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The importance of tumor necrosis factor (TNF)-alpha and the TNF receptor gene polymorphisms in the etipathogenesis of inflammatory bowel disease (IBD) has not been elucidated. DNA from peripheral blood samples was obtained from 124 patients with Crohn's disease (CD), 106 patients with ulcerative colitis (UC), and 111 unrelated healthy controls. We examined two single nucleotide polymorphisms (SNPs) of the TNF-alpha gene, TNF (–308 G/A and –238 G/A), an SNP of the TNF receptor superfamily member 1A gene, TNFRSF1A(also known as TNFR1), at codon 12 in exon 1 (CCA/CCG), and two SNPs of the 1B gene, TNFRSF1B (also known as TNFR2), (1466 A/G and 1493 C/T). There was a difference in the carrier frequency for haplotype AG (–308 A, –238 G) between UC patients and the controls (OR=4.76, 95% CI=1.53–14.74, P

The complete nucleotide sequence of the mitochondrial genome of the Oriental white stork, Ciconia boyciana, has been determined from captive storks by a novel method incorporating Long PCR and shotgun sequencing. 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes were identified as in other vertebrate mitochondrial genomes. The position and direction of the NADH6 and tRNA-Glu genes were the same as previously reported for avian mitochondrial genomes. A 71 bp direct repeat and long CAAA repeat sequences were found at the 3' end of the D-loop region, together with SCB-1, SCB-2, SCB-3, and three TAS sequences. Direct sequencing of the PCR fragments in the D-loop region in 26 captive Oriental white storks originating from Japan, China, and Russia revealed nucleotide differences at 18 sites along 1,248 bp, and a total of nine haplotypes have been identified. It was found that one pair of individuals in the Japanese captive breeding program were of the same haplotype, suggesting that they were caught from the same nest. The pair has since been dissolved in consideration of the possibility of inbreeding depression.

The radioresistant N10 and parental KB cell lines were examined for the expression of human DNA repair genes which were related to the repair of radiation-induced DNA damage by northern blot analysis using five kinds of DNA probes (XRCC1, XRCC3, XRCC5, RAD51, RAD52). In the unirradiated condition, N10 cells showed higher expression of XRCC1, XRCC3 and RAD51 mRNA than did KB cells. The X-irradiation induced a time-dependent increase in the mRNA levels of XRCC3 and RAD51 in both cell lines with a maximum at 2 h postirradiation. The XRCC1 mRNA in N10 was maintained at the same level even after irradiation, whereas that in KB was decreased after irradiation. There was no difference in the expression of XRCC5 and RAD52 mRNA between N10 and KB cells in both unirradiated and irradiated conditions. From these findings, it was suggested that XRCC1, XRCC3 and RAD51 contribute to the radioresistance in cell line N10.

Treatment of HuH-7 human hepatocellular carcinoma (HCC) cells with 1-10 mM sodium butyrate (SB) resulted in growth inhibition in a dose-dependent manner. At 3 mM and higher concentrations, SB caused nuclear fragmentation and DNA ladder formation characteristic of apoptosis. In the treated cells, the expression of p21 (WAFI/CIPI) increased and that of alpha-fetoprotein (AFP) decreased. These characteristic changes were also observed with 5 other human HCC cell lines with or without mutation of the p53 gene. The ability of these cells to form colonies in soft agar was suppressed by either pretreating the cells with SB prior to soft agar plating or incubating untreated cells in SB-containing soft agar. Direct injection of SB into tumors developed from HuH-7 cells in nude mice resulted in an increase in the p21 level, a decrease in the tumor size and an increase in the survival time of mice. When the inoculation of HuH-7 cells into nude mice was immediately followed by subcutaneous injection of SB, development of tumors was either significantly delayed or completely suppressed. These results suggest that SB induces cellular differentiation and suppresses growth and tumorigenicity of HCC cells in vitro and in viva by a mechanism independent of p53 but possibly dependent on p21.

A gene (designated ldcC) mapped at 4.6 min on the Escherichia coli chromosome codes for a protein of 713 amino acids (aa) that shows strong similarities in both size and amino-acid sequence (69% identical residues and 85% conserved residues) to lysine decarboxylase (LDC) from E. coli (CadA, acid-inducible LDC, 715 aa) or from Hafnia alvei (739 aa). A pUC18 derivative carrying the ldcC gene conferred high LDC activities on an E. coli strain devoid of the functional cadA gene, even when the bacteria were grown under non-inducing conditions at physiological pH. Thus, the gene encodes another lysine decarboxylase, probably a constitutively expressed enzyme, the presence of which was suggested from the previous observations that low LDC activities were detectable in cadA- mutant and non-induced wild-type cells.

The Fas antigen (Fas/APO-l/CD95) has been shown to induce apoptosis when bound to a monoclonal anti-Fas antibody or Fas ligands. Recently, a new soluble human Fas isoform which lacks the transmembrane domain due to alternative splicing has been isolated; however, no mouse Fas isoforms have been reported so far. Analysis of Fas transcripts by RT-PCR detected no Fas transcripts corresponding to the human soluble Fas isoform in mouse thymus, spleen, and liver. However, we detected a new isoform with a 117-bp deletion in the second exon in various mouse tissues and cell lines. This isoform, termed truncated Fas (T-Fas), can be generated by alternative splicing and lacks the N-terminal portion of the extracellular domain just after the signal sequence. Since the deletion involves the first cysteine-rich motif believed to be necessary for binding to the Fas ligand, the T-Fas protein may lack the ability to induce apoptosis. The expression of T-Fas relative to that of the normal Fas varies considerably among mouse tissues and cell lines, suggesting preferential transcription of the T-Fas isoform in certain cell types.

Leber's hereditary optic neuropathy (LHON) is a disorder considered to be due to a mutation in the mitochondrial DNA (mtDNA). The authors analyzed the mtDNA genotype of peripheral blood leukocytes from two family members with LHON, and further analyzed mtDNA in various tissues originating from the separate germ layers (skin, hair, leukocyte, muscle, arachnoid, CSF cells, and buccal mucosa) in two LHON patients. A G-to-A transition of mtDNA np 11778 (Wallace mutation) was detected by the elimination of a SfaN I site and the acquisition of a Maelll site in all individuals and tissues examined.

