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| 2. |
- Stenman, U H, et al.
(author)
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Summary report of the TD-3 workshop: characterization of 83 antibodies against prostate-specific antigen
- 1999
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In: Tumor Biology. - : Springer Science and Business Media LLC. - 1423-0380 .- 1010-4283. ; 20:Suppl. 1, s. 1-12
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Journal article (peer-reviewed)abstract
- Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86-91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158-163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3-11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.
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| 4. |
- O'Neill, E, et al.
(author)
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Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR.
- 1998
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In: Molecular Microbiology. - 0950-382X .- 1365-2958. ; 28:1
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Journal article (peer-reviewed)abstract
- The Pseudomonas-derived sigma54-dependent regulator DmpR has an amino-terminal A-domain controlling the specificity of activation by aromatic effectors, a central C-domain mediating an ATPase activity essential for transcriptional activation and a carboxy-terminal D-domain involved in DNA binding. In the presence of aromatic effectors, the DmpR protein promotes transcription from the -24, -12 Po promoter controlling the expression of specialized (methyl)phenol catabolic enzymes. Previous analysis of DmpR has led to a model in which the A-domain acts as an interdomain repressor of DmpR's ATPase and transcriptional promoting property until specific aromatic effectors are bound. Here, the autonomous nature of the A-domain in exerting its biological functions has been dissected by expressing portions of DmpR as independent polypeptides. The A-domain of DmpR is shown to be both necessary and sufficient to bind phenol. Analysis of phenol binding suggests one binding site per monomer of DmpR, with a dissociation constant of 16 microM. The A-domain is also shown to have specific affinity for the C-domain and to repress the C-domain mediated ATPase activity in vitro autonomously. However, physical uncoupling of the A-domain from the remainder of the regulator results in a system that does not respond to aromatics by its normal derepression mechanism. The mechanistic implications of aromatic non-responsiveness of autonomously expressed A-domain, despite its demonstrated ability to bind phenol, are discussed.
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| 5. |
- Ng, L C, et al.
(author)
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Aromatic effector activation of the NtrC-like transcriptional regulator PhhR limits the catabolic potential of the (methyl)phenol degradative pathway it controls.
- 1995
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In: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 177:6
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Journal article (peer-reviewed)abstract
- Pseudomonas putida P35X (NCIB 9869) metabolizes phenol and monomethylphenols via a chromosomally encoded meta-cleavage pathway. We have recently described a 13.4-kb fragment of the chromosome that codes for the first eight genes of the catabolic pathway and a divergently transcribed positive regulator, phhR. The eight structural genes lie in an operon, the phh operon, downstream of a -24 TGGC, -12 TTGC promoter sequence. Promoters of this class are recognized by RNA polymerase that utilizes the alternative sigma 54 factor encoded by rpoN (ntrA) and are positively regulated by activators of the NtrC family. In this study, we have identified the coding region for the 63-kDa PhhR gene product by nucleotide sequencing of a 2,040-bp region and polypeptide analysis. PhhR was found to have homology with the NtrC family of transcriptional activators, in particular with DmpR, the pVI150-encoded regulator of (methyl)phenol catabolism by Pseudomonas sp. strain CF600. By using a luciferase reporter system, PhhR alone was shown to be sufficient to activate transcription from the phh operon promoter in an RpoN+ background but not an RpoN- background. Luciferase reporter systems were also used to directly compare the aromatic effector profiles of PhhR and DmpR. Evidence that the difference in the growth substrate ranges of strains P35X and CF600 is due to the effector activation specificities of the regulators of these systems rather than the substrate specificities of the catabolic enzymes is presented.
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| 10. |
- Coulibaly, S, et al.
(author)
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Modulation of invasive properties of murine squamous carcinoma cells by heterologous expression of cathepsin B and cystatin C
- 1999
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In: International Journal of Cancer. - 0020-7136. ; 83:4, s. 526-531
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Journal article (peer-reviewed)abstract
- Murine SCC-VII squamous carcinoma cells have the capacity to penetrate reconstituted basement membranes (Matrigel) in vitro. The invasion of Matrigel layers by SCC-VII cells was significantly reduced by E-64, a specific inhibitor of lysosomal cysteine proteinases. The cathepsin-B-selective E-64 derivative, CA-074, inhibited penetration of Matrigel by SCC-VII cells to the same extent, indicating a major role for this particular lysosomal enzyme in extracellular-matrix degradation during squamous-carcinoma-cell invasion. SCC-VII cells were stably transfected with a cDNA encoding human procathepsin B, in an attempt to modulate the invasive properties of the cell line. The transfected cells expressed the heterologous gene, secreted increased amounts of procathepsin B and displayed enhanced invasive potential. In vivo, the activity of cathepsin B is strictly regulated by endogenous inhibitors. SCC-VII cells were therefore also stably transfected with a cDNA encoding human cystatin C, the most potent cysteine-proteinase inhibitor in mammalian tissues. The expression of this transgene resulted in the production of active recombinant cystatin C and a pronounced reduction in Matrigel invasion. These studies demonstrate that the invasive properties of squamous-cell carcinomas can be changed by modulation of the balance between cathepsin B and its endogenous inhibitors, and provide further evidence for the involvement of this lysosomal cysteine proteinase in tumour invasion and metastasis.
