Large-scale prospective genome-wide association study of oxaliplatin in stage II/III colon cancer and neuropathy.

BACKGROUND: The severity of oxaliplatin (L-OHP)-induced peripheral sensory neuropathy (PSN) exhibits substantial interpatient variability, and some patients suffer from long-term, persisting PSN. To identify single-nucleotide polymorphisms (SNPs) predicting L-OHP-induced PSN using a genome-wide association study (GWAS) approach. PATIENTS AND METHODS: A large prospective GWAS including 1379 patients with stage II/III colon cancer who received L-OHP-based adjuvant chemotherapy (mFOLFOX6/CAPOX) under the phase II (JOIN/JFMC41) or the phase III (ACHIVE/JFMC47) trial. Firstly, GWAS comparison of worst grade PSN (grade 0/1 versus 2/3) was carried out. Next, to minimize the impact of ambiguity in PSN grading, extreme PSN phenotypes were selected and analyzed by GWAS. SNPs that could predict time to recovery from PSN were also evaluated. In addition, SNPs associated with L-OHP-induced allergic reactions (AR) and time to disease recurrence were explored. RESULTS: No SNPs exceeded the genome-wide significance (P < 5.0 × 10-8) in either GWAS comparison of worst grade PSN, extreme PSN phenotypes, or time to recovery from PSN. An association study focusing on AR or time to disease recurrence also failed to reveal any significant SNPs. CONCLUSION: Our results highlight the challenges of utilizing SNPs for predicting susceptibility to L-OHP-induced PSN in daily clinical practice.

Landfill leachate sludge use as soil additive prior and after electrocoagulation treatment: A cytological assessment using CHO-k1 cells.

Electrocoagulation has recently attracted attention as a potential technique for treating toxic effluents due to its versatility and environmental compatibility, generating a residue chemically suitable to be used as a soil additive. In the present study, landfill leachate sludge hazardous effects were investigated prior and after electrocoagulation process using in vitro assays with the mammalian cells CHO-k1. An integrated strategy for risk assessment was used to correctly estimate the possible adverse landfill leachate sludge effects on human health and ecosystem. Electrocoagulation process proved to be an effective treatment due to possibility to improve effluent adverse characteristics and produce sludge with potential to be used as soil additive. Despite low cytoxicity, the residue presented genotoxic and mutagenic effects, indicating a capacity to induce genetic damages, probably due to induction of polyploidization process in cells. The observed effects demand an improvement of waste management methods for reduce negative risks of landfill leachate sludge application.

Large-scale prospective pharmacogenomics study of oxaliplatin-induced neuropathy in colon cancer patients enrolled in the JFMC41-1001-C2 (JOIN Trial).

BACKGROUND: Peripheral sensory neuropathy (PSN) is a dose-limiting toxicity of oxaliplatin-based chemotherapy. Several genetic markers have been shown to predict oxaliplatin-induced PSN; however, results remain to be validated in a large-scale and prospective pharmacogenomics study. PATIENTS AND METHODS: Among 882 patients enrolled in the JFMC41-1001-C2 (JOIN trial), which was designed to investigate the tolerability of adjuvant-modified FOLFOX6 (mFOLFOX6) in Japanese Patients with stage II or III colon cancers undergoing curative resection, 465 patients were eligible for this pharmacogenomics analysis. Twelve single-nucleotide polymorphisms (SNPs) were selected based on published data. The effect of each genotype on time to PSN onset was evaluated in all patients (n = 465) using the Cox proportional hazard model. For the association analysis between severity of PSN and 12 SNP markers, 84 patients who failed to complete 12 cycles of mFOLFOX6 from grade 0/1 PSN group were excluded because the termination of the protocol treatment had been caused by reasons other than PSN. RESULTS: Comparison of grade 0/1 PSN with grade 2/3 PSN or grade 3 PSN showed no significant associations with any of the 12 SNP markers after adjustment for total dose of oxaliplatin. Time-to-onset analysis also failed to reveal any significant differences. CONCLUSIONS: Our large-scale and prospective pharmacogenomics study of Japanese patients receiving protocol treatment of adjuvant mFOLFOX6 could not verify a role for any of the 12 SNP markers reported as being significantly associated with PSN. Considering the OR observed in this study (range: 0.76-1.89), further evaluation of these 12 SNP markers in the context of L-OHP-induced PSN is unlikely to be clinically informative.

