Flooding tolerance in interspecific introgression lines containing chromosome segments from teosinte (Zea nicaraguensis) in maize (Zea mays subsp. mays).

BACKGROUND AND AIMS: Nicaraguan teosinte (Zea nicaraguensis), a species found in frequently flooded areas, provides useful germplasm for breeding flooding-tolerant maize (Z. mays subsp. mays). The objective of this study was to select flooding-tolerant lines using a library of introgression lines (ILs), each containing a chromosome segment from Z. nicaraguensis in the maize inbred line Mi29. METHODS: To produce the ILs, a single F1 plant derived from a cross between maize Mi29 and Z. nicaraguensis was backcrossed to Mi29 three times, self-pollinated four times and genotyped using simple sequence repeat markers. Flooding tolerance was evaluated at the seedling stage under reducing soil conditions. KEY RESULTS: By backcrossing and selfing, a series of 45 ILs were developed covering nearly the entire maize genome. Five flooding-tolerant lines were identified from among the ILs by evaluating leaf injury. Among these, line IL#18, containing a Z. nicaraguensis chromosome segment on the long arm of chromosome 4, showed the greatest tolerance to flooding, suggesting the presence of a major quantitative trait locus (QTL) in that region. The presence of the QTL was verified by examining flooding tolerance in a population segregating for the candidate region of chromosome 4. There was no significant relationship between the capacity to form constitutive aerenchyma and flooding tolerance in the ILs, indicating the presence of other factors related to flooding tolerance under reducing soil conditions. CONCLUSIONS: A flooding-tolerant genotype, IL#18, was identified; this genotype should be useful for maize breeding. In addition, because the chromosome segments of Z. nicaraguensis in the ILs cover nearly the entire genome and Z. nicaraguensis possesses several unique traits related to flooding tolerance, the ILs should be valuable material for additional QTL detection and the development of flooding-tolerant maize lines.

Haplotypes of aldosterone synthase (CYP11B2) gene in the general population of Japan: the Ohasama study.

Since the identification of a chimeric aldosterone synthase which induces mendelian hypertension, polymorphisms in aldosterone synthase (CYP11B2) has been one of major targets for molecular analyses in association with hypertension. To date, four polymorphic variants of CYP11B2, -344T/C at promoter region, a gene conversion in intron 2, 2713A/G (in exon 3) which converts from Lys to Arg at codon 173 (K173R), and 4986T/C (in exon7) which converts from Val to Ala at codon 386 (V386A), have been identified in Caucasian population. Then, linkage disequilibrium between -344T/C polymorphism and a gene conversion in intron 2 or K173R mutation has been described, suggesting the presence of genetic haplotypes in Caucasians. Since the presence of a gene conversion in intron 2 or V386A mutation was still unknown in the Japanese population, all these polymorphisms were examined together to determine the CYP11B2 haplotypes of Japanese, using DNA samples from 1290 participants of the Ohasama study, who represent the general population of a rural community of northern Japan. Molecular analyses demon- strated the presence of a gene conversion of intron 2, but the absence of V386A mutation in Japanese population. The complete linkage disequilibrium between -344T/C polymorphism and K173R mutation was noted. Although -344T allele was linked either with a gene conversion in intron 2 or with normal intron 2, -344C allele was completely linked with normal intron 2. These results indicate the presence of 3 allelic haplotypes of CYP11B2, -344C with normal intron 2 and 173R, -344T with normal intron 2 and 173K, and -344T with converted intron 2 and 173K, in the general Japanese population. The frequency (total 1.0) was 0.35, 0.53, and 0.12, respectively. The presence of allelic haplotypes is considered to be an additional genetic information to individual polymorphism of CYP11B2 to determine the linkage between CYP11B2 polymorphisms and hypertension.

Aldosterone synthase gene (CYP11B2) C-334T polymorphism, ambulatory blood pressure and nocturnal decline in blood pressure in the general Japanese population: the Ohasama Study.

