A mouse molecular mimic of human vascular adhesion protein-1

Human vascular adhesion protein-1 (VAP-1) is an endothelial sialoglycoprotein which exists in forms of Mr 90000 and 170000 and mediates lymphocyte binding to vessels under shear. VAP-1 is functionally defined by an inhibitory mouse mAb 1B2. A large-scale immunoaffinity purification of VAP-1 from human tonsil lysates was performed to determine the protein sequence for VAP-1 cDNA cloning. A dominant protein of molecular weight 90000 was obtained which yielded an N-terminal sequence of 20 amino acids which bore no significant identity to any protein sequence in the data banks. A mouse mAb (5B11) against a synthetic peptide from this sequence was raised and found to stain tissues in an identical manner to mAb 1B2, to inhibit lymphocyte adhesion to endothelial cells and to recognize VAP-1. Later, the N-terminal sequence obtained from the 1B2 immunoprecipitations was found to be identical to a mouse cyclophilin C associated protein (mCyCAP) subsequently published by others. We show here by several criteria at the protein and DNA level that VAP-1 is distinct from mCyCAP. Moreover, we elucidate the mechanism which results in binding of mCyCAP to mAb 1B2 during antibody synthesis in hybridoma cells and the sequelae of co-precipitation of mCyCAP during the immunoaffinity chromatography. Binding of mCyCAP to a mouse mAb has not been described before and suggests a new function for this molecule in immunoglobulin synthesis and/or secretion. Moreover, these data indicate that the N-terminal peptide of mCyCAP is a molecular mimic of a functionally important epitope of VAP-1.     

Cloning and characterization of mouse vascular adhesion protein-1 reveals a novel molecule with enzymatic activity

Human vascular adhesion protein-1 (VAP-1) is a sialylated endothelial cell adhesion molecule mediating the initial L-selectin-independent interactions between lymphocytes and endothelial cells in man. In this work we cloned and characterized mouse VAP-1 (mVAP-1) and produced an anti-mVAP-1 mAb against a recombinant mVAP-1 fusion protein. The isolated cDNA encodes a novel 84.5-kDa mouse molecule. The anti-mVAP-1 mAb stained high endothelial venules in peripheral lymph nodes, and smooth muscle cells and lamina propria vessels in gut. During immunoblotting, this anti-mVAP-1 mAb recognized a 110/220-kDa Ag, suggesting that mVAP-1 is a dimer. Since mVAP-1 has significant sequence identity to members of a family of enzymes called the copper-containing amine oxidases, we showed that mVAP-1 possesses monoamine oxidase activity. Thus, mVAP-1 is the first mouse membrane-bound amine oxidase identified at the molecular level. Based on the 83% identity between the isolated cDNA and human VAP-1 cDNA, the expression pattern, the molecular mass, and the enzyme activity against monoamines, the cloned molecule represents a mouse homologue of human VAP-1. Cloning of mVAP-1 provides a valuable tool for in vivo studies of the significance of VAP-1 for lymphocyte-endothelial cell interactions and of the possible relationship between leukocyte adhesion and amine oxidase activity.     

Isolation and characterization of a gene from the DiGeorge chromosomal region homologous to the mouse Tbx1 gene

Given the higher proportion of manufactured foods now available which meet current dietary recommendations, the food supply in developed countries like Australia could be said to be "healthier". Yet the "health" of the diet is often achieved at the expense of the "health" of the environment since ecological problems created a current food production and distribution methods remain unaddressed. Further, nutritional modifications which produce foods that are low in fat, sugar, salt and high in fibre do not necessarily address the concerns consumers have about the food supply. An emphasis solely on the physical health of populations, through improved diet, is out of keeping with current views on health which recognise the importance of overall well-being. Through the development of the concept of "sustaining gastronomy", consumers, food manufacturers and producers, and food regulators can better address the problems inherent in the food system, including those of an environmental nature.     

Isolation and chromosomal mapping of a mouse homolog of the Batten disease gene CLN3

We describe the isolation and chromosomal mapping of a mouse homolog of the Batten disease gene, CLN3. Like its human counterpart, the mouse cDNA contains an open reading frame of 1314 bp encoding a predicted protein product of 438 amino acids. The mouse and human coding regions are 82 and 85% identical at the nucleic acid and amino acid levels, respectively. The mouse gene maps to distal Chromosome 7, in a region containing genes whose homologs are on human chromosome 16p12, where CLN3 maps. Isolation of a mouse CLN3 homolog will facilitate the creation of a mouse model of Batten disease.     