Postcomparison of [(18) F]-fluorodeoxyglucose uptake in the brain after short-term bright light exposure and no intervention.

OBJECTIVE: Bright light therapy is widely used as the treatment of choice for seasonal affective disorder. Nonetheless, our understanding of the mechanisms of bright light is limited and it is important to investigate the mechanisms. The purpose of this study is to examine the hypothesis that bright light exposure may increase [(18) F]-fluorodeoxyglucose (FDG) uptake in olfactory bulb and/or hippocampus which may be associated neurogenesis in the human brain. METHOD: A randomized controlled trial comparing 5-day bright light exposure + environmental light (bright light exposure group) with environmental light alone (no intervention group) was performed for 55 participants in a university hospital. The uptake of [(18) F]FDG in olfactory bulb and hippocampus using FDG positron emission tomography was compared between two groups. RESULTS: There was a significant increase of uptake in both right and left olfactory bulb for bright light exposure group vs. no intervention group. After adjustment of log-transformed illuminance, there remained a significant increase of uptake in the right olfactory bulb. CONCLUSION: The present findings suggest a possibility that 5-day bright light exposure may increase [(18) F]FDG in the right olfactory bulb of the human brain, suggesting a possibility of neurogenesis. Further studies are warranted to directly confirm this possibility.

Modeling the nutrient removal process in aerobic granular sludge system by coupling the reactor- and granule-scale models.

We developed a model for nutrient removal in an aerobic granular sludge system. This model can quantitatively describe the start-up of the system by coupling a model for studying the population dynamics of the granules in the reactor (reactor-scale model) and a model for studying the microbial community structure in the granules (granule-scale model). The reactor-scale model is used for simulation for 10 days from the start, during which the granule size is relatively small; the granule-scale model is used after Day 10. The present approach proposes the output data of the reactor-scale model after 10 days as initial conditions for the granule-scale model. The constructed model satisfactorily describes experimental data in various spatial and temporal scales, which were obtained in this study by performing the anaerobic-aerobic-anoxic cycles using a sequencing batch reactor. Simulations using this model quantitatively predicted that the stability of nutrient removal process depended largely on the dissolved oxygen (DO) concentration, and the DO setpoint adaptation could improve the nutrient removal performance.

Inflammation enhanced brain-derived neurotrophic factor-induced suppression of the voltage-gated potassium currents in small-diameter trigeminal ganglion neurons projecting to the trigeminal nucleus interpolaris/caudalis transition zone.

We recently indicated that brain-derived neurotrophic factor (BDNF) enhances the excitability of small-diameter trigeminal ganglion (TRG) neurons projecting onto the trigeminal nucleus interpolaris/caudalis (Vi/Vc) transition zone via a paracrine mechanism following masetter muscle (MM) inflammation. The present study investigated whether modulation of voltage-gated potassium (K) channels by BDNF contributes to this hyperexcitability effect. To induce inflammation we injected complete Freund's adjuvant (CFA) into the MM. The escape threshold from mechanical stimulation applied to skin above the inflamed MM was significantly lower than in naïve rats. TRG neurons innervating the site of inflammation were subsequently identified by fluorogold (FG) labeling, and microbeads (MB) were used to label neurons projecting specifically to the Vi/Vc region. BDNF significantly decreased the total, transient (IA), and sustained (IK) currents in FG-/MB-labeled small-diameter TRG neurons under voltage-clamp conditions in naïve and inflamed rats. The magnitude of inhibition of IA and IK currents by BDNF in FG-/MB-labeled TRG neurons was significantly greater in inflamed rats than in naïve rats, and BDNF inhibited IA to a significantly greater extent than IK. Furthermore, co-administration of K252a, a tyrosine kinase inhibitor, abolished the suppression of IA and IK currents by BDNF. These results suggested that the inhibitory effects of BDNF on IA and IK currents in small-diameter TRG neurons projecting onto the Vi/Vc potentiate neuronal excitability, and in turn, contribute to MM inflammatory hyperalgesia. These findings support the development of voltage-gated K(+) channel openers and tyrosine kinase inhibitors as potential therapeutic agents for the treatment of trigeminal inflammatory hyperalgesia.

