Development of Neospora caninum cultured with human serum in vitro and in vivo

Because there has been no report of symptomatic Neospora caninum infection in humans, we examined the effect of human serum on the parasite's growth in either a bovine angioendothelial cell or Caco-2 cell culture in vitro and in immunocompromised mice in vivo. There was no difference in intracellular parasite numbers between cells incubated with human serum at 24 hr after challenge and those incubated with fetal bovine serum (FBS), which has no titer for the anti-N. caninum agglutination antibody test. Serum of sheep infected with N. caninum, which has the anti-N. caninum antibody, reduced the numbers of the intracellular parasite significantly. We also showed that there was no inhibitory effect on the intracellular multiplication of the parasite in cells incubated with human serum through incorporation of 3H-uracil. CB-17 scid mice administered human serum daily and challenged with N. caninum died on day 20 or 22 after challenge, when large numbers of parasite clusters were found in the brain, oviduct, adrenal gland, lung, stomach, spleen, skeletal muscle, pancreas, and mesenteric lymph nodes. Scid mice administered FBS survived until the end of the experiment. These results suggest that adult human serum may have no inhibitory effect on the development of N. caninum in vitro and in vivo.     

Axenic cultivation of Entamoeba dispar in newly designed yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium

Yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium that allows axenic cultivation of Entamoeba dispar was designed based on casein-free yeast extract-iron-serum (YI-S) medium, and the usefulness of the medium was assessed. The main differences from YI-S medium are replacement of glucose by gluconic acid, addition of dihydroxyacetone and D-galacturonic acid monohydrate, and sterilization by filtration. This medium promoted the axenic growth of 5 strains of E. dispar (2 strains of nonhuman primate isolates and 3 strains of human isolates). In addition, to clarify the biological basis for the growth of E. dispar in this medium, analyses of relevant enzymes on the glycolytic pathway of the amoebae as well as of the protozoans that are the best culture supplement for amoebae are being performed.     

Irradiation of mammalian cultured cells with a collimated heavy-ion microbeam

As the first step for the analysis of the biological effect of heavy charged-particle radiation, we established a method for the irradiation of individual cells with a heavy-ion microbeam apparatus at JAERI-Takasaki. CHO-K1 cells attached on a thin film of an ion track detector, CR-39, were automatically detected under a fluorescence microscope and irradiated individually with an 40Ar13+ ion (11.5 MeV/nucleon, LET 1260 keV/microm) microbeam. Without killing the irradiated cells, trajectories of irradiated ions were visualized as etch pits by treatment of the CR-39 with an alkaline-ethanol solution at 37 degrees C. The exact positions of ion hits were determined by overlaying images of both cells and etch pits. The cells that were irradiated with argon ions showed a reduced growth in postirradiation observations. Moreover, a single hit of an argon ion to the cell nucleus resulted in strong growth inhibition. These results tell us that our verified irradiation method enables us to start a precise study of the effects of high-LET radiation on cells.     

Dosing-time-dependent differences in lipopolysaccharide-induced liver injury in rats

Dosing-time-dependent differences in lipopolysaccharide (LPS)-induced liver injury were examined in rats housed under a 12 h light:dark (LD) cycle. LPS (5 mg/kg) was intravenously injected into different groups of rats at 2, 14, or 20 h after light on (HALO). Elevations in serum liver enzymes after 14 HALO were significantly greater than those after 2 HALO. These parameters were lower in rats given LPS at 20 HALO, compared to 14 HALO. The number of polymorphonuclear cells (PMN) in the liver and the amount of hepatic myeloperoxidase activity, which reflects the number of PMN in liver tissues, was significantly greater in the 14 than in the 2 HALO group. In addition, hepatic interleukin-6 (IL-6) production in the 14 HALO group was enhanced compared to that in the 2 HALO trial. These results suggest that LPS-induced liver injury is greater during the early active than during the early resting period. Dosing-time-dependent variation in the accumulation of PMN in the liver and, potentially, subsequent IL-6 production in liver tissues might be involved in this phenomenon.     

Evaluation of liver and peripheral blood micronucleus assays with 9 chemicals using young rats. A study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Japanese Environmental Mutagen Society (JEMS)-Mammalian Mutagenicity Study Group (MMS)

We conducted simultaneous liver and peripheral blood micronucleus assays in young rats with seven rodent hepatocarcinogens-4,4'-methylenedianiline (MDA), quinoline, o-toluidine, 4-chloro-o-phenylenediamine (CPDA), dimethylnitrosamine (DMN), p-dimethylaminoazobenzene (DAB), and di(2-ethylhexyl)phthalate (DEHP)-and two mutagenic chemicals-kojic acid and methylmethanesulfonate (MMS). Quinoline, DMN, and DAB were positive in the liver assay, while o-toluidine, kojic acid, DAB, and MMS were positive in the peripheral blood assay. o-Toluidine, kojic acid, and DAB are reportedly negative in mouse bone marrow micronucleus assays, indicating a species difference. Our results revealed a correlation between micronucleus induction in hepatocytes and hepatocarcinogenicity. This technique can be useful for the detection of micronucleus-inducing chemicals that require metabolic activation, and it enables simultaneous comparison of the micronucleus-inducing potential of chemicals in the liver and peripheral blood in the same individual.     

