Chromosome elimination in the interspecific hybrid medaka between Oryzias latipes and O. hubbsi.

An interspecific hybrid medaka (rice fish) between Oryzias latipes and O. hubbsi is embryonically lethal. To gain an insight into the cellular and molecular mechanisms that cause the abnormalities occurring in the hybrid medaka, we investigated the behavior of chromosomes and the expression patterns of proteins responsible for the chromosome behavior. The number of chromosomes in the hybrid embryos gradually decreased to nearly half, since abnormal cell division with lagging chromosomes at anaphase eliminated the chromosomes from the cells. The chromosome lagging occurred at the first cleavage and continued throughout embryogenesis even after the midblastula transition. Fluorescent in-situ hybridization analyses revealed that the chromosomes derived from O. hubbsi are preferentially eliminated in both O. latipes-hubbsi and O. hubbsi-latipes embryos. Whole-mount immunocytochemical analyses using antibodies against alpha-tubulin, gamma-tubulin, inner centromere protein, Cdc20, Mad2, phospho-histone H3 and cohesin subunits (SMC1alpha, SMC3 and Rad21) showed that the expression patterns of these proteins in the hybrid embryos are similar to those in the wild-type embryos, except for phospho-histone H3. Phospho-histone H3 present on chromosomes at metaphase was lost from normally separated chromosomes at anaphase, whereas it still existed on lagging chromosomes at anaphase, indicating that the lagging chromosomes remain in the metaphase state even when the cell has proceeded to the anaphase state. On the basis of these findings, we discuss the cellular and molecular mechanisms of chromosome elimination in the hybrid medaka.

Proliferative activity of intratumoral CD8(+) T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumor immunity.

Tumor-infiltrating lymphocytes, particularly CD8(+) T cells, could be a manifestation of antitumor immunity. We clinicopathologically analyzed the biological significance of tumor-infiltrating lymphocytes in 221 patients with renal cell carcinoma without preoperative treatments. More abundant infiltration of tumor tissue not only by CD8(+) but also CD4(+) T cells was associated with shorter survival of the patients, because of the positive correlation between the number of lymphocytes and representative tumor grade factors. This suggests that immune cell reactions are more pronounced as the tumor grade/biological malignancy progresses, probably because of increased antigenicity of tumor cells. We next analyzed the proliferative activity of CD8(+) T cells that infiltrated in tumor cell nests, which could also reflect antitumor immunity. Higher labeling index of Ki-67, a proliferation-associated antigen, among CD8(+) T cells in contact to tumor cells was associated with a longer survival by both uni- and multivariate analyses. Our data in human renal cell carcinoma suggest that infiltration of tumor tissue by T cells itself does not denote the efficacy of antitumor immunity because of its dependence on the biological malignancy of tumor cells, but infiltration of tumor tissue by CD8(+) T cells bearing more pronounced proliferative activity could reflect effective antitumor immunity. This concept would be important for future immunotherapy of human cancer.

Correlation of genetic etiology with response to beta-adrenergic blockade among symptomatic patients with familial long-QT syndrome.

Mutations in any of the five genes KCNQ1, KCNH2, KCNE1, KCNE2, and SCN5A can be responsible for familial long QT syndrome (LQTS), an arrhythmogenic disorder that entails a high risk of sudden death. beta-Adrenergic blocking agents are the first therapeutic choice, and 80% of patients treated with these agents show symptomatic relief; however the remaining 20% do not respond well. We previously performed a nationwide analysis of familial long QT syndrome (LQTS) in Japan and identified 32 mutations in the KCNQ1 and KCNH2 genes. In the present retrospective study, we found that patients carrying mutations in the KCNQ1 gene responded better to beta-adrenergic blocking agents than those with KCNH2 mutations (12 of 13 vs 1 of 5; P = 0.0077, Fisher's exact test). This is a good example of the power of genetic diagnosis to direct the selection of appropriate therapy for patients with diseases of heterogeneous genetic etiology.

