Wheat Gluten Protein Analysis

Wheat Gluten Protein Analysis Wheat is one of the most important cereal crops and its end-products like breads, noodles, pasta and other baked products are consumed globally and have become staple diet. The viscoelastic properties of wheat dough are primarily dependent upon the interaction of gluten proteins. Gluten proteins consist of gliadins, which provide viscous property to wheat dough, and glutenins, which contribute towards elasticity of the dough (Ciaffi et al., 1996). Storage protein deposition is affected by environmental conditions during the grain development period (Randall Moss, 1990; Lukow McVetty, 1991). For controlling the variation in wheat flour, it is imperative that the regulatory factors responsible for formation, folding and polymerization of gluten proteins should be studied. In nature, folding of proteins is mediated by an array of proteins that act as molecular chaperones or foldases (Fischer and Schmid, 1999). The wheat gluten proteins are proline rich (10-30%) (Van-Dijk et al., 1997) and about 6% of all Xaa-Pro (Xaa: other bulky amino groups preceding proline) peptide bonds show the cis conformation. Peptidyl prolyl cis-trans isomerases (PPIases) are the only enzymes known to catalyse cis-trans isomerisation of peptidyl prolyl bonds which is a rate-limiting step in protein folding (Fischer et al., 1989). Understanding the role of PPIases in gluten protein deposition in wheat could help in developing strategies for manipulating the storage proteins desired for different food products by breeding and/or genetic engineering strategies. Peptidyl-prolyl cis-trans isomerases comprise of three distinct classes of proteins- cyclophilins, which bind to the immunosuppressive drug cyclosporin A (CsA) (Handshumacher et al., 1984); FK506-binding proteins (FKBPs), which bind the macrolide drugs FK506 and rapamycin (Harding et al., 1989); and the parvulin family (Dolonski and Heitman, 1997). Due to their drug binding activities, cyclophilins and FKBPs are also known as immunophilins. The FKBPs are conserved in all organisms from prokaryotes to higher plants and mammals (Gasser et al., 1990). Rice genome is reported to contain largest number of FKBP members (Ahn et al., 2010). FKBPs, beside folding of proteins, are also involved in many other cellular processes such as cell signalling (Luan et al., 1998), protein complex formation (Pratt and Toft, 1997; Reynold et al., 1999), regulation of plant growth and development (Geisler et al., 2004), stress response (Kurek et al., 1999; Yu et al., 2012) and in redox control of photosynt hesis (Gupta et al., 2002; Gopalan et al., 2004). Two multidomain FKBPs, FKBP73 and FKBP77, were cloned earlier from wheat (Avezier et al., 1998). These proteins were also demonstrated to play role in signal transduction through their interaction with mammalian p23 and plant HSP90 (Owens-Grillo et al., 1996; Reddy et al., 1998). Recently, genes encoding three single-domain wheat FKBPs, TaFKBP13, TaFKBP16-1 and TaFKBP16-3 were cloned and characterized by Gollan et al. (2011). TaFKBP13 was the first active lumenal FKBP reported in cereals, whereas, TaFKBP16-1 and TaFKBP16-3 did not show any PPIase activity (Gollan et al., 2011). These FKBPs were also implicated in assembly of photosytem complexes and thylakoid membrane complexes (Gollan et al., 2011). It is evident that information on FKBPs which have been cloned and characterized from wheat is limited (Aviezer et al., 1998; Bhave et al., 2011). Further, their role in gluten protein deposition has also not been explored as yet. Theref ore, the present study was carried out with the following objectives. To analyze differences in deposition of gluten storage protein in grains at different stages of development in Indian wheat cultivars having varied protein content. Developmental changes in total PPIase activity and its correlation with storage protein deposition. To study the contribution of cyclophilins and FKBPs towards total PPIase activity in developing grains by inhibition assays employing cyclosporin A and FK506 as specific inhibitors, respectively. Cloning and characterization of FKBP genes and their expression analysis. Salient findings of the study Different hexaploid wheat (Triticum aestivum) cultivars (GLUPRO, LOKI, HPW89), which varied in their protein content, were selected for this study. The grains were harvested at different stages of development viz. 8, 12, 16, 20, 25 days post anthesis (DPA) and maturation. The isolation and separation of different storage protein fractions from the wheat grains pose a challenge due to their cross contamination. Therefore, different methods, which were reported earlier by Osborne (1924) and Fu and Saperstein (1996) were tried. These methods did not result in isolation of pure fractions of gliadins and glutenins from the grains of cultivars used in this study. However, the method reported by DuPont (2005) resulted in highest recovery of different protein fractions with minimal cross-contamination. The reducing SDS-PAGE analysis demonstrated that the accumulation of gliadins in the cultivars of wheat included in this study was affected by the developmental stage of the grain. Present stu dy also demonstrated that accumulation of high molecular weight subunits of glutenins (HMW-GSs) was also cultivar- and stage dependent. The profile of high molecular weight subunits of glutenins (LMW-GSs) was not altered significantly after 16 DPA in any of the three cultivars. Contrary to gliadins and glutenins, the albumins in the present study did not show any significant inter-cultivar variability. Further, the accumulation of albumins in all the three cultivars started after 12 DPA and increased up to maturation. The different albumins may consist of proteins involved in important cellular functions like protein folding, plant defence mechanism, stress response, etc. (Merlino et al., 2009) and, therefore, must be conserved in nature, which explains the lack of intercultivar variation in the three cultivars analysed in this study. Developmental regulation of PPIases in wheat grains has been reported for cyclophilin (Grimwade et al., 1996) and FKBP73 (Aviezer et al., 1998) at transcript and protein level, respectively. Expression studies of PPIases at activity level are however lacking also important because the transcript levels may not always culminate in higher levels of protein or activity due to post-transcriptional regulation (Arnholdt-Schmitt, 2004). Therefore, to elucidate the role of PPIase genes in accumulation of storage proteins in wheat grain, PPIase assays were performed by using crude protein extract of