Electroacupuncture inhibits cyclooxygenase-2 up-regulation in rat spinal cord after spinal nerve ligation.

Electroacupuncture (EA) has long been used to treat pain including neuropathic pain, but its mechanisms remain to be delineated. Since cyclooxygenase-2 (COX-2) has been reported to increase in the spinal dorsal horn following spinal nerve ligation (SNL) and it may play a role in the neuropathic pain, we hereby tested the hypothesis that EA may affect COX-2 expression and hence neuropathic nociception after SNL. The results showed that EA (2 Hz) can significantly reduce mechanical and thermal hypersensitivity following lumbar L5 SNL in rats. Immunostaining demonstrated suppression of COX-2 expression in the spinal L4-L6 dorsal horn after EA. The present results suggest that EA may alleviate neuropathic hypersensitivity by, at least partially, inhibiting COX-2 expression in the spinal cord.

Effects of alpha-tocopherol on metmyoglobin formation and reduction in beef from cattle fed soybean or cottonseed meal diets.

Hereford-Angus crossbred heifers were fed a cottonseed meal-based diet containing gossypol (14 mg free gossypol x kg body wt(-1) x d(-1); CSM), a soybean meal-based diet (SBM), or alpha-tocopherol-supplemented diets (4,036 IU vitamin E x heifer(-1) x d(-1) for 90 d; CSM+E and SBM+E). The effects of diet on color stability and aerobic metmyoglobin reducing ability of beef longissimus lumborum (LL) and psoas major (PM) were evaluated. The CSM containing gossypol did not affect alpha-tocopherol concentration, a* value, or hue angle value of beef muscles obtained from control or vitamin E-supplemented cattle compared to their SBM counterparts. Vitamin E supplementation increased endogenous alpha-tocopherol concentrations and color stability in LL and PM muscles compared with controls from either diet (P < .05). In the aerobic metmyoglobin reducing ability study, LL and PM muscles were stored in 1% O2:99% N2 (a pigment-oxidizing atmosphere) for 48 h and subsequently stored aerobically for an additional 48 h. Within the LL, alpha-tocopherol supplementation delayed metmyoglobin formation in LL exposed to 1% O2 (P < .05). Within the PM, no differences in metmyoglobin formation were found between controls and vitamin E treatments in SBM or CSM diets. Relative aerobic metmyoglobin reduction was the same (P > .05) in LL and PM muscles within SBM or CSM diets for control and vitamin E treatments. Alpha-tocopherol did not seem to affect metmyoglobin aerobic reducing ability in LL and PM muscles.

Beef color update: the role for vitamin E.

Dietary supplementation of livestock with vitamin E results in improved quality of meat subsequently obtained from these animals. The effect is especially noteworthy in cattle, in which the primary effects are delayed discoloration and lipid oxidation. A threshold level of alpha-tocopherol in muscle ensures a detectable effect; dietary strategies for attaining this threshold must consider tocopherol status of cattle arriving at the feedyard and duration and level of supplementation. The alpha-tocopherol concentration in muscle must be determined before proper interpretation of experimental results can be made. Muscles vary in their color stability, and this relative difference is not changed by vitamin E supplementation. Several in vitro models have been used to characterize the interaction between alpha-tocopherol, lipid oxidation, and oxymyoglobin oxidation. Alpha-tocopherol seems to exert its color-stabilizing effect by indirectly delaying oxymyoglobin oxidation via direct inhibition of lipid oxidation. However, recent results demonstrating a protective effect of alpha-tocopherol toward oxymyoglobin in low-oxygen atmospheres indicate that additional mechanisms may exist. A better understanding of the fundamental bases for protection of water-soluble myoglobin by lipid-soluble alpha-tocopherol is needed to optimize this beneficial effect.

Molecular characterization of the murine Hif-1 alpha locus.

Hypoxia inducible factor 1 alpha (HIF-1 alpha) is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor that mediates certain cellular responses to low oxygen tension, iron chelators, Co2+, Ni2+, Mg2+, and low intracellular glucose concentration. Upon exposure to the above conditions, HIF-1 alpha is upregulated and heterodimerizes with the Ah receptor nuclear translocator (ARNT, also known as HIF-1 beta), the heterodimeric complex binds TACGTG-containing genomic enhancer elements, and activates transcription of target genes. As a first step in developing genetic models to study the biology related to cellular hypoxia, we have cloned the murine HIF-1 alpha cDNA, determined the tissue-specific expression of its mRNA, functionally analyzed its protein product, and characterized its promoter and its genomic structure. A comparison between the murine and human HIF-1 alpha protein sequence reveals 95%, 99%, and 83% identity in the bHLH, PAS, and variable domains, respectively. RNAse protection assays demonstrate that in adult mice, the mHIF-1 alpha mRNA is expressed at high levels in kidney, heart, brain, thymus, and placenta, with moderate expression in liver, spleen, testis, and lung and much lower expression in skeletal muscle testis. Northern blot analysis indicates that the mRNA of the murine HIF-1 alpha is transcribed in two forms, a major 4-kb species and a minor 5-kb species; both are present in all tissues examined. The Hif-1 alpha promoter is GC rich, does not have a TATA element near its transcriptional start site, and does not respond to hypoxia or Co2+. The mHIF-1 alpha structural gene is composed of 15 exons. The splice junction sites within the bHLH and the PAS domains of HIF-1 alpha gene are highly conserved with respect to a number of previously characterized members of the bHLH-PAS superfamily. However, unlike other bHLH-PAS genes, where the variable domain is encoded by 2 exons, the variable region of the mHIF-1 alpha gene is encoded by 7 exons. Furthermore, most of these splice junction sites in the variable region are conserved with that of HIF-2 alpha, a recently cloned hypoxia-responsive bHLH-PAS protein (also known as MOP2, EPAS1, and HLF). These data suggest that HIF-1 alpha, along with HIF-2 alpha, represents a new subclass of the bHLH-PAS superfamily.

