ICAAR, a novel member of a new family of transmembrane, tyrosine phosphatase-like proteins

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA-2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTM1) gene. ICAAR, IA-2 and IA-2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36.     

Characterization of a second member of the sentrin family of ubiquitin-like proteins

Sentrin is a novel ubiquitin-like protein that can be conjugated to other proteins in a manner analogous to ubiquitination. Two additional cDNA sequences that encode proteins highly homologous to sentrin have been reported to GenBankTM. It is not known whether these sentrin-like proteins could also function as protein modifiers. In this report, a second member of the sentrin family was characterized in detail. Sentrin-2 is a 95-amino acid polypeptide that is 46% identical and 66% homologous to sentrin-1. Northern blot analysis showed that the sentrin-2 message was expressed in all tissues, but was barely detectable in the liver and placenta. The ability of sentrin-2 to conjugate to other proteins was tested by expressing hemagglutinin epitope-tagged sentrin-2 in COS cells. Western blot analysis showed that sentrin-2 could be transferred to other proteins in a pattern similar to that of sentrin-1 conjugation and had similar C-terminal processing. We further showed that both sentrin-1 and sentrin-2 could covalently modify RanGAP1, a Ran GTPase-activating protein critically involved in nuclear transport. Immunocytochemical analysis showed that sentrin-2 derivatives were highly enriched in the nucleus. Taken together, our results demonstrate that sentrin-2 is another protein modifier for the sentrinization pathway.     

Identification and characterization of a novel member of the EXT gene family, EXTL2

Recently, two homologous genes, EXT1 and EXT2, with a putative tumor suppressor function have been described. Mutations in both genes are responsible for multiple exostosis syndrome (EXT), an autosomal dominant condition characterized by the presence of multiple osteochondromas, bony excrescences that sometimes undergo malignant transformation to chondrosarcoma. This family of EXT genes has been extended by the identification of an EXT-like (EXTL) gene showing a high degree of homology with the EXT genes. We report here a second EXT-like gene (EXTL2) which is homologous to the EXT and EXTL genes. EXTL2 consists of 5 exons encoding an ubiquitously expressed protein of 330 amino acids. In addition, a putative pseudogene, EXTL2P was also identified. The EXTL2 gene was assigned to chromosome 1p11-p12, whereas EXTL2P was mapped on chromosome 2q24-q31.     

Cloning and expression analysis of a novel salicylate suppressible gene, Hs-CUL-3, a member of cullin/Cdc53 family

By using a mRNA differential display technique to search for salicylate suppressible genes, we identified a cDNA in human foreskin fibroblasts, which by GenBankTM DNA data base search shows sequence homology to the recently reported cullin/Cdc53 (CUL) family genes, especially CUL-3. We have cloned the full-length human CUL-3 (Hs-CUL-3) cDNA. It encodes a 768-amino acid polypeptide and has a predicted molecular weight of 88,939. The amino acid sequence of Hs-CUL-3 shows 46% homology to that of its Caenorhabditis elegans ortholog, Ce-CUL-3, and 27 and 23% to that of Hs-CUL-1 and Hs-CUL-2, respectively. Northern blot analysis showed that phorbol 12-myristate 13-acetate increased the expression of Hs-CUL-3 mRNA in a concentration- and time-dependent manner, and this increase was inhibited by sodium salicylate. Hs-CUL-3 widely expressed in human tissues and its expression in cultured COLO205 colon cancer cells was increased when compared with that in normal colon cells. It is likely that Hs-CUL-3 is involved in cell proliferation control.     

The C. elegans vulval induction gene lin-2 encodes a member of the MAGUK family of cell junction proteins

The study objective was, firstly, to investigate whether anaesthesia with induced arterial hypotension would cause leakage of a biochemical marker of neuronal injury, adenylate kinase (AK), into the cerebrospinal fluid (CSF). (Definition: arterial hypotension = mean arterial pressure (MAP) 50-65 mmHg during > or = 10 min). Secondly, a subgroup of patients was examined with a limited battery of psychometric tests. Patients, scheduled for orthognathic surgery, were allocated to either hypotension (n = 20) or normotension (n = 20). Seventeen patients were subjected to psychometry. Arterial blood pressure was recorded continuously and controlled by adjustments of the administered concentration of the inhalational anaesthetic isoflurane. Fentanyl, an opioid, was given equally in both groups. A lumbar puncture was performed approximately 20 h post-operatively for a CSF sample, later analysed for AK activity. Neuropsychological tests were performed the day before surgery and the fourteenth day postoperatively. The CSF-AK value was pathologically increased ( > 0.040 U/L) in 24 patients (65%), of whom 9 were normotensive. There was no significant difference between the CSF-AK values in the hypotensive and normotensive groups, mean values were 0.082 (s.d. 0.051) and 0.066 (s.d. 0.059) U/L, respectively. The overall correlation between the 10 min MAP levels and the CSF-AK values was close to zero. In the pilot neuropsychological investigation some abnormalities were observed, indicating clinically significant adverse effects in four hypotensive patients, of whom two displayed pathologically increased CSF-AK values. At the group level, the correlation between the changes in psychometry and the measured CSF-AK values was poor. Increases in CSF-AK activities may be a non-specific occurrence in the perioperative interval, possibly indicating an adverse effect on the brain. Arterial hypotension could not be proven to explain the CSF-AK outcome.     

