Brachytic 1 of barley (Hordeum vulgare L.) encodes the α subunit of heterotrimeric G protein.

Physiological and molecular biological analysis of the dwarf barley (Hordeum vulgare L.) mutant brachytic 1 (brh1) was conducted. The root responses of brh1 to brassinolide were weaker than those of wild type, but the responses of leaf segments of dark-grown plants were not. Responses of brh1 to gibberellin A3 were similar to or slightly stronger than those of wild type. Endogenous levels of these hormones in young seedlings were not clearly different between brh1 and wild type. Skotomorphogeneses of brh1 were similar to those of wild type. Some of these physiological characteristics of brh1 resemble those of the dwarf rice (Oryza sativa L.) mutant daikoku (dwarf1; d1), whose dwarfism is caused by a mutation in the heterotrimeric G protein α (Gα) subunit. A database search indicated that the barley Gα gene is located near the locus where Brh1 has already been genetically mapped. Sequences of the Gα gene and cDNAs of five brh1 alleles contained substitutions and a deletion that lead to the production of abnormal Gα proteins. These results indicate that the phenotype of brh1, similarly to that of d1, is caused by mutations in an orthologous gene encoding Gα.

An alternative mechanism for cleistogamy in barley.

Cleistogamy in barley is genetically determined by the presence of the recessive allele cly1, but the dominant allele at the linked locus Cly2 is epistatic over cly1. Although the molecular basis for cly1 action is well understood, that of Cly2 is not. Here we show that anther non-extrusion can occur not just when the lodicules fail to expand adequately (a trait which is fully determined by the allelic state at the cly1 locus), but by the premature timing of anthesis before the spike has emerged from the boot. The transcription of HvAP2 at cly1 is unaffected by the timing of anthesis. Where this occurs prematurely, by the time that the spike has emerged from the boot, the lodicules have already become shrunken and have lost the capacity to push the lemma and palea apart. Premature anthesis appears to be governed by a dominant gene, probably Cly2. Of the three phases of development of a non-cleistogamous barley floret (spike emergence from the boot, floret gaping induced by lodicule expansion and anther extrusion), genetic variation is available regarding at least the former two.

Roles and regulation of cytokinins in tomato fruit development.

Cytokinins (CKs) are thought to play important roles in fruit development, especially cell division. However, the mechanisms and regulation of CK activity have not been well investigated. This study analysed CK concentrations and expression of genes involved in CK metabolism in developing tomato (Solanum lycopersicum) ovaries. The concentrations of CK ribosides and isopentenyladenine and the transcript levels of the CK biosynthetic genes SlIPT3, SlIPT4, SlLOG6, and SlLOG8 were high at anthesis and decreased immediately afterward. In contrast, trans-zeatin concentration and the transcript levels of the CK biosynthetic genes SlIPT1, SlIPT2, SlCYP735A1, SlCYP735A2, and SlLOG2 increased after anthesis. The expression of type-A response regulator genes was high in tomato ovaries from pre-anthesis to early post-anthesis stages. These results suggest that the CK signal transduction pathway is active in the cell division phase of fruit development. This study also investigated the effect of CK application on fruit set and development. Application of a synthetic CK, N-(2-chloro-pyridin-4-yl)-N'-phenylurea (CPPU), to unpollinated tomato ovaries induced parthenocarpic fruit development. The CPPU-induced parthenocarpic fruits were smaller than pollinated fruits, because of reduction of pericarp cell size rather than reduced cell number. Thus, CPPU-induced parthenocarpy was attributable to the promotion of cell division, not cell expansion. Overall, the results provide evidence that CKs are involved in cell division during development of tomato fruit.

An unusual spliced variant of DELLA protein, a negative regulator of gibberellin signaling, in lettuce.

