Melatonin, a pineal secretory product with antioxidant properties, protects against cisplatin-induced nephrotoxicity in rats.

In an attempt to define the role of the pineal secretory melatonin and an analogue, 6-hydroxymelatonin (6-OHM), in limiting oxidative stress, the present study investigated the cisplatin (CP)-induced alteration in the renal antioxidant system and nephroprotection with the two indolamines. Melatonin (5 mg/kg), 6-OHM (5 mg/kg), or an equal volume of saline were administered intraperitoneally (i.p.) to male Sprague Dawley rats 30 min prior to an i.p. injection of CP (7 mg/kg). After CP treatment, the animals each received indolamine or saline every day and were sacrificed 3 or 5 days later and plasma as well as kidney were collected. Both plasma creatinine and blood urea nitrogen increased significantly following CP administration alone; these values decreased significantly with melatonin co-treatment of CP-treated rats. In the kidney, CP decreased the levels of GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio, an index directly related to oxidative stress. When animals were treated with melatonin, the reduction in the GSH/GSSG ratio was prevented. Treatment of CP-enhanced lipid peroxidation in the kidney was again prevented in animals treated with melatonin. The activity of the antioxidant enzyme, glutathione peroxidase (GSH-Px), decreased as a result of CP administration, which was restored to control levels with melatonin co-treatment. Upon histological analysis, damage to the proximal tubular cells was seen in the kidneys of CP-treated rats; these changes were prevented by melatonin treatment. 6-OHM has been shown to have some antioxidative capacity, however, the protective effects of 6-OHM against CP-induced nephrotoxicity were less than those of melatonin. The residual platinum concentration in the kidney of melatonin co-treated rats was significantly lower than that of rats treated with CP alone. It is concluded that administration of CP imposes a severe oxidative stress to renal tissue and melatonin confers protection against the oxidative damage associated with CP. This mechanism may be reasonably attributed to its radical scavenging activity, to its GSH-Px activating property, and/or to its regulatory activity for renal function.

Prevention of nephrotoxicity of cisplatin by repeated oral administration of ebselen in rats.

The ability of ebselen, which exhibits glutathione peroxidase (GSH-Px)-like activity, to prevent cisplatin (CDDP)-induced nephrotoxicity was examined in rats. CDDP (6 mg/kg [20 micromol/kg] body weight) was injected intraperitoneally. In subgroups, daily ebselen doses of 2.75 (10 micromol), 5.5 (20 micromol), or 11.0 mg (40 micromol)/kg body weight were administrated orally 1 hour prior to CDDP treatment. Treatment with CDDP alone resulted in significantly increased plasma creatinine (Cr) and blood urea nitrogen (BUN) levels. Repeated administration of 5.5 and 11.0 mg/kg ebselen prevented the CDDP-induced elevation of plasma Cr and BUN levels and protected against kidney damage. Relative to controls, rat that received CDDP treatment displayed a decreased ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), an indicator directly related to oxidative stress, and elevated malondialdehyde (MDA) levels in the kidney. In comparison with controls, activity of GSH-Px activity, which antioxidant enzyme, was also reduced in the kidney of rats treated with CDDP. Repeated administration of 5.5 or 11.0 mg/kg ebselen prevented CDDP-induced alteration of GSH/GSSG ratios, MDA levels, and GSH-Px activity; however, no protection against CDDP was observed with administration of 2.75 mg/kg ebselen. Effective protection of CDDP-induced nephrotoxicity with ebselen was observed only when the molar amount of each daily ebselen treatment equaled or exceeded

The superiority of water-diluted 0.25% to neat 1% lidocaine for trigger-point injections in myofascial pain syndrome: a prospective, randomized, double-blinded trial.

Trigger-point injection with a mixture of commercially available 1% lidocaine in sterile distilled water at a ratio of 1:3 compared with 1% lidocaine alone resulted in better efficacy and less injection pain. This simple procedure may be suitable for treatments of a wide range of myofascial pain syndromes.

Global brain ischemia produced by clamping left subclavian artery and bicarotid trunk in the rabbit.

Thirty-five rabbits were divided randomly into 5 groups: sham operation, 10 minutes clamping bicarotid trunk (partial ischemia, PI), and 3 groups of 5, 7, and 10 minutes clamping left subclavian artery and bicarotid trunk (global ischemia, GI). Systolic arterial pressure increased slightly in the PI group, but doubled in the GI groups during clamping. Heart rate did not change in the PI group, but decreased transiently in the GI groups during clamping. Brain temperature decreased gradually in the GI groups during clamping, but did not change in the PI group. Necrotic changes were present 96 hours later in approximately 50% of the hippocampal CA1 cells in the GI groups, but in none of the cells in the PI and sham operation groups. The present results may indicate that clamping left subclavian artery and bicarotid trunk in the rabbit brings about global brain ischemia.

