Allelotype of renal cell carcinoma.

Several recent studies based on restriction fragment length polymorphism analysis have supported the concept that the accumulation of multiple genetic alterations converts a normal cell to a malignant cell. Activation of oncogenes and/or inactivation of tumor suppressor genes have been observed during tumor progression in colorectal cancer, lung cancer, and breast cancer. To investigate the possibility that multiple genes are altered during the progression of renal cell carcinoma, we have used restriction fragment length polymorphism markers throughout the genome to test for loss of heterozygosity in 38 renal cell carcinomas. Nearly 64% of the tumors had lost heterozygosity on the short arm of chromosome 3. We also observed loss of heterozygosity averaging about 30% at informative loci on six other chromosomal arms (chromosomes 5q, 6q, 10q, 11q, 17p, and 19p). These results lead us to suspect the existence of several tumor suppressor genes associated with carcinogenesis of renal cell carcinoma.

Cloning and characterization of four types of cDNA encoding myeloperoxidase from human monocytic leukemia cell line, SKM-1.

Four cDNA clones encoding myeloperoxidase were isolated from a cDNA library of monocytic leukemia SKM-1 cells. The sequences of two of them were identical to those of cDNA clones previously isolated from a HL-60 cell cDNA library. The sequences of the other two cDNA clones, MP-S34 and MP-S29, differed from those previously described. There was a deletion of 57 bp in the MP-S34 sequence, which was generated by partially skipping exon 9. MP-S29 had a 171 bp deletion, which was generated by completely skipping exon 10. Thus MP-S34 and MP-S29 encoded polypeptides lacking 19 and 57 amino acids, respectively. Both deletions were located on the sequence encoding the heavy subunit. These results indicate that the heterogeneity of the heavy subunit of MPO observed in leukocytes or leukemia could be in part produced by partial or complete skipping of an exon.

The monocytic cell line SKM-1 strongly expresses the myeloperoxidase gene.

A newly established human monocytic cell line, SKM-1, showed strong expression of myeloperoxidase mRNA, to the same extent as in HL-60 cells. We studied the cell morphology and myeloperoxidase expression of this cell line, which was established from a patient with myelodysplastic syndrome who had an abnormal chromosome on the upstream region of 17p13. Electron micrographs showed the cells to have a fragile and irregular cell surface. SKM-1 cells were peroxidase-positive. About 60% of myeloperoxidase (MPO) was released to the culture fluid from SKM-1 cells but only a few percent of MPO was released from HL-60 cells into the culture fluid. The predominant mRNA size of SKM-1 myeloperoxidase was 3.3 kb although there was a smaller size as well. Fluorescent in situ hybridization of MPO mRNA showed strong staining in 5% to 10% of SKM-1 cells and of bone marrow cells from patients with myelogenous leukemia, while all cells from HL-60 were positive.

Prenatal diagnosis of oculocutaneous albinism by analysis of the fetal tyrosinase gene.

Tyrosinase-negative oculocutaneous albinism, the most severe subtype of a heterogeneous group of albinism, is an autosomal recessive trait caused by mutations in the tyrosinase gene. Prenatal diagnosis had been made previously only by evaluating fetal skin obtained by biopsy, an invasive procedure that cannot be performed earlier than 19 weeks of gestation. A pregnant mother of a 9-year-old Japanese boy with tyrosinase-negative oculocutaneous albinism wanted a prenatal diagnosis. Polymerase chain reaction amplification and allele-specific oligonucleotide hybridization revealed that the child is homozygous and the parents heterozygous for the pathologic mutation of the tyrosinase gene in exon 2 (single base insertion) but not for the one in exon 1. Prenatal diagnosis was made by analyzing the tyrosinase gene in fetal cells obtained by amniocentesis at 14 weeks of gestation, which demonstrated that the fetus was heterozygous for mutant tyrosinase gene. Pregnancy was therefore continued and a normal male infant was born. This procedure, the analysis of the fetal genomic tyrosinase DNA, is a rapid and reliable approach to the prenatal diagnosis of oculocutaneous albinism at a relatively early stage of pregnancy and is safer and less invasive than previous methods using fetal skin biopsy.

Three different GB virus C/hepatitis G virus genotypes. Phylogenetic analysis and a genotyping assay based on restriction fragment length polymorphism.

