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H Wacker

Publications and source records attributed to H Wacker.

At least 19 recordsLinked to original sources
PubMed1999

Antigenic phenotyping of lymphoid cells and B cell gene rearrangement in type B gastritis and in gastritis not associated with Helicobacter pylori colonization.

Marginal-zone B cells of the mucosa-associated lymphoid tissue (MALT) are the normal counterpart of the neoplastic cells in MALT lymphoma. In both cases these lymphocytes express surface immunoglobulins, but are negative when stained for B cell associated antigens like CD10 and CD23. Furthermore, the B cell gene rearrangement has been found in Helicobacter pylori associated chronic gastritis and in extranodal type of marginal-zone lymphoma. The aim of this study was to quantify the number of IgM-, CD10-, and CD23-positive lymphocytes in patients with type B gastritis and to compare the results with the antigen profile of mononuclear cells in patients with gastritis not associated with H. pylori. Additionally, the immunoglobulin heavy-chain (IgH) gene rearrangement in H. pylori positive and H. pylori negative gastritis was studied. From 23 patients with a positive urease test and/or histologically proven H. pylori infection and chronic gastritis and from 22 patients with H. pylori negative chronic gastritis mucosa biopsy specimens were taken. Single-cell suspensions were obtained following enzymatic digestion. For immunocytochemistry, an alkaline phosphatase-antialkaline phosphatase method was applied. IgH gene rearrangement in formalin-fixed, paraffin-embedded specimens was determined by polymerase chain reaction in 11 patients with chronic gastritis. An increase in mu-positive plasma cells and B lymphocytes was detected in patients with H. pylori positive gastritis as compared with patients with H. pylori negative gastritis (10.0 vs. 3.9%, p < 0.001, and 4.3 vs. 1.6%, p < 0.01, respectively). In both groups, the proportion of CD10- and CD23-positive lymphocytes was <1%. IgH gene rearrangement was not restricted to type B gastritis; single bands were also present in 3 of 7 patients with H. pylori negative chronic gastritis. Our finding of IgH gene rearrangement in some of the patients with H. pylori negative chronic gastritis indicates that additional factors may be critical for these genotypical changes and for the pathogenesis of gastric MALT lymphoma.

Adolescent↗
PubMed1997

"Adult only" esterase/phospholipase A of the small-intestine brush border membrane: isolation, identification of the catalytic site, and biosynthesis.

We have isolated and investigated an esterase/phospholipase A (EC 3.1.1) which is an intrinsic protein of the small-intestine brush border membrane of adult but not of preweaning rabbits. This enzyme had been referred to previously as AdRabB [Boll, W., Schmid-Chanda, T., Semenza, G., & Mantei, N. (1993) J. Biol. Chem. 268, 12901-12911]. Its amino acid sequence shows four extensive, homologous repeats between the signal sequence and a hydrophobic stretch in the C-terminal region. We have identified a serine residue (Ser400) in the (unexpectedly) single catalytic site and indicate that this esterase is likely to operate by way of a Ser-His-Asp/Glu triad. This esterase/phospholipase A is synthesized as a single polypeptide chain of 170-180 kDa, agreeing well with the size deduced from its cognate cDNA sequence [Boll, W., et al. (1993) J. Biol. Chem. 268, 12901-12911], and it undergoes (at least) N-glycosylation. Once it has reached the brush border membrane, it is subjected in vivo to two types of proteolytic processing, presumably by pancreatic protease(s): a split after Arg263, with loss of the first N-terminal repeat, and two or more cleavages much farther downstream but still located in the luminal bulk of the protein; these splits lead to multiple bands of approximately 140, 125, 100, and 90 kDa.

Amino Acid Sequence↗
PubMed1996

Comorbidity and boundaries of affective disorders with anxiety disorders and substance misuse: results of an international task force.

Associations between affective disorders, anxiety disorders, and substance use disorders were examined in epidemiological studies conducted in Germany, Switzerland, Puerto Rico, and the mainland US. There was a remarkable degree of similarity across studies in the magnitude and type of specific disorders associated with the affective disorders. Comorbidity with affective disorders was greater for the anxiety disorders than for substance misuse. Panic disorder was the subtype of anxiety that was most highly comorbid with depression. Social phobia was the specific phobic type with the strongest association with the affective disorders. The magnitude of associations between substance misuse and affective disorders generally was quite low and less consistent across sites. No major differences were found in the patterns of comorbidity by gender or age group, affective subtype or prevalence period. The onset of anxiety disorders generally preceded that of depression, whereas alcohol misuse was equally likely to pre-or post-date the onset of affective disorders. Finally, comorbidity was associated with an elevation in treatment rates across all sites, confirming Berkson's paradox on an international level.

