Comet assay in murine bone-marrow cell line (FDC-P2).

The comet assay, also known as the single cell gel electrophoresis (SCGE) assay, is a rapid, simple, visual and sensitive technique for measuring DNA damage in mammalian cells. In the present study, Methyl methanesulfonate (MMS), 4-Nitrosoquinoline-Oxide (4NQO), Cyclophosphamide (CPA), and Benzo(a)pyrene (BP)-induced DNA damage was assayed in vitro in a murine bone-marrow cell line (FDC-P2), with or without an activation mixture (rat liver S9). All compounds caused significant DNA damage. With MMS and 4NQO, the frequency of comet tails, scored manually under a fluorescence microscope, increased dose-dependently, and reached a maximum of 53.2 and 74.8% respectively. Three parameters indicating DNA damage in the comet assay with the two-layer method, tail length, %DNA in tail, and tail moment, calculated using the automated image analysis software "Comet Analyzer v1.5" increased with all compounds. With MMS and 4NQO, all parameters increased at concentrations over 40 and 0.25 micromol/L, respectively. The in vitro comet assay with rat liver S9 could detect DNA damage caused by the metabolites of CPA and BP. The comet assay using the two-layer method is easy and efficient, and so can be conducted on a routine as basis. The assay with FDC-P2 cells was highly sensitive in detecting DNA damage with the frequency of comet tails, tail moment, %DNA in tail and tail length as indicators of the damage. Metabolism-mediated DNA damage could be detected with the addition of a rat S9 mixture at a final concentration of 6% for 6h exposure.

Comparative characterization study of microporous carbons by HRTEM image analysis and gas adsorption.

The present work presents a useful comparison of micropore size distributions (MPSDs) obtained from gas adsorption and image analysis of high-resolution transmission electron micrographs. It is shown that the MPSD obtained for a chemical activated carbon is concordant with that obtained from CO2 adsorption. In addition, this technique has allowed us to obtain the MPSD of a carbon molecular sieve (CMS) prepared in our laboratory by a copyrolysis process, which could only be characterized by CO2 adsorption at 273 K (not by N2 adsorption at 77 K due to diffusional problems). The MPSD obtained by high-resolution transmission electron microscopy (HRTEM) for the CMS is wider than that obtained by CO2 adsorption, suggesting that HRTEM is detecting the closed porosity existing in this sample, which is not accessible to gas adsorption. The existence of closed porosity in the CMS is explained considering the preparation method used. Thus, HRTEM combined with image analysis seems to be useful for structural analysis of narrow micropores including closed porosity.

Effects of milk products fermented by Bifidobacterium longum on blood lipids in rats and healthy adult male volunteers.

The effects of milk products fermented by Bifidobacterium longum strain BL1, a probiotic strain, on blood lipids in rats and humans were studied. Rats were fed a cholesterol-enriched experimental diet, supplemented with lyophilized powders of 1) acid milk (control), 2) milk fermented with a mixed culture of ordinary yogurt starters composed of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (SL), and 3) bifidobacterium milk fermented with the probiotic B. longum strain BL1, respectively. The bifidobacterium milk feeding brought about significant lowering of the serum concentrations of total cholesterol, low-density lipoprotein cholesterol, and triglycerides, in comparison with the control, while no change in high-density lipoprotein cholesterol concentration was observed. On the other hand, supplementation with SL milk resulted in only slight, nonsignificant decreases in serum lipid concentrations in comparison with the control. In the human study, 32 subjects with serum total cholesterol ranging from 220 to 280 mg/dl were randomly assigned to two treatments: 1) intake of a low-fat drinking yogurt prepared with ordinary yogurt starters composed of S. thermophilus and L. delbrueckii subsp. bulgaricus (P-group) and 2) intake of a low-fat drinking yogurt prepared with the two ordinary yogurt starters plus B. longum strain BL1 (B-group). After intake for 4 wk at 3 x 100 ml/day, reduction of serum total cholesterol was observed in approximately half of the B-group subjects; a particularly significant decrease in serum total cholesterol was found among subjects with moderate hypercholesterolemia (serum total cholesterol > 240 mg/dl). However, the serum lipid concentrations in the P-group subjects were almost stable during the experimental periods. The present results indicate the potential of the probiotic B. longum strain BL1 in serum lipid improvement.

Effects of n-3 polyunsaturated fatty acids on dermatitis in NC/Nga mice.

The administration of n-3 polyunsaturated fatty acids (PUFAs) is known to be effective against allergic diseases by suppressing the production of eicosanoids derived from arachidonic acid. To investigate the mechanisms and efficacy of n-3 PUFA treatment in patients with atopic dermatitis (AD), we administered four different formulas of alpha-linolenic acid for 6 weeks in an AD model using NC/Nga mice. According to the doses of alpha-linolenic acid given, the levels of alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid in the red blood cell membranes increased while the levels of linoleic acid and arachidonic acid decreased. However, there was no significant difference among the four dose groups in clinical skin severity score, histopathological findings of skin lesions, or levels of total plasma IgE. Moreover, there was no significant difference in the production of leukotriene B(4) and Leukotriene C(4) from skin lesions after stimulation with A23187 among the groups, although the production of prostaglandin E(2) (PGE(2)) was significantly reduced and skin blood flow in the ear was significantly higher in the group given the highest dose of alpha-linolenic acid. Our results suggest that the administration of alpha-linolenic acid can change the fatty acid composition, PGE(2) production, and skin blood flow but may not prevent the development of dermatitis in NC/Nga mice.