OBJECTIVE: The C-344T polymorphism in the 5'-flanking region of the aldosterone synthase (CYP11B2) gene has been suggested to be associated with hypertension and disturbed circadian blood pressure (BP) rhythm through its effect on aldosterone synthesis. However, previous findings on this topic have been inconsistent. DESIGN: A cross-sectional study. SUBJECTS AND METHODS: We investigated the CYP11B2 C-344T genotype in 802 subjects, aged 40 and over, in a Japanese community, who gave written informed consent and were monitored for 24 h ambulatory BP. RESULTS: The frequencies of the CC, CT, and TT genotypes in these Japanese subjects were 0.14, 0.44, and 0.42, showing a higher frequency of the T allele (0.64) than in Caucasians. Although there was no significant difference in 24 h ambulatory BP levels among the genotypes, the nocturnal decline in BP was significantly greater in the CC homozygous subjects than in other subjects (P = 0.0065 for systolic and P = 0.031 for diastolic decline in nocturnal BP). Detailed analyses demonstrated that this association was significant only in aged (60 years and over) or male subjects. The prevalence of previous cardiovascular disease was significantly less in these subjects with the CC genotype than in those with the TC and TT genotypes, although age, body mass index, male gender, smoking, use of alcohol and antihypertensive medication did not differ among the three genotypes. There was no significant difference among the three genotypes in biochemical and hormonal parameters. CONCLUSION: Although the C-344 T polymorphism of CYP11B2 did not directly influence the level of 24 h BP, the CC genotype was associated with decreased nocturnal BP in elderly or male Japanese. Since prevalence of previous cardiovascular disease was significantly less in homozygous CC subjects, greater nocturnal BP decline in this genotype appears to be beneficial in the circadian BP rhythm.

Induction of apoptosis in melanoma cell lines by p53 and its related proteins.

Melanoma cells rarely contain mutant p53 and hardly undergo apoptosis by wild-type p53. By using recombinant adenoviruses that express p53 or p53-related p51A or p73beta, we tested their apoptotic activities in melanoma cells. Yeast functional assay revealed a mutation of p53 at the 258th codon (AAA [K] instead of GAA [E]) in one cell line, 70W, out of six human melanoma cell lines analyzed (SK-mel-23, SK-mel-24, SK-mel-118, TXM18, 70W, and G361). Adenovirus-mediated transfer of p53, p51A, and/or p73beta suppressed growth and induced apoptotic DNA fragmentation of SK-mel-23, SK-mel-118, and 70W cells. Interestingly, p51A induced DNA fragmentation in them more significantly than p53 and p73beta. By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family members. Apoptotic Bax and antiapoptotic Bcl-2 were not significantly upregulated or downregulated by expression of p53, p51A, or p73beta, except for p53-expressing 70W cells, which contained a larger amount of Bax protein than LacZ-expressing cells. Activation of caspase-3 was demonstrated only in p51A-expressing SK-mel-118 cells. We show here that p51A can mediate apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and p73beta. It is also suggested that in melanoma cells (i) cellular target protein(s) other than Bcl-2 and Bax might be responsible for induction of p51A-mediated apoptosis and (ii) caspase-3 is not always involved in the apoptosis by p53 family members.

High throughput detection of drug-metabolizing enzyme polymorphisms by allele-specific fluorogenic 5' nuclease chain reaction assay.

We have developed an allele-specific fluorogenic 5' nuclease chain reaction assay for detecting polymorphisms in the following human drug-metabolizing enzyme genes: CYP2C9 (CYP2C9*2 and *3), CYP2C19 (CYP2C19*2 and *3), CYP2D6 (CYP2D6*4, *10, *14, *18, and *21(C8)), N-acetyltransferase 2 (NAT2*5B, *6A, and *7B), thiopurine methyltransferase (TPMT*3C), and aldehyde dehydrogenase2 (ALDH2*2). This method is a marriage of two emerging technologies, the use of allele-specific amplification primers for target DNA and hybridization of the TaqMan probe. The TaqMan probe is labeled with both a fluorescent reporter dye and a quencher dye. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. All assays are performed using a single thermocycling protocol. This genotyping method is rapid and highly sensitive and yields a high throughput. It could be applied toward automated large-scale genotyping.

Detection assay of rare variants of the thiopurine methyltransferase gene by PCR-RFLP using a mismatch primer in a Japanese population.

Thiopurine methyltransferase (TPMT) catalyzes the metabolism of important drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. The identification and frequency distributions of several variant TPMT alleles (TPMT*2--*8) have been described recently in many ethnic groups. We have recently demonstrated that TPMT*3C is the most common allele in Japanese subjects; however, it remains to be elucidated whether TPMT*4--*8 variants also exist in Japanese subjects. To detect polymorphisms in the TPMT gene (TPMT*4--*8), we have developed a mismatch polymerase chain reaction and restriction fragment length polymorphism method and conducted a population study of Japanese subjects. Genotyping of these variant forms was carried out in 192 Japanese healthy volunteers. The TPMT*4, TPMT*5, TPMT*6, TPMT*7, and TPMT*8 variants were not detected in any of the samples analyzed. This study provides the first analysis of the TPMT*4--*8 variants in a sample of the Japanese population and indicates that TPMT*4--*8 variants do not occur or are rare alleles in this population.

Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population.

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. The molecular basis for the genetic polymorphism of TPMT has been established for European Caucasians, African-Americans, Southwest Asians and Chinese, but it remains to be elucidated in Japanese populations. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in Japanese samples (n=192) using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 0.8% of the samples (three heterozygotes). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the samples analyzed. This study provides the first analysis of TPMT mutant allele frequency in a sample of Japanese population and indicates that TPMT*3C is the most common allele in Japanese subjects.

Gene therapy for canine alpha-L-iduronidase deficiency: in utero adoptive transfer of genetically corrected hematopoietic progenitors results in engraftment but not amelioration of disease.

Canine alpha-L-iduronidase (iduronidase) deficiency is a model of the human lysosomal storage disorder mucopolysaccharidosis type I (MPS I). We used this canine model to evaluate the therapeutic potential of hematopoietic stem cell (HSC) gene therapy for enzyme deficiencies. In previous studies, iduronidase-deficient dogs infused with autologous marrow cells genetically modified to express iduronidase had long-term engraftment with provirally marked cells, but there was no evidence of proviral iduronidase expression or clinical improvement. The presence of humoral and cellular immune responses against iduronidase apparently abrogated the therapeutic potential of HSC gene therapy in these experiments. To evaluate HSC gene therapy for canine MPS I in the absence of a confounding immune response, we have now performed in utero adoptive transfer of iduronidase-transduced MPS I marrow cells into preimmune fetal pups. In three separate experiments, 17 midgestation fetal pups were injected with 0.5-1.5 x 10(7) normal or MPS I allogeneic long-term marrow culture (LTMC) cells transduced with neo(r)- or iduronidase-containing retroviral vectors. Nine normal and three MPS I pups survived the neonatal period and demonstrated engraftment of provirally marked progenitors at levels of up to 12% for up to 12 months. However, the proportion of provirally marked circulating leukocytes was approximately 1%. Neither iduronidase enzyme nor proviral-specific transcripts were detected in blood or marrow leukocytes of any MPS I dog. Humoral immune responses to iduronidase were not detected in neonates, even after "boosting" with autologous iduronidase-transduced LTMC cells. All MPS I dogs died at 8-11 months of age from complications of MPS I disease with no evidence of amelioration of MPS I disease. Our results suggest that iduronidase-transduced primitive hematopoietic progenitors can engraft in fetal recipients, contribute to hematopoiesis, and induce immunologic nonresponsiveness to iduronidase in MPS I dogs. However, the therapeutic potential of HSC gene transfer in this model of iduronidase deficiency appears to be limited by poor maintenance of proviral iduronidase gene expression and relatively low levels of genetically corrected circulating leukocytes.

Nontargeted stable integration of recombinant adeno-associated virus into human leukemia and lymphoma cell lines as evaluated by fluorescence in situ hybridization.

A number of studies on human epithelial cells of varying origin have demonstrated integration of recombinant adeno-associated virus (AAV) vectors into a variety of chromosomes compared with the site-specific integration on chromosome 19 predominantly observed for wild-type (wt) AAV. We have constructed a recombinant AAV (rAAV) vector and tested the integration into hematopoietic cells, using the human acute myeloid leukemia cell line AML5 and the human non-Hodgkin's lymphoma cell line OCI-LY18 as targets. The integration sites were visualized by fluorescence in situ hybridization (FISH). Positive signals were observed for chromosomes 1, 2, 3, 8, 14, 15, 19, and Y. The majority of cells demonstrated integration into one specific site. A minority showed simultaneous integration into more than one chromosome. The frequency of observed integrations was not uniformly distributed among chromosomes; for instance, in AML5 chromosome 2 seemed to be favored. Colony-derived AML5 clones bore unique integration patterns indicating successful transduction of clonogenic progenitor cells with high proliferative potential. The integration was stable and observed for more than 12 months after transduction. FISH has been shown to be a powerful tool for detailed analyses of rAAV integration patterns and can be used to evaluate targets and transduction conditions.

Adoptive transfer of genetically modified human hematopoietic stem cells into preimmune canine fetuses.

