Molecular genetic analysis of QTLs for ferulic acid content in dried straw of rice (Oryza sativa L.).

Phenolic acids are secondary metabolic organic compounds produced by plants and often are mentioned as allelochemicals. This study was conducted to determine the genetic basis controlling the ferulic acid content of rice straw in a recombinant inbred (RI) population derived from a cross between a japonica variety, Asominori, with a higher content of ferulic acid, and an indica variety, IR24, with a lower content, using 289 RFLP markers. Continuous distributions and transgressive segregations of ferulic acid content were observed in the RI population, which showed that ferulic acid content in rice straw was quantitatively inherited. Single marker analysis and composite interval mapping identified three quantitative trait loci (QTLs) for ferulic acid content with LOD values of 2.03 (chromosome 3), 3.16 (chromosome 6), and 3.06 (chromosome 7); all three had increased additive effects (13.5, 18.3, and 18.1 microg g(-1)) from the Asominori parent and accounted for 5.5, 16.9, and 12.8% of total phenotypic variation, respectively. This is the first report on the identification of QTLs associated with ferulic acid and their chromosomal localization on the molecular map of rice. The tightly linked molecular markers that flank the QTLs might be useful in breeding and selection of varieties with higher phenolic acid content.

Assessment of groundwater contamination by nitrate leaching from intensive vegetable cultivation using geographical information system.

This study employed the Geographical Information System (GIS) technology to investigate nitrate contamination of groundwater by agrochemical fertilizers in the Kakamigahara Heights, Gifu Prefecture, central Japan. Thematic information and chemical data of groundwater from the Heights were analyzed in a GIS environment to study the extent and variation of nitrate contamination and to establish spatial relationships with responsible land use types. The high and correlated concentrations of Ca(2+), Mg(2+), SO(4)(2-), and NO(3)(-) reflected the polluted nature of the unconfined highly permeable Kakamigahara aquifer. Ninety percent of the water samples showed nitrate concentrations above the human affected value (3 mg/l NO(3)(-)), while more than 30% have exceeded the maximum acceptable level (44 mg/l NO(3)(-)) according to Japan regulations. The spatial analyses indicated that groundwater contamination by nitrate is closely associated with one specific land use class, the "vegetable fields". The nitrate concentration of groundwater under vegetable fields was significantly higher than that under urban land or paddy fields. Most of the unacceptable nitrate levels were encountered in boreholes assigned to "vegetable fields" but a few were also found in boreholes allotted to "urban" class. Therefore, the vegetable fields were considered the principal source of nitrate contamination of groundwater in the Kakamigahara. However, contamination from urban sources is also possible.

Natural denitrification in the Kakamigahara groundwater basin, Gifu prefecture, central Japan.

Although nitrate is recognized as the most common groundwater contaminant due to growing anthropogenic sources, such as agriculture in particular, its adverse effects on human and animal health are debatable. The current issue, however, is to control and reduce nitrate contamination with regards to the long residence time of groundwater within aquifers. Denitrification has recently been recognized for its ability to reduce high nitrate concentrations in groundwater. The Kakamigahara groundwater basin, Gifu prefecture, Japan, witnessed rising levels of nitrate (>12 mg/l NO(3)-N) originating from agricultural sources. Chemical analyses for the determination of major constituents of groundwater and delta(15)N of residual nitrate were performed on representative groundwater samples in order to fulfill two main objectives. One is to investigate the current situation of nitrate groundwater pollution. The second objective is to determine whether the denitrification is a potential natural mechanism, which eliminates nitrate pollution in the Kakamigahara aquifer. Agricultural nitrate contamination of groundwater was obvious from characteristically high concentrations of Ca(2+), Mg(2+), NO(3)(-) and SO(4)(2-). High nitrate concentrations were found on the eastern side of the basin in association with vegetable cultivation fields, and decreased gradually towards the west of the basin along the direction of groundwater flow. The decrease of nitrate concentration was conveniently coupled with increase of HCO(3)(-) (the heterotrophic denitrification product), pH and delta(15)N of residual nitrate (due to isotopic fractionation) from east to west. Therefore, denitrification in situ is continuously removing nitrate from the Kakamigahara groundwater system.