Adolescent↗
PubMed1995

Characterization of lipid exchange proteins isolated from small intestinal brush border membrane.

Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. They differ widely in apparent molecular weight and catalyze cholesterol, phosphatidylcholine, and phosphatidylinositol exchange between two populations of small unilamellar lipid vesicles. In order to determine their interrelations, the smallest water-soluble lipid exchange protein was purified to homogeneity by gel filtration on Sephadex G-75 and cation exchange chromatography on Mono S. It is a basic protein of apparent molecular mass of 13 +/- 0.5 kDa. The purified protein was used to raise polyclonal antibodies. Polyclonal antibodies were also produced against a lipid exchange protein of apparent molecular mass of 100-120 kDa. By comparing lipid exchange, lipid binding, and immunological properties of the water-soluble lipid exchange proteins it can be shown that the 13-kDa (peak 3) protein is related to the 100-120 kDa (peak 1) protein; the properties of these two proteins are different from those of the peak 2 lipid exchange protein of apparent molecular mass of 22 kDa. Based on the immunological cross-reactivity observed between the 13 and 100-120 kDa and the lipid binding properties of these two proteins, a working hypothesis is proposed: both proteins are probably part of an integral membrane protein of the brush border membrane that facilitates cholesterol and phosphatidylcholine absorption in this membrane. Evidence derived from immunogold labeling of BBMV supports the notion that this protein is located on the external (luminal) side of the brush border membrane. The analogous behavior of rabbit and human small intestinal brush border membrane in terms of lipid absorption and the release of water-soluble lipid exchange proteins is discussed.

Animals↗
PubMed1992

Location of the two catalytic sites in intestinal lactase-phlorizin hydrolase. Comparison with sucrase-isomaltase and with other glycosidases, the membrane anchor of lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase was isolated by immunoadsorption chromatography from rabbit brush-border membrane vesicles. Inactivation of the enzyme with [3H]conduritol-B-epoxide, a covalent active site-directed inhibitor, labeled glutamates at positions 1271 and 1747. Glu1271 was assigned to lactase, Glu1747 to phlorizin hydrolase activity. In contrast, the nucleophiles in the active sites of sucrase-isomaltase are aspartates (Asp505 and Asp1394). Asp505 is a part of the isomaltase active site and is localized on the larger subunit, which carries the membrane anchor also, while Asp1394 is a part of the active of sucrase. Alignment of these 2 nucleophilic Glu residues in lactase-phlorizin hydrolase and of their flanking regions with published sequences of several other beta-glycosidases allows the classification of the configuration retaining glycosidases into two major families: the "Asp" and the "Glu" glycosidases, depending on the carboxylate presumed to interact with the putative oxocarbonium ion in the transition state. We offer some predictions as to the Glu acting as the nucleophile in the active site of some glycosidases. By hydrophobic photolabeling, the membrane-spanning domain of lactase-phlorizin hydrolase was directly localized in the carboxyl-terminal region thus confirming this enzyme as a monotopic type I protein (i.e. with Nout-Cin orientation) of the brush-border membranes. A simplified version of the Me2+ precipitation method to efficiently and simply prepare brush-border membrane vesicles is also reported.

1-Deoxynojirimycin↗
PubMed1990

Rabbit small intestinal trehalase. Purification, cDNA cloning, expression, and verification of glycosylphosphatidylinositol anchoring.

alpha,alpha-Trehalase (EC 3.2.1.28), an intrinsic protein of intestinal brush-border membranes, was purified to homogeneity from rabbits. Partial amino acid sequences were determined. Two degenerate oligonucleotides based on the sequence of a CNBr peptide were employed in a polymerase chain reaction to amplify a 71-base pair fragment of trehalase DNA with rabbit intestine cDNA as a starting template. This fragment was used as a hybridization probe to isolate full length trehalase clones from a rabbit intestine cDNA bank. Sequence analysis revealed that trehalase comprises 578 amino acids, contains at the amino terminus a typical cleavable signal sequence, at the carboxyl terminus a rather hydrophobic region typical of proteins anchored via glycosylphosphatidylinositol, and four potential N-glycosylation sites. Trehalase has no sequence homologies with other sequenced brush-border glycosidases. Northern blot analysis revealed a 1.9-kilobase trehalase mRNA in small intestine and kidney, smaller amounts in liver, and none in lung. Southern blot analysis indicated the gene has a length of 20 kilobase pairs or less. Injection into Xenopus laevis oocytes of mRNA synthesized in vitro from a trehalase template resulted in the expression of trehalase activity several hundredfold above background. The trehalase activity was membrane-bound and could be solubilized upon digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. This strongly suggests that rabbit small intestinal trehalase is anchored via glycosylphosphatidylinositol also when expressed in X. laevis oocytes.