To develop a surrogate model system for assaying gene transfer into human hematopoietic stem cells (HSCs) with in vivo repopulating potential, we injected human marrow cells transduced with a reporter retroviral vector in long-term marrow cultures (LTMCs), into the yolk sacs of preimmune canine fetuses. Of eight mid-gestation fetuses injected through the exteriorized uterine wall and under ultrasound guidance, seven were born alive. One puppy died in the neonatal period accidentally. The remaining six puppies are all healthy at 31 months of age. There was no evidence for graft-versus-host disease or any untoward effects of in utero adoptive transfer of transduced human LTMC cells. All puppies were chimeras. Human cells, detected by fluorescence in situ hybridization, were present in blood, declining from 38% to 0.05% between 10 and 44 weeks after birth. Corresponding numbers for marrow were from 20% to 0.05%. Human cells were also detected in assays of hematopoietic cell progenitors and in stimulated blood cultures. All six puppies were positive for the presence of proviral DNA at various time-points after birth. In three dogs, provirus was detected up to 41 weeks after birth in blood or marrow, and in one dog up to 49 weeks in blood. These data support the further development of this large-animal model system for studies of human hematopoiesis.

Retroviral-mediated transfer and expression of the multidrug resistance protein 1 gene (MRP1) protect human hematopoietic cells from antineoplastic drugs.

Multidrug resistance protein (MRP1) is a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily that confers multidrug resistance. The transfer and expression of the MRP1 gene in human hematopoietic stem cells may be a useful alternative to multidrug resistance (MDR1) gene transfer for protection from the myelosuppressive effects of chemotherapy in cancer patients. We constructed a gibbon ape leukemia virus packaging cell line (PG13) using the human MRP1 cDNA in a Moloney murine leukemia virus (MoMuLV) backbone containing a modified LTR. This PG13-based cell line, designated MRP1-PG13, produces retroviral vectors bearing the MRP1 gene at a titer of 1.7x10(5) viral particles/ml. Transduction of the human leukemic cell line K562 showed that viral MRP1-PG13 supernatants routinely transfer the MRP1 gene to approximately 35% of target K562 cells, of which at least one third are capable of proliferating in the presence of otherwise toxic concentrations of etoposide. Southern blot analyses indicated that most clones had only one proviral integration. Northern blot analysis of expanded K562 clones showed the presence of a major full-length approximately 8-kb MRP1 transcript as well as a minor approximately 6-kb transcript in all clones. Flow cytometric analysis of the producer cells and clones of transduced K562 cells demonstrated significantly increased MRP1 expression in these cells (approximately 30-fold increase). Human bone marrow mononuclear cells and CD34+ cells were also transduced with MRP1-PG13 supernatants on fibronectin-coated culture flasks in the presence of SCF, IL-3, and IL-6. PCR analysis of individual hematopoietic colonies in methylcellulose cultures demonstrated proviral DNA in approximately 10% of unselected human hematopoietic progenitor cells cultured from nonsorted mononuclear cell samples and in up to approximately 75% of progenitors when CD34-enriched cell populations were targeted. To assess functional MRP1 gene expression, normal human hematopoietic progenitors and K562 cells were cultured in methylcellulose assays containing vincristine or etoposide. All transduced samples gave rise to approximately 10% drug-resistant colonies, which were shown to be provirus-positive by PCR. Our studies document the development of an amphotropic MRP1 retroviral vector producer cell line and pave the way for large animal and preclinical studies of chemoprotection by MRP1 gene transfer.

High serum levels of granulocyte-macrophage colony-stimulating factor in patients with liver cirrhosis and granulocytopenia.

Leucopenia is often observed in patients with liver cirrhosis. We measured levels of serum granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with liver cirrhosis by a sensitive enzyme linked immunosorbent assay. Eight out of 22 patients with liver cirrhosis had detectable serum GM-CSF (range, 55 to 245 pg/ml:mean, 135 pg/ml). Serum GM-CSF was detected in all patients with a granulocyte count below 2.0 x 10(9)/l, but in only one patient with a granulocyte count above 2.0 x 10(9)/l. Haemoglobin concentration, platelet count, serum albumin, total bilirubin and aminotransferase levels did not correlate with serum GM-CSF levels. These findings may reflect a feedback mechanism between the number of circulating granulocytes and serum GM-CSF levels in patients with cirrhosis.

[Study on the inhibitory effect of oral granisetron against nausea/vomiting induced by cytosine arabinoside containing chemotherapy for tumors in the hematopoietic organs].

