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PubMed1996

Chitin-binding proteins from cowpea (Vigna unguiculata) seeds.

Vicilins (7S storage proteins) from cowpea (Vigna unguiculata) and other legume seeds were shown to bind to chitin, to regenerated chitin (fully acetylated chitin) and to chitosan (deacetylated chitin). Adsorbed vicilins were desorbed from these matrices by acetic and hydrochloric acids and by highly polymerized soluble chitosan. Proteins such as the lectin of common bean (PHA), soybean trypsin inhibitor (Kunitz), a beta-1,3-glucanase from cowpea seeds, bovine pancreatic alpha-chymotrypsin, chicken ovalbumin, serum albumin and rabbit gamma-globulin did not bind. The present result is the first description of vicilin binding to chitin but other proteins, such as wheat germ agglutinin (WGA), a lectin that contains the so called "chitin-binding domain", and a chitinase isolated from cowpea seeds, which are involved in the defense mechanisms of plants against insects and fungi, were also shown to bind to chitin as previously reported. The binding of vicilins to chitin is probably effected not through a "chitin-binding domain" because they do not share this sequence with the defense-related proteins cited above. We propose that this association of vicilins with chitin may be related to the effect of variant vicilins on the development of Callosobruchus maculatus (bruchid) in resistant cowpea seeds.

Chemical Fractionation↗
PubMed2000

Arabidopsis thaliana germin-like proteins: common and specific features point to a variety of functions.

Germin-like proteins (GLPs) are ubiquitous plant proteins encoded by diverse multigene families. It is not known whether they share germin's unusual biochemical properties and oxalate oxidase activity. Using specific antibodies, we have studied three GLPs (AtGER1. AtGER2 and AtGER3) in Arabidopsis thaliana (L.) Heynh. as well as in transgenic tobacco (Nicotiana tabacum L.) plants overexpressing these proteins. Like wheat ( Triticum aestivum L.) germin, these Arabidopsis GLPs are associated with the extracellular matrix (ECM) and they also seem to exist as two glycosylated isoforms. However, none of them is an oxalate oxidase. Although GLPs display several conserved features, each has its specific characteristics. Both AtGER2 and AtGER3 are oligomeric proteins that share germin's resistance to pepsin and to dissociation by heat and SDS. In contrast, AtGER1 seems to exist as a monomer. The GLPs may interact with the ECM in a variety of ways, since each is efficiently extracted by different conditions. In addition, germins and GLPs all bind Cibacron Blue, a dye often but not exclusively used for the purification of enzymes having nucleotide cofactors. In the case of AtGER2, binding to the dye is so tight that it almost allows a one-step purification of this protein. The variety of sequences, expression patterns and biochemical features indicates that GLPs could be a class of receptors localized in the ECM and involved in physiological and developmental processes as well as stress response.

Arabidopsis↗
PubMed2006

Standard assays do not predict the efficiency of commercial cellulase preparations towards plant materials.

Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex process involving the concerted action of exo/endocellulases and cellobiases yielding glucose and xylanases yielding xylooligomers and xylose. An overview of commonly measured cellulase-, cellobiase-, and xylanase-activity, using respectively filter paper, cellobiose, and AZCL-dyed xylan as a substrate of 14 commercially available enzyme preparations from several suppliers is presented. In addition to these standardized tests, the enzyme-efficiency of degrading native substrates was studied. Grass and wheat bran were fractionated into a water unsoluble fraction (WUS), which was free of oligosaccharides and starch. Additionally, cellulose- and xylan-rich fractions were prepared by alkaline extraction of the WUS and were enzymatically digested. Hereby, the capability of cellulose and xylan conversion of the commercial enzyme preparations tested was measured. The results obtained showed that there was a large difference in the performance of the fourteen enzyme samples. Comparing all results, it was concluded that the choice of an enzyme preparation is more dependent on the characteristics of the substrate rather than on standard enzyme-activities measured.

Biological Assay↗
PubMed2001

A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family.

A universal primer (Sprimer: 5'-GGX AAY AAY AGY GGX CAZ CC-3', X = A, G, C or T; Y = T or C; Z = A or G), designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3'-terminal genome regions from infected plant samples representing 21 different viruses in the family. Sequencing of some of the fragments (c. 1.7 kb) showed that the type strain (ATTC PV-107) of Oat necrotic mottle virus is not a distinct species in the genus Rymovirus, but is synonymous with Brome streak mosaic virus (genus Tritimovirus) and that Celery mosaic virus is a distinct member of the genus Potyvirus not closely related to any other sequenced species. Potyviruses infecting crops in China were also investigated, showing that viruses on cowpea and maize in Hangzhou, Zhejiang province were respectively Bean common mosaic virus and Sugarcane mosaic virus and that one on garlic in Nanjing, Jiangsu province was Onion yellow dwarf virus. Fragments were also sequenced from Chinese isolates of Lettuce mosaic virus and Soybean mosaic virus (from Hangzhou), Turnip mosaic virus (2 different isolates from Zhejiang province) and RNA1 of Wheat yellow mosaic virus (from Rongcheng, Shandong province).

