P2X(3) is a novel ATP-gated cation channel that is selectively expressed by small-diameter sensory neurones in rodents, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. We have studied, for the first time, P2X(3) immunoreactivity in human inflammatory bowel disease, using Western blotting and immunohistochemistry. A major 66-kDa specific protein was found by Western blotting in all colon extracts. In the inflamed group there was a significant two-fold increase in the relative optical density of the 66-kDa band (21.2 +/- 3.1; n=8) compared to controls (11.4 +/- 3.7; n=8; P=0.009). In the control colon, P2X(3)-immunoreactive neurones were scattered throughout the myenteric and submucosal plexuses, with some neurones showing immunopositive axons/dendrites. The pattern of immunostaining was similar to the neuronal marker peripherin. In general, the intensity of the staining was greater in myenteric than submucosal neurones. The number of P2X(3)-immunoreactive neurones was significantly increased in the myenteric plexus of inflamed colon compared to controls (n=13; P=0.01). In humans, unlike rodents, P2X(3) is thus not restricted to sensory neurones. Increased P2X(3) in inflamed intestine suggests a potential role in dysmotility and pain, for which it represents a new therapeutic target.

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The complete amino acid sequence for an atype gliadin protein of wheat (Triticum aestivum Linnaeus) endosperm has been derived from a cloned cDNA sequence. An additional cDNA clone that corresponds to about 75% of a similar a-type gliadin has been sequenced and shows some important differences. About 97% of the composite sequence of A-gliadin (an a-type gliadin fraction) has also been obtained by direct amino acid sequencing. This sequence shows a high degree of similarity with amino acid sequences derived from both cDNA clones and is virtually identical to one of them. On the basis of sequence information, after loss of the signal sequence, the mature a-type gliadins may be divided into five different domains, two of which may have evolved from an ancestral gliadin gene, whereas the remaining three contain repeating sequences that may have developed independently.The gliadins constitute a major fraction of the storage proteins of hexaploid (6x, AABBDD) wheat grain (Triticum aestivum Linnaeus) (1). They are important dietary proteins and determinants of dough and bread-baking quality but are limited in nutritional quality by low levels of lysine (1,2). In addition, gliadins, or peptides derived from them during digestion, initiate damage to the absorptive epithelium of the small intestine to produce the consequent symptoms of celiac disease in susceptible individuals (3, 4).Gliadins, which are synthesized on membrane-bound ribosomes in the endosperm cells during grain development (5,6), are separated into 35-50 components by two-dimensional electrophoresis (7,8); all components are rich in glutamine (30-50 mol %) and proline (15-30 mol %). On the basis of NH2-terminal amino acid sequences, gliadins may be grouped into three subfamilies (9, 10), a-type, -type, and wtype. Corresponding gene subfamilies are located on the short arms of homoeologous-group-1 chromosomes for y type and cw-type gliadins or on the short arm of group-6 chromosomes for a-type gliadins (11).We report here the primary sequences of a-type gliadins determined by DNA sequencing of complementary DNA (cDNA) clones and by amino acid sequencing of A-gliadin (an aggregable type of a-gliadin) (1). The a-type gliadins possess a unique protein structure that includes a signal sequence (absent from the mature proteins) plus five peptide domains, each having a distinctive amino acid composition. MATERIALS AND METHODSConstruction of a Wheat cDNA Library. Poly(A)+ RNA from developing wheat grain of the cultivar 'Cheyenne' was prepared and fractionated as described by Okita and Greene (6). Double-stranded cDNA was prepared by a modification (12) of the method of Wickens et al. (13) and inserted into the unique Pst I site of pBR322 by oligo(dG)-oligo(dC) tailing (14). The recombined cDNA plasmids were then used to transform Escherichia coli RR1.Identification of a-Type Gliadin cDNA Plasmids. Colony hybridization (15) was performed with 32P-labeled, reversetranscribed wheat endosperm poly(A)+ RNA. Hybrid-selected translation and NaDodSO4/polyacrylami...

The formation of the alpha 1,4 glucosidic linkages of bacterial glycogen occurs first by synthesis of ADPglucose from ATP and alpha glucose 1-P and then transfer of the glucose moiety from the formed sugar nucleotide to a pre-existing glucan primer. Unlike mammalian glycogen synthesis, regulation occurs at the synthesis of the sugar nucleotide. Generally glycolytic intermediates activate ADPglucose synthesis while AMP, ADP and/or Pi inhibit ADPglucose synthesis. A variation of activator specificity is is seen when the enzyme is isolated from different bacteria and is thought to be related to the predominant type of carbon assimilation or dissimilation pathways present in the particular organism. Evidence indicating that the allosteric activation effects observed in vitro are physiologically pertinent for the regulation of glycogen synthesis is reviewed. The recent experiments in identifying the allosteric activator site of the Escherichia coli ADPglucose pyrophosphorylase as well as other chemical modification studies identifying amino acid residues essential for allosteric activation and for catalytic activity are discussed. Evidence is also presented for the covalent modification of the Rhodopseudomonas sphaeroides ADPglucose pyrophosphorylase by bromopyruvate at its allosteric activator site. Regulation of the biosynthesis of glycogen also occurs at the genetic level and the current evidence for the existence of a glycogen operon is presented. In addition the current studies concerning the cloning of the DNA region containing the Escherichia coli structural genes coding for the glycogen biosynthetic enzymes as well as the nucleotide sequence of the E. coli ADPglucose pyrophosphorylase are presented.

Abstract:We have monitored glial cell line-derived neurotrophic factor (GDNF) secretion from rat C6 glioblastoma cells by ELISA. Representative cytokines, neurotrophins, growth factors, neuropeptides, and pharmacological agents were tested for their ability to modulate GDNF release.