Selective separation of cadmium from cobalt, copper, iron (III) and zinc by water-based two-phase system of tetrabutylammonium bromide.

An aqueous two-phase system of tetrabutylammonim. bromide (TBAB) and (NH(4))(2)SO(4)mixture is proposed for the selective extraction of trace Cd(2+) from large amounts of Co(2+), Cu(2+), Fe(3+) and Zn(2+). Transparent two-phase system is prepared by mixing 3 ml of 1.0 mol/l TBAB, and 2 ml of sample solution and 1.1g of (NH(4))(2)SO(4), the two-phase system thus obtained is of 1.5 ml upper phase and 4.1 ml bottom phase. TBAB was distributed between the upper and bottom phases respectively, but the concentration in upper phase is much higher than that of the bottom phase. The results showed that Cd(2+) is selectively extracted into the upper phase in the pH ranges 1.0-9.0, while Co(2+) Cu(2+) and Fe(3+) ions were little extracted (<1%) at pH 3.0. Zinc ion was extracted to upper phase by about 24%, but it did not interfere the extraction of Cd(2+). The interaction between CdBr(4)(2-) and TBA(+) plays an important role in the extraction process.

Highly sensitive chelating agents for histochemical staining of rare earth metals.

In tissues, bromopyridylazo-diethylaminophenol has been found to be capable of staining very small amounts of rare earth metals, particularly praseodymium, terbium, dysprosium, holmium, ytterbium, and lutetium. Differentiation of a target metal from interfering metals was achieved using masking agents, polyphosphates and aminopolycarboxylic acids.

The study of the partitioning mechanism of methyl orange in an aqueous two-phase system.

An aqueous two-phase system of dodecyl triethylammonium bromide (C(12)NE, cationic surfactant) and sodium dodecyl sulfate (SDS, anionic surfactant) mixture is proposed for the extraction of some dyes and porphyrin compounds. Transparent two phase-systems are formed when the surfactant concentrations and C(12)NE/SDS ratios are in certain regions. In this study, the aqueous two phase-systems were prepared by mixing 0.1 mol l(-1) C(12)NE and SDS with a molar ratio of 1.7:1.0. The results showed that negatively charged chlorophyll (sodium copper chlorophyllin) and positively charged dye (methyl violet) were efficiently extracted into the upper phase. The negatively charged methyl orange (pH>7) was moved into the upper phase mostly while amphoteric methyl orange (pH<3) was distributed in the two phases uniformly. Except for hydrophobic force, charge interaction between solute and surfactant also play an important role in the extraction process.

Expression of p21WAF1/CIP1 in colorectal adenomas and adenocarcinomas and its correlation with p53 protein expression.

The expression of p53-inducible cyclin-dependent kinase inhibitor, p21WAF1/CIP1 in non-neoplastic mucosa, adenoma and adenocarcinoma of the colorectum was examined by immunohistochemistry and western blotting and its relation with the expression of p53 protein was analyzed. Non-neoplastic epithelial cells at the surface area showing no proliferative activity expressed p21WAF1/CIP1. The expression of p21WAF1/CIP1 was immunohistochemically detected in 55% (206/377) of the adenomas and 66% (190/289) of the adenocarcinomas, respectively. The incidence of strongly positive cases was significantly higher in the adenocarcinomas (27%) than in the adenomas (18%) (P < 0.05). The incidence of cases with strong p21WAF1/CIP1 expression was higher in stages 0, 1 and 2 carcinomas than in stages 3 and 4 carcinomas (P < 0.05). A decrease in the incidence of cases with strong expression was detected in carcinomas invading deeper than muscularis propria. The incidence of strongly positive cases was significantly lower in carcinomas with lymph node metastasis than those without metastasis (P < 0.05). The expression of p21 as well as p53 detected by western blotting was compatible with the results of immunohistochemistry in most cases examined. However, there was no significant correlation between the expression of p21WAF1/CIP1 and the abnormal accumulation of p53. These findings overall suggest that: (i) the physiological expression of p21WAF1/CIP1 may be associated with cellular senescence of colorectal mucosa; (ii) reduced expression of p21WAF1/CIP1 may participate in the progression of colorectal carcinoma; and (iii) p53-independent pathway may be considerably involved in the induction of p21WAF1/CIP1.