Clinical dose and adverse effects of topical steroids in daily management of atopic dermatitis.

BACKGROUND: Topical steroids are used as the first-line therapy for atopic dermatitis. OBJECTIVES: To determine the clinical doses of topical steroids for the daily treatment of atopic dermatitis in clinics and to elucidate their adverse effects. PATIENTS AND METHODS: A multicentre retrospective analysis of a series of 1271 patients (210 infants, 546 children, and 515 adolescents and adults) with atopic dermatitis. RESULTS: Less than 89.5 g, 135 g and 304 g of topical steroid were applied in 90% of the patients in the infant, childhood, and adolescent and adult AD groups, respectively, on the entire body during the 6-month treatment period. The majority of patients were controlled well; however, 7% of infant, 10% of childhood and 19% of adolescent and adult patients remained in a very severe or severe state or experienced exacerbation even though they applied larger amounts of topical steroids. With regard to adverse effects, the incidence of telangiectasia on cheeks tended to increase in patients who had a longer duration of disease and who applied more than 20 g to the face during the 6-month treatment period. The steroid-induced atrophy of the antecubital and popliteal fossae was more frequently observed in males than in females. CONCLUSIONS: Topical steroids are useful for treating atopic dermatitis, but a substantial percentage of patients cannot be satisfactorily treated with topical steroids. For such patients, adjustments of dose and rank of topical steroids and other therapeutic adjuncts are necessary.

Proton relay in a one-dimensional hydrogen-bonded chain composed of water molecules and a squaric acid derivative.

The crystal structure of a hydrated crystal of bis(squaryl)biphenyl (BSQB*4H2O), in which two squaric acid moieties are connected with a 4,4'-biphenyl unit, was characterized by the presence of a one-dimensional hydrogen-bonded chain composed of BSQB and water molecules. X-ray crystallographic analysis showed that BSQB exists in a dianion form and that, on average, two of the four water molecules are protonated. The enhanced temperature dependence of the thermal parameters of the oxygen atoms of the water molecules suggested dynamic disorder of the water molecules. The solid-state magic angle spinning deuterium NMR spectrum of BSQB*4D2O revealed that deuterons are exchanged between heavy water molecules and oxonium ions with an exchange rate of ca. 700 Hz around 250 K and that deuterons start to migrate in a hydrogen-bonded cluster of water molecules. Ac dielectric measurements were also used to examine the dynamic process in the hydrated crystal. The dielectric permittivity of the crystal dramatically increased above 250 K with a distinct frequency dependence (epsilon' = 4.7 x 10(4) at 340 K and 1 kHz). The frequency dependence of tan delta at 290 K exhibited a maximum at 3.0 kHz, and this maximum shifted to lower frequencies when the temperature of the crystal decreased. These experimental results suggested that in the one-dimensional hydrogen-bonded chain of BSQB*4H2O a proton relay between oxonium ions and water molecules occurred within a cluster of four water molecules and that the relay was transmitted to the adjacent cluster mediated by the modulation of the negative charge distribution of the BSQB dianion. These phenomena were interpreted as the solitonic migration of the charged domain boundaries along the one-dimensional hydrogen-bonded chain.

Hepatitis B virus harboring nucleotide deletions in the core promoter region and genotype B correlate with low viral replication activity in anti-HBe positive carriers.