Four new human renal cell carcinoma cell lines expressing globo-series gangliosides.

Clinicopathological studies revealed that monosialosyl galactosyl globoside (MSGG) and disialosyl galactosyl globoside (DSGG) expressed by renal cell carcinoma (RCC) are one of the biochemical indicator related to the metastatic potential. The present study examines the characteristics of four new human RCC cell lines and compares the expression of MSGG and DSGG among them using TLC immunostaining and flow cytometry. TOS-1 and TOS-2 were derived from metastatic subcutaneous tissues. TOS-3 and TOS-3LN were derived from the primary lesion and from metastatic lymph nodes respectively. Monolayer culture, light microscopy and electron microscopy of these cells showed that these cell lines were derived from RCC. TLC immunostaining and flow cytometric analysis revealed increased levels of MSGG in TOS-2 and TOS-3LN, and increased DSGG in TOS-1 and TOS-3LN. These cell lines would be useful for functional studies of globo-series ganglioside expressed by RCC.

Cloning and sequence analysis of hydroxyquinol 1,2-dioxygenase gene in 2,4,6-trichlorophenol-degrading Ralstonia pickettii DTP0602 and characterization of its product.

A gene encoding hydroxyquinol 1,2-dioxygenase was cloned from 2,4,6-trichlorophenol-degrading Ralstonia (Pseudomonas) pickettii strain DTP0602. Cell-free extracts of Escherichia coli containing a cloned 1.4-kb StuI-XhoI DNA fragment of R. pickettii DTP0602 hydroxyquinol 1,2-dioxygenase converted hydroxyquinol into maleylacetate and also degraded 6-chlorohydroxyquinol. The 1.4-kb DNA fragment contained one open reading frame (designated hadC) composed of 948 nucleotides. The molecular mass of 34,591 deduced from the gene product (HadC) was in agreement with the size (35 kDa) of the purified HadC protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of HadC exhibited high homology to that of the hydroxyquinol 1,2-dioxygenase of 2,4,5-trichlorophenoxyacetic acid-degrading Burkholderia cepacia AC1100 (Daubaras, D. L. et al., Appl. Environ. Microbiol., 61, 1279-1289, 1995). The active enzyme had a molecular mass of 68 kDa, suggesting that it is functional as a homodimer. The enzyme also catalyzed the oxidation of pyrogallol and 3-methylcatechol, possible intermediates in the degradation of 2,4,6-trichlorophenol, in addition to 6-chlorohydroxyquinol and hydroxyquinol. The dioxygenase catalyzed both ortho- and meta-cleavage of 3-methylcatechol.

[Cerebellar infarction due to vertebral artery dissection in a girl].

We report here a case of vertebral artery dissection, which is rare in childhood. A 12-year-old, previous healthy girl was admitted to our hospital with symptoms of vertigo, tinnitus, hearing loss, nausea and vomiting. Although there was neither higher cortical dysfunction, motor weakness, sensory disturbance nor slurred speech. She could not stand up because of severe vertigo. Cranial magnetic resonance imaging (MRI) revealed a subacute cerebellar infarct. A left vertebral artery angiogram on the hospital day 3 demonstrated a sharp narrowing at the C1-C2 level. After an anticoagulant therapy for about 2 weeks, all the symptoms disappeared except for mild tinnitus. Two months later, a left vertebral artery angiogram showed an abrupt occlusion at the C1 level. MRI T1-weighted images demonstrated a thrombus within the false lumen of the dissected vessels. A flow void revealed the patency of the residual true lumen. From these findings, we made a diagnosis of vertebral artery dissection, which was considered to have caused cerebellar infarction. The patient was mostly normal at discharge, and 100 mg/day of aspirin has been given until present.

Effects of short duration morning bright light in healthy elderly. II: sleep and motor activity.

