Cloning and characterization of the human galanin GALR2 receptor

We present the molecular cloning and characterization of the human galanin receptor, hGALR2. hGALR2 shares 85%, 39%, and 57% amino acid identities to rGALR2, hGALR1, and hGALR3, respectively. hGALR2, along with rGALR2, can be distinguished from the other cloned galanin receptors by a tolerance for both N-terminal extension and C-terminal deletion of galanin, as well as by a primary signaling mechanism involving phosphatidyl inositol hydrolysis and calcium mobilization. By RT-PCR, GALR2 mRNA was abundant in human hippocampus, hypothalamus, heart, kidney, liver, and small intestine. A weak GALR2 mRNA signal was detected in human retina, and no signal was detected in cerebral cortex, lung, spleen, stomach, or pituitary.     

Structural organization and chromosomal localization of the human ribosomal protein L9 gene

Monoclonal antibodies (MoAbs) raised against Trypanosoma cruzi microsomal fraction (Mc) and cross-reactive with mammalian tissues were used to evaluate the ability of cross-reactive T. cruzi antigens to induce an immune response in Chagas' disease. Thus, we studied the ability of sera from Chagas' disease patients (CDP) with different degrees of cardiac dysfunction to block the immune recognition of these MoAb to the target antigen determining for each serum an inhibition index (II). By means of this approach we inferred that blocking of monoclonal antibody binding to T. cruzi microsomes by subjects' serum represents antibodies with the same reactivity. After serological and medical examinations, individuals were separated into the following groups: Chagas' disease patients without manifest cardiac involvement (CDP-0), CDP with suspected or borderline cardiac disease (CDP-1), CDP with moderate myocardial dysfunction (CDP-2), CDP with overt cardiac dysfunction (CDP-3) and controls including healthy subjects (HS) and patients with idiopathic myocarditis (IMP). The reactivity between MoAb 5F2 and its target antigen was significantly (p < 0.05) inhibited by sera from CDP irrespective of the clinical stage [CDP: n = 46, 50 +/- 20, mean II +/- SD: control: n = 16, 18 +/- 8]. Moreover, 5F2 was able to distinguish (p < 0.05) sera from CDP with mild disease (CDP clinical grade 0/1: n = 26, 34 +/- 18) from that of CDP with severe disease (CDP clinical grade 2/3: n = 20, 67 +/- 7). Moreover, the inhibitory capacity of sera from asymptomatic CDP (CDP-0) correlated with patients age (r = 0.66, p < 0.05). CDP-0 below or equal 40 years of age had results (n = 15, 25 +/- 13) comparable (p > 0.05) to that of controls while mean inhibition of CDP-0 over 40 years of age (n = 5, 60 +/- 5) was indistinguishable (p > 0.05) from that of patients with severe disease. Competitive assay with MoAb 5A9B11 also showed significant differences (p < 0.05) between sera from CDP (n = 46, 46 +/- 24) and controls (n = 13, 5 +/- 5). On the contrary, the differences observed between CDP with different cardiac involvement was not significant (mild: n = 26, 31 +/- 22; severe: n = 20, 66 +/- 11). However a thorough study of data from asymptomatic sera revealed the existence of two levels of reactivity, with low and high capacity to inhibit the reaction of 5A9B11 against Mc. On the contrary, CDP sera showed a blocking activity for 1A10C11 comparable to that of controls (CDP: n = 25, 19 +/- 9; control: n = 12, 14 +/- 6). Some cross-reactive MoAbs recognized epitopes partially composed of carbohydrates. Interestingly, 5F2 and 5A9B11 epitopes did not appear to have carbohydrates moieties. In summary, immunoinhibition assays revealed differences in the immune response of chronic chagasic patients against parasite epitopes. These results have opened the possibility to identify a prognosis marker of the disease suggesting the clinical utility of monitoring levels of these anti-Mc antibodies in patients with chronic Chagas' disease.     

Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3

Galanin is a 29- or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K(D) = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively.     

