Biochemical and hematologic effects of polyvinylpyrrolidone-wrapped fullerene C60 after oral administration.

The fullerene C60 is used in consumer products such as cosmetics owing to its antioxidative effects and is being developed for nanomedical applications. However, knowledge regarding the safety of fullerene C60, especially after oral administration, is sparse. Here, we examined the safety of fullerene C60 in mice after 7 d of exposure to orally administered polyvinylpyrrolidone (PVP)-wrapped fullerene C60 (PVP-fullerene C60). Mice treated with PVP-fullerene C60 showed few changes in the plasma levels of various markers of kidney and liver injury and experienced no significant hematologic effects. Furthermore, the histology of the colon of PVP-fullerene C60-treated mice was indistinguishable from that of control mice. These results suggest that PVP-fullerene C60 lacks toxicity after high-dose oral administration and indicate that PVP-fullerene C60 can be considered safe for oral medication. These data provide basic information that likely will facilitate the production of safe and effective forms of fullerene C60.

Biological safety of liposome-fullerene consisting of hydrogenated lecithin, glycine soja sterols, and fullerene-C60 upon photocytotoxicity and bacterial reverse mutagenicity.

Various water-soluble derivatives of fullerene-C60 (C60) have been developed as detoxifiers for reactive oxygen species (ROS), whereas C60 incorporated in liposome (Lpsm) has not been reported yet. We prepared the liposome-fullerene (0.2% aqueous phase, Lpsm-Flln) which was composed of hydrogenated lecithin, glycine soja (soybean) sterols, and C60 in the weight ratio of 89.7:10:0.3, then examined the photocytotoxicity and bacterial reverse mutagenicity, as comparing with the Lpsm containing no C60. Photocytoxicity of Lpsm-Flln or Lpsm was examined using Balb/3T3 fibroblastic cells at graded doses of 0.49-1000 microg/mL under the condition of UVA- or sham-irradiation. The cells were irradiated with UVA (5 J/cm2, 320-400 nm, lambda max = 360 nm) at room temperature for 50 min. The resultant cell viability (% of control) did not decrease dose-dependently to 50% or less regardless of the UVA-irradiation. These results show that Lpsm-Flln or Lpsm does not possess photocytotoxicity to Balb/3T3 fibroblasts, and Lpsm-Flln may not exert a UVA-catalytic ROS-increasing action. A possibility for the reverse mutation by Lpsm-Flln or Lpsm was examined on four histidine-demanding strains of Salmonella typhimurium and a tryptophan-demanding strain of Escherichia coli. As for the dosages of Lpsm-Flln or Lpsm (313-5000 microg/plate), the dose-dependency of the number of reverse mutation colonies of each strain did not show a twice or more difference versus the negative control regardless of the metabolic activation, and, in contrast, marked differences for five positive controls (sodium azide, N-ethyl-N'-nitro-N-nitrosoguanidine, 2-nitrofluorene, 9-aminoacridine, and 2-aminoanthracene). The growth inhibition of bacterial strains and the deposition of Lpsm-Flln or Lpsm were not found. As a result, the bacterial reverse mutagenicity of Lpsm-Flln or Lpsm was judged to be negative under the conditions of this test. Thus, Lpsm-Flln and Lpsm may not give any significant biological toxic effects, such as photocytotoxicity and bacterial reverse mutagenicity.

[Extremely elderly patient in whom the pacing lead was implanted via the femoral vein].

We report on an extremely elderly patient in whom we were unable to insert a pacing lead via the subclavian or internal jugular vein because of a superior vena cava obstruction; we instead inserted the pacing lead via the femoral vein. The patient was a 98-year-old male. Thirty-nine years previously, pacemaker implantation was performed for complete atrioventricular block. Afterwards, pacemaker replacement and reimplantation had been performed a total of 15 times. The patient was recently admitted because of pacing failure. Pacemaker replacement was performed, but pacing was not possible because of disconnection of the pacing lead. Insertion of a new pacing lead was attempted via both subclavian veins and the right jugular vein but failed; this approach was abandoned and temporary pacing was done. Superior vena cava obstruction was noted on chest computed tomography (CT), and pacing lead insertion through the superior vena cava was deemed unfeasible. Myocardial electrode implantation was also considered, but general anesthesia was deemed problematic because of the patient's extreme age. A pacing lead was inserted via the right femoral vein, and the generator was implanted in the right lower abdomen. Postoperative pacing was satisfactory.