Apiaceae↗
PubMed1978

The development of the concept of dietary fiber in human nutrition.

Fundamental studies of the laxative action of wheat bran were undertaken in the United States in the early decades of the 20th century. Walker in South Africa extended these studies among African blacks and later suggested that cereal fiber protected them against certain metabolic disorders. Trowell in Uganda elaborated this concept with regard to the rarity of common noninfective diseases of the colon. Another stream of inquiry stemmed from the hypothesis of Cleave who postulated that the presence of refined sugar, and to a lesser extent white flour, caused many metabolic diseases, while the loss of fiber caused certain colonic disorders. Meanwhile Burkitt had collected massive evidence of the rarity of appendicitis and many venous disorders in rural Africa and parts of Asia. In 1972 Trowell proposed a new physiological definition of fiber in terms of the residue of plant foods that resisted digestion by alimentary enzymes of man. Southgate has proposed chemical methods to analyze the components of dietary fiber: cellulose, hemicellulose, and lignin.

Cellulose↗
PubMed1991

Chemoreception for an insoluble nonvolatile substance: starch taste?

Substances that are insoluble in both water and lipids, such as starch, are commonly assumed to be tasteless. Starch was suspended in water with a viscous gum. Rats given a choice of fluid containing starch and the same fluid without starch consistently preferred fluids containing starch. Rats were able to detect as little as 0.5% starch from several species of plants (corn, rice, wheat, and potato). In contrast, rats ignored comparable concentrations of cellulose suspended in water. Rats were also capable of choosing the fluid containing higher levels of starch when given a choice of 1 vs. 2% starch or 0.5 vs. 1% starch. This ability to detect starch did not appear to be mediated by salivary alpha-amylase because 1) raw starch is highly resistant to hydrolysis by salivary amylase, 2) starch preference was not correlated with the susceptibility of the starch to hydrolysis by salivary amylase, and 3) starch preference was not blocked by partial or extensive desalivation. Attempts to extract impurities with either organic solvents or water did not provide any evidence that such impurities contribute to starch preference. These experiments point to a seemingly novel form of chemoreception that could be useful to animals that need to identify starch-rich foods.

Amylases↗
PubMed1992

Identification, partial purification, and characterization of two guanosine triphosphate-binding proteins associated with insulin receptors.

We have previously suggested that at least two different G-proteins are involved in mediating insulin receptor functions. Here we identify and partially purify two G-proteins with apparent molecular masses of 41 and 67 kilodaltons (kDa) that interact with insulin receptors in rat adipocytes and human placenta. Treatment of isolated rat adipocytes with insulin inhibited pertussis toxin-catalyzed ADP-ribosylation of a 41-kDa G-protein in subsequently isolated plasma membranes by 30.2 +/- 3.0% and in partially purified insulin receptor preparations by 35.6 +/- 5.7%. There was no associated decrease in the concentration of the 41-kDa G-protein in the plasma membranes, as determined by immunoblot with a common G alpha antibody. The common G alpha antibody also recognized a 67-kDa protein in the plasma membranes, the concentration of which was not affected by insulin. However, the 67-kDa protein was enriched in partially purified solubilized insulin receptor preparations. Two similar, 41- and 67-kDa G-proteins were identified in the wheat germ-purified insulin receptor preparations obtained from human placenta. Removal of these two G-proteins from insulin receptor preparations results in loss of the ability of insulin to stimulate receptor kinase activity. Addition of a fraction enriched with 41- and 67-kDa G-proteins to the G-protein-depleted insulin receptor restores the insulin sensitivity of the insulin receptor kinase activity. Furthermore, addition of G-protein-depleted insulin receptors to the fraction containing partially purified 41- and 67-kDa G-proteins enhances pertussis toxin-catalyzed ADP-ribosylation of the 41-kDa G-protein. These results indicate that either the 41- or 67-kDa G-protein, or both, interact with the insulin receptor mediating insulin receptor kinase activity. Such mutual interaction and regulation between the insulin receptor and G-proteins could be an important component of the signal transduction mechanism for insulin.

Adenosine Diphosphate Ribose↗
PubMed1981

Effect of heating rate and freezing and reheating of corn and wheat starch-water dispersions.