Efeito fungitóxico do óleo essencial de aroeira da praia (Schinus terebinthifolius RADDI) sobre Colletotrichum gloeosporioides / Fungitoxic effect of essential oil from aroeira (Schinus terebinthifolius RADDI) on Colletotrichum gloeosporioides

Neste trabalho foi avaliado o efeito do óleo essencial do fruto de Schinus terebinthifolius sobre o crescimento micelial do fungo Colletotrichum gloeosporioides in vitro, e no desenvolvimento da antracnose no período de pós-colheita em mamões. As diferentes concentrações de óleo foram diluídas em Tween 80 a 8%. No experimento in vitro foram preparados meios de cultura BDA nas concentrações de 0,05; 0,10; 0,25 e 0,50% do óleo essencial. O controle negativo foi realizado apenas com meio BDA e o controle solvente com meio BDA e Tween 80 a 8%. A inibição do crescimento do fungo foi diretamente proporcional à quantidade do óleo e a maior inibição encontrada foi de 79,07% na concentração de óleo de 0,50%. No experimento in vivo os frutos do mamoeiro foram inoculados com o fungo em quatro tratamentos: com biofilme; com biofilme mais 0,50% do óleo; com fungicida Prochloraz e frutos controle. Embora o tratamento com óleo tenha sido eficiente contra o fungo, não foi indicado comercialmente, pois apresentou valores elevados de perda de massa fresca, de firmeza, e também sintomas de fitotoxidade. O óleo tem propriedade antifúngica contra C. gloeosporioides in vitro e in vivo, contudo, não é recomendado para o mamão em função da fitotoxidez.

This study evaluated the effect of essential oil from Schinus terebinthifolius fruit on the mycelial growth of the fungus Colletotrichum gloeosporioides in vitro and on the anthracnose development during the postharvest period of papaya fruits. The different oil concentrations were diluted in 8% Tween 80. For the in vitro experiment, PDA culture media were prepared at the concentrations of 0.05, 0.10, 0.25 and 0.50% essential oil. Negative control was prepared with PDA medium alone, while solvent control was prepared with PDA medium and 8% Tween 80. Fungal growth inhibition was directly proportional to the oil amount and the greatest inhibition was 79.07% at 0.50% oil concentration. For the in vivo experiment, papaya fruits were inoculated with the fungus in four treatments: with biofilm, with biofilm plus 0.50% oil, with the fungicide Prochloraz and control fruits. Although treatment with oil was efficient against the fungus, it was not commercially recommended since it presented high values of loss of fresh mass and firmness, as well as phytotoxicity symptoms. The oil has antifungal property against C. gloeosporioides both in vitro and in vivo; however, it is not recommended for papaya fruits due to its phytotoxicity.

Super-high-dose islet transplantation is associated with high SUITO index and prolonged insulin independence: a case report.