BACKGROUND: Emergence of anti-HBe following seroconversion of HBe antigen indicates reduced hepatitis B virus (HBV) replication in the liver and low infectivity in the natural course of infection. However, some patients show continued replication or reactivation even in the presence of anti-HBe. OBJECTIVE: To clarify the cause of HBV replication, we investigated genotype differences and mutations in the core promoter and precore region in relation to virus titer. STUDY DESIGN: Using quantification of HBV DNA, nucleotide sequencing of the core promoter and precore region, and genotyping with the S gene by restriction fragment length polymorphism (RFLP), we analyzed sera of 26 anti-HBe positive carriers (28 serum samples). RESULTS: Various mutations were detected including C to T point mutation at nt 1653, A to T and G to A contiguous point mutations at nt 1762 and 1764 in the core promoter region, and G to A point mutation at nt 1896 in the precore region, but no common mutations were detected that were directly related to the virus titer from earlier reported mutations. In contrast, the mean titer of genotype B virus was 1.5 x 10(5) copies per ml and that of mutant HBV of genotype C having 8 base pairs (8-bp) deletion (nt 1768-1775) in the core promoter region was 7.9 x 10(4) copies per ml (mean titer). These titers showed commonly lower than that of genotype C virus without 8-bp deletion (median titer 5.0 x 10(6) copies per ml). Transition of genotype from C to B after viral reactivation and reduction of proportion of 8-bp deletion mutant at reactivation period was observed in a patient who demonstrated exacerbation of liver dysfunction due to immunosuppressive therapy and increased viral replication. CONCLUSIONS: These results confirm those of our earlier study describing low replication ability of 8-bp deletion mutant HBV in vitro, and also indicate that the presence of genotype B correlates with reduced titer of HBV.

Induction of CD54 and CD58 expression in cultured human endothelial cells by beta-interferon with or without hyperthermia in vitro.

Human endothelial cells were treated by beta-interferon with or without hyperthermia at 42 degrees C for 90 min in vitro to investigate whether these modalities were able to increase the expression of either CD54 or CD58 on the surface of the endothelial cells. The results were that the population of the endothelial cells expressing both CD54 and CD58 increased 4 days after the treatment with beta-interferon, which was independent of hyperthermia. In contrast, the primarily isolated peripheral lymphocytes from a patient with malignant melanoma (disease free state) or normal individuals responded to neither beta-interferon nor hyperthermia in terms of the expression of CD54 or CD58. These results indicate that beta-interferon may activate endothelial cells to lead to the successive activation of the other immune cells in vivo.

Effects of cetirizine and epinastine on the skin response to histamine iontophoresis.

Epinastine and cetirizine are second-generation, nonsedating and long-lasting antihistamines that are now frequently used for the allergic disorders. We have examined the inhibitory effects of these two drugs on the histamine-induced flare and wheal responses using iontophoresis at 1, 2, 4, 8 and 24 h after the oral administration by a double-blind, cross-over and placebo-controlled study. Both cetirizine and epinastine significantly inhibited the histamine-induced flare and wheal responses at 2 h after the oral administration when compared with placebo. The inhibitory effects of cetirizine and epinastine on the flare response lasted long until at 24 h, however, epinastine was less potent than cetirizine. The inhibitory effects on the wheal response was also clearly and significantly evident at 2-8 h by cetirizine and epinastine. At 24 h cetirizine only showed the significant inhibition on the histamine-induced wheal response. In contrast, epinastine seemed to exhibit the inhibitory capacity earlier than did cetirizine. The inhibitory action of the drugs on the histamine-induced wheal response peaked at 4 h after the oral administration. The histamine-induced itch sensation was also markedly or completely suppressed at 2-8 h by the drugs. Thus, both drugs exhibited the potent and long-lasting antihistamine activity on the skin responses induced by histamine iontophoresis.

CD1a+, CD3+, CD4+, CD8+, CD68+ and cutaneous lymphocyte-associated antigen-positive cells in Bowen's disease.

BACKGROUND: Bowen's disease (BD) is a squamous cell carcinoma in situ that rarely invades into the underlying dermis. However, little is known about its immunohistology. Objectives To evaluate the relationship between the cytological properties of the tumour cells in BD and the host immune response. METHODS: We examined the expression of p53, proliferating cell nuclear antigen (PCNA) and Ki67 antigen, and the number of mitotic cells, together with the number of intratumoral and dermal infiltrating CD1a+, CD3+, CD4+, CD8+, CD68+ and cutaneous lymphocyte-associated antigen (CLA)+ cells in 18 cases of genital BD. RESULTS: When compared with normal genital skin (n = 10), there was a significantly higher number of mitotic cells as well as higher expression of p53+, PCNA+ and Ki67+ cells in BD. There was significant mutual correlation between CD3+, CD4+ and CD68+ cells in the tumoral epidermis. The number of CD1a+ Langerhans cells significantly decreased in BD epidermis; however, dermal CD1a+ cells were increased. Interestingly, numbers of dermal CD1a+ cells significantly correlated with those of intratumoral CD3+, CD4+ and CD68+ cells. In situ hybridization for human papillomavirus (HPV) demonstrated that HPV-infected BD had significantly less infiltration of intratumoral CD3+ cells and CLA+ cells. CONCLUSIONS: The present data suggest that dermal CD1a+ cells may participate in the immune surveillance and that HPV infection may interfere with the intratumoral infiltration of CLA+ cells in BD.

