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W Zimmermann

Publications and source records attributed to W Zimmermann.

At least 55 records · Page 3Linked to original sources
PubMed1998

High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca - substrate specificity in glycosyl hydrolase family 5.

BACKGROUND: . beta-Mannanases hydrolyse the O-glycosidic bonds in mannan, a hemicellulose constituent of plants. These enzymes have potential use in pulp and paper production and are of significant biotechnological interest. Thermostable beta-mannanases would be particularly useful due to their high temperature optimum and broad pH tolerance. The thermophilic actinomycete Thermomonospora fusca secretes at least one beta-mannanase (molecular mass 38 kDa) with a temperature optimum of 80 degreesC. No three-dimensional structure of a mannan-degrading enzyme has been reported until now. RESULTS: . The crystal structure of the thermostable beta-mannanase from T. fusca has been determined by the multiple isomorphous replacement method and refined to 1.5 A resolution. In addition to the native enzyme, the structures of the mannotriose- and mannohexaose-bound forms of the enzyme have been determined to resolutions of 1.9 A and 1.6 A, respectively. CONCLUSIONS: . Analysis of the -1 subsite of T. fusca mannanase reveals neither a favourable interaction towards the axial HO-C(2) nor a discrimination against the equatorial hydroxyl group of gluco-configurated substrates. We propose that selectivity arises from two possible mechanisms: a hydrophobic interaction of the substrate with Val263, conserved in family 5 bacterial mannanases, which discriminates between the different conformations of the hydroxymethyl group in native mannan and cellulose; and/or a specific interaction between Asp259 and the axial hydroxyl group at the C(2) of the substrate in the -2 subsite. Compared with the catalytic clefts of family 5 cellulases, the groove of T. fusca mannanase has a strongly reduced number of aromatic residues providing platforms for stacking with the substrate. This deletion of every second platform is in good agreement with the orientation of the axial hydroxyl groups in mannan.

Actinomycetales↗
PubMed1998

Transcriptional regulation of the non-specific cross-reacting antigen gene, a member of the carcinoembryonic antigen gene family up-regulated in colorectal carcinomas.

Human non-specific cross-reacting antigen (NCA), a close relative of the tumor marker human carcinoembryonic antigen (CEA), is also an in vitro homotypic intercellular adhesion molecule capable of inhibiting differentiation when expressed ectopically by myoblasts. Moreover, NCA appears to be overexpressed at the transcriptional level to a greater extent and more frequently in colorectal carcinomas than CEA. This study examines the transcriptional control mechanisms responsible for orchestrating NCA expression. The region within 284 bp upstream of the translational start site of the NCA gene was found to be capable of directing high levels of expression in functional promoter assays. Footprinting experiments identified three cis-acting elements and mobility-shift assays revealed that the first of these elements is bound by the upstream stimulating factors USF1 and USF2 while the other two are bound by the stimulatory proteins Sp1 and Sp3. No cis-acting elements corresponding to CEA footprint FP4 or the silencer CEA FP5 were detected in the NCA promoter, which may contribute to the differential expression of NCA versus CEA in tumorigenesis.

Antigens, Neoplasm↗
PubMed1998

Mice transgenic for the human CGM6 gene express its product, the granulocyte marker CD66b, exclusively in granulocytes.

The nonspecific cross-reacting antigen-95 (NCA-95/CD66b), is a member of the human carcinoembryonic antigen (CEA) family encoded by the CGM6 gene that is exclusively expressed in neutrophils and eosinophils. No murine counter-part is known to exist. We have analyzed a cosmid containing the complete CGM6 gene. The coding sequence is contained within six exons spanning a 16.5 kb region. The main transcriptional start site was mapped to a tight cluster between nucleotides -95 and -101 relative to the translational start site. As with other members of the CEA gene family, no typical TATA or CAAT-box sequences were found in the CGM6 gene. Transgenic mice were established with the cosmid insert. CD66b expression is first seen in the fetal liver on day 12.5 of mouse embryonic development, and it first appears in the bone marrow at day 17.5. Northern blot analysis showed that CD66b transcripts are confined to the bone marrow of adult mice, whereas immunohistochemistry also showed CD66b-positive granulocytes in the spleen, thymus, and lungs. FACScan analyses of bone marrow and spleen cells showed CD66b expression to be exclusive to granulocytes. Thus, all the elements necessary for regulating granulocyte-specific expression are present within this cosmid clone. These mice could provide a model for transplantation and for inflammation studies using CD66b as a granulocyte-specific marker.