Characterisation of a new human and murine member of the DnaJ family of proteins

We report the characterisation of a human gene, designated MCG18 (multiple endocrine neoplasia type 1 candidate gene 18), that encodes a new member of the DnaJ family of proteins. Database searches indicate that MCG18 also has the locus name HSPF2. MCG18 lies 250bp centromeric of the VRF/VEGFB gene on chromosome 11q13. The MCG18 cDNA is predicted to encode a 241 amino acid product that has partial homology to Escherichia coli dnaJ in that it contains the J domain. However, MCG18 has greatest similarity to a functionally undefined protein from Caenorhabditis elegans, both of which are predicted to have a membrane-spanning region adjacent to their J domains. The cDNA encoding the murine homolog (Mcg18) was also cloned and sequenced, and the encoded protein shares 81% similarity to MCG18. The coding region of MCG18 is interrupted by 4 introns and the mRNA is expressed as a 1.4kb message in all tissues examined, including those derived from the breast, ovary, bladder, lung and keratinocytes.     

Molecular analysis of the human immunoglobulin V lambda II gene family

The 8.12 idiotype characterizes a subpopulation of anti-DNA antibodies in patients with systemic lupus erythematosus (SLE). The idiotype is present on lambda light chains and has previously been shown to be exclusively encoded by V lambda II light chains. RFLP analysis of the V lambda II gene family has shown the family to consist of 10 to 15 members. Thus far, the sequences of seven V lambda II germline genes are reported in the literature with one of these a pseudogene. To identify the V lambda II genes that encode 8.12 positive antibodies and to further characterize the V lambda II family, germline V lambda II clones were derived from a patient with SLE. Two libraries were constructed: a genomic DNA library and a library of PCR-derived V lambda II gene products obtained using a conserved V lambda II leader region primer and a primer for the nonamer region 3' of the coding sequence. We now describe seven new germline genes, two of which are pseudogenes. Comparison of V lambda II germline genes to sequences of 8.12 positive light chains produced by EBV-transformed B cell lines show that all 8.12 positive light chains are encoded by a limited number of highly homologous members of the V lambda II family. 8.12 negative V lambda II encoded light chains also derive from a limited number of V lambda II genes, suggesting that only a subset of the apparently available V lambda II genes are commonly expressed.     

Substrate induction and glucose repression of maltose utilization by Streptomyces coelicolor A3(2) is controlled by malR, a member of the lacl-galR family of regulatory genes

The rat treated with bile duct ligation (BDL) and furan is a unique animal model of massive bile ductular hyperplasia in which normal liver parenchyma is largely replaced with well-differentiated proliferated bile ductules. We have now developed a simple cell isolation procedure to obtain and culture viable bile ductular epithelial cells in high numbers and with a high degree of purity from the livers of BDL/furan-treated rats. Primary monolayer cell cultures were readily established when the isolated bile ductular epithelial cells were cultured in plastic tissue culture wells coated with rat tail tendon type I collagen plus bovine plasma fibronectin. Under these conditions, epidermal growth factor (EGF) was mitogenic for the cultured cells, and they retained phenotypic features typical of hyperplastic bile ductular epithelium but did not show evidence of ductal morphogenesis in vitro. In contrast, when the isolated bile ductular cells were cultured for 7 to 16 days in the presence of 25 ng EGF/mL and 10% fetal bovine serum on type I collagen gels, they formed into branching ductal structures whose ultrastructural features very closely resembled those of polarized hyperplastic bile ductules/ducts in vivo. Histological preparations of these gel cultures further showed that numerous ductal structures with defined lumens were present. Phenotypically, these ductal structures were completely surrounded by a thickened basement membrane that was strongly immunoreactive for laminin. Like their in vivo biliary cell counterparts, the epithelial cells comprising these ductal structures in culture also exhibited strong immunocytochemical staining reactions for cytokeratins 8 and 19, for glutathione S-transferase pi 7, and for luminal gamma-glutamyl transpeptidase, but they did not express immunoreactive albumin or alpha-fetoprotein. Occasional epithelial cells of the ductal structures, when examined in 10-day-old primary gel culture, showed strong nuclear staining for incorporated 5-bromo-2'deoxyuridine, indicating active cell proliferation. Our results support the development of a novel biliary epithelial cell culture model that has the potential of serving as a powerful tool for investigation of factors that regulate hyperplastic bile ductular morphogenesis, cell proliferation, and polarized cell functions in a structural form in vitro that mimics that of hyperplastic bile ductules induced in vivo.     