developing grains, and activity was estimated by a coupled enzyme assay method using chymotrypsin for cleaving the test peptide N-succinyl-ala-ala-pro-phe-p-nitroanilidine (Fischer et al., 1984). Principal Component Analysis (PCA) revealed that PPIase activity in cvs. HPW 89 and GLUPRO was related to the accumulation of gliadins. The presence of PPIase activity at different stages of grain develop ment in all the cultivars and its close association with storage proteins indicated that these enzyme(s) may be playing an important role in deposition of storage proteins in wheat. PPIase activity of FKBPs and cyclophilins is inhibited by immunosuppressant drugs FK506 and CsA, respectively (Harding et al., 1989). Since no cross inhibition by the two drugs is reported (Harding et al., 1989), we, therefore, employed CsA and FK506 as specific inhibitors to determine the contribution of these two classes of proteins to total grain PPIase activity. Except at 25 DPA in LOK I, the PPIase activity at all stages of grain development in the three cultivars was almost totally inhibited by CsA. These observations, thus, suggest that PPIase activity in the grains, except at 25 DPA in LOK-1, was primarily due to cyclophilins. Since FK506-inhibitable activity in the crude protein extracts of the three cultivars was negligible, therefore, to further investigate the reason for this observation, cloning of FKBP genes, which are expressed in the developing grains, was attempted. Sequence of an active FKBP type-1 domain of wFKBP73 (accession number X86903.1) comprising of 95 (50-1 45) amino acid (a.a.) residues (Blecher et al., 1996) was used as a query, which resulted in identification of hundreds of different putative FKBP sequences in T. aestivum. These sequences were retrieved from NCBI and subjected to TBLASTn using TIGR Plant Transcript Assemblies database (TADB; http://plantta.jcvi.org/) for wheat. Of the several retrieved sequences from TIGR, three different cDNAs, TaFKBP15-1, TaFKBP16-1 and TaFKBP20-1, which showed longest open reading frame (ORFs), were selected for cloning using the RNA isolated from the developing grains harvested at 16 DPA. The study successfully resulted in cloning of three FKBP genes from Indian wheat. Bioinformatics analysis of the cloned cDNAs revealed that TaFKBP16-1 consists of an ORF of 408 bp encoding a protein of 135 a.a. residues with molecular weight (M.W.) and pI of 15.26 kDa and 5.75, respectively. The 561 bp and 477 bp ORFs of TaFKBP20-1 and TaFKBP15-1, respectively, were predicted to encode proteins of 186 and 157 a.a. residues, respectively, with M.W. and pI of 19.95 kDa and 6.77, and 16.61 kDa and 8.96, respectively. In silico analysis of a.a. sequences of the cloned TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1 revealed that the FKBP domains architecture, though conserved in these proteins, also show variability observed in their secondary structures. Further, analysis of signal peptide using different online tools predicted localization of TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1 to nucleus, possibly cytosol and ER, respectively. Compared to human homologue, hFKBP12, both TaFKBP15-1 and TaFKBP20-1 showed presence of all the essential residues (Y26, F36, F46, W59, Y82 and F99) required for PPIase activity, as compared to only three (Y26, Y82 and F99) in TaFKBP16-1. TaFKBP15-1 is 40% and 38% similar to TaFKBP16-1 and TaFKBP20-1, respectively, whereas, TaFKBP20-1 is 30% similar to TaFKBP16-1. The variability observed in these FKBPs in wheat suggests that these proteins may be playing specific roles in the cells, which need to be investigated further. The recombinant TaFKBP20-1, TaFKBP16-1 and TaFKBP15-1 proteins were expressed in E. coli BL21-CodonPlus(DE3)pLysS and purified by Ni-NTA affinity chromatography. The recombinant nature of the purified proteins was validated by immunoblotting studies using anti-His antibody, which resulted in detection of the specific bands corresponding to the respective purified FKBP proteins. For biochemical characterization of three proteins, PPIase assays were performed. None of the three purified FKBP proteins showed any detectable PPIase activity since the first order rate constant (0.013 s-1) in presence of up to 2 Â µg of each of the three purified proteins was similar to the first order rate constant (0.0135 s-1) observed for the uncatalysed control (in the absence of protein). To determine the reasons for lack of PPIase activity, despite the presence of conserved a.a. residues, TaFKBP20-1 and TaFKBP16-1 were subjected to chymotrypsin susceptibility assay, which is used for determining the PPIase activity. The assays revealed that both the proteins were cleaved by chymotrypsin which could be one of the reasons for the absence of PPIase activity in the two proteins. Lack of detectable activity inTaFKBP15-1, however, could be because of improper refolding due to the use of urea which was employed for solubilization of this protein during purification. FKBPs in plants have been implicated in various stress responses (Kurek et al., 1999; Sharma and Singh, 2003; Magiri et al., 2006). Ca2+ is one of the most important secondary messengers in eukaryotes, which plays an important role in different signal transduction pathways under stress conditions (Reddy, 2001). A number of multi-domain FKBPs viz., MzFKBP66, AtFKBP62, AtFKBP65, wFKBP73 and wFKBP77 have been reported to interact with CaM in plants (Vucich and Gasser 1996; Hueros et al., 1998; Kurek et al., 2002; Aviezer-Hagai et al. 2007). We, therefore, also analysed the CaM-binding property of the cloned FKBPs. CaM gel-overlay assay demonstrated that of the three proteins, only the purified TaFKBP15-1 interacted with CaM, which was dependent on the presence of Ca2+. Real-time PCR analysis of TaFKBP20-1 and TaFKBP15-1 in developing grains of wheat revealed that expression of these genes is regulated developmentally- and is cultivar-dependent. The lack of PPIase activity observed for TaFKBP16-1, TaFKBP20-1 and TaFKBP15-1 indicates that ability to catalyse cis-trans isomerisation of peptidyl prolyl bond may not be a conserved feature of plant FKBPs, since other plant orthologues viz., TaFKBP16-1, TaFKBP16-3, AtFKBP20-2, AtFKBP42were also found to be inactive (Gollan et al., 2011; Lima et al., 2006, Edvardsson et al., 2007; Kamphausen et al., 2002). However, despite lack of PPIase activity, these FKBPs may be involved in other cellular functions such as cell signalling, stress response, photosynthesis and plant development, as reported for other orthologues (Sigal and Dumnot, 1992; Geisler et al., 2003; Gollan et al., 2011; Lima et al., 2006).