Effect of dried bonito (katsuobushi) and some of its components on GABAA receptors.

Katsuobushi, a popular Japanese food additive and traditional flavour enhancer, is produced from a fish, bonito, by a variety of processes, including boiling, sun drying, smoking and mould culturing. Aqueous katsuobushi (AK), which is produced from katsuobushi powder by extraction with water, and some of its aroma components, such as 2-ethyl-3-methylpyrazine and phenol derivatives, potentiated dose-dependently the response of the GABAA receptors expressed in Xenopus oocytes. When AK, 2-ethyl-3-methylpyrazine or 3-methoxyphenol were injected into mice prior to an intraperitoneal administration of pentobarbital, the pentobarbital-induced sleeping time increased. In an elevated plus maze test, intraperitoneal administration of 2-ethyl-3-methylpyrazine to mice increased significantly both the number of entries into the open arms and the duration of stay in the open arms, indicating anti-anxiety activity. Katsuobushi and its aroma components may modulate human mood or consciousness through acting on GABAA receptors in the brain.

Evaluation of soil bacterial biomass using environmental DNA extracted by slow-stirring method.

A simple and rapid method (slow-stirring method) for extracting environmental DNA (eDNA) from soils was constructed by physical mild stirring with chemical treatment. eDNA was extracted efficiently with minimal damage from various kinds of soil. The amount of eDNA and soil bacterial biomass showed a linear proportional relation [Y=(1.70x10(8))X, r2=0.96], indicating that bacterial biomass could be evaluated by quantifying levels of eDNA. Consequently, the average bacterial biomass in an agricultural field was calculated as 5.95x10(9) cells/g sample, approximately 10-100 times higher than that in non- and oil-polluted fields.

[Stenting in obstruction of superior vena cava; clinical experience with the self-expanding endovascular prosthesis].

From August 1997 to December 2002, 14 consecutive patients with superior vena cava syndrome with the self-expanding endovascular prosthesis. Diagnoses were adenocarcinoma in 6, small cell carcinoma in 4, squamous cell carcinoma in 1, metastatic lung cancer in 2, and invasive thymoma in 1. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were measured on their admission and perioperative period. Expecting only 1 patient complete symptomatically relieved within 3 days of stent implantation. Superior vena cava pressure or radial pressure of the stent was sufficient to relieve obstruction. Preoperative ANP level were normal, BNP level were increased. Postoperatively both ANP level and BNP level were slightly increased under intravenous dopamine hydrochloride. Implantation of the self-expanding stent endovascular prosthesis for superior vena cava syndrome provides rapid symptomatic relief and improves the patient's quality of life.

Kinetic analyses of alcohol-induced potentiation of the response of GABA(A) receptors composed of alpha(1) and beta(1) subunits.

To investigate the kinetics of both the potentiation and desensitization of the response of ionotropic GABA receptors (GABA(A) receptors) in the presence of various compounds, we expressed receptors composed of alpha(1) and beta(1) subunits by injecting cells with the cRNAs synthesized from cloned bovine GABA(A) receptor cDNAs and measured the electrical responses of the cells electrophysiologically with or without the compounds. The potentiation of the GABA(A) receptor-mediated response was quantitatively analyzed using a simple model with the assumption that the receptors have two identical binding sites for GABA molecules with a dissociation constant of K(1), and one potentiation site for the compound with a dissociation constant of K(p), and that the binding of the compound to the potentiation site only increases the affinity of the GABA binding sites, changing K(1) to K(1p). The estimated K(p) and K(1p) were dependent on the functional groups and the chain length of the compounds. These results could be satisfactorily analyzed using this simple model. The potentiation of the GABA(A) receptor-mediated response by the components of essential oils used for aromatherapy was also examined. These compounds accelerated the decay of the response, possibly due to desensitization of the receptors, which was also analyzed on the basis of the model.

Effects of bisphenol A and its derivatives on the response of GABA(A) receptors expressed in Xenopus oocytes.