DELLA proteins are negative regulators of the signaling of gibberellin (GA), a phytohormone regulating plant growth. DELLA degradation is triggered by its interaction with GID1, a soluble GA receptor, in the presence of bioactive GA. We isolated cDNA from a spliced variant of LsDELLA1 mRNA in lettuce, and named it LsDELLA1sv. It was deduced that LsDELLA1sv encodes truncated LsDELLA1, which has DELLA and VHYNP motifs at the N terminus but lacks part of the C-terminal GRAS domain. The recombinant LsDELLA1sv protein interacted with both Arabidopsis GID1 and lettuce GID1s in the presence of GA. A yeast two-hybrid assay suggested that LsDELLA1sv interacted with LsDELLA1. The ratio of LsDELLA1sv to LsDELLA1 transcripts was higher in flower samples at the late reproductive stage and seed samples (dry seeds and imbibed seeds) than in the other organ samples examined. This study suggests that LsDELLA1sv is a possible modulator of GA signaling in lettuce.

Isolation and functional characterization of the FLOWERING LOCUS T homolog, the LsFT gene, in lettuce.

High temperature-induced bolting of lettuce is undesirable agriculturally, making it important to find the mechanism governing the transition from vegetative to reproductive growth. FLOWERING LOCUS T (FT) genes play important roles in the induction of flowering in several plant species. To clarify floral induction in lettuce, we isolated the FT gene (LsFT) from lettuce. Sequence analysis and phylogenetic relationships of LsFT revealed considerable homology to FT genes of Arabidopsis, tomato, and other species. LsFT induced early flowering in transgenic Arabidopsis, but was not completely effective compared to AtFT. LsFT mRNA was abundant in the largest leaves under flowering-inducible conditions (higher temperatures). Gene expression was correlated with flower differentiation of the shoot apical meristem. Our results suggest that LsFT is a putative FT homolog in lettuce that regulates flower transition, similar to its homolog in Arabidopsis. This is the first information on the lettuce floral gene for elucidating regulation of the flowering transition in lettuce.

Cleistogamous flowering in barley arises from the suppression of microRNA-guided HvAP2 mRNA cleavage.

The cleistogamous flower sheds its pollen before opening, forcing plants with this habit to be almost entirely autogamous. Cleistogamy also provides a means of escape from cereal head blight infection and minimizes pollen-mediated gene flow. The lodicule in cleistogamous barley is atrophied. We have isolated cleistogamy 1 (Cly1) by positional cloning and show that it encodes a transcription factor containing two AP2 domains and a putative microRNA miR172 targeting site, which is an ortholog of Arabidopsis thaliana AP2. The expression of Cly1 was concentrated within the lodicule primordia. We established a perfect association between a synonymous nucleotide substitution at the miR172 targeting site and cleistogamy. Cleavage of mRNA directed by miR172 was detectable only in a noncleistogamous background. We conclude that the miR172-derived down-regulation of Cly1 promotes the development of the lodicules, thereby ensuring noncleistogamy, although the single nucleotide change at the miR172 targeting site results in the failure of the lodicules to develop properly, producing the cleistogamous phenotype.

[A case of advanced gastric cancer that a medical therapy effect was provided grade 3 by a histopathological diagnosis after long-term chemotherapy].

A 64-year-old man underwent long-term chemotherapy for advanced gastric cancer with para-aortic lymph node metastasis (cT3 N3 M0 P0, cStage IV). S-1 (120 mg/day) was orally administered for 14 days and CDDP (60 mg/m2) was administered 10 times by intravenous drip on day 8. Next, paclitaxel (PTX 80 mg/m2) was administered 12 times by intravenous drip on days 1, 8 and 15. After 10 S-1/CDDP courses and 12 paclitaxel courses, the lymph node swelling decreased in size, but the tumor increased. CPT-11 (100 mg/m2) was administered as third-line therapy by intravenous drip on days 1, 8, 15 and 22. A partial response (PR) was obtained after 2 courses, but due to severe pyloric stenosis, a food passage disorder appeared. We performed distal gastrectomy with D2 lymph node dissection. The histological diagnosis revealed a complete disappearance of cancer cells in the stomach and lymph nodes.