✓ Calphostin C acts at the regulatory domain as a highly selective inhibitor of protein kinase C (PKC), and staurosporine acts at the catalytic domain as a nonspecific PKC inhibitor. The authors investigated the capacity of calphostin C and staurosporine to promote apoptotic fragmentation of DNA in four human glioma cell lines. The exposure of glioma cell lines to 100 nM calphostin C for 2 to 8 hours induced a decrease in particulate PKC activities and exposure for 16 to 24 hours produced a concentration-dependent increase in internucleosomal DNA cleavage on agarose gel electrophoresis. In addition, the human glioma cells showed the classic morphological features of apoptosis: cell shrinkage, nuclear condensation, and the formation of apoptotic bodies. A 24-hour exposure to staurosporine failed to induce internucleosomal DNA fragmentation at concentrations generally used to achieve maximum inhibition of enzyme activity (50 nM) but promoted fragmentation at considerably higher concentration (more than 200 nM). Deoxyribonucleic acid fragments obtained from cells exposed to 100 nM calphostin C for 16 to 24 hours possessed predominantly 59-phosphate termini, consistent with the action of a Ca++/Mg++-dependent endonuclease. Northern and Western blot analyses revealed that the exposure to 100 nM calphostin C for 4 hours failed to alterbcl-2 transcript and protein, but exposure for more than 8 hours decreased the amount ofbcl-2 transcript and protein. Together, these observations suggest that calphostin C is capable of inducing apoptotic DNA fragmentation and cell death in a highly concentration dependent manner in human glioma cells and that the apoptosis is closely associated with the decrease in transcription and translation ofbcl-2.

Frequencies of polymorphisms in the rhodopsin gene of Japanese retinitis pigmentosa and normal individuals

There are three isoforms of calcitonin receptors (CTRs). The first type (type 1) has 479 amino acids in rat and 482 amino acids in porcine. The second type (type 2) has a 39 amino acid insertion in the second extracellular domain. The third type (type 3) has a 16 amino acid insertion in the first intracellular domain. The human CTR which has been isolated from a human ovarian carcinoma cell line belongs to type 3. Using RT-PCR with primers whose sequences correspond to the human CTR cDNA, we analyzed the expression of the CTR transcripts in seven human tumor cell lines and surgical specimens. The CTR m-RNA were found in all samples. Transcripts which were 48bp shorter than that of the type 3 human CTR were detected, but not transcripts of type 2 and type 3. Since the structure of this CTR is same as that of the type 1 rat and porcine CTRs, we termed it the human type 1 CTR. PCR studies using human genomic DNA as a template revealed that the 48bp sequence constitutes an exon. These results indicate that the type 1 and the type 3 human CTRs are generated by alternative splicing and a majority of human CTR transcripts is type 1.

In order to test the hypothesis that esophageal achalasia may be due to neurotropic viral damage to the esophageal myenteric plexus, esophageal tissue with or without achalasia was analyzed by polymerase chain reaction for the presence of human herpes virus DNA or measles virus RNA. The DNA and RNA were extracted from the esophageal muscle of 12 patients with achalasia and six patients with upper esophageal carcinoma. Peripheral blood mononuclear cells from eight adult volunteers and two samples of umbilical blood mononuclear cells were also used as controls. PCR amplification with a pair of primers specific for herpes simplex type 1 and 2 viruses identified 92-bp fragments in nearly all specimens, including those without achalasia. Each 92-bp fragment was confirmed to be identical to a single herpes simplex virus sequence by automated DNA sequence analysis. No amplification for five other herpes viruses or measles virus was detected. Therefore, a specific viral etiology for achalasia was not identified in this study.

In order to confirm engraftment of sibling-donor marrow cells or lekuemia relapse from the patient leukemic cells, DNA from nucleated cells in peripheral blood or bone-marrow fluid from patients who had received bone marrow transplantation (BMT) was examined using the MCT118 gene (D1S80) (a variable number of tandem repeats: VNTR) and 6 other genes and 9 restriction enzymes before and after BMT. In 5 of 10 patients (Case 1, 3, 4, 7, and 9) a difference between the DNA from donor cells and recipient cells was confirmed using the MCT118 gene, and engraftment of the donor marrow cells could be demonstrated. In Case 7, not only the engraftment of donor cells but also the leukemia relapse from recipient cells were confirmed using MCT118. Among the remaining 5 cases, Case 5, 8, and 10, the engraftment of donor cells was confirmed using the COLA2A1 gne (12q14.3). In Case 5, and 10, the engraftment could be detected using a gene in the CA2 locus (8g22). In Case 2, the presence of cells from the sibling was identified with the THRB gene (3p24) alone. In Case 6, the presence of cells from the donor could be confirmed using the DMD gene (Xp21.3-21.1) alone. In Case 8, the AK1 gene (9q34.1-34.2) could identified the difference between DNA from donor cells and recipient cells only, but the difference could also be detected using the COLA2A1 gene. In none of the cases, the INS gene (11p15.5) could identify the difference between the DNA from donor and recipient cells. Although only 10 cases were investigated, the findings suggest the use of first the MCT118 gene, then the COLA2A1 gene and the other genes when trying to determine engraftment of donor marrow cells or leukemia relapse after BMT.

Fas antigen mRNA induction in the brain was examined using a transient global cerebral ischemia model in BALB/C mice. Northern blot analysis revealed little Fas antigen mRNA expression in the brains of sham-operated mice. A marked induction of Fas mRNA expression was detected in the brains of mice 6 h after 30 min of cerebral ischemia. These results suggest a possible apoptotic mechanism for cell death, mediated by the Fas antigen, in postischemic brain.

CD34+DR- and CD34+DR+ cells were isolated from the marrow mononuclear cells of five patients with chronic myelogenous leukemia (CML) carrying the Philadelphia (Ph) chromosome. Analysis of bcr/abl hybrid mRNA in individual colonies from a single cell using reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated the presence or absence of the hybrid mRNA. For patient 1 in the chronic phase of CML, the hybrid mRNA was detected in all colonies derived from CD34+DR+ and CD34+DR- hemopoietic progenitors. In contrast, for patient 2 in the chronic phase of CML, the mRNA was detected in all individual colonies from CD34+DR+ progenitors but not in any from CD34+DR- progenitors. For patient 3 in the chronic phase of CML, the mRNA was detected in all individual colonies from CD34+DR+ but in only some of the colonies from CD34+DR- progenitors. For patients 4 and 5 in the acute crisis of CML, the mRNA was found in a portion of colonies from CD34+DR+ and CD34+DR- progenitors. These results indicated that normal clones can persist in CD34+DR- progenitors in some patients with CML, even when chromosome analysis detects the Ph chromosome in all metaphases of bone marrow cells.