INTRODUCTION: One of the current issues of clinical islet transplantation is the difficulty to achieve a prolonged insulin-free status. Functional islet mass gradually decreased after transplantation. We developed the SUITO index, which reflects engrafted islet mass. The SUITO (Secretory Unit of Islet Transplant Objects) index more than 26.0 is associated with an insulin-free status. In this study, we have experienced that super-high-dose islet transplantation maintained insulin-free status and a high SUITO index for a prolonged period. MATERIALS AND METHODS: Two islet isolations were performed in February 2007 and January 2008. Ductal injections were performed at the procurement site using the ET-Kyoto solution and pancreata preserved by a two-layer method. Islets were isolated using the modified Ricordi method. Both isolated islets were transplanted into a type 1 diabetic patient. Efficacy of islet transplantation was assessed by the amount of insulin requirements and SUITO index. RESULTS: Islet yields were 514,467 islet equivalents (IE) and 872,174 IE, with purities of 49% and 85% for the first and second islet transplantations, respectively. The patient received a total of 24,327 IE/kg body weight. The immunosuppression was based on the Edmonton protocol. After the second islet transplantation, the average SUITO index for the following 1 month was 48.5, and the patient became insulin-free. At postoperative day 1006, the SUITO index was 44.6 and the patient maintained an insulin-free status with excellent glycemic control. CONCLUSION: Super-high-dose islet transplantation was associated with an high SUITO index and prolonged insulin independence.

Postnatal development of the corticothalamic projections from the visual association cortex to the lateralis posterior complex in the cat.

The postnatal development of the corticothalamic projection from the lateral suprasylvian cortex (LS) to the lateral medialis-suprageniculate nucleus (LM-Sg) of the cat thalamus was assessed by means of the anterograde tracer biocytin. In the adult, two types of corticothalamic fibers were found: type I established a network of fine fibers present throughout the LM-Sg, it was characterized by a linear sequence of small (less than 0.5 microm in diameter), single terminal boutons making contact mainly with thin dendrites and/or dendritic spines. Type II, found less frequently, gave off short, side branches near axon terminals and formed clusters of 5-10 large terminal boutons (0.5-1.5 microm in diameter), making contact predominately with medium-sized dendrites and/or vesicle-containing profiles, forming a synaptic glomerulus. At birth (P0), anterogradely-labeled fibers were found in the LM-Sg as in adults. In the early postnatal period (until P6) as well as around the time of eye-opening (P7-P10) to P21, neonatal fibers were largely unbranched many of them having axons tipped with growth cones. Axon terminals containing synaptic vesicles were rarely observed but when present these exhibited considerable variation in their morphological appearance of synapses. Thus, it was not possible to categorize them into the two types of axons which characterize the adult. After P25, terminal swellings bearing a close resemblance to those of type II fibers begin to appear. In this way, the main two corticothalamic fiber types could be identified. These findings demonstrate that significant postnatal changes occur in the synaptology of corticothalamic fibers in the LM-Sg, particularly with the maturation of type II fibers.

Enhanced excitability of rat trigeminal root ganglion neurons via decrease in A-type potassium currents following temporomandibular joint inflammation.

The aim of the present study was to investigate the effect of temporomandibular joint inflammation on the excitability of trigeminal root ganglion neurons innervating the temporomandibular joint using a perforated patch-clamp technique. Inflammation was induced by injection of complete Freund's adjuvant into the rat temporomandibular joint. The threshold for escape from mechanical stimulation in the temporomandibular joint-inflamed rats was significantly lower than that in control rats. Fluorogold labeling was used to identify the trigeminal root ganglion neurons innervating the site of inflammation. When voltage-clamp (V(h)=-60 mV) conditions were applied to these Fluorogold-labeled small diameter trigeminal root ganglion neurons (<30 mum), voltage-dependent transient K(+) current densities were significantly reduced in the inflamed rats compared with controls. In addition, the voltage-dependence of inactivation of the voltage-dependent transient K(+) current was negatively shifted in the labeled temporomandibular joint-inflamed trigeminal root ganglion neurons. Furthermore, temporomandibular joint inflammation significantly reduced the threshold current and significantly increased action potential firings evoked at two-fold threshold in the Fluorogold-labeled small trigeminal root ganglion neurons. Application of 4-aminopyridine (0.5mM) to control trigeminal root ganglion neurons mimicked the changes in the firing properties observed after complete Freund's adjuvant treatment. Together, these results suggest that temporomandibular joint inflammation increases the excitability of trigeminal root ganglion neurons innervating temporomandibular joint by suppressing voltage-dependent transient K(+) current via a leftward shift in the inactivation curve. These changes may contribute to trigeminal inflammatory allodynia in temporomandibular joint disorder.

