Apple pectin affects the efficacy of epigallocatechin gallate on oral sucrose tolerance test in adult mice.

Epigallocatechin gallate (EGCg), a dietary polyphenol and a major tea catechin, is a known sucrase inhibitor. Since dietary pectin is known to modulate some of the functions of the gastrointestinal tract, we investigated whether it could specifically affect the efficacy of EGCg on an oral sucrose tolerance test in mice. Male Crj:CD-1 (ICR) mice (seven weeks old) were randomly divided into two groups and fed a 5 % apple pectin (PE) or 5 % cellulose (CE) diet (control diet) for 28 days. After the experimental diet period, all mice were fasted overnight. A volume of 0.2 mL EGCg (20 mg/mL) was orally administered to all the mice by stainless steel feeding needle via injection syringe and a sucrose tolerance test was performed. The blood glucose levels were measured in blood collected from the tail vein using the OneTouch® Ultra® blood glucose monitoring system. Blood glucose levels at 30 minutes and 60 minutes after sucrose loading in the PE group were significantly higher than initial blood glucose levels. However, blood glucose levels at 30 minutes, 60 minutes, and 120 minutes after sucrose loading in the CE group were not significantly higher than initial blood glucose levels. After laparotomy, plasma lipids were also measured. Plasma triglyceride concentrations were significantly greater in the PE group than in the CE (control) group. This demonstrates that dietary pectin can affect the efficacy of EGCg on the oral sucrose tolerance test in mice.

Effect of pectin enhancement on plasma quercetin and fecal flora in rutin-supplemented mice.

Few reports have considered the effects of dietary fiber on plasma quercetin and the intestinal flora. We investigated the effects of pectin on the plasma and fecal flora of mice fed a diet supplemented with the quercetin glycoside rutin. Male mice were randomly divided into 2 groups, which were fed a pectin-rutin (PR) or cellulose-rutin (CR) diet for 14 d. Plasma quercetin and isorhamnetin metabolites were measured by high-performance liquid chromatography. Feces were immediately processed with bacteriological procedures. The fecal flora was investigated. Plasma quercetin and isorhamnetin concentrations were significantly higher in the PR diet group, as was the plasma isorhamnetin/quercetin ratio. The composition of the intestinal flora differed between the 2 dietary groups. The total number of fecal bacteria was significantly larger in the PR group, in which most types of bacteria were more abundant, with the exceptions of bifidobacteria, fusiform-shaped bacteria, and staphylococci. The lower gut seemed to be the major absorption site for rutin. Pectin might thus enhance the bioavailability of quercetin from rutin by altering the metabolic activity of the intestinal flora and/or gut physiological function.

Cytotoxic and DNA damage-inducing activities of low molecular weight phenols from rhubarb.

Six new phenol (anthraquinone or stilbene) glycosides with an acyl group at 6-position of the glucopyranose moiety were isolated from rhubarb (the roots of Rheum palmatum) cultivated in Japan, together with 22 known compounds. Most of these compounds were evaluated for cytotoxic activity against tumor and normal cells and for induction of DNA damage by spore rec-assay. Among them, emodin and aloe-emodin showed higher cytotoxic activities against human oral squamous cell carcinoma (HSC-2) and salivary gland tumor (HSG) cell lines than against normal human gingival fibroblasts (HGF). Chrysophanol 8-O-beta-(6'-acetyl)glucopyranoside, 4-(4'-hydroxyphenyl)-2-butanone 4'-O-beta-D-(2"-O-galloyl-6"-O-cinnamoyl) glucopyranoside, and 6"-O-(4'''-hydroxybenzoyl) resveratroloside exhibited relatively higher cytotoxic activities against all these cells. The other glycosides of anthraquinone or stilbene showed weaker cytotoxic activity against these tumor cell lines, but may be considered as cancer chemopreventive agents. Spore rec-assay with a recombination deficient mutant of Bacillus subtilis M45 demonstrated the DNA damage-inducing activity of emodin and aloe-emodin 15-O-beta-D-glucopyranoside among, rhubarb phenols.