The authors conducted a comparative study on the rapid diagnosis of cytomegalovirus (CMV) interstitial pneumonitis by 4 different methods. Among them, the polymerase chain reaction (PCR) method was found to achieve the most rapid diagnosis of CMV infection. The clinical usefulness of making a diagnosis from the serum antibody titer and viral isolation was questionable as compared with the PCR method. Therefore, early diagnosis of CMV infection by PCR may be promising especially for the compromised patients with hematological diseases.

The human cholinesterase (ChE) gene from a patient with acholinesterasemia was cloned and analyzed. By using ChE cDNA as a probe, four independent clones were isolated from a genomic library constructed from the patient's DNA. Sequencing analysis of all of the four clones revealed that exon 2 of the ChE gene was disrupted by a 342-base-pair (bp) insertion of Alu element, including a poly(A) tract of 38 bp, which showed 93% sequence homology with a current type of human Alu consensus sequence. Southern blot analysis showed that the Alu insertion occurred in both alleles of the patient and was inherited in the patient's family. This Alu insertion was flanked by 15-bp of target site duplication in exon 2 corresponding to positions 1062-1076 of ChE cDNA, indicating that an Alu element could have been integrated by retrotransposition. Thus, this case provides an important clue to the mechanism of inactivation of a gene by integration of a retrotransposon.

A fragment encoding proline-activating domain (grs 2-pro) of gramicidin S synthetase 2 (GS 2) was found in an 8.1-kilobase pairs (kb) DNA fragment of Bacillus brevis Nagano, which contained the full length of GS 1 gene (grs 1). The clones designated GS719 and GS708, which expressed gramicidin S synthetase 1, were elucidated to express immunoreactive proteins to GS 2 antibodies with approximate molecular weights of 115,000, 105,000 (GS719), and 110,000 (GS708). The partial purification of the gene products of these clones was carried out using DEAE-Sepharose CL-6B column chromatography. The immunoreactive proteins to GS 2 antibodies were separated from gramicidin S synthetase 1 protein and had specific proline-dependent ATP-32PPi exchange activity. The nucleotide sequence for the proline-activating domain in the 8.1-kb insert was determined. This fragment was 2,879 base pairs long, and encoded 959 amino acids. The calculated molecular weight of 111,671 was consistent with the apparent molecular weight of 115,000 found in SDS-PAGE of the immunoreactive products to GS 2 antibodies. The open reading frame for this protein followed grs 1 gene, though two were separated by a 73-base pair noncoding sequence, and remained open to the end.(ABSTRACT TRUNCATED AT 250 WORDS)

Interspecies difference in expression of the c-myc gene between two human and three rat glioma cell lines was studied with use of a human c-myc probe. The c-myc deoxyribonucleic acid (DNA) fragments detected at higher stringency in Southern blotting, showed a difference in size and gene copy number between human and rat glioma cells. The c-myc transcript was detected at both higher and lower stringencies in Northern blotting in human glioma cells, whereas it was demonstrated only at lower stringency in rat glioma cells, and the c-myc transcript was seen in cytoplasms of both glioma cells by in situ hybridization. The c-myc protein, if examined with anti-human c-myc protein monoclonal antibody, was observed as two separated components in Western blotting and localized immunocytochemically in nuclei in human glioma cells, whereas it was detected as three separate forms in Western blotting and shown in both nuclei and cytoplasm in rat glioma cells. The above discrepancy in manifestation of c-myc DNA fragments, transcript and protein could be due to the difference in nucleotide sequence of c-myc gene between human and rat glioma cells.

One of two main FL-amnion cell alkaline phosphatase (AP), the fast migrating one (FL-AP F ) has been reported to be identical to Kasahara isoenzyme (K.I.), which occurs preferentially in sera of patients with primary hepatoma. We purified FL-AP F of which the apparent molecular weight was 135 000 by gel filtration, and that of the subunit was 62000 on SDS/PAGE, indicating homodimeric structure of FL-AP F . FL-AP F was found to react with monoclonal antibody against adult intestinal AP, but not with monoclonal antibody to placental AP. We isolated FL-AP F cDNA clone from FL-amnion cells, of which cDNA was 2525 base pairs in length. Nucleotide sequence of the coding region and the 3' untranslated region was identical to the sequence of human adult intestinal AP cDNA. But the untranslated region of the 5' end of the isolated clone was slightly longer than that of intestinal AP. Hence, FL-AP F (K.I.) may occur by altered glycosylation of intestinal AP.

✓ Two human glioma cell lines were examined for multidrug resistance (MDR). A vincristine (VCR)-resistant glioma cell line showed a cross resistance to Adriamycin (doxorubicin, ADR) and etoposide (VP-16) to varying extents, suggesting the presence of MDR; the resistance to VCR was considerably decreased by calcium entry blockers. On the other hand, another VCR-sensitive glioma cell line exhibited no cross resistance to ADR or VP-16. Double minute chromosomes and homogeneously staining regions as well as clonal aberrations of chromosome 7 were not observed in cytogenetic studies of multidrug-resistant and multidrug-sensitive glioma cell lines. In Northern and Southern blot analyses, MDR gene 1 (MDR1) messenger ribonucleic acid (mRNA) was shown to be overexpressed without any amplification of the MDR1 gene in multidrug-resistant glioma cell lines as compared to multidrug-sensitive glioma cell lines. It would be reasonable to suggest that amplification of the MDR1 gene may not be a sine qua non for acquisition of MDR and that the MDR1 mRNA level may be well correlated with the extent of MDR.

