Molecular and cellular characterizations of a cDNA clone encoding a novel isozyme of aldehyde dehydrogenase from rice.

Aldehyde dehydrogenases (ALDHs) are a group of enzymes catalyzing the conversion of aldehydes to the corresponding acids. In mammals and yeasts, at least two isozymes of ALDH are known to be involved in ethanol metabolism (cytosolic ALDH1 and mitochondrial ALDH2). Although mitochondrial ALDH isozymes have previously been identified in several plants, such as maize and tobacco, it is unclear whether cytosolic ALDH isozymes also exist in plants. In this study, we identified and characterized a cDNA clone encoding aldehyde dehydrogenase (ALDH1a) from rice (Oryza sativa L. cv. Nipponbare). The open reading frame of this clone did not contain a typical mitochondrial targeting signal. Analysis of the subcellular localization of ALDH1a using green fluorescent protein (GFP) suggested that ALDH1a is a cytosolic enzyme rather than a mitochondrial enzyme. A genomic Southern hybridization indicated that sequences homologous to the ALDH1a gene are present in at least two regions of the rice genome. Amplification by RT-PCR showed that ALDH1a is expressed strongly in roots, but not in leaves, of rice seedlings, suggesting that ALDH1a functions in roots.

Characterization and expression of the genes for cytochrome c oxidase subunit VIb (COX6b) from rice and Arabidopsis thaliana.

Many of the subunits of cytochrome c oxidase (COX) in the mitochondria of higher plants are encoded by nuclear genes. These genes are less characterized compared to mitochondrial-encoded genes. We previously isolated a cDNA encoding COX6b (designated OsCOX6b1 in this study) from the rice nuclear genome and analyzed its expression. The deduced protein had an extended N-terminus compared with human and yeast COX6b proteins. In this study, we identified another COX6b gene (OsCOX6b2) in rice and revealed that it was actually expressed. The deduced protein of this gene did not have an extended N-terminus and had about the same size as the human and yeast proteins. Genomic Southern hybridization analysis revealed that there was at least one OsCOX6b-homologus sequences in the rice genome other than OsCOX6b1 and OsCOX6b2. Furthermore, we identified three COX6b genes in a dicotyledonous plant, Arabidopsis thaliana. One of these genes (AtCOX6b1) was relatively long, with a length similar to that of OsCOX6b1, and the other two (AtCOX6b2 and AtCOX6b3) were shorter, with lengths similar to the length of OsCOX6b2. Genomic Southern hybridization analysis indicated there were no additional COX6b genes in the Arabidopsis genome. The coding regions of OsCOX6b1 and AtCOX6b1 were separated by four introns and those of OsCOX6b2, AtCOX6b2 and AtCOX6b3 were separated by three introns. A Northern hybridization analysis showed that OsCOX6b1, AtCOX6b1 and AtCOX6b3 were expressed in all organs examined, although with some differences in the amount of expression among the organs. OsCOX6b2 and AtCOX6b2 were strongly expressed in roots but most of the transcripts of AtCOX6b2 were degraded. The evolution of COX6b genes from rice and Arabidopsis is discussed.

Transcript levels of tandem-arranged alternative oxidase genes in rice are increased by low temperature.

We identified two genes for alternative oxidase (AOX) from rice. One AOX gene (designated AOX1a) is located approx. 1.9 kb downstream of another AOX gene (designated AOX1b). Comparison of the genomic and cDNA sequences of the two AOX genes showed that the AOX1a gene is interrupted by three introns, as are AOX genes of other plants. On the other hand, two introns are inserted in the AOX1b gene. The predicted AOX1a and AOX1b precursor proteins consist of 332 and 335 amino acid residues, respectively. A genomic Southern hybridization analysis indicated that rice has several AOX genes other than the two tandem-arranged AOX genes. Steady-state mRNA levels of both of the genes for AOX1a and AOX1b were increased under low temperature (4 degrees C). However, no difference in the pattern of induction of transcription between the genes for AOX1a and AOX1b was observed.