Treatment of glioblastoma by direct inoculation of concentrated high titer-recombinant retrovirus carrying the herpes simplex virus thymidine kinase gene.

We prepared retroviruses carrying the lacZ gene or herpes simplex virus thymidine kinase (HTK) gene with titers of 1.4-2.5 x 10(11) colony-forming units (cfu)/ml, and stereotaxically inoculated only 3 microliters of the retroviruses into a mouse glioma model. This resulted in highly efficient transduction in vivo. The transduced glioma cells migrated far from the implantation site, potentiating the induction of the remarkable bystander effect. Following repetitive ganciclovir (GCV) intraperitoneal injection, effective killing of glioma cells in the mouse brain was observed. The transduction efficiency was nearly as high as that observed for the implantation of high-titer retrovirus-producing fibroblasts. Eighty per cent of brain tumor-bearing mice were completely cured by our treatment protocol using concentrated HTK-harboring retroviruses. Our results suggest that repeated inoculations of high-titer retroviruses carrying the HTK gene followed by GCV treatment may be a promising strategy for the clinical treatment of malignant gliomas. To achieve further safety in the gene therapy of glioma, genes abundantly expressed in human glioblastoma were searched by the Serial Analysis of Gene Expression (SAGE) technique. Among the top-147 most expressed tags in glioblastoma, we found a tag, TTTTGGGTAT, originated from an unidentified gene, which was not detected in human astrocyte cultures. Real-time quantitative RT-PCR showed that MAGE-E1 expression was 2.6-15 fold enriched in glioblastoma relative to human astrocytes. Expressed Sequence Tags (ESTs) containing this tag were homologous to melanoma-associated antigen gene (MAGE) family, and this new cDNA, named MAGE-E1, was cloned by 5'-rapid amplification of cDNA ends (RACE) technique. MAGE-E1 expression was enriched in glioblastoma and low in other cancers, and MAGE-E1 expression was detected only in brain and ovary among normal tissues. These results indicate that MAGE-E1 is a novel and glioma-specific member of MAGE family, which can be applied to glioma-specific gene transduction.

Pod-1/Capsulin shows a sex- and stage-dependent expression pattern in the mouse gonad development and represses expression of Ad4BP/SF-1.

Mammalian sex-determination and differentiation are controlled by several genes, such as Sry, Sox-9, Dax-1 and Mullerian inhibiting substance (MIS), but their upstream and downstream genes are largely unknown. Ad4BP/SF-1, encoding a zinc finger transcription factor, plays important roles in gonadogenesis. Disruption of this gene caused disappearance of the urogenital system including the gonad. Ad4BP/SF-1, however, is also involved in the sex differentiation of the gonad at later stages, such as the regulation of steroid hormones and MIS. Pod-1/Capsulin, a member of basic helix-loop-helix transcription factors, is expressed in a pattern closely related but mostly complimentary to that of the Ad4BP/SF-1 expression in the developing gonad. In the co-transfection experiment using cultured cells, overexpression of Pod-1/Capsulin repressed expression of a reporter gene that carried the upstream regulatory region of the Ad4BP/SF-1 gene. Furthermore, forced expression of Pod-1/Capsulin repressed expression of Ad4BP/SF-1 in the Leydig cell-derived I-10 cells. These results suggest that Pod-1/Capsulin may play important roles in the development and sex differentiation of the mammalian gonad via transcriptional regulation of Ad4BP/SF-1.

Effect of dietary cardiac glycosides on blood pressure regulation in rats.