Adherent cells (AdCs) in blood from normal volunteers produced granulocyte-macrophage (GM) colony-stimulating activity (CSA) in the presence of 10 ng/ml of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) in vitro. GM-CSA produced by adherent cells in the presence of GM-CSF reached a plateau level on day 6. Colonies stimulated by adherent cell-conditioned medium (AdC-CM-GM-CSF), which had been harvested after 6 days of incubation of AdCs with rhGM-CSF, were granulocyte predominant. When phagocyte-depleted marrow mononuclear cells (PD-M-MNCs) were cultured with AdC-CM-GM-CSF and anti-rabbit serum against rhGM-CSF, 99% of the colonies on day 7 were exclusively composed of neutrophils. When 2 X 10(4) PD-M-MNCs were cultured in a medium containing AdC-CM-GM-CSF, AdC-CM-GM-CSF + anti-GM-CSF, AdC-CM-GM-CSF + anti-G-CSF, or AdC-CM-GM-CSF + both of the anti-bodies, the PD-M-MNCs formed (mean +/- SD) 100 +/- 2.0%, 64.3 +/- 2.5%, 38.6 +/- 0.4%, and 6.0 +/- 0.4% GM colonies, respectively. Furthermore, northern blot analysis revealed that AdCs incubated with 10 ng/ml of rhGM-CSF for 6 h expressed much more mRNA of G-CSF than those without the CSF. These data indicated that AdCs in blood produce G-CSF in the presence of GM-CSF.

A conserved DNA region among the subspecies of koala, of mitochondrial cytochrome b gene, was employed to analyze the genetic variation among 3 available subspecies of koala. This conserved sequence, 307 bp DNA, was sequenced using polymerase chain reaction and direct DNA sequencing technique. Substitutions in the nucleotide sequences were observed, with which koalas can be divided into 3 DNA haplotypes subspecies, but the molecular data provided inconsistency with current classification of the 3 subspecies of koala.

Summary Background Atopy is a common syndrome underlying asthma, rhinitis, and eczema, and is characterised by high immunoglobulin E (IgE) responses to common antigens. IgE and mast-cell chymase (MCC–a serine protease secreted by skin mast cells) have a key role in atopic or allergic inflammation of the skin. The gene for MCC is located within a cluster of genes for cellular proteases on chromosome 14q11·2. We aimed to identify variants of MCC and another gene within this complex, and assess whether there is a genetic association between variants of MCC and atopic disorders–particularly eczema. Methods We randomly selected 100 controls and recruited patients-100 in each group–with atopic asthma, non-atopic asthma, atopic rhinitis, and atopic eczema. PCR amplification was used to test genomic DNA for an association between allelic polymorphisms in MCC and a flanking gene (CGL1, for the cathepsin-G-like protein) on chromosome 14q11 and asthma, rhinitis, and eczema. Findings We found a significant association between a BstXI polymorphism in MCC and eczema (odds ratio 2 17 [95% CM 21-3 88], p=0 009), but no association with atopic asthma, rhinitis, or non-atopic asthma. There was no association between an MboII polymorphism in CGL1 and any of the atopic disorders. Interpretation These findings suggest that variants of MCC may be one source of genetic risk for eczema.

It is thought that malignant tumors occur through interactions of multiple environmental factors and a personal genetic factor. A normal somatic cell having an intrinsic function is able to acquire the characteristics of a malignant cell under the influence of many factors. A small percentage of all tumors have obvious familial aggregation. These entities are called familial cancer. The familial cancer syndrome is well defined for colorectal cancer, breast cancer, endocrine neoplasia, and so on. Traits of familial tumors are sequentially inherited by offspring through gametes in a Mendelian fashion, most commonly in an autosomal-dominant manner. Carcinogenesis requires multiple genetic events. A patient with a familial tumor is ahead of an individual without any germline mutation in the carcinogenesis process. In such a situation, patients frequently suffer from multiple malignant tumors at a young age. It is well known that three major genes are closely related to the cell cycle and tumorigenesis. These gene types are protooncogenes, tumor suppressor genes, and DNA mismatch repair genes. Proto-oncogenes function to accelerate cells during the G1 or growth phase of the cell cycle. Tumor suppressor genes act as blocks against cell growth and proliferation. Inactivation of tumor suppressor genes requires alterations in both alleles. These phenomena are known as Knudson's two-hits theory. However, DNA mismatch repair genes are known as caretaker genes and correct mismatch pair generation during DNA replication. Germline mutation of DNA mismatch repair genes causes hereditary nonpolyposis colorectal cancer. The tumor phenotype from patients with hereditary nonpolyposis colorectal cancer is demonstrated to be microsatellite instability positive.

Background. The etiology of inflammatory bowel disease, which includes ulcerative colitis and Crohn’s disease, has not yet been made clear. However, inflammatory bowel disease is recognized as a multifactorial disease, and innate genetic factors might contribute to the pathogenesis. Cytokine genes are thought to be important in inflammatory bowel disease. Recently, interleukin 18, cloned as a novel proinflammatory cytokine, has been implicated in inflammatory bowel disease, especially Crohn’s disease. Methods. To identify germline mutations in patients with inflammatory bowel disease, the entire coding region of IL18 was examined using a DNA sequencing procedure. Results. No functional mutations were found, but a novel single nucleotide polymorphism (SNP) was identified as TCA/ TCC at codon 35. In patients with Crohn’s disease, the frequency of TCC allele carriers was significantly higher than in healthy controls (χ2 = 9.35, P = 0.002229, OR = 2.58, 95% CI = 1.39–4.80). Also, the magnitude of the association was more remarkable in females (χ 2 = 16.36, P = 0.000052, OR = 8.17, 95% CI = 2.73–24.41). The TCC allele at codon 35 of IL18 may increase the risk for Crohn’s disease, especially in females. Conclusions. IL18 is probably one of several genes that determine susceptibility to Crohn’s disease.

PROBLEM: Interleukin-18 (IL-18) strongly induces interferon-gamma production and is produced not only by types of immune cells but also by types of non-immune cells. Ovulation is thought to be an inflammation-like reaction in which many pro-inflammatory cytokines are involved. We investigated whether IL-18 is involved in the functions of ovary. METHOD OF STUDY: The 4-week-old immature female mice were examined for IL-18 and IL-18 receptor (IL-18R) expression on their ovaries under stimulation with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) by immunohistochemical staining, Western blotting and reverse transcript-polymerase chain reaction. The IL-18R was blocked by the injection of anti-IL-18R monoclonal antibody to immature mice during PMSG-hCG stimulation, and the number of ovulated ova was counted. RESULTS: The expression of both proteins and mRNA of IL-18 and IL-18R were very low in immature ovaries before stimulation, but after PMSG injection both IL-18 and IL-18R increased dramatically in theca cells and reached a maximum level at the peri-ovulatory phase then slightly lowered, but still kept a high level during the luteal phase in the corpus luteum. The treatment of IL-18R monoclonal antibody to the mice during ovarian stimulation reduced the number of ovulated ova and inhibited the expansion of cumulus cells surrounding the ovum. CONCLUSION: IL-18 and IL-18R play roles in various kinds of function of ovary.