Subjective sleep feelings and motor activity were measured in seven healthy elderly subjects for 6 days. The subjects were exposed to bright light (6000 lux) for 30 min in the morning or instructed to sit in front of a desktop lighting device without light. The average level of motor activity during the night was significantly decreased in the bright light condition, compared with the controlled condition. However, daytime motor activity did not show significant differences between the two conditions. From these findings, even a short duration of morning bright light is effective in maintaining sleep without changing daytime activity.

Efficacy of interferons in treating children with chronic hepatitis C.

UNLABELLED: Twenty-two children with chronic hepatitis serologically positive for hepatitis C virus (HCV) were treated with interferon-alpha (IFN-alpha). Liver biopsy showed chronic active hepatitis in 13 and chronic persistent hepatitis in 9 patients. A sustained clearance of HCV was observed in 8/22 children 12 months after the administration of IFN-alpha for 26 weeks, associated with normalization of HCV core antibody. Of these eight patients six had HCV genotype III and two HCV genotype II or IV. Hepatitis relapsed in seven other patients after completion of IFN-alpha with an increase in HCV core antibody titre, five with HCV genotype II, and two with HCV genotype III or IV. A second course of IFN-alpha suppressed the reactivation of HCV in all seven patients. Three of seven responders who relapsed after the first course remained negative for HCV RNA 12 months after their second course of IFN-alpha. However, the remaining four patients with HCV genotype II again relapsed after completing their second course of IFN-alpha. Seven children with the HCV genotype II resistant to IFN, including 8 weeks of IFN-beta administration, and showed no significant reduction in HCV core antibody titre. CONCLUSION: The genotype of HCV (III) and a reduction in the core antibody titre appear to be useful parameters for predicting the response to IFN-alpha therapy.

Four novel KVLQT1 and four novel HERG mutations in familial long-QT syndrome.

BACKGROUND: Familial long-QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur due to ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far. We investigated mutations of these genes in LQTS families. METHODS AND RESULTS: Thirty-two Japanese families with LQTS were brought together for screening for mutations. Genomic DNA from each proband was examined by the polymerase chain reaction-single-strand conformation polymorphism technique followed by direct DNA sequencing. In four of the families, comprising 16 patients, mutations were identified in KVLQT1; five other families (9 patients) segregated mutant alleles of HERG. All 25 of these patients carried the specific mutations present in their respective families, and none of 80 normal individuals carried these alleles. Mutations were confirmed by endonuclease digestion or hybridization of mutant allele-specific oligonucleotides. No mutation in SCN5A was found in any family. CONCLUSIONS: We identified nine different mutations among 32 families with LQTS. Eight of these were novel and account for 25% of all types of mutations reported to date. Such a variety of mutations makes it difficult to screen high-risk groups using simple methods such as endonuclease digestion or mutant allele-specific amplification.

Combination chemotherapy for malignant paraganglioma.

Treatment with a combination chemotherapeutic regimen consisting of cyclophosphamide, vincristine, and dacarbazine for malignant paraganglioma with hepatic metastasis is reported. A 51-year-old male presented with tumors in the retroperitoneal space and liver. The patient was diagnosed as having paraganglioma based on elevated levels of serum neuron-specific enolase, urinary catecholamine and vanillylmandelic acid, and on histological findings of the liver specimen. The patient was treated with this combination chemotherapy in repeated 21-day cycles. Temporary improvement in laboratory findings and a 20% reduction in the size of the hepatic masses were observed without severe adverse effects.

Effects of growth factors and gut hormones on proliferation of primary cultured gastric mucous cells of guinea pig.

