Developmental expression of a novel murine homeobox gene (Chx10): evidence for roles in determination of the neuroretina and inner nuclear layer.

Few potential regulatory proteins of vertebrate retinal development have been identified. We describe a 39 kDa murine polypeptide (Chx10) with a homeodomain 82% identical to that of the nematode protein ceh-10. In the developing mouse, the Chx10 transcript is expressed throughout the anterior optic vesicle and all neuroblasts of the optic cup. In the mature retina, the Chx10 protein is restricted to the inner nuclear layer, in which its expression decreases from the outer to the inner margin. Chx10 transcripts are also detected in regions of the developing thalamus, hindbrain, and ventral spinal cord. The data suggest that Chx10 plays critical roles in the formation of the neuroretina and in the development and maintenance of the inner nuclear layer.

Cloning and characterization of additional members of the G protein-coupled receptor family.

A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.

Dopamine D4 receptor variant, D4GLYCINE194, in Africans, but not in Caucasians: no association with schizophrenia.

Because antipsychotic drugs selectively block dopamine receptors and since dopamine D4 receptors are elevated sixfold in postmortem schizophrenia brain, we searched for possible abnormalities in the coding region of the genomic DNA sequence for the dopamine D4 receptor in control and schizophrenia tissues. The DNA sequence for the first 250 bases of exon 3 of this receptor was examined in the genomic DNA from 296 control individuals and 58 schizophrenics. Twenty-three out of 183 control blacks (12.6%) and 3 out of 24 (12.5%) schizophrenic blacks revealed a replacement of T by G, predicting a substitution of valine by glycine at amino acid position 194. The identical prevalence of 12.5% indicates that the variant is not associated with schizophrenia. The amino acid replacement occurs one amino acid away from a serine amino acid which is critical for the attachment of dopamine. None of the 147 Caucasians (113 controls; 34 schizophrenics) revealed this variant, termed D4GLYCINE194.

Dopamine D4 receptor variant in Africans, D4valine194glycine, is insensitive to dopamine and clozapine: report of a homozygous individual.

The D4Valine194Glycine receptor is a variant of the dopamine D4 receptor and is found in 12.5% of the Afro-Caribbean population. Glycine replaces valine at a position one amino acid away from a serine which is critical for the attachment of dopamine. To determine whether this mutation had an effect on the properties of the dopamine D4 receptor, we constructed this variant and tested the sensitivity of the expressed protein with various drugs. We found that the variant receptor was two orders of magnitude less sensitive to dopamine, clozapine and olanzapine. The variant receptor was insensitive to guanine nucleotide, indicating the absence of a high-affinity state or functional state. The one 15-year-old individual found homozygous for this variant also had sickle cell disease. The patient revealed an overall pattern of low weight and no axillary or pubic hair.

The human dopamine D2(Longer) receptor has a high-affinity state and inhibits adenylyl cyclase.

Brain dopamine D2 receptors are the main targets for antipsychotic and anti-Parkinsonian drugs. The dopamine D2 receptor has three forms, D2(Short), D2(Long) and D2(Longer). D2(Longer) is a newly found splice variant which contains two additional amino acids (valine and glutamine) in the third cytoplasmic loop of the receptor. To determine whether D2(Longer) was functional, the cDNA was transfected into CHO cells. D2(Longer) revealed a high-affinity state for dopamine ( approximately 1.5 nM), and mediated dopamine-inhibited adenylyl cyclase.

Schizophrenia: elevated mRNA for dopamine D2(Longer) receptors in frontal cortex.

Because dopamine D2 receptors are the primary targets for antipsychotic drugs, including clozapine and quetiapine, and because some studies have found D2 receptors to be elevated in schizophrenia, we examined the mRNA of three forms of the D2 receptor, particularly the new form of the dopamine D2 receptor, D2(Longer), in post-mortem brains from patients who died with schizophrenia. Using quantitative competitive RT-PCR (reverse transcriptase-polymerase chain reaction), the D2(Longer) mRNA was higher in the frontal cortex, compared to control tissues. The mRNA concentration of D2(Long) and D2(Short) was also higher in the frontal cortex, compared to control tissues. Although most of the schizophrenia patients had received different antipsychotic drugs for varying periods of time, the mRNA of D2(Longer), as well as that for D2(Long) and D2(Short), in such medicated tissues was similar to that in a frontal cortex tissue from a patient who had reliably never received antipsychotic drugs. It is possible, therefore, that the elevation of the mRNAs for D2(Longer), D2(Long) and D2(Short) in the frontal cortex may be related to the disease of schizophrenia itself.

New dopamine receptor, D2(Longer), with unique TG splice site, in human brain.

Brain dopamine receptor agonists alleviate the signs of Parkinson's disease, while dopamine receptor antagonists alleviate hallucinations and delusions in psychosis. The dopamine type 2 receptor (or D2) is blocked by antipsychotic drugs, including even the "atypical" drugs such as clozapine or remoxipride, in direct relation to their clinical potencies. Compared to the long form of the D2 receptor (D2(Long)), the short form (D2(Short)) may be three times more sensitive to benzamide antipsychotic drugs. Hence, it is essential to identify additional variants of dopamine receptors for which more selective antipsychotic drugs can be found. Although no family linkage has been found between the D2 receptor and schizophrenia, there can be brain region abnormalities in the RNA transcript expression of dopamine receptors. Therefore, in order to identify variant dopamine D2 receptors, we searched for mutations in the RNA transcripts for the dopamine D2 receptor in the striatum of post-mortem brains from individuals who died with psychosis, including schizophrenia. A new splice variant of the D2 receptor, D2(Longer), with a unique TG splice site, was found in one control brain and in two psychotic brains.