We investigated the antiemetic effect, safety and usefulness of granisetron tablet on nausea/vomiting induced by cytosine arabinoside (Ara-C) in the chemotherapy for tumors in the hematopoietic organs. Out of 52 cases with malignant tumors in the hematopoietic organs including acute leukemia, 30 in granisetron group had no antiemetic treatment, were evaluated for the clinical efficacy of granisetron and 22 in control group. Their chemotherapies were combination therapy with Ara-C and daunorubicin (DNR), Ara-C and mitoxantrone (MIT), or Ara-C and etoposide (VP-16). In the trial, the dosage of granisetron tablet was 2 mg once a day, and the drug was given before each chemotherapy for 6 consecutive days. In clinical efficacy the effective rate of granisetron (the percentage of cases in which the trial drug was assessed as "Remarkably effective" or "Effective") was more than 80% on each day of administration. There was no adverse event. As the abnormal laboratory test value, only 1 case tested positive in urine protein, whose causal relation to the trial drug was judged as "Unassessable". Granisetron was judged as "Safe" in 31 out of 32 cases (96.9%). In terms of usefulness, the drug was rated "Extremely useful" or "Useful" in 26 out of 30 cases (86.7%). The above results have shown that granisetron tablet, when administered orally once daily at a dose of 2 mg, has an excellent antiemetic effect, and is a safe and useful drug.

Primary papillary adenocarcinoma in the third portion of the duodenum.

We herein report a rare case of papillary adenocarcinoma which occurred in the third portion of the duodenum. The patient was a 62-year-old Japanese male who was admitted due to vomiting and right lower abdominal pain. No abnormal findings were found in the laboratory examinations. After a diagnosis of primary duodenal carcinoma was made by radiologic, endoscopic and ultrasonographic studies, a pancreatoduodenectomy was performed. The histology of the resected specimen revealed papillary adenocarcinoma, with invasion reaching to the pancreatic body. Some characteristic features of the disease are also reviewed.

[A case of Fitz-Hugh-Curtis syndrome confirmed by laparoscopy].

A 25-year-old Japanese female was admitted to the Department of Surgery in Kyushu Koseinenkin Hospital because of serious right hypochondralgia. Gastrofiberscopy, abdominal ultrasonography, intravenous pyelography and irrigoscopy did not reveal the origin of the pain, and she was introduced to the Department of Internal Medicine. Because enzyme immunoassay of the uterine cervical specimen in the Department of Urology showed positive chlamydial antigen, we suspected her of perihepatitis induced by Chlamydia trachomatis (Fitz-Hugh-Curtis syndrome). Laparoscopy revealed typical violin string adhesions between the anterior surface of the liver and the corresponding parietal peritoneum, and the diagnosis was confirmed. After an administration of Ofloxacin was started, the symptom disappeared completely. It is considered to be important to remember this syndrome when examining a young women with right hypochondralgia.

[Five cases of Crow-Fukase syndrome].

We presented five cases of Crow-Fukase syndrome. Plasma cell hyperplasia or dyscrasia in bone marrow were recognized in all cases and localized bone lesion was seen in three cases. Thyroid dysfunction was seen in three cases; hyperthyroidism in one case and hypothyroidism in two cases, which was considered to be one of the characteristics though it has seldom been described in this disease. Two of four cases treated with prednisolone had good responses but two cases treated with interferon had no effect.

Serum granulocyte colony-stimulating factor levels in umbilical cord blood of normal full-term neonates.

We measured granulocyte colony-stimulating factor (G-CSF) levels in cord blood of 59 normal full-term neonates immediately after birth and the subsequent changes in G-CSF levels of 16 cases by our modified enzyme-linked immunosorbent assay (ELISA) for G-CSF. Ten out of 59 cases examined (17%) showed G-CSF levels in cord blood after delivery between 20 and 57 pg/ml, although in the remaining cases, the G-CSF levels were below 20 pg/ml, which was our minimal detection level. A direct relationship between G-CSF levels and white blood cell count, absolute granulocyte numbers in cord blood, gestation age or weight was not observed. Although G-CSF levels in cord blood after delivery remained below 20 pg/ml in 5 cases out of 16 tested, those in the remaining 11 cases (69%) subsequently became elevated after delivery. The peak G-CSF level in cord blood after delivery ranged from 26-364 pg/ml, and the time of it was between 4.5 and 18 h. As G-CSF level per wet weight of placenta was high (124 x 29 pg/ml), these subsequent elevations of G-CSF in cord blood after delivery may result from a gradual influx from the placenta.