In this study, evaluation of scanning electron micrographs emphasized that a heated starch-water dispersion is sensitive not only because of the type of starch but also to the mode of heating, cooling, and reheating. Differences in granule appearance were seen with both corn and wheat starch-water dispersions when they were heated at different rates. Both freezing and reheating influenced the starch-water dispersions. Microwave oven reheating appeared to yield a product with more retrogradation of the starch component. For an understanding of the effect of temperature-time stresses on this common ingredient, starch, much more study is needed.

Freezing↗
PubMed1988

The projection of the medial and posterior articular nerves of the cat's knee to the spinal cord.

We studied the spinal projections of the medial and posterior articular nerves (MAN and PAN) of the knee joint in the cat with the aid of the transganglionic transport of horseradish peroxidase. The afferent fibers of the MAN entered the spinal cord via the lumbar dorsal roots L5 and L6 and those of the PAN entered via the dorsal roots L6 and L7. Within the dorsal root ganglia, most labeled neurons had small to medium diameters. A relatively higher number of medium-size cell bodies were labeled from the PAN than from the MAN. In the spinal cord labeled MAN afferent fibers and terminations were most dense in the L5 and L6 segments, and those of the PAN were most dense in L6 and L7, that is, in the respective segments of entry. Labeled afferent fibers from both nerves projected rostrally at least as far as L1 and caudally as far as S2. Labeled fibers were found in Lissauer's tract as well as in the dorsal column immediately adjacent to the dorsal horn. In the spinal gray matter, both nerves had two main projection fields, one in the cap of the dorsal horn in lamina I, the other in the deep dorsal horn in laminae V-VI and the dorsal part of lamina VII. Both nerves, but particularly the PAN, projected to the medial portion of Clarke's column. No projection was found to laminae II, III, and IV of the dorsal horn or to the ventral horn. Since these findings parallel observations on hindlimb muscle afferent fibers, the present data support the existence of a common pattern for the central distribution of deep somatic afferent fibers.

Afferent Pathways↗
PubMed1983

Inhibition of aminoacyl-tRNA synthetases by p-chloroamphetamine and its role in protein synthesis inhibition.

p-Chloroamphetamine inhibited to some degree all amino acid-dependent pyrophosphate-exchange activities which could be detected in a rabbit reticulocyte extract. A detailed kinetic analysis of the reaction catalyzed by reticulocyte leucyl-tRNA synthetase demonstrated that the inhibitor affected only amino acid binding. Less rigorous studies of other synthetases from both rabbit reticulocyte and Escherichia coli could be similarly interpreted, suggesting that this compound interacts in a common manner with these several enzymes. The contribution of such effects to the inhibition of protein synthesis by the drug was investigated using cell-free translation systems in which rates of amino acid incorporation were limited to varying degrees by the synthesis and availability of aminoacyl-tRNA. In a wheat germ system programmed with globin mRNA, in which levels of amino acids and aminoacyl-tRNAs were shown to limit the rate of protein synthesis, the inhibition produced by p-chloroamphetamine could be partially reversed by increasing the concentration of the limiting amino acid. In a reticulocyte lysate, in which amino acid concentrations were not limiting, inhibition failed to show an amino acid-reversible component. Thus, while the inhibition of aminoacyl-tRNA synthetases by amphetamines can be shown in some cases to play a role in the effects of these compounds on in vitro protein synthesis, other sites of interference with initiation and/or elongation reactions may predominate, depending on the construction of the system under study.

Amino Acyl-tRNA Synthetases↗
PubMed1997

Homology modelling of an antimicrobial protein, Ace-AMP1, from lipid transfer protein structures.

BACKGROUND: Plant nonspecific lipid transfer proteins (ns-LTPs) are small basic proteins that facilitate lipid shuttling between membranes in vitro. The function of ns-LTPs in vivo is still unknown. It has been suggested, in relation to their lipid binding ability, that they may be involved in cutin formation. Alternatively, they may act in the plant defence system against pathogenic agents. Ace-AMP1 is an antimicrobial protein extracted from onion seed that shows sequence homology with ns-LTPs but that is unable to transfer lipids. We have recently determined the three-dimensional structure of wheat and maize ns-LTPs. In order to compare the structural features of Ace-AMP1 and ns-LTPs, we have used the comparative modelling software MODELLER to predict the structure of Ace-AMP1. RESULTS: The global fold of Ace-AMP1 is very similar to those of ns-LTPs, involving four helices and a C-terminal tail without secondary structure elements. The structure of maize and wheat ns-LTP is characterized by the existence of a tunnel-like hydrophobic cavity in which a lipid molecule can be inserted. In the Ace-AMP1 structure, this cavity is blocked by a number of bulky residues. Similarly, the electrostatic potential contours of ns-LTPs show some common features that were not observed in Ace-AMP1. CONCLUSIONS: Although Ace-AMP1 displays a similar global fold to ns-LTPs, it does not present a hydrophobic cavity, which may explain why Ace-AMP1 cannot shuttle lipids between membranes in vitro. The large differences in the electrostatic properties of Ace-AMP1 and ns-LTPs suggest a different mode of interaction with membranes.

