Comparing the efficacy of stimbiotic and a combination of xylanase and beta-glucanase, in broilers fed wheat-barley based diets with high or low AME.

A stimbiotic is defined as a product that stimulates a fiber-degrading microbiome to increase fiber fermentability. The aim of this study was to examine if it is more advantageous to feed a stimbiotic (xylanase + xylo-oligosaccharides [STB]) or a combination of xylanase and beta-glucanase (Xyl + BG) to broilers fed wheat-barley based diets with differing AME levels. Cobb 500 broilers (n = 480, 80 birds per treatment) were fed 6 dietary treatments in a 2 × 3 factorial arrangement; 2 AME levels, 'High' or 'Low', which differed by 100 kcal ME/kg, and 3 additive supplementations, with no supplemental additives, STB or Xyl + BG. Diets were fed as 3 phases, starter (d 0-14), grower (d 14-21) and finisher (d 21-35). On bird age d 14, 21 and 35, total pen body weight and feed intake were determined, and feed conversion ratio corrected for mortality (cFCR) was calculated. On d 21 and d 35 ileal viscosity and beta-glucan content and caecal SCFA concentration were determined. Additive suplementation had no impact on cFCR in birds fed the low AME diet, but in birds fed the high AME diet the cFCR value was reduced in the presence of the additives (P = 0.001 and P = 0.015, at d 14-21 and d 21-35, respectively). At d 21, cecal SCFA concentration was consistently higher (P = 0.015), and ileal beta-glucan level lower (P = 0.002), in birds fed the diet supplemented with STB compared to those without additives. At d 35, ileal viscosity was lower in birds fed STB compared to those fed the diet without supplementation of additives, irrespective of diet AME level (P = 0.017). These results suggest that both STB and Xyl + BG ameliorate the antinutritive effects of the non-starch polysaccharides (NSP) present in wheat-barley based diets, resulting in improved bird performance. However, supplementation with STB induces a comparatively greater positive effect on NSP hydrolysis and SCFA production.

Supra-nutritional vitamin E supplementation for 28 days before slaughter maximises muscle vitamin E concentration in finisher pigs.

A 4 × 3 factorial experiment (n=8 pigs per treatment combination) was conducted with 96 female Landrace × Large White pigs to examine the required level of dietary vitamin E and optimum feeding duration before slaughter to maximise muscle vitamin E content in the Longissimus thoracis et lumborum (LTL) muscle. The respective factors were four dietary levels of vitamin E (supplemented as dl-α-tocopheryl acetate; 35, 300, 500, and 700 IU/kg) and three feeding durations (14, 28 and 42 days before slaughter). Vitamin E concentration in the LTL was maximised at 6 mg/kg, which was achieved by feeding a 700 IU vitamin E diet for 28 days before slaughter (P<0.001). There was no further increase in the vitamin E content of the LTL by feeding the high vitamin E diet more than 28 days before slaughter.

Vitamin E and omega-3 fatty acids independently attenuate plasma concentrations of proinflammatory cytokines and prostaglandin E3 in Escherichia coli lipopolysaccharide-challenged growing-finishing pigs.

This study tested the hypothesis that vitamin E (Vit E) and omega-3 fatty acids will additively attenuate the production of proinflammatory cytokines and PGE2 in immune system­stimulated growing­finishing pigs. A total of 80 mixed sex pigs weighing 50.7 ± 0.76 kg (mean ± SE) were blocked and stratified based on sex and BW to a 2 × 2 factorial design with the respective factors being 1) without and with 300 IU Vit E and 2) without and with 25% replacement of tallow to linseed oil as a source of n-3 fatty acids. Each treatment consisted of 4 replicate pens with 5 pigs (3 barrows and 2 gilts) per pen. All pigs were challenged with an intramuscular injection of Escherichia coli lipopolysaccharide (LPS; O111:B4) twice weekly over the 6-wk experiment. After LPS challenge, pigs fed a diet supplemented with n-3 fatty acids had fewer (P < 0.05) white blood cells and tended to show both a reduced (P < 0.10) proportion of lymphocytes and IgG concentration compared with pigs fed a diet without any supplements. Supplementation of n-3 fatty acids reduced (P < 0.01 and P < 0.05) serum concentrations of cortisol and tumor necrosis factor α (TNF-α), respectively. The serum concentration of PGE2 was decreased (P < 0.05) with supplementation of both Vit E and n-3 fatty acids; however, the extent of the reduction was greater (P < 0.001) in pigs fed an n-3 fatty acid­supplemented diet. However, there were no additive effects of the combined supplementation of Vit E and n-3 fatty acids on serum concentrations of proinflammatory cytokines and PGE2. The results suggest that n-3 fatty acids independently attenuate production of TNF-α and PGE2 in immune system­stimulated growing­finishing pigs.

