Neural substrates, experimental evidences and functional hypothesis of acupuncture mechanisms.

OBJECTIVES: Although acupuncture therapy has demonstrated itself to be effective in several clinical areas, the underlying mechanisms of acupuncture in general and the analgesic effect in particular are, however, still not clearly delineated. We, therefore, have studied acupuncture analgesic effect through fMRI and proposed a hypothesis, based on the obtained result, which will enlighten the central role of the brain in acupuncture therapy. METHODS: The proposed model, termed as a broad sense hypothalamus-pituitary-adrenal (BS-HPA) axis, was based on our observed neuroimaging results. The model incorporates the stress-induced HPA axis model together with neuro-immune interaction including the cholinergic anti-inflammatory model. RESULTS: The obtained results coupled with accumulating evidence suggest that the central nervous system is essential for the processing of these effects via its modulation of the autonomic nervous system, neuroimmune system and hormonal regulation. CONCLUSIONS: Based on our fMRI study, it appears that understanding the effects of acupuncture within a neuroscience-based framework is vital. Further, we have proposed the broad sense-HPA axis hypothesis which incorporates the experimental results.

Effect of dietary linoleate/alpha-linolenate balance on the brain lipid composition, reproductive outcome and behavior of rats during their prenatal and postnatal development.

The effect of the dietary linoleate (LA)/alpha-linolenate (LNA) balance during development on the brain lipid composition, reproductive outcome and behavior of rats was studied. Female rats were fed on experimental diets during pregnancy and the resulting pups for 16 weeks. The dietary LA/LNA ratios were 1.07 (LA1), 2.64 (LA2), 4.45 (LA3), 7.68 (LA4) and 10.35 (LA5). The relative content of docosahexaenoate (DHA) in the brain of pups tended to increase with decreasing LA/LNA ratio at 0 and 3 weeks, while the level of DHA was maintained constant at 16 weeks regardless of the dietary LA/LNA ratio. The learning ability was measured at 12 weeks of age, and there was no difference among the groups. In an open field test, the exploratory index was significantly lower in the LA1 group than in the LA2 group. The LA1 group had a smaller litter size and lower survival rate than the other groups. We conclude that if the diet contained appropriate amounts and balance of LA and LNA, it was possible for rats to synthesize an appropriate amount of DHA and have normal behavioral activity without DHA supplementation.

The physical map of the chloroplast DNA from Korean ginseng (Panax ginseng C.A. Meyer).

To compare the gene order of the chloroplast genome among dicotyledonous plants, we constructed a physical map of chloroplast DNA (cpDNA) of Korean ginseng (Panax ginseng C.A. Meyer) with four restriction enzymes, BamHI, HindIII, EcoRI, and PstI. The restriction enzyme recognition sites of the physical map were also confirmed by Southern hybridization of total ginseng cpDNA with homologous and heterologous probes. The cpDNA of Korean ginseng was determined as a circular molecule with a total size of about 154 kb, which contain two inverted repeats of 23 kb each that disrupt the rest of the molecule into a large (90 kb) and a small single copy region (18 kb). The genome structure of Korean ginseng cpDNA was similar in size and gene order to that of tobacco cpDNA. The cpDNA of Korean and American ginseng (P. quinquefolius) showed very similar restriction patterns.

Enzyme-substrate intermediate at a specific lysine residue is required for deoxyhypusine synthesis. The role of Lys329 in human deoxyhypusine synthase.

Deoxyhypusine synthase catalyzes the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine] in eukaryotic translation initiation factor 5A. We recently reported biochemical evidence for a covalent enzyme-substrate intermediate involving a specific lysine residue (Lys329) in human deoxyhypusine synthase (Wolff, E. C., Folk, J. E., and Park, M. H. (1997) J. Biol. Chem. 272, 15865-15871). In an effort to evaluate the role of this enzyme-substrate intermediate in catalysis, we carried out site-directed mutagenesis (Lys to Arg and/or Ala) of the conserved lysine residues in human deoxyhypusine synthase. A drastic reduction in enzyme intermediate formation and enzymatic activities was observed with mutant proteins with substitution at Lys287 but not with those with mutations at residues 141, 156, 205, 212, 226, 251, or 338. Lys to Ala or Lys to Arg substitution at Lys329 totally abolished covalent enzyme-substrate intermediate formation and deoxyhypusine synthesis activity, indicating that Lys329 is the unique site for the enzyme intermediate and that it is absolutely required for deoxyhypusine synthesis in the eukaryotic translation initiation factor 5A precursor. The K329A mutant showed spermidine cleavage activity ( approximately 6% of the wild type enzyme) suggesting that in contrast to deoxyhypusine synthesis, spermidine cleavage can occur without enzyme intermediate formation.