Amino Acid Sequence↗
PubMed1998

Meiosis of wheat x lymegrass hybrids.

Meiosis was examined in pollen mother cells of F1 hybrids made from crosses between wheat (Triticum aestivum) and lymegrass (Leymus arenarius and L. mollis). Fluorescence genomic in situ hybridization detected pairing between wheat and lymegrass chromosomes during prophase I and metaphase I. Such pairing, when resulting in bivalent formation, was likely to yield correct disjunction, and hence intergenomic recombination could be incorporated into the gametes. Bivalents in these hybrids, however, were more frequently formed between chromosomes of the same parental origin. Univalents were common, whereas multivalents were not clearly detected. Meiotic behaviour in some cells was not totally aberrant, and this may have accounted for the presence of normal pollen. The results are discussed in relation to intergenomic pairing, meiotic behaviour in wide-hybrids and genome relationships, including the Leymus genome origin.

Hybridization, Genetic↗
PubMed2004

Glycemia and insulinemia in healthy subjects after lactose-equivalent meals of milk and other food proteins: the role of plasma amino acids and incretins.

BACKGROUND: Milk products deviate from other carbohydrate-containing foods in that they produce high insulin responses, despite their low GI. The insulinotropic mechanism of milk has not been elucidated. OBJECTIVE: The objective was to evaluate the effect of common dietary sources of animal or vegetable proteins on concentrations of postprandial blood glucose, insulin, amino acids, and incretin hormones [glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1] in healthy subjects. DESIGN: Twelve healthy volunteers were served test meals consisting of reconstituted milk, cheese, whey, cod, and wheat gluten with equivalent amounts of lactose. An equicarbohydrate load of white-wheat bread was used as a reference meal. RESULTS: A correlation was found between postprandial insulin responses and early increments in plasma amino acids; the strongest correlations were seen for leucine, valine, lysine, and isoleucine. A correlation was also obtained between responses of insulin and GIP concentrations. Reconstituted milk powder and whey had substantially lower postprandial glucose areas under the curve (AUCs) than did the bread reference (-62% and -57%, respectively). Whey meal was accompanied by higher AUCs for insulin (90%) and GIP (54%). CONCLUSIONS: It can be concluded that food proteins differ in their capacity to stimulate insulin release, possibly by differently affecting the early release of incretin hormones and insulinotropic amino acids. Milk proteins have insulinotropic properties; the whey fraction contains the predominating insulin secretagogue.

Adult↗
PubMed1988

Human serum antibodies reactive with dietary proteins. Antigenic specificity.

The antigenic specificity of human serum IgG antibodies reactive with common dietary proteins has been evaluated by competitive binding using a solid-phase immunoassay (ELISA). Antibodies reactive with bovine milk antigens were shown to be reactive predominantly with casein, rather than alpha-lactalbumin, beta-lactoglobulin, gamma-globulin or albumin. Furthermore, sera containing antibodies reactive with bovine casein, wheat gliadin and chicken ovalbumin showed competitive binding only by each respective dietary protein antigen. IgG4 antibodies specifically reactive with ovalbumin, gliadin or casein were also not cross-reactive in competitive binding studies. Furthermore, both IgG2 and IgG4 anti-milk antibodies showed significant inhibition only with bovine casein, and not with alpha-lactalbumin or beta-lactoglobulin. These data are relevant to concepts regarding the immunobiological role of antibodies of the IgG4 isotype.

Adult↗
PubMed2001

Alternative medicine. Achieving balance between herbal remedies and medical therapy.

The case patient was taking multiple herbal preparations as well as the prescription hypnotic zolpidem. The combination was probably increasing the patient's confusion, agitation, and aggression. The treatment team reached a compromise with the daughter after providing her with education and support. They continued the wheat germ oil and a multivitamin supplement, which appeared safe, even if of limited value. The patient continued taking valproate, 125 mg bid, which reduced her physical aggression and improved resistance to care. All other herbal remedies and zolpidem were discontinued. Balancing traditional therapies with requests for herbal remedies is a common challenge for physicians. The most successful intervention occurs when doctors familiarize themselves with herbal preparations and educate patients and families about the treatments.