Transcript levels of the nuclear-encoded respiratory genes in rice decrease by oxygen deprivation: evidence for involvement of calcium in expression of the alternative oxidase 1a gene.

We investigated the effect of oxygen on the expressions of respiratory genes encoded in the nuclear and mitochondrial genomes of rice (Oryza sativa L.). Hypoxic treatment decreased the transcript levels of nuclear-encoded, but not mitochondrial-encoded respiratory genes. The effects of ruthenium red (an inhibitor of Ca(2+) fluxes from organelles) and/or CaCl(2) on plants under hypoxic conditions suggested that Ca(2+) is a physiological transducer of a low-oxygen signaling pathway for expression of the alternative oxidase 1a gene (AOX1a), but not for expressions of genes involved in the cytochrome respiratory pathway, in rice.

A novel plant nuclear gene encoding chloroplast ribosomal protein S9 has a transit peptide related to that of rice chloroplast ribosomal protein L12.

We have cloned a novel nuclear gene for a ribosomal protein of rice and Arabidopsis that is like the bacterial ribosomal protein S9. To determine the subcellular localization of the gene product, we fused the N-terminal region and green fluorescent protein and expressed it transiently in rice seedlings. Localized fluorescence was detectable only in chloroplasts, indicating that this nuclear gene encodes chloroplast ribosomal protein S9. The N-terminal region of rice ribosomal protein S9 was found to have a high sequence similarity to the transit peptide region of the rice chloroplast ribosomal protein L12, suggesting that these transit peptides have a common lineage.

Flow cytometry based on heterogeneity index score compared with urine cytology to evaluate their diagnostic efficacy in bladder tumor.

Flow cytometric (FCM) examination of DNA distribution based on heterogeneity index score (HIS) and ploidy pattern and of bladder irrigation specimens were compared with conventional urine cytologic examinations to evaluate the diagnostic efficacy of each method. Of 56 patients with histologically proved bladder tumors, 24 (43%) had positive urine cytology, 41 (73%) had positive FCM, and 45 (80%) had positive urine cytology and/or positive FCM. When bladder tumors were graded histologically, 8 of 11 patients (73%) with grade I bladder carcinoma, 22 of 28 patients (79%) with grade II, and 15 of 17 patients (88%) with grade III had positive urine cytology and/or positive FCM. Of the 28 patients with grade II, 12 with positive urine cytologic results had higher mean HIS (117.5) when compared with 16 (61.1) in whom urine cytology was negative. Of those who continued to have positive FCM in the face of negative findings on cystoscopic and urine cytologic examinations during follow-up, 3 patients eventually were found to have tumors (2 distal ureter, 1 bladder). These results indicate that FCM examinations for DNA distribution of bladder irrigation specimens are as useful as conventional urine cytology in the management of bladder tumors, can be more sensitive for detection and monitoring of the disease, and can contribute further to accurate diagnosis of the disease when combined with conventional urine cytology.

One-stage total cystectomy and ileal loop diversion in patients over eighty years' old with bladder carcinoma. Pre- and postoperative functional reserve of various organs.

During the period from 1976 to 1981, 364 patients with bladder carcinoma were seen at the Keio University Hospital. Extensive preoperative investigation of pulmonary, cardiovascular, and renal function was obtained in all patients. Of the 12 patients studied, 9 underwent a one-stage total cystectomy and ileal loop diversion and the remaining 3 a two-stage procedure. Of the 9 patients, decreased FEV 1.0 per cent by spirometry was noted in 5, ECG abnormality such as bundle branch blocks in 8, and diminished creatinine clearance ranging from 28 to 68 ml/min were observed in all 9. Major postoperative complications included pyelonephritis in 2 patients, pneumonia in 1, pelvic abscess in 2, renal insufficiency in 3, and paralytic ileus in 2. There was no immediate postoperative death. In these elderly patients, functional reserve of the lung, heart, and kidney is less than optimal and is further decreased by major surgical procedures. Therefore, total cystectomy in the elderly patients is justifiable only in a selected group of patients, when functional status of the vital organ is thoroughly worked up and prophylactic and therapeutic measures are instituted promptly if indicated.