Role of capsaicin-sensitive primary afferent inputs from the masseter muscle in the C1 spinal neurons responding to tooth-pulp stimulation in rats.

The aim of the present study was to demonstrate the convergence of inputs from masseter muscle (MM) and tooth pulp (TP) onto C1 spinal neurons and to determine whether the afferent fibers express the functional vanilloid receptor (VR1). Extracellular single-unit recordings were made from 61 C1 units responding to TP electrical stimulation with a constant temporal relationship to a digastric electromyogram signal in pentobarbital anesthetized rats. Eighty-four percent of C1 neurons responding to TP stimulation also responded to the ipsilateral MM stimulation. Of these neurons, 61% were considered to be afferent inputs from Adelta-fibers and the remaining units (39%) were C-fibers, based on calculation of the nerve conduction velocity. Intramuscular injection of capsaicin (0.05 and 0.1%) produced a reduction in a MM-induced C1 neuronal activity in a dose-dependent manner and this effect was antagonized by pretreatment with an antagonist of VR1, capsazepine. Some of these units were also excited by noxious heat stimulation (> 43 degrees C). The trigeminal root ganglion (TRG) neurons that innervated the MM were retrogradely labeled with Fluorogold (FG) and the small-diameter FG-labeled TRG neurons expressed the immunoreactivity for VR1. After intramuscular mustard oil injection (noxious chemical stimulation), the C1 neuronal activity induced by both touch and pinch stimuli was enhanced and their receptive field sizes were significantly expanded. These changes were reversed within 15-20 min. These results suggest that there may be the convergence of noxious afferents inputs from the MM and TP afferents on the same C1 neurons in rats, and that the afferent fibers expressing the functional VR1 may contribute to the hyperalgesia and/or referred pain associated with temporomandibular joint disorder.

Fucoidan derived from Cladosiphon okamuranus Tokida ameliorates murine chronic colitis through the down-regulation of interleukin-6 production on colonic epithelial cells.

Our previous study indicated that the interleukin (IL)-6/STAT-3 signal was up-regulated in inflammatory bowel disease (IBD) in both humans and animal models. We also discovered phosphorylated STAT-3 in the nucleus of the colonic epithelial cells in IBD mice. Intestinal epithelial cells (IEC) have been shown to secrete IL-6. Therefore, the secretion of IL-6 from IEC may be one of the mechanisms of STAT-3 phosphorylation in IEC during the pathogenesis of IBD, and inhibition of IL-6 production by IEC may be beneficial in preventing IBD. We examined the preventative effect of various types of fucoidans on IL-6 production in a lipopolysaccharide (LPS)-stimulated murine colonic epithelial cells line, CMT-93, in vitro. We also determined in vivo the effect of fucoidans on murine chronic colitis induced with dextran sodium sulphate. Among fucoidans, those from Cladosiphon okamuranus Tokida and Kjellmaniella crassifolia inhibited IL-6 production in CMT-93 cells with the down-regulation of NF-kappaB nuclear translocation. Analysis of the effect of fucoidan on murine colitis in vivo showed that the disease activity index and myeloperoxidase activity decreased in mice fed Cladosiphon fucoidan, but not Fucus fucoidan. Cytokine profiles in colonic lamina propria indicated that the synthesis of interferon (IFN)-gamma and IL-6 decreased and that of IL-10 and transforming growth factor (TGF)-beta increased in mice fed Cladosiphon fucoidan, compared with mice fed a standard diet or Fucus fucoidan. The levels of IL-6 mRNA in colonic epithelial cells was lower in colitis-induced Balb/c mice fed Cladosiphon fucoidan than those fed a standard diet. Fucoidan improves murine chronic colitis by down-regulating the synthesis of IL-6 in the colonic epithelial cells. Fucoidan derived from C. o. Tokida may be useful as a dietary substance for the patients with inflammatory bowel disease.