The endogenous level of GA(1) is upregulated by high temperature during stem elongation in lettuce through LsGA3ox1 expression.

Bolting of lettuce is promoted by high temperatures. Gibberellins (GAs) play an important role in the bolting of several plant species, and it has been reported that exogenous GAs induce bolting and early flowering in lettuce. To clarify the role of GAs in this process, we examined the expression of genes involved in GA metabolism (LsGA20ox-1 and -2, LsGA3ox-1 and -2, and LsGA2ox-1 and -2) and endogenous GAs in lettuce stems. Quantitative reverse-transcription PCR indicated that the expression of a GA 3-oxidase gene, LsGA3ox1, is significantly upregulated by high (35/25 degrees C) temperature compared to low (25/15 degrees C) temperature, whereas transcription of the GA 20-oxidase gene, which is upregulated in long-day conditions in arabidopsis and spinach during bolting, is not clearly affected. Quantification of GA by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) showed that high temperature also upregulates the content of GA(1), a bioactive GA in lettuce. Our results suggest that LsGA3ox1 is a candidate for the gene responsible for the increase in GA(1) during lettuce bolting at high temperatures.

The efficacy and toxicity of FOLFOX regimen (a combination of leucovorin and fluorouracil with oxaliplatin) as first-line treatment of metastatic colorectal cancer.

BACKGROUND: Oxaliplatin is the third-generation platinum complex to be active in advanced colorectal cancer. In Japan, it was approved in April 2005. FOLFOX regimen(a combination of leucovorin and fluorouracil with oxaliplatin) has been one of the standard chemotherapy regimens for metastatic colorectal cancer. METHODS: To evaluate the efficacy and feasibility of FOLFOX4 and mFOLFOX6 regimens as first-line chemotherapy in Japanese patients with metastatic colorectal cancer, 23 consecutive patients with histologically confirmed colorectal cancer who were treated between June 2005 and August 2007 were investigated in this retrospective study. FOLFOX4 was used for treatment in 13 patients(57%), and mFOLFOX6 was in 10 patients(43%). RESULTS: The objective response rate was 50.0%. The median survival time was 17.4 months. The median number of cycles was 8.0, with a median relative dose intensity of 74.5% for oxaliplatin. Grade 3 or 4 hematological toxicities were leukocytopenia in four patients, and neutropenia in 12 patients, while non-hematological toxicities such as nausea, anorexia and sensory neuropathy occurred in only one patient each adverse event. No treatment-related deaths occurred. CONCLUSION: FOLFOX regimen has good efficacy in Japanese patients with metastatic colorectal cancer as first-line chemotherapy, with an acceptable overall toxicity profile.

[A case of advanced gastric cancer effectively treated with paclitaxel/S-1 as neoadjuvant chemotherapy].

We report a patient with advanced gastric cancer responding remarkably to neoadjuvant combination chemotherapy consisting of paclitaxel and S-1. The patient was a 65-year-old female who had large type 2 advanced gastric cancer with severe lymph node metastasis(cT3, cN3, cH0, cP0, cM0, cStage IV), treated with paclitaxel/S-1 as neoadjuvant chemotherapy. After the second course, according to UGI, gastroscope and CT findings, a significant tumor reduction was obtained. Distal gastrectomy with D2 nodal dissection were performed. The histological diagnosis was pT1, pN1, pStage IB. The histological effect of main tumor was judged to be Grade 2. The patient has now been in good health without a recurrence for 10 months after surgery. This case suggests that neoadjuvant chemotherapy with paclitaxel/ S-1 is a potential regimen for advanced gastric cancer.

[Second malignancy after gastrectomy for early gastric cancer-is there any evidence that adjuvant chemotherapy for early gastric cancer causes a second malignancy ?].

