Kan-ichi Nakagawara

Structure, chromosomal localization and expression of mouse genes encoding type III Reg, RegIIIα, RegIIIβ, RegIIIγ

Abstract Reg (regenerating gene), first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet β-cells. Recently, it has been revealed that Reg and Reg-related genes constitute a multigene family, Reg family, which consists of three subtypes (type I, II, III) based on the primary structures of the encoded proteins of the genes. In mouse, type I and type II Reg genes (i.e. RegI and RegII gene) have so far been isolated. In the present study, the complete nucleotide (nt) sequences of the cDNAs and genes encoding murine type III Reg (regenerating gene product), RegIIIα, RegIIIβ and RegIIIγ were determined. RegIIIα, RegIIIβ and RegIIIγ encode 175-, 175- and 174-amino acid (aa) proteins, respectively, with 60–70% homology. All three genes are composed of six exons and five introns spanning approx. 3 kb, and exhibit distinctive structural features unique for members of the Reg gene family. All the mouse Reg genes, RegIIIα, RegIIIβ, RegIIIγ, RegI and RegII, are assigned to the adjacent site of chromosome 6C by fluorescence in situ hybridization (FISH). RegIIIα, RegIIIβ and RegIIIγ were expressed weakly in pancreas, strongly in intestinal tract, but not in hyperplastic islets, whereas both RegI and RegII were expressed in hyperplastic islets. These results suggest that genes of the mouse Reg family are derived from a common ancestor gene by several gene duplications, and have obtained divergency in expression and function in the process of genetic evolution.

Isolation, structural determination and expression of a novel reg gene, human reg Iβ

We have isolated a novel human gene and cDNA encoding a member of the regI proteins, regI beta. The gene encodes a 166-amino acid protein which has 22 amino acid substitutions in comparison with the previously isolated human reg protein, regI alpha. RegI beta was expressed only in pancreas, whereas regI alpha was expressed in kidney and stomach as well as in pancreas.

Human REG family genes are tandemly ordered in a 95-kilobase region of chromosome 2p12

Reg, first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet β‐cells. Recently, it has been revealed that Reg and Reg‐related genes constitute a multigene family, the Reg family. In human, the four REG family genes, i.e., REG Iα, REG Iβ, REG‐related sequence (RS) and HIPIPAP, have so far been isolated. In this study, we analyzed YAC clones containing the four genes and performed two‐color FISH to determine the map order of the genes. The human REG family genes are tandemly ordered in the 95‐kbp DNA region of chromosome 2p12 as follows: 2cen‐HIPIPAP‐RS‐REG Iα‐REG Iβ‐ptel.

Assignment of CD38, the gene encoding human leukocyte antigen CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase), to chromosome 4p15

CD38 has been used as a phenotype marker of lymphocyte differentiation. Recently, we have demonstrated that cyclic ADP-ribose can be synthesized and hydrolyzed by CD38 and acts as a second messenger in insulin secretion from pancreatic beta-cells. We have mapped the CD38 gene to human chromosome 4p15 by fluorescence in situ hybridization.

RhoN, a novel small GTP-binding protein expressed predominantly in neurons and hepatic stellate cells

A cDNA encoding a novel member of the small molecular weight GTP-binding protein (small G-protein) superfamily was cloned from rat spinal cord. The deduced amino acid sequence was highly homologous with those of so-far-known Rho proteins. Rho proteins were reported to alter many important cellular functions including formation of both actin stress fibers and focal adhesions. RNA blot hybridization and in situ hybridization analyses indicated that the novel small G-protein is expressed specifically in neurons in the brain and spinal cord and also in hepatic stellate cells. Based on the sequence similarity and neuron-specific expression in the brain, this protein was named RhoN. Unlike classical Rho proteins, RhoN was not susceptible to the ADP-ribosylation reaction by C3 botulinum toxin. Accordingly, RhoN seemed to be specifically involved in neuronal and hepatic functions as a C3 toxin-insensitive member of the Rho subfamily. Then, a mouse genomic DNA segment containing the RhoN gene was cloned. The locus was mapped on the mouse chromosome 11C-D. The sequence data showed that the protein-coding sequence for RhoN is divided by 4 introns, and that the defined 5 exons may encode intramolecular domains serving for different functions.

Telomerase activity in human chorionic villi and placenta determined by TRAP and in situ TRAP assay

Telomerase activity (TA) was analysed in human chorionic villi and placenta in normal and abnormal pregnancy using the telomeric repeat amplification protocol (TRAP) and in situ TRAP assay. Twenty chorionic villi specimens and 25 placenta specimens from normal pregnancies were examined as well as placenta specimens from 10 cases of intrauterine growth retardation (IUGR; nine asymmetric and one symmetric). TA was detected in 18 of the 20 (90 per cent) chorionic villi specimens and in 18 of the 25 (72 per cent) placenta specimens from normal pregnancy. However, no or only weak TA was exhibited in the placenta specimens of the nine asymmetric IUGR cases. In situ TRAP assay detected TA in trophoblastic cells from normal pregnancy, but not in trophoblastic cells from cases of asymmetric IUGR.