Cloning, expression and chromosomal localization of Wnt-13, a novel member of the Wnt gene family

The Wnt genes, encoding structurally-related secreted glycoproteins, are implicated in mammary carcinogenesis induced by mouse mammary tumor virus. In search of the Wnt gene(s) expressed in human gastric cancer, a WTGC1 cDNA fragment sharing 66.9% amino-acid homology with human and mouse Wnt-2 was isolated by degenerate polymerase chain reaction. The human gene corresponding to WTGC1 was designated as Wnt-13 and overlapping Wnt-13 cDNAs were cloned. Nucleotide sequence analysis indicated that the Wnt-13 gene encodes the protein of 372 amino acids, including a signal peptide, two potential N-glycosylation sites and 24 cystein residues highly conserved among members of the Wnt gene family. The Wnt-13 mRNA of 2.5 kb in size was detected in heart, brain, placenta, lung, prostate, testis, ovary, small intestine and colon of adult human and also in brain, lung and kidney of fetal human. Among various cancer cell lines, the Wnt-13 mRNA was detected in HeLa (cervical cancer), MKN28 and MKN74 (gastric cancer). The Wnt-13 gene has been mapped to human chromosome 1p13. These results suggest that the Wnt-13 gene may be involved in normal human development or differentiation as well as in human carcinogenesis.     

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.     

SOX20, a new member of the SOX gene family, is located on chromosome 17p13

SOX genes share a high sequence identity with the HMG box present in the testis determining gene SRY. We have identified a HMG box-like sequence motif on six contiguous cosmids, which cross-hybridize to a SOX9 cDNA probe. A data base search revealed a high similarity of the deduced amino acid sequence to the human SOX12 and the murine Sox16 HMG domains. The cosmids were assigned to chromosome 17p13 by FISH analysis.     

Identification and sequence analysis of Treponema pallidum tprJ, a member of a polymorphic multigene family

This study examines occupational health and safety provision from farmers' perspectives, to address the question 'Are farmers' health and safety needs being met?' Given that farmers encounter a variety of health and safety risks in the course of their daily work, and that available statistics clearly indicate they are a high risk group, a review of the literature suggests that this area has attracted little research attention. No study has critically examined the system of occupational health and safety provision for farmers and no attempts have been made to elicit farmers' perspectives on the subject. A telephone survey using a questionnaire divided into four sections, involving a random sample of 150 farmers from the counties of Cumbria, Cheshire and Cambridgeshire in England, found that farmers considered the system of occupational health and safety provision to be inadequate and that their occupational health and safety needs were not being met. Recommendations for improvements are made based on the results of the survey.     

Isolation and characterization of LRP6, a novel member of the low density lipoprotein receptor gene family

A novel member of the low density lipoprotein receptor (LDLR) gene family has been identified and characterized. This gene, termed LDL receptor-related protein 6 (LRP6), encodes a transmembrane protein which has 71% identity and is structurally similar to the protein encoded by LRP5, a proposed candidate gene for type 1 diabetes located on human chromosome 11q13. LRP6 maps to human chromosome 12p11-p13. Mouse Lrp6 encodes a protein that has 98% identity to human LRP6 and maps to chromosome 6. Unlike other members of the LDLR family, LRP6 and LRP5 display a unique pattern of four epidermal growth factor (EGF) and three LDLR repeats in the extracellular domain. The cytoplasmic domain of LRP6 is not similar to other members of the LDLR family, while comparison with LRP5 reveals proline-rich motifs that may mediate protein-protein interactions. Thus, it is likely that LRP6 and LRP5 comprise a new class of the LDLR family.     

The human LARGE gene from 22q12.3-q13.1 is a new, distinct member of the glycosyltransferase gene family

Meningioma, a tumor of the meninges covering the central nervous system, shows frequent loss of material from human chromosome 22. Homozygous and heterozygous deletions in meningiomas defined a candidate region of >1 Mbp in 22q12.3-q13.1 and directed us to gene cloning in this segment. We characterized a new member of the N-acetylglucosaminyltransferase gene family, the LARGE gene. It occupies >664 kilobases and is one of the largest human genes. The predicted 756-aa N-acetylglucosaminyltransferase encoded by LARGE displays features that are absent in other glycosyltransferases. The human like-acetylglucosaminyltransferase polypeptide is much longer and contains putative coiled-coil domains. We characterized the mouse LARGE ortholog, which encodes a protein 97.75% identical with the human counterpart. Both genes reveal ubiquitous expression as assessed by Northern blot analysis and in situ histochemistry. Chromosomal mapping of the mouse gene reveals that mouse chromosome 8C1 corresponds to human 22q12.3-q13.1. Abnormal glycosylation of proteins and glycosphingolipids has been shown as a mechanism behind an increased potential of tumor formation and/or progression. Human tumors overexpress ganglioside GD3 (NeuAcalpha2,8NeuAcalpha2, 3Galbeta1,4Glc-Cer), which in meningiomas correlates with deletions on chromosome 22. It is the first time that a glycosyltransferase gene is involved in tumor-specific genomic rearrangements. An abnormal function of the human like-acetylglucosaminyltransferase protein may be linked to the development/progression of meningioma by altering the composition of gangliosides and/or by effect(s) on other glycosylated molecules in tumor cells.