Animals↗
PubMed1998

Production of cyclomaltononaose (delta-cyclodextrin) by cyclodextrin glycosyltransferases from Bacillus spp. and bacterial isolates.

The conversion of soluble starch to cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD) and cyclomaltononaose (delta-CD) by cyclodextrin glycosyltransferases (E.C. 2.4.1.19) from Bacillus spp. and bacterial isolates was studied. The results show that delta-CD was formed by all the enzymes investigated in the range of 5%-11.5% of the total amount of alpha-, beta-, gamma-, and delta-CD produced.

Bacillus↗
PubMed1998

[Continuous serologic study and sanitation inspection of Sarcoptes scabiei var. suis infection: preliminary results].

Pig mange was investigated serologically with ELISA using Chekit Sarcoptest. The test consists of a homogenized antigen of Sarcoptes scabiei var. vulpes. A total of 356 sows sera samples were collected from 16 mange free breeding units and 19 units with clinical manifestation of mange infestation. The specificity of the test was 99.34% and the mean sensitivity value was 62%. This test will allow in the future not only the rapid and certain diagnosis of pig mange but also the screening of the pig breeding units after eradication of the disease.

Animals↗
PubMed1997

cea5, a structurally divergent member of the murine carcinoembryonic antigen gene family, is exclusively expressed during early placental development in trophoblast giant cells.

The carcinoembryonic antigen (CEA) gene family encodes a large family of glycoproteins. Some are probably involved in the homeostasis/development of epithelial cells and granulocyte activation, while others e.g. the pregnancy-specific glycoproteins, are expressed in the placenta and are essential for a positive outcome of pregnancy. In this paper, we have characterized cea5, a member of the murine CEA gene family. RNase protection and in situ hybridization analyses revealed that Cea5 mRNA is exclusively synthesized in primary and secondary trophoblast giant cells of the placenta only during early stages of development. Full-length Cea5 cDNA was obtained by a reverse transcription-polymerase chain reaction using day 10.5 post-coitum placental RNA. The 1.6-kilobase pair (kb) Cea5 mRNA encodes a secreted glycoprotein with a predicted size of 30 kDa. It is composed of a leader peptide (L), one immunoglobulin (Ig) variable or N, and one Ig constant-like or A domain. This domain organization is unique within the human and murine CEA families. Two overlapping cosmid clones covering 54 kb of the cea5 gene locus were mapped. cea5 consists of three exons (L, N, A/3'-untranslated region exon) located within a 4-kb region. rnCGM2, the rat cea5 counterpart, exhibits the same restricted expression pattern. This together with their exceptional conservation within the rat and murine CEA families and their absence from the human CEA family suggests that cea5 and rnCGM2 are of functional importance for rodent placental development.

Amino Acid Sequence↗
PubMed1997

Expression of transgenic carcinoembryonic antigen (CEA) in tumor-prone mice: an animal model for CEA-directed tumor immunotherapy.

Carcinoembryonic antigen (CEA) is a tumor marker for the most common forms of adenocarcinomas. We have previously described C57BL/6 mice transgenic for the complete human CEA gene. Compared with humans, these mice reveal a conserved spatiotemporal CEA expression pattern. To establish animal models for CEA-targeted tumor immunotherapy, we have crossed CEA transgenic mice with mice that are genetically predisposed to tumor development. These immunocompetent animals should allow optimization of immunotherapy strategies for maximal destruction of tumor tissues with minimal damage to CEA-expressing normal tissues. To develop a breast tumor model, CEA transgenic mice were cross-bred with mice transgenic for the rat neu protooncogene controlled by the mouse mammary tumor virus long terminal repeat. Female offspring developed poorly differentiated breast tumors, none of which, however, expressed CEA. As a model for colorectal tumors, mice bearing a mutation in the Apc gene (Min mice) and the CEA transgene developed multiple intestinal adenomas with strong CEA expression in all tumor cells. CEA expression had no significant effect on tumor growth. Occasional, well-differentiated breast adenocarcinomas in female offspring expressed CEA focally in tumor cells lining pseudolumina. Cross-breeding Apc(Min/+) mice with neu transgenic mice did not reveal a synergistic effect on the kinetics of breast tumor formation. Finally, CEA transgenic mice crossbred with mice transgenic for the SV40 large T antigen regulated by the surfactant protein-C promoter, developed multiple lung adenocarcinomas that revealed a mosaic CEA expression pattern.