RNA editing of transcripts of the gene for apocytochrome b (cob) in rice mitochondria.

RNA editing was examined in rice mitochondrial apocytochrome b (cob) transcripts. Nineteen C-U conversions were found, and most of them changed the polypeptide sequence encoded by genomic DNA sequence. Evidence for partial and excess editing was also found.

Identification of cDNA encoding cytochrome c oxidase subunit 5c (COX5c) from rice: comparison of its expression with nuclear-encoded and mitochondrial-encoded COX genes.

Little is presently known about the nuclear-encoded genes for cytochrome c oxidase (COX) in higher plants. In rice, only the nuclear-encoded COX5b gene has been reported. To understand the relationship between the expression of nuclear-encoded and mitochondrial-encoded COX genes in rice, we first characterized a cDNA encoding one of the other nuclear COX genes, COX5c, which encodes 63 amino acids. The deduced amino acid sequence of COX5c from rice was highly homologous to that from sweet potato. Genomic Southern hybridization indicated that the rice COX5c subunit is encoded by a single copy of the COX5c gene. Furthermore, we compared the expression patterns of the nuclear-encoded COX5c and COX5b genes with the expression pattern of the mitochondrial-encoded COX1 gene among several organs by Northern blot analysis. The results suggested that regulatory systems of expression between the nuclear-encoded and the mitochondrial-encoded COX genes are different among different organs in rice.

The gene for alternative oxidase-2 (AOX2) from Arabidopsis thaliana consists of five exons unlike other AOX genes and is transcribed at an early stage during germination.

We investigated the expressions of genes for alternative oxidase (AOX1a, AOX1b, AOX1c and AOX2) and genes for cytochrome c oxidase (COX5b and COX6b) during germination of Arabidopsis thaliana, and examined oxygen uptakes of the alternative respiration and the cytochrome respiration in imbibed Arabidopsis seeds. A Northern blot analysis showed that AOX2 mRNA has already accumulated in dry seeds and subsequently decreased, whereas accumulation ofAOX1a mRNA was less abundant from 0 hours to 48 hours after imbibition and then increased. The increase of the capacity of the alternative pathway appeared to be dependent on the expressions of both AOX2 and AOX1a. On the other hand, steady-state mRNA levels of COX5b and COX6b were gradually increased during germination, and the capacity of the cytochrome pathway was correlated with the increase of expressions of the COX genes. Antimycin A, the respiratory inhibitor, strongly increased the expression of AOX1a but had no effect on the expression of AOX2. A 5'RACE analysis showed that AOX2 consists of five exons, which is different from the case of most AOX genes identified so far. Analysis of subcellular localization of AOX2 using green fluorescent protein indicated that the AOX2 protein is imported into the mitochondria.

Antibody-mediated insulin resistance treated by cessation of insulin administration.

A 45-year-old Japanese man was referred to our hospital because of hyperglycemia despite the administration of as much as 120 U/day of human insulin. He had no history of injecting animal insulin. Free insulin was below 5 microU/ml, but a high titer of total insulin (about 3,000 microU/ml) was observed, suggesting the presence of antibodies against human insulin. Scatchard analysis showed an increased insulin binding capacity in the plasma characterized by a higher affinity for insulin. He was successfully treated by cessation of insulin administration. A Scatchard analysis series showed that a reduction in the insulin binding capacity of antibodies paralleled the improvement in glycemic control.

[A comparative study between cefodizime (CDZM) and cefotaxime (CTX) in respiratory tract infections].