The common causes of post-operative death in early gastric cancer are other diseases and second malignancy. In Japan, adjuvant chemotherapy using oral administration of 5-fluorouracil is widely accepted in spite of no statistical evidence until the presentation by Kinoshita in 2005. In 1999, Fujimoto mentioned that adjuvant chemotherapy and chemo-immune-therapy cause a second malignancy. Adjuvant chemotherapy was once the standard treatment for early gastric cancer patients. So, we investigated the relation between adjuvant chemotherapy and second malignancy for early gastric cancer patients. As a result, adjuvant chemotherapy using 5-fluorouracil(Tegafur)did was not cause second malignancy under multivariate analysis. The most important factor in second malignancy with post-operative early gastric cancer patients was heredity(cancer within first-degree relatives). From this point of view, early gastric patients after gastrectomy and endoscopic treatment are necessary in regular examinations to detect other organ cancers and remnant gastric cancer, especially in patients with hereditary factors.

[A case of advanced gastric cancer with peritoneal metastasis that responded to paclitaxel plus 5-FU therapy].

We report a case in which paclitaxel (PTX) plus 5-FU therapy was remarkably effective for advanced gastric cancer with peritoneal metastasis. The patient was a 41-year-old woman who had type 4 gastric cancer with peritoneal metastasis. PTX plus 5-FU therapy was performed. The regimen included 600 mg/body/day of 5-FU by continuous iv administration from day 1 to 5 and consequent administration of PTX (90 mg/body) on days 8, 15 and 22 for 28 days repetitively. After 4 courses were completed, abdominal CT revealed that ascites had disappeared and the stomach wall had become thinner. Thus,we considered the patient had a partial response, and performed total gastrectomy. After the operation, PTX plus 5-FU therapy was used for 8 courses with the same regimen. No serious adverse event was observed,and the patient maintained good QOL throughout the treatment. No sign of progressive disease was seen for 12 months after the operation. However, 18 months after beginning the treatment, peritoneal metastasis became worse, and she died 19 months after treatment had begun. Considering the effectiveness and mild toxicity, PTX plus 5-FU therapy is thought to be useful for advanced gastric cancer.

Gastric cancer treated in 1991 in Japan: data analysis of nationwide registry.

The Japanese Gastric Cancer Association Registration Committee reported the treatment results and causes of death of patients with primary gastric cancer treated in 1991 at the leading hospitals in Japan. Data of 8851 patients with primary gastric cancer were collected from 113 hospitals, and data of 7935 patients with gastric resection were finally analyzed. The lost-to-follow-up rate was 6.9%; the direct death rate was 1.0%. The cumulative 5-year survival rate (5YSR) of all the patients was 68.2%; 89.9% for Stage I, 69.1% for Stage II, 43.5% for Stage III, and 9.9% for Stage IV. Characteristic findings of the analyzed data were (1) high proportion of early-stage cancer, (2) high resection rate, (3) low mortality rate, (4) low incidence of upper-third cancer, (5) poor treatment results in cases with scirrhous cancer, infiltrating growth, and marked lymphatic or venous invasion, and (6) predominance of systematic (D2) and extended lymphadenectomies possibly resulting in reducing local recurrence and improving survivals.

Field studies on the regulation of abscisic acid content and germinability during grain development of barley: molecular and chemical analysis of pre-harvest sprouting.

To investigate whether the regulation of abscisic acid (ABA) content was related to germinability during grain development, two cDNAs for 9-cis-epoxycarotenoid dioxygenase (HvNCED1 and HvNCED2) and one cDNA for ABA 8'-hydroxylase (HvCYP707A1), which are enzymes thought to catalyse key regulatory steps in ABA biosynthesis and catabolism, respectively, were cloned from barley (Hordeum vulgare L.). Expression and ABA-quantification analysis in embryo revealed that HvNCED2 is responsible for a significant increase in ABA levels during the early to middle stages of grain development, and HvCYP707A1 is responsible for a rapid decrease in ABA level thereafter. The change in the embryonic ABA content of imbibing grains following dormancy release is likely to reflect changes in the expression patterns of HvNCEDs and HvCYP707A1. A major change between dormant and after-ripened grains occurred in HvCYP707A1; the increased expression of HvCYP707A1 in response to imbibition, followed by a rapid ABA decrease and a high germination percentage, was observed in the after-ripened grains, but not in the dormant grains. Under field conditions, HvNCED2 showed the same expression level and pattern during grain development in 2002, 2003, and 2004, indicating that HvNCED2 expression is regulated in a growth-dependent manner in the grains. By contrast, HvNCED1 and HvCYP707A1 showed a different expression pattern in each year, indicating that the expression of these genes is affected by environmental conditions during grain development. The varied expression levels of these genes during grain development and imbibition, which would have effects on the activity of ABA biosynthesis and catabolism, might be reflected, in part, in the germinability in field-grown barley.