The 5'-untranslated region (5'-UTR) sequences of 33 GB virus C/hepatitis G virus (GBV-C/HGV) obtained from different geographic areas were determined through reverse-transcription polymerase chain reaction and dideoxy chain termination sequencing, the alignment of sequences, the estimation of the number of nucleotide substitution per site, and construction of phylogenetic trees. The 5'-UTR of GBV-HGV was found to be heterogeneous, with 70.9-99.5% homology. Three distinct phylogenetic branches were observed consistently in all phylogenetic trees. GBV-C is the prototype for one, HGV for another, and there is a new branch which consisted of GBV-C/HGV isolates from Asia. Genotype-specific restriction sites for the restriction enzymes, ScrFI and BsmFI, were identified, and a simple restriction fragment polymorphism analysis was developed for genotyping. These data provide evidence that GBV-C/HGV consists of three different genotypes. Our simple genotyping assay will also provide a tool for epidemiological studies of GBV-C/HGV infection.

Enhanced hepatocyte growth factor expression associated with prolonged rat hepatic allograft survival in recipients pretreated with donor-specific blood.

BACKGROUND: Pretransplantation injection of freshly heparinized donor blood (donor-specific blood transfusion, or DST) significantly prolongs the survival of hepatic allografts from ACI(RT1a) to LEW(RT1l) rats. We investigated hepatocyte growth factor (HGF) expression in rat hepatic allografts of recipients pretreated with or without DST. METHODS: The levels of HGF mRNA and protein in hepatic allografts were determined after transplantation. The localization of HGF+ cells was identified with a rat anti-HGF monoclonal antibody. RESULTS: Plasma HGF concentrations in transplanted rats treated with DST were significantly and persistently increased compared to untreated rats with hepatic allografts. The number of HGF+ cells in hepatic allografts of recipients pretreated with DST on day 14 was significantly greater than that in allografts of untreated recipients on day 7. HGF+ cells were also found in the marginal zone and red pulp of recipient spleens. Northern blot analysis revealed the presence of three HGF+ cell phenotypes: HGF+ED1+, HGF+ED2+, and HGF+ED1-ED2-. Most HGF+ cells were ED1-ED2-. In situ hybridization demonstrated HGF mRNA in the mononuclear cells in the portal and sinusoidal areas as well as the marginal zone and red pulp in both DST-treated and untreated recipient spleens. CONCLUSIONS: Enhanced HGF expression in rat hepatic allografts is associated with immunologic unresponsiveness induced by DST.

Genotype of GB virus C/hepatitis G virus by molecular evolutionary analysis.

GB virus C/hepatitis G virus is a newly described virus. Classification of GB virus C/hepatitis G virus into genotypes has not been established. We analyzed nucleotide sequences within the 5' untranslated region of GB virus C/hepatitis G virus isolates and segregated these isolates into genotypes. Twenty serum samples with GB virus C/hepatitis G virus RNA from Australia, Cameroon, the Congo, Japan, Mongolia, and Bangladesh were studied. Reverse transcription and polymerase chain reaction were used to obtain GB virus C/hepatitis G virus RNA. After nucleotide sequences from the 5' untranslated region were determined, 68 nucleotide sequences, including 48 previously reported sequences, were analyzed by molecular evolutionary methods. The phylogenetic tree of the 5' untranslated region showed that all strains could be divided into three major genotypes, GB type (type 1), HG type (type 2), and Asian type (type 3). Bootstrap analysis indicated that the strains could be divided into three major genotypes but could not be further subdivided. Moreover, frequency histograms of pairwise distances between nucleotide sequences demonstrated only one peak. These result indicated that GB virus C/hepatitis G virus can be classified into three major genotypes, GB type (type 1), HG type (type 2), and Asian type (type 3), and should not be divided into minor subtypes.

GB virus C/hepatitis G virus infection among Japanese patients with chronic liver diseases and blood donors.

