The correlation between feed-intake cycle and nutritional zinc-deficient status in rats

The characteristic cyclic variation in feed intake of rats fed a Zn-deficient diet (Mills et al, Am J Clin Nutr 22: 1240-1249 (1969)) followed a Cosinor curve, as determined by computer analysis (Tamaki et al, Br J Nutr 73: 711-722 (1995)). The values of amplitude for the feed-intake cycle had a positive correlation to their own day-to-day variations and to the correlation value of their own simulated cycles (r2 = 0.764, df = 50, p < 0.001 and r2 = 0.682, df = 50, p < 0.001, respectively). The cyclic variation in feed intake was accompanied by a cyclic variation in body-weight change in rats fed the Zn-deficient diet, and cyclic variation in body-weight change occurred similarly in pair-fed control rats. There were no differences in the mesors of body-weight change cycles of Zn-deficient rats and pair-fed control rats (Zn-deficient rats: 2.5 +/- 1.0 g/d, pair-fed rats: 2.8 +/- 1.0 g/d, mean +/- SD, df = 18, t = -0.674, ND). Rats fed the Zn-deficient diet were given different amounts of Zn supplementation by daily subcutaneous injection. The amplitude of the feed-intake cycle was decreased with increasing Zn supplementation (r2 = 0.919, df = 5, p < 0.001). The concentration of Zn for the appearance of the feed-intake cycle was estimated to be 71.6 +/- 6.6 micrograms/d per rat. The Zn level in the serum showed a significant decrease in the Zn-deficient diet groups, but the supplement of Zn did not vary in the Zn-deficient rats injected with up to 47.3 micrograms/d per rat. From these results, an analysis of the feed-intake cycle allowed us to estimate the quantitative Zn-deficient status of rats.     

Effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase inhibitor, on cholesterol esterification and secretions of apolipoprotein B-containing lipoprotein and bile acids in HepG2

We studied the effect of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl) ureido]methyl]cyclohexane), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on intracellular cholesterol esterification and the secretion of apolipoprotein B100 (apoB)-containing lipoprotein and bile acids in the human hepatoma cell line HepG2. NTE-122 markably inhibited [3H]oleate incorporation into cholesteryl esters in HepG2 cells incubated with 5 microg/ml 25-hydroxycholesterol as a stimulus for ACAT (IC50=6.0 nM). On the other hand, NTE-122 did not affect [3H]oleate incorporation into triglycerides and phospholipids and [14C]acetate incorporation into cholesterol. The stimulation of ACAT by 25-hydroxycholesterol caused significant increases in the secretion of radiolabeled cholesteryl esters, radiolabeled triglycerides and apoB mass. NTE-122 pronouncedly inhibited the secretion of radiolabeled cholesteryl esters in proportion to the inhibition of cellular cholesterol esterification, and it significantly reduced the secretion of radiolabeled triglycerides and apoB mass in HepG2 cells incubated with 25-hydroxycholesterol. Furthermore, NTE-122 increased the secretion of bile acids synthesized from [14C]-cholesterol. These results suggest that NTE-122 is capable of exhibiting anti-hyperlipidemic effects by reducing both the cholesterol content and the amount of secreted very low-density lipoprotein and enhancing the excretion of bile acid from the liver.     

Effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase inhibitor, on cholesterol esterification and high-density lipoprotein-induced cholesterol efflux in macrophages

We investigated the effects of a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), on ACAT activities in macrophages originating from several species and high-density lipoprotein (HDL)-induced cholesterol efflux in phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells. NTE-122 inhibited cell-free ACAT activities in human PMA-treated THP-1 cells and mouse J774.1 cells with IC50 values of 0.88 and 360 nM, respectively. NTE-122 competively inhibited the ACAT activity in PMA-treated THP-1 cells. NTE-122 also inhibited cellular ACAT activities in PMA-treated THP-1 cells, rat peritoneal macrophages and J774.1 cells with IC50 values of 3.5, 84 and 6800 nM, respectively. Furthermore, NTE-122 prevented cholesterol accumulation in PMA-treated THP-1 cells incubated with acetylated low density lipoprotein, simultaneously with HDL, while it caused accumulation of a significant amount of free cholesterol in the absence and even in the presence of HDL. NTE-122 also enhanced HDL-induced cholesterol efflux from established foam cells converted from PMA-treated THP-1 cells. These results suggest that NTE-122, capable of inhibiting macrophage ACAT activity in humans more strongly than those in the other species, exhibits anti-atherogenic effects by preventing the foam cell formation and enhancing the foam cell regression in humans.     

Seasonal variation of saponins, sucurose and monosaccharides in cultivated ginseng roots

Yield of methanolic extract of fresh roots of Ginseng harvested in winter was found to be more than two-fold grater than from roots collected in summer; this remarkable increase is mainly due to the large increase of sucrose in roots in winter. On the other hand, biologically active dammarane-saponins in the roots increase in summer. These results indicate that roots should be harvested in summer for the production of high quality Ginseng extracts.     