Almost completely homogenous gastric mucous epithelial cells of guinea pigs were grown to confluence in the presence of 10% fetal calf serum (FCS). FCS, epidermal growth factor (EGF), and insulin significantly increased 5-bromo-2'-deoxyuridine (BrdU) uptake by the cells and EGF together with insulin increased the cells' [3H] thymidine uptake. Basic fibroblast growth factor (bFGF) enhanced EGF-induced DNA synthesis by the cells, but vasoactive intestinal peptide (VIP), secretin, prostaglandin E2 (PGE2), and dibutyryl cyclic AMP (dbcAMP) neither induced DNA synthesis nor enhanced the effect of EGF on DNA synthesis by the cells. Gastrin, cholecystokinin-octapeptide (CCK8), and carbamylcholine chloride (CCh) also did not enhance the effect of EGF on DNA synthesis. 125I-EGF, 125I-bFGF, and 125I-gastrin binding to the gastric mucous cells revealed the presence of high-affinity receptors for EGF and bFGF, but not for gastrin. Northern blot analysis showed the expression of EGF receptor mRNA, but not gastrin receptor mRNA. These results suggest that EGF, insulin, and bFGF may cooperatively regulate gastric mucous cell growth, but that gastrin and other gastrointestinal hormones do not have a direct stimulatory effect on mucous cell growth in the guinea pig.

DNA ploidy heterogeneity in early and advanced gastric cancers.

To evaluate the clinical utility of flow cytometric DNA analysis in gastric cancers, four or more fresh tissue specimens were systematically taken from gastric cancers in 127 consecutive patients including 68 early cancers. DNA ploidy and its variation in individual tumors were determined, and the data were related to clinicopathologic findings. DNA aneuploidy was detected frequently (84.3%) irrespective of tumor progression and correlated significantly with histologic grade (G1-2 [89.6%] vs. G3-4 [76.0%], P < 0.05). DNA ploidy heterogeneity was found in 67.7% of tumors and correlated with invasion depth (mucosa [40.5%] vs. submucosa-serosa [81.2%], P < 0.001), regional lymph node metastases (negative [58.4%] vs. positive [82.0%], P < 0.01), and stage grouping (I [58.8%] vs. II-IV [86.0%], P < 0.01). The maximum DNA index of a tumor correlated significantly with invasion depth (mucosa [1.16, median] vs. submucosa [1.82], P < 0.01) and lymph node metastases (negative [1.22] vs. positive [1.86], P < 0.001). The DNA index of the subpopulation that was the most widely distributed within the tumor was significantly associated with lymph node metastases (negative [1.14, median] vs. positive [1.44], P < 0.001) and histologic grade (G1-2 [1.37] vs. G3-4 [1.12], P < 0.001). More than 80% of the diploid and/or single aneuploid stemline tumors were stage I, whereas more than half of diploid and multiple aneuploid stemline tumors were stage IV. Variation in DNA ploidy rather than presence of DNA aneuploidy correlates best with progression of gastric cancer.

The sign of Leser-Trelat associated with esophageal carcinoma.

A 79-year-old woman was admitted to our hospital with complaints of dysphagia and multiple verrucous papules that had developed over the previous year. The diagnosis of esophageal carcinoma was based on upper gastrointestinal radiography and endoscopic examination with biopsy. The clinical syndrome was consistent with the sign of Leser-Trelat associated with esophageal carcinoma. Although radiation therapy and chemotherapy were undertaken, the patient died 8 months later because of the sign of Leser-Trelat in association with squamous cell esophageal carcinoma.

Induction of cyclooxygenase protein and stimulation of prostaglandin E2 release by epidermal growth factor in cultured guinea pig gastric mucous cells.

The present study was undertaken to investigate whether epidermal growth factor (EGF) could stimulate prostaglandin E2 release, and if so, by what mechanism EGF would exert such an effect in gastric mucosal cells. In cultured guinea pig gastric mucous cells, EGF dose-dependently stimulated prostaglandin E2 release, with maximal stimulation observed at 10 ng/ml. EGF stimulated an increase in cyclooxygenase activity, which was reduced by protein synthesis inhibitor, actinomycin D, and cycloheximide. EGF also stimulated the enzyme protein synthesis estimated by Western blot analysis, whereas EGF did not stimulate phospholipase A2 activity. These results suggest that such an effect of EGF of de novo synthesis of cyclooxygenase protein and prostaglandin E2 release may be involved at least in part in the mechanism of EGF-induced local regulation of gastric mucosal integrity.