The mouse Id2 and Id4 genes: structural organization and chromosomal localization

The development of an antigen presentation system based on the plum pox potyvirus (PPV) is here described. The amino-terminal part of PPV capsid protein was chosen as the site for expression of foreign antigenic peptides. Modifications in this site were engineered to avoid the capability of natural transmission by aphids of this PPV vector. As a first practical attempt, different forms of an antigenic peptide (single and tandem repetition) from the VP2 capsid protein of canine parvovirus (CPV) were expressed. Both chimeras are able to infect Nicotiana clevelandii plants with similar characteristics to wild-type virus and remain genetically stable after several plant passages. The antigenicity of purified chimeric virions was demonstrated, proving the suitability of this system for diagnostic purposes. Moreover, mice and rabbits immunized with chimeric virions developed CPV-specific antibodies, which showed neutralizing activity.     

Characterization of human and mouse rod cGMP phosphodiesterase delta subunit (PDE6D) and chromosomal localization of the human gene

Unmethylated bacterial DNA containing a high frequency of the CpG motif, is mitogenic and induces T-cell independent, murine B-cell proliferation. These stimulatory effects are also induced by synthetic oligonucleotides that contain one or more unmethylated CpG dinucleotides (CpG oligo). Such mitogenicity is not seen with highly methylated vertebrate DNA, which has a lower prevalence of the CpG motif than bacterial DNA. Due to their stimulatory effects, CpG oligo have been proposed for use as vaccine adjuvants. In order to determine if a synthetic CpG oligo that was stimulatory for B-cell proliferation could augment the murine antibody response to protective bacterial polysaccharide epitopes (Pseudomonas aeruginosa LPS-O polysaccharide side chain; high-molecular-weight polysaccharide or high-MW PS), BALB/c mice were injected with mitogenic doses of CpG oligo simultaneously with high-MW PS, and antibody titers were measured by ELISA weekly for 4 weeks. Controls received PBS, a nonstimulatory control oligo plus PS, CpG alone, or PS alone. Despite evidence of B-cell mitogenicity and an increase in total IgM in CpG oligo-treated mice, CpG oligo treatment plus PS significantly decreased the high-MW PS antibody response compared to PS alone. The blunting of the anti-PS antibody response could be eliminated by vaccinating the animals with PS prior to CpG oligo. We conclude that despite in vitro and in vivo evidence of B-cell proliferation, this CpG oligo reduces PS-specific antibody responses in an animal model when given simultaneously with a bacterial polysaccharide. Based on results in this model, oligonucleotides containing stimulatory unmethylated CpG dinucleotides may not be useful adjuvants when given simultaneously with bacterial PS vaccines.     

Genomic organization and chromosomal localization of the Itm2a gene

BACKGROUND: Pemphigus vulgaris is a potentially life-threatening autoimmune disease. Although combination therapies with prednisone and azathioprine are usually effective in controlling the disease, some patients either do not respond to this treatment or show early relapses. OBJECTIVE: To find out whether mycophenolate mofetil would be an effective drug in controlling pemphigus vulgaris in patients who failed initial treatment with azathioprine and prednisone. RESULTS: Twelve patients who were initially diagnosed as having pemphigus vulgaris and had relapsed while undergoing treatment with azathioprine (1.5-2 mg/kg of body weight) and prednisolone (2 mg/kg of body weight) subsequently received combination therapy with mycophenolate mofetil (2 x 1 g/d) and prednisolone (2 mg/kg of body weight per day). Eleven of the 12 patients responded to therapy and showed no relapse of their disease even after tapering of the steroid dose. One patient did not respond. Toxic effects were low with only mild gastrointestinal symptoms in 5 patients and mild lymphopenia (World Health Organization grade I) in 9 patients. During the 9- to 12-month follow-up, none of the 11 patients showed reappearance of pemphigus lesions. CONCLUSION: Treatment of pemphigus vulgaris with mycophenolate is a safe and effective treatment.     

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.     