The clinical efficacy and safety of Cefodizime (CDZM), a new cephem antibiotic, was objectively compared with that of Cefotaxime (CTX) in patients with respiratory infections under a well-controlled comparative study. Patients were administered CDZM or CTX by drip infusion b.i.d. for 14 days in principle at a daily dose of two grams. The parameters assessed were clinical efficacy, safety and clinical usefulness. The following results were obtained: 1. On the basis of committee judgement the clinical efficacy rate was 78.1% (125/160) for the CDZM group, 82.7% (124/150) for the CTX group, and no significant difference was observed between the two drug groups. On the other hand, on the basis of judgement by physicians in charge, the clinical efficacy rate was 83.1% (133/160) for the CDZM group, 83.9% (125/149) for the CTX group, and no significant difference was observed between the two groups. 2. The corresponding figures for patients with pneumonia and pulmonary suppuration were 82.4% (70/85) for the CDZM group, 79.7% (59/74) for the CTX group and no significant difference was observed according to the committee judgement. The judgement by physicians in charge also revealed 83.5% (71/85) for the CDZM group and 82.2% (60/73) for the CTX group. No significant difference was noted between the two groups. While, the committee judgement for the clinical efficacy in patients with chronic respiratory tract infections showed 73.3% (55/75) for the CDZM group, 85.5% (65/76) for the CTX group, and no significant difference was observed between the two drug groups. The corresponding figures were 82.7% (62/75) for the CDZM group, 85.5% (65/76) for the CTX group, and no significant difference was observed between the two drug groups on the judgement by physicians in charge. Furthermore, the clinical efficacy of both drugs on chronic respiratory tract infections was assessed according to "Criteria for Evaluation of Clinical Efficacy of Chemotherapeutics on Chronic Respiratory Tract Infection". It was 76.8% (53/69) for the CDZM group, 76.3% (58/76) for the CTX group, and no significant difference was observed between the two groups. 3. The bacteriological eradication rate of causative pathogens was 92.4% out of 66 patients treated with CDZM and 95.5% out of 67 patients treated with CTX in whom judgement was possible. No significant difference was observed between the two drug groups. 4. The adverse reactions occurred in 4 (2.2%) patients for the CDZM group and 8 (4.5%) patients for the CTX group respectively, with no significant inter-group difference in frequency of these reactions for all cases.(ABSTRACT TRUNCATED AT 400 WORDS)

[Priapism with malignant lymphoma: a case report].

A case with priapism in a 66-year-old male due to malignant lymphoma is reported. Physical examination revealed an erected penis and enlarged lymph nodes in left neck and bilateral inguinal areas. Rectal digital examination of prostate revealed an irregular surface and swollen hard left lobe. Open biopsy of the bladder and vesicostomy were carried out. Several nodular lesions in the trigone and in the left wall were excised. Pathologically the diagnosis was malignant lymphoma, diffuse, large cell type and clinical stage was stage IV. CHOP therapy with bleomycin and methotrexate was performed. Priapism disappeared 7 days after the initiation of the chemotherapy, but impotence has been noticed after the chemotherapy for more than one year. This will be the first case with priapism due to malignant lymphoma reported in Japan so far.

[Establishment of two renal cancer cell lines].

Two renal cell carcinoma (RCC) cell lines, JMSU2 and JMSU3, derived from the primary sites of mixed cell type and spindle cell type RCC, respectively, have been established and maintained for 31 and 22 months. Karyotypic analysis revealed human karyotypes with modal numbers of 84 and 55, respectively. Consistent chromosomal abnormalities were 1p+, 3p-, 6q- or 8p- in the JMSU2 cells and 1p-, inv (5p + q-) or loss of sex chromosome in the JMSU3 cells. Electron microscopy revealed abundant glycogen granules, lipid droplets and microvilli. The JMSU3 cells transplanted to nude mice produced tumors with a spindle cell pattern similar to that of the original tumor. High concentrations of cytokines, such as interleukin-6 (145,000 pg/ml), interleukin-8 (35,300 pg/ml) and granulocyte-colony stimulating factor (6,340 pg/ml), were detected in the culture supernatant of the JMSU3 cells. Interleukin-1 beta (IL-1 beta) dose-dependently inhibited the proliferation of the JMSU2 and JMSU3 cells in culture. Tumor cytotoxic factor/hepatocyte growth factor (TCF/HGF) dose-dependently enhanced JMSU3 cell proliferation, but suppressed JMSU2 cell proliferation. These findings suggest that IL-1 beta and TCF/HGF have regulatory roles in the proliferation of RCC.