Synthesis and biological activity of 1-N-[4-(substituted)amidino and guanidino-2-hydroxybutyryl]kanamycins A and B

The synthesis and biological properties of 1-N-[4-(substituted)amidino and guanidino-2-hydroxybutyryl]kanamycins A and B are described. Reaction of 3,3",6'-tri-N-tert-butoxycarbonyl-amikacin with an appropriate amidinating or guanidinating reagent and subsequent deblocking gave a series of amikacin derivatives having an amidino or guanidino group on the 4"'-position. The corresponding kanamycin B analogs were also prepared by a similar procedure. Among these derivatives, 1-N-(4-formamidino- and guanidino-2-hydroxybutyryl)kanamycins A (7a and 7k) and B (11 and 14) exhibited antibacterial activity similar to the corresponding 4-amino analogs. The nephrotoxic potential of selected compounds is also briefly discussed.     

Specific binding of phorbol esters to Friend erythroleukemia cells -- general properties, down regulation and relationship to cell differentiation

Specific and saturable binding sites for [20-³H]phorbol 12,13-dibutyrate([³H]PDBu) were demonstrated in intact Friend erythroleukemiacells (FELC), in which inducible erythroid differentiation isreversibly inhibited by phorbol esters. The binding of [³H]PDButo intact cells was maximal within only 15 min of incubationat 37°C, after which there was a gradual decrease; bindingat 4°C however, was a slow process, requiring > 180 minfor maximal binding. A Scatchard analysis showed that the dissociationconstant for binding of [³H]PDBu is 8.3 nM; at saturation, 1.75x 10⁵ molecules of [³H]PDBu are bound per cell. The bindingof [³H]PDBu is blocked by 12-O-tetradecanoyl phorbol-13-acetate,phorbol 12,13-didecanoate, mezerein, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and resiniferatoxin, but not by phorbol or4-phorbol 12,13-didecanoate. There was, in general, a good correlationbetween the potency of these agents in inhibiting [³H]PDBu bindingand their activity in promoting tumors on mouse skin. Inducersof differentiation, such as hexamethylene bisacetamide, dimethylsulfoxide and butyric acid, as well as inhibitors of cell differentiation,dexamethasone and local anesthetics, did not significantly blockthe binding of [³H]PDBu to intact FELC. When FELC were inducedto differentiate with 4 mM hexamethylene bisacetamide (80% ofcells were benzidine-positive), a slight decrease (10–20%)in the number of binding sites at saturation was seen, but thedissociation constant was not changed. When the cells were preculturedwith non-radioactive phorbol esters, a significant decreasein [³H]PDBu binding was observed, suggesting a homologous downregulation of phorbol ester receptors. Scatchard analysis indicatedthat the decrease in [³H]PDBu binding was due to a decreasein the number of binding sites and not to a change in affinity.Such specific phorbol ester binding sites might mediate a numberof biochemical and biological effects of phorbol esters on FELC.     

Induction of erythroleukemia cell adhesion by plant diterpene tumour promoters: a quantitative study and correlation with in vivo activities

A potent tumour promoter on mouse skin, phorbol-9-myristate-9a-acetate,induces certain clones of Friend erythroleukemia cells to becomeadhesive to the surface of tissue culture dishes, whereas inthe absence of this compound, these cells grow in suspension.We have quantitatively tested 20 other phorbol esters and relatedcompounds for this effect. When the results are expressed asthe concentrations of compounds which show half-maximum effecton cell adhesion, the decreasing order of potency is: phorbol-9-myristate-9a-acetate(3.6 x 10^–10M) gnilatimacrin > milliamine A phorbol-9,9a-didecanoate mezerein gnidilatin ingenol-3,20-dibenzoate > phorbolol-9-myristate-9a-acetate> phorbol-9,9a-dibutyrate phorbol-9,9a-dibenzoate > 4a-O-methyl-phorbol-9-myristate-9a-acetate> phorbol-9-myristate-9a-acetate-3-aldehyde > phorbol-9,9a-diacetate> 2,3-dihydrophorbol-9-myristate-9a-acetate. Phorbol, 4a-phorbol-9,9a-didecanoate,phorbol-3,9,9a-triacetate, phorbol-9-myristate, phorhol-9-monoacetateand phorbol-9a-monoacetate were inactive in this assay whentested at concentrations as high as 1 µg/ml (10^–6M).None of these 20 compounds induced adhesion when they were testedwith a variant clone of Friend erythroleukemia cells which isresistant to the induction of adhesion and several other effectsof phorbol-myristate-acetate. When the relative potencies ofthese compounds in the adhesion assay were compared to availablein vivo data on tumour promoting activity on mouse skin, therewas, in general, a good qualitative correlation. A better butnot perfect quantitative correlation was obtained when the resultsfrom the adhesion assay were compared with reported inflammatoryactivity on mouse ear. When several other tumour promoters andcocarcinogens which differ structurally from the phorbol estersand related plant diterpenes were tested, none of these inducedadhesion in this assay.     

