A 13-week oral dose subchronic toxicity study of gardenia yellow containing geniposide in rats.

Gardenia yellow powders A, B and C, containing geniposide at 0.284%, 0.938% and 2.783%, respectively, were administered orally to male and female SD rats as 3% feed admixtures for 13-weeks to evaluate any potential toxicity. Mean geniposide intake values were 5.72, 18.9 and 56.3mg/kg/day in groups receiving these feed admixtures, respectively. All animals survived the duration of the study. The following findings were evident in the gardenia yellow C group: chromatouria, slightly increased plasma total bilirubin, blackish brown discoloration of the kidneys and liver, brown pigments in the proximal tubular epithelium of the kidneys. Slightly increased plasma total bilirubin was considered to be due to interference of metabolite of geniposide with the system of measurement and not to be a toxic effect since there were no related changes in histopathology of the liver or in any blood chemistry parameters. Other findings were limited to pigmentations or discolorations attributable to metabolites of geniposide. No treatment-related effects were evident on body weight, food consumption, ophthalmology, hematology or organ weights in any group. Therefore, it was concluded that 3-month ingestion of the gardenia yellow powder containing geniposide at 2.783% (approximately 60 mg/kg/day as geniposide intake) does not cause any severe toxic effects.

Molecular identification and characterization of two medium-chain acyl-CoA synthetases, MACS1 and the Sa gene product.

In this study, we identified and characterized two murine cDNAs encoding medium-chain acyl-CoA synthetase (MACS). One, designated MACS1, is a novel protein and the other the product of the Sa gene (Sa protein), which is preferentially expressed in spontaneously hypertensive rats. Based on the murine MACS1 sequence, we also identified the location and organization of the human MACS1 gene, showing that the human MACS1 and Sa genes are located in the opposite transcriptional direction within a 150-kilobase region on chromosome 16p13.1. Murine MACS1 and Sa protein were overexpressed in COS cells, purified to homogeneity, and characterized. Among C4-C16 fatty acids, MACS1 preferentially utilizes octanoate, whereas isobutyrate is the most preferred fatty acid among C2-C6 fatty acids for Sa protein. Like Sa gene transcript, MACS1 mRNA was detected mainly in the liver and kidney. Subcellular fractionation revealed that both MACS1 and Sa protein are localized in the mitochondrial matrix. (14)C-Fatty acid incorporation studies indicated that acyl-CoAs produced by MACS1 and Sa protein are utilized mainly for oxidation.

Various cooking methods and the flavonoid content in onion.

Onion is a major source of flavonoids and is cooked in various ways in the world. The major flavonoids in onion are two quercetin glycosides, quercetin 4'-O-beta-glucoside (Q4'G) and quercetin 3,4'-O-beta-diglucosides (Q3,4'G), which are recognized as bioactive substances that are good for our health. We have investigated the effect of cooking procedures on the content of antioxidants. We selected quercetin conjugates, total phenol compounds, and ascorbic acid to estimate the amount of flavonoid ingestion from onion. We examined the following cooking methods: boiling, frying with oil and butter, and microwave cooking. Various cooking methods do not consider the degradation of quercetin conjugates when cooking onion. Microwave cooking without water better retains flavonoids and ascorbic acid. Frying does not affect flavonoid intake. The boiling of onion leads to about 30% loss of quercetin glycosides, which transfers to the boiling water. At that time, the effect of additives on the quercetin conjugates is different according to the compounds. The hydrolysis of quercetin glycosides for daily cooking might occur with the addition of seasonings such as glutamic acid. Additional ferrous ions accelerated the loss of flavonoids.

Bilirubin adsorption therapy and subsequent liver transplantation cured severe bilirubin encephalopathy in a long-term survival patient with Crigler-Najjar disease type I.

Crigler-Najjar disease (CN) type I is characterized by persistent unconjugated hyperbilirubinemia from birth. The male patient here was diagnosed with this disease as a neonate and had been treated by phototherapy. At age 16 he suddenly developed generalized convulsions, followed by impaired cognitive function. The serum level of bilirubin was extremely high (total bilirubin: 41.7 mg/dl) and there were no other detectable causes responsible for the metabolic encephalopathy. He received bilirubin adsorption therapy several times, and the bilirubin encephalopathy improved in response to the fall in the serum level of bilirubin. After this he underwent a successful liver transplantation in Australia, and recovery of his mental faculties was satisfactory. Within the subsequent 3 years epileptic abnormal discharges on the electroencephalogram disappeared. Phototherapy alone can not prevent the rise in the serum level of bilirubin in adolescent or adult patients with CN type I, therefore such patients tend to experience life-threatening bilirubin encephalopathy. To save patients with the acute onset type of bilirubin encephalopathy, sufficient bilirubin adsorption followed by liver transplantation appears to be the most recommended therapeutic approach.

Glycine prevents apoptosis of rat sinusoidal endothelial cells caused by deprivation of vascular endothelial growth factor.

