Identification of the molecular defect in a family with spondyloepiphyseal dysplasia.

Spondyloepiphyseal dysplasias (SED) are a heterogeneous group of inherited disorders characterized by disproportionate short stature and pleiotropic involvement of the skeletal and ocular systems. Evidence has suggested that SED may result from structural defects in type II collagen. To confirm the validity of this hypothesis, the structure of the "candidate" type II collagen gene (COL2A1) has been directly examined in a relatively large SED family. Coarse scanning of the gene by Southern blot hybridization identified an abnormal restriction pattern in one of the affected members of the kindred. Analysis of selected genomic fragments, amplified by the polymerase chain reaction, precisely localized the molecular defect and demonstrated that all affected family members carried the same heterozygous single-exon deletion. As a consequence of the mutation, nearly 90 percent of the assembled type II collagen homotrimers are expected to contain one or more procollagen subunits harboring an interstitial deletion of 36 amino acids in the triple helical domain.

Retinoic acid down-regulates the expression of the vasoactive intestinal polypeptide receptor type-1 in human breast carcinoma cell lines.

Four breast carcinoma cell lines (T47D, ZR-75-1, MDA-MB-231, and MCF-7) were tested for regulation of the expression of vasoactive intestinal polypeptide receptor type-1 (VIP-R1). In all four cell lines, retinoic acid (RA) treatment caused a fast and marked decrease in VIP-R1 mRNA level as examined by Northern blots. Cycloheximide pretreatment attenuated the effect from 3- to 2-fold, indicating that existing proteins can mediate the decreasing effect of RA, but to attain the maximal effect new protein synthesis might be needed. Transcriptional inhibition with Actinomyocin D showed that RA did not influence the VIP-R1 mRNA half-life, indicating that the decreasing effect of RA on the mRNA level is due to transcriptional inhibition. In agreement with the observations on mRNA level, we found that the VIP receptor number was reduced 3-fold from 88 to 32 fmol/10(6) cells in T47D cells and from 222 to 73 fmol/10(6) cells in MDA-MB-231 cells upon RA treatment for 72 h. The promoter and 5'-flanking region of the VIP-R1 gene were cloned from a human placental cosmid library, and 2.5 kb were sequenced to search for regulatory elements. Our results, therefore, imply that the regulation of VIP-R1 gene expression by RA could have a role in human mammary tumor biology.

Deletion mapping of chromosome 3p in human uterine cervical cancer.

Deletion mapping of chromosome 3p was performed on 47 cases of human uterine cervical cancer using 24 polymorphic DNA markers including five inter-Alu DNA markers and two NotI-boundary cosmid markers obtained in our laboratory. The most likely order of these 24 polymorphic DNA markers was determined as being cen-[D3S4, H8]-D3S693-D3S659-D3S30-D3S687-[D3S2, UR9, UR47]-J36-J17-GNAI2B-D3F15S2-D3S643- D3S32-D3S23-D3S686-H35-UR189-D3S685-D3S 11 - D3S12-THRB-D3S22-pter, based on the data from radiation hybrid mapping genetic linkage analysis and in situ hybridization. Loss of heterozygosity (LOH) at one or more loci on chromosome 3p was detected in 21 of 47 cases (45%). Four tumors showed partial or interstitial deletions, and the common region of LOH in these tumors was 3p13-p21.1 between the D3S30 marker and the D3S2 marker. Candidates for tumor-suppressor genes, APEH, D8, GNA12B, ZNF35, RARB, THRB and RAFI, were all mapped outside of the common region in uterine cervical cancer. However, this region is commonly deleted in carcinoma of the lung, breast and kidney, and encompasses the breakpoint of the (3;8) translocation in hereditary renal cell carcinoma. This result indicates the presence of a novel tumor-suppressor gene in the region of 3p13-p21.1, which is involved in the development of several human cancers.

BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.

We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozygous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

Cloning and expression of cytokine-inducible nitric oxide synthase cDNA from rat islets of Langerhans.

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the beta-cells of interleukin (IL)-1 beta-exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokine-exposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH beta-cells cultured in the absence of cytokines. Addition of IL-1 beta alone or in combination with tumor necrosis factor-alpha induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured as nitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokine-exposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the L-arginine analogs, N omega-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as a series of spots with the expected molecular mass of 131 kDa and pI values in the range of 6.8 to 7.0. In conclusion, the IL-1 beta-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.

