Neuroprotective effects of alpha-tocopherol on oxidative stress in rat striatal cultures

Oxidative stress caused by an increase in free radicals plays an important role in neuronal death. We investigated the effects of alpha-tocopherol on oxidative stress-induced cytotoxicity using primary cultures of rat striatal neurons. alpha-Tocopherol at concentrations of 1-10 microM significantly prevented cytotoxicity induced by superoxide radical (O(2(-)) donor, 1,1'-dimethyl-4,4'-bipyridium dichloride (paraquat). In contrast, alpha-tocopherol did not affect the cytotoxicity of hydrogen peroxide (H(2)O(2)), which enhances hydroxyl radical (.OH) formation by metal-catalyzed Fenton reactions. alpha-Tocopherol significantly inhibited the cytotoxicity of nitric oxide (NO) donors, S-nitrosocysteine and 3-morpholinosydnonimine (SIN-1). alpha-Tocopherol showed potent protection against cytotoxicity induced by L-buthionine-[S,R]-sulfoximine (BSO), which causes depletion of intracellular glutathione. Moreover, alpha-tocopherol afforded a moderate but significant inhibition of cytotoxicity induced by a non-specific protein kinase inhibitor, staurosporine, which is known to induce apoptosis in many types of cells including neurons. These results suggest that alpha-tocopherol protects striatal neurons by the reduction of oxidative stress, presumably by decreasing intracellular O(2)(-) levels, and at least partly by the inhibition of apoptosis.     

Anti-angiogenesis effects of borrelidin are mediated through distinct pathways: threonyl-tRNA synthetase and caspases are independently involved in suppression of proliferation and induction of apoptosis in endothelial cells

Borrelidin, an antibiotic with anti-angiogenic activity, not only suppresses new capillary tube formation, but also collapses formed capillary tubes in a rat aorta culture model. Since it selectively inhibits threonyl-tRNA synthetase, we examined the effect of threonine on its anti-angiogenic activity. We found that a high concentration of threonine (1 mM) attenuated the ability of borrelidin to inhibit both capillary tube formation in the rat aorta culture model and human umbilical vein endothelial cells (HUVEC) proliferation, yet did not affect the ability of borrelidin to collapse formed capillary tubes or to induce apoptosis in HUVEC. Borrelidin activated caspase-3 and -8, and inhibitors of both caspase-3 and -8 suppressed borrelidin-induced apoptosis in HUVEC. Taken together, these data suggest that the anti-angiogenic effects of borrelidin are mediated through at least two mechanisms, i.e. one threonine-dependent and the other threonine-independent, and borrelidin induces apoptosis in endothelial cells via the caspase-8/-3 pathway.     

Protective effects of glutathione on 5-fluorouracil-induced myelosuppression in mice

The protective effects of glutathione (GSH) administration on myelosuppression induced by 5-fluorouracil (5-FU) were investigated in female BALB/c mice. Animals were allocated to four groups (16 mice/group). GSH was given orally at a dose of 800 mg/kg to groups 3 and 4 for 21 consecutive days (day 0 to day 20). 5-FU was repeatedly administered at a dose of 40 mg/kg to groups 2 and 3 for 1 week (day 7 to day 13) by gavage. Group 3 served as a combined treatment group and group 1 as a non-treated control group. The total observation period was 3 weeks. Body weight was measured once a week. A decrease in body weight due to 5-FU treatment was observed in groups 2 and 3 on day 14. Although the body weight in group 2 had not increased by 1-week after cessation of 5-FU treatment, the value in group 3 markedly recovered. Hematology, total nucleated myelocyte count and histopathology of bone marrow were carried out on day 14 and day 21. In groups 2 and 3, these examinations showed thrombocytopenia, leukopenia, reticulocytopenia and myelosuppression on day 14. However, platelets and bone marrow were less affected in group 3 than in group 2. On day 21, the thrombocytopenia in groups 2 and 3 was resolved. The myelosuppression, leukopenia and reticulocytopenia resolved in group 3, but not in group 2. Although simple microcytic anemia occurred delayed on day 21, it was less severe in group 3 than in group 2. Therefore, GSH may have preventive effects against 5-FU-induced hematopoietic toxicity, and accelerate recovery after cessation of 5-FU treatment.     