Effect of diallyl disulfide on acute gastric mucosal damage induced by alcohol in rats.

This study investigated the gastroprotective effects of diallyl disulfide (DADS), a secondary organosulfur compound derived from garlic (Allium sativum L.) on experimental model of ethanol (EtOH)-induced gastric ulcer in rats. The antiulcerogenic activity of DADS was evaluated by gross/histopathological inspection, pro-inflammatory cytokines, and lipid peroxidation with antioxidant enzyme activities in the stomach. DADS (100 mg/kg) was administered by oral gavage 2 h prior to EtOH treatment (5 ml/kg). The animals were killed 1 h after receiving EtOH treatment. Pretreatment with DADS attenuated EtOH-induced gastric mucosal injury, as evidenced by decreased severity of hemorrhagic lesions and gastric ulcer index upon visual inspection. DADS also prevented histopathological alterations and gastric apoptotic changes caused by EtOH. An increase in tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase was observed in the gastric tissues of EtOH-treated rats that coincided with increased serum TNF-α and interleukin 6 levels. In contrast, DADS effectively suppressed production of pro-inflammatory mediators induced by EtOH. Furthermore, DADS prevented the formation of gastric malondialdehyde and the depletion of reduced glutathione content and restored antioxidant enzyme activities, such as catalase, glutathione peroxidase, and glutathione reductase in the gastric tissues of EtOH-treated rats. These results indicate that DADS prevents gastric mucosal damage induced by acute EtOH administration in rats and that the protective effects of DADS may be due to its potent antioxidant and anti-inflammatory activities.

Ecklonia cava promotes hair growth.

BACKGROUND: Previous studies have reported the protective effects on skin elasticity of the edible marine seaweed Ecklonia cava, which acts through regulation of both antioxidative and anti-inflammatory responses. AIM: We evaluated the effect of E. cava and one of its components, dioxinodehydroeckol, on hair-shaft growth in cultured human hair follicles and on hair growth in mice. METHODS: The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to check cell viability of human dermal papilla cells (DPCs) and outer root sheath (ORS) cells after treatment with E. cava and its metabolite, dioxinodehydroeckol. Hair-shaft growth was measured using the in vitro hair-follicle organ-culture system, in the presence or absence of E. cava and dioxinodehydroeckol. Anagen induction activity was examined by topical application of E. cava to the dorsal skin of C57BL/6 mice. Insulin-like growth factor (IGF)-1 expression was measured by reverse transcriptase PCR and ELISA. RESULTS: The proliferation activity was found to be highest for the ethyl acetate-soluble fraction of E. cava (EAFE) in DPCs and in ORS cells. Treatment with EAFE resulted in elongation of the hair shaft in cultured human hair follicles, and promoted transition of the hair cycle from the telogen to the anagen phase in the dorsal skin of C57BL/6 mice. In addition, EAFE induced an increase in IGF-1 expression in DPCs. Dioxinodehydroeckol, a component of E. cava, induced elongation of the hair shaft, an increase in proliferation of DPCs and ORS cells, and an increase in expression of IGF-1 in DPCs. CONCLUSIONS: These results suggest that E. cava containing dioxinodehydroeckol promotes hair growth through stimulation of DPCs and ORS cells.

A ß-cyclodextrin, polyethyleneimine and silk fibroin hydrogel containing Centella asiatica extract and hydrocortisone acetate: releasing properties and in vivo efficacy for healing of pressure sores.