Amino Acid Sequence↗
PubMed1988

Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme.

We report the primary structures of human and rabbit brush border membrane beta-glycosidase complexes (pre-pro-lactase-phlorizin hydrolase, or pre-pro-LPH, EC 3.2.1.23-62), as deduced from cDNA sequences. The human and rabbit primary translation products contain 1927 and 1926 amino acids respectively. Based on the data, as well as on peptide sequences and further biochemical data, we conclude that the proteins comprise five domains: (i) a cleaved signal sequence of 19 amino acids; (ii) a large 'pro' portion of 847 amino acids (rabbit), none of which appears in mature, membrane-bound LPH; (iii) the mature LPH, which contains both the lactase and phlorizin hydrolase activities in a single polypeptide chain; (iv) a membrane-spanning hydrophobic segment near the carboxy terminus, which serves as membrane anchor; and (v) a short hydrophilic segment at the carboxy terminus, which must be cytosolic (i.e. the protein has an Nout-Cin orientation). The genes have a 4-fold internal homology, suggesting that they evolved by two cycles of partial gene duplication. This repetition also implies that parts of the 'pro' portion are very similar to parts of mature LPH, and hence that the 'pro' portion may be a water-soluble beta-glycosidase with another cellular location than LPH. Our results have implications for the decline of LPH after weaning and for human adult-type alactasia, and for the evolutionary history of LPH.

Amino Acid Sequence↗
PubMed1986

Cell-free synthesis, membrane integration, and glycosylation of pro-sucrase-isomaltase.

Cell-free translation of total RNA from rabbit intestinal mucosa in a rabbit reticulocyte lysate, after immunoprecipitation with antibodies directed against sucrase-isomaltase, yielded a polypeptide of 200 kDa, which was identified as pro-sucrase-isomaltase. Addition of dog pancreatic microsomal vesicles to the translation system resulted in the appearance of an additional 220-kDa polypeptide. The 220-kDa polypeptide was associated with the membranes in a way that made it inaccessible to proteolysis; this protection was abolished by lytic detergent concentrations, indicating that the polypeptide was segregated into the microsomal vesicle. The 220-kDa polypeptide was glycosylated as evidenced by it being bound to concanavalin A-Sepharose and eluted with alpha-methyl-D-mannopyranoside. The increase in apparent molecular mass (approximately 20 kDa) of the primary translation product upon translocation was due to the addition of carbohydrate; treatment of the 220-kDa polypeptide with endo-beta-N-acetylglucosaminidase H increased its electrophoretic mobility to that of the 200-kDa polypeptide which was obtained in the absence of membranes. Partial N-terminal amino acid sequence of a translation product labeled with [3H]Leu in the absence of membranes revealed that Leu was incorporated into identical positions as in the final (pro)-sucrase-isomaltase, thus indicating the lack of a transient signal peptide.

Animals↗
PubMed1984

A two-active site one-polypeptide enzyme: the isomaltase from sea lion small intestinal brush-border membrane. Its possible phylogenetic relationship with sucrase-isomaltase.

The enzyme responsible for all of the isomaltase activity and much of the maltase activity in the small intestine of the Californian sea lion (Zalophus californianus) was isolated by detergent solubilization of the brush-border membrane, followed by immunoadsorption chromatography using antibodies directed against rabbit sucrase-isomaltase. In 0.1% Triton X-100, sea lion isomaltase occurs as a monomer of Mr = 245,000 and is composed of a single polypeptide chain. As judged from the stoichiometry of the covalent binding of the affinity label, conduritol-B-epoxide, this polypeptide chain carries two enzymatically active sites; they are apparently identical and do not show either positive or negative cooperativity. In addition to cross-reacting immunologically with rabbit sucrase-isomaltase, sea lion isomaltase has similar overall enzymatic properties, with the exception of not hydrolyzing sucrose. The Alaskan fur seal (Collarhinus ursinus) has a two-active site isomaltase; however, in contrast to the sea lion, this animal is endowed with a small but significant sucrase activity. Along with (fully active) pro-sucrase-isomaltase, sea lion isomaltase is one of the very few examples of enzymes with more than one active site on a single polypeptide chain acting "in parallel" (rather than "in series"). Furthermore, this enzyme triggers some interesting questions on the phylogenetical pedigree of small intestinal sucrase-isomaltase.