Human ERRgamma, a third member of the estrogen receptor-related receptor (ERR) subfamily of orphan nuclear receptors: tissue-specific isoforms are expressed during development and in the adult.

The nuclear receptor protein superfamily is a large group of transcription factors involved in many aspects of animal development, tissue differentiation, and homeostasis in the higher eukaryotes. A subfamily of receptors, ERRalpha and beta (estrogen receptor-related receptor alpha and beta), closely related to the ER, were among the first orphan nuclear receptors identified. These receptors can bind DNA as monomers and are thought to activate transcription constitutively, unaffected by beta-estradiol. Studies of the expression patterns of ERRalpha and gene disruption experiments of ERRbeta indicate that they play an important role in the development and differentiation of specific tissues in the mouse. In this work we demonstrate the existence in humans of a third member of this subfamily of receptors, termed ERRgamma, which is highly expressed in a number of diverse fetal and adult tissues including brain, kidney, pancreas, and placenta. The ERRgamma mRNA is highly alternatively spliced at the 5'-end, giving rise to a number of tissue-specific RNA species, some of which code for protein isoforms differing in the N-terminal region. Like ERRalpha and beta, ERRgamma binds as a monomer to an ERRE. A GAL4-ERRgamma fusion protein activates transcription in a ligand-independent manner in transfected HEK293 cells to a greater degree than either the GAL4-ERRalpha or -beta fusion proteins.

Negative cooperativity in the insulin-like growth factor-I receptor and a chimeric IGF-I/insulin receptor.

Insulin and insulin-like growth factor-I (IGF-I) share a spectrum of metabolic and growth-promoting effects, mediated through homologous receptors that belong to the tyrosine kinase family. The dissociation rate of insulin from its receptor is affected by negative cooperativity, i.e. accelerates with increased receptor occupancy. The dose-response curve for the acceleration of tracer dissociation by unlabeled insulin has a distinct bell-shaped curve, with a progressive slowing down at insulin concentrations greater than 100 nM. The kinetics of the IGF-I interaction with its receptor has not been studied in such detail. In the present work, we report that while the IGF-I receptor exhibits negative cooperativity like the insulin receptor, the concentration dependence of the dissociation kinetics is distinct from that of native human insulin by not being bell-shaped, but monophasic like that of insulin analogues mutated at the hexamer-forming surface; it is changed to an insulin-type curve by substitution of IGF-I receptor's sequence including residues 382-565 with the homologous insulin receptor domain. The data suggest that like insulin, IGF-I has a bivalent binding mode and crosslinks two distinct areas of the two alpha subunits that are close, but distinct from the equivalent insulin receptor binding sites.

Hyperproinsulinemia in a three-generation Caucasian family due to mutant proinsulin (Arg65-His) not associated with imparied glucose tolerance: the contribution of mutant proinsulin to insulin bioactivity.

Familial hyperproinsulinemia is a genetic abnormality characterized by an increased proportion of proinsulin immunoreactivity in the circulation due to mutations affecting the posttranslational processing of proinsulin. In affected Japanese families, this has been associated with noninsulin-dependent diabetes mellitus or impaired glucose tolerance. A three-generation Caucasian family with hyperproinsulinemia was identified through unexplained hyperinsulinemia in a normal volunteer participating in a metabolic study. High pressure liquid chromatography analysis of fasting plasma revealed a major peak eluting close to the position of proinsulin. Direct sequencing of the proinsulin gene exon 3 showed a heterozygous point mutation (CGT-->CAT) resulting in the substitution of Arg-->His in position 65 (corresponding to the AC cleavage site) in the index case, his mother, and his maternal grandmother. Using specific enzyme-linked immunosorbent assay methods to quantify insulin and proinsulin (including its conversion intermediates), the impact of this mutation on B cell secretion and glucose tolerance was studied. All affected subjects had normal oral glucose tolerance. In the basal state and after oral glucose administration, their proinsulin responses were immense, but intact insulin responses were slightly reduced. However, when calculating insulin bioactivity by assuming 9% activity for mutant Arg65-->His proinsulin, responses in affected subjects were comparable to those in normal subjects. In conclusion, our data demonstrate hyperproinsulinemia in a three-generation Caucasian family due to heterozygous mutant Arg65-->His proinsulin. This was not associated with impaired glucose tolerance. These results suggest that this mutation in the heterozygous state per se does not affect glucose tolerance and that the biological activity of mutant proinsulin contributes to glucose homeostasis in this family. The association of the same mutation with impaired glucose tolerance or diabetes in previous studies may be the result of selection bias or associated conditions (e.g. the genetic background of the kindreds examined).

Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins.

We report the characterization of three novel members of the KRAB-domain containing C2-H2 zinc finger family (ZNF133, 136 and 140). KRAB (Krüppel-associated box) is an evolutionarily conserved protein domain found N-terminally with respect to the zinc finger repeats that encodes the DNA binding domain. ZNF133 and ZNF140 have both the KRAB A- and KRAB B-boxes present at their N-terminus, whereas ZNF136 contains only the KRAB A-box. We have previously demonstrated that the KRAB domains derived from ZNF133 and ZNF140 are potent transcriptional repression domains [Margolin et al. (1994) Proc. Natl. Acad. Sci. USA 91, 4509-4513]. The KRAB domain from ZNF136, containing only subdomain A, is a considerable weaker suppression domain; however, when fused to the heterologous KRAB B subdomain of ZNF10 (KOX1) the two subdomains from a KRAB domain which induces repression as potently as previously reported KRAB domains.

A simple assay for the detection of antibodies to endocrine islet cell surface antigens.

A simple and sensitive immunoradiometric assay for the detection of islet cell surface antibodies (CIRMA) has been developed. Live, transformed islet cells derived from a liver metastasis of a transplantable islet cell tumor were grown in removable microtiter wells and incubated with antibody. Cell-bound antibodies were quantitated using 125I-labelled second antibodies. The assay was used to detect islet cell antibodies present in sera from non-diabetic and diabetic BB rats and proved to be particularly effective for screening hybridoma supernatants in order to identify monoclonal antibodies recognizing islet cell surface antigens.

Studies of relationships between variation of the human G protein-coupled receptor 40 Gene and Type 2 diabetes and insulin release.

AIMS: Recently, a novel human G protein-coupled receptor 40 (GPR40), which is predominantly expressed in pancreatic islets, was shown to mediate an amplifying effect of long-chain fatty acids on glucose-induced insulin secretion. The present aim was to examine the coding region of GPR40 for variation and to assess whether identified variants confer an increased risk of Type 2 diabetes or altered insulin release. METHODS: Mutation analysis was performed in 43 patients with Type 2 diabetes, 18 normal glucose-tolerant subjects, and 3 maturity-onset of diabetes in the young (MODY) X patients using direct sequencing. Genotyping was performed using polymerase chain reaction (PCR)-generated primer extension products analysis by high throughput chip-based mass spectrometry (MALDI-TOF). The potential impact of GPR40 mutations on [(3)H]-myo-inositol turnover was estimated in COS-7 cells after stimulation with various concentrations of 5,8,11-eicosatriynoic acid. RESULTS: Two nucleotide substitutions, an Arg211His polymorphism and a rare Asp175Asn mutation, were identified. Both variants showed EC(50) values similar to the wild type. However, the maximal efficacy of the rare Asp175Asn was 39% lower compared with the wild type (P = 0.01). The Arg211His polymorphism had a similar allele frequency among 1384 Type 2 diabetic patients [MAF%; 23.4 (95% CI: 21.8-25.0)] and 4424 middle-aged glucose-tolerant subjects [24.1% (23.2-25.0)]. A genotype-quantitative trait study of 5597 non-diabetic, middle-aged subjects from the Inter99 cohort showed no significant differences in oral glucose tolerance test (OGTT)-derived estimates of insulin release between carriers of various GPR40 genotypes. CONCLUSIONS: Variations in the coding region of GPR40 do not appear to be associated with Type 2 diabetes or insulin release alterations.

Flow cytometric analysis of monoclonal islet cell surface antibodies reacting with RINm5AH insulinoma cells.