Overexpression of a petunia zinc-finger gene alters cytokinin metabolism and plant forms.

Shoot branching and plant height are among the key factors that define the overall architecture of plants. We found that overexpression of a cDNA for a zinc-finger protein of petunia, designated Lateral shoot-Inducing Factor (LIF), in transgenic petunia plants resulted in a dramatic increase in lateral shoots and reduced plant height. LIF overexpression also caused a decrease in the number of cells in the stem, leaf, and flower, accompanied by enlargement of cells. trans-Zeatin was decreased while N6-(Delta2-isopentenyl)adenine was increased in the leaves of LIF-overexpressed petunia. Most of the riboside, ribotide, and glucoside forms were also increased. Expression analysis using a LIF::GUS fusion gene and RT-PCR suggested that LIF is specifically expressed around the bases of axillary buds and weakly in basal part of flowers in wild-type petunia. GFP-LIF-GUS fusion proteins were translocated into the nucleus when transiently expressed in onion epidermal cells. LIF overexpression resulted in enhanced branching also in tobacco and Arabidopsis, indicating the conservation of the response to LIF overexpression among dicotyledonous plants. On the basis of these results we discuss about possible functions of LIF.

PIVKA-II-producing advanced gastric cancer.

We describe the case of a 68-year-old man with primary advanced adenocarcinoma of the stomach, who displayed extremely high plasma levels of protein induced by vitamin K antagonist (PIVKA)-II (15 600 mAU/ml) and normal levels of alphafetoprotein (AFP) (4 ng/ml). Ultrasonography and dynamic computed tomography ruled out hepatocellular carcinoma (HCC) or liver metastasis. After preoperative chemotherapy, pancreatico-spleno total gastrectomy with D2 lymphadenectomy was performed. Postoperatively, plasma levels of PIVKA-II returned to within the normal range (29 mAU/ml). Microscopic examination revealed stomach adenocarcinoma showing various histological types, such as moderately to poorly differentiated mucinous adenocarcinoma, but hepatoid differentiation of gastric adenocarcinoma was not detected. Localization of PIVKA-II and AFP within tumor cells was demonstrated by immunohistochemical staining using monoclonal antibodies. These results indicate that tumor cells from gastric cancer may produce PIVKA-II. Some cases of PIVKA-II- and AFP-producing advanced gastric cancer with liver metastasis have been reported, but this is the first report of gastric cancer without liver metastasis producing PIVKA-II alone.

DNA polymerase theta is preferentially expressed in lymphoid tissues and upregulated in human cancers.