Pancreatic endocrine hormones Essay

Energy, in the form of sugar, is transported in the blood. It is carried throughout the body and into all cells to produce ATP. ATP is needed for all cellular activity of the body. It is essential that the blood can maintain the body’s fuel at a constant level (homeostasis) regardless of how long it has been since the last meal. There are three main organs that regulate the control of blood sugar: the pancreas, the liver and the adrenal glands. The pancreas produces hormones called insulin and glucagon. These hormones work antagonistically to maintain blood sugar levels that are neither too low or too high. The adrenal gland plays a key function in making sure blood sugar levels are high enough. The liver helps with sugar metabolism by creating insulin receptor sites. After a meal, insulin directs the flow of nutrients. This promotes fuel storage in the liver, adipose tissue and in muscles. The flow of nutrients during fasting is influenced by glucagon. Once glycogen stores are depleted, muscle protein is degraded, and amino acids are used for gluconeogenesis in the liver. Triglycerides stored in adipose tissue are broken down under the fasting condition. The concentration of glucose in the blood rises rapidly after the ingestion of glucose ( in a high carbohydrate meal). Insulin carries out its function and starts to bring blood glucose concentrations back down to normal, then this removes the stimulus that tells the beta cells to secrete the insulin in the first place. As a result, the beta cells become less and less stimulated and so the rate of secretion of insulin declines in parallel to the rate of decline in blood glucose concentration. This mechanism is referred to as negative feedback.

