The effect of Am-80, one of retinoids derivatives on experimental allergic encephalomyelitis in rats.

Experimental allergic encephalomyelitis (EAE) is an autoimmune model with inflammation and demyelination in the central nervous system, which resembles the human demyelinating disorder, multiple sclerosis (MS). In this study, we investigated the effect of Am-80, a synthetic retinoid, on EAE in DA rats. DA rats immunized with complete Freund's adjuvant (CFA) supplemented with myelin basic protein (MBP) developed severe EAE which reached the peak 12 to 14 days after immunization. Am-80 and prednisolone administered orally for 12 days after immunization diminished the clinical symptoms and infiltration of inflammatory cells in a dose dependent manner. However, after stopping administration, EAE recurred in DA rats treated with Am-80, but not with prednisolone. The different responses between Am-80 and prednisolone were not due to the difference in the tolerability to the MBP because both inhibited the delayed-type hypersensitivity response to MBP only during administration. To investigate the mechanism how Am-80 alone delayed the response, the expressional levels of mRNA for interleukin-6 (IL-6), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in spinal cord were examined. Transcriptional levels of IL-6, IFN-gamma and TNF-alpha were parallel with the clinical symptoms of the disease in Am-80-treated rats, that is, expressional levels of their mRNA were diminished during the administration of Am-80, which then increased as soon as the administration was stopped. Among them, the expression of IL-6 mRNA was more rapidly and highly relapsed than that of the other two cytokines mRNA. However, prednisolone attenuated transcriptions of all these cytokines throughout the experiment. Therefore, these findings suggested that the inhibition of EAE is, in part, related to the inhibition of IL-6 production. However, there are many possible mechanism in the suppression of EAE by Am-80, further experiments will be necessary.

Effect of Am-80, a synthetic derivative of retinoid, on experimental arthritis in mice.

Am-80 is a newly snythesized retinoid with the structure of one aromatic amide among retinobenzoic acids. It exhibits specific biological activities of retinoic acid such as the activation of cellular differentiation and proliferation. We investigated the effect of Am-80 on collagen-induced arthritis (CIA) in mice and the immunopharmacological action on the production of several cytokines in the in vitro and in vivo models. Am-80, at doses of 0.3, 1 and 3 mg/kg, significantly inhibited the severity and development of the arthritis index, progression of foot pad swelling, bone damage and histopathological alterations. Am-80 also inhibited the production of anti-type II collagen (CII) IgG antibody, but did not affect the delayed-type hypersensitivity (DTH) response in arthritic mice. To determine the inhibitory mechanism of Am-80, we studied the effect of Am-80 on the production of cytokines. Am-80 did not affect the production of IFN-gamma by Th1 cells (1E10.H2 cells) and IL-4 by Th2 cells (D