Acylated anthocyanins from the violet-blue flowers of Orychophragonus violaceus

Three acylated cyanidin 3-sambubioside-5-glucosides (1-3) were isolated from the violet-blue flowers of Orychophragonus violaceus, and their structures were determined by chemical and spectroscopic methods. Two of those acylated anthocyanins (1 and 3) were cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-xylopyranosyl)-6-O-(4-O-(beta-D-glucopyranosyl)-trans-acyl)-beta-D-glucopyranoside]-5-O-(6-O-malonyl-beta-D-glucopyranoside)s, in which the acyl groups were p-coumaric acid for 1, and sinapic acid for 3, respectively. The last anthocyanin 2 was cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-xylopyranosyl)-6-O-(4-O-(beta-D-glucopyranosyl)-trans-feruloyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside. In these flowers, the anthocyanins 2 and 3 were present as dominant pigments, and 1 was obtained in rather small amounts.     

Synthesis and characterization of a novel reagent containing dansyl group, which specifically alkylates sulfhydryl group: an example of application for protein chemistry

We have synthesized a novel reagent containing dansyl group, iodoacethyl dansylcadaverine (IADC), which specifically alkylates sulfhydryl groups. The carboxyl group of iodoacetic acid was activated with dicyclohexylcarbodiimide and was condensed with amino group of dansylcadaverine. Purity and chemical structure of IADC was confirmed with mass spectrometry (MS) and NMR. IADC alkylated GSH but not GSSG, which was confirmed by MS. The reactivity of IADC with proteins was also investigated with Western blotting using anti-dansyl antibody. IADC reacted only with sulfhydryl-containing proteins. The specificity of the interaction of IADC with sulfhydryl groups in proteins was confirmed by adding excessive amount of a well-known sulfhydryl-specific reagent, 5, 5'-dithiobis(2-nitrobenzoic acid), which led to a complete inhibition. To show the usefulness of IADC, the cysteines in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken muscle were modified with this reagent, and GAPDH was then digested by lysyl endopeptidase. The peptides generated from digestion of IADC-incorporated GAPDH were applied to an anti-dansyl immunoaffinity column. The peptide fragments bound and eluted from the column were separated by HPLC, and the amino acid sequence of each peptide was analyzed, and peptide was identified as the one containing a Cys residue(s). These data showed that IADC is a useful reagent to specifically identify the positions of a Cys residue(s) in proteins.     

Role of Rho-kinase in reexpansion pulmonary edema in rabbits

Reexpansion of a collapsed lung increases the microvascular permeability and causes reexpansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its target Rho-kinase (ROCK) regulate endothelial permeability, although their roles in reexpansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue-to-plasma (T/P) and bronchoalveolar lavage (BAL) fluid-to-plasma (B/P) ratios with (125)I-labeled albumin. After intratracheal instillation of (125)I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of (125)I-albumin in plasma/total amount of instilled (125)I-albumin. T/P, B/P, and PL index were significantly increased in the reexpanded lung. These increases were attenuated by pretreatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the reexpanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H(2)O(2)-induced increase in permeability and mitigated the morphological changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with reexpansion pulmonary edema.     

Superoxide dismutase (SOD) as a potential inhibitory mediator of inflammation via neutrophil apoptosis

Superoxide dismutase (SOD) is supposed to be an effective agent for neutrophil-mediated inflammation in the area of critical medicine. We investigated the involvement of SOD in the regulation of neutrophil apoptosis. Exogenously added SOD effectively induced neutrophil apoptosis, and the fluorescence patterns determined using annexin-V and the 7-AAD were similar to those seen in Fas-mediated neutrophil apoptosis. Neutrophils are short-lived leukocytes that need to be removed safely by apoptosis. The clearance of apoptotic neutrophils from sites of inflammation is a crucial determinant of the resolution of inflammation. Catalase inhibited the neutrophil apoptosis and caspase-3 activation. Spontaneous apoptosis, hydrogen peroxide and anti-Fas antibody-induced apoptosis of neutrophils were accelerated in Down's syndrome patients, in whom the SOD gene is overexpressed. Hydrogen peroxide was thought to be a possible major mediator of ROS-induced neutrophil apoptosis in caspase-dependent manner. Neutrophil apoptosis represents a crucial step in the mechanism governing the resolution of inflammation and has been suggested as a possible target for the control of neutrophil-mediated tissue injury. SOD may be a potential inhibitory mediator of neutrophil-mediated inflammation.