[Peripheral keratinizing ameloblastoma in a dwarf rabbit (Oryctolagus cuniculus f. dom.)]. / Peripheres keratinisierendes Ameloblastom bei einem Zwergkaninchen (Oryctolagus cuniculus f. dom.).

A 3.5-year-old, male, neutered dwarf rabbit was presented with a history of a fast-growing gingival mass at the maxilla. The neoplasm was surgically completely excised. Histopathologically, an expansively growing, multilobulated, partially cystic, peripheral, keratinizing ameloblastoma was diagnosed. The immunohistochemical phenotyping of the tumour cells resulted in cytoplasmic labelling with various pan-cytokeratin antibodies. The cytokeratins 5/6, 7, 10 and 14 were expressed variably. Cytokeratin 20 was not detected. Vimentin was expressed in the cytoplasm of mesenchymal cells of the tumour stroma. In addition, in the nuclei of approximately 10% of the tumour cells the protein of the tumour suppressor gene p53 was expressed while in approximately 5% the proliferation marker Ki67 was expressed. Odontogenic tumours should be considered as a differential diagnosis of oral masses in rabbits.

Genetic analysis of the LGI/Epitempin gene family in sporadic and familial lateral temporal lobe epilepsy.

Mutations in the LGI1/Epitempin gene cause autosomal dominant lateral temporal lobe epilepsy (ADLTE), a partial epilepsy characterized by the presence of auditory seizures. However, not all the pedigrees with a phenotype consistent with ADLTE show mutations in LGI1/Epitempin, or evidence for linkage to the 10q24 locus. Other authors as well as ourselves have found an internal repeat (EPTP, pfam# PF03736) that allowed the identification of three other genes sharing a sequence and structural similarity with LGI1/Epitempin. In this work, we present the sequencing of these genes in a set of ADLTE families without mutations in both LGI1/Epitempin and sporadic cases. No analyzed polymorphisms modified susceptibility in either the familial or sporadic forms of this partial epilepsy.

Identification and characterization of a novel human brain-specific gene, homologous to S. scrofa tmp83.5, in the chromosome 10q24 critical region for temporal lobe epilepsy and spastic paraplegia.

We describe the structure, genomic organization, and some transcription features of a human brain-specific gene previously localized to the genomic region involved in temporal lobe epilepsy and spastic paraplegia on chromosome 10q24. The gene, which consists of six exons disseminated over 16 kb of genomic DNA, is highly homologous to the porcine tmp83.5 gene and encodes a putative transmembrane protein of 141 amino acids. Unlike its porcine homolog, from which two mRNAs with different 5'-sequences are transcribed, the human gene apparently encodes three mRNA species with 3'-untranslated regions of different sizes. Mutation analysis of its coding sequence in families affected with temporal lobe epilepsy or spastic paraplegia linked to 10q24 do not support the involvement of this gene in either diseases.

Molecular cloning of Xp11 breakpoints in two unrelated mentally retarded females with X;autosome translocations.

Mental retardation is a very common and extremely heterogeneous disorder that affects about 3% of the human population. Its molecular basis is largely unknown, but many loci have been mapped to the X chromosome. We report on two mentally retarded females with X;autosome translocations and breakpoints in Xp11, viz., t(X;17)(p11;p13) and t(X;20)(p11;q13). (Fiber-) FISH analysis assigned the breakpoints to different subbands, Xp11.4 and Xp11.23, separated by approximately 8 Mb. High-resolution mapping of the X- chromosome breakpoints using Southern blot hybridization resulted in the isolation of breakpoint-spanning genomic subclones of 3 kb and 0. 5 kb. The Xp11.4 breakpoint is contained within a single copy sequence, whereas the Xp11.23 breakpoint sequence resembles an L1 repetitive element. Several expressed sequences map close to the breakpoints, but none was found to be inactivated. Therefore, mechanisms other than disruption of X-chromosome genes likely cause the phenotypes.

Comparative genome sequence analysis of the Bpa/Str region in mouse and Man.