Improvement of human faecal flora-associated mouse model for evaluation of the functional foods.

AIMS: Animal models are required for evaluation of the functional foods such as pro/prebiotics exerting effects through the metabolism of the intestinal microflora. The object of this study was to establish new human flora-associated mice reflecting the environment of the human intestinal tract. METHODS AND RESULTS: We inoculated a human faecal suspension into segmented filamentous bacteria (SFB) monoassociated mice as a model system. In both human flora (HF) and SFB-associated mouse (HF-SFB mouse), intestinal characteristics such as the composition of intraepithelial lymphocytes, the expression of major histocompatibility complex (MHC) class II molecules and the number of immunoglobulin A-producing cells in the mucosa was closer to those of conventionally reared mice than was case with human flora-associated mice (HF mice) lacking SFB. Several predominant bacterial groups except lactobacilli in human flora were found in faeces of HF-SFB mice. Lactobacilli established small populations in the gut of HF-SFB mice when administered before inoculation with the human flora. Faecal enzymatic activities and organic acid concentration of HF-SFB mice proportionally reflected those of the donor subject. CONCLUSION: We established a new human flora-associated mouse (HF-SFB mouse), in which intestinal characteristics are normally developed and their major microbial composition reflect the human. SIGNIFICANCE AND IMPACT OF THE STUDY: HF-SFB mice are a valuable model for studying pro/prebiotic effects on the human intestine.

Cimetidine increases survival of colorectal cancer patients with high levels of sialyl Lewis-X and sialyl Lewis-A epitope expression on tumour cells.

Cimetidine has been shown to have beneficial effects in colorectal cancer patients. In this study, a total of 64 colorectal cancer patients who received curative operation were examined for the effects of cimetidine treatment on survival and recurrence. The cimetidine group was given 800 mg day(-1) of cimetidine orally together with 200 mg day(-1) of 5-fluorouracil, while the control group received 5-fluorouracil alone. The treatment was initiated 2 weeks after the operation and terminated after 1 year. Robust beneficial effects of cimetidine were noted: the 10-year survival rate of the cimetidine group was 84.6% whereas that of control group was 49.8% (P<0.0001). According to our previous observations that cimetidine blocked the expression of E-selectin on vascular endothelium and inhibited the adhesion of cancer cells to the endothelium, we have further stratified the patients according to the expression levels of sialyl Lewis antigens X (sL(x)) and A (sL(a)). We found that cimetidine treatment was particularly effective in patients whose tumour had higher sL(x) and sL(a) antigen levels. For example, the 10-year cumulative survival rate of the cimetidine group with higher CSLEX staining, recognizing sL(x), of tumours was 95.5%, whereas that of control group was 35.1% (P=0.0001). In contrast, in the group of patients with no or low levels CSLEX staining, cimetidine did not show significant beneficial effect (the 10-year survival rate of the cimetidine group was 70.0% and that of control group was 85.7% (P=n.s.)). These results clearly indicate that cimetidine treatment dramatically improved survival in colorectal cancer patients with tumour cells expressing high levels of sL(x) and sL(a).

cDNA cloning of calcineurin heterosubunits from the pheromone gland of the silkmoth, Bombyx mori.