Adenocarcinoma↗
PubMed1997

Down-regulation of carcinoembryonic antigen family member 2 expression is an early event in colorectal tumorigenesis.

Carcinoembryonic antigen gene family member 2 (CGM2), a member of the carcinoembryonic antigen (CEA) family, is expressed in normal colon and rectum but is down-regulated in colorectal adenocarcinomas. In situ hybridization studies demonstrate that CGM2 expression is limited to epithelial cells in the upper third of the crypts. Two other CEA family members, biliary glycoprotein (BGP) and nonspecific cross-reacting antigen (NCA), are similarly expressed, whereas CEA transcripts were found down to the base of the crypts but were less predominant in the upper region. Only low CGM2 and BGP mRNA levels were seen in colorectal tumors. CEA mRNA was expressed at an equivalent level in normal epithelia and in tumor cells, whereas NCA transcript levels were upregulated in tumor cells. Monoclonal antibodies that recognize the CGM2 protein reveal its presence on the apical membranes of epithelial cells in the upper third of the crypts but its absence from colorectal tumors, which do express the CEA and NCA-50/90 proteins. The newly cloned CGM2 3'-untranslated region was used to probe RNAs from adenomas, colorectal tumors at different stages of progression, and liver metastases of colorectal adenocarcinomas. This showed that CGM2 is already down-regulated in adenomas when compared to normal mucosae. The CGM2 expression pattern along with its sequence homology to BGP suggests a similar tumor suppressor function for CGM2.

Adenocarcinoma↗
PubMed1997

Neurological disease and encephalitis in cats experimentally infected with Borna disease virus.

Barrier-bred cats were inoculated intracerebrally with either the rabbit-adapted Borna disease virus (BDV) strain V or a newly isolated feline BDV, obtained from a cat with natural staggering disease (SD). Three out of eight inoculated cats developed neurological signs and non-suppurative encephalitis; all three recovered from the acute stage of disease. Sero-conversion and the development of neutralizing antibodies occurred in all of the virus-inoculated cats. In addition, cats inoculated with feline BDV showed an early peripheral T cell response not present in cats inoculated with BDV strain V, suggesting that the feline virus exerted a more vigorous effect on the immune system. Using immunohistochemistry and a reverse transcriptase-polymerase chain reaction assay, BDV-specific antigen and nucleic acid could be demonstrated in brain samples from each cat with encephalitis, showing that incomplete viral clearance was probably responsible for the maintenance of inflammation. The successful induction of neurological signs and encephalitis in one cat infected with feline BDV, together with the detection of BDV-specific antigen and nucleic acid in the brain, provides strong evidence for the notion that BDV is the etiological agent behind feline SD.

Animals↗
PubMed1997

Highly specific monoclonal antibody demonstrates that pregnancy-specific glycoprotein (PSG) is limited to syncytiotrophoblast in human early and term placenta.

Pregnancy specific glycoproteins (PSG) in humans constitute a family of 11 closely related glycoproteins (PSG1-8, PSG11-13) of unknown function(s), which are produced in large amounts by the placenta. As a step toward understanding the biology of PSG, specific monoclonal antibodies (mAbs) against PSG were developed and used to investigate the ultrastructural localization of PSG in the early and term placenta and in first trimester decidua. One mAb, BAP-3, was found to react with all six individually expressed PSGs representing five alternatively spliced forms, but not with any of the seven expressed members of the carcinoembryonic antigen (CEA) subfamily. The BAP-3 epitope is located in the PSG B2 domain. Using the BAP-3 mAb, PSGs were found to be expressed exclusively by the syncytiotrophoblast of first trimester and term villi. The intensity of the staining was much higher in early than in term placenta. All three main cellular compartments involved in the biosynthesis pathway of secreted proteins, i.e. rough endoplasmic reticulum, the Golgi complex and secretory vesicles, were stained for PSG. A second PSG-reactive mAb, BAP-1, also stained the apical plasma membrane of some glandular epithelial cells in first trimester decidua in addition to syncytiotrophoblast. This staining was most likely due to cross-reactivity with biliary glycoprotein (BGP).