Structure and expression of mitsugumin29 gene1

Recently mitsugumin29 unique to the triad junction in skeletal muscle was identified as a novel member of the synaptophysin family; the members of this family have four transmembrane segments and are distributed on intracellular vesicles. In this study, we isolated and analyzed mouse mitsugumin29 cDNA and genomic DNA containing the gene. The mitsugumin29 gene mapped to the mouse chromosome 3 F3‐H2 is closely related to the synaptophysin gene in exon‐intron organization, which indicates their intimate relationship in molecular evolution. RNA blot hybridization and immunoblot analysis revealed that mitsugumin29 is expressed abundantly in skeletal muscle and at lower levels in the kidney. Immunofluorescence microscopy demonstrated that mitsugumin29 exists specifically in cytoplasmic regions of the proximal and distal tubule cells in the kidney. The results obtained may suggest that mitsugumin29 is involved in the formation of specialized endoplasmic reticulum systems in skeletal muscle and renal tubule cells.

Pancreatic β cell proliferation by intermittent hypoxia via up-regulation of Reg family genes and HGF gene

AIMS Although accumulating evidence suggests the associations between sleep apnea syndrome (SAS) and type 2 diabetes, the direct effect of intermittent hypoxia (IH) on pancreatic β cell proliferation remains a missing piece of the puzzle. MAIN METHODS Rat RINm5F β cells, hamster HIT-T15 β cells, and human 1.1B4 β cells were exposed to normoxia (21% O2, 5% CO2, and balance N2), to sustained hypoxia (SH: 1% O2, 5% CO2, and balance N2), or to intermittent hypoxia (IH: 64 cycles of 5 min SH and 10 min normoxia) for 24 h. After the treatment, cellular proliferation and apoptosis were measured by WST-8 assay and TUNEL method, respectively. The expression of regenerating gene (Reg) family, interleukin (IL)-6, and hepatocyte growth factor (HGF) was determined by real-time RT-PCR. KEY FINDINGS The cellular proliferation of HIT-T15, RINm5F and 1.1B4 cells by IH was significantly increased, whereas apoptosis of these cells was unchanged. Real-time RT-PCR revealed that the mRNA levels of Reg family genes, IL-6, a typical Reg family gene inducer, and HGF, an inhibitor of high-concentration of Reg protein-induced apoptosis, were increased in IH-treated cells. In addition, siRNAs against rat Reg family genes except for PAP I/Reg 2 attenuated IH-induced β cell proliferation. SIGNIFICANCE IH stress stimulates pancreatic β cell to induce IL-6 gene expression. By the IL-6 stimulation, β cells over-express Reg family genes as well as HGF gene. Reg family proteins stimulate β cell proliferation and HGF inhibits apoptosis of β cells. As a result, β cell numbers are increased by IH.

Involvement of autoimmunity to REG, a regeneration factor, in patients with primary Sjögren's syndrome

The regenerating gene (Reg) was isolated originally as a gene specifically over‐expressed in regenerating pancreatic islets and constitute a growth factor family. Reg gene product (Reg) is important in the pathophysiology of various human inflammatory diseases. Recently, the possible involvement of human REG in the regeneration of salivary ductal epithelial cells of patients with primary Sjögrens syndrome (SS) was reported. However, the expression of the REG family genes in minor salivary glands (MSG) and the occurrence of anti‐REG Iα autoantibodies in SS patients were obscured. In this study, we examined the expression of REG family genes in the MSG of SS and screened anti‐REG Iα autoantibodies in SS. The mRNA levels of REG family genes in MSG were quantified using real‐time reverse transcription–polymerase chain reaction (RT–PCR) and REG Iα expression in the MSG was analysed by immunohistochemistry. The mRNA level of REG Iα in the MSG of SS patients was significantly higher than that of control. REG Iα protein was expressed highly in SS ductal epithelial cells. Anti‐REG Iα autoantibodies in the sera were found in 11% of SS. All the MSG in the anti‐REG Iα autoantibody‐positive group showed REG Iα expression, whereas only 40% showed REG Iα expression in the anti‐REG Iα autoantibody‐negative group. The anti‐REG Iα autoantibody‐positive group showed significantly lower saliva secretion and a higher ratio of grade 4 (by Rubin–Holt) in sialography. These data suggest strongly that autoimmunity to REG Iα might play a role in the degeneration of MSG ductal epithelial cells in primary SS.

Up-regulation of cyr61 in vascular smooth muscle cells of spontaneously hypertensive rats

In the present study, we applied a fluorescent differential display method to mRNAs from aortae of spontaneously hypertensive rats (SHRs), stroke-prone spontaneously hypertensive rats (SPSHRs), and their parental strain, Wistar Kyoto rats (WKYRs), to identify the genes involved in the development of hypertension. Through this screen we came across a gene that is consistently up-regulated in hypertensive rats. Nucleotide sequence determination of the corresponding cDNA revealed that the gene is the rat orthologue of cyr61. Northern blot analysis showed that cyr61 expression increases in SHR and SPSHR before the onset of hypertension and is sustained thereafter at higher levels than in age-matched WKYRs. In situ hybridization analysis demonstrated that cyr61 is expressed strongly in smooth muscle cells (SMCs) in media of SHR and SPSHR but not WKYR aorta. Fluorescent in situ hybridization mapped the cyr61 gene to rat chromosome 1p12–13, which is located in close proximity to a recently defined quantitative trait locus including NHE3 Na+/H+ exchanger. Overexpression of the cyr61 gene in stably transfected rat SMC line A7r5 caused rather inhibitory effects on the proliferation and DNA and protein synthesis. Our results thus demonstrate for the first time that cyr61 can also act as a growth inhibitor in SMC of genetically hypertensive rats. This may reveal a new route for investigation of the pathogenesis of hypertension.