Validation of an LC-MS/MS method for the quantitation of phytosterols derived from Aloe vera gel.

A method to quantitate five minor phytosterols named Aloe sterols identified from Aloe vera gel was validated using AVGP (Aloe vera gel powder) as the sample. To measure the Aloe sterols content, AVGP was extracted with chloroform/methanol (2:1, v/v) and analyzed by liquid chromatography-tandem mass spectrometry. The calibration curve revealed a high coefficient of determination (>0.999). The limit of quantification was 2.3-4.1 ng/mL. Average recoveries ranged from 95 to 105%. The intra-day and inter-day precision were 2.6-6.4% and 3.8-7.3%, respectively, confirming good method precision. Aloe sterols were also quantified in AVGE (Aloe vera gel extract) using this method. We showed that the composition ratio of each Aloe sterol in AVGP did not change in AVGE. Additionally, we measured the concentration of Aloe sterols in the capsule containing AVGE, and confirmed that it was stable even after 1 year of storage. In conclusion, a quantification method was established to simultaneously measure multiple plant sterols with similar structures. • A quantification method to simultaneously measure several plant sterols with similar structures was established. • Results from the intra-day precision and the inter-day precision confirmed good precision. • This method can be applied to processed raw materials and/or foods in long-term storage.

Toxicological evaluation of ß-Galactosidase enzyme produced by Papiliotrema terrestris.

ß-Galactosidase (EC 3.2.1.23) is a glycoside hydrolase that catalyzes the release of sugar monomers from ß-galactoside oligosaccharides via hydrolysis of glycosidic bonds, and has potential uses in the food industry. The safety of this enzyme and its production organism, Papiliotrema terrestris (P. terrestris), are described herein. P. terrestris was non-pathogenic upon intravenous (IV) administration of 2.4 × 108 cfu and oral administration of 1.3 × 109 cfu. Genotoxic results for the enzyme concentrate were negative in a bacterial reverse mutation test (Ames test) and chromosome aberration test in cultured Chinese hamster lung fibroblast (CHL/IU) cells. In a 13-week oral gavage study in Sprague-Dawley rats, no adverse effects were observed in any of the tested group and a No Observed Adverse Effects Level (NOAEL) of 2000 mg/kg bw/day [total organic solids (TOS) 1800 mg/kg bw/day)] was established, which was the highest dosage tested. Allergenicity sequence analysis revealed no evidence suggesting that ß-galactosidase is an allergen. The data presented in this study support the conclusion that ß-galactosidase produced from P. terrestris is safe for use in food production.

Intraosseous schwannoma of the cervical vertebral body: A case report and review of the literature.

Intraosseous schwannomas (IOS) of non-sacral vertebra are extremely rare; only 14 cases were reported previously. We described a case of IOS involving a cervical vertebral body, successfully treated by surgical resection, with a review of the literature and discussion of this extremely rare tumour.

Retinopathy online challenge: automatic detection of microaneurysms in digital color fundus photographs.

The detection of microaneurysms in digital color fundus photographs is a critical first step in automated screening for diabetic retinopathy (DR), a common complication of diabetes. To accomplish this detection numerous methods have been published in the past but none of these was compared with each other on the same data. In this work we present the results of the first international microaneurysm detection competition, organized in the context of the Retinopathy Online Challenge (ROC), a multiyear online competition for various aspects of DR detection. For this competition, we compare the results of five different methods, produced by five different teams of researchers on the same set of data. The evaluation was performed in a uniform manner using an algorithm presented in this work. The set of data used for the competition consisted of 50 training images with available reference standard and 50 test images where the reference standard was withheld by the organizers (M. Niemeijer, B. van Ginneken, and M. D. Abràmoff). The results obtained on the test data was submitted through a website after which standardized evaluation software was used to determine the performance of each of the methods. A human expert detected microaneurysms in the test set to allow comparison with the performance of the automatic methods. The overall results show that microaneurysm detection is a challenging task for both the automatic methods as well as the human expert. There is room for improvement as the best performing system does not reach the performance of the human expert. The data associated with the ROC microaneurysm detection competition will remain publicly available and the website will continue accepting submissions.

Multifaceted effects of selective inhibitor of phosphodiesterase III, cilostazol, for cerebral vasospasm after subarachnoid hemorrhage in a dog model.