Pheromone biosynthesis activating neuropeptide (PBAN) stimulates the step of fatty acyl reduction in the pheromone biosynthetic pathway of the silkmoth, Bombyx mori. It has been suggested that the intracellular signal transduction of PBAN in B. mori involves Ca(2+), calmodulin, and calcineurin (also known as protein phosphatase 2B). We have cloned two cDNAs encoding calcineurin heterosubunits from a pheromone gland cDNA library of B. mori. The 2,996-bp clone predicts a 495-amino acid protein homologous to the catalytic subunit calcineurin A (CnA) with a molecular mass of 55,968. The deduced amino acid sequence well conserves the calcineurin B (CnB)-binding domain and two subdomains, a calmodulin-binding and an autoinhibitory domain, showing 77-85% and 82% identities to the isoforms of Drosophila melanogaster CnA and human CnA, respectively. On the other hand, the 820-bp clone predicts a 170-amino acid protein homologous to the regulatory subunit CnB with a molecular mass of 19,357. The deduced amino acid sequence well conserves four EF-hand type calcium-binding structures, showing 95% and about 85% identities to D. melanogaster CnB and mammalian CnBs, respectively. A yeast two-hybrid system has demonstrated the molecular interaction between B. mori CnA and CnB. Northern blot analyses revealed that both CnA and CnB genes were expressed in various larval and adult tissues of B. mori. Both transcripts detected in the pheromone gland three days before adult eclosion increased by the day before eclosion and the mRNA levels were found to be high even two days after adult eclosion. Immunohistochemical analysis has revealed that B. mori calcineurin is localized in the cytoplasm of the pheromone-producing cells.

[Pollen surveys in Sapporo for 6 years during 1992 to 1997].

We carried out pollen surveys for 6 years from 1992 to 1997. The method of pollen survey studied was based on the standardization board of air born pollen survey and pollen information in Japan. The pollen samples were collected in Nishioka-area of Toyohira-ku, and Kawazoe-area of Minami-ku in Sapporo. As the pollen sample device. IS Rotary pollen trap was used at Nishioka-area during 1992 to 1994 and at Kawazoe-area in 1993, and Durham's pollen trap was used at Kawazoe-area during 1995 to 1997. The yearly pollen counts were highly fluctuated in trees, particularly Yew (Taxus spp.), Alder (Alnus spp.), Birch (Betula spp.), Pine (Pinus spp.), Fir (Abies spp.), than in Grasses (Gramineae) and Weeds (Polygonaceae, Plantago spp., Artemisia spp., Chenopodiaceae). In addition, the starting date of pollination was widely fluctuated year by year in trees. For instance, the pollen of Birch was confirmed on April 13 in 1993, as the first day in Nishioka-area, while it was on April 28 in 1994. It should be considered that the pollen counts and the starting date of pollination were highly dependent on the weather condition of the previous year and the current year.

Peflin and ALG-2, members of the penta-EF-hand protein family, form a heterodimer that dissociates in a Ca2+-dependent manner.

Peflin, a newly identified 30-kDa Ca(2+)-binding protein, belongs to the penta-EF-hand (PEF) protein family, which includes the calpain small subunit, sorcin, grancalcin, and ALG-2 (apoptosis-linked gene 2). We prepared a monoclonal antibody against human peflin. The antibody immunoprecipitated a 22-kDa protein as well as the 30-kDa protein from the lysate of Jurkat cells. Western blotting of the immunoprecipitates revealed that the 22-kDa protein corresponds to ALG-2. This was confirmed by Western blotting of the immunoprecipitates of epitope-tagged peflin or ALG-2 whose cDNA expression constructs were transfected to human embryonic kidney (HEK) 293 cells. Gel filtration of the cytosolic fraction of Jurkat cells revealed co-elution of peflin and ALG-2 in fractions eluting earlier than recombinant ALG-2, further supporting the notion of heterodimerization of the two PEF proteins. Surprisingly, peflin dissociated from ALG-2 in the presence of Ca(2+). Peflin and ALG-2 co-localized in the cytoplasm, but ALG-2 was also detected in the nuclei as revealed by immunofluorescent staining and subcellular fractionation. Peflin was recovered in the cytosolic fraction in the absence of Ca(2+) but in the membrane/cytoskeletal fraction in the presence of Ca(2+). These results suggest that peflin has features common to those of other PEF proteins (dimerization and translocation to membranes) and may modulate the function of ALG-2 in Ca(2+) signaling.