BACKGROUND: Pressure sores are lesions caused by impaired blood flow. Conventional dressings can absorb exudates, but do not promote wound healing. A hydrogel composed of ß-cyclodextrin (ß-CD), polyethyleneimine (PEI) and silk fibroin (SF) was assessed for use in healing of pressure sores. METHODS: The hydrogel was prepared by crosslinking ß-CD-grafted PEI and SF using epichlorohydrin. The gel was then immersed in an aqueous solution of Centella asiatica extract (CAE) 0.7 mg/mL and/or hydrocortisone acetate (HCA) 0.5 mg/mL. The in vivo pressure sore-healing efficacy of the dry gel (with or without the drugs) was investigated in terms of the hyperplasia of epidermis and the number of neutrophils in the skin tissue. RESULTS: The specific loading of CAE was 0.0091 g/g of dry gel. The percentage of CAE released at 24 h at pH 3.0, 5.0 and 7.4 was approximately 63.9%, 55.0% and 44.4%, respectively. This pH-dependent release is possibly due to the degree of gel swelling, which decreased with increasing pH. The specific loading of HCA was 0.0050 g/g dry gel, and the percentage release of HCA at 24 h was around 20% at all three pH points. It is likely that HCA release is independent of pH. HCA is a hydrophobic compound, and therefore the release of HCA is affected by the partitioning of HCA between the ß-CD cavity and the bulk water phase, but not by the degree of swelling of the hydrogel. The pressure sores treated with the hydrogel healed in 6 days, compared with 10 days for controls. CONCLUSIONS: In this study, a ß-CD/PEI/SF hydrogel containing CAE and HCA reduced the healing time for pressure sores.

Whole body protein deposition and plasma amino acid profiles in growing and/or finishing pigs fed increasing levels of sulfur amino acids with and without Escherichia coli lipopolysaccharide challenge.

A split plot experiment with 72 male pigs weighing 52.9 ± 0.39 kg (mean ± SEM) was conducted to examine AA partitioning and body protein deposition (PD) in response to increasing dietary sulfur amino acids (SAA) with or without immune system (IS) activation. The main plot was with and without IS activation, and 4 diets containing different amounts of standardized ileal digestible (SID) SAA (SAA to Lys ratios of 0.45, 0.55, 0.65 and 0.75) were the subplots. Activation of IS was achieved by intramuscular injection of Escherichia coli lipopolysaccharides (LPS; serotype 055:B5, Sigma; 30 µg/kg BW) every Monday and Thursday, with control pigs injected with sterile saline. Maximum body PD, measured by dual-energy X-ray absorptiometry (DXA), and minimum plasma urea content were achieved at SID SAA:Lys ratio of 0.55 in saline-injected pigs but were achieved at a SID SAA:Lys ratio of 0.75 in IS-activated pigs. Immune system activation increased rectal temperature (P < 0.05), plasma haptoglobin (1.1 vs. 2.0 mg/mL; P < 0.001), and the proportion of neutrophils (0.39 vs. 0.42; P < 0.05) and decreased serum albumin content (38.4 vs. 36.8 g/L; P < 0.01). Increasing dietary SAA had no effects on these variables. Immune system-activated pigs had lower levels of homocysteine (Hcy; P < 0.001) and a lower Ser content (P < 0.05). Results showed that increasing dietary SAA as DL-methionine in growing and/or finishing pigs altered plasma AA contents, and that use efficiency of the AA was improved when greater levels of SAA were supplemented in IS-activated pigs.

Effect of polyacetylenic acids from Prunella vulgaris on various plant pathogens.

AIMS: This study is aiming at characterizing antifungal substances from the methanol extract of Prunella vulgaris and at investigating those substances' antifungal and antioomycete activities against various plant pathogens. METHODS AND RESULTS: Two polyacetylenic acids were isolated from P. vulgaris as active principles and identified as octadeca-9,11,13-triynoic acid and trans-octadec-13-ene-9,11-diynoic acid. These two compounds inhibited the growth of Magnaporthe oryzae, Rhizoctonia solani, Phytophthora infestans, Sclerotinia sclerotiorum, Fusarium oxysporum f. sp. raphani, and Phytophthora capsici. In addition, these two compounds and the wettable powder-type formulation of an n-hexane fraction of P. vulgaris significantly suppressed the development of rice blast, tomato late blight, wheat leaf rust, and red pepper anthracnose. CONCLUSIONS: These data show that the extract of P. vulgaris and two polyacetylenic acids possess antifungal and antioomycete activities against a broad spectrum of tested plant pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the occurrence of octadeca-9,11,13-triynoic acid and trans-octadec-13-ene-9,11-diynoic acid in P. vulgaris and their efficacy against plant diseases. The crude extract containing the two polyacetylenic acids can be used as a natural fungicide for the control of various plant diseases.