BACKGROUND: Selective inhibition of phosphodiesterase type III (PDE III) may be involved in the pathophysiology of vasospasm and a PDE III inhibitor, cilostazol, is thus expected to attenuate vasospasm after subarachnoid hemorrhage (SAH). We tested the therapeutic effects of cilostazol on angiographic and morphological vasospasm. METHOD: Twenty-one mongrel dogs were divided into 4 groups: (1) control (n = 3); (2) SAH (n = 6); (3) SAH with low-dose treatment (n = 6), and (4) SAH with high-dose treatment (n = 6). We used the established double-hemorrhage model of SAH achieved by injecting autologous blood. Angiography was performed on day 0 and day 7. The animals were euthanized after a second angiogram, and Western blotting was performed to analyze phenotypic changes in smooth muscle cells of the basilar artery. The basilar artery was sectioned for immunohistochemistry of SM1, SM2 and SMemb to analyze phenotypic changes (SM1, SM2 for the contractile type of smooth muscle myosin heavy chain and SMemb for the synthetic type). Intact endothelial cells were counted under a microscope. RESULTS: Severe vasospasm was obtained in the SAH group (42 +/- 1%). Cilostazol attenuated angiographic vasospasm in both treatment groups (63 +/- 2 and 74 +/- 4%, respectively). Prevention of endothelial damage and phenotypic changes in smooth muscle cells were observed in both treatment groups (p < 0.05 vs. control, ANOVA). CONCLUSION: Cilostazol attenuates vasospasm following SAH in dogs by suppressing phenotypic changes in the basilar artery and preventing endothelial damage. Therefore, we anticipate that cilostazol may be useful in the management of vasospasm.

Asymptomatic non-functioning ectopic pituitary adenoma in the suprasellar region: a case report.

Asymptomatic non-functioning ectopic pituitary adenomas (EPAs) are extremely rare. To our knowledge, this is the first case report of an asymptomatic non-functioning EPA in the suprasellar region treated by cranio-orbital skull base surgery. EPAs should be considered in the differential diagnosis and histological confirmation is necessary.

Heme synthase (ferrochelatase) catalyzes the removal of iron from heme and demetalation of metalloporphyrins.

The red pigments in meat products, including cooked cured ham, arise from the reaction of myoglobin with nitric oxide generated from exogenous nitrite. Since carcinogenic nitrosoamines may be generated by the treatment of meats with nitrite, the production of nitrite-free meat products is an attractive alternative. Raw dry-cured (Parma) hams are produced by the treatment of meats with salts other than nitrite. Analysis of pigments in raw dry-cured hams reveals that the main pigment is zinc protoporphyrin, suggesting that the conversion of heme to zinc protoporphyrin occurs via an iron-removal reaction from myoglobin heme during the processing of raw hams. Purification of the iron-removal enzyme showed that it was identical to ferrochelatase. Recombinant ferrochelatase in combination with NADH-cytochrome b5 reductase catalyzed NADH-dependent iron-removal reaction from hemin and hemoproteins. Metal ions such as zinc and cobalt were also removed from the corresponding metalloporphyrins. The addition of zinc ions led to the formation of zinc protoporphyrin. In cultured cells, the conversion of zinc mesoporphyrin to mesoheme was observed to be dependent on ferrochelatase and could be markedly induced during erythroid differentiation. This is the first demonstration of a new enzyme reaction, the reverse reaction of ferrochelatase, which may contribute to a new route of the recycling of protoporphyrin and heme in cells.

Involvement of 101F6, a homologue of cytochrome b561, in the reduction of ferric ions.

We have isolated and characterized a small transmembrane protein, called 101F6, showing high sequence homology to cytochrome b(561), a protein containing two binding sites for haem. The newly identified 101F6 contains six membrane spanning domains in which conserved histidine residues are located, and has a molecular mass of 25 kDa. When the haem-binding with bacterial expressed 101F6 was examined, the protein bound haem and the deletion of one histidine residue at 149 caused a loss of the binding. 101F6 mRNA was expressed widely in various tissues, and especially abundant in liver, kidney and lung. It was also expressed in several cultured cell lines. The protein expressed from the 101F6 cDNA in Balb/3T3 cells was about 25 kDa in size and was localized in small vesicles, including endosomes and endoplasmic reticulum of the perinuclear region. Comparison of the location of 101F6 with that of transferrin receptor-1 revealed that the localization of 101F6 in small vesicles was not always the same as the localization of transferrin receptor-1, but was similar to that of haem oxygenase-1. The other homologue to cytochrome b(561), SDR-2 was also expressed in the small vesicles similar to the location of 101F6. Finally, reduction of ferric ions as well as of azo-dye increased with 101F6- or SDR-2-expressing cells. Thus, both 101F6 and SDR-2 were localized in small vesicles of cells and played roles in the reduction of ferric ions.

Studies on the non-pathogenicity of Chryseobacterium proteolyticum and on the safety of the enzyme: protein-glutaminase.