DNA polymerase theta (Pol theta) is a recently identified family A polymerase that contains an intrinsic helicase domain. Drosophila Pol theta mutants are hypersensitive to bifunctional DNA crosslinking agents and exhibit an elevated frequency of spontaneous chromosomal aberrations, suggesting a role for Pol theta in repair of DNA interstrand crosslinks and in the general maintenance of genome stability. To investigate a possible involvement of Pol theta in tumorigenesis, we have examined its expression in various normal and malignant tissues. Paired tumor and adjacent nontumorous tissues from patients with lung (n = 27), stomach (n = 28) and colon (n = 26) cancer, as well as 26 normal human tissues, were examined for Pol theta expression by RT-PCR, Northern or Western blot analysis. Pol theta was predominantly expressed in primary lymphoid organs including the fetal liver, thymus and bone marrow where lymphocyte progenitors undergo V(D)J rearrangements of their antigen receptor genes. In addition, Pol theta expression was upregulated in germinal center B cells, in which class switch recombination of the immunoglobulin genes occurs. Examination of Pol theta expression in matched cancer specimens revealed that Pol theta was barely detectable in the nontumorous tissues but was upregulated in 17 of 27 (63%) lung, 11 of 28 (39%) stomach and 20 of 26 (77%) colon cancers. Moreover, patients with high levels of Pol theta expression had a significantly poorer clinical outcome compared with those expressing low levels of Pol theta. These results implicate that Pol theta may have a specialized function in lymphocytes and that its overexpression may contribute to tumor progression.

[Present condition of home care treatment at Shinshiro Municipal Hospital].

Shinshiro Municipal Hospital is situated in a mountainous region of Oku-Mikawa area at the East side of Aichi prefecture. Because of this geographical location, there are relatively higher numbers of elderly patients living alone in comparison to other regions of Japan. Our hospital consists of a general ward (255 beds), recuperation area combined with a medical care ward (46 beds), and a visiting nurse care unit. We have intended to strengthen a team medical care system for patients from time of admission to after discharge treatment by use of our unique hospital arrangement under the detailed coordination of associates from the different wards. However, many of the elderly patients living alone will not choose a home care treatment due to constricted circumstances. Therefore, we established a 24-hour care system in order to actively promote a visiting nurse care for patients at home. Regardless of our efforts in helping patients, it is still hard to deal with problems of elderly patients living alone. We assume that the problems associated with elderly patients living alone could be improved by enhancing each care unit's coordination. In any event, we have been trying to solve problems associated with patients since the visiting nurse care unit was started in April 2003. Our intention is to offer a home medical care in which patients can be treated with safety as well as at ease.

A semidwarf phenotype of barley uzu results from a nucleotide substitution in the gene encoding a putative brassinosteroid receptor.

Brassinosteroids (BRs) play important roles throughout plant growth and development. Despite the importance of clarifying the mechanism of BR-related growth regulation in cereal crops, BR-related cereal mutants have been identified only in rice (Oryza sativa). We previously found that semidwarf barley (Hordeum vulgare) accessions carrying the "uzu" gene, called "uzu" barley in Japan, are non-responding for brassinolide (BL). We then performed chemical and molecular analyses to clarify the mechanisms of uzu dwarfism using isogenic line pairs of uzu gene. The response of the uzu line to BL was significantly lower than that of its corresponding normal line. Measurement of BRs showed that the uzu line accumulates BRs, similar to known BR-insensitive mutants. The marker synteny of rice and barley chromosomes suggests that the uzu gene may be homologous to rice D61, a rice homolog of Arabidopsis BR-insensitive 1 (BRI1), encoding a BR-receptor protein. A barley homolog of BRI1, HvBRI1, was isolated by using degenerate primers. A comparison of HvBRI1 sequences in uzu and normal barley varieties showed that the uzu phenotype is correlated with a single nucleotide substitution. This substitution results in an amino acid change at a highly conserved residue in the kinase domain of the BR-receptor protein. These results may indicate that uzu dwarfism is caused by the missense mutation in HvBRI1. The uzu gene is being introduced into all hull-less barley cultivars in Japan as an effective dwarf gene for practical use, and this is the first report about an agronomically important mutation related to BRs.

Recurrence in jejunal pouch after proximal gastrectomy for early upper gastric cancer.

Recently, jejunal pouch reconstruction after proximal gastrectomy has been a common procedure in Japan. However, according to our investigation, there are no reports of recurrence in the jejunal pouch. We experienced a patient with a recurrence in the jejunal pouch after proximal gastrectomy for early upper gastric cancer. In view of the pathological findings and the recurrence site, we suspected an implantation on the staple-line made for the jejunal pouch.