High dosage expression of a zinc finger protein, Grt1, suppresses a mutant of fission yeast slp1(+), a homolog of CDC20/p55CDC/Fizzy.

Selective proteolysis at and after the onset of anaphase is a key cell cycle event required for sister chromatid separation as well as for exit from mitosis. It requires ubiquitination of substrates by Anaphase Promoting Complex(APC)/Cyclosome. Slp1, a WD-repeat protein, is a putative activator for APC in fission yeast. With another WD- repeat protein, Ste9/Srw1, it is thought to promote the proteolysis in a substrate-specific manner. We report here characterization of a temperature-sensitive (ts) slp1 mutant and its high-dosage suppressor, grt1(+). In cells arrested in metaphase, wild-type Slp1 was preferentially found in a complex with hyperphosphorylated Cut9 (subunit of APC), whereas the ts Slp1 protein, lacking the last 113 amino acids, failed to interact with Cut9. The temperature sensitivity was suppressed by high dosage expression of a zinc finger protein, Grt1. The ts slp1 mutant was unable to maintain the normal level of Grt1 protein. The reduction in the Grt1 level may be a primary defect since high dosage expression of grt1(+) rescues the slp1 mutant. The grt1-suppression had an additive effect to ste9 and wee1-50, both of which partially suppress the ts slp1 mutant. Therefore, grt1(+) would define an independent pathway that facilitates the function of Slp1.

Expression of thymidylate synthase in relation to survival and chemosensitivity in gastric cancer patients.

Expression of thymidylate synthase (TS) has been studied as a prognostic factor and mechanism of drug resistance in gastric cancers. The relationship between TS expression in surgically resected specimens and clinicopathological factors was examined in 216 gastric cancer patients. Immunohistochemical demonstration of the protein was achieved using an anti-TS polyclonal antibody. Positive TS staining was observed in 50 patients (23.1%). Lymph node metastasis was more frequent in patients with TS-positive tumors than in those with TS-negative tumors (P<0.01). Patients were followed for more than 5 years and survival was examined. In 163 patients who received fluorouracil (FU)-based chemotherapy, the overall 5-year survival rate was 41.8% for patients with TS-positive tumors and 57.0% for patients with TS-negative tumors (P<0.01). In the 53 patients who did not receive chemotherapy, these figures were 25.6% and 79.5%, respectively (P<0.05). In patients with T3 gastric cancer who were treated with curative gastrectomy, however, FU-based chemotherapy did not affect survival of either patients with TS-positive tumors or with TS-negative tumors. Multivariate analysis also revealed TS expression to be a significant variable for predicting postoperative survival (P<0.05). These results indicate that TS expression can be used as an independent prognostic factor for patients with gastric cancer. However, TS expression is not a major predictor of the efficacy of FU-based chemotherapy.

Novel antiarthritic agents with 1,2-isothiazolidine-1,1-dioxide (gamma-sultam) skeleton: cytokine suppressive dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase.

Various 1,2-isothiazolidine-1,1-dioxide (gamma-sultam) derivatives containing an antioxidant moiety, 2,6-di-tert-butylphenol substituent, were prepared. Some compounds, which have a lower alkyl group at the 2-position of the gamma-sultam skeleton, showed potent inhibitory effects on both cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LO), as well as production of interleukin (IL)-1 in in vitro assays. They also proved to be effective in several animal arthritic models without any ulcerogenic activities. Among these compounds, (E)-(5)-(3,5-di-tert-butyl-4-hydroxybenzylidene)-2-ethyl-1, 2-isothiazolidine-1,1-dioxide (S-2474) was selected as an antiarthritic drug candidate and is now under clinical trials. The structure-activity relationships (SAR) examined and some pharmacological evaluations are described.