Inhibition of proliferation of gastric epithelial cells by a cyclooxygenase 2 inhibitor, JTE522, is also mediated by a PGE2-independent pathway.

BACKGROUND: Cyclooxygenase-2 (COX-2) is one of the rate-limiting enzymes for prostaglandin synthesis from arachidonic acid. Although it is known that inhibition of cyclooxygenase activity delays ulcer healing, the regulatory relationship between COX-2 and its metabolites in gastric epithelial cell proliferation is not well known. AIM: To investigate whether COX-2 has an effect on gastric mucosal cell proliferation and further studied whether such effect is mediated only by prostaglandin E2 (PGE2), a representative metabolite of arachidonates in the gastric mucosa. METHODS: Artificial wounds of defined area size were created on complete monolayer cell sheets of isolated rat gastric epithelial cells and rat gastric cell line RGM1 under the addition of arachidonic acid or a COX-2 selective inhibitor, JTE522. Repair of wounds was assessed by monitoring wound size, with cell proliferation detected using 5-bromodeoxyuridine staining. Quantity of secreted PGE2 was measured by enzyme immunoassay. RESULTS: Stimulation of foetal calf serum increased the expression of COX-2 protein and inhibition of COX-2 retarded wound healing with reduction of cell proliferation. Arachidonic acid increased PGE2 production and accelerated restoration. Combination of JTE522 and arachidonic acid resulted in a marked retardation of wound healing compared to the control, but JTE522 did not completely suppress the increase in cellular PGE2 content following the addition of arachidonate. CONCLUSIONS: The difference in the effects of JTE522 on PGE2 production and on wound healing suggest that the involvement of COX-2 in gastric epithelial cell proliferation is not mediated solely by PGE2.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 2). Testicular toxicity in rats treated orally with ethinylestradiol for 2 weeks.

Parameters of ethinylestradiol-induced testicular toxicity were evaluated with organ weight determination, histopathological examination and quantitative morphometry. Male Sprague-Dawley rats were administered ethinylestradiol orally at 3 or 10 mg/kg/day for 2 weeks and 3 mg/kg/day for 4 weeks. Final body weights in all treated groups were lower than in the respective control group. Decreased absolute and/or relative organ weights of epididymides, prostate, seminal vesicles and testes were observed in all treated groups. In the testes, apoptosis of round spermatids, atrophy of seminiferous tubules, exfoliation of spermatids or spermatocytes, and vacuolar degeneration of Sertoli cells were only observed with 4 weeks treatment. Apoptosis of pachytene spermatocytes and atrophy of Leydig cells were also more marked in the 4 week treated group than after 2 weeks. Therefore, degenerative histopathological changes in testes were more remarkable after 4 weeks treatment than in the 2 weeks treatment groups. However, retention of spermatids was less after 4 weeks treatment and the TUNEL index, calculated as the number of TUNEL-positive spermatocytes or spermatids, was increased in all treated groups. These results suggest that ethinylestradiol-induced testicular toxicity can be detected in male rats administered the compound for 2 weeks and that the TUNEL method for in situ detection of apoptosis is effective for evaluation of testicular toxicity.

Brain metastasis from malignant pancreatic somatostatinoma. Case report.

Somatostatinomas are rare endocrine tumors that are located primarily in the pancreas. Metastases are seen most frequently in the liver and lymph nodes. The authors present the case of a 63-year-old man who had a malignant somatostatinoma of pancreatic tail origin that metastasized to the brain 10 years after diagnosis of the primary tumor. The metastatic brain lesions were totally removed and the patient is alive without tumor recurrence 12.3 years after the initial diagnosis. To our knowledge, this case represents the first documentation of brain metastasis from a malignant somatostatinoma, as well as the longest survival time of a patient with a somatostatinoma.

[Development of hen's egg with low antigenicity for allergic patients].

We tried to prepare hen's egg with low antigenicity for allergy patients. First, we examined the effects of pH, heating temperature and time on antigenicity of egg. Dried whole egg solid (DES) was dissolved in the buffer solution adjusted at various pH and heated at 120 degrees C for 10, 20 and 40 min. As a result the antigenicity didn't significantly decrease. Next, DES was treated with succinic anhydride, sodium hydroxide and was heated. The antigenicity of the resultant modified whole egg solid (MES) was much lower than that of heated whole egg solid. Antigenicity was measured precisely by enzyme immunoassay, inhibition ELISA, RAST inhibition and PCA. It was found that the antigenicity of MES was decreased less than 1/1000 of that of DES. Then SDS-polyacryl-amide gel electrophoresis and gel filtration were performed to clarify the degree of the change of egg proteins. The bands and peaks which corresponded to ovalbumin and ovomucoid known to be major allergenic proteins responsible for egg allergy disappeared and new bands and peaks were detected.

Hypervolemic hypotension in surgical treatment of intracranial aneurysm.

Fifty cases of intracranial aneurysms were operated upon under hypervolemic hypotension. The cerebral metabolism was maintained within normal range. The dissection and clipping of the aneurysm were considerably easier, and a premature rupture of the aneurysm could be handled without difficulty. The advantages and the surgical results are discussed.