Protein-glutaminase (PG) is a protein-deamidating enzyme produced from the microorganism Chryseobacterium proteolyticum strain 9670. Food safety studies were conducted on both the enzyme and the production organism. The strain was evaluated for pathogenicity and toxigenicity by intravenous and oral inoculation studies in Slc:ICR male SPF mice. The results demonstrate that the tested C. proteolyticum strain is of very low pathogenicity comparable to known food source bacterial strains and is very unlikely to demonstrate any pathogenicity in animals or humans. The level of endotoxin is very low and typical of the endotoxin levels in drinking water and teas. A 90-day study of PG, conducted in Sprague-Dawley rats, showed no adverse effects due to the enzyme up to dose levels of 2500 mg/kg-bw/day (NOAEL). Details of the study are presented, including, organ and body weights, histological findings, and blood and urine chemistry. Additionally, bacterial reverse mutation test (Ames test) and chromosomal aberration test using mammalian established cell line were conducted, resulting in the absence of mutagenicity in PG.

The antioxidant role of a reagent, 2',7'-dichlorodihydrofluorescin diacetate, detecting reactive-oxygen species and blocking the induction of heme oxygenase-1 and preventing cytotoxicity.

Heme oxygenase-1 (HO-1) degrades heme into biliverdin, iron and CO. The enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli, and is induced in response to reactive oxygen species (ROS). 2',7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) is a common reagent used to detect ROS by the oxidation of 2',7'-dichlorodihydrofluorescin (DCFH) to fluorescent dichlorodihydrofluorescein. We previously found that rapid oxidation of DCFH occurred with heme-compounds as well as ROS [Ohashi, T. et al. (2002) FEBS Lett. 511, 21-27], and then examined the effect of DCFH-DA on the induction of HO-1 expression by arsenite, cadmium and hemin, which induce oxidative stress and cytotoxicity. We found suppression of the arsenite-, cadmium- and hemin-dependent induction of HO-1 with DCFH-DA. The suppression occurred at the transcriptional level since the promoter activity of the Maf-recognition site of the HO-1 gene decreased with the DCFH-DA treatment. DCFH abolished the phosphorylation of extracellular signal-regulated kinase, the nuclear translocation of a transcriptional activator Nrf2, and cell death. An antioxidant, N-acetylcysteine (NAC), also suppressed the induction by arsenite and cadmium, but not that by hemin, indicating that DCFH blocked a different site in the stress signal pathway from NAC. Considering that the oxidation of DCFH diminishes ROS generated by various stressors, our findings provide a potential strategy for protection of cells from toxic insults using DCFH-like molecules.

Cloning and nucleotide sequence of carbaryl hydrolase gene (cahA) from Arthrobacter sp. RC100.

A carbaryl hydrolase gene (cahA) encoded on the plasmid pRC1 in Arthrobacter sp. RC100 was cloned and sequenced. The entire region of the deduced amino acid sequence was found to be homologous to that of an amidase family. Parts of the consensus sequences of the amidase gene have been identified in CahA from strain RC100. CahA was overexpressed in Escherichia coli JM109, and the enzyme was purified to homogeneity by protamine sulfate treatment, ammonium sulfate precipitation, and hydrophobic and anion-exchange chromatographies. The purified enzyme showed hydrolase activity toward 1-naphthylacetamide and isobutyramide but showed no activity toward 1-naphthylacetate. This is the first report of an amidase that is able to hydrolyze N-methylcarbamate pesticides.

Low- and high-level expressions of heme oxygenase-1 in cultured cells under uninduced conditions.

Heme oxygenase-1 (HO-1) degrades heme into biliverdin, iron, and CO. The enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. We examined the regulation of HO-1 expression in culture cells under uninduced conditions. Observations by in situ hybridization and immunostaining showed that in cultured mouse fibroblast Balb/3T3 cells not subjected to treatment, 10-15% of cells highly expressed HO-1. The similar pattern of the expression of HO-1 was observed with mouse embryo liver BNL-CL2 cells and Chinese hamster ovary cells. The marked expression of HO-1 was related to the activation of stress-activated protein kinase and to the expression of cyclooxygenase (Cox)-2. When the cells were treated with arachidonic acid, a precursor of prostaglandin, induction of HO-1 in the HO-1-expressing cells but not in the low-expressing cells occurred. This increase was abrogated by the treatment with the Cox inhibitors, indomethacin, and dexamethasone. Neither prostaglandin H2, E2 nor F2a induced HO-1 expression. These results suggest that some cells respond to the cellular stress and intermediates of prostaglandin biosynthesis may act as endogenous stressors to induce HO-1.

[Influence of conversion from forest to pasture on visual effect of landscape].