Diets containing inulin but not lupins help to prevent swine dysentery in experimentally challenged pigs.

Swine dysentery is a contagious mucohemorrhagic diarrheal disease caused by the intestinal spirochete Brachyspira hyodysenteriae that colonizes and induces inflammation of the cecum and colon. It has been reported that a diet containing chicory root and sweet lupin can prevent swine dysentery. This experiment was conducted to test the hypothesis that inulin in the chicory root rather than galactans in lupins was responsible for protective effects. An experiment with a 2 × 2 factorial arrangement of treatments was undertaken using pigs fed barley- and triticale-based diets, with the main effects being protein source [185 g/kg of canola meal (decreased galactans) or 220 g/kg of lupins (greater galactans)] and inulin supplementation (0 or 80 g/kg). Forty Large White × Landrace pigs weighing 21 ± 3 kg, with 10 pigs per diet, were allowed to adapt to the diets for 2 wk, and then each pig was challenged orally 4 times with a broth culture containing B. hyodysenteriae on consecutive days. Pigs were killed when they showed clinical signs of dysentery or 6 wk postchallenge. Pigs fed diets without inulin had 8.3 times greater risk (P = 0.017) of developing swine dysentery and were 16 times more likely (P = 0.004) to have colon contents that were culture-positive for B. hyodysenteriae, compared with the pigs fed a diet with 80 g/kg of inulin. Diets containing lupins did not prevent pigs from developing clinical swine dysentery; however, inclusion of lupins or inulin or both in the diets delayed the onset of disease compared with the diet based mainly on canola meal (P < 0.05). Diet did not influence the total concentration of organic acids in the ileum, cecum, or upper and lower colon; however, the molar proportions of the organic acids were influenced (P < 0.05). Consequently the pH values in the cecum, and upper and lower colon were not influenced (P > 0.05) by diet. However the pH values of the ileal digesta were decreased in pigs fed the diet with both lupins and inulin compared with the diet containing only lupins (P < 0.05). In conclusion, this study shows that diets supplemented with highly fermentable carbohydrates from inulin protected pigs against developing swine dysentery.

Effects of neolignans from the stem bark of Magnolia obovata on plant pathogenic fungi.

AIMS: To characterize antifungal principles from the methanol extract of Magnolia obovata and to evaluate their antifungal activities against various plant pathogenic fungi. METHODS AND RESULTS: Four neolignans were isolated from stem bark of M. obovata as antifungal principles and identified as magnolol, honokiol, 4-methoxyhonokiol and obovatol. In mycelial growth inhibition assay, both magnolol and honokiol displayed more potent antifungal activity than 4-methoxyhonokiol and obovatol. Both magnolol and honokiol showed similar in vivo antifungal spectrum against seven plant diseases tested; both compounds effectively suppressed the development of rice blast, tomato late blight, wheat leaf rust and red pepper anthracnose. 4-Methoxyhonokiol and obovatol were highly active to only rice blast and wheat leaf rust respectively. CONCLUSIONS: The extract of M. obovata and four neolignans had potent in vivo antifungal activities against plant pathogenic fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Neolignans from Magnolia spp. can be used and suggested as a novel antifungal lead compound for the development of new fungicide and directly as a natural fungicide for the control of plant diseases such as rice blast and wheat leaf rust.

Mycofumigation with Oxyporus latemarginatus EF069 for control of postharvest apple decay and Rhizoctonia root rot on moth orchid.

AIMS: To characterize the volatile antifungal compound produced by Oxyporus latemarginatus EF069 and to examine in vitro and in vivo fumigation activity of the fungus. METHODS AND RESULTS: An antifungal volatile-producing strain, O. latemarginatus EF069 inhibited the mycelial growth of Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum f. sp. lycopersici, and Rhizoctonia solani by mycofumigation. An antifungal volatile compound was isolated from the hexane extract of wheat bran-rice hull cultures of O. latemarginatus EF069 by repeated silica gel column chromatography and identified as 5-pentyl-2-furaldehyde (PTF). The purified PTF inhibited mycelial growth of R. solani in a dose-dependent manner. The mycofumigation with solid cultures of EF069 also reduced effectively the development of postharvest apple decay caused by B. cinerea and Rhizoctonia root rot of moth orchid caused by R. solani. CONCLUSIONS: Oxyporus latemarginatus EF069 showed in vitro and in vivo fumigation activity against plant pathogenic fungi by producing 5-pentyl-2-furaldehyde. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxyporus latemarginatus EF069 producing an antifungal volatile compound may be used as a biofumigant for the control of fungal plant diseases.