Recently, a novel hepatitis virus, GB virus C/hepatitis G virus (GBV-C/HGV), has been isolated. To elucidate the seroprevalence of chronic GBV-C/HGV infection in Japan and the phylogenetic relationship between Japanese strains and the strains previously reported, serum GBV-C/HGV RNA was detected by reverse transcription polymerase chain reaction (RT-PCR) in 203 patients with chronic liver diseases and 200 samples of voluntary blood donors. RT-PCR was performed with primers derived from the 5'-untranslated region which were conserved between GBV-C and HGV and distant from other flaviviruses including hepatitis C virus (HCV). The nucleotide sequences were determined by the dideoxy chain termination method. The phylogenetic analysis was performed by the neighbor-joining method. In 10 (4.7%) of 203 patients with chronic liver diseases and in 1 (0.5%) of 200 blood donor samples, serum GBV-C/HGV RNA was detected. Of 10 patients, 9 patients were positive for anti-HCV and negative for HBsAg, and 1 patient was positive for HBsAg and negative for anti-HCV. The phylogenetic analysis indicated that there were three major groups which were group 1 (GBV-C), group 2 (HGV), and group 3 (a group of Japanese strains). These data indicated that (1) there was a low prevalence of GBV-C/HGV infection in Japanese patients with chronic liver diseases, (2) a high proportion of patients with GBV-C/HGV infection had chronic HCV infection however, and (3) there were at least three groups in strains of GBV-C/HGV.

Levels of telomerase catalytic subunit mRNA as a predictor of potential malignancy.

This study investigated the relationship between the levels of human telomerase reverse transcriptase (hTERT) mRNA and that of telomerase activity in hepatocellular carcinoma (HCC). A significant correlation between hTERT mRNA expression and telomerase activity by transfecting the gene encoding hTERT into telomerase-negative human fibroblast cells has clearly been demonstrated. However, the relationship between levels of telomerase activity and that of hTERT mRNA has yet to be elucidated. In this study, the levels of hTERT mRNA were analyzed in 24 HCC patients by real-time PCR. And the intensity of telomerase activity was analyzed by fluorescence-based TRAP method. The difference of hTERT mRNA level was highly significant between tumor tissues and non-cancerous liver tissues. And there were significant correlations between the levels of hTERT mRNA and that of telomerase activity (r=0.751) in tumor tissues. We observed a strong correlation between levels of hTERT mRNA and that of telomerase activity in HCC. Our results suggest that the levels of hTERT mRNA would be useful in genetic diagnostic tests, instead of telomerase activity, to screen at-risk patients of HCC in human liver tissues.

Alteration of the CDKN2A gene in pancreatic cancers: Is it a late event in the progression of pancreatic cancer?

Various genetic changes are involved in progression of various cancers. We examined alterations (deletion, sequence abnormalities, methylation) of the CDKN2A gene in cell lines and tumor tissues of pancreatic cancers. Some alterations of this gene were found in all the 12 cell lines examined. In the primary lesions of pancreatic cancers, homozygous or hemizygous deletion were found in 8 of 24 ductal carcinoma and 4 of 9 other types of carcinomas. It appears that there is an association between the alteration of this gene and tumor size, regional lymph node metastasis and hematogenous distant metastasis in the ductal carcinoma, but not in the other types of carcinomas. All the 5 liver metastatic lesions of the ductal carcinoma examined revealed homozygous or hemizygous deletion and 3 bp deletion. These results suggest that inactivation of the CDKN2A gene occurs more frequently in cell lines than in pancreatic cancer tissues. Such genetic events on the CDKN2A gene may play an important role possibly at a later step in the progression of pancreatic ductal carcinoma.

Extra Y chromosome in T-cell acute lymphoblastic leukemia.

We describe a 66-year-old man who developed T-cell acute lymphoblastic leukemia (ALL) with a sole clonal chromosomal abnormality of 47,XY,+Y. Leukemic cells were positive for CD2, CD7, terminal deoxynucleotidyl transferase and cytoplasmic CD3. T-cell receptor beta, gamma, and delta genes remained germline configurations. The bone marrow aspirate was 47,XY,+Y in all metaphase cells observed. The patient achieved complete remission by chemotherapy, and the bone marrow cells and the phytohemagglutinin stimulated peripheral blood lymphocytes showed a normal karyotype of 46,XY at that time. This fact suggests that an extra Y chromosome may be a kind of new chromosomal abnormality of T-cell ALL.

Telomerase reverse transcriptase mRNA expression and telomerase activity in hepatocellular carcinoma.