Geographical information system (GIS) was applied to quantitatively evaluate the influence of conversion from forest to pasture on the visual effect of the landscape by the viewpoint of landscape conservation. The visible area of the intermediate zone was larger than that of both the near and far zones and hence, the influence of the conversion was the biggest on the visual effect of the intermediate zone. Therefore, intermediate zone is the most important part in landscape conservation. Analyses on the visibility of forest on the background of pasture showed that most parts of visible forest in all the zones located at elevations 400-600 m, but rarely distributed on slopes as steep as 25 degrees. The measures in landscape management and conservation were also discussed in the sense of increasing the visual effect of pasture landscape.

Galectin-1 induces cell adhesion to the extracellular matrix and apoptosis of non-adherent human colon cancer Colo201 cells.

To isolate cDNAs for molecules involved in cell adhesion to the extracellular matrix, expression cloning with non-adherent colon cancer Colo201 cells was carried out. Four positive clones were isolated and, when sequenced, one was found to be galectin-1, a beta-galactoside-binding protein. When cultured on fibronectin-, laminin-, and collagen-coated and non-coated dishes, the adherent galectin-1 cDNA-transfected Colo201 cells increased and spread somewhat. Immunofluorescence staining revealed that galectin-1 was expressed inside and outside of Colo201 cells. The adhesion was dependent on the carbohydrate-recognition domain of galectin-1 since lactose inhibited the adhesion and exogenously-added galectin-1 caused the adhesion. PD58059, an inhibitor of mitogen-activated protein kinase, or LY294002, a phosphoinositide 3-OH kinase inhibitor, decreased the adhesion. Furthermore, the expression of galectin-1 in Colo201 cells induced apoptotic cell death, while exogenously-added galectin-1 did not cause apoptosis. These results indicate that galectin-1 plays a role in both cell-matrix interactions and the inhibition of Colo201 cell proliferation, and suggest that galectin-1 expressed in cells could be associated with apoptosis.

Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: formation of the fluorescein is independent of the generation of reactive oxygen species.

Oxidative stress and the generation of reactive oxygen species (ROS) have been implicated in the pathogenesis of cellular damage. These events have usually been reported in terms of oxidation of a reporter molecule such as 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA). Treatment of HeLa cells with hemin or metalloporphyrins resulted in a rapid oxidation of DCFH in a time- and dose-dependent manner. This oxidation was inhibited by treatment of the cells with a large amount of superoxide dismutase and catalase, which is different from observations that these enzymes had no effect on the induction of heme oxygenase-1, a stress-induced protein, in hemin-treated cells. To examine the possibility that the oxidation of DCFH is independent of the generation of ROS, the oxidation was measured using hemoglobin-synthesizing erythroleukemia K562 cells. When K562 cells were treated with delta-aminolevulinic acid, a precursor of heme, oxidation of DCFH increased depending on the heme content in cells. Then DCFH-DA was oxidized directly with heme, hemoglobin, myoglobin and cytochrome c. These results suggest that oxidation of DCFH is not always related to the generation of ROS but may be related to heme content in cells.

A zinc-finger protein, PLAGL2, induces the expression of a proapoptotic protein Nip3, leading to cellular apoptosis.

Pleomorphic adenomas gene-like 2 (PLAGL2) protein containing seven C(2)H(2) zinc finger motifs exhibits DNA binding and transcriptional activation activity and is expressed in response to hypoxia or iron deficiency. To identify the target genes of PLAGL2, we transfected mouse PLAGL2 cDNA into Balb/c3T3 fibroblasts and neuroblastoma Neuro2a cells. Both cells were induced to undergo apoptosis by the expression of PLAGL2 as judged by assays of TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling), DNA fragmentation, propidium iodide staining, and the binding of annexin V to the cell surface. The treatment of the cells with an iron chelator, desferrioxamine, resulted in the induction of apoptosis with a concomitant accumulation of PLAGL2 in the nucleus. The expression of PLAGL2 in Balb/c3T3 cells led to the mRNA expression of a proapoptotic factor, Nip3, which can dimerize with Bcl-2. Nip3 mRNA was also induced in desferrioxamine-treated cells. Furthermore, the Nip3 promoter containing a hypoxia-responsive element was activated by PLAGL2, independent of hypoxia-inducible factor-1 (HIF-1). The transfection of antisense oligonucleotide to mouse Nip3 mRNA into PLAGL2-expressing cells led to a decrease in apoptotic cells compared with sense oligonucleotide-transfected cells. Despite the activation of DNA-HIF-1 binding activity under hypoxic conditions, neither an accumulation of HIF-1 alpha nor the activation of HIF-1 was observed following the expression of PLAGL2. These results indicate that PLAGL2 is located downstream of HIF-1 and suggest that PLAGL2 functions as a tumor suppressor in association with HIF-1.