Amylolytic hydrolysis of native starch granules affected by granule surface area.

Initial stage of hydrolysis of native starch granules with various amylolytic enzymes, alpha-amylase from Bacillus subtilis, glucoamylase I (GA-I) and II (GA-II) from Aspergillus niger, and beta-amylase from sweet potato showed that the reaction was apparently affected by a specific surface area of the starch granules. The ratios of the reciprocal of initial velocity of each amylolytic hydrolysis for native potato and maize starch to that for rice with the amylolytic enzymes were nearly equivalent to the ratio of surface area per mass of the 2 starch granules to that of rice, that is, 6.94 and 2.25, respectively. Thus, the reciprocal of initial velocity of each enzymatic hydrolysis as expressed in a Lineweaver-Burk plot was a linear function of the reciprocal of surface area for each starch granule. As a result, it is concluded that amylolytic hydrolysis of native starch granules is governed by the specific surface area, not by the mass concentration, of each granule.

Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans.

AIMS: To isolate and identify antioomycete substances from Fusarium oxysporum EF119 against Phytophthora infestans and to investigate their antimicrobial activities against various plant pathogenic bacteria, oomycetes and true fungi. METHODS AND RESULTS: Two antioomycete substances were isolated from liquid cultures of F. oxysporum EF119, which shows a potent disease control efficacy against tomato late blight caused by P. infestans. They were identified as bikaverin and fusaric acid by mass and nuclear magnetic resonance spectral analyses. They inhibited the mycelial growth of plant pathogenic oomycetes and fungi. Fusaric acid also effectively suppressed the cell growth of various plant pathogenic bacteria, but bikaverin was virtually inactive. Treatment with bikaverin at 300 microg ml(-1) suppressed the development of tomato late blight by 71%. Fusaric acid provided effective control against tomato late blight and wheat leaf rust over 67% at concentrations more than 100 microg ml(-1). CONCLUSIONS: Both bikaverin and fusaric acid showed in vitro and in vivo antioomycete activity against P. infestans. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium oxysporum EF119 producing both bikaverin and fusaric acid may be used as a biocontrol agent against tomato late blight caused by P. infestans.

4-Week repeated intravenous dose toxicity study of a new camptothecin anticancer agent CKD-602 in dogs.

CKD-602 is a new camptothecin derivative antitumor agent with a formula (7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin) developed by Chong Kun Dang Pharmaceutical Company in Korea. In the present study, the subacute toxicity of CKD-602 was investigated after 4-week repeated intravenous administration of the test chemical in beagle dogs. The test chemical was administered intravenously at dose levels of 0, 0.001, 0.005, or 0.01 mg/kg/day for 4 weeks to male and female dogs (n = 3 for male and female dogs for each dose). During the test period, clinical signs, mortality, body weights, food and water consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights and histopathology were examined. In the high dose group, an increase in the incidence of abnormal clinical signs and a decrease in food and water intake and body weight gain were observed in both sexes. Hematological investigations revealed decreased white blood cells (WBC) in both sexes and reduced red blood cells (RBC), hemoglobin and hematocrit in females. Histopathological examinations revealed an increase in the incidence of atrophy of the sternal and femoral marrow and spleen in both sexes and atrophy of the thymus and mesenteric lymph node in males. No treatment-related adverse effects were observed in both sexes of the low and middle dose groups. In conclusion, the 4-week repeated intravenous dose of CKD-602 to beagle dogs caused increases in the clinical signs and histopathological changes, and decreases in the body weight gain, food and water intake, RBC, hemoglobin, hematocrit and WBC at the dose level of 0.01 mg/kg/day. In the present experimental conditions, the target organs were determined to be bone marrow, blood cells, spleen, thymus, and mesenteric lymph node. The no-observed-adverse-effect levels (NOAEL) for males and females were considered to be 0.005 mg/kg/day, respectively.