The progress of human and mouse genome sequencing programs presages the possibility of systematic cross-species comparison of the two genomes as a powerful tool for gene and regulatory element identification. As the opportunities to perform comparative sequence analysis emerge, it is important to develop parameters for such analyses and to examine the outcomes of cross-species comparison. Our analysis used gene prediction and a database search of 430 kb of genomic sequence covering the Bpa/Str region of the mouse X chromosome, and 745 kb of genomic sequence from the homologous human X chromosome region. We identified 11 genes in mouse and 13 genes and two pseudogenes in human. In addition, we compared the mouse and human sequences using pairwise alignment and searches for evolutionary conserved regions (ECRs) exceeding a defined threshold of sequence identity. This approach aided the identification of at least four further putative conserved genes in the region. Comparative sequencing revealed that this region is a mosaic in evolutionary terms, with considerably more rearrangement between the two species than realized previously from comparative mapping studies. Surprisingly, this region showed an extremely high LINE and low SINE content, low G+C content, and yet a relatively high gene density, in contrast to the low gene density usually associated with such regions.

Differential gene expression by endothelial cells in distinct angiogenic states.

Angiogenesis is a complex process that can be regarded as a series of sequential events comprising a variety of tissue cells. The major problem when studying angiogenesis in vitro is the lack of a model system mimicking the various aspects of the process in vivo. In this study we have used two in vitro models, each representing different and distinct aspects of angiogenesis. Differentially expressed genes in the two culture forms were identified using the suppression subtractive hybridization technique to prepare subtracted cDNA libraries. This was followed by a differential hybridization screen to pick up overexpressed clones. Using comparative multiplex RT-PCR we confirmed the differential expression and showed differences up to 14-fold. We identified a broad range of genes already known to play an important role during angiogenesis like Flt1 or TIE2. Furthermore several known genes are put into the context of endothelial cell differentiation, which up to now have not been described as being relevant to angiogenesis, like NrCAM, Claudin14, BMP-6, PEA-15 and PINCH. With ADAMTS4 and hADAMTS1/METH-1 we further extended the set of matrix metalloproteases expressed and regulated by endothelial cells.

A genome-wide survey of RAS transformation targets.

An important aspect of multi-step tumorigenesis is the mutational activation of genes of the RAS family, particularly in sporadic cancers of the pancreas, colon, lung and myeloid system. RAS genes encode small GTP-binding proteins that affect gene expression in a global way by acting as major switches in signal transduction processes, coupling extracellular signals with transcription factors. Oncogenic forms of RAS are locked in their active state and transduce signals essential for transformation, angiogenesis, invasion and metastasis via downstream pathways involving the RAF/MEK/ERK cascade of cytoplasmic kinases, the small GTP-binding proteins RAC and RHO, phosphatidylinositol 3-kinase and others. We have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, to contrast differential gene expression profiles in immortalized, non-tumorigenic rat embryo fibroblasts and in HRAS- transformed cells. Sequence and expression analysis of more than 1,200 subtracted cDNA fragments revealed transcriptional stimulation or repression of 104 ESTs, 45 novel sequences and 244 known genes in HRAS- transformed cells compared with normal cells. Furthermore, we identified common and distinct targets in cells transformed by mutant HRAS, KRAS and NRAS, as well as 61 putative target genes controlled by the RAF/MEK/ERK pathway in reverted cells treated with the MEK-specific inhibitor PD 98059.

Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues.

A four-step procedure for the efficient and systematic mining of whole EST libraries for differentially expressed genes is presented. After eliminating redundant entries from the EST library under investigation (step 1), contigs of maximal length are built upon each remaining EST using about 4 000 000 public and proprietary ESTs (step 2). These putative genes are compared against a database comprising ESTs from 16 different tissues (both normal and tumour affected) to determine whether or not they are differentially expressed (step 3; electronic northern). Fisher's exact test is used to assess the significance of differential expression. In step 4, an attempt is made to characterise the contigs obtained in the assembly through database comparison. A case study of the CGAP library NCI_CGAP_Br1.1, a library made from three (well, moderately, and poorly differentiated) invasive ductal breast tumours (2126 ESTs in total) was carried out. Of the maximal contigs, 139 were found to be significantly (alpha = 0.05) over-expressed in breast tumour tissue, while 13 appeared to be down-regulated.