Skeletal muscle growth of oMTla-oGH transgenic mice.

Forty-eight transgenic mice carrying an ovine metallothionein 1a-ovine growth hormone (oMTla-oGH) transgene and 48 littermate control mice were used to investigate the effect of GH transgene on the growth and biochemical characteristics of skeletal muscle. Transgene expression was initiated in the transgenic mice by the addition of zinc sulfate to the water at 21 d of age; control mice were also supplemented with zinc sulfate. These mice were maintained on zinc sulfate until 84 d of age. Groups of mice (16 controls, 16 transgenics) were killed at 21, 42 and 84 d of age and muscles from the hind leg were dissected, weighed and analyzed. At 84 d, male transgenics were 32% heavier than controls, while female transgenics were 47% heavier. Transgenic mice of both sexes had smaller (p < 0.01) muscles than controls at weaning (21 d). In spite of significantly heavier body weights of transgenic mice at 84 d of age, there were no significant differences in muscle weights. This was due to a significantly lower (p < 0.01) proportion of muscle, expressed as percentages of body weights, in transgenic mice compared with controls. Higher DNA and RNA concentrations at 42 d of age and elevated cathepsins C and H activities at 42 and 84 d of age indicate that muscle protein metabolism is more active in transgenic mice, which are growing at a greater rate than controls from weaning to 84 d of age. The fact that oMTla-oGH transgenic mice inherently have a lower proportion of muscle, compared to controls and that this proportion does not change in spite of transgene activation and 30 fold increase in plasma GH levels, suggests the hypothesis that muscle growth may be controlled by locally produced IGFs, which are essentially independent of circulating GH concentrations.

Diamine and triamine analogs and derivatives as inhibitors of deoxyhypusine synthase: synthesis and biological activity.

Deoxyhypusine synthase catalyzes the initial step in the posttranslational formation of the amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine] in eukaryotic initiation factor 5A (eIF-5A). eIF-5A and its hypusine modification are believed to be essential for cell growth. A number of compounds related to diamines and triamines were synthesized and tested as inhibitors of this enzyme. The findings indicate that the long chain triamines 2a and 2b and their guanyl derivatives 3a, 3b, 4a, and 4b exert inhibition by binding to enzyme through only a portion of their structures at any one time. The inhibition exhibited by N-ethyl-1,7-diaminoheptane 20 and its guanyl derivative 21 supports this notion and is evidence for participation of the secondary amino group in binding to enzyme. There is preliminary evidence that amidino and isothiuronium groups may also serve as basic centers for binding to enzyme. Few of the compounds tested here were comparable in inhibitory potency to 1-guanidino-7-aminoheptane (GC7) the most effective known inhibitor of deoxhypusine synthase, and none proved nearly as efficient as GC7 in inhibiting the enzyme in Chinese hamster ovary cells. Hence, unlike the antiproliferative effect of GC7, for which there is evidence of cause by interference with deoxhypusine synthase catalysis (Park, M. H.; Wolff, E. C.; Lee, Y. B.; Folk, J. E. J. Biol. Chem. 269, 1994, 27827-27832), the effective growth arrest exerted by several of the newly synthesized compounds cannot be attributed to inhibition of hypusine synthesis.

Antiproliferative effects of inhibitors of deoxyhypusine synthase. Inhibition of growth of Chinese hamster ovary cells by guanyl diamines.

Certain guanyl diamines are effective inhibitors of deoxyhypusine synthase (Jakus, J., Wolff, E. C., Park, M. H., and Folk, J. E. (1993) J. Biol. Chem. 268, 13151-13159), the first enzyme involved in the biosynthesis of the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Evidence that hypusine is implicated in cell growth prompted this study of the cellular effects of these inhibitors. In Chinese hamster ovary (CHO) cells, inhibition of hypusine biosynthesis followed by progressive arrest in cellular proliferation was observed with both N-mono- and N,N'-bisguanyl derivatives of 1,6-diaminohexane, 1,7-diaminoheptane, and 1,8-diaminooctane. Cells treated with these compounds showed no significant change in polyamine distribution, suggesting that the observed growth inhibition is not mediated through an interference with polyamine metabolism. N1-guanyl-1,7-diaminoheptane, the most potent inhibitor of deoxyhypusine synthase both in vitro and in cells, exhibited the highest antiproliferative activity toward CHO cells. No early cytotoxic effects were observed with this inhibitor, and its antiproliferative activity appeared to be reversible. Transport studies showed that N1-guanyl-1,7-diaminoheptane is actively taken up by the polyamine transport system. Mutant CHO cells defective in polyamine transport were found to be resistant to growth inhibition by this compound. The findings suggest that the antiproliferative effect of N1-guanyl-1,7-diaminoheptane is exerted intracellularly through inhibition of hypusine synthesis.

