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 Sjögren's 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.

Microsatellite instability and alternative genetic pathway in intrahepatic cholangiocarcinoma.

BACKGROUND/AIMS: Intrahepatic cholangiocarcinoma (ICC) arises from intrahepatic bile duct epithelium and is the second most prevalent among primary liver cancers. The aim of this study was to clarify the mechanism of cholangiocarcinogenesis. METHODS: We studied the incidence of microsatellite instability (MSI) involving eight highly polymorphic microsatellite markers and alternations of the K-ras, p53 and mdm-2 genes in human ICC tissues. Overexpression of mdm-2 oncoprotein was also immunohistochemically studied. RESULTS: Of all 65 cases examined, K-ras gene mutation was found in three cases (4.6%) at codon 12. Analysis of p53 alterations was performed in 28 cases including 22 frozen samples and mutations were found in three cases (10.7%). Overexpression of mdm-2 protein was observed in 25 (41.7%) out of 60 cases analyzed. In 22 frozen samples, seven (31.8%) cases showed mdm-2 amplification and four (18.2%) cases revealed MSI-positive phenotype. Among the cases analyzed, all the tumors with mdm-2 amplification/overexpression harbored the wild-type p53 gene and all the microsatellite instability-positive cases were from mass-forming (MF) + periductal-infiltrating (PI) subtype. CONCLUSIONS: These results suggest that mdm-2 plays a role, which might be partially through inhibiting p53 activity, in cholangiocarcinogenesis and that M

Characterization of human junctophilin subtype genes.

Junctophilin (JP) subtypes, namely JP-1, 2, and 3, have been currently identified in excitable cells and constitute a novel family of junctional membrane complex proteins. Our studies have suggested that JPs take part in the formation of junctional membrane complexes by spanning the membrane of the intracellular Ca(2+) store and interacting with the cell-surface membrane. In this report we describe the primary structures, genomic organization, and tissue distribution of human JP subtypes. By cloning and analyzing human genomic DNA segments, the protein-coding sequence interrupted with four introns was defined in each JP gene. The deduced human JP subtypes shared characteristic structural features with their rabbit and mouse counterparts. Genomic mapping demonstrated that JP genes do not cluster on the human genome. RNA blot hybridization indicated that tissue-specific expression patterns of JP genes in human are essentially the same as those in mouse; skeletal muscle contained both JP-1 and JP-2 mRNAs, the heart predominantly expressed JP-2 mRNA, and the brain specifically contained JP-3 mRNA. In the light of this, we propose intramolecular domains of JP subtypes based on the structural and functional characteristics.

Telomerase activity Simplification of assay and detection in bladder tumor and urinary exfoliated cells.

Detection of telomerase activity can differentiate malignant from benign cells. However, the original telomeric repeat amplification protocol (TRAP) methods had a number of limitations including a radioisotope labeling [α(32)P] dCTP [α(32)P] dGTP system. We developed digoxigenin labeled CX primer to detect telomerase activity without using radioisotope and attempted to detect telomerase activity of bladder tumor and exfoliated cells in bladder cancer patients. Telomerase activity was detected in 5 (71%) of 7 patients diagnosed with grade 1, 31 (97%) of 32 grade 2, and 11 (100%) of 11 grade 3 bladder tumors. In urinary exfoliated cells, 32 (82%) of 39 grades 1 or 2 bladder tumors were positive for telomerase activity but 20 (51%) of 39 were positive for urinary cytology (P < 0.01). Ten (91%) of 11 of grade 3 tumors were positive for telomerase activity and 11 (100%) of 11 were positive urinary cytology. Three of 100 noncancerous patients were positive for telomerase activity. Sensitivity, specificity, and positive predictive value of telomerase activity assay in urinary exfoliated cells were 84%, 97%, and 93%, respectively. Telomerase activity may be a useful diagnostic marker to detect the existence of immortal cancer cells in the urine.

The study of PSA gene expression on urogenital cell lines.

PURPOSE: Reverse transcriptase-polymerase chain reaction (RT-PCR) offers a potentially more sensitive assay for detecting cells expressing prostate-specific antigen (PSA) mRNA in peripheral circulation. But the sensitivity and specificity are variable depending on the position of the PSA amplification. To increase sensitivity and specificity, the whole PSA cDNA (1466 bp) was separated into eight different parts. METHODS: We examined RT-PCR on 12 urogenital cell lines, including three prostate cancer (LNCaP, PC3, DU145), five human renal cell carcinoma (SMKT-R3, TOS-1, TOS-2, R4, ACHN), two urinary bladder cancer (YTS-1, KK-47) and two testicular cancer (NEC8, NEC14) cell lines. The sizes of the eight fragmented PSA used in the experiment were PSA-1 (1-257bp), PSA-2 (1-322bp), PSA-3 ( 172-507bp), PSA-4 (172-851 bp), PSA-5 (595-1347 bp), PSA-6 (682 967 bp), PSA-7 (682-1347 bp) and PSA-8 (863-1466 bp). RESULTS: All cell lines had positive signals from PSA-6, PSA-7 and PSA-8. The positive signals from PSA-1, PSA-2 and PSA-3 were detected in some other cell lines in addition to the three prostate cancer cell lines. Only LNCaP which produces the PSA protein had a positive signal from PSA-5. PC3 and DU145 (which do not produce PSA) and LNCaP had a positive signal from PSA-4. Therefore, the inner primer PSA-4' (578-782 bp) used to increase sensitivity and specificity. Nested RT-PCR on the 12 cell lines, using the PSA-4 and 4' primers, detected more clear bands in the three prostate cancer cells. CONCLUSION: Nested RT-PCR using PSA-4 (outer primer) and PSA-4' (inner primer) may be useful for detecting prostate cancer cells in the peripheral blood.