Animals↗
PubMed1997

Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system.

A method has been developed for culturing cardiac myocytes in a collagen matrix to produce a coherently contracting 3-dimensional model heart tissue that allows direct measurement of isometric contractile force. Embryonic chick cardiomyocytes were mixed with collagen solution and allowed to gel between two Velcro-coated glass tubes. During culture, the cardiomyocytes formed spontaneously beating cardiac myocyte-populated matrices (CMPMs) anchored at opposite ends to the Velcro-covered tubes through which they could be attached to a force measuring system. Immunohistochemistry and electron microscopy revealed a highly organized tissue-like structure of alpha-actin and alpha-tropomyosin-positive cardiac myocytes exhibiting typical cross-striation, sarcomeric myofilaments, intercalated discs, desmosomes, and tight junctions. Force measurements of paced or unpaced CMPMs were performed in organ baths after 6-11 days of cultivation and were stable for up to 24 h. Force increased with frequency between 0.8 and 2.0 Hz (positive "staircase"), increasing rest length (Starling mechanism), and increasing extracellular calcium. The utility of this system as a test bed for genetic manipulation was demonstrated by infecting the CMPMs with a recombinant beta-galactosidase-carrying adenovirus. Transduction efficiency increased from about 5% (MOI 0.1) to about 50% (MOI 100). CMPMs display more physiological characteristics of intact heart tissue than monolayer cultures. This approach, simpler and faster than generation of transgenic animals, should allow functional consequences of genetic or pharmacological manipulation of cardiomyocytes in vitro to be studied under highly controlled conditions.

Actins↗
PubMed1996

Coordinate expression of splice variants of the murine pregnancy-specific glycoprotein (PSG) gene family during placental development.

The human and murine pregnancy-specific glycoprotein (PSG) gene families encode a large number of closely related proteins which are abundantly expressed in the fetal trophoblast and secreted into the maternal circulation. Although the presence of a well conserved tripeptide sequence His or Arg-Gly-Asp or Glu or Lys (H/RGD/E/K) similar to the RGD motif found in extracellular matrix proteins hints towards a possible interaction with integrin-type receptors, the function of this group of proteins related to the carcinoembryonic antigen family is still unknown. It is also not clear whether the various members of the PSG family exert the same function. Here we describe the cloning of two splice variants of Cea4 (Cea4a, Cea4b), a murine PSG family member, which lacks the RGD-related consensus motif. Cea4a, like most of the other rodent PSG members, is composed of three immunoglobulin (Ig) variable-like domains (N1-N3) and and one Ig constant-like domain (A). In contrast, Cea4b lacks the N2 domain (N1N3A), demonstrating for the first time that PSG isoforms produced by alternative splicing also exist in mice. The mRNAs coding for Cea4a and Cea4b exhibit the same expression kinetics during placental development as found for two other murine PSGs, Cea2 and Cea3, which contain the RGD-like motif. Expression starts after day 12.5 of embryonic development (E12.5) and maximum steady-state levels are reached around E15.5-E17.5 as determined by RNase protection analyses. At E17.5, PSG transcripts can be detected exclusively in the spongiotrophoblast of the placenta. In addition, PCR analyses revealed that Cea2, Cea3, and Cea4 transcripts are also found in RNA from a pool of embryos (E12-E15) but are absent from a number of adult tissues tested (kidney, lung, testis, ovary, liver, brain, thymus, heart, spleen). These results indicate that the various PSG isoforms exert their function(s) at the same time during placental and embryonic development.