Aged↗
PubMed2005

Gluten: a two-edged sword. Immunopathogenesis of celiac disease.

Celiac disease (CD) is a small intestinal disorder caused by adaptive and innate immune responses triggered by the gluten proteins present in wheat. In the intestine, gluten is partially degraded and modified, which results in gluten peptides that bind with high affinity to HLA-DQ2 or HLA-DQ8 and trigger an inflammatory T cell response. Simultaneously, gluten exposure leads to increased production of IL15, which induces the expression of NKG2D on intraepithelial lymphocytes and its ligand MICA on epithelial cells, leading to epithelial cell destruction. The gluten-specific T cell response results in the production of antibodies against tissue transglutaminase and these are specific indicators of disease. CD is one of the most common inherited diseases, the HLA-DQ locus being the major contributing genetic factor. However, as the inheritance does not follow a Mendelian segregation pattern, multiple other genes, each with relative weak effect, contribute to disease development. An important role for environmental factors, however, can not be ignored as the concordance rate in monozygous twins is considerably less than 100%. The identification of these environmental factors and susceptibility genes may allow a better understanding of disease etiology and provide diagnostic and prognostic markers. The current treatment for CD consists of a life-long gluten-free diet. Although long thought to be impossible, recent results suggest that the development of nontoxic wheat varieties may be feasible, which would aid disease prevention and provide an alternative food source for patients.

Autoantibodies↗
PubMed1996

Wheat germ agglutinin-reactive chains of giant hemoglobin from the polychaete Perinereis aibuhitensis.

Wheat germ agglutinin-reactive chains of multisubunit extracellular hemoglobin from the polychaete Perinereis aibuhitensis were identified to clarify the carbohydrate gluing which is the carbohydrate-dependent supramolecular architecture of the hemoglobin (Ebina S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371). Electron microscope micrographs of Perinereis hemoglobin showed a characteristic shape of two-tiered hexagonal rings whose diameter and height were determined to be 29.4 +/- 1.7 nm and 20.0 +/- 1.8 nm, respectively. Four types of globins and two types of linkers were isolated from the giant hemoglobin by reverse-phase chromatography and SDS-PAGE. These constituents showed similar NH2-terminal sequences as those previously reported for corresponding chains of Tylorrhynchus hemoglobin (Suzuki T. and Gotoh T. (1986) J. Biol. Chem. 261, 9257-9267; Suzuki T. et al. (1990) J. Biol. Chem. 265, 12168-12177). Thus, each globin of Perinereis hemoglobin was identified in terms of amino acid sequence homology and designated using names common to Tylorrhynchus hemoglobin, namely, a, A, b, and B. The linkers were stained by horseradish peroxidase (HRP)-lectins and PAS staining kits, indicating the presence of carbohydrate oligomers. Lectin staining was also significantly positive to globins a and A, which belong to strain A, but negative to globins b and B, which belong to strain B. Results showed that linkers and globins of strain A had a site in a carbohydrate oligomer to which wheat germ agglutinin (WGA) could bind. On the other hand, an alignment between known amino acid sequences of annelid globins and linkers and the sequences of lectins revealed that only the domain of the cysteine-rich motif in linkers has a homology with WGA-type lectins. The results of this study clarify the structuring mechanism of a supramolecule by lectin-like binding, called carbohydrate gluing.

Amino Acid Sequence↗
PubMed1988

Survey of alpha-amylase activity and isoamylases in autopsy tissue.

We quantified total amylase and its isoenzymes in 22 different human tissues obtained at autopsy. Isoenzymes were separated by use of wheat-germ inhibition (WI) and electrophoresis on cellulose acetate (CA) and agarose (AG). Mean (+/- SD) total activity was highest in salivary glands (parotid 1710 +/- 897 U/g, submandibular 605 +/- 354 U/g), and pancreas (258 +/- 137 U/g). All other tissues contained 100- to 1000-fold less amylase. As assessed with WI, pancreas, jejunum, liver, placenta, testis, skeletal muscle, and spleen contained more than 90% pancreatic isoamylase. Salivary glands and thyroid contained more than 90% salivary isoamylase. All other tissues contained a mixture of the two isoenzymes. CA and AG often produced different results. For both CA and AG the most common pancreatic isoforms were P2 and S1. Salivary gland homogenates demonstrated a band migrating in the P3 position on CA. We conclude that both types of amylase isoenzymes can be found in tissues other than salivary gland and pancreas, but that their low total amylase concentrations diminish their clinical importance.

Electrophoresis, Polyacrylamide Gel↗