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 and proliferative activity in placenta from women with and without fetal growth restriction.

OBJECTIVE: To analyze telomerase and proliferative activity in placenta from women with and without fetal growth restriction (FGR). METHODS: Telomerase activity was analyzed in 30 first-trimester chorionic villi specimens (group A) and in 28 second- and third-trimester placenta specimens (group B) from women without FGR. Telomerase activity also was analyzed in 11 placenta specimens from women with asymmetric FGR (group C). The proliferative activity of these 69 specimens was assessed by immunohistochemical staining, using the MIB-1 monoclonal antibody. RESULTS: Telomerase activity was detected in 28 (93.3%) of 30 chorionic villi specimens and in 18 (64.3%) of 28 placenta specimens without FGR. In contrast, no telomerase activity was exhibited in the placenta specimens from any of the 11 women with asymmetric FGR by telomeric repeat amplification protocol assay. Telomerase activity also was detected by in situ telomeric repeat amplification protocol assay in trophoblastic cells from women without FGR but not in trophoblastic cells from women with asymmetric FGR. Thus, telomerase activity was detected significantly more often in groups A and B than in group C (P < .01). The rate of proliferative activity, evident as positive MIB-1 staining in trophoblastic cells, in groups A and B (28.1+/-1.7% and 7.0 +/-2.9%, respectively) was significantly higher than that in group C (1.9+/-0.6%; P < .01). CONCLUSION: Telomerase and proliferative activity were minimal in placenta from women with asymmetrical FGR, suggesting placental senescence with asymmetrical FGR.

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 gene.

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.

Structural organization and chromosomal localization of the mouse tesk1 (testis-specific protein kinase 1) gene.

TESK1 (testis-specific protein kinase 1) is a protein serine-threonine kinase, containing characteristic structural features composed of an N-terminal kinase domain and a C-terminal proline-rich domain. Tesk1 mRNA is predominantly expressed in testicular germ cells, and developmental changes of expression in mouse testis suggest a role for this kinase in spermatogenesis. In the present study, we isolated and determined the overall sequence of the mouse Tesk1 gene, which spans 6.1 kilobases (kb) and contains 10 exons and 9 introns. The protein kinase domain is located in exons 1-9, while the proline-rich domain is in exons 9 and 10. The deduced 627 amino acid sequence of mouse TESK1 shows 97% and 94% identity with the rat and human TESK1, respectively. Sequence of the 5'-flanking and -untranslated region is devoid of a TATA box, but does contain several potential binding sites for transcription factors, including Sp1, AP-1, c-Myc, SRY and CREM (cyclic AMP-responsive element modulator). As CREM is implicated in the activation of several male germ cell-specific genes, it is suggested that the expression of the Tesk1 gene is under the control of CREM transcription activity. The Tesk1 gene was mapped to mouse chromosome 4A5-C1 by fluorescence in situ hybridization.

Structure, chromosomal localization and expression of mouse genes encoding type III Reg, RegIII alpha, RegIII beta, RegIII gamma.

Reg (regenerating gene), first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet beta-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), RegIII alpha, RegIII beta and RegIII gamma were determined. RegIII alpha, RegIII beta and RegIII gamma 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, RegIII alpha, RegIII beta, RegIII gamma, RegI and RegII, are assigned to the adjacent site of chromosome 6C by fluorescence in situ hybridization (FISH). RegIII alpha, RegIII beta and RegIII gamma 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.

Functional analysis and chromosomal gene assignment of rat kidney prostaglandin EP3 receptor.

We have reported two isoformes of rat prostaglandin EP3 receptor with their different carboxyl-terminal tails (rEP3A and rEP3B receptors), which are derived by alternative RNA splicing, and both receptors have been shown to be localized to renal distal tubules. In the present study, we characterized the signal transduction system of rat kidney EP3 receptors either in a renal cell line mimicking renal distal tubule cells, TKC2, or in COS-7 cells by functional expression of these receptors. We also examined the chromosomal localization of the EP3 receptor gene by fluorescence in situ hybridization (FISH). In TKC2 cells, vasopressin (AVP, 10(-7) M), prostaglandin (PG) E2 (10(-7) M), or forskolin (10(-8) M) markedly stimulated cyclic AMP formation. Overexpression of the rEP3A receptor significantly attenuated the AVP-, PGE2- or forskolin-induced cyclic AMP formation, whereas there was no change with rEP3B receptor expression. On the other hand, in COS-7 cells transfected with rEP3A receptor cDNA, PGE2 (10(-7) M) did not affect cytosolic free calcium concentration ([Ca2+]i), whereas transfection of rEP3B receptor cDNA evoked PGE2-induced increases in [Ca2+]i. Moreover, we have revealed that the rEP3 receptor gene is localized to rat chromosome 2q44-45. In conclusion, rEP3A or rEP3B receptor is suggested as a mediator of the natriuretic/diuretic action of PGE2 in renal distal tubules via a decrease in cyclic AMP formation or an increase in [Ca2+]i, respectively. Information of the gene assignment of rat EP3 receptor to rat chromosome 2q44-45 is useful for further analysis of the role of EP3 receptor in genetically hypertensive rat models.