Squamous cell carcinoma antigen (SCC-Ag) is produced by the two almost identical, tandemly arrayed genes, SCCA1 and SCCA2. In this study, we investigated the mechanism of increased expression of SCC-Ag in a cell line SCCMM derived from an aggressive adenoid SCC with high titer of SCC-Ag in the patient serum. The differential polymerase chain reaction using specific primers for SCCA1 and SCCA2 revealed no gene amplification in SCCMM. However, RT-PCR demonstrated that levels of SCCA1 and SCCA2 mRNAs in SCCMM were 80- and 120-fold higher than those in CaSki as a reference SCC cell, respectively. Western blot analysis showed that the levels of these two SCC-Ag proteins in SCCMM were 15-fold higher than those in CaSki, suggesting that expression of the SCC-Ag in SCCMM was controlled at both the transcriptional and post-transcriptional levels. To investigate highly malignant character of SCCMM, expression of cell cycle regulatory proteins was investigated by Western blotting. Cyclin E and cyclin B1 were expressed at approximately 100-fold higher levels in SCCMM than in CaSki, but Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) were expressed at low levels and p16 and p19 ink4 CDKIs were not detected. These results suggest that the aggressive growth of SCCMM is due, at least in part, to large increases in cyclin E and cyclin B1 expression with low levels of CDKIs.

In order to detect chimerism, fluorescence in situ hybridization (FISH) and cytogenetic analyses were performed on bone marrow cells from 47 patients with hematological malignancies following allogeneic hematopoietic cell transplant (HCT). The dual-color XY, major Bcr-Abl (M-Bcr-Abl), and specific alpha-satellite probes were used for sex-mismatched HCT, chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS) cases with karyotypic abnormalities before HCT, respectively. Donor cells were found using FISH analysis in all 32 cases examined within 2 months following HCT, confirming engraftment. In six cases, however, cytogenetic analysis failed to detect donor cells due to lack of metaphases. Relapse occurred in four of the six cases in which mixed chimerism was detected using FISH analysis after 6 months of HCT. In contrast, after 12 months of HCT, no relapse was found in 24 patients without host cells. For two patients with mixed chimerism, gradual reduction of immunosuppressants or donor lymphocyte infusion resulted in the disappearance of host cells as analyzed using FISH analysis. In three extramedullary relapse cases, however, cytogenetic relapse preceded morphological and FISH relapse. These findings suggest that FISH analysis is more useful for detecting residual host cells after HCT, and the combination of FISH and cytogenetic analyses provide a more detailed evaluation for HCT patients. The results also indicate that monitoring of mixed chimerism using FISH analysis after 6 months of HCT is important for allowing the early detection of hematological relapse.

Aneuploidy and hyperploidy are often detected in malignant melanoma by cytogenetic analysis and flow cytometric analysis of DNA content. To determine the ploidy of cells in surgical specimens of melanin-producing tumors of Japanese patients, we performed fluorescence in situ hybridization (FISH) using touch smear technique to count the number of chromosomes 18 and X + Y in interphase nuclei using alpha-satellite DNA probes, D18Z1, DXZ1 and DYZ3. A normal melanocyte strain showed two D18Z1 and two [DXZ1+DYZ3] signals per nucleus, indicating 2N, and a malignant melanoma cell line showed 4 per nucleus, indicating 4N, consistent with results of cytogenetic and flow cytometric analyses. Therefore we employed this FISH method to analyze ploidy of surgical specimens. Specimens obtained from 8 patients with nevus cell nevus showed 2 FISH signals per nucleus. On the other hand, in all specimens obtained from 8 patients with malignant melanoma (6 primary and 2 metastatic melanoma), 65-90% of cells exhibited 4 signals per nucleus, indicating 4N. Histopathologically, 50-70% of cells were identified as malignant melanoma cells, indicating that our FISH method is effective to detect melanoma cells in tissue. We also analyzed allelic loss of the p53 gene by FISH with a p53 locus-specific probe and mutation of the p53 gene by immunostaining since mutation and deletion of the p53 gene may cause hyperploidy. All specimens except one obtained from a case with young-onset metastatic melanoma exhibited no allelic losses or negative p53 staining, showing the p53 gene was intact. These results indicate that tetraploidy, not caused by p53 mutation or deletion, is commonly found in malignant melanoma of Japanese patients. It is also suggested that there is no positive relationship between tetraploidy and poorer prognosis, and mutation and allelic loss of the p53 gene might be markers of aggressive form of malignant melanoma.

We examined the expression and the regulation of p21 waf1 and p27 kip1 cdk inhibitors in P19 mouse embryonal carcinoma (EC) cells following treatment with all- trans retinoic acid (ATRA) to induce neuronal differentiation. The levels of p27 mRNA and protein increased within 24 h of treatment with ATRA, reaching a plateau 4–5 days later prior to neurite formation. In contrast, levels of p21 expression remained low until after neurites were extensively formed. Induction of muscle differentiation from P19 cells by treatment with dimethyl sulfoxide caused only transient increases in p27 levels. In a mutant P19 cell line, RAC65, treatment with ATRA induced neither p27 accumulation nor neuronal differentiation, but p21 mRNA expression increased markedly. In contrast, treatment of RAC65 cells with 9- cis retinoic acid induced both p27 expression and neuronal differentiation. Correlation between p27 expression and neuronal differentiation was also observed in NT2/D1 human EC cells. Luciferase reporter assays showed that p27 promoter activity increased in ATRA-treated cells, consistent with the elevation of p27 mRNA levels. Arrest of neuronal differentiation of P19 cells by okadaic acid resulted in inhibition of p27 expression, whereas p21 mRNA expression was greatly enhanced. Conversely, inhibition of p27 expression by antisense p27 oligonucleotides resulted in blockade of neuronal differentiation. Taken together, these results strongly suggest that the expression of p27 is indispensable for neuronal differentiation of EC cells.