A serotonin-4 receptor-like pseudogene in humans.

During a search for new G-protein-linked receptors for dopamine and serotonin, we found a serotonin-4 receptor-like pseudogene. This receptor-like pseudogene is intronless, contains an in-frame stop codon following transmembrane-3, and has two one-nucleotide insertions between transmembrane-5 and -6 regions which alter the reading frame. The predicted amino acid sequence of the human pseudogene is about 35% identical with that of the rat serotonin-4 receptor.

A human serotonin-7 receptor pseudogene.

Although the serotonin-7 receptor was cloned several years ago, its localization in brain tissues remains confusing because of the existence of a related expressed pseudogene, the sequence of which has not hitherto been reported. During the course of searching for related receptor genes, we also searched for this pseudogene to determine its sequence. Human genomic DNA was screened for dopamine and serotonin receptor-like genes, using the polymerase chain reaction method and degenerate oligonucleotide primers based on the similar sequences in the transmembrane-6 and -7 regions of the serotonin-5A, the serotonin-7, and the dopamine D2, D3 and D4 receptors. This resulted in one of the clones having a 115 bp fragment, of which 89% of the bases were identical to the transmembrane-6 and -7 regions of the serotonin-7 receptor sequence. The fragment was radiolabelled and used to screen a human fetal brain cDNA library. A novel cDNA clone of 1326 bp was isolated. Based on the nucleotide sequence, 88% of the bases in this sequence of the pseudogene are identical to the human serotonin-7 receptor coding sequence. However, compared to the serotonin-7 receptor DNA sequence, the pseudogene sequence has nucleotide deletions and insertions, resulting in frame-shifts and stop codons. It was concluded that this sequence represented a pseudogene related to the serotonin-7 receptor gene.

Dopamine D2 receptor promoter polymorphism: no association with schizophrenia.

A functional polymorphism in the promoter region of the dopamine D2 receptor gene, the -141C Del allele, which may be associated with schizophrenia susceptibility, has previously been described in a Japanese sample. The present study was done in order to examine whether such an association would also be found in a North American schizophrenia patient population. However, analysis of the -141C Del allele frequency in the present group of schizophrenia patients (n = 50) and control subjects (n = 51) did not identify any significant differences. These data support the recent reports on German and British subjects that this genetic variation in the 5'-flanking region of the dopamine D2 receptor gene does not play a major role in the genetic predisposition to schizophrenia.

Curcumin enhances the immunosuppressive activity of cyclosporine in rat cardiac allografts and in mixed lymphocyte reactions.

Curcumin (CCM; diferuoylmethane) is a dietary pigment in curry with known antineoplastic and anti-inflammatory effects. The immunosuppressive effects of CCM were studied in (1) rat heterotopic cardiac transplant models, using Brown-Norway (BN, RT1(n)) hearts to WKY (RT1(u)) hosts or Buffalo (BUF, RT1(b)) hearts to Wistar-Furth (WF, RT1(u)) hosts, (2) reverse transcriptase-polymerase chain reaction analysis of cytokines from transplanted specimens, and (3) mixed lymphocyte reactions (MLR). In the BN-to-WKY model, CCM alone significantly increased the mean survival time (MST) to 20.5 to 24.5 days, as compared to 9.1 days among nontreated controls. The combination of CCM and subtherapeutic doses of CsA produced further prolongation of the MST to 28.5 to 35.6 days, better than that of CCM or CsA alone (P <.05). In a BUF-to-WF model, CCM alone did not increased the MST, unless it was combined with subtherapeutic doses of CsA, wherein two thirds of the grafts survived for more than 60 days (P <.05 as compared to either treatment group). Cytokine analysis revealed significantly reduced expression of interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and granzyme B in the day 3 specimens of the CCM and CCM CsA-treated allografts compared with the nontreated allograft controls. MLRs using the two MHC-incompatible rat strains (BNxWKY) showed an effect of increasing concentrations of CCM and/or CsA, which by combination index (CI) analysis showed a synergistic effect (CI = 0.22 to 0.81). This study for the first time demonstrates the effectiveness of CCM as a novel adjuvant immunosuppressant with cyclosporine both in vivo and in vitro.

The human serotonin-7 receptor pseudogene: variation and chromosome location.

We report a variation of the pseudogene for the serotonin-7 receptor in human DNA. Human genomic DNA was amplified, using the polymerase chain reaction method and degenerate oligonucleotide primers for serotonin receptor-like genes. A novel gene DNA sequence of 1325 bp was found. Based on nucleotides, this gene is 88% identical to the serotonin-7 receptor coding sequence. Compared with the previously known serotonin-7 receptor pseudogene, this pseudogene has 1 nucleotide deletion and 4 nucleotide mutations. The gene is located on human chromosome 12 at 12p12.3-p13.2.