Structure and chromosomal mapping of the mouse P2X3 gene

Since arthritis induced by Mycobacterium products (adjuvant) in rats is considered to be immunologically driven, the objective of the present study was to determine if the immunosuppressor drug cyclosporin could affect hindpaw edema and joint hyperalgesia simultaneously. Female Holtzman rats (140-170 g) presented hyperalgesia and edema on the 8th and 12th day following adjuvant injection. Daily systemic (oral or intramuscular) administration of cyclosporin (0.5-5.0 mg kg (-1) day (-1)) or dexamethasone (0.01-0.1 mg kg (-1) day (-1)) for 15 days starting on day zero dose-dependently inhibited the hindpaw edema and hyperalgesia in arthritic rats. However, hyperalgesia but not edema could be detected two days after cyclosporin withdrawal. We concluded that a) the continuous presence of cyclosporin is essential to reduce the development of joint hyperalgesia and that b) different mechanisms underlie the appearance of hyperalgesia and edema in this model. The intracerebroventricular (i.c.v.) administration of 5-50-fold smaller doses of cyclosporin (1.5-150 micrograms/day) or dexamethasone (15 micrograms/day) also reduced the arthritic hindpaw edema and hyperalgesia. Peripheral blood from animals injected with effective systemic cyclosporin doses showed detectable levels of the drug, whereas peripheral blood from those injected with i.c.v. cyclosporin did not, as measured by specific RIA. Our results indicate that cyclosporin administered by the central route is as effective as by the systemic route to reduce joint hyperalgesia and hindpaw edema in arthritic rats. The antiarthritic effect induced by low doses of cyclosporin in the central nervous system (CNS) could be explored to avoid it often associated systemic side effects during chronic therapy. However, the mechanism(s) involved in the antiarthritic response to cyclosporin in the CNS remain to be elucidated.     

Structure of the murine CD156 gene, characterization of its promoter, and chromosomal location

The murine cell surface antigen mCD156 is a glycoprotein that is expressed in monocytic cell lines and consists of a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, and an epidermal growth factor-like domain in the extracellular region. The mCD156 gene is composed of 24 exons and 23 introns and spans approximately 14 kilobases. The first exon encodes most of the signal peptide sequence, and the transmembrane region is encoded by a single exon (19). In contrast, the other regions are composed of multiple exons. Of these, exons 7-12 and 12-15 encode a metalloprotease domain and a disintegrin domain, respectively. Sequence analysis of the 5'-flanking DNA revealed many potential regulatory motifs. Chloramphenicol acetyltransferase analysis demonstrated that nucleotides at positions -183, -334, and -623 contained cis-acting enhancing elements in a mouse monocytic cell line, aHINS-B3. Nucleotides at positions -183 and -390 contained elements responsible for lipopolysaccharide (LPS) inducibility, although several other 5'-flanking regions were also involved in LPS responsiveness. Regions -202, -507, and -659 play a role in interferon-gamma inducibility. Some of the potential regulatory motifs and other unknown cis elements may be involved in the constitutive expression, and LPS and interferon-gamma inducibilities. The mCD156 gene was mapped to chromosome 7, region F3-F4.     

Sequence, structure, and chromosomal mapping of the mouse Lgals6 gene, encoding galectin-6

In the accompanying paper (Gitt, M. A., Colnot, C., Poirier, F., and Barondes, S. H., and Leffler, H. (1998) J. Biol. Chem. 273, 2954-2960), we reported that mouse gastrointestinal tract specifically expresses two closely related galectins, galectins-4 and -6, each with two carbohydrate recognition domains in the same peptide. Here, we report the isolation, characterization, and chromosomal mapping of the complete mouse Lgals6 gene, which encodes galectin-6, and of a fragment of a distinct gene, Lgals4, which encodes galectin-4. The coding sequence of galectin-6 is specified by eight exons. The upstream region contains two putative promoters. Both Lgals6 and the closely related Lgals4 are clustered together about 3.2 centimorgans proximal to the apoE gene on mouse chromosome 7. The syntenic human region is 19q13.1-13.3.     

Isolation, mapping, and functional expression of the mouse X chromosome glycerol kinase gene

Glycerol kinase (Gyk) participates in the metabolism of endogenously derived and dietary glycerol. Deficiency of the human enzyme activity is an X-linked recessive disorder with a clinical picture varying from childhood metabolic crisis to asymptomatic adults incidentally identified by hyperlipidemia screening (pseudohypertriglyceridemia). Gyk is a member of a small group of kinases termed ambiquitous enzymes that are found in the cytosol or as membrane-bound enzymes associated with the voltage-dependent anion channel of the mitochondrial outer membrane. It was recently reported that in humans there are X-linked and autosomal copies of Gyk sequences, both apparently functional genes and processed pseudogenes. To understand the role of Gyk in normal metabolism and the variable clinical features seen with Gyk deficiency, we have characterized the mouse Gyk gene. We present the sequence of a full-length mouse Gyk cDNA that is alternatively spliced in brain. The Gyk gene was mapped to the mouse X chromosome by both fluorescence in situ hybridization and an interspecies backcross panel, demonstrating conservation of synteny with dmd. To confirm the functional identity of the cDNA, transient transfection of the cDNA into COS7 cells was shown to cause a marked elevation in glycerol kinase activity.     