Clonality of hematopoietic cells on a smale scale (nanogram amounts of DNA) can be detected by X-chromosome inactivation using the polymerase chain reaction (PCR). The human androgen-receptor gene (HUMARA) has a polymorphic short tandem repeat (STR), and has generally been used for clonality analysis since heterozygosity for the gene occurs in 90% of caucasian females. We examined heterozygosity of the STR on HUMARA in 110 Japanese females and found heterozygosity in 74 of 110 (67%). To examine for hematologic clonality in females with HUMARA homozygosity, we used a primer specific for a novel polymorphic STR site between DXS15 and DXS134 (DXS15-134) on Xq28. Heterozygosity for this site was found in 50 of 110 females (46%). Clonality of the hematopoietic cells was detected in 91 of 110 females (83%) using PCR of either the STR sites on HUMARA or DXS15-134. The X-inactivation patterns using PCR of DXS15-134 corresponded exactly with those obtained using PCR of HUMARA in 18 females who were heterozygous for both DXS15-134 and HUMARA. Using PCR of DXS15-134, we examined the clonality of bone marrow cells separated by flow cytometry in a patient with erythroleukemia (M6). Clonality was found not only in myeloid lineage cells but also in B lymphocytes. The clonality assay for DXS15-134 may be useful to assess for clonality of hematopoietic cells in the Japanese population, when combined with the HUMARA assay.

Characteristics of karyotypes were analyzed in de novo acute myeloid leukemia (AML) with trilineage myelodysplasia (AML/TMDS) at initial diagnosis and compared with myelodysplastic syndrome (MDS) cases that had evolved to AML (MDS/AML). Abnormal karyotypes were seen in 11 of 19 patients with AML/TMDS and 13 of 16 MDS/AML cases. Trisomy 8 was observed in 3 AML/TMDS cases as a sole anomaly and was also present in 3 MDS/AML cases but not as a sole finding. Although MDS/AML frequently displayed monosomies or long-arm deletions of chromosome 5, 7 and 9, only one case exhibited long-arm deletion (of chromosome 7) in AML/TMDS. Two or more chromosome aberrations were found in some cases in both groups. These findings suggest that AML/TMDS had passed through several preleukemic stages at diagnosis, as has been well documented in MDS and MDS/AML. Additionally, clonal evolution may have already occurred in AML/TMDS, as MDS transformed to AML is associated with clonal evolution.

Point mutations of the K-ras gene at codon 12 are often detected in the pancreatic juice of patients with pancreatic cancer. Detection of these mutations may, thus, have diagnostic implications. K-ras mutations may also have diagnostic potential for other biliary tumors. We sought to detect K-ras mutations in DNA obtained from bile in patients with biliary tract cancers, pancreatic cancer and benign biliary disease but who had obstructive jaundice. In 35 patients, bile was collected during percutaneous transhepatic choledocal drainage (PTCD) catheters. K-ras gene mutations at codon 12 in the samples were examined using mutant-allele-specific-amplification (MASA). We compared these results with cytological analyses of bile. K-ras mutations at codon 12 in bile were detected in 11 of 14 (79%) of the patients with biliary duct cancer, 3 of 9 (33%) with pancreatic cancer but not in patients with gallbladder cancer (n=3), papilla of Vater's cancer (n=3) or benign biliary diseases (n=6). In the patients, where cytological evaluation did not reveal malignant cells, K-ras mutations in bile were detected in 5 of 7 (71%) patients with biliary duct cancer and 2 of 5 (40%) with pancreatic cancer. This approach, when used in conjunction with bile cytology, may improve the yield in diagnosing suspected malignant tumors of the pancreatic-biliary system.

In order to investigate the radioresistance mechanism of human carcinoma cells, we measured intracellular manganese‐ (Mn‐) and copper/zinc‐ (Cu/Zn‐) superoxide dismutases (SODs), glutathi‐one (GSH) and poly(ADF‐ribose) polymerase (PARP) in radioresistant N10 and its parental KB cell lines. The Mn‐SOD level was 1.3‐fold less in N10 than in KB, but Mn‐SOD was induced at 1.3 to 1.5‐fold higher level in N10 than in KB by X‐irradiation (4‐ Gy). Cu/Zn‐SOD in N10 showed a higher level than that in KB both without and with irradiation. In addition, N10 had a 1.65‐fold higher GSH level than did KB and became radiosensitive on treatment with buthionine sulfoximine, an inhibitor of GSH. Furthermore, PARP mRNA was highly expressed in N10 as compared to KB under unirradiated conditions. X‐Irradiation reduced the PARP mRNA level in KB in a time‐dependent manner, whereas the PARP mRNA level in N10 was still high at 6 h postirradiation. Assay for PARP activity demonstrated an approximately 3‐fold higher activity in N10 than in KB under unirradiated conditions. X‐Irradiation caused a rapid induction of PARP activity within 1 h in both cell lines, but treatment of cells with nicotinamide, a PARP inhibitor, markedly reduced the enzyme induction in N10, but not in KB, and potentiated the radiosensitivity in N10. These factors may all contribute to the radioresistance of the N10 cell line.

Villous tissues from 30 spontaneous abortions and the same number of artificial abortions were obtained and analysed for the frequency of polyploid cells. Single cell suspensions were made from these tissues without culture and the ploidy of > 100 cells was analysed. Trisomies of chromosomes 17 and 4 have rarely been reported in villous cells of spontaneous abortions, suggesting that the presence of more than three copies of chromosomes 17 and 4 per cell indicates polyploidy. The number of chromosomes 17 and 4 was detected by fluorescence in-situ hybridization analysis using centromeric probes D17Z1 and D4Z1. Most villous cells from cases of spontaneous and artificial abortions had two D17Z1 or D4Z1 signals per cell, with very small percentages of cells (0.5 +/- 0.4%) showing three signals per cell. However, in four cases of spontaneous abortions, 2-12% of cells had three D17Z1 or D4Z1 signals per cell. This indicates the presence of triploid cells in these cases of spontaneous abortion, at a significantly higher frequency compared to artificial or the remaining 26 cases of spontaneous abortion. In addition, three cases contained 0.2-0.4% of cells showing six signals, indicating that these cells were dividing triploid cells. The low frequency of mosaicism reported here would not be detectable by conventional chromosomal analysis.