To investigate the possible physiological significance of dietary cardiac glycosides in blood pressure regulation, the blood pressure of normal Sprague Dawley rats raised on a regular diet, which naturally contains large amounts of Na+-pump inhibitors, was compared with that of rats on a purified synthetic diet, which contains no Na+-pump specific inhibitors, and with that of rats on a synthetic diet supplemented with 10 microg x mL(-1) ouabain or 10 microg x mL- convallatoxin in the drinking water. After 6 weeks on the synthetic diet, the systolic blood pressure in the synthetic diet group was significantly elevated (145 +/- 5 vs. 128 +/- 4 mmHg, P < 0.05). At 10 weeks it reached a plateau (154 +/- 3 vs. 122 +/- 3 mmHg, P < 0.05). Plasma renin activity and Na+ level were significantly higher in animals fed synthetic diets than in the regular diet group (P < 0.01). Administration of either losartan or lisinopril or a switch to a low salt synthetic diet (0.03% sodium) normalized the synthetic diet-induced high blood pressure. Supplementation of the synthetic diet with the cardiac glycosides delayed the onset of the increase in blood pressure for 4 weeks. Plasma aldosterone levels were approximately doubled in the cardiac glycoside-treated groups. Higher plasma Na+ levels and hematocrit values present in the synthetic diet group were normalized by the glycoside supplements. These results suggest that supplemental dietary cardiac glycosides exert bidirectional effects on blood pressure regulation through actions that modulate extracellular fluid and electrolyte balance.

In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene.

We aimed to introduce foreign DNA into spermatogenic cells in the testis by injection of the DNA encoding jellyfish fluorescent proteins, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) into the seminiferous tubules and in vivo electroporation. We obtained fluorescent spermatozoa only when using the gene of the YFP protein fused to a mitochondrial localization signal peptide. Intracytoplasmic injection into oocytes of these spermatozoa gave fluorescent fetuses and pups. Almost all of the individuals produced from fluorescent spermatozoa were transgenic. We confirmed integration of the gene into chromosomes and its transmission into offspring. This is the first report of gene transfer into germ cells and subsequent production of transgenic offspring.

A method for detection of a cytokine and its mRNA in the central nervous system of the developing rat.

We report here an effective and concise method to determine the localization of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, and its mRNA in the central nervous system of pre- and postnatal rats. This method allows for double staining to demonstrate localization of different molecules on the same tissue specimen at the levels of mRNA and proteins by in situ hybridization and immunohistochemistry, respectively. Additionally, the present method gives results more quickly than the conventional isotopic techniques. By use of this method, we carried out immunohistochemistry with an anti-rat MIF polyclonal antibody and demonstrated positive staining using the avidin-biotin complex method (ABC method). To detect its mRNA, we performed nonradioactive in situ hybridization using a digoxigenin (DIG)-labeled RNA probe prepared from a full length fragment of rat MIF cDNA. MIF was strongly expressed in the telencephalon on embryonic day 16. Non-radioactive in situ hybridization with a DIG-labeled RNA probe as well as the immunohistochemistry described here could be applicable to characterize localization of mRNA and proteins of different molecules on the same tissue specimen.

[Analysis of 209 PCB congeners by high separation gas chromatography/low resolution mass spectrometer].

All PCB congeners were analyzed by high separation gas chromatography/low resolution mass spectrometer, using 209 PCB congeners as standards. They were separated into 169 peaks, including 28 peaks containing 2 PCBs and 8 peaks containing 3 PCBs. The rice oil samples of Fukuoka and Taiwan Poisonings were analyzed for PCB congeners. They showed 115 separated peaks of PCB congeners. Total PCB concentrations were 879 and 769 ppm in 2 samples of Fukuoka rice oil and 57 and 83 ppm in 2 samples of Taiwan rice oil.

[Analysis of all PCB congeners in breast milk and blood of Yusho patients].