Localization of the mouse protein serine/threonine phosphatase 2C beta gene to chromosome 17E 4-5.

Protein phosphatase 2C (PP2C) is one of four major classes of protein serine/threonine phosphatase and is considered to have a role in signal transduction of stress responses. It has two isotypes, alpha and beta, encoded by different genes. In this study, the mouse PP2C beta gene was mapped by in situ hybridization to chromosome 17E 4-5.

Assignment of the gene for rat thromboxane receptor (Tbxa2r) to chromosome 7q11 by fluorescence in situ hybridization.

Thromboxane plays physiological and pathophysiological roles in many tissues. Recently, we cloned a cDNA for rat kidney thromboxane receptor (Tbxa2r) and showed that Tbxa2r is expressed in the renal glomerulus, vasculature, and transitional cell epithelium of renal pelvis. Here, we map the gene for this receptor (Tbxa2r) to rat chromosome 7q11 by fluorescence in situ hybridization.

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 beta-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 1 alpha, REG 1 beta, REG-related sequence (RS) and HIP/PAP, 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-HIP/PAP-RS-REG I alpha-REG I beta-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.

Structure and chromosomal mapping of genes for the mouse kappa-opioid receptor and an opioid receptor homologue (MOR-C).

Recent cDNA cloning studies have defined four members of the opioid receptor family, i.e., delta-, mu- and kappa-subtypes, and an opioid receptor homologue for unknown ligands. In this report, we isolated and analyzed mouse genomic DNA segments containing the kappa-opioid receptor gene and a gene for the opioid receptor homologue (designated as MOR-C). The genes are closely related each other in exon-intron organization, suggesting their evolutional relationship. Using in situ hybridization, we show that the kappa-opioid receptor gene and the MOR-C gene map to mouse chromosome 1A2-3 and 2H2-4, respectively.

Isolation, structural determination and expression of a novel reg gene, human regI beta.

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.

Structure, chromosomal localization, and expression of mouse reg genes, reg I and reg II. A novel type of reg gene, reg II, exists in the mouse genome.

We previously identified a gene, reg (i.e. regenerating gene), in the screening of a regenerating islet-derived cDNA library of rat (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114), and isolated a human reg cDNA and gene (Watanabe, T., Yonekura, H., Terazono, K., Yamamoto, H., and Okamoto, H. (1990) J. Biol. Chem. 265, 7432-7439); the rat and human cDNAs encode 165- and 166-amino acid proteins, respectively. Until now, it was thought that there is a single locus for Reg protein in the mammalian genome. In this study, we isolated two distinct cDNAs and genes, one of which was a mouse homologue to rat and human reg gene, the other a novel type of reg gene. We designated them reg I and reg II, respectively. The two proteins encoded by these genes share 76% amino acid sequence identity with each other. Both genes span about 3 kilobase pairs, and the genomic organization of six exons and five introns is conserved between them. Chromosomal mapping studies indicate that the reg I gene is localized on mouse chromosome 12, whereas the reg II gene is localized on chromosome 3. By Northern blot analysis, both reg I and reg II mRNAs are detected in the normal pancreas and hyperplastic islets of aurothioglucose-treated mice, but not in the normal islets. It is remarkable that in the gallbladder reg I is expressed, but reg II is not.

Effects of antiepileptic drugs and organic brain damage on SCE frequency in cerebral palsies.

In order to assess the genotoxic effects of antiepileptic drugs (AEDs), analysis of sister chromatid exchanges (SCEs) was performed in lymphocytes of three groups of males; epileptics with AED therapy, non-epileptics without AED therapy and healthy controls. The epileptics and non-epileptics were cases of severe cerebral palsies due to perinatal asphyxia. Possible confounding factors of SCE frequencies, such as age, smoking habit, drug usage other than AED, recent history of viral infection, etc. were controlled. The frequency of SCE per cell was 4.63 +/- 0.71 (mean +/- S.D.) in epileptics, 4.70 +/- 0.89 in non-epileptics and 3.84 +/- 0.56 in healthy controls. SCEs in both epileptics and non-epileptics were significantly higher (p less than 0.05) than those in controls. There was no significant difference of SCE frequency between epileptics and non-epileptics. These results suggested that no mutagenic effect of AED could be demonstrated as revealed by SCEs, and organic brain damage per se might influence the baseline SCE frequency. The possible explanations for such observations are discussed.