Epstein-Barr virus (EBV)-transformed B cell lines that produce human antisperm antibodies were established using peripheral blood lymphocytes (PBLs) from infertile women with sperm immobilizing antibodies in their sera. We obtained three stable cell populations (designated B1, B2, D5) of transformed PBLs originating from three different patients. They produced IgM sperm-reacting antibodies directed against the tail of live, methanol-fixed and NaIO4-treated human spermatozoa. The established antisperm antibodies recognized noncarbohydrate sperm membrane antigens with different specificity and distribution in the male reproductive system. Antisperm antibody-B2 corresponding antigen appears to be specific for the male reproductive system. This antigen is excreted from the epithelial cells of the ductus epididymidis and bound to the spermatozoa in the lumen of the ductus. Antisperm antibodies B1 and D5 corresponding antigens were expressed on the spermatozoa in the seminiferous tubules and were common to the secretions of the ductus epididymidis, prostate and some other somatic organs. The cDNA of the immunoglobulin heavy chain genes were analyzed by reverse transcription polymerase chain reaction (RT-PCR) using RNA extracted from these clones. The immunoglobulin heavy chain cDNA sequences of these antisperm antibodies showed extremely high homology to previously reported immunoglobulin germline DNA sequences, implying that these antisperm antibodies might be natural autoantibodies rather than antibodies stimulated by external antigen.

The expression of mRNA for the Fas antigen, a membrane-associated protein mediating apoptosis, was localized by in situ hybridization histochemistry in murine brains following 30 min of global cerebral ischemia. Six hours following the ischemia, many labeled cells were detected anew throughout the brain. The hybridization was seen in the small neural cells and in the cells along the walls of the ventricles and vessels, and became undetectable 24 h following the ischemia. These results suggest that the Fas antigen is expressed in the neuron, glia and periventricular cells of the post-ischemic brain.

We characterized two novel mutations of the galactose-1-phosphate uridyltransferase (GALT) gene intwo Japanese patients with GALT deficiency and identified N314D and R333W mutations, previouslyfound in Caucasians. One novel missense mutation was an G-to-A transition in exon 8, resulting in thesubstitution of arginine by histidine at the codon 231 (R231H). GALT activity of the R231H mutantconstruct was reduced to 15% of normal controls in a COS cell expression system. The other was asplicing mutation, an A-to-G transition at the 38th nucleotide in exon 3 (318AG), resulting in a38-bp deletion in the GALT cDNA by activating a cryptic splice acceptor site. In seven Japanesefamilies (14 alleles for classic form and one allele for Duarte variant) with GALT deficiency, the R231H and 318AG mutations were found only on both alleles of the proband. The N314D and R333W mutations were found on one allele each. The Q188R was prevalent in the United States butnot in Japanese patients. The N314D mutation was associated with the Duarte variant in Japanesepersons, as well as in the United States. We speculate that classic galactosemia mutations appear todiffer between Japanese and Caucasian patients. Our limited data set on galactosemia mutations in Japanese suggests that the N314D GALT mutation encoding the Duarte variant arose before Asianand Caucasian people diverged and that classic galactosemia mutations arose and/or accumulated afterthe divergence of Asian and Caucasian populations. © 1995 Wiley-Liss, Inc.

Atopy, characterised by allergic asthma and rhinitis, is due to increased IgE responses to common aeroallergens. An Oxford group has described maternal inheritance of atopy, where there is significant linkage between IgE responsiveness and a VNTR marker D11S97 and a CA microsatellite within a candidate gene, the high affinity IgE receptor beta subunit (Fc epsilon RI beta), on chromosome 11q. Attempts at independent replication have produced conflicting results. We therefore recruited 270 atopic asthmatic probands in a Japanese community population for genetic linkage analysis. Four families, each with more than 15 meioses and a clear phenotype for atopy, were selected for genetic analysis. Atopy was defined as presence of all of raised total IgE, positive RAST and skin tests to three or more aeroallergens; non-atopy, as absence of all these criteria. Linkage analysis showed a maximum two-point lod score of 9.35 for D11S97 and Fc epsilon RI beta under the assumption of unequal rates of maternal and paternal recombination. Two families showed close genetic linkage with Fc epsilon RI beta with a pattern of maternal inheritance. These results from a Japanese population provide further evidence for genetic linkage between severe atopy and chromosome 11q13 and the likelihood of genomic imprinting at the locus.

A deficiency in low Km aldehyde dehydrogenase (ALDH2) is regarded as the main factor responsible for "Oriental flushing" and other symptoms due to alcohol sensitivity. In this study, the relationship of the ALDH2 genotype to alcohol-associated symptoms and drinking behavior was investigated in 524 Japanese workers, using a new, rapid, and nonisotopic polymerase chain reaction (PCR) method. Differences in the frequency of alcohol-associated manifestations between the normal homozygote and the other deficient types were apparent. In addition, among the ALDH2-deficient individuals, the atypical homozygote was obviously more hypersensitive to alcohol than the heterozygote, judging from the frequency of flushing or other drinking-associated manifestations with a small dose of alcohol. Drinking frequency also apparently decreased in the following order: typical homozygote, heterozygote, atypical homozygote. Similarly, mean amounts of alcohol consumption also decreased in the same order, although considerable variation existed within the typical homozygote and the heterozygote group. In contrast, neither the manifestations nor the drinking behavior were, in general, influenced by polymorphism of the alcohol dehydrogenase beta-subunit (ADH2) gene in males. These findings further indicate the important contribution of the ALDH2 genotype to alcohol sensitivity in Orientals.