Vergence disorders in patients with spinocerebellar ataxia 3/Machado-Joseph disease: a synoptophore study

Diplopia, a common symptom in spinocerebellar ataxia 3/Machado-Joseph disease (SCA3/MJD) cases, is not always due to asymmetric ophthalmoplegia. We found a Japanese SCA3/MJD family, in which three patients clearly had an impairment of divergence eye movement. We thus quantitatively examined the vergence ranges in eight Japanese SCA3/MJD cases using the synoptophore test. An impairment of the vergence eye movements was found in all patients, and the vergence impairment pattern, but not the ophthalmoplegia pattern, was found to be compatible with the diplopia pattern. The diplopia in SCA3/MJD cases is, therefore, attributed, at least in part, to the impairment of the vergence eye movements.     

Flow cytometric differentiation of Asian and Western types of multiple sclerosis, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and hyperIgEaemic myelitis by analyses of memory CD4 positive T cell subsets and NK cell subsets

We examined the alterations of memory CD4(+) T cell subsets bearing surface receptors linked to either Th1 or Th2 cytokine production as well as natural killer (NK) cell subsets by three color flow cytometry in the peripheral blood from 36 patients with clinically definite multiple sclerosis (MS), 27 patients with HAM/TSP, 13 patients with hyperIgEaemic myelitis who had mite antigen-specific IgE and 25 healthy controls (HC). The patients with MS were clinically classified into an optico-spinal form of MS (Asian type, MS-A) and the conventional form of MS (Western type, MS-W). MS-A showed a significant increase of CD4(+)CD45RA(-)CCR5(+) cells (Th1 cells) through the relapse and remission phases in comparison to HC, while MS-W showed a significant increase of CD4(+)CD45RO(+)CD62L(-) cells (Th1 cells) only at the relapse phase. HAM/TSP showed a significant increase of CCR5(+) and CD62L(-) memory CD4(+) T cells as well as CD30(+) memory CD4(+) T cells (Th2 cells) in comparison to HC. On the other hand, a selective increase of CD4(+)CD45RO(+)CD30(+) cells was found in hyperIgEaemic myelitis. The percentage of mature NK cells (CD3(-)CD16(+)CD56(+) cells) as well as double negative T cells (CD3(+)CD4(-)CD8(-) cells) decreased significantly in HAM/TSP in comparison to HC. Our findings therefore suggest a flow cytometric analysis of Th1/Th2-associated markers on memory CD4(+) T cells as well as NK cell subsets to be useful for differentiating various inflammatory neurologic conditions.     

Th1 dominance in HAM/TSP and the optico-spinal form of multiple sclerosis versus Th2 dominance in mite antigen-specific IgE myelitis

To clarify the Th1/Th2 balance in spinal cord inflammation, we used ELISA to measure the total and allergen-specific IgE in 69 patients with clinically definite multiple sclerosis (MS), including 24 patients with the optico-spinal form of MS, 45 with HAM/TSP, 30 HTLV-I carriers without HAM/TSP, 40 patients with acute myelitis, 43 with neurodegenerative disorders, and 42 healthy subjects, and flow cytometry to study the intracellular IFNgamma-positive versus IL-4-positive cell ratio (intracellular IFNgamma/IL-4 ratio) in peripheral blood CD4(+) T cells in 40 patients with MS, including 17 patients with the optico-spinal form of MS, 23 with HAM/TSP, 22 with acute myelitis, 23 with neurodegenerative disorders, and 36 healthy subjects. Patients with HAM/TSP showed a significantly higher intracellular IFNgamma/IL-4 ratio, lower IL-4(+)/IFN-gamma(-) cell percentages, lower total IgE level, and lower frequency of cedar pollen-specific IgE than did the controls. The patients with optico-spinal MS showed a significantly higher intracellular IFNgamma/IL-4 ratio and higher IL-4(-)/IFN-gamma(+) cell percentages than the controls even at remission or in the convalescence phase. In contrast, in the patients with acute myelitis, the total serum IgE level and the frequency of mite antigen-specific IgE were significantly elevated in comparison to the controls, while those having mite antigen-specific IgE myelitis showed a significantly lower IFNgamma/IL-4 ratio in the CD4(+) T cells in comparison to the controls. These findings suggest that the Th1 cell response is predominant in HAM/TSP and optico-spinal MS, whereas the Th2 cell response is predominant in mite antigen-specific IgE myelitis.