Developmental toxicity assessment of the new fluoroquinolone antibacterial DW-116 in rabbits.

DW-116 is a newly developed fluoroquinolone antibacterial with a broad spectrum against both Gram-positive and Gram-negative bacteria. We have reported recently that DW-116 is embryotoxic and teratogenic in rats. The present study was conducted to investigate the teratogenicity of DW-116, together with maternal toxicity and developmental toxicity using New Zealand White rabbits. The test chemical was administered by gavage to pregnant rabbits from gestational day (GD) 6 through to GD 18 at dose levels of 0, 5, 19.5 and 76.1 mg kg(-1) day(-1). All does were subjected to caesarean section on day 28 of gestation and their foetuses were examined for external, visceral and skeletal abnormalities. In the 76.1 mg kg(-1) group, a minimal maternal toxicity, as evidenced by decreased body weight gain during treatment period, was observed in pregnant rabbits. Significant embryo-foetal toxicity, including increased number of foetal deaths and delayed foetal ossification, was seen. However, no treatment-related morphological changes were detected in foetal external, visceral and skeletal examinations. There were no adverse effects on either pregnant dams or embryo-foetal development at 19.5 and 5 mg kg(-1). It was concluded that administration of DW-116 during the major organogenetic period in rabbits produced decreased maternal body weight gain, increased number of foetal deaths and foetal developmental delay but no evidence of teratogenicity. The no-observed-adverse-effect levels (NOAELs) of DW-116 are considered to be 19.5 mg kg(-1) day(-1) for does and embryo-foetuses, respectively.

Subacute toxicity evaluation of a new camptothecin anticancer agent CKD-602 administered by intravenous injection to rats.

The subacute toxicity of a new camptothecin anticancer agent, CKD-602, was investigated after 4-week repeated intravenous administration of the chemical in Sprague-Dawley rats. The test chemical was administered intravenously to rats at dose levels of 0, 0.003, 0.013, or 0.067 mg/kg/day for males and 0, 0.004, 0.018, or 0.089 mg/kg/day for females. At the end of the treatment period, 10 rats/sex/group were sacrificed. The remaining 5 rats/sex in the vehicle control and high dose groups continued the study without treatment for 2 weeks (recovery period). During the test period, clinical signs, mortality, body weights, food and water consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights, and histopathology were examined. In both sexes of the high dose group, an increase in the incidence of abnormal clinical signs and paleness of the eyes, a reduction in the body weight gain, food consumption and urine protein, and an increase in the water consumption were observed. Hematological investigations revealed a decrease in the red blood cells, hemoglobin and hematocrit and an increase in the mean corpuscular volume, mean corpuscular hemoglobin, platelets, and reticulocytes in a dose-dependent manner. Serum total cholesterol and total protein values were lower in females than those of controls, but not in males. An increase in the heart and liver weights and a decrease in the thymus weight were also found. Histopathological alterations included an increase in the incidence of atrophy of the sternal marrow, atrophy, fibrosis and mast cell hyperplasia of the femoral marrow, atrophy of the white pulp and extramedullary hematopoiesis of the spleen, atrophy of the thymus, auricular hypertrophy of the heart, extramedullary hematopoiesis and centriacinar telangiectasis of the liver, follicular degeneration of the ovary, and inflammation of the tail. The major treatment-related effects were not recovered at the end of 2-week recovery period. There were no adverse effects in the low and middle dose groups of both genders. In the present experimental conditions, the target organs were determined to be bone marrow, blood cells, spleen, liver, thymus, and heart. The no-observed-adverse-effect level was considered to be 0.013 mg/kg/day for males and 0.018 mg/kg/day for females.

Cold accumulation of SCOF-1 transcripts is associated with transcriptional activation and mRNA stability.