Human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit of human telomerase. To clarify the clinical significance of hTERT mRNA in hepatocellular carcinoma (HCC), we investigated the relationship between telomerase activity and hTERT mRNA in human HCC and non-HCC tissues. The hTERT mRNA was detected in 17 (89.47%) of 19 livers with HCC and in 4 (21.05%) of 19 noncancerous tissues from these livers. Telomerase activity was detected in 17 of the 19 tumor tissues (89.47%) and in 4 of the 19 nontumor tissues (21.05%). The hTERT mRNA was detected in all tissues that were telomerase-positive and it was undetected in all tissues that were telomerase-negative. The correlation between the expression of hTERT mRNA and human telomerase activity in this study indicates that hTERT mRNA could be useful to diagnose cancer. Also, as telomerase production may be under the control of hTERT mRNA, the possibility is great that noncancerous liver tissue with chronic liver diseases acquires HCC when the hTERT mRNA is positive.

Detection of the most common mutation of adenine phosphoribosyltransferase deficiency among Japanese by a non-radioactive method.

About 79% of all the Japanese patients with adenine phosphoribosyltransferase (APRT) deficiency have been estimated to possess at least one APRT*J allele with a substitution of ACG for ATG at codon 136. We developed a non-radioactive method for diagnosing genotypes of this disease. Part of the genomic DNA including the mutation site of the APRT*J allele was amplified using polymerase chain reaction and the amplified product was dot-blotted onto nylon membranes and then hybridized with either APRT*J-specific or non-APRT*J-specific synthetic oligonucleotides labelled at the 5' termini with biotin in the presence of non-labelled competitive synthetic sequences. The temperature was gradually decreased during the hybridization. When competitive sequences were omitted, difference in the intensity of the hybridization between APRT*J-containing and non-containing samples was not sufficiently clear to differentiate the genotypes. When an excess amount of competitive sequences was added in addition to biotin-labelled oligonucleotides, this method effectively differentiated samples containing only APRT*J alleles from those containing only non-APRT*J alleles. The present method was also useful to differentiate samples with both APRT*J and non-APRT*J alleles from those having only either of the alleles. An equivalent procedure using competitive sequence for hybridization and gradually decreasing the temperature will be useful for detecting point mutations in other genes.

Genetic diagnostic test of hepatocellular carcinoma by telomerase catalytic subunit mRNA.

This study investigated the relationship between telomerase activity and telomere length and between telomerase reverse transcriptase (hTERT) mRNA and telomere length. Both cancerous and non-cancerous tissues were studied in individuals with hepatic carcinoma. In this study, the telomere length in HCC livers had a wide range, no clear significant correlation was found between hTERT mRNA and telomere length. Telomerase activity was more strongly correlated with hTERT mRNA than with telomere length. The correlation between hTERT mRNA and telomerase activity shown here indicates that hTERT mRNA has potential for cancer diagnosis.

Effect of maternal separation on feeding behavior of rats in later life.

Effects of maternal separation on feeding behavior, particularly on rebound hyperphagia, in adult rats were examined. Time-restricted scheduled feeding (2 h per day for 6 days), was given at the age of 3, 6, 9 or 12 weeks in rats that were maternal separated from postnatal days (PD) 1-21 and control rats. Following the time-restricted scheduled feeding, rats were fed freely for 24 h (rebound hyperphagia). Body weight, daily normal food consumption and food consumption during time-restricted scheduled feeding and rebound hyperphagia were measured. Body weight of 3-week-old maternally separated rats were less than those of control rats. There was no significant difference in normal daily food consumption. Food consumption during rebound hyperphagia was significantly increased in 6- to 9-week-old female maternally separated rats, but there was no difference observed in males. Postnatal maternal separation enhanced rebound hyperphagia of female rats in later life. These results indicate that postnatal maternal separation made rats more vulnerable to the development of abnormal feeding behavior in response to food restriction in later life.

Analysis of herpes virus group (DNA) from cerebrospinal fluid in vogt-koyanagi-harada disease.