[Demonstration of interleukin-1 (IL-1) as an autocrine growth inhibitor in renal cancer cell line, TC-1].

In this report, we demonstrated that Interleukin-6 (IL-6) production could be induced by stimulating renal cell carcinoma cell lines, namely ACHN, Caki-1 and TC-1 cells with Interleukin-1 beta (IL-1 beta). IL-1 beta had no effects on cell proliferation in ACHN cells. However, IL-1 beta could suppress cell proliferation in Caki-1 and TC-1 cells. Flow cytometric cell cycle analysis by double staining method with propidium iodide and Proliferating cell nuclear antigen (PCNA) disclosed IL-1 beta caused cell accumulation at G1 phase. Fine granules were visualized in perinuclear area of TC-1 cells treated with IL-1 beta under microscopy. High electron density granules and spherically dilated rough endoplasmic reticula were observed by electron microscopic examinations. In TC-1 cell culture, IL-1 beta excretion into the supernatant was demonstrated by bioassay and ELISA. These results suggest that IL-1 beta functions as an "autocrine growth inhibitor" against TC-1 cells. Half-maximal inhibition of IL-1 beta and IFN-alpha was 6.5 pg/ml, and 720 U/ml, respectively for TC-1 proliferation and combination of these cytokines showed enhanced activity in cell growth inhibition.

[Lymphadenectomy in the operative treatment of renal pelvic and ureteral carcinoma].

We evaluated 18 patients with renal pelvic and ureteral carcinoma focusing on the operative treatment. All the patients underwent nephroureterectomy and partial cystectomy, except for two patients, with CIS of the ureter and renal insufficiency, and one with invasive bladder carcinoma, in whom partial ureterectomy and total cystectomy were performed, respectively. Lymphadenectomy were performed in all the patients and resected lymph nodes were from renal hilus, para-aorta or vena cava, according to the affected side, intra aorto-caval and all the pelvic nodes in the affected side. Lymph node metastases were found in 4 patients (22%), 2 in the primary and distant nodes and the other in the regional nodes only. Skipped lesion of the lymph node metastasis was not rare and lymph node dissection from the renal hilus to the pelvis on the affected side should be included in the operative treatment of renal and ureteral carcinoma.

[Continent urinary reservoir (CUR) for supravesical diversion. Pouchmetric properties of the colonic CUR and role of detubularization].

Eight patients underwent pouchmetry at 12 months after creation of the colonic continent urinary reservoir (CUR) for supravesical diversion. Pouchmetry revealed a volume-dependent tonic and phasic increase in pouch pressure in 3 patients who had undergone pouch construction using the partially detubularized right colon (Heineke-Mikulicz type closure after splitting the transverse and ascending colon at the antimesenteric border), whereas the volume-dependent phasic increase in pouch pressure was much less remarkable in 5 patients whose pouch was constructed using the totally detubularized right colon (Heineke-Mikulicz type closure after splitting the whole colonic segment including the cecum). The maximum pouch pressure at the pouch volume of 400 to 500 ml was significantly higher (p less than 0.05) in the partial detubularization group (46 +/- 6.0 cmH2O, mean +/- S.D.) than in the total detubularization group (12 +/- 1.6 cmH2O). In creation of the low-pressure pouch using the right colon, detubularization should extend to the whole portion of the isolated colonic segment.