Genomic structure and chromosomal localization of the human interleukin 15 gene (IL-15)

The morphologic and functional characteristics of cultured hair follicle dermal papilla (DP) cells, dermal sheath (DS) cells and interstitial dermal fibroblasts (DF cells) derived from human scalp tissue are compared. DP and DS cells, but not DF cells, showed aggregative behavior at a preconfluent density. All three types of cells stained positive for type I collagen, type IV collagen, laminin and heparan sulfate proteoglycan. Only DP and DS cells expressed smooth muscle alpha-actin. DP and DS cells also synthesized more glycosaminoglycans (GAG) than DF cells, while there was no significant difference between DP and DS cells in GAG synthesis. Ultrastructurally, 7 out of 10 strains of DP and 2 out of 10 strains of DS cells were found to form intranuclear rodlets, while none of the 10 strains of DF cells examined formed intranuclear rodlets. The conditioned medium of the three types of cells was collected and tested for the presence of interleukin (IL)-1 beta, tumor necrosis factor (TGF)-beta 2, IL-6, platelet-derived growth factor-AB, epidermal growth factor, b-FGF, GM-CSF, insulin-like growth factor (IGF)-1 and HGF (hepatocyte growth factor) by ELISA or RIA. Among the tested cytokines and growth factors, TGF-beta 2, IL-6 and IGF-I were detectable in at least some conditioned media. The others were undetectable. There was no significant difference in the production of IL-6 and IGF-I among the three types of cells. In contrast, DP cells produced the highest levels of TGF-beta 2, DS cells produced intermediate levels of TGF-beta 2, and DF cells produced the lowest levels of TGF-beta 2. DP and DS cells are morphologically and functionally different from the nonfollicular, interstitial DF cells. Moreover, the presence of some minor biologic differences between DP and DS cells suggests that they represent follicular mesenchymal cells in different functional or differentiation states.     

The chromosomal distribution of mouse odorant receptor genes

Odorant receptors (ORs) on nasal olfactory sensory neurons are encoded by a large multigene family. Each member of the family is expressed in a small percentage of neurons that are confined to one of several spatial zones in the nose but are randomly distributed throughout that zone. This pattern of expression suggests that when the sensory neuron selects which OR gene to express it may be confined to a particular zonal gene set of several hundred OR genes but select from among the members of that set via a stochastic mechanism. Both locus-dependent and locus-independent models of OR gene choice have been proposed. To investigate the feasibility of these models, we determined the chromosomal locations of 21 OR genes expressed in four different spatial zones. We found that OR genes are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Our studies show that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raise the possibility that OR gene choice may be locus-independent or involve consecutive stochastic choices.     

Dihydropyrimidinase deficiency: structural organization, chromosomal localization, and mutation analysis of the human dihydropyrimidinase gene

Dihydropyrimidinase (DHP) deficiency (MIM 222748) is characterized by dihydropyrimidinuria and is associated with a variable clinical phenotype. This disease might be associated with a risk of 5-fluorouracil toxicity, although no cases have been reported. We present here both the molecular characterization of the human DHP gene and, for the first time, the mutations causing DHP deficiency. The human DHP gene spans >80 kb and consists of 10 exons. It has been assigned to 8q22, by FISH. We performed mutation analysis of genomic DNA in one symptomatic and five asymptomatic individuals presenting with dihydropyrimidinuria. We identified one frameshift mutation and five missense mutations. Two related Japanese adult subjects were homozygous for the Q334R substitution, whereas two other, unrelated Japanese infant subjects were heterozygous for the same mutation, but this mutation is not common in the Japanese population. A Caucasian pediatric patient exhibiting epileptic attacks, dysmorphic features, and severe developmental delay was homozygous for W360R. Using a eukaryotic expression system, we showed that all mutations reduced enzyme activity significantly, indicating that these are crucial DHP deficiency-causing mutations. There was no significant difference, in residual activity, between mutations observed in the symptomatic and those observed in the asymptomatic individuals.     