The efficacy of the "BURP" maneuver during a difficult laryngoscopy.

The displacement of the larynx in the three specific directions (a) posteriorly against the cervical vertebrae, (b) superiorly as possible, and (c) slightly laterally to the right have been reported and named the "BURP" maneuver. We evaluated the efficacy of the BURP maneuver in improving visualization of the larynx. Six hundred thirty patients without obvious malformation of the head and neck participated in this study. We divided the degree of visualization of the larynx using laryngoscopy into five grades and compared the visualization of the larynx using the BURP maneuver with that of laryngoscopy with and without simple laryngeal pressure ("Back"). The maneuver of Back and BURP significantly improved the laryngoscopic visualization from initial inspection. The BURP maneuver also significantly improved the visualization compared with the Back maneuver. We concluded that the BURP maneuver improved the visualization of the larynx more easily than simple back pressure on the larynx.

Growth inhibitory effect of interferon-beta is associated with the induction of cyclin-dependent kinase inhibitor p27Kip1 in a human gastric carcinoma cell line.

The effect of IFN-beta on cell growth and the mechanism of growth modulation was examined in human gastric carcinoma cell lines. IFN-beta inhibited the cell growth of TMK-1 cells, whereas it did not affect the growth of the other three cell lines (MKN-7, -28, and -45). The number of apoptotic cells in IFN-beta-treated TMK-1 cells was 2-fold the number of those in control TMK-1 cells, whereas the induction of apoptosis was not observed in IFN-beta-treated MKN-28 cells. IFN-beta enhanced the expression of cyclin-dependent kinase inhibitor p27Kip1 at mRNA and protein levels. The increased p27Kip1 bound preferentially to CDK6. The phosphorylation level of retinoblastoma protein in TMK-1 cells was reduced by IFN-beta treatment. IFN-beta did not affect the expression of cell cycle regulators in MKN-28 cells. These results suggest that the induction of p27Kip1 by IFN-beta might confer inhibition of cell growth, leading to subsequent apoptosis in TMK-1 cells.

Expression of cyclin-dependent kinase inhibitor p21WAF1/CIP1 in non-neoplastic mucosa and neoplasia of the stomach: relationship with p53 status and proliferative activity.

The expression of the p53-inducible cyclin-dependent kinase inhibitor p21WAF1/CIP1 in non-neoplastic mucosa, adenoma, and adenocarcinoma of the stomach was examined immunohistochemically and its relationship with p53 expression and proliferative activity was analysed. In normal gastric mucosa as well as in intestinal metaplasia the epithelial cells at the surface which showed no proliferative activity expressed p21WAF1/CIP1, whereas the cells in the deep area of the glands expressing Ki-67 did not. In the neoplastic lesions, the expression of p21WAF1/CIP1 was detected in 78 per cent (112/144) of the adenomas and 76 per cent (262/343) of the adenocarcinomas. The incidence of p21WAF1/CIP1 expression did not differ among histological types of gastric carcinoma. The strong expression of p21WAF1/CIP1 was more frequently observed in carcinomas invading into submucosa or in cases of stages 2, 3, and 4 than in carcinomas limited to the mucosa or in stage 1 cases. The incidence of strongly positive cases was higher in carcinomas with lymph node metastasis than in those without metastasis. There was no apparent correlation between the expression of p21WAF1/CIP1 and the abnormal accumulation of p53 or with proliferative activity measured by Ki-67 expression. These findings overall suggest that p21WAF1/CIP1 might be associated with the senescence of non-neoplastic gastric epithelial cells; that a p53-independent pathway might be substantially involved in the induction of p21WAF1/CIP1 in gastric neoplasia; and that the proliferative activity of gastric cancer might not be solely dependent on control of the cell cycle by p21WAF1/CIP1.

Genetic status and expression of the cyclin-dependent kinase inhibitors in human gastric carcinoma cell lines.