Two monoclonal islet cell surface antibodies, isolated following immunization with human pancreatic islets, were analyzed for reactivity with the insulin-producing rat islet tumor RINm5AH cell line using computer assisted flow cytometric analysis. The antibody binding was quantified utilizing the gating facilities, single and dual parameter analysis of this system which are not available in conventional assays for cell surface antibody determination. The two antibodies specifically bind to the RINm5AH cells, although the dual parameter analysis demonstrated binding to only a subpopulation.

Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAs identify putative candidate genes for developmental and malignant disorders.

We have isolated and chromosomally fine-mapped 16 novel genes belonging to the human zinc finger Krüppel family (ZNF131-140, 142, 143, 148, 151, 154, and 155), including 1 of the GLI type (ZNF143) and 3 containing a KRAB (Krüppel-associated box) segment (ZNF133, 136, and 140). Based on their map position, several of these ZNF genes are putative candidate genes for both developmental and malignant disorders: ZNF138, ZNF139, and ZNF143 were localized to 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4, regions involved in deletions and/or translocations associated with Williams syndrome, split hand and foot disease (SHFD1), and Beckwith-Wiedemann syndrome, respectively. ZNF133 was localized to 20p11.2, close to, but probably distinct from, the region deleted in Alagille syndrome. Zinc finger genes mapping to regions commonly deleted in solid tumors included ZNF132, 134, 135, 137, 154, and 155, all located on 19q13 (thyroid adenoma), and ZNF151, at 1p36.1-p36.2 (neuroblastoma, colon cancer, and other tumors). In addition, several of the ZNFs mapped to regions implicated in recurrent chromosomal rearrangements in hematological malignancies (ZNF139, 7q21.3-q22.1; ZNF148, 3q21-q22; ZNF151, 1p36.1-p36.2). The study indicates that the number of ZNF genes in human is large and that systematic isolation and mapping of ZNF genes is a straightforward approach for the identification of novel candidate disease genes.

Monoclonal antibodies against pancreatic islet-cell-surface antigens selected by flow cytofluorometry.

BALB/c mice were immunized with human islets of Langerhans, and spleen cells from two mice, found to develop cell-surface antibodies against insulin-producing rat islet tumour RIN-5F cells, were fused with mouse myeloma cells. Antibody-producing hybrids were cloned on the basis of their production of surface antibodies reactive with paraformaldehyde-fixed RIN-5F cells by indirect immunofluorescence analysis in the fluorescence-activated cell sorter. Among 236 primary clones, eight stable cell lines producing islet-cell-surface antibodies were eventually cloned. Antibody 2G3 (IgM) reacted with viable normal rat islet cells and high insulin-producing rat islet tumour RIN5-A2 cells, while 3G3 (IgM) only reacted with RIN5-A2 cells. Antibody beta B1 (IgG1) reacted with all islet cells tested and detected an Mr21k component in immunoblotting experiments with RIN-5AH cell plasma membrane proteins electrophoretically transferred to nitrocellulose filters. Antibody 7F6 (IgM) reacted with all islet and non-islet cells tested and detected bands of Mr 66k and 27k by immunoblotting. Antibodies gamma B3, gamma B6, gamma C2, and 6B1 (all IgM) showed varying degrees of binding to different islet cells, but reacted only weakly with non-islet human cells. It is concluded that monoclonal antibodies against pancreatic islet cells may define specific endocrine islet-cell-surface determinants.

Mining of assembled expressed sequence tag (EST) data for protein families: application to the G protein-coupled receptor superfamily.

The availability of large expressed sequence tag (EST) databases has led to a revolution in the way new genes are identified. Mining of these databases using known protein sequences as queries is a powerful technique for discovering orthologous and paralogous genes. The scientist is often confronted, however, by an enormous amount of search output owing to the inherent redundancy of EST data. In addition, high search sensitivity often cannot be achieved using only a single member of a protein superfamily as a query. In this paper a technique for addressing both of these issues is described. Assembled EST databases are queried with every member of a protein superfamily, the results are integrated and false positives are pruned from the set. The result is a set of assemblies enriched in members of the protein superfamily under consideration. The technique is applied to the G protein-coupled receptor (GPCR) superfamily in the construction of a GPCR Resource. A novel full-length human GPCR identified from the GPCR Resource is presented, illustrating the utility of the method.