Pigmentary retinopathy with nephrotic syndrome, Ménétrier's disease, and diabetes mellitus.

A patient with pigmentary retinopathy, nephrotic syndrome, Ménétrier's disease, and diabetes mellitus is presented. Other complications were congestive heart failure, hypothyroidism, hypertension, and hypertriglyceridemia. Hypogenitalism was also suspected. Pigmentary retinopathy is known to associate with many systemic diseases, which are classified into several syndromes. This case superficially resembles Alström's disease due to the common characteristics of pigmentary retinopathy, diabetes mellitus, renal disease, and hypogenitalism. But clinically and histologically, there are distinct differences. To our knowledge, this association has never been reported.

EGF stimulates both cyclooxygenase activity and cell proliferation of cultured guinea pig gastric mucous cells.

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin all dose-dependently stimulated [3H]-thymidine incorporation into guinea pig gastric mucous cells cultured in vitro. On the other hand, other growth factors, e.g., platelet-derived growth factor (PDGF) and gastrointestinal hormones, such as gastrin, had no effect on DNA synthesis in these cells. Exposure of the cells to EGF at concentrations which stimulated DNA synthesis caused increases in both prostaglandin (PG) E2 release and cyclooxygenase (COX) enzyme protein synthesis, as evaluated by Western blot analysis. These results suggest that such increases in DNA synthesis and PGE2 release may be involved, at least in part, in the mechanism of EGF-induced local regulation of gastric mucosal integrity.

[Clinical significance of DNA ploidy heterogeneity in early gastric cancers].

To evaluate the clinical significance of DNA ploidy heterogeneity (DH), four or more fresh tissue specimens were obtained from a tumor in 68 resected early gastric cancers. DNA content was measured by flow cytometry and the presence of DH was prospectively investigated. The incidence of DH correlated to invasion depth (m < sm), lymph vessel invasion (negative < positive) and tumor size (10 mm or less in diameter < more than 10). When the criteria of indication for minimum surgery were determined as the intramucosal cancer without n, ly and v factor, 85% of contraindication cases demonstrated DH. These results indicate that DH is a useful marker of tumor progression in early gastric cancer and will be an aid for determining indications for minimum resection.

[Flow cytometric analysis of DNA ploidy in intramucosal gastric carcinoma].

In order to investigate whether or not DNA ploidy was altered in intramucosal gastric carcinomas, nuclear DNA content of biopsy specimen was measured using flow cytometry in 38 intramucosal carcinomas. DNA aneuploidy was detected in 27 of 38 lesions (71.1%), and noted more frequently in differentiated carcinomas than in undifferentiated ones (83.0% vs. 20.0%, p < 0.01). There was no significant relationship between the frequency of DNA aneuploidy and macroscopical type or tumor size. DNA aneuploidy was even found in two of three minute carcinomas (5 mm or less in diameter). DNA indices showed 1.2 or lower values in 40% of the lesions with DNA aneuploidy. The average value of DNA index was significantly larger in depressed type than in elevated type (p < 0.01). In conclusion, DNA ploidy is altered in most differentiated intramucosal carcinomas. A high resolution method is essential for accurate determination of DNA ploidy in intramucosal carcinomas, especially elevated ones.

[Flow cytometric DNA analysis of colorectal carcinoma in adenoma].

We evaluated the DNA ploidy in 23 lesions of colorectal carcinoma in adenoma (CIA) and 90 adenomas without carcinomas by flow cytometry using fresh samples. DNA ploidy of carcinoma and adenoma components were assessed, respectively, with 17 paraffin-embedded samples of CIAs. The incidence of DNA aneuploidy (AP) was significantly higher in CIAs than in adenomas (47.8% vs. 12.2%, p < 0.01). Even in adenoma components of CIAs, AP tended to be found more frequent than in adenomas (41.2% vs. 12.2%). The incidence of AP in adenoma components was similar to that in carcinoma components (35.3%) in CIAs. In conclusion, DNA aneuploidy in adenomas may be a marker of malignant potential.