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PubMed1990

Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis.

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain alpha-, beta-, and gamma-zein and alpha-, beta-, and gamma-coixin. The alpha-coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa alpha-zeins. Like the alpha-zeins, the C1 and C2 alpha-coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to gamma-coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of gamma-zein and represents 15% of the total coixin. The beta-zein fraction was composed of a major 17 kDa protein band, while the beta-coixin fraction consisted of a mixture of alpha- and gamma-coixins. Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa alpha-zein, as did C4 and C5 antisera. The antiserum against gamma-coixin showed strong cross-reaction with gamma-zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa alpha-zeins as well as the 28 and 16 kDa gamma-zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa alpha-zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Southern↗

PubMed1991

Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley.

A 1420 bp genomic fragment (lambda-hor1-17) encompassing a Hor-1 gene encoding a C-hordein polypeptide is presented. The deduced amino acid sequence is 261 residues long. It comprises a 20 amino acid signal peptide, unique NH2- and COOH-terminal regions and a coding region comprised of pentapeptide (PQQPY) and octapeptide (PQQPFPQQ) repeat motifs. The 431 bp of 5' non-coding region contains a 'TATA box' at -105, a 'CACA box' (-181 to -201) and a -300 prolamin element. In the 3' non-coding region there are two putative polyadenylation signals located 88 and 142 bp downstream of the stop codon. The structure of lambda-hor1-17 is compared with that of another gene (lambda-hor1-14) encoding a C-hordein polypeptide, which contains an amber codon interrupting the ORF. A functional assay in which the 5' non-coding regions of the two genes were fused to the beta-glucuronidase (GUS) gene demonstrated that both genes were transcriptionally active and that circa 430 bp of the C-hordein promoters were sufficient to drive the expression of the GUS gene in developing barley endosperms. It also demonstrated that both promoters had transcriptional efficiencies comparable with that of the 35S CaMV promoter. The in vitro translation of the coding region of lambda-hor1-14 in the wheat germ system showed that the premature stop codon could be partially suppressed. The suppression was also demonstrated in a transient expression assay in vivo using isolated barley endosperms.

Amino Acid Sequence↗

PubMed1996

Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.

Proteins from Lupinus albus L. cv. Rio Maior seeds were fractionated according to solubility criteria. Patterns of concanavalin A (ConA)-binding polypeptides from the different classes, albumins, globulins, glutelins and prolamins, were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands of apparent molecular masses of 29 and 23.5 kDa with glutelin solubility characteristics bound the lectin. The 23.5-kDa band was separated by two-dimensional electrophoresis into two components: one glycosylated and heterogeneous with an isoelectric point of approx. 10 (designated as G23) and another, not detected with ConA, precipitating in the first dimension. The amino acid and hexosamine analysis of G23 showed that it is particularly rich in Gly (11.2%), Glx (10.0%), Ser (9.0%), Leu (8.2%), Asx (7.5%), and Pro (6.7%) and that it has a considerable content of the sulphur-containing amino acids Met (2.0%) and Cys (5.8%) and contains glucosamine. The determined N-terminal amino acid sequence of G23 was: 1KG(R)V5KGTGD10(T)PXXV15XLY(N)R20T, and this had no significant similarity to any of the amino acid sequences contained in the data bank SWISS-PROT 26. The glycoprotein G23 was completely deglycosylated with peptide-N-glycosidase F, yielding a homogeneous 21-kDa polypeptide composed of approximately 191 amino acids. The structures of the major N-linked neutral oligosaccharides of G23, determined by exoglycosidase sequencing, were as follows: Man alpha 2Man alpha 6(Man alpha 3) Man alpha 6(Man alpha 2Man alpha 2Man alpha 3)Man beta 4GlcNAc beta 4 GlcNAc (13%); +/- Man alpha 2Man alpha 6(Man alpha 3)Man alpha 6(+/- Man alpha 2 Man alpha 2 Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (29%); Man alpha 6(Man alpha 3) Man alpha 6(Man alpha 2Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (13%); Man alpha 6(Man alpha 3)Man alpha 6(Man alpha 3)Man beta 4GlcNAc beta 4GlcNAc (16%); Man alpha 6(Man alpha 3)(Xyl beta 2)Man beta 4GlcNAc beta 4GlcNAc (28%). Changes in G23 abundance during seed development, germination and seedling growth were monitored with a specific antibody. The glycoprotein G23 started to accumulate appreciably during seed formation between the 40th and the 50th days after anthesis and was detected following seed imbibition, until the 9th day in cotyledons, the 2nd day in roots and the 4th day in hypocotyls and leaves.

Amino Acid Sequence↗

PubMed1990

An immunodominant site of gamma-zein1 is in the region of tandem hexapeptide repeats.