A pharmacokinetic study with the high-dose anticancer agent menadione in rabbits.

The aim of this investigation was to assess the pharmacokinetic properties of high-dose menadione (VK3), as an anticancer agent, in plasma and red blood cells (RBCs) in rabbits. An extremely high dose of 75 mg menadiol sodium diphosphate (Synkayvite) was intravenously injected. HPLC analysis was applied to measure the major metabolite, menadione, VK3. The kinetic properties of VK3 in both plasma and red blood cells showed a short elimination half-life, high clearance, and large volume of distribution in plasma and RBCs. The mean elimination t1/2 values of menadione in plasma and in RBCs were 27.17 +/- 10.49 min and 35.22 +/- 11.82 min, respectively. The plasma clearance (CL/F) of VK3 was 0.822 +/- 0.254 L min-1. The systemic clearance in RBCs was 0.407 +/- 0.152 L min-1. The apparent volume of distribution (Vd/F) in plasma was 30.833 +/- 12.835 L and that in RBCs 20.488 +/- 9.401 L. The plasma AUC was 32.453 +/- 9.785 micrograms min mL-1 and that of RBCs 67.219 +/- 24.449 micrograms min mL-1. Menadiol was rapidly biotransformed to menadione in blood. The formation rate constant (kf) of menadione in plasma was 0.589 +/- 0.246 min-1, and that of RBCs 1.520 +/- 1.345 min-1. Through this study the estimated menadione dosage needed to maintain a plasma level of 1 microgram mL-1 for anticancer purposes was 19.7 mg kg-1 every hour.

Dietary vitamin E effect on color stability and sensory assessment of spoilage in three beef muscles.

Effects of dietary vitamin E supplementation (1204 IU/head/day) for 122 days on color stability and microbial load on beef m. longissimus lumborum (LL), m. gluteus medius (GM) and m. psoas major (PM) were studied by subjective and objective evaluation. Color stability of these muscles followed the order LL > GM > PM (p < 0.05). Vitamin E-treated LL, GM and PM showed less metmyoglobin formation, higher a(∗) values and lower hue angle values than controls during storage at 4 °C (p < 0.05). Sensory evaluation demonstrated that panelists preferred the appearance of vitamin E-treated LL, GM and PM beef steaks. Vitamin E supplementation did not affect total microbial load on LL, PM and GM and did not influence panelists' olfactory assessment of microbial spoilage of beef. Endogenous α-tocopherol concentration and lipid stability of microsomal fractions of LL, GM and PM were greater (p < 0.05) in vitamin E-treated muscles relative to controls. There was no muscle effect on the pro-oxidant activity of microsomes towards oxymyoglobin oxidation (p > 0.05). Oxymyoglobin stability was greater in the presence of microsomal fractions obtained from vitamin E-treated muscle than in those from controls. Dietary vitamin E supplementation delayed oxymyoglobin oxidation in LL, PM and GM muscle and increased the color shelf-life of these muscles without affecting total microbial load.

Lipiodol uptake and retention by human hepatoma cells.

Lipodol has important diagnostic and therapeutic uses in hepatoma. However, the mechanisms of its selective, prolonged retention in hepatoma cells is not well understood. Therefore, using oil-red O, light and electron microscopy and neutron activation analysis we have determined that HepG2 cells are characterized by lipiodol deposition and emulsification on the cell surface, action uptake of lipodol by endocytosis, and prolonged intracellular retention. These findings may have major clinical significance in the development of a new treatment for hepatoma patients.

Determination of anticancer drug vitamin K3 in plasma by high-performance liquid chromatography.