To study the effects of bisphenol-A (BPA) known to have estrogenic actions, and its derivatives, 3,5-dimethylphenol (DMP) and p-t-butylphenol (TBP), on ionotropic gamma-aminobutyric acid (GABA) receptors, GABA(A) receptors were expressed in Xenopus oocytes by injecting both poly(A)+ RNA prepared from rat whole brain and cRNAs synthesized from cloned cDNAs of alpha1 and beta1 subunit of the bovine receptors, and their electrical responses were measured by the voltage clamping method. BPA caused the potentiation and inhibition of the former receptor-responses, while it caused only inhibition of the latter ones. In the presence of low concentrations of GABA, DMP and TBP potentiated the responses of both receptors. DMP and TBP also increased the rate of decay of the response, possibly by desensitization of the receptors when GABA solution was continuously bath-applied. Diethyl terephthalate (DTP), which is also known to have estrogenic actions, had little effect on both the responses and the decay of both receptors.

Cloning of a cDNA for a constitutive NRT1 transporter from soybean and comparison of gene expression of soybean NRT1 transporters.

We have isolated a cDNA for a putative transporter, named GmNRT1-3, in the NRT1 family from soybean. It was predicted to have a similar topological structure not only to both GmNRT1-1 and GmNRT1-2 reported previously, but also to other members of the family. Two other cDNAs isolated have parts of the sequence for putative NRT1 transporters, GmNRT1-4 and GmNRT1-5, suggesting that at least five NRT1 transporters occur in soybean. These GmNRT1 genes and the GmNRT2 gene, encoding a soybean NRT2 nitrate transporter, showed different expression patterns to each other under various nitrogen conditions. Specifically, GmNRT1-3 was constitutively expressed in both roots and leaves, while GmNRT1-2 was gradually expressed as the roots developed in the presence of ammonium as a nitrogen source, but not in the presence of both ammonium and nitrate. Based on these results, we discussed the possible regulation in the expression and role of these transporters in nitrate uptake.

Genomic DNA analysis of thyrotropin receptor in a family with hereditary hyperthyroidism.

Mutations of the thyrotropin receptor (TSH-R) gene have been reported in some cases of hyperthyroidism. We report a case of a family that had a high incidence of hyperthyroidism (6/13) which strongly suggested hereditary factors. We then analyzed whether the family had mutations of the TSH-R gene. No significant mutations in exon 10 of the TSH-R gene were found in the patient by restriction fragment length polymorphism analysis and polymerase chain reaction direct sequencing, when compared with those with 4 normal subjects and 2 patients with Graves' disease. Unknown mutations in the extracellular region of the receptor or other genes in this family remain to be studied.

Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions.

BACKGROUND: Sphingomonas paucimobilis SYK-6 utilizes an extradiol-type catecholic dioxygenase, the LigAB enzyme (a protocatechuate 4,5-dioxygenase), to oxidize protocatechuate (or 3,4-dihydroxybenzoic acid, PCA). The enzyme belongs to the family of class III extradiol-type catecholic dioxygenases catalyzing the ring-opening reaction of protocatechuate and related compounds. The primary structure of LigAB suggests that the enzyme has no evolutionary relationship with the family of class II extradiol-type catecholic dioxygenases. Both the class II and class III enzymes utilize a non-heme ferrous center for adding dioxygen to the substrate. By elucidating the structure of LigAB, we aimed to provide a structural basis for discussing the function of class III enzymes. RESULTS: The crystal structure of substrate-free LigAB was solved at 2.2 A resolution. The molecule is an alpha2beta2 tetramer. The active site contains a non-heme iron coordinated by His12, His61, Glu242, and a water molecule located in a deep cleft of the beta subunit, which is covered by the alpha subunit. Because of the apparent oxidation of the Fe ion into the nonphysiological Fe(III) state, we could also solve the structure of LigAB complexed with a substrate, PCA. The iron coordination sphere in this complex is a distorted tetragonal bipyramid with one ligand missing, which is presumed to be the O2-binding site. CONCLUSIONS: The structure of LigAB is completely different from those of the class II extradiol-type dioxygenases exemplified by the BphC enzyme, a 2,3-dihydroxybiphenyl 1,2-dioxygenase from a Pseudomonas species. Thus, as already implicated by the primary structures, no evolutionary relationship exists between the class II and III enzymes. However, the two classes of enzymes share many geometrical characteristics with respect to the nature of the iron coordination sphere and the position of a putative catalytic base, strongly suggesting a common catalytic mechanism.