Apoptosis of sinusoidal endothelial cells (SECs) is one of the initial events in the development of ischemia-reperfusion injury of the liver. Glycine has been shown to diminish ischemia-reperfusion injury in the liver and improve graft survival in the rat liver transplantation model. Here, we investigated the effect of glycine on apoptosis of primary cultured rat SECs induced by vascular endothelial growth factor (VEGF) deprivation. Isolated rat SECs were cultured in EBM-2 medium supplemented with 10% fetal bovine serum (FBS) and growth factors including 20 ng/mL VEGF for 3 days. SECs at 3 days of culture showed spindle-like shapes; however, cells started shrinking and detaching from dishes by VEGF deprivation. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-uridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining in these conditions. Control SECs contained only a few percent of TUNEL-positive cells; however, they started increasing 4 hours after VEGF deprivation, and the percentage of TUNEL-positive cells reached about 50% at 8 hours and almost 100% at 16 hours after VEGF deprivation. Interestingly, this increase in TUNEL-positive cells after VEGF deprivation was prevented significantly when glycine (1-10 mmol/L) was added to the medium, the levels being around 60% of VEGF deprivation without glycine. Furthermore, strychnine (1 micromol/L), a glycine receptor antagonist, inhibited this effect of glycine, suggesting the possible involvement of the glycine receptor/chloride channel in the mechanism. Moreover, Bcl-2 protein levels in SECs were decreased 8 hours after VEGF deprivation, which was prevented almost completely by glycine. It is concluded that glycine prevents apoptosis of primary cultured SECs under VEGF deprivation.

Enantioselective uptake of amino acid with overoxidized polypyrrole colloid templated with L-lactate.

An overoxidized polypyrrole colloid, which can recognise enantiomers of amino acids has been prepared by a newly developed molecular imprinting technique. A polypyrrole colloid, which had been polymerised from a mixture of pyrrole (monomer), polyvinylpyrrolidone (steric stabiliser), peroxodisulfate (oxidant) and L-lactate (dopant), was overoxidized to create a dopant-complementary cavity. The enantioselectivity of the overoxidized colloid was evaluated by comparing the uptake of L-alanine and L-cysteine with that of the respective D-enantiomers. The L/D uptake ratios for these amino acids were about 2, while phenylalanine showed suppressed uptake for both the enantiomers. The absence of phenylalanine uptake can be explained in terms of the molecular size, which is too large to be accommodated by the cavity created by L-lactate. In contrast, a colloid templated with L-phenyllactate took up L-phenylalanine with a higher enantioselectivity of about 7. A colloid templated with L-lactate was applied to surface chirality analysis through enantioselective adsorption on cysteine-modified gold surfaces. Quartz microbalance experiments and scanning electron microscope observation of the gold surface revealed that the colloidal particle has higher affinity to a surface modified with L-cysteine than to one modified with D-cysteine.

Awareness of and attitude of elderly subjects regarding health care and welfare in rapidly ageing population in Japan.

OBJECTIVES: We aimed to obtain information on the degree of knowledge and understanding about the current systems of health care and welfare held by the elderly, in order to achieve comprehensiveness in family practice. METHOD: We conducted a study on the awareness of healthy elderly persons by direct interview. The study was carried out in Kuni Village in a remote mountainous region in Japan, where the elderly population accounts for 24.8% of the total population. The subjects were self-dependent in their daily living activities and were aged 65 years and older. RESULTS: The subjects' knowledge of health care and welfare systems was generally good, and the degree of their utilization of these systems was also good. But 83.3% of those who did not want to utilize the welfare system indicated their preference to depend on their family for support. CONCLUSION: Family physicians must endeavour to offer comprehensive care to their patients by including these systems for rapidly ageing communities.

Isolation and mapping of a human zinc finger gene (ZNF188) homologous to ZNF187, a serum-response-element binding protein.

From a human pancreas cDNA library we isolated and characterized a novel zinc finger gene encoding a protein homologous to ZNF187, a serum response element-binding protein. The full-length cDNA contained an open reading frame of 1,686 nucleotides encoding a predicted 562-amino-acid peptide that included an ATP-GTP binding site and seven C2H2 zinc finger domains. The consensus sequence of the C2H2 domains (CX2CX3FX5LX2HX3H) is common in the SRE-binding region present in Drosophila Krüppel proteins. An alternatively spliced form of the transcript found in the cDNA library lacked both the ATP-GTP binding site and any C2H2 zinc finger domains. We localized this gene (ZNF188) to chromosome band 7q22.1-->q22.3 by fluorescence in situ hybridization.

Determination of bradykinin-(1-5) in inflammatory exudate by a new ELISA as a reliable indicator of bradykinin generation.