Deregulation of cyclin, cyclin-dependent kinases (CDKs) and their inhibitors could have a pivotal role in the development of diverse human cancers. We examined the genetic status and the expression of CDK inhibitors (p21, p27, p16 and p15), CDK2 and cyclins (A, D1 and E) in eight gastric carcinoma cell lines, in comparison with the status of p53 gene alterations. All the cell lines (except MKN-28) that contained a p53 gene abnormality expressed very low or undetectable levels of p21 mRNA, while the cell lines (MKN-45 and -74) with wild-type p53 gene expressed high levels of p21 mRNA. An inverse correlation was found between the level of p21 mRNA and the expression of mRNAs for CDK2 and G1 cyclins. MKN-28 was an exception; it contained mutated p53, and expressed mRNAs for p21, CDK2 and G1 cyclins at high levels. Only MKN-45 and -74, with wild-type p53, expressed considerable levels of p21 protein. Homozygous deletion of the p16 and p15 genes was detected in two (MKN-45 and HSC-39) of the eight gastric carcinoma cell lines, p16 protein was not expressed in three cell lines (MKN-28, MKN-74 and KATO-III), as well as MKN-45 and HSC-39. Rearrangement of the p15 gene was found in TMK-1. Rearrangement of the p27 gene was detected in MKN-45, although the expression of p27 protein was well preserved in all the gastric carcinoma cell lines. The expression of pRb was also preserved in all the cell lines except KATO-III. No obvious correlation was observed between the p53 gene status and the expression of p27 and p16. These findings suggest that abnormal regulation of CDK2/cyclins and CDK inhibitors might be involved in deregulated growth of gastric carcinomas.

Inhibition of cell growth by transforming growth factor beta 1 is associated with p53-independent induction of p21 in gastric carcinoma cells.

Cell cycle regulators such as cyclins, cyclin-dependent kinases (cdks) and their inhibitors control the growth of cells. SDI1/CIP1/WAF1/p21 is a potent inhibitor of G1 cdks, whose expression is induced by wild-type p53. To elucidate the mechanism of growth inhibition by transforming growth factor beta 1 (TGFbeta 1), we examined the effect of TGFbeta 1 on the expression of p21, G1 cyclins and cdks by human gastric cancer cell lines. TGFbeta 1 induced p21 expression and subsequently suppressed cdk2 kinase activity, followed by a reduction in phosphorylation of the product of the retinoblastoma tumor suppressor gene in TMK-1 cells, which are responsive to TGFbeta 1. Coimmunoprecipitation analysis demonstrated that TGFbeta 1 increased the level of p21 protein present in complexes with cdk2. In contrast, TGFbeta 1 did not induce p21 in TGFbeta 1-resistant MKN-28 cells. TGFbeta 1 did not affect the levels of p53 mRNA and protein in TMK-1 and MKN-28 cells, which contain mutated p53 genes. These mutated p53 complementary DNAs, when overexpressed, failed to activate transcription from the p21 promoter. Furthermore, TGFbeta 1 caused a reduction in the steady-state level of cyclin A protein concomitantly with inhibition of cdk2 kinase activity in TMK-1 cells. These results suggest that the growth inhibition of tumor cells by TGFbeta 1 is associated with p53-independent induction of p21, subsequent suppression of cdk activity and a decrease in cyclin A protein in TMK-1 cells.

Expression of cyclin E in human gastric adenomas and adenocarcinomas: correlation with proliferative activity and p53 status.

The expression of cyclin E in human gastric adenomas and adenocarcinomas was examined immunohistochemically to elucidate the role of cyclin E in stomach carcinogenesis. The expression of cyclin E was detected in 49% (90/182) of the adenomas and 59% (260/439) of the adenocarcinomas. The incidence of strongly positive cases (overexpression of cyclin E) was significantly higher in the adenocarcinomas (29%; 128/439) than in the adenomas (4%; 8/182) (p < 0.01). The incidence of the cyclin E expression showed a tendency to be higher in deeply invasive carcinomas and in the cases with lymph node metastasis, while the incidence did not differ among histological types. The expression of cyclin E was significantly correlated with the proliferative activity of the tumor cells measured by KI-67 antigen expression (p < 0.01). It was also correlated with the abnormal accumulation of p53 protein in the tumor cells (p < 0.01). These results suggest that overexpression of cyclin E and subsequent deregulation of the cell cycle may confer the development and progression of the gastric carcinomas.

Genetic status of p53 and induction of apoptosis by radiation or isoflavones in human gastric carcinoma cell lines.

We have shown previously that various human cancer cell lines undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation or isoflavones. Here, we assessed the role of p53 gene in cell cycle and apoptosis following treatment of 11 gastric carcinoma cell lines with gamma-rays, genistein, biochanin A, or daidzein. Cell survival was measured by trypan blue staining, and apoptosis was assessed by fluorochrome staining. The rate of cell survival and apoptosis of the cells by gamma-irradiation or isoflavones did not correlate with p53 gene abnormalities. Flow cytometric measurement of DNA content demonstrated that while gamma-irradiation and genistein induced G(2) arrest, biochanin A and daidzein blocked the cell cycle of all carcinoma cells at G(1) phase. At multiple time points following irradiation, G(2) arrest was observed at 12-16 h in the wild-type and mutant p53 cell lines. Induction of p53 and p21 proteins was not observed in wild-type p53 lines after exposure to gamma-irradiation or isoflavones by Western blotting. Moreover, transfection of the wild-type p53 gene into MKN-1 cells failed to induce G(1) arrest by gamma-irradiation and genistein. Based on these results, we hypothesize that gastric cancer cells may possess a signal pathway which is different from the usual mechanisms of the p53-mediated DNA damage response in normal or hematopoietic tumor cells.