Tetra-n-butylammonium bis(1,2-dimercaptoethene-1, 2-dicarbonitrilato-S,S')nickel(III)

The crystal structure of the title compound, (C(16)H(36)N)[Ni(C(4)N(2)S(2))(2)], shows stacking of the dimerized anions, surrounded by columns of cations.

[Radiosurgery for cystic metastatic brain tumor].

Stereotactic radiosurgery (SRS) for brain metastasis results in a high local control rate. But cystic metastatic tumor should have been a contraindication for SRS. Because it is often found that a cyst is too large to be irradiated, the tumor does not exist in the center of the irradiation field. Between 1995 and 1998, 8 consecutive patients underwent linear accelerator-based SRS for cystic brain metastases identified by computed tomography or magnetic resonance image scan. Stereotactic cyst aspiration is carried out after placement of the BRW frame under local anesthesia. All of the patients except one were confirmed to have sufficient reduction of the cysts. 5-7 days after stereotactic cyst aspiration, SRS was performed. The dose range was 25-30 Gy. In follow-up MRI, local recurrences and enlargement of cysts were not noted. Six patients with neurological symptoms recuperated satisfactorily. Median survival was 30 weeks from the date of radiosurgery. All of the patients died and the causes of death were related with the primary lesion. We conclude that our technique, a combination of stereotactic cyst aspiration and SRS is an effective measure which leads to palliation of neurologic symptoms and is a low risk treatment for patients with cystic brain metastasis.

Cutaneous CD30 (Ki-1)-positive anaplastic large cell lymphoma preceded by Hodgkin's disease.

A 38-year-old female was diagnosed as Hodgkin's disease of the axillar lymph nodes, nodular sclerosis type, as evidenced by the presence of Reed-Sternberg cells positive for CD30 and CD15 and negative for CD3, CD20, and CD45. She achieved complete remission after combination chemotherapy. Two years later, she noticed a red papule on her public area without any lymph node involvement. The biopsy specimens showed diffuse proliferation of large-sized atypical lymphoid cells positive for CD30 and CD45, and negative for CD3, CD20 and CD15. These findings were mostly compatible with CD30 (Ki-1)-positive anaplastic large cell lymphoma (Ki-1 lymphoma). Our case is considered to be cutaneous Ki-1 lymphoma preceded by Hodgkin's disease.

Reduced transcription and progeny virus production of hepatitis B virus containing an 8-bp deletion in basic core promoter.

We have demonstrated previously the presence of an 8-bp deletion mutant, spanning from nt. 1768 to nt. 1775 in the basic core promoter region of hepatitis B virus (HBV) in patients with anti-HBe positive asymptomatic phase before developing acute exacerbation after immunosuppressive treatment. The transcription and progeny virus production activities of the mutant were examined by transfection of the recombinant plasmid [pUC Del(2)] containing the head-to-tail dimer DNA of the mutant into HepG2 cells. The amounts of hepatitis B surface antigen (HBsAg) and HBe antigens secreted into the culture medium were markedly reduced. Southern blotting of DNAs extracted from the culture medium also showed reduced mutant activity to produce progeny virus. Northern blotting and RNase protection assay of RNAs extracted from transfected cells demonstrated that the transcription of both precore mRNA and pregenome RNA was reduced significantly compared to that of wild-type HBV. The promoter activity examined by transfection of the CAT plasmid containing deletion mutant DNA was much lower than that of wild type. Co-transfection experiments, however, of the CAT plasmid containing wild-type DNA with pUC Del(2) reduced CAT activity induced by wild-type, suggesting that truncated X protein produced by the mutant does not possess a sufficient transactivating activity. Gel shift assay using HepG2 nuclear extract and a probe containing four TA-rich regions in CP and various competitors suggested that the lack of the third TA-rich region was responsible for the transcription reduction of precore mRNA and pregenome RNA. The possible mechanisms are discussed.