Generation of free radicals during the death of Saccharomyces cerevisiae caused by lipid hydroperoxide.

The exposure of Saccharomyces cerevisiae cells to 13-L-hydroperoxylinoleic acid (LOOH) caused their death, the degree of which was dependent on the growth phase of the cells. Pre-application of ethanol, hydrogen peroxide (H2O2) and LOOH to S. cerevisiae cells reduced the effect of LOOH on the cells, showing the transient cross adaptation to LOOH. Antioxidants such as N,N',-diphenyl-p-phenylenediamine (DPPD), melatonin and vitamin E, and inhibitors of permeability transition of mitochondria, cyclosporin A and trifluoperazine, inhibited the LOOH-triggered cell death, while an inhibitor of glutathione synthetase, buthionine sulfoximine (BSO), enhanced the cell death by LOOH. Reactive oxygen species (ROS) were detected by flow cytometry, using the ROS-specific fluorescent indicator. A ferric iron chelator, deferoxamine, inhibited the LOOH-triggered cell death, and peroxyl radicals (LOO.) were detected by a spin trapping method. These reactive radicals possibly induced the death of S. cerevisiae cells. However, the DNA fragmentation characteristic of apoptosis was not observed in S. cerevisiae cells after exposure to LOOH, staurosporine, dexamethasone or etoposide, which have been reported to cause apoptosis in mammalian cells.

Potentiation of GABAA receptors expressed in Xenopus oocytes by perfume and phytoncid.

To study the effects of perfume and phytoncid on GABAA receptors, ionotropic GABAA receptors were expressed in Xenopus oocytes by injecting mRNAs that had been prepared from rat whole brain. Essential oil, perfume and such phytoncid as leaf alcohol, hinokitiol, pinene, eugenol, citronellol and citronellal potentiated the response in the presence of GABA at low concentrations (10 and 30 microM), possibly because they bound to the potentiation-site in GABAA receptors and increased the affinity of GABA to the receptors. Since it is known that the potentiation of GABAA receptors by benzodiazepine, barbiturate, steroids and anesthetics induces the anxiolytic, anticonvulsant and sedative activity or anesthetic effect, these results suggest the possibility that the intake of perfume or phytoncid through the lungs, the skin or the intestines modulates the neural transmission in the brain through ionotropic GABAA receptors and changes the frame of the human mind, as alcohol or tobacco does.

Serum lipoprotein(a) and apolipoprotein(a) phenotypes in patients with rheumatoid arthritis.

OBJECTIVE: To determine serum lipoprotein(a) (Lp[a]) concentrations and to analyze the apolipoprotein(a) (Apo[a]) phenotype in patients with rheumatoid arthritis (RA). METHODS: The subjects included 131 patients with RA and 200 healthy control subjects. Serum Lp(a) concentrations were measured by enzyme-linked immunosorbent assay, and the Apo(a) phenotype was determined by immunoblotting. HLA-DR typing was also done. RESULTS: The mean serum Lp(a) level was significantly higher (P < 0.001) in the RA patients (27.5 mg/dl) than in the controls (15.0 mg/dl). The S3 allele was found in 70.0% of the patients versus 39.5% of the controls (P < 0.001). There was no significant difference in HLA-DR4 positivity between patients with and without the S3 phenotype. CONCLUSION: The serum Lp(a) level was increased in patients with RA, possibly partly because of S3 phenotype predominance.

Sacral perineurial cyst with ossification of the arachnoid membrane.

A case of sacral perineurial cyst with ossification of the arachnoid membrane discovered intraoperatively is reported. We are not aware of any similar cases in the literature.

Circadian variation of urinary type I collagen crosslinked C-telopeptide and free and peptide-bound forms of pyridinium crosslinks.