Cervical sympathectomy affects adrenocorticotropic hormone and thyroid-stimulating hormone in rats.

To examine the effects of bilateral cervical sympathectomy on the secretion of adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), growth hormone (GH), and prolactin (PRL), 18 male rats were divided into three groups: control (Cont), sham operation (Sham), and bilateral cervical sympathectomy (Symp). All rats were kept under a normal circadian rhythm for 2 weeks. Subsequently, blood was collected and plasma ACTH as well as serum TSH, GH, and PRL levels were measured. The difference in ACTH levels between the Cont and Sham groups was not significant, but ACTH levels in the Symp group were significantly higher than those in the other groups. The difference in TSH levels between the Cont and Sham groups was also not significant, but TSH levels in the Symp group were significantly lower than those in the Cont group. There were no statistically significant differences in GH and PRL levels among these groups. The present results suggest that cervical sympathectomy in the rat increases ACTH secretion and decreases TSH secretion in the pituitary. These effects seem to be due to a mildly increased secretion of melatonin in the pineal body that probably in turn increases corticotropin-releasing factor (CRF) secretion and decreases thyrotropin-releasing hormone (TRH) secretion in the hypothalamus. Extrapolation of these findings to humans suggests that longterm and repeated stellate ganglion block would affect the pituitary secretions of ACTH and TSH.

Use of cerium oxide (CeO(2)) as a packing material for the chromatographic separation of C(60) and C(70) fullerenes.

Cerium oxide (CeO(2)) was tested as a packing material in liquid chromatography for the separation of C(60) and C(70) fullerenes. The separation of C(60) and C(70) fullerenes could be achieved within 20 min by using pure n-hexane as the mobile phase. Furthermore, some higher fullerenes could also be separated in less than 40 min. The peak area was reproducible to a large extent. The separation of fullerenes by liquid chromatography on CeO(2) is shown to be an effective method for their isolation in large amounts. The column efficiency of the CeO(2) column was compared with commercial silica gel and ODS columns. The main advantage of the CeO(2) column is its ability to separate large amounts of fullerenes (C(60) and C(70)) in toluene.

Concurrent amplification of cyclin E and CDK2 genes in colorectal carcinomas.

The genetic changes of cyclin A, DI, E and CDK2 were examined in human colorectal carcinomas by Southern-blot analysis. Gene amplification of cyclin E was detected in 5 of 53 (9.4%) primary colorectal carcinoma tissues. Interestingly, in 3 of 5 tumors showing cyclin E gene amplification, the CDK2 gene was amplified simultaneously with rearrangements. No obvious correlation was detected between gene amplification and clinicopathological features of colorectal carcinomas. Out of 7 colon carcinoma cell lines, 2 showed gene amplification of cyclin E without gene amplification of CDK2. No amplification of cyclin A or DI gene was found in any of the colorectal carcinoma tissues or colon carcinoma cell lines. Our results suggest that the concurrent amplification of cyclin E and CDK2 genes may play a role in colorectal carcinogenesis.

Frequent amplification of the cyclin E gene in human gastric carcinomas.

We searched for genetic alterations of the cyclin D1 and cyclin E genes in 45 human gastric carcinoma tissues. Expression of cyclin E mRNA and protein was also analyzed in eight of them by Northern and Western blots and immunohistochemical staining. The cyclin E gene was amplified 3-10 fold in seven gastric cancer tissues (15.6%), of which six were advanced gastric cancers. All of the cases with the cyclin E gene amplification displayed lymph node metastasis. Moreover, the case with the gene amplification overexpressed the cyclin E mRNA and protein. One of eight gastric cancer cell lines, MKN-7, shared the cyclin E gene amplification, and all of the gastric cancer cell lines expressed high levels of the cyclin E mRNA and protein even without gene amplification. Amplification of the cyclin D1 gene was not observed in any of the gastric carcinoma tissues or gastric carcinoma cell lines. These results suggest that the gene amplification and overexpression of cyclin E play an important role in the abnormal growth and progression of gastric carcinoma.