Alternative Splicing↗
PubMed1996

Crystallization and preliminary crystallographic analysis of two beta-mannanase isoforms from Thermomonospora fusca KW3.

Three beta-mannanase isoforms were isolated from the supernatant of a thermophilic actinomycete culture from Thermomonospora fusca KW3. Two of the isoforms (Q1, Q 1.1) were crystallized by the hanging-drop method at room temperature using ammonium sulfate as a precipitant. The isoforms form rod-shaped colorless crystals. Both belong to the orthorhombic space group P2(1)2(1)2(1). The cell dimensions are a = 46.7, b = 61.1, and c = 128.2 A for isoform Q1, and a = 43.8, b = 46.2, and c = 132.8 A for isoform Q1.1. The asymmetric unit of either isoform contains one mannanase molecule. Native data have been collected to 2.2 A resolution for Q1 and to 1.65 A resolution for Q1.1 using synchrotron radiation.

Journal Article↗
PubMed1996

Detailed comparative map of human chromosome 19q and related regions of the mouse genome.

One of the larger contiguous blocks of mouse-human genomic homology includes the proximal portion of mouse chromosome 7 and the long arm of human chromosome 19. Previous studies have demonstrated the close relationship between the two regions, but have also indicated significant rearrangements in the relative orders of homologous mouse and human genes. Here we present the genetic locations of the homologs of 42 human chromosome 19q markers in the mouse, with an emphasis on genes also included in the human chromosome 19 physical map. Our results demonstrate that despite an overall inversion of sequences relative to the centromere, apparent "transpositions" of three gene-rich segments, and a local inversion of markers mapping near the 19q telomere, gene content, order, and spacing are remarkably well conserved throughout the lengths of these related mouse and human regions. Although most human 19q markers have remained genetically linked in mouse, one small human segment forms a separate region of homology between human chromosome 19q and mouse chromosome 17. Three of the four rearrangements of mouse versus human 19q sequences involve segments that are located directly adjacent to each other in 19q13.3-q13.4, suggesting either the coincident occurrence of these events or their common association with unstable DNA sequences. These data permit an unusually in-depth examination of this large region of mouse-human genomic homology and provide an important new tool to aid in the mapping of genes and associated phenotypes in both species.

Animals↗
PubMed1995

The cea10 gene encodes a secreted member of the murine carcinoembryonic antigen family and is expressed in the placenta, gastrointestinal tract and bone marrow.

Although members of the carcinoembryonic antigen (CEA) family have been shown to convey cell adhesion in vitro, their location in some tissues contradicts such a function. As a basis for investigating their in vivo functions, we are characterizing the mouse CEA family. This paper describes the structure and expression of a new murine family member, cea10. Two full-length cDNA clones were isolated from a mouse colon library, whose deduced protein sequence comprises two immunoglobulin variable-like N-domains, directly followed by a short C-terminal domain indicating that this molecule is secreted. Although this domain organization suggests a closer relationship to the murine pregnancy-specific glycoproteins (PSG), which form a subgroup within the CEA family, sequence comparisons place Cea10 within the CEA subgroup. Overlapping cosmid clones containing the complete cea10 locus were mapped and the exons determined. No A2-like exon, characteristic for all other members of the murine CEA family, could be found. Sequences of the promoter and the first exon showed remarkably high similarity to the corresponding regions of bgp1 and bgp2, two other members of the murine CEA subgroup. Consensus sequences for two transcription factors (USF and an AP-2-like factor) that bind to the human BGP gene promoter were also present in the cea10 promoter and possibly convey expression of these genes in epithelial cells. RNase protection assays revealed transcriptional activity of cea10 in the colon and early placenta (10.5-12.5-day embryos) and to a lower extent in the small intestine, cecum, stomach, salivary glands and bone marrow. As some other CEA family members are deregulated in tumors, we quantified the expression levels of Cea10 transcripts in colonic mucosa and in adenomatous polyps of Min/+ mice. No differences in the steady-state levels of Cea10 mRNA could be found, suggesting that the Cea10 protein does not play a role in early tumor development. Taken together, Cea10 combines characteristic features of both CEA and PSG subgroup members in its structure and expression pattern.

Amino Acid Sequence↗