Inactivating mutations and overexpression of BCL10, a caspase recruitment domain-containing gene, in MALT lymphoma with t(1;14)(p22;q32).

Mucosa-associated lymphoid tissue (MALT) lymphomas most frequently involve the gastrointestinal tract and are the most common subset of extranodal non-Hodgkin lymphoma (NHL). Here we describe overexpression of BCL10, a novel apoptotic signalling gene that encodes an amino-terminal caspase recruitment domain (CARD), in MALT lymphomas due to the recurrent t(1;14)(p22;q32). BCL10 cDNAs from t(1;14)-positive MALT tumours contained a variety of mutations, most resulting in truncations either in or carboxy terminal to the CARD. Wild-type BCL10 activated NF-kappaB but induced apoptosis of MCF7 and 293 cells. CARD-truncation mutants were unable to induce cell death or activate NF-kappaB, whereas mutants with C-terminal truncations retained NF-kappaB activation but did not induce apoptosis. Mutant BCL10 overexpression might have a twofold lymphomagenic effect: loss of BCL10 pro-apoptosis may confer a survival advantage to MALT B-cells, and constitutive NF-kappaB activation may provide both anti-apoptotic and proliferative signals mediated via its transcriptional targets.

Positional cloning of the gene for X-linked retinitis pigmentosa 2.

X-linked retinitis pigmentosa (XLRP) results from mutations in at least two different loci, designated RP2 and RP3, located at Xp11.3 and Xp21.1, respectively. The RP3 gene was recently isolated by positional cloning, whereas the RP2 locus was mapped genetically to a 5-cM interval. We have screened this region for genomic rearrangements by the YAC representation hybridization (YRH) technique and detected a LINE1 (L1) insertion in one XLRP patient. The L1 retrotransposition occurred in an intron of a novel gene that consisted of five exons and encoded a polypeptide of 350 amino acids. Subsequently, nonsense, missense and frameshift mutations, as well as two small deletions, were identified in six additional patients. The predicted gene product shows homology with human cofactor C, a protein involved in the ultimate step of beta-tubulin folding. Our data provide evidence that mutations in this gene, designated RP2, are responsible for progressive retinal degeneration.

Molecular characterization of a common fragile site (FRA7H) on human chromosome 7 by the cloning of a simian virus 40 integration site.

Common fragile sites are chromosomal loci prone to breakage and rearrangement, hypothesized to provide targets for foreign DNA integration. We cloned a simian virus 40 integration site and showed by fluorescent in situ hybridization analysis that the integration event had occurred within a common aphidicolin-induced fragile site on human chromosome 7, FRA7H. A region of 161 kb spanning FRA7H was defined and sequenced. Several regions with a potential unusual DNA structure, including high-flexibility, low-stability, and non-B-DNA-forming sequences were identified in this region. We performed a similar analysis on the published FRA3B sequence and the putative partial FRA7G, which also revealed an impressive cluster of regions with high flexibility and low stability. Thus, these unusual DNA characteristics are possibly intrinsic properties of common fragile sites that may affect their replication and condensation as well as organization, and may lead to fragility.

Genomic structure of a novel LIM domain gene (ZNF185) in Xq28 and comparisons with the orthologous murine transcript.

Construction of a transcript map in the DXS52 region in Xq28 had previously led to the isolation of a cDNA with a LIM zinc finger domain in the carboxyl terminus. In parallel, the orthologous murine transcript was isolated from the syntenic region. The human and mouse cDNAs have been designated ZNF185 and Zfp185, respectively. By integrating the cDNA sequence with the cosmid-derived genomic sequence the exon-intron structure of the 3' end of the ZNF185 gene was resolved. Comparative sequence analyses of the human genomic sequence with the full-length murine cDNA facilitated prediction of the 5' end of the gene. The selective expression of three transcripts corresponding to the ZNF185 gene and a related gene was shown by Northern and Southern blots. In situ hybridizations revealed a nonubiquitous and stage-specific expression of Zfp185, especially in differentiating connective tissue. Since LIM proteins regulate cellular proliferation and/or differentiation by diverse mechanisms, and some have directly been associated with disease, conceivably ZNF185 may represent a candidate for a disease-causing gene linked to Xq28. Knowledge of the genomic structure will permit detailed mutation analyses.