Germline mutations in patients with familial adenomatous polyposis were analyzed by polymerase chain reaction (PCR) amplification of the adenomatous polyposis coli gene. PCR products from heterozygous patients for deletions of this gene formed four distinct bands on polyacrylamide gel electrophoresis. The four fragments were subsequently purified and both strands of each fragment were directly sequenced, using an automated DNA sequencer and the same primers as those for PCR amplification. It was found that the two slower migrating fragments were "bulge" heteroduplexes, while the other two were homoduplexes made up of two wild-type strands and two deletion-mutant strands, respectively. The sites of deletions in the adenomatous polyposis coli gene could be exactly determined in four of the five patients. In an attempt to identify deletion-carriers of familial adenomatous polyposis at the presymptomatic stage, a family study was also carried out, and two children were found to have the same mutations as those of their affected parents. The direct sequencing of heteroduplex fragments generated during PCR amplification is a potentially useful method for detecting mutations of not only the adenomatous polyposis coli gene but also many other genes of genetic diseases.

To examine whether the dosage effect of germ-line mutations in patients with familial adenomatous polyposis (FAP) is sufficient to cause colorectal adenomas, or an additional somatic mutation of the normal allele is required as well, we have investigated somatic mutations of the APC gene in multiple adenomas developed in one FAP patient. In addition to a 5-bp deletion of one allele present constitutionally in this patient, the normal APC allele had been lost in five of seven DNA samples extracted from small adenomas (< 3 mm in diameter) with mild or moderate atypia. This result indicates that the inactivation of both alleles of the APC gene is probably essential for the development of an early-stage adenoma, in agreement with the two-hit mutational model underlying the concept of tumor suppressor genes.

The frequency and distribution of eight mutations (R111X, IVS4nt-1, Y204C, R243Q, IVS7nt-2, W326X, Y356X, and R413P) in the phenylalanine hydroxylase gene of Orientals in Japan and Korea were examined by allele-specific oligonucleotide hybridization. The mutant alleles comprised 54 and 55% of the phenylketonuria (PKU) chromosomes examined in 36 patients in Japan and 10 patients in Korea, respectively. The spectrum of PKU mutations in Japan was similar to that in China, particularly in northern China, but different from that in Korea. The IVS4nt-1 mutation had a high frequency in Korea and southern China, due to the result of the founder effect and genetic drift. The R413P mutation, which may have originated in the regions surrounding the Baikal, expanded to northern China and Japan. We did not find Caucasian mutations in the Japanese or Korean PKU chromosomes. Thus, PKU mutations occurred after racial divergence between Caucasians and Mongoloids, and there were different founding populations for PKU in the two populations.

A new cell line, designated TC-YIK, was established from YIK-1 tumor cells, derived from argyrophil small cell carcinoma (ASCC) of the uterine cervix, and serially heterotransplanted into nude mice, integrating human papillomavirus type 16 (HPV16) DNA. The population doubling time of TC-YIK was approximately 21.6 hours at the 119th subculture. Subcutaneous injection of 1 x 10(8) TC-YIK cells into nude mice yielded a solid tumor. The cytologic appearance of TC-YIK was similar to that of YIK-1. The TC-YIK cells contained argyrophil granules and neurosecretory granules in the cytoplasm and showed positive immunohistochemical staining for neuron-specific enolase, serotonin, and chromogranin. Thus, TC-YIK retained the histochemical characteristics of ASCC. The TC-YIK cells contained HPV16 DNA in a multiple-copy integrated form and actively transcribed the integrated HPV16 genome. Amplification of the c-myc oncogene was observed in the TC-YIK cells. These data suggest that TC-YIK is a useful in vitro experimental model of ASCC and that HPV16 and c-myc may play some role in the genesis of this malignant tumor and/or maintenance of the transformed TC-YIK phenotype.

An isolated case of Duchenne muscular dystrophy (DMD) in a female who has a deletion of the DMD locus is described. This patient was a 26-year-old woman born to unrelated, healthy parents. She was initially examined at age 6 because of a waddling gait. At age 15, pseudohypertrophy of calves and pes equinus were observed along with proximal muscular weakness and wasting. Her serum creatine kinase level was high and histological evidence of muscular dystrophy was apparent on muscle biopsy. The patient was ambulant at age 15 and progression of motor disability has been slow. Chromosomal studies revealed a normal karyotype, and mental retardation is moderate. DNA analysis at age 26 revealed that she has a deletion of DMD cDNA 8 mapped within Xp21 and is heterozygous for the deletion. Since diagnosis of DMD is now dependent on the evidence of mutation or deletion at Xp21, this patient is thought to have a form of DMD. Expression of the DMD gene in the heterozygous state might be due to random but unequal lyonization.

Five families with familial inherited TSH deficiency, reported to date, were examined for the TSH beta gene at the nucleotide level. The first family carries a single base substitution in the 29th codon which lies in the so-called CAGYC region; GCA (glycine) is replaced by AGA (arginine). This substitution induces conformational changes of the beta-polypeptide which make it unable to associate with the alpha-subunit. This mutation generates a new cleavage site for a restriction endonuclease MaeI, a new marker that can be used for DNA diagnosis. The second and third families were found to carry the same nucleotide substitution. Also, all three families were associated with an additional single base substitution in intron 2 as a polymorphic change, suggesting that these three families may have originated from the same single founder from Shikoku Island in Japan. The nucleotide sequence from the fourth and fifth families showed no alterations in the TSH beta gene from the about -200 basepair up-stream region to the polyadenylation site.

Cell hybrids (BIM) were produced between human neuroblastoma cells (IMR-32) and thymidine auxotrophs (B3T) of rat nerve-like cells (B103) in order to obtain cell lines undergoing stable neuronal differentiation. BIM cells exhibited the growth properties of partial transformation: 1) When the cell growth reached a plateau, BIM cells ceased to proliferate and expressed a differentiated phenotype. The shape of the cells changed from flat to round and they extended neurites. 2) When cultured in methylcellulose, BIM cells formed colonies, indicating that BIM cells have the ability of anchorage-independent growth. By Southern blot analysis, BIM cells had both human and rat types of N-myc genes. The human N-myc genes were amplified, but the extent of the amplification was lower in BIM cells than that in the parental cell line IMR-32. The rat N-myc gene was detected at a similar level in BIM, B3T, B103, and rat fibroblastic cells 3Y1. Therefore, the decrease in amplification of human N-myc genes may be involved in the properties of partial reverse-transformation in BIM cells. When treated with various drugs such as db-cAMP, forskolin, and cAMP with isobutyl-methylxanthine, BIM cells expressed a nerve-like phenotype. These findings indicate that cell hybridization yielded partial normalization of transformed nerve-like cells.