Synthetic vitamin K3 (VK3, 2-methyl-1,4-naphthoquinone, or menadione) has been found to exhibit antitumor activity against various human cancer cells at relative high dose. Parallel to our study on the mechanism of VK3 action and for future clinical trials in Taiwan, we developed a simple, sensitive and accurate high-performance liquid chromatographic method for the determination of VK3 in biological fluids. VK3 was extracted from the plasma samples with n-hexane. The chromatographic separation employed an ODS analytical column (5 microns, 250 x 4.6 mm I.D.) with a mobile phase of methanol-water (70:30, v/v) and UV detection at 265 nm. On completely drying of the extraction solution, n-hexane, by a stream of nitrogen, menadione was lost to a great extent. Methanol (70%, 200 microliters) was added to the extraction solvent after extraction and centrifugation to prevent the loss of menadione. The absolute recovery was 82.4 +/- 7.69% (n = 7). The within-day and between-day calibration curves of VK3 in plasma in the ranges of interest (0.01-10.00 micrograms/ml; 0.01-5.00 micrograms/ml) showed good linearity (r > 0.999) and acceptable precision. The limit of quantitation of VK3 was 10 ng/ml in plasma. This method has been successfully applied to a pilot pharmacokinetic study of VK3 in rabbits receiving an intravenous high-dose bolus injection of 75 mg menadiol sodium diphosphate (Synkayvite). The pharmacokinetic properties of menadione could be described adequately by an open two-compartment model. The mean half-life of menadiol (transformation to menadione) was 2.60 +/- 0.12 min.(ABSTRACT TRUNCATED AT 250 WORDS)

I-131-lipiodol cytotoxicity in hepatoma cells.

Hepatoma is a common cancer in Taiwan. New effective treatment for hepatoma patients is urgently needed. Encouraging results of I-131-lipiodol treatment for hepatoma with minimal toxicity have been recently reported. The mechanism of lipiodol targeting and retention by hepatoma are not well understood. The cellular interaction of lipiodol and the cytotoxic effects of I-131-lipiodol on hepatoma cells were investigated in this study. HepG2 cells were cultured with lipiodol, and untreated HepG2 cells were used as the control. Changes of cellular morphology were accessed by light and electron microscopy. The uptake and retention of lipiodol by HepG2 cells were studied by phase contrast microscopy and neutron activation analysis. HepG2 cells were cultured with I-131-lipiodol varying from 0.12 microCi to 120 microCi. The cytotoxic effect of I-131-lipiodol was evaluated by the surviving fraction of HepG2 cells. Changes in cellular morphology was examined by light microscopy. Results indicated that HepG2 cells were capable of active uptake of large amounts of lipiodol by endocytosis and prolonged intra-cellular retention associated with the formation of many bulging cytoplasmic extensions. I-131-lipiodol was highly cytotoxic to HepG2 cells. There was a steep dose response relationship, and the effective dose (LD50) was 1.2 microCi (480 rads). The cytotoxic effects of I-131-lipiodol were associated with pleomorphism of HepG2 cells, an increase in cell size and nuclear-cytoplasmic ratio, an increase in the size and number of nuclei, and vacuolation of the cytoplasm around the nuclear regions. Multiple nucleoli, fragmentation and segregation and ring shaped changes of nucleoli were also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary carnosine on plasma and tissue antioxidant concentrations and on lipid oxidation in rat skeletal muscle.

The effect of dietary carnosine supplementation on plasma and tissue carnosine and alpha-tocopherol concentrations and on the formation of thiobarbituric acid reactive substances (TBARS) in rat skeletal muscle homogenates was evaluated. Plasma, heart, liver and hind leg muscle was obtained from rats fed basal semipurified diets or basal diets containing carnosine (0.0875%), alpha-tocopheryl acetate (50 ppm), or carnosine (0.0875%) plus alpha-tocopheryl acetate (50 ppm). Dietary carnosine supplementation did not increase carnosine concentrations in heart, liver and skeletal muscle. Dietary supplementation with both carnosine and alpha-tocopherol increased carnosine concentrations in liver 1.56, 1.51- and 1.51-fold as compared with diets lacking carnosine, alpha-tocopherol or both carnosine and alpha-tocopherol, respectively. Dietary supplementation with both carnosine and alpha-tocopherol also increased alpha-tocopherol concentrations in heart and liver 1-38-fold and 1.68-fold, respectively, as compared to supplementation with alpha-tocopherol alone. Dietary supplementation with carnosine, alpha-tocopherol or both carnosine and alpha-tocopherol was effective in decreasing the formation of TBARS in rat skeletal muscle homogenate, with dietary alpha-tocopherol and alpha-tocopherol plus carnosine being more effective than dietary carnosine alone. The data suggest that dietary supplementation with carnosine and alpha-tocopherol modulates some tissue carnosine and alpha-tocopherol concentrations and the formation of TBARS in rat skeletal muscle homogenates.

Preparation of [131I]lipiodol as a hepatoma therapeutic agent.

An isotopic exchange method was used to label lipiodol with 131I. The labelling efficiency was > 92.5%, and the radiochemical purity of [131I]lipiodol was above 98% as determined by ITLC. The influencing factors e.g. the heating temperature, reaction time, pH and storage conditions were studied and the optimum conditions were determined. In a pilot study injecting [131I]lipiodol for the treatment of hepatoma, about 70% of hepatoma patients had a response to the treatment with a reduction of alpha-fetoprotein and decrease of hepatoma sizes. The overall median survival was 9 months (range 2-17 months).