Breast milk and blood of Yusho patients were analyzed for polychlorinated biphenyl (PCB) congeners by High separation gas chromatography/Low resolution mass spectrometer. Seventy-one and forty-nine PCB congeners were identified and quantified in the breast milk and blood, respectively. Total PCB concentrations (Whole base) in breast milk of 2 Yusho patients were 69.9 and 15.1 ppb, respectively, being 11.6 and 2.5 times higher than those of 4 control breast milk. Average total PCB concentration of 5.0 ppb in whole blood of 13 Yusho patients was 3.6 times higher than those of control persons. Fourteen PCB congeners, such as 99, 117, 130, 137, 138, 156, 157, 164, 170, 171, 172, 189, 191 and 195, were particularly higher concentration in breast milk and blood of Yusho patients than in those of control persons. Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and coplanar PCBs were also analyzed in the breast milk of Yusho patients and control persons. Large portions, 83 and 74%, of Dioxin toxic equivalency (TEQ) in the 2 Yusho breast milk were consisted of TEQ of 2,3,4,7,8-pentaCDF only, while total TEQ in the breast milk of control persons was consisted of PCDFs 48%. PCDDs 29% and coplanar PCBs 23%.

Effects of guar gum and cellulose on cecal enzyme activity and cecal short-chain fatty acids in young and aged mice.

The effects of cellulose or guar gum on cecal enzyme activity and cecal short-chain fatty acids (SCFAs) in young and aged mice were studied. Male Crj:CD-1 (ICR) mice were fed an MF diet for 4 (young mice) or 23 months (aged mice). The MF diet was then replaced with a semisynthetic diet supplemented with 5% guar gum or 5% cellulose. The mice were fed the guar gum or cellulose diet for 3 weeks. There was no significant difference in cecal content between the two diet groups. There were no significant differences in total short-chain fatty acid production between the young mice fed the cellulose and those fed the guar gum diet, and between the aged mice fed the cellulose and guar gum diet. There were significant differences in cecal enzyme activity between the young mice fed the cellulose and those fed the guar gum diet. Beta-glucuronidase activity was significantly higher in the young mice fed the guar gum diet than in those fed the cellulose diet. There were also significant differences in cecal enzyme activity between the aged mice fed the cellulose diet and those fed the guar gum diet. Beta-glucuronidase activity was significantly higher in the aged mice fed the guar gum diet than in these fed the cellulose diet. Beta-glucosidase activity was significantly lower in the aged mice fed the guar gum diet than in those fed the cellulose diet. The effect of cellulose on the microflora between the young and aged mice might be different from the effect of guar gum. The degree of adaptation to the diet of microflora in young and aged mice fed the cellulose diet might differ from that in those fed the guar gum diet. The higher enzyme activities of microflora in aged animals compared to young animals, might have some relation with the incidence of colon cancer in aged animals.

Organochlorine compounds from a terrestrial higher plant: structures and origin of chlorinated orcinol derivatives from diseased bulbs of Lilium maximowiczii.

Seven chlorine-containing orcinol derivatives (2-8) and orcinol (9) have been isolated from diseased bulbs of the edible lily Lilium maximowiczii, and their structures have been elucidated. Six of the chlorinated orcinol derivatives (2, 4-8) showed antifungal activity. Because organochlorine compounds are rare in terrestrial higher plants, their biosynthetic origin was examined. These compounds were shown to be induced in intact bulb scales by UV irradiation or by inoculation with the pathogenic fungus Fusarium oxysporum f. sp. lilii. Biosynthetic studies suggested that these "natural organochlorine pesticides" are produced by enzymatic chlorination of orcinol (9) with chloroperoxidase and hydrogen peroxide, which are both induced in the plant tissue under stress conditions.

Effect of the long-term feeding of dietary lipids on the learning ability, fatty acid composition of brain stem phospholipids and synaptic membrane fluidity in adult mice: a comparison of sardine oil diet with palm oil diet.

The effect of 12 month feeding of 5% palm oil or sardine oil diet on the maze-learning ability, fatty acid composition of brain stem phospholipids and synaptic membrane fluidity in mice was studied. The time required to reach the maze exit and the number of times that a mouse strayed into blind alleys in the maze were measured three times every 4 days. The time and number of mice fed on the sardine oil diet were less than those of animals fed on the palm oil diet in the first and second trials. The results of fatty acid composition analysis of brain stem phosphatidylethanolamine showed that the percentage of docosahexaenoic acid (22:6, n-3; DHA) was higher, but the arachidonic acid (20:4, n-6; AA) and docosatetraenoic acid (22:4, n-6; DTA) were lower in the sardine oil diet fed-mice than in the palm oil diet fed-animals. Moreover, the microviscosity of the synaptic plasma membrane in the sardine oil diet group was lower than that in the palm oil diet group. These results suggest that the adult mice fed on the sardine oil diet for a long period maintain higher levels of docosahe xaenoic acid in brain phospholipids, synaptic membrane fluidity and maze-learning ability than animals fed on the palm oil diet.