Characterization and chromosomal mapping of two pseudogenes of the mouse Pafaha/Lis1 gene: retrointegration hotspots in the mouse genome

This study examines the relationship between trainees' conjugal family experience, current intergenerational family relationships, and the client's perception of the therapeutic alliance. Participants were 74 first practicum family therapy trainees, representing two family therapy programs, and 90 clients. Results indicated a moderately significant relationship between conjugal family experience and trainees' reported intergenerational intimacy with parents. Additionally, clients whose therapists had conjugal family experience reported a slightly more favorable therapeutic alliance than clients whose therapists did not have conjugal family experience. Additionally, trainees with conjugal family experience reported more current intimacy and individuation than nonconjugal trainees and felt less intimidated by their parents.     

Structure of the gene encoding the ubiquitin-conjugating enzyme Ubcm4, characterization of its promoter, and chromosomal location

An improved method for the simultaneous determination of underivatized biogenic amines, cadaverine, putrescine, spermidine, histamine, tyramine and some amino acids precursors, histidine and tyrosine, in food products, based on ion-exchange chromatography (IC) with integrated pulsed amperometric detection (IPAD) has been developed. The method was successfully used for the analysis of biogenic amines and amino acids in food both of vegetable (kiwi, Actinidia chinensis) and animal origin, (fish, pilchard), as well as in fermented foods, such as cheese (Emmenthal) and dry sausages (salami). The method was also successfully used to study the changes in biogenic amines during the ripening of dry fermented sausages (salami). The analytes were extracted from foods with perchloric acid and the extracts were purified by liquid-liquid partition using n-hexane. Determination of biogenic amines was performed through cation-exchange chromatography with isocratic elution and IPAD. The detection limits for the analytes under investigation were found to range from 1.25 to 2.50 ng, at a signal-to-noise ratio of 3:1. Average recoveries ranged from 85.5 to 97.4% and R.S.D. values ranged from 3.4 to 8.8. The proposed method offers a number of advantages over our previous IPAD method, such as the application to a larger number of analytes and matrices, a simpler extraction procedure and clean-up, isocratic elution using low acid and base concentrations, an improved chromatographic separation and a lower detection limit.     

Structure, promoter analysis and chromosomal assignment of the human APEX gene

Measures of functional vision are needed to assess elderly low vision patients, their success in using devices, and their ability to manage outside the treatment setting. A new test devised to measure functional ability through the performance of everyday tasks was administered to 94 patients who had acuities of 20/100 or worse in their better eye. Consisting of three versions and four subtests: spot reading, short-term text reading, identifying paper currency and clock reading, the test used standardized items and was timed. In a multiple regression model predicting test performance higher scores were associated with better near acuity (P = .002), higher education (P = .022) and higher levels of self-reported visual skills (P = .072). These predictors plus distance acuity, age and sex only accounted for 35 percent of the variance in test scores. Repeated administration of the test to a different group of 21 patients showed the test to be reliable (intraclass correlation = .85, P < .01) and to have no practice or version effects or differences between raters. This new test may be useful for natural history studies and clinical trials involving low vision patients but further evaluation of its sensitivity to change over time is required.     

In situ localization and chromosomal mapping of the AG1 (Dmp1) gene

Dentinogenesis is being used as a model for understanding the biomineralization process. The odontoblasts synthesize a structural matrix comprised of Type I collagen fibrils which define the basic architecture of the tissue. The odontoblasts also synthesize and deliver a number of dentin-specific acidic macromolecules into the extracellular compartment. These acidic macromolecules may be involved in regulating the ordered deposition of hydroxyapatite crystals within the matrix. AG1 is the first tooth-specific acidic macromolecule to have been cloned and sequenced. To identify which cells of the rat incisor pulp/odontoblast complex were responsible for synthesis of AG1, in situ hybridization was used. Digoxigenin labeled sense and anti-sense AG1 riboprobes were prepared. The AG1 mRNA was found to be expressed in the mature secretory odontoblasts. Neither pulp cells nor pre-odontoblasts showed any staining with the anti-sense probes. Chromosomal localization studies placed the AG1 gene on mouse chromosome 5q21, in tight linkage with Fgf5. AG1 has been renamed Dmp1 (dentin matrix protein 1) in accordance with present chromosomal nomenclature. Mouse 5q21 corresponds to the 4q21 locus in humans. This is the locus for the human tooth mineralization disorder dentinogenesis imperfecta Type II (DI-II). These data suggest that the Dmp1 gene is involved in mineralization and is a candidate gene for DI-II.