[A case of teratoma of the testis with retroperitoneal lymph nodes involved with malignant teratoma after chemotherapy].

This report describes a case of teratoma with retroperitoneal lymph nodes involved with malignant teratoma (enteric adenocarcinoma) after extensive chemotherapy for the original testicular cancer. A 18-year-old man with a mixed cell tumor (embryonal carcinoma+teratoma+yolk sac tumor) received three courses of VAB-6 chemotherapy for bulky mass following inguinal orchiectomy. He was referred to Tochigi Cancer Center for treatment of a residual mass. He was treated with resection of the mass combined with left nephrectomy due to severe adhesion and pathological diagnosis of the resected lymph node was mature teratoma with a massive necrotic tissue. Two courses of BEP chemotherapy were given to the patient following the surgery. Six months after completion of chemotherapy, a retroperitoneal mass of 1.5 cm in diameter, was detected by CT scan. Standard retroperitoneal lymph node dissection was performed and the pathological diagnosis of the lymph node was teratoma with malignant transformation containing enteric adenocarcinoma. Teratomatous portion of the primary lesion was precisely re-examined and adenocarcinoma, similar histology to the retroperitoneal mass, was identified. He received two courses of EAP chemotherapy (Cis-platin+etoposide+doxorubicin) as an adjuvant chemotherapy following the surgery and he is alive with no evidence of recurrence for 21 months. Presence of non germ cell malignancy after chemotherapy in testicular cancer has been regarded as a rare phenomenon. Flow cytometric DNA analyses of both embryonal carcinoma and teratoma in the primary lesion, mature teratoma and teratoma with malignant transformation of the retroperitoneal lymph node disclosed that these tumors were all aneuploid tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

[Production of a lymphocyte chemotactic factor by human renal cancer cells].

Tumor infiltrating lymphocyte (TIL) is commonly observed in renal cell carcinoma (RCC) tissues. First step of lymphocyte accumulation in cancerous tissues is the lymphocyte migration toward cancer cells. However, no conclusion has been drawn which cytokine is involved in TIL of RCC. The purpose of this study is the identification of a lymphocyte chemotactic factor (chemokine) produced by a newly established RCC cell line. A new human renal cancer cell line (TC-2) was established from a primary site of a 52-year-old man. A marked lymphocyte infiltration was noticed at the cancer tissue. A tissue culture has been continued for 24 months. Flow cytometric analysis of this cell line revealed DNA aneuploidy. A human karyotype, with a modal number of 72, and consistent abnormalities, such as 4q+ and 5q-, were demonstrated by Giemsa banding analysis. Approximately 2.9 fold of lymphocyte chemotactic activity was detected in the culture supernatant of TC-2 cells (TC-2CM) as measured by in vitro migration assay. Sixty percent of this activity was abrogated by adding the neutralizing antibody against interleukin-8 (IL-8) into TC-2CM. Analysis of surface markers of migrating lymphocytes disclosed that lymphocytes expressing CD3, CD8 and CD16 phenotype predominantly showed migration. These results suggested that chemotactic activity for lymphocytes derived from TC-2 cells was partly IL-8.

[A case of retroperitoneal dedifferentiated liposarcoma with tumor thrombus in the inferior vena cava].

We experienced a case of retroperitoneal dedifferentiated liposarcoma with tumor thrombus in the inferior vena cava. A 45-year-old man was pointed out a retroperitoneal mass on ultrasound examination. He was referred to the Department of Urology, Tochigi Cancer Center for critical evaluation. Image diagnostic procedures showed a retroperitoneal mass surrounded by loose tissue and large thrombus in the inferior vena cava. The tumor was removed surgically with right lobe of the liver and segment of the inferior vena cava. Histological examination revealed the tumor composed of the peripheral well differentiated liposarcoma and the central dedifferentiated liposarcoma. Characteristic radiological findings of retroperitoneal liposarcoma with dedifferentiation could be pointed out.