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| 14. |
- Göthe, Sten, et al.
(author)
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Mice devoid of all known thyroid hormone receptors are viable but exhibit disorders of the pituitary-thyroid axis, growth, and bone maturation.
- 1999
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In: Genes & development. - : Cold Spring Harbor Laboratory. - 0890-9369. ; 13:10, s. 1329-41
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Journal article (peer-reviewed)abstract
- Thyroid hormone (T3) has widespread functions in development and homeostasis, although the receptor pathways by which this diversity arises are unclear. Deletion of the T3 receptors TRalpha1 or TRbeta individually reveals only a small proportion of the phenotypes that arise in hypothyroidism, implying that additional pathways must exist. Here, we demonstrate that mice lacking both TRalpha1 and TRbeta (TRalpha1(-/-)beta-/-) display a novel array of phenotypes not found in single receptor-deficient mice, including an extremely hyperactive pituitary-thyroid axis, poor female fertility and retarded growth and bone maturation. These results establish that major T3 actions are mediated by common pathways in which TRalpha1 and TRbeta cooperate with or substitute for each other. Thus, varying the balance of use of TRalpha1 and TRbeta individually or in combination facilitates control of an extended spectrum of T3 actions. There was no evidence for any previously unidentified T3 receptors in TRalpha1(-/-)beta-/- mouse tissues. Compared to the debilitating symptoms of severe hypothyroidism, the milder overall phenotype of TRalpha1(-/-)beta-/- mice, lacking all known T3 receptors, indicates divergent consequences for hormone versus receptor deficiency. These distinctions suggest that T3-independent actions of T3 receptors, demonstrated previously in vitro, may be a significant function in vivo.
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| 19. |
- Ng, L C, et al.
(author)
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Genetic evidence for interdomain regulation of the phenol-responsive final sigma54-dependent activator DmpR.
- 1996
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 271:29
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Journal article (peer-reviewed)abstract
- The final sigma54-dependent DmpR activator regulates transcription of the dmp operon that encodes the enzymes for catabolism of (methyl)phenols. DmpR is expressed constitutively, but its transcriptional promoting activity is controlled positively in direct response to the presence of aromatic pathway substrates (effectors). DmpR has a distinct domain structure with the amino-terminal A-domain controlling the specificity of activation of the regulator by aromatic effectors (signal reception), a central C-domain mediating an ATPase activity essential for transcriptional activation, and a carboxyl-terminal D-domain involved in DNA binding. Deletion of the A-domain has been shown previously to result in an effector-independent transcriptional activator with constitutive ATPase activity. These results, in conjunction with the location of mutations within the A- and C-domains which exhibit an effector-independent (semiconstitutive) property, have led to a working model in which the A-domain serves to mask the ATPase and transcriptional promoting activity of the C-domain in the absence of effectors. To investigate the mechanism by which the A-domain exerts its repressive effect, we developed a genetic system to select positively for intramolecular second site revertants of DmpR. The results demonstrate (i) that mutations within the A-domain can suppress the semiconstitutive activity of C-domain located mutations and vice versa; (ii) that the C-domain located mutations do not influence the intrinsic ATPase and transcriptional promoting property of the C-domain in the absence of the A-domain; and (iii) that semiconstitutive mutations of the A- and C-domain have an additive effect. Taken together these results support a model in which the A-domain represses the function(s) of the C-domain by direct interactions between residues of the two domains.
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| 21. |
- Sturegård, Erik, et al.
(author)
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Severe gastritis in guinea-pigs infected with Helicobacter pylori
- 1998
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In: Journal of Medical Microbiology. - 0022-2615. ; 47:12, s. 1123-1129
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Journal article (peer-reviewed)abstract
- An appropriate animal model is essential to study Helicobacter pylori infection. The aim of this study was to investigate if H. pylori can colonise the guinea-pig stomach and whether the infection causes gastritis and a serological response similar to that observed in man. Guinea-pigs were infected either with fresh H. pylori isolates from human gastric biopsies or with a guinea-pig passaged strain. When the animals were killed, 3 and 7 weeks after inoculation, samples were taken for culture, histopathology and serology. H. pylori was cultured from 22 of 29 challenged animals. All culture-positive animals exhibited a specific immune response against H. pylori antigens in Western blotting and gastritis in histopathological examination. Antibody titres in enzyme immunoassay were elevated among animals challenged with H. pylori. The inflammatory response was graded as severe in most animals and consisted of both polymorphonuclear leucocytes and lymphocytes. Erosion of the gastric epithelium was found in infected animals. These results suggest that the guinea-pig is suitable for studying H. pylori-associated diseases. Moreover, guinea-pigs are probably more similar to man than any other small laboratory animal as regards gastric anatomy and physiology.
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