The immunochemical data from studies with polyclonal antisera to gamma-zein1, the 27 kD component of the maize prolamin, indicated that the region containing 8 tandem repeats of the sequence PPPVHL is an immunodominant site. In one case, the entire antibody repertoire of an antiserum recognized epitope(s) within this region. Three 17-mer oligopeptides corresponding to the predicted antigenic epitopes of gamma-zein1 were synthesized and reacted with three different anti-gamma-zein1 sera in order to map antigenic sites in the intact protein. These antisera yielded positive reactions with a 17-mer peptide (peptide 37), which was not in a hydrophilic maximum but derived from the repeat region. The same antisera gave little or no reaction with other peptides (peptides 38 and 39), both of which were in a hydrophilic maximum. In addition, an antiserum to peptide 37 reacted strongly with both the homologous antigen and the intact gamma-zein1. Peptide 37 also blocked the binding of antisera to gamma-zein1 in competition assays. Subsequently, the shorter 6-mer (peptide 82) and 12-mer (peptide 80) versions of peptide 37 were synthesized, and both reacted with anti-peptide 37 serum and also with each of the three anti-gamma-zein1 sera. In these reactions and in competition assays, the reactivity and the blocking ability increased in proportion to the length of the peptide. Based on these data, it was concluded that the repeat region of gamma-zein1 is the site of one or more continuous immunodominant epitopes. The data also suggest that the repeat region is exposed on the surface of the folded protein and probably occur as a mobile, random coil.

Amino Acid Sequence↗

PubMed1994

Changes in the carbohydrates and nitrogenous components during germination of proso millet, Panicum miliaceum.

Since germination has been found to increase the nutritive qualities of grains, proso millet, a minor millet, was allowed to germinate for 1-7 days and analysed for protein fractions, free amino acids, lysine, tryptophan, methionine, non-protein nitrogen, starch, and sugars. Overnight soaking and germination up to 7 days significantly increased the free amino acids and total sugars while the content of dry weight and starch decreased. Increases in albumin and globulin and large decreases in prolamin accompanied sprouting. The percent protein in germinated grains was higher than in the initial grain as a result of dry matter loss during germination. Further, there was an increase in lysine, tryptophan and non-protein nitrogen contents during germination. No change was noticed in methionine content.

Carbohydrate Metabolism↗

PubMed1994

Immunological analysis of serum for buckwheat fed celiac patients.

Buckwheat (Fagopyrum esculentum, Moench), a herbaceous plant of the Polygonaceae family, does not have any affinity for the Gramineae family. It is commercially rated as a cereal, which has led to some misunderstanding concerning its use in gluten induced enteropathy or celiac disease. The effect of buckwheat flour ingestion by celiac patients was evaluated through the indirect immunofluorescence technique. Samples of serum were collected 30 days after the flour ingestion. Cryostat performed cuts of wheat and buckwheat grains were accomplished in order to compare the reactivity of their proteins and serum of celiac and normal patients. The assay revealed that the buckwheat flour presents no toxicity for the celiac patient and no anti-protein antibodies formation in the grain was observed for serum dilutions of 1:2 up to 1:32 with 0.15 M NaCl solution in 0.01 M phosphate buffer, pH 7.2 throughout the 30 day assay period. The results of the immunological assays showed that the buckwheat flour does not present toxic prolamines to celiac disease patients.

Antibodies↗

PubMed1982

Toxicity of different wheat gliadins in coeliac disease.

To determine the toxic effect of different gliadins on coeliac patients, which has been variably assessed in the literature, wheat prolamines (gliadin) were separated into the main fractions alpha-, beta-, gamma-, and omega-gliadins by chromatography on Sulfopropyl Sephadex C-50. The chemical compositions of the gliands were characterized by polyacrylamide gel electrophoresis, amino-acid analysis, determination of amide nitrogen and peptide maps. The peptide fractions B2 and B3 were isolated from the gliadins by a peptic tryptic digestion, ultrafiltration and gel filtration on Sephadex G-50. The gliadins and the peptide fractions were examined for coeliac activity by immunological tests (LIF tests) and by organ-culture tests. The results show that the peptide fractions are generally more active than their respective gliadins. The peptide fractions of all gliadins have a coeliac-specific toxic effect; their activities correlate with the chemical composition of the gliadins.

Amino Acids↗

PubMed1995

A chemical study of the protein fractions of Tarragona hazelnuts (Corylus avellana).

The protein contents of six Tarragona hazelnuts varieties were determined. The protein contents (g protein per 100 g hazelnuts) were between 12.1 g/100 g and 18.8 g/100 g, according to variety; a mean discrepancy of 2.3 g/100 g was detected between data from the same variety grown with irrigation and using a dry-farming method. Percentages of albumins, globulins, glutelins and prolamines were also determined. In all varieties albumins and globulins were the major fractions, averaging about 86.58% of the total protein content.