This study was performed to investigate the circadian variation of urinary CrossLaps (CTx), which was the type I collagen peptide released during bone matrix degradation, and peptide-bound and free forms of urinary pyridinium crosslinks. Urine was obtained during the 24 h of the study in seven separate collections as follows: from 23:00 h to the first void (FV) followed by FV at 11:00, 11:00-14:00, 14:00-17:00, 17:00-20:00, 20:00-23:00, and 23:00 h to FV the next morning. Total, free, and peptide-bound pyridinoline (Pyr) and deoxypyridinoline (Dpyr) excretion measured by high-performance liquid chromatography (HPLC) and CTx measured by enzyme-linked immunosorbent assay in nine premenopausal women aged 22-40 years and nine osteoporotic women aged 65-83 years was analyzed. Among three parameters of Pyr measured by HPLC, a significant day and night difference was found only in total Pyr (21.9% higher at night than during the day in premenopausal women and 24.0% in osteoporotic women, whereas no significant day and night variation was found in free and peptide-bound Pyr in either group. In contrast, total and peptide-bound Dpyr were significantly (37.9% and 66.9%) higher at night than those during the day in premenopausal women (38.0%) and osteoporotic women (48.8%). For free Dpyr, there were no day and night differences in the two groups. The day and night variances were significantly greater in peptide-bound Dpyr than with total Dpyr in both groups. In urinary CTx, a significant circadian variation with a peak at night and a nadir at 17:00 h was found (p < 0.0001) (premenopausal was 54.0% higher at night than during the day; osteoporotic was 38.4%. In conclusion, urinary CTx represented remarkable circadian variation compared with urinary pyridinium crosslinks measured by HPLC. Furthermore, free pyridinium crosslinks did not undergo a circadian variation. Peptide-bound crosslinks might contribute mostly to the circadian variation of total excretion of pyridinium crosslinks.

Characterization of the gntT gene encoding a high-affinity gluconate permease in Escherichia coli.

We characterized the gntT gene encoding a high-affinity gluconate permease of Escherichia coli K-12. Primer extension and lacZ-operon fusion analyses revealed that gntT has one strong and two weak promoters, all of which are regulated positively by cAMP-CRP and negatively by GntR. The weak promoters became constitutive when separated from the upstream region including the strong promoter that overlaps a putative GntR-binding sequence. Gluconate-specific uptake activity was observed with cells harboring the gntT plasmid clone, which was enhanced by the presence of gntK encoding gluconate kinase.

Changes in mitochondrial membrane potential during oxidative stress-induced apoptosis in PC12 cells.

We examined the effects of various types of oxidative stress on cell survival and on mitochondrial membrane potential (delta psi m) in PC12 cells transfected with BCL-2. Several types of oxidative stress such as exposure to hydrogen peroxide, 13-L-hydroperoxylinoleic acid, and xanthine + xanthine oxidase triggered apoptotic nuclear condensation and DNA fragmentation in normal PC12 cells. These types of oxidative stress induced significant increases in level of reactive oxygen species (ROS) before cell death. By contrast, BCL-2 prevented the apoptosis induced by these oxidative stresses. However, BCL-2 did not reduce ROS levels, indicating that it functions downstream of ROS generation. We measured delta psi m as a potential target of ROS during oxidative stress-induced cell death. Hydrogen peroxide, 13-L-hydroperoxylinoleic acid, and xanthine + xanthine oxidase induced a significant loss of delta psi m simultaneously with cell death. BCL-2 prevented the decrease in delta psi m as well as apoptosis induced by oxidative stress. These observations suggest that the oxidative stress triggers apoptosis associated with both increased generation of ROS and decreases in level of delta psi m and that BCL-2 prevents cell death as well as delta psi m but not ROS production.

The sugar specificity of Na+/glucose cotransporter from rat jejunum.

A cDNA for a Na+/glucose cotransporter was cloned from rat jejunum cDNA library. This transporter was expressed in Xenopus oocytes by injection of cRNA synthesized from the cDNA, and the transporter ability was electrophysiologically examined. The cotransporter had a very narrow sugar specificity. Only D-glucose, D-galactose, and some of their derivatives elicited significant electrical responses. These results of sugar specificity were compared with those of the H+/hexose cotransporter of Chlorella. Dose-response relationships of several sugars followed a simple Michaelis-Menten type of kinetics. Both Vm and Km were dependent on the sugars. Not only the affinity of sugars to the cotransporter but also the rate of conformational change of the cotransporter loaded with the sugar and Na+, which translocates them from outside to inside, possibly depends on the sugar structure. The rate-limiting step of the transportation may be the conformational change, i.e., isomerization, of the cotransporter that translocates both the sugar and Na+ from outside to inside.