The mouse histone H1 genes: gene organization and differential regulation

There are six mouse histone H1 genes present in the histone gene cluster on mouse chromosome 13. These genes encode five histone H1 variants expressed in somatic cells, H1a to H1e, and the testis-specific H1t histone. Two of the genes that have not been assigned previously to the five somatic H1 subtypes have been identified as encoding the H1b and H1d subtypes. Three of the H1 genes, H1a, H1c and H1t, are present on an 80 kb segment of DNA that contains nine core histone genes. Two others, H1d and H1e, are present in a second patch, while the H1b gene is at least 500 kb away in a patch containing 14 core histone genes. The histone H1 genes are differentially expressed. All five genes for the somatic histone H1 proteins are expressed in exponentially growing cells. However, the levels of H1a, H1b and H1d mRNAs are greatly reduced in cells that are terminally differentiated or arrested in G0, while the H1c and H1e mRNAs continue to be expressed. In addition to the major RNA that ends at the stem-loop, the H1c gene expresses a longer, polyadenylated mRNA in differentiated cells, although in varying amounts. None of the other histone H1 genes encodes detectable amounts of polyadenylated mRNAs.     

Sequence analysis of the cDNA for the human casein kinase I delta (CSNK1D) gene and its chromosomal localization

A cDNA clone coding for human casein kinase I (CK1) has been isolated and sequenced. The insert of 1911 bp contained an open reading frame of 415 amino acids. The entire amino acid sequence of human CK1 was 97% homologous to that of rat CK1 delta, and their sequences in the kinase domain (284 amino acid residues) were completely identical, predicting that the obtained cDNA is for a human homolog of the CK1 delta isoform (CSNKID). The considerable similarity in the amino acid sequence of the kinase domain of human CK1 delta to the Saccharomyces cerevisiae CK1, HRR25 (66%), and to the Saccharomyces pombe CK1, HHP1 (78%), which are involved in the repair of DNA strand break, supports the speculation that human CK1 delta might also act in DNA metabolism through excision and recombinational repair. The human CK1 delta gene was mapped to chromosome 17q25.2-q25.3 by fluorescence in situ hybridization and polymerase chain reaction analysis of the human/rodent hybrid cell panels.     

The exon-intron structure and chromosomal localization of the mouse macrophage mannose receptor gene Mrc1: identification of a Ricin-like domain at the N-terminus of the receptor

The macrophage mannose receptor is a transmembrane protein that is expressed on the surface of mature macrophages. The ectodomain of the receptor contains multiple domains, eight of which belong to the calcium-dependent C-type lectin family. The mannose receptor binds to carbohydrate polymers that have a high content of mannose. This property allows this protein to function as a phagocytic receptor that participates in first-line host defense against invading microorganisms. In this paper we describe the intron-exon structure of the mouse macrophage mannose receptor gene which was found to span at least 70 kilobases. We also report the localization of this gene, termed Mrc1, to mouse Chromosome 2. Like its human counterpart, Mrc1 contains 30 exons and 29 introns. A protein module that resembles a subdomain of the B chain of the plant lectin Ricin has been found within the N-terminal cysteine-rich domain of the mannose receptor.     

Sequence, genomic organization, and chromosomal location of the mouse Müllerian-inhibiting substance type II receptor gene

We have determined the sequence and structure of the mouse Müllerian-inhibiting substance (MIS) type II receptor gene. Sequence comparisons demonstrate that the mouse, rat, rabbit, and human MIS type II receptors are highly conserved. The mouse MIS type II receptor gene is encoded by 11 exons and spans approximately 9-kb. Only half of the intron/exon boundaries of its kinase domain are conserved in comparison to the kinase domain of the related activin type II receptor. Whereas the activin type II receptor gene contains large introns (> 40-kb), the largest intron of the MIS type II receptor gene is only 4.3-kb. The MIS type II receptor gene (Amhr) is closely linked to Hoxc on mouse chromosome 15. Knowledge of the sequence and genomic structure of Amhr provides important information for the genetic manipulation of the Amhr locus.