Albumins↗

PubMed1981

[Progress in human pancreas transplantation (author's transl)].

Between July 1980 and March 1981, 7 simultaneous pancreas and kidney transplants were performed in patients with juvenile diabetes and renal insufficiency. A duct-occluded (Prolamine) segmental pancreas was placed into the peritoneal cavity with vascular anastomoses to the left iliac vessels. Three transplantations were unsuccessful because of a damaged transplant, and one was lost because of venous thrombosis. Three patients with a maximal follow-up of 9 1/2 months have normal blood sugar despite lack of exogenous insulin and despite prednisone intake.

Adult↗

PubMed1980

[Partial duodenopancreatectomy and intraoperative occlusion of the pancreatic tail for chronic pancreatitis (authors' translation)].

Report on 81 partial duodenopancreatectomies for chronic pancreatitis. To prevent a postoperative pancreatic fistula and the recurrence of pancreatitis the duct system of the pancreatic tail was occluded with a solution of prolamine. This occlusion leads to an isolate 'burning out' of the exocrine parenchyma. The operative mortality was 1.2%. Up to now a recurrence of chronic pancreatitis was not observed. The later results within an observation period of 28 months are favourable.

Chronic Disease↗

PubMed1982

A chemical study of the protein fraction of Mediterranean sweet almond varieties (Prunus amygdalus).

The protein contents and amino-acid compositions of five Mediterranean almond varieties were determined. The protein contents were between 18 and 24 g/100 g of almond, according to variety. The content of each amino-acid, expressed in g/100 g of protein, was similar in all varieties. The essential amino-acid composition was compared with the FAO pattern of amino-acid requirements and to the protein of milk and eggs. Percentages of albumins, globulins, glutelins and prolamines were also determined.

Amino Acids↗

PubMed1983

[Tryptophan content of cereal proteins].

The tryptophan contents of the albumin, globulin, prolamine and glutelin fractions from wheat, rye, barley, oat, sorghum, rice and maize were estimated by HPLC after alkaline hydrolysis. Complete amino acid compositions, including the amide contents, are given for all these protein fractions.

Amino Acids↗

PubMed2000

Celiac disease--a worldwide problem.

Celiac disease and dermatitis herpetiformis are caused by the alcohol soluble fractions of wheat, barley, and rye. Reliable serological tests are available for both mass and risk group screening and recent epidemiological studies on celiac disease suggest that the prevalence varies between 1:100-300 in different continents. The clinical manifestations of the disease has changed in the West and the classical symptomatic cases represent only approximately 1/7th of all diagnosed cases. Symptoms such as, anemia, short stature, dental enamel defect or osteoporosis can be the only manifestations of the atypical disease. There is an increased prevalence of celiac disease in patients with autoimmune diseases. Recent data suggest that there is a correlation between the prevalence of autoimmune diseases and the number of years that an individual consumes gluten-containing foods. Genetic studies revealed a high prevalence of certain HLA antigens in celiac patients, however, there is likelihood that non-HIA genes are also important in the pathomechanism. An interesting new development is the recognition of tissue transglutaminase (tTG), an enzyme that probably forms an autoantigen with gluten. It is generally accepted that antibodies to tTG are identical to the previously described antiendomysium antibodies. Whether or not tTG is responsible for the initiation of an immunoreaction against prolamines or just exacerbates the immune response is a subject of further investigations.

Age of Onset↗

PubMed2003

Expression of the gamma-zein protein of maize in seeds of transgenic barley: effects on grain composition and properties.

A cDNA clone encoding the gamma-zein protein of maize was expressed in developing grain of barley using the starchy endosperm cell-specific promoter from the wheat Glu-1D-1 (HMW subunit 1Dx5) gene. Seven transgenic lines were recovered from 226 bombarded immature embryos, of which two were sterile and four tetraploid, while five were shown to express the gamma-zein protein based on western blotting. Southern blot analysis showed the presence of between about three and twelve transgene insertions. Detailed comparative studies of five null and five homozygous transformed sub-lines from transgenic line A showed that gamma-zein accounted for over 4% of the total prolamin fraction, corresponding to about 1.9% of the total grain N. Comparison of the proteins present in the gel protein fraction demonstrated that the gamma-zein was incorporated into polymers, as in maize. However, there was no effect on grain hardness measured using the Perten Single Kernel Characterisation System or on the vitreousness measured by visual inspection. This contrasts with the situation in maize where a clear association with vitreousness has been reported.

Gene Transfer Techniques↗

PubMed2003

In situ Western hybridization: a new, highly sensitive technique to detect foreign and endogenous protein distribution in rice seeds.