Inactivation and Aggregation of β-Galactosidase in Lyophilized Formulation Described by Kohlrausch-Williams-Watts Stretched Exponential Function

Purpose. To examine whether the empirical Kohlrausch-Williams-Watts (KWW) equation is applicable not only to protein aggregation but also to protein denaturation in lyophilized formulations. Lyophilized -galactosidase (-GA) formulations containing polyvinylalcohol and methylcellulose were used as model formulations. The possibility of predicting storage stability based on the temperature dependence of the estimated parameters of inactivation/aggregation—time constant () and its distribution () is discussed. Methods. Protein aggregation in lyophilized -GA formulations at 10-70°C and 6-43% relative humidity was determined as a function of time by size exclusion chromatography. Enzyme activity was also determined using 2-nitrophenyl--D-galactopyranoside as a substrate. Results. Inactivation and aggregation of -GA were describable with the empirical KWW equation, regardless of whether the temperature was above or below the NMR relaxation-based critical mobility temperature (T_mc) or whether protein molecules with different degrees of deformation resulting from stresses during lyophilization exist in the formulation. The estimated parameter for protein aggregation decreased rapidly as temperature increased beyond T_mc because the mobility of polymer molecules increased in the initial stages of glass transition. The time required for 10% enzyme to aggregate (t₉₀) calculated from the and parameters exhibited a change in temperature dependence gradient near T_mc. In contrast, t₉₀ for protein inactivation exhibited temperature dependence patterns varying with the excipients. Conclusions. The t₉₀ calculated from the estimated and parameters was found to be a useful parameter for evaluating the stability of lyophilized -GA formulations. The prediction of t₉₀ by extrapolation was possible in the temperature range in which did not rapidly vary with temperature.     

Effect of gelatins on human cancer cells in vitro

Three types of gelatins were tested for their antiproliferative activities in vitro against three human tumor cell lines (K-562; erythroleukemia, HCT-15; colon carcinoma, AGS; gastric carcinoma) with viable cell count and tritium-thymidine ((3)H-TdR) uptake by those cells. Porcine skin (PS) gelatin exerted the strongest antiproliferative activity of all three gelatins. Bovine bone (BB) gelatin did not exert such an activity. PS gelatin exerted antiproliferative activity against K-562 cells also in a serum-free medium. The serum-free medium contains two growth factors, insulin and transferrin, as well as nutrients. The activity of PS gelatin was not interfered by addition of insulin and transferrin to the medium. Effect of diluting a K-562 cell-concentration on the activity of PS gelatin was tested. Diluting the cell concentration did not affect the activity of PS gelatin. Moreover, the conditioned medium in which K-562 cells had been cultured did not stimulate the proliferation of K-562 cells. In conclusion, PS gelatin suppress the proliferation of human tumor cell lines in vitro. The antiproliferative activity of PS gelatin might not be attributed to trapping growth factors or autocrine mediators.     

Human papillomaviruses in the normal oral cavity of children in Japan

The purpose of this study was to determine the frequency of human papillomavirus (HPV) infections in the normal oral cavity of children in Japan. Oral squamous cell specimens were collected from 77 children (44 boys and 33 girls), aged 3 and 5 years. Extracted DNA was evaluated for HPV infections by polymerase chain reaction (PCR) methods, using consensus primers for the L1 region, specific primers, and direct DNA sequencing analysis. Thirty-seven of 77 specimens (48.1%) were positive for HPV DNA. Positive rates of boys and girls in all specimens were 28.3 (22/77) and 19.5 (15/77)%, respectively. The positive rate in 3-year-old children was 45.2 (14/31)%, and positive rates in boys and girls were 52.6 (10/19) and 33.3 (4/12)%, respectively. The positive rate in 5-year-old children was 50.0 (23/46)%, and positive rates in boys and girls were 48.0 (12/25) and 52.4 (11/21)%, respectively. HPV types were determined by specific PCR and direct DNA sequencing analysis. Frequent HPV types in the specimens of all children were HPV-16 (11/37; 29.7%),-1 (6/37; 16.2%),-2 (6/37; 16.2%),−75 (6/37; 16.2%). The results of the present investigation indicate that many HPVs, including HPV-16 (a high-risk type for cancer), are present in the oral cavity of 3- and 5-year-old children. It is suggested, therefore, that the oral cavity is already a reservoir of HPVs in childhood where later HPV-associated diseases, such as oral cancer and other oral lesions, may develop.     