Cytokines and chemokines in the epidermis.

Keratinocytes (KC) produce a vast repertoire of cytokines, including interleukins, growth factors, colony stimulating factors, and chemokines. Under normal conditions, most of them are not synthesized or remain in the cytoplasm, but external stimuli, such as trauma, bacterial infections, chemical substances, or ultraviolet irradiation induce the production and release of these cytokines from KC. KC-derived cytokines regulate the immune and inflammatory responses through their receptors on KC, Langerhans cells, dermal fibroblasts and endothelial cells, and infiltrating T-cells.

Sequential analyses of the mutations in the core upstream and precore regions of hepatitis B virus genome in anti-HBe positive-carriers developing acute exacerbation.

The nucleotide sequences of the core upstream and precore regions (371 nucleotide length, nt. 1604-1974) of hepatitis B virus (HBV) were analysed sequentially in three subjects who were positive serologically for anti-HBe and had acute clinical exacerbation after immunosuppressive treatment. These patients were asymptomatic HBV carriers before therapy. The results revealed that the mutant with an 8-bp deletion (nt. 1768-1775) located in the basic core promoter region was dominant in the asymptomatic HBV carrier phase in two of three subjects. After exacerbation, however, such mutant clones possessing 8-bp deletion disappeared or decreased in number and were replaced by the clones possessing a precore stop codon mutation G to A (nt. 1896) or by the clones possessing additional contiguous point mutations A to T (nt. 1762) and G to A (nt. 1764) and a new point mutation C to T (nt. 1653). Possible relationships between acute exacerbation of liver function accompanied by mutation and the transition of the dominant clones were discussed.

[Changes in metabolism and peritumoral circulation after radiosurgery of metastatic brain tumors: evaluation by three dimensional dual isotope SPECT].

Concurrent use of two different isotopes, 201T1C1 and 123I-IMP, in SPECT is useful in separative evaluation of tumor metabolism and peritumoral circulation. Three dimensional SPECT employed in our study has an obvious advantage over two dimensional SPECT for its accurate imaging of tumors and peritumoural areas. Changes of tumor metabolism and regional circulation in peritumoral edematous tissues were investigated by fused 3-D SPECT images using 201T1C1 and 123I-IMP. In this study, the volume of isotope accumulative and isotope defective regions were measured. Fusion of SPECT images was performed by the use of panning visualization software; Application Visualization System Medical View (K.G.T.). The threshold of 3-D rendering was determined by conforming the volume of the hemisphere and of the tumor estimated on CT to the volume of 123I-IMP and 201T1C1 accumulating area respectively. Accumulative volume of 201T1C1 in the tumor decreased remarkably at 7 days after radiosurgery (p < 0.01). Defective volume of peritumoral hypoperfusion was measured on 3-D SPECT. The average volume was 80.5 + 32.5cm3 before radiosurgery. It decreased by approximately 60% at 7 days after radiosurgery (p < 0.05). Analysis of 3-D SPECT images using two different isotope tracers is reliable and useful to evaluate early the changes of metabolism and peritumoral circulation in or around intracerebral tumors.

Experimental vasospasm produced without blood cell components--hypothesis for the development of cerebral vasospasm.

A canine model of cerebral vasospasm using noncellular blood material (fibrin glue) was designed to investigate the effect of cerebrospinal fluid obstruction. The arachnoid membrane covering the cerebral arteries in the basal cistern was dissected and fibrin glue was applied to the adventitial surface of the arteries in three groups of animals. In Group 1, the arachnoid membrane was extensively dissected and fibrin glue was widely applied to the cerebral arteries. In Group 2, the dissection and coating was less extensive. Group 3 was a control group in which the arachnoid membrane was dissected but fibrin glue was not applied. Cerebral angiography 1 week later clearly demonstrated vasospasm in all six dogs in Group 1 and in four of six dogs in Group 2. Vasospasm did not occur in Group 3. The dogs were sacrificed and the arteries in the basal cistern were removed. Histological investigation showed typical findings of vasospasm and inertness of fibrin glue to the tissue. Cerebral vasospasm can be induced by a noncellular material from the blood densely applied to the arterial surface suggesting that obstruction of cerebrospinal fluid circulation around the artery may be important in the development of cerebral vasospasm.