Alterations of lymphocyte subsets in patients with recurrent fetal wastage positive for antiphospholipid antibodies treated with Chinese herbal medicine.

The alterations of peripheral blood lymphocyte subsets were sequentially analyzed in patients with recurrent fetal wastage who were treated with the Chinese herbal medicine, Sairei-to (Chan ling-Tan) for positive antiphospholipid antibodies to analyze the underlying mechanisms of the therapy. The titer of antiphospholipid antibodies was significantly decreased by administration of Sairei-to at one and two months after commencement of treatment and in the newly pregnant state compared with that before administration. The percentage of CD19-positive cells significantly decreased at two months after commencement of Sairei-to treatment and at the newly pregnant state compared with that before administration in successful pregnancies. The percentage of CD4-positive cells significantly increased two months after commencement of Sairei-to treatment compared with that before administration in both successful pregnancies and total cases. The CD4/CD8 ratio was increased significantly after two months administration of Sairei-to in both successful pregnancies and total cases. Thus, it is suggested that the administration of Sairei-to might induce the predominance of CD4-positive cells in parallel with the suppression of antiphospholipid antibodies. Moreover, the suppression of CD19-positive cells (B cells) was induced by administration of Sairei-to which might be involved in successful continuation of pregnancy in the patients.

Effects of pectin on jejunal and ileal morphology and ultrastructure in adult mice.

The effects of pectin on jejunal and ileal morphology and ultrastructure were studied using adult male mice fed a semisynthetic diet containing 8% (w/w) cellulose or pectin for 30 days. No significant differences in the jejunal villus height between the 2 groups were found, but the jejunal crypt depth, and both the ileal villus height and crypt depth of the mice fed the pectin diet were significantly greater than those of the mice fed the cellulose diet. There were evident ultrastructural differences in the jejunal absorptive cells between 2 dietary groups: numerous intercellular spaces were observed in the jejunal absorptive cells of the mice fed the pectin diet, but not the cellulose diet. Moreover, the ileal absorptive cells of the mice fed the pectin diet contained numerous peroxisomes, whereas there were few in these cells of mice fed the cellulose diet. The functional characteristics of the ileum of the mice fed the pectin diet might be different from those fed the cellulose diet.

Immunosuppressive therapy for recurrent aborters with positive antiphospholipid antibodies and alteration of 6ketoPGF1 alpha/TXB2 ratio.

Twelve women (13 pregnancies) with antiphospholipid antibodies (APA) who had suffered from two or more recurrent spontaneous abortions or fetal deaths and had successful pregnancy outcomes after immunosuppressive therapy were studied. APA titers were determined by enzyme-linked immunosorbent assay against cardiolipin, phosphatidyl serine and phosphatidyl inositol. Plasma levels of 6ketoprostaglandin F1 alpha (6ketoPGF1 alpha) and thromboxane B2 (TXB2) were determined by radioimmunoassay. All of the 13 pregnancies resulted in term delivery. None of the 13 patients suffered from pregnancy-induced hypertension, and only one showed intrauterine growth retardation. A significant decrease of APA titer was observed after immunosuppressive therapy. The 6ketoPGF1 alpha/TXB2 ratios before the therapy, after it and at the 1st, 2nd and 3rd trimesters of pregnancy were 0.62 +/- 0.398, 0.88 +/- 0.106, 0.84 +/- 0.550, 1.25 +/- 0.834 and 0.67 +/- 0.413, respectively. The ratio at the 2nd trimester was significantly higher than that before the therapy (P < 0.05, paired t-test, n = 9). The results indicate that the immunosuppressive therapy affected the physiological balance between thromboxane A2 and prostacyclin, and improved clinical symptoms such as recurrent fetal wastage.