In order to detect herpes virus group DNA including that of the Epstein-Barr virus (EBV) in patients with Vogt-Koyanagi-Harada disease (VKH), the authors employed the polymerase chain reaction (PCR) procedure using DNA from cerebrospinal fluid (CSF) obtained from patients with VKH. Method. Seven CSF samples were obtained from six definite, active VKH cases and DNA was isolated. DNA fragments containing parts of herpes simplex virus (HSV), herpes zoster virus (VZV), cytomegalo virus (CMV), EBV and human herpes virus type 6 (HHV-6) sequences were amplified by PCR. Results. No DNA fragment corresponding to the DNA sequence of the herpes virus group was detected. Conclusion. Our results suggest that the herpes virus group does not have a close association with the cause of VKH.

[Detection of the mutation responsible for adenine phosphoribosyltransferase deficiency among Japanese patients].

Adenine phosphoribosyltransferase (APRT) deficiency causes 2,8-dihydroxyadenine(DHA) urolithiasis and renal failure. Recently, two different common mutations were identified; one was APRT* J with a substitution of ACG for ATG at codon 136, called "Japanese-type", another was APRT* Q0 with TGA for TGG at codon 98. Approximately 98% of all Japanese patients with this disorder have been estimated to have these mutations APRT* J (approximately 80%) and/or APRT*Q0 (approximately 20%). We developed a diagnostic method to detect these genotypes. After gene amplification by PCR, target DNA was hybridized with a biotinylated specific probe in the presence of the non-labelled competitive probe on a dot-blotted membrane. To detect the APRT* J (or APRT* Q0) mutation, the biotinylated APRT* J (or APRT* Q0) probe and non-labelled normal probe for the same region were used as specific and competitive probes, respectively. After incubation at 60 degrees C for 30 min, the temperature was gradually decreased from 60 degrees C to 40 degrees C during 120 min, and then incubation was continued at 40 degrees C for 30 min. By using method, we were able to omit the posthybridization process, and the detecting signal was clear and highly specific. This method is useful for detecting point mutations in other genes.

[Determination of human IgG subclass by EIA].

The human IgG subclasses have specific characteristics in response to respective antigens, and determination of their characteristics is important in analysis of infectious, autoimmune and other diseases. We developed quantitative EIA (ELISA) assays for Japanese human IgG subclasses with excellent reproducibilities using monoclonal antibodies. The assays enable determinations between 0.4 ng/ml and 2,000 ng/ml. The serum IgG subclass levels (mean+SD) in healthy adults are as follows: IgG1, 5.93 + 2.07 mg/ml (59.94%); IgG2, 3.36 + 1.84 mg/ml (32.97%); IgG3, 0.30 + 0.19 mg/ml (3.07%); and IgG4, 0.22 + 0.15 mg/ml (2.34%). Excellent correlation was obtained between total IgG level measured by EIA using anti-whole human serum and the sum of the four IgG subclasses (IgG1+IgG2+IgG3+IgG4). Samples from every patient with M protein of IgG1 and IgG2 types shown by M band on immunoelectrophoresis also had the highest level in four IgG subclasses.

[Detection of human parvovirus B19 DNA using PCR and serological test using ELISA for diagnosis of infection].

The development of a polymerase chain reaction (PCR) method for detection of human parvovirus B19 DNA and serological test of clinical specimens using ELISA is described. Of 43 serum (or plasma) samples, 29 were found to be positive for B19 DNA using the PCR, anti-IgM was detected in 16 specimens and anti-IgG in 25. In all specimens confirmed to contain B19-specific IgM, the presence of B19 DNA was demonstrated. Furthermore, B19 DNA was demonstrated in a patient with B19 infection for a longer period of time than was anti-IgM. In addition, we discuss the diagnosis of B19 infection using anti-IgG, in the acute stage and convalescent stage. In fetal infection, the PCR method provides sufficient detection of B19 DNA in cord blood, amniotic fluid, ascitic fluid, and fetal pericardial effusion and pleural effusion.

[Identification and differentiation of the human herpes virus group using the PCR method].

Six kinds of human herpes viruses have been identified and classified on the basis of structure and characteristics. We studied the identification and classification of these types using PCR to amplify the virus-specific DNA sequences. This method showed higher sensitivity than the conventional method of virus isolation and culture for HSV and CMV detection. For each positive control, the viral DNA was amplified only when the complementary primers themselves were used. PCR apparently detects only the activated virus, because normal controls were negative when this method was used. Therefore, the present method is thought to closely reflect viral activation and infectious diseases in patients with latent infections.