Animals↗
PubMed1983

Biosynthesis and assembly of the largest and major intrinsic polypeptide of the small intestinal brush borders.

The sucrase-isomaltase complex (SI) of the small intestinal brush border membrane accounts for approximately 9-10% of the intrinsic protein. The isomaltase subunit alone interacts with the membrane directly, via a highly hydrophobic segment at its N-terminal region. This segment has a helical conformation for more than 85% and crosses the membrane twice, the N-terminus being located at the outer, luminal side of the membrane. The sucrase subunit is attached to the membrane solely via its interactions with the isomaltase subunit. The sucrase-isomaltase complex is synthesized as a single, very long (Mr approximately 260 000) polypeptide chain (pro-SI, carrying the two sites of sucrase and isomaltase in an already enzymically active form), with the isomaltase portion corresponding to the N-terminal part of pro-SI. Pro-SI is processed into 'final' SI by pancreatic proteases. Recently the cell-free translation of pro-SI has been achieved in vitro. From a detailed knowledge of the anchoring of SI (and pro-SI) in the membrane it has been possible to suggest one particular mechanism as the most likely for the synthesis, insertion and assembly of pro-SI.

Amino Acid Sequence↗
PubMed1982

N-Terminal sequences of pig intestinal sucrase-isomaltase and pro-sucrase--isomaltase. Implications for the biosynthesis and membrane insertion of pro-sucrase--isomaltase.

The hog sucrase-isomaltase complex is anchored to the small-intestinal brush border membrane, as in the rabbit, via a hydrophobic segment located in the N-terminal region of the isomaltase subunit. The immediate precursor of the 'final' sucrase-isomaltase (i.e., pro-sucrase-isomaltase as prepared from adult hogs whose pancreas had been disconnected from the duodenum) is an amphiphilic single polypeptide chain of Mr 260000-265000. Its N-terminal sequence is virtually identical with (not merely homologous to) the corresponding region of the isomaltase subunit of 'final' sucrase-isomaltase. This shows that the isomaltase portion of pro-sucrase-isomaltase is the N-terminal 'half' of the precursor polypeptide chain. Thus the succession of domains in pro-sucrase-isomaltase and its mode of anchoring in the membrane could be deduced. On this basis a likely mechanism of biosynthesis and insertion is proposed.

Amino Acid Sequence↗
PubMed1982

Biosynthesis of sucrase-isomaltase. Purification and NH2-terminal amino acid sequence of the rat sucrase-isomaltase precursor (pro-sucrase-isomaltase) from fetal intestinal transplants.

The dimeric enzyme sucrase-isomaltase (a complex of sucrose alpha-glucohydrolase, EC 3.2.1.48 and oligo-1,6-glucosidase (dextrin 6 alpha-D-glucanohydrolase), EC 3.2.1.10) of the rat small intestinal microvillus membrane is synthesized as a single chain enzymatically active precursor protein. This precursor (called pro-sucrase-isomaltase) was purified from fetal intestinal transplants in which sucrase-isomaltase was found almost exclusively in the uncleaved precursor form. A two-step procedure was developed using monoclonal antibody affinity chromatography on protein A Sepharose CL-4B followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequence of purified pro-sucrase-isomaltase was identical with that of the isolated isomaltase subunit which possesses the membrane anchor for the mature enzyme complex but differed from the NH2-terminal sequence of the sucrase subunit. This identity shows that the isomaltase domain comprising the membrane anchor is synthesized prior to the bulk of the protein destined to be localized on the luminal side of the microvillus membrane. A model is proposed for the mode of membrane assembly and the subsequent cleavage of pro-sucrase-isomaltase into its mature subunits.

Amino Acid Sequence↗
PubMed1981

[Stepwise combination of skeletal scintigram and x-rays in the improvement and rationalisation of skeletal metastases detection (author's transl)].

Skeletal scintigraphy with 99mTc phosphate complexes allows the early detection of sites of pathological activity in bones. High sensitivity of this procedure (93%) contrasts with low specificity (59%). Scintigraphically-located sites are uncharacteristic and can imply various diagnoses and require radiodiagnostic investigation. The stepwise application of both methods is recommended, according to tumour type and clinical picture in order to avoid unnecessary and costly duplication of investigation procedures. Thus, the combination of the high specificity of skeletal radiology and the high sensitivity of scintigraphy aids the early detection of skeletal metastases, whereby whole-body skeletal scintigraphy forms the basis for such investigations.

Adenocarcinoma↗