We have developed a highly sensitive in situ Western hybridization technique to study tissue-specific expression of foreign and endogenous genes in transgenic and non-transformed rice seed. The expression pattern of the soybean ferritin gene directed by a rice glutelin gene promoter, GluB-1, in transgenic rice seed revealed by this method was exactly the same as that revealed by immunological tissue printing but much clearer than the latter, and corresponded well to the results of GluB-1 promoter characterization studies. This method provides an alternative choice for studying the tissue-specific expression of a promoter, omitting the complicated transgenic procedure. The method can also be used to study the expression and accumulation pattern of endogenous genes, such as glutelin and prolamine genes, in non-transformed plants.

Blotting, Western↗

PubMed1998

Lysine-rich gamma-zeins are secreted in transgenic Arabidopsis plants.

We have previously shown that the maize (Zea mays L.) storage prolamine gamma-zein, accumulates in endoplasmic reticulum-derived protein bodies in transgenic plants of Arabidopsis thaliana (L.) ecotype R + P. The retention of gamma-zein in the endoplasmic reticulum was found to be mediated by structural features contained in the polypeptide, an N-terminal proline-rich and a C-terminal cysteine-rich domain which were necessary for the correct retention and assembly of gamma-zein within protein bodies (M.I. Geli et al., 1994, Plant Cell 6: 1911-1922). In the present work we incorporated in the gamma-zein gene lysine-rich coding sequences which were positioned after the N-terminal proline-rich domain and at five amino-acid residues from the C-terminus. The targeting of lysine-rich gamma-zeins was analyzed by expression of chimeric genes regulated by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic Arabidopsis plants. The lysine-rich gamma-zeins were detected by immunoblotting and we found that these proteins were modified posttranslationally to reach their mature form. Subcellular fractionation and immunocytochemical studies demonstrated that glycosylated lysine-rich gamma-zeins were secreted to the cell wall of transgenic Arabidopsis leaf cells.

Arabidopsis↗

PubMed2003

Superiority of combined chemo-embolization and portal infusion with 5-fluorouracil over locoregional infusion concepts in Novikoff hepatoma-bearing rats.

The aim of this study was to investigate experimentally whether there is a superior effect of the combination of hepatic artery chemo-embolization with portal vein infusion over either of the two treatment modalities alone. Novikoff hepatoma cells transplanted under the liver capsule of Sprague Dawley rats were used as a model. Tumor growth was assessed at 7 and 21 days after tumor inoculation. The prolamine solution Ethibloc was employed for embolization, and 5-fluorouracil was used as a chemotherapeutic agent for both infusion and chemo-embolization. All arterial treatment modalities were administered in a super-selective manner. There was no intolerable toxicity after dosages of 55 to 125 mg 5-fluorouracil/kg body weight. With regard to therapeutic efficacy the results show that embolization is an effective therapeutic means for inducing tumor necrosis in selected liver areas. As a consequence, the ranking of all treatment modalities was based on the combined evaluation of tumor size and extent of tumor necrosis. According to this evaluation, hepatic artery chemo-embolization was superior to the respective type of infusion (P<0.01). In addition, the combination of both modalities in the form of hepatic artery chemo-embolization and portal vein infusion was effective in destroying more than 97% of vital tumor tissue (P<0.01). These results suggest the need for a comparative clinical study.

Animals↗

PubMed2006

A novel cis-acting element, ESP, contributes to high-level endosperm-specific expression in an oat globulin promoter.

To examine the genetic controls of endosperm (ES) specificity, several cereal seed storage protein (SSP) promoters were isolated and studied using a transient expression analysis system. An oat globulin promoter (AsGlo1) capable of driving strong ES-specific expression in barley and wheat was identified. Progressive 5' deletions and cis element mutations demonstrated that the mechanism of specificity in the AsGlo1 promoter was distinct from that observed in glutelin and prolamin promoters. A novel interrupted palindromic sequence, ACATGTCATCATGT, was required for ES specificity and substantially contributed to expression strength of the AsGlo1 promoter. This sequence was termed the endosperm specificity palindrome (ESP) element. The GCN4 element, which has previously been shown to be required for ES specificity in cereal SSP promoters, had a quantitative role but was not required for tissue specificity. The 960-bp AsGlo1 promoter and a 251-bp deletion containing the ESP element also drove ES-specific expression in stably transformed barley. Reporter gene protein accumulated at very high levels (10% of total soluble protein) in ES tissues of plants transformed with an AsGlo1:GFP construct. Expression strength and tissue specificity were maintained over five transgenic generations. These attributes make the AsGlo1 promoter an ideal promoter for biotechnology applications. In conjunction with previous findings, our data demonstrate that there is more than one genetically distinct mechanism by which ES specificity can be achieved in cereal SSP promoters, and also suggest that there is redundancy between transcriptional and post-transcriptional tissue specificity mechanisms in cereal globulin genes.

Avena↗