We have developed an ELISA for BK-(1-5) (Arg1-Pro2-Pro3-Gly4-Phe5). In rat carrageenin-induced pleurisy, in which a plasma exudation peak was observed 5 h after carrageenin, BK levels in the exudates were negligible (< 60 pg/rat). BK-(1-7) (des-Phe8-Arg9-BK) was detectable (900-400 pg/rat) over the entire course of the inflammation. However, a larger amount of BK-(1-5) was detectable in association with the increase in plasma exudation, showing a peak (8800 +/- 1200 pg/rat) 3 h after carrageenin. Bromelain (10 mg/kg, i.v.) and soy bean trypsin inhibitor (0.3 mg/rat, intra-pleural) significantly reduced BK-(1-5) levels (by 60-93%, 3, 7 and 19 h after carrageenin) and plasma exudation rates (by 61-74%, 3 and 7 h after carrageenin). Dexamethasone (0.3 mg/kg, i.p.) reduced BK-(1-5) levels (by 78%) and decreased plasma exudation (by 70%) 3 h after carrageenin. In nasal allergy patients, antigen challenge of nasal mucosa elevated BK-(1-5) levels and active kallikrein levels in nasal washes. These results verify that BK-(1-5) determined by ELISA is a good indicator for release of kinins in vivo.

Identification of basic fibroblast growth factor-like immunoreactivity in panax ginseng extract: investigation of its molecular properties.

Basic fibroblast growth factor (bFGF)-like immunoreactivity was detected in extracts of Panax ginseng root by using a sensitive two-site enzyme immunoassay specific for human bFGF (hbFGF). In an investigation of the molecular properties of this bFGF-like molecule (bFGF-LI), the bFGF-LI and hbFGF were found to be equivalent with respect to antigenicity, molecular weight, isoelectric point, affinity for binding to heparin, and mitogenic activity toward BALB/c3T3 fibroblasts. The identification of this bFGF-LI molecule in Panax ginseng root helps to explain various activities of the traditional Chinese medicine ginseng.

Cloning, functional expression and tissue distribution of human alpha 1c-adrenoceptor splice variants.

We report the cloning and characterization of two isoforms of human alpha 1c-adrenoceptor cDNA (alpha 1c-2, alpha 1c-3). These isoforms are generated by alternative splicing and differ from the clone we previously isolated (alpha 1c-1) in their length and sequences of the C-terminal domain. Tissue distribution of mRNAs showed that these variants co-express with alpha 1c-1 in the human heart, liver, cerebellum and cerebrum. Despite the structural differences, functional experiments in transfected CHO cells showed that the three isoforms have similar ligand binding properties, and all couple with phospholipase C/Ca2+ signaling pathway.

Topical application of cyclosporine on guinea pig allergic contact dermatitis.

Topical cyclosporine applied to the test site substantially inhibited the elicitation reaction of contact sensitivity in the guinea pigs previously sensitized with 2,4-dinitrochlorobenzene (DNCB). This suppressive effect of the drug was short lived and reversible. Cyclosporine was not effective when given six hours or later after antigenic challenge to the test site. Cyclosporine had no effect on the toxic contact reaction in normal animals either to croton oil or to DNCB in high concentration. Cyclosporine applied topically to the challenge site also resulted in a reduction of retest and flare-up reactions of contact sensitivity to DNCB, but did not affect the production of generalized rash in the same animals. These results indicate that in the future local topical application of cyclosporine may make treatment of human cutaneous immune-mediated disorders a possibility without serious side effects.

Mechanisms of adverse effect of air-oxidized soy bean oil-feeding in rats.

In order to clarify the mechanism of the adverse effects of air-oxidized oil feeding, the effects of dietary soy bean oil on body weight gain, food consumption, fecal consistency and gastrointestinal functions were investigated in rats. Feeding of oxidized soy bean oil with a peroxide value above 350 produced significant reductions in body weight gain, food consumption and intestinal sucrase activity, and severe diarrhea. These adverse effects were prevented with the concurrent feeding of Gobo dietary fiber and were rapidly eliminated within a day after switching to the diet containing the unoxidized soy bean oil. Furthermore, the remarkable release of sucrase from the small intestine was observed on perfusion of the jejunum with Ringer's bicarbonate solution containing the oxidized soy bean oil at the 1% level. These findings suggest that the adverse effects occurring after air-oxidized oil feeding are due to a disturbance of gastrointestinal functions, and raise the possibility that the primary cause might be solubilization or exfoliation of the brush border membrane.

Immunocytochemical identification of CRF in the human hypothalamus.

Immunoreactive ovine corticotropin-releasing factor (CRF) was revealed in the cell bodies of parvocellular neurons in the paraventricular nucleus of the human hypothalamus by an immunocytochemical technique. The immunoreactivity was negative in magnocellular neurons. Immunoreactive nerve fibers were found in the subependymal layers of the third ventricle and in the perivascular space of the primary plexus in the lower hypothalamus. No CRF immunoreactive cell bodies were found in the supraoptic nucleus.