Enhancement of urinary bladder carcinogenesis by combined treatment with benzyl isothiocyanate and N-butyl-N-(4-hydroxybutyl)nitrosamine in rats after initiation

Previously we reported that benzyl isothiocyanate (BITC) strongly enhanced rat urinary bladder carcinogenesis after initiation with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), while potently inhibiting BBN-induction of lesions when given simultaneously with the carcinogen. In the present experiment, the effects of simultaneous treatment with BITC and low-dose BBN on the postinitiation period of rat urinary bladder carcinogenesis were examined. After treatment with 500 ppm BBN for 4 weeks for initiation, groups of 20, 6-week-old, F344 male rats were given 25 ppm BBN alone, basal diet alone, or 100 or 1000 ppm BITC in the diet together with or without 25 ppm BBN in their drinking water for 36 weeks and then killed for autopsy. Further groups consisting of 10 rats each were similarly given BITC or the basal diet together with or without 25 ppm BBN, without initiation treatment. In the initiated groups receiving subsequent BBN exposure, papillary and nodular hyperplasia, dysplasia and carcinoma incidences were significantly increased, and they were further increased by the combined treatment with 100 and 1000 ppm BITC in a dosedependent manner. In the non-initiation groups, carcinomas were only observed in a single rat in each of the BBN-treated control and BBN/BITC 100 ppm treatment groups. The results indicate that simultaneous treatment with BITC and a low dose of BBN does not inhibit, but rather enhances rat urinary bladder carcinogenesis after appropriate initiation, and further suggest that BITC may be a human risk factor, at least in high-risk populations.     

Successful Laparoscopic Right Gastroepiploic Aneurysmectomy: Report of a Case

A 75-year-old woman presented with a pulsatile, movable mass, about 5cm in diameter, in her lower abdomen. Abdominal ultrasonography revealed a circular mass with a variable hypo- and isoechoic border and a hypoechoic center. Color Doppler echography showed blood flow in the hypoechoic center, which was strongly enhanced on contrast-enhanced computed tomography. Based on these findings, we diagnosed a splanchnic artery aneurysm; however, celiac arteriography, performed twice, could not definitively identify it. An operation was performed under the tentative diagnosis of an aneurysm of the superior mesenteric artery or the gastroepiploic artery. On laparoscopic exploration, a globe-shaped mass, about 5cm in diameter, was found in the right side of the greater omentum, which was diagnosed as an aneurysm of the right gastroepiploic artery. We resected the aneurysm laparoscopically and the patient had an uneventful postoperative course. Thus, laparoscopic surgery was effective for this patient who required no vascular reconstruction.     

Pain processing within the primary somatosensory cortex in humans

To investigate the processing of noxious stimuli within the primary somatosensory cortex (SI), we recorded magnetoencephalography following noxious epidermal electrical stimulation (ES) and innocuous transcutaneous electrical stimulation (TS) applied to the dorsum of the left hand. TS activated two sources sequentially within SI: one in the posterior bank of the central sulcus and another in the crown of the postcentral gyrus, corresponding to Brodmann's areas 3b and 1, respectively. Activities from area 3b consisted of 20- and 30-ms responses. Activities from area 1 consisted of three components peaking at 26, 36 and 49 ms. ES activated one source within SI whose location and orientation were similar to those of the TS-activated area 1 source. Activities from this source consisted of three components peaking at 88, 98 and 109 ms, later by 60 ms than the corresponding TS responses. ES and TS subsequently activated a similar region in the upper bank of the sylvian fissure, corresponding to the secondary somatosensory cortex (SII). The onset latency of the SII activity following ES (109 ms) was later by 29 ms than that of the first SI response (80 ms). Likewise, the onset latency of SII activity following TS (52 ms) was later by 35 ms than that of area 1 of SI (17 ms). Therefore, our results showed that the processing of noxious and innocuous stimuli is similar with respect to the source locations and activation timings within SI and SII except that there were no detectable activations within area 3b following noxious stimulation.