The augmenting effect of OK432-stimulated B cells on the in vitro generation of anti-tumor cytotoxic T lymphocytes from tumor-draining lymph node cells: the possible role of interleukin-12.

Effect of a local injection with a streptococcal preparation OK432 on the in vitro generation of anti-tumor cytotoxic T lymphocytes (CTLs) from tumor-draining lymph nodes (LN) was investigated. A peritumoral injection with OK432 on days 2, 4, 6 and 8 significantly increased both the total cell number and the proportion of B cells in the draining LN cells on day 10 after a subcutaneous inoculation with B16 melanoma. In an in vitro proliferative assay, OK432 showed a stimulatory effect on both normal splenic T and B cells. In a cytolytic assay, the OK432-injected B16-draining LN cells showed a higher level of anti-B16 CTL activity than the B16-draining LN cells after in vitro restimulation. This augmenting effect of OK432 was dependent on the B cells. Moreover, nonadherent cells from the OK432-injected B16-draining LN cells showed a low but significantly higher level of anti-B16 CTL activity than those from the B 16-draining LN cells after in vitro restimulation, whereas this augmenting effect of OK432 was abolished by the in vitro addition of anti-interleukin (IL)-12 monoclonal antibody. Collectively, these findings suggest that the augmenting effect of a local injection with OK432 on the potential of tumor-draining LN cells to turn into anti-tumor CTLs after in vitro restimulation was at least in part due to IL-12 derived from the OK432-stimulated B cells.

Genotyping of hepatitis C virus in the Dominican Republic.

Genotyping of hepatitis C virus (HCV) of liver disease patients in the Dominican Republic was performed. Eighty-four samples positive for HCV antibody, which were confirmed by ELISA, particle agglutination, and recombinant immunoblot assay III tests, were subjected to HCV genotyping by polymerase chain reaction using type-specific primers located in the nonstructural protein 5 region. Of the 84 samples tested, 50 (59%) were found to have genotype 1a/I and this genotype was the most frequent type detected in the present study. The numbers of isolates of genotypes 1b/II, 2a/III, 2b/IV, and 3a/V were three (3.6%) six (7.1%), two (2.4%), and two 2.4%), respectively. The number of samples having mixed genotype populations was 16 (19%). The possible causes of the high prevalence of genotype 1a/I in the Dominican Republic compared with other countries and of the high detection ratio of samples having mixed genotypes are discussed.

The concurrent administration of OK432 augments the antitumor vaccination effect with tumor cells by sustaining locally infiltrating natural killer cells.

The effect of a local injection with a streptococcal preparation OK432 on the antitumor vaccination with tumor cells was investigated. Natural killer (NK) cells, which were detected by anti-NK1.1 monoclonal antibody (mAb), increased in the peritoneal exudate cells after an intraperitoneal (i.p.) injection with syngeneic B16 melanoma cells. Furthermore, a concurrent i.p. injection with OK432 efficiently sustained the locally infiltrating NK cells. The OK432 treatment also sustained the augmented NK and lymphokine-activated killer activities in the peritoneal exudate cells. This treatment also increased the ability of the locally infiltrating NK cells to produce interferon gamma in response to the tumor cells. In addition, the concurrent i.p. injection with OK432 in combination with the tumor cells enhanced the capacity of the spleen cells to turn into anti-(B16 melanoma) cytotoxic T lymphocytes after in vitro restimulation. This augmenting effect of OK432 was dependent on NK cells. Moreover, the concurrent injection with OK432 at the time of anti-tumor vaccination significantly enhanced the protective immunity against B16 melanoma at the rechallenge. Taken together, these findings indicate that a concurrent local injection with OK432 in combination with tumor cells efficiently augments the antitumor vaccination effect, in part, by sustaining the locally infiltrating activated NK cells.