Effects of iodinated fatty acid ester on human hepatocellular carcinoma cells.

The interaction between Lipiodol and cells was studied by treating Lipiodol in a human hepatocellular carcinoma cell line(Hep) and mouse fibroblast cell line (L929). Irregular, sustained radioactivity was released from both cell lines shortly after incubation in the radioiodinated Lipiodol mixed media. Lipiodol droplets were found to be firmly attached to the cells following the incubation and these cells were strongly positive for fat stains. The radioiodinated Lipiodol demonstrated the same behavior of accumulation within the cell and on the cell membrane. Although the amount of Lipiodol attached was almost equal in both of the cell lines, the final amount accumulated in the cells was larger in the Hep cells. The accumulation of Lipiodol within the cell and on the cell membrane may play a significant role for its selective targeting and its prolonged retention in the solid tumor.

Distribution of Lipiodol in hepatocellular carcinoma.

Intrahepatic distribution of Lipiodol and I-131 Lipiodol infused via the hepatic arteries was evaluated in six patients with HCC who had undergone hepatic lobectomy or segmentectomy. CT scan and gamma camera radiograph confirmed that the oily contrast material or I-131 radioactivity accumulated selectively in the tumor over a long period. One to two thirds of the tumor mass appeared necrotic, although the extent tended to be larger in the case of radioactive Lipiodol infusion. The tumor cells contained numerous lipid globules within the cytoplasm. Also, oil red 0 stain demonstrated that the individual tumor cells had non-globular lipid on their surface. In conclusion, Lipiodol leaks out of the vascular spaces to attach to the cancer cell membrane as a non-globular lipid as well as to enter the cancer cells as a globular lipid. This phenomenon specific to cancer cells suggests a biochemical membrane change which may have occurred during carcinogenesis, causing alteration of membrane transport and cell death.

Adjuvant treatment of operable stomach cancer with polyadenylic.polyuridylic acid in addition to chemotherapeutic agents: a preliminary report.

A randomized trial of polyadenylic.polyuridylic acid [poly(A).poly(U)] in addition to chemotherapy was undertaken in patients with stomach cancer following curative gastrectomy. They were randomized into a group of 108 patients receiving chemotherapy plus poly(A).poly(U) and a control group of 116 patients receiving chemotherapy alone. Chemotherapy consisted of injections of 5-fluorouracil, 12 mg/kg once weekly and adriamycin, 40 mg/m2 once every 3 weeks, continuously after operation. Poly(A).poly(U) was infused in a 100 mg dose, once a week six times from 5 days after the first injection of chemotherapeutic agents and 6 months later in a half dose similarly. At 55 months after initiation of the trial, the mean follow-up periods were 24 months for both groups. It has been revealed that patients who received the combined treatment postoperatively showed a lesser mortality and lower rate of recurrence, both reflecting significant increases in overall (P less than 0.05) and relapse-free (P less than 0.02) survivals as compared to those who received chemotherapy alone. This effect is more pronounced in patients having moderately advanced lymphnode involvement (N1) than in patients without (N0) or more advanced (N2) involvement. Thus, poly(A).poly(U) appears to be an effective agent when used postoperatively with chemotherapy in stomach cancers.

Adjuvant treatment of operable stomach cancer with polyadenylic.polyuridylic acid in addition to chemotherapeutic agents. Differential effect on natural killer cell and antibody-dependent cellular cytotoxicity.

Peripheral blood lymphocytes of operable stomach cancer patients were evaluated sequentially for their natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities before and after chemotherapy in association with polyadenylic.polyuridylic acid [poly(A).poly(U)]. Their cytotoxicity was measured by 4 h-chromium release assays, using human K562 and sensitized murine L1210 cells as targets for assays of NK and ADCC respectively. The mean NK cytotoxicity of 89 patients before treatment was significantly lower than that of the 18 sex- and age-matched healthy controls, whereas assays of ADCC showed similar levels of cytotoxicity in both groups. Patients who had received postoperative chemotherapy (5 fluorouracil, 12 mg/kg and adriamycin, 40 mg/M2) once, had, 5 days after injection, NK cytotoxicity levels similar to those before treatment. For these patients, an additional administration of poly(A).poly(U) (100 mg) resulted, 2 days later, in a significant increase in the levels of NK cytotoxicity without affecting the levels of ADCC. Repeated injections of poly(A).poly(U) alternated with chemotherapy induced, consistently, exclusive enhancement of NK activity after each injection. These results suggest that the effector cells for NK and ADCC activities are of functionally different cell populations.