.0～12.5 g· L-1.在10.0和12.5 g·L-12个盐浓度下,66份高粱种质材料的相对存活率、相对地上部鲜质量和相对株高的差异均达显著水平,表明不同品种的耐盐碱性不同.其中,‘三尺三’为高度耐盐碱品种,‘MN-2735’等16个品种为耐盐碱品种,‘EARLY HONEY’等32个品种为中等耐盐碱品种,‘Tx-622B’等16个品种为盐碱敏感品种,‘MN-4588’为高度盐碱敏感品种.苏丹草类型高粱一般具有较高的耐盐碱性,而保持系对盐碱较为敏感.