Cold acclimation enhances the transcription of several cold regulated (COR) genes. However, little is known about whether the elevation of the transcriptional level of the COR genes is due to transcriptional activation, or mRNA stability by a low temperature. Recently, we cloned a novel cold-inducible zinc finger protein gene from soybean, SCOF-1, which may function as a positive regulator of the COR gene expression . Here we report that the elevation of the SCOF-1 transcript level by cold stress is associated with both transcriptional activation and post-transcriptional mRNA stability under a low temperature. A nuclear run-on assay reveals that cold acclimation elevates the SCOF-1 transcript about three-fold compared to that of non-acclimated soybean nuclei. Furthermore, SCOF-1 transcripts increased substantially by a low temperature in transgenic tobacco plants that constitutively expressed SCOF-1 under the control of a constitutive cauliflower mosaic virus (CaMV) 35S promoter. When a transcription inhibitor, cordycepin, was treated with the deacclimating soybean cell, the decay level of the SCOF-1 transcripts was delayed significantly. This suggests that it may affect de novo protein synthesis, which degrades the SCOF-1 mRNA at room temperature. In addition, a secondary structure may be involved in the mRNA stability of SCOF-1 under a low temperature.

Inhibitory effect of interleukin-1alpha-induced apoptosis by Polygala tenuifolia in Hep G2 cells.

A human hepatoma cell line, Hep G2 cells are reliable for the study of alcohol-induced hepatotoxicity. In this study, we investigated the effect of an aqueous extract of Polygala tenuifolia WILLDENOW (Polygalaceae) roots (PTAE) on ethanol (EtOH)-induced cytotoxicity in Hep G2 cells. PTAE (0.01-1 microg/ml) dose-dependently inhibited the EtOH-induced interleukin-1alpha (IL-1alpha) secretion. PTAE (0.01-1 microg/ml) also inhibited the EtOH- and IL-1alpha-induced cytotoxicity. Furthermore, we found that PTAE inhibited the IL-1alpha-induced apoptosis of Hep G2 cells. These results suggest that PTAE may prevent the EtOH-induced cytotoxicity through inhibition of the apoptosis of Hep G2 cells.

Cloning and characterization of the 5'-flanking region for the human topoisomerase III gene.

The human DNA topoisomerase III (hTOP3) gene encodes a topoisomerase homologous to the Escherichia coli DNA topoisomerase I subfamily. To understand the mechanisms responsible for regulating hTOP3 expression, we have cloned the 5'-flanking region of the gene coding for the hTOP3 and analyzed its promoter activity. The presence of a single transcription initiation site was suggested by primer extension analysis. The hTOP3 gene promoter is moderately high in GC content and lacks a canonical TATA box, suggesting that hTOP3 promoter has overall similarity to promoters of a number of housekeeping genes. Examination of the promoter sequence indicated the presence of four Sp-1 consensus binding sequences and a putative initiator element surrounding the transcription initiation site. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 52-base pair region from -326 to -275 upstream of the transcription initiation site includes a positive cis-acting element(s) for the efficient expression of hTOP3 gene. On the basis of gel mobility shift and supershift assays, we demonstrated that both YY1 and USF1 transcription factors can bind to the 52-base pair region. When HeLa cells were transiently transfected with a mutant construct which had disabled both YY1- and USF1-binding sites, the luciferase activity was greatly reduced, suggesting that these binding elements play a functional role in the basal activation of the hTOP3 promoter. Transfection studies with mutations that selectively impaired YY1 or USF1 binding suggested that both YY1 and USF1 function as activators in the hTOP3 promoter.

Cloning of a cDNA encoding phospholipase D from Pimpinella brachycarpa.

Phospholipase D (PLD, EC 3.1.4.4) has been known to be related to various cellular processes in plants. To gain an understanding of the property of the enzyme in Pimpinella brachycarpa, the cDNA of the enzyme was isolated by PCR with degenerate primers, cDNA library screening, and 5' RACE. The full-length PLD cDNA is 2859 bp long and contains an open reading frame of 2424 bp coding for a polypeptide of 808 amino acids. The deduced enzyme has a calculated molecular mass of 91.7 kDa and pI of 5.86. The percent identity and similarity values of P. brachycarpa PLD with those of other PLDs in plants are 70 approximately 78 and 84 approximately 95, respectively. It was identified that PLD from P. brachycarpa has HQKIVVVD and HAKMMIVD sequences which were homologous with a duplicated HXKXXXXD motif that has been conserved in PLDs from plants, animals, and yeast. Based on the analysis of amino acid similarity, it is believed that PLD from P. brachycarpa is an alpha form which is distinct from PLD beta reported recently. The N-terminus is homologous to the C2 domain which is present in a number of different proteins involved in signal transduction and membrane trafficking in animals. Southern and northern blot analyses indicated that PLD was expressed from one copy of PLD gene in the genome of P. brachycarpa.