Species specificity of anticoagulant activity of activated human protein C: involvement of factor V as well as protein S.

Activated protein C (APC) possesses species specificity in its anticoagulant activity. Human APC exerts only weak activity in rat plasma compared with that in human plasma. The present study was undertaken to estimate the difference in interaction of human and rat factors with human APC and to assess the cause of the species specificity. Human or rat protein S (PS), factor V, or factor VIII was used to supplement human plasma depleted of each respective factor, and the anticoagulant activity of human APC was measured in term of the elongation of activated partial thromboplastin time (APTT). The activity of human APC in rat PS- or factor V-supplemented plasma was weaker than that in the human PS- or factor V-supplemented plasma. Furthermore, using purified human and rat factor V, human APC showed weaker inactivation of rat factor V than human factor V. Equal anticoagulant activity was observed in human or rat factor VIII-supplemented plasma. And there was a little difference in the interaction of APC with its inhibitors in human or rat plasma during a few minutes of incubation as judged by measurement of residual activity by an enzyme capture assay. From these results factor V as well as PS seems to play a major role in the species specificity of APC.

Grade II astrocytoma visualized by technetium-99m-ECD SPECT.

We present a case of primary brain tumor demonstrating increased uptake of 99mTc-ECD. Astrocytoma (Grade II) showed significantly increased cerebral blood perfusion on dynamic images and homogeneously increased uptake on static images with 99mTc-ECD brain SPECT. There seems to be some difference in perfusion and mechanism of tumor uptake among the cerebral blood flow imaging agents (99mTc-ECD, 99mTc-HMPAO and [123I]-IMP) and 201TI-chloride.

Treatment for patients with recurrent abortion with positive antiphospholipid antibodies using a traditional Chinese herbal medicine.

Twelve patients with recurrent abortion who had shown positive antiphospholipid antibodies were treated through the administration of a Japanese modified traditional Chinese herbal medicine Sairei-To (Chan ling-Tang) The patients had experienced a total of 27 spontaneous abortions in their previous pregnancies and had no other pregnancy history except for one patient. The patients were treated with 9.0 g of Sairei-To per day before their next pregnancy. The positive value of antiphospholipid antibodies returned to negative in 9 patients out of 12 patients through the treatment. Out of 12 patients, in 10 patients, their new pregnancy continued uneventfully and delivered an offspring (Success rate: 83.3%). Thus, the current treatment was considered to be an effective therapy for patients with recurrent abortion who were found to be positive for antiphospholipid antibodies.

Effects of docosahexaenoic acid and sardine oil diets on the ultrastructure of jejunal absorptive cells in adult mice.

The influence of docosahexaenoic acid (DHA) and sardine oil diets on the ultrastructure of jejunal absorptive cells was studied. Adult male Crj:CD-1 (ICR) mice were fed a fat-free semisynthetic diet supplemented with 5% (by weight) purified DHA ethyl ester, refined sardine oil, or palm oil. The mice received the DHA or palm oil diets for 7 days (groups 1 and 2) and the refined sardine oil or palm oil diets for 30 days (groups 3 and 4). There were significant ultrastructural changes in the jejunal absorptive cells between the mice fed on the palm oil diet and those receiving the DHA and sardine oil diets. The endoplasmic reticulum and Golgi apparatus of some jejunal absorptive cells in the mice fed on the palm oil diet for 7 and 30 days developed vacuolation on the upper site of the nucleus. In contrast, many granules, which appeared to be lipid droplets, were observed in the endoplasmic reticulum and Golgi apparatus of the jejunal absorptive cells in the DHA and sardine oil diet groups. These results suggest that ultrastructural differences in the jejunal absorptive cells between mice in the omega-3 fatty acid and palm oil diet groups may be associated with the changes in lipid metabolism.