Internet Management

Internet Corporation for Assigned names and Numbers (ICANN) is the non-governmental organization that is primarily responsible for making key technical decisions related to the internet. Â  ICANN was established in 1998 in California, United States.Previously, it was Internet Assigned Numbers Authority (IANA), run by US government, which performed these tasks(ICANN,n.d.).ccording to the Joint project agreement between US government and ICANN, the responsibilities of ICANN are to ensure transparency and accountability in matters related to technical coordination of internet addresses, security of root name servers, and taking part in Government Advisory Committee.For public, what it essentially does is that it takes appropriate steps to coordinate unique addresses of websites. Control of content on internet, stopping of spam, and access to internet do not come under its responsibilities(ICANN,n.d.).2. Why is this organization so controversial?Ever since the IANA’s tasks were t ransferred to ICANN, the controversy over its workings began. Â  ICANN is mainly criticized for its unaccountability and secretive workings. It is not subject to the kind of accountability that other non- governmental organizations are. Some critics also argue that ICANN has disempowered its employees to challenge it workings through California’s laws(ICANN,n.d.).In the beginning years, directors were appointed after a community of internet users voted them to positions. But after sometime, that rule was eliminated and now the directors are not appointed through a democratic process. ICANN defended itself by saying that they were restructuring themselves to come up to the expectations of modern internet. But, critics do not agree. So till this day, ICANN is an organization which is subject to minimal accountability.It is also being criticized for not letting governments, United Nations, and International Telecommunication Union to exercise appropriate level of power.3. What are one or two recent decisions this organization has recently come under criticism for?On 25th June, ICANN introduced new rules with regard to website domains. Previously, a website owner could get a website that ended in .com, .org etc. But, now they have more choice. They can buy an address which can end in any way such as, .khi, .apple etc. Â  Many people have criticized this move because they believe that there is no need for more competition in this market. Some also contend that there is no demand for additional domains.Another major concern regarding this program is the high cost of getting a gTLD. According to ITworld.com, ICANN hopes to get $185,000 from organizations for gTLD and an additional $75,000 per year more to keep it. So, the cost is enormous. Governments of Paris and New York, who plan to get the new gTLD, also say that the cost of getting a new gTLD is way too high (ICANN,n.d.).4. Why do you think so many people generally are unaware about who controls the int ernet?One simple reason for people not being aware of who controls internet is that people are not interested to know about it. Many of the ordinary web surfers are mainly concerned with visiting their favorite websites and not with knowing about the controllers of internet.Although owners of the website buy domains for themselves yet they do not know about who controls internet. One reason for this can be that many owners of websites are part- timers. So, they do not have enough time to consider these matters.

The Theoretical And Methodological Perspectives Of...

In the comparison of the theoretical and methodological perspectives of radicals Karl Marx and Friedrich Engels, with the more liberal teachings of Emile Durkheim and Max Weber, there must be an understanding that essentially they were all intellects of the period of the Enlightenment. The philosophical basis of the Enlightenment was that human beings are substantially perfectible. This meant that human beings could be taught things and that there was never an end to the capacity of what could be achieved by a human being. Furthermore, what caused such delays in their intellectual progress were the inequalities of society, which were a consequence that was leftover from the feudal emphasis of faith and tradition. The principles†¦show more content†¦According to Karl Marx, capital is goods that are allotted for investments to increase overall profit. The ability to acquire capital comes from the accrual of resources through barter and trade. In a cycle labeled by Marx as â₠¬Å"M-C-M†, capital starts off simply as money. After acquiring money, a capitalist then finds a way to transform the money into a commodity to increase their profits. After such instance the commodities in turn will be used to purchase more goods such as machinery and labor. By turning the profits into commodities, the capitalist is able to produce more goods and in turn that in to more cash flow and increasingly more money. Consistent with Marx’s teachings, wealth is not really about how much money you have, but how many commodities that you have accumulated. Marx states that the effort of the labor that is produced by individuals is not a determinant in the value of the product produced. All of the workers are considered to be of equal value in the beginning and are only differentiated when they function on different levels. Therefore, the value of said product is not determined by the labor hours of the individual, but by how many hours are necessary for the product ion of a commodity. The average worker usually works the necessary value to support their family. The typical worker works the equivalent value that is needed in order to support his family and return to work. While, in exchange the capitalist receives the surplus