Cloning of collagenase 3 from the synovial membrane and its expression in rheumatoid arthritis and osteoarthritis.

OBJECTIVE: To analyze synovial membrane of patients with rheumatoid arthritis (RA) for the expression of unknown matrix metalloproteinases (MMP). METHODS: Degenerate oligonucleotides corresponding to highly conserved regions of the MMP gene family and the rapid amplification of cDNA ends (RACE) method have been used to search for new members of this gene family. MMP gene expression has been characterized by Northern blot analysis. RESULTS: We cloned a MMP cDNA from the synovial membrane that is completely identical to the recently published collagenase 3 cDNA derived from a human breast cancer cDNA library (Freije, et al: J Biol Chem 1994;269:16766-73). Collagenase 3 is expressed in parallel with interstitial collagenase and stromelysin 1 in RA and osteoarthritis (OA). Collagenase 3 gene expression was not detected in several normal human tissues. CONCLUSION: The expression of collagenase 3 in the synovial membrane in RA and OA suggests its involvement in articular tissue degradation.

cDNA sequence and mRNA tissue distribution of a novel human matrix metalloproteinase with a potential transmembrane segment.

The complementary DNA sequence of a novel matrix metalloproteinase was isolated from a human lung cDNA library. It consists of 3530 bp and encodes a polypeptide of 669 amino acids. In comparison to other matrix metalloproteinases, the deduced sequence of the amino acid chain exhibits closest similarity to a recently discovered membrane-type matrix metalloproteinase of 582 amino acids. Likewise, it is composed of a signal peptide, a prodomain, a catalytic domain, a hemopexin-homologous domain and a C-terminal domain. Furthermore, the novel matrix metalloproteinase shares a similar activation site with its 582-amino-acid homologue, an insertion of eight amino acids in the catalytic domain and a tract of more than 20 hydrophobic amino acids near the C-terminus. The hydrophobic structure in the C-terminal domain suggests that the novel matrix metalloproteinase is also membrane bound. When lung cell membrane fractions were probed in immunoblots with polyclonal antibodies against a recombinant fragment of the 669-amino-acid chain, a protein of M(r) 72,000 reacted preferentially with the antibodies. Northern-blot analysis demonstrated quite different tissue distributions of mRNA for the two membrane-type matrix metalloproteinases. While mRNA for the 582-amino-acid enzyme was found predominantly in lung, placenta, kidney, ovary, intestine, prostate and spleen, mRNA for the 669-amino-acid enzyme appeared to be synthesized preferentially in liver, placenta, testis, colon and intestine. Substantial amounts of the latter mRNA were also detected in pancreas, kidney, lung, heart and skeletal muscle.

Tissue-type plasminogen activator mutants imitating urokinase in the peptide link between kringle and protease domains and at selected sites within the protease domain.

Tissue-type plasminogen activator (tPA) mutants which, at selected amino acid positions, mimic urokinase-type plasminogen activator (uPA) were expressed in Chinese hamster ovary cells and examined for their catalytic properties. In one series of mutants, the dipeptide Ser262 Thr263 between kringle 2 and the protease domain of tPA was (a) replaced by an Ala residue, (b) lengthened by additional Ser and Ala residues, (c) exchanged for the 16-amino-acid link between kringle and protease domains of uPA and an additional Ala residue. The activities of the latter two mutants toward plasminogen were, in the absence of fibrin, 3-5-fold higher and, in the presence of fibrin, comparable to or lower than the activity of tPA. The kinetic data suggest a short interdomain peptide in tPA as most favorable for high fibrin stimulation of tPA activity. In a second series of mutant, selected amino acid residues of the tPA protease domain were replaced by residues of the homologous uPA domain. Positions chosen for exchange are either close to the active site or are part of a tPA-specific insertion in the variable region preceding the active-site Ser residue. Compared to authentic tPA, protease-domain mutants exhibited 7.3-424-fold lower activities toward plasminogen, mainly due to lower kcat values. Km values differed only moderately. A mutant containing an additional hydroxyl group at the S1 site, tPA A473S, had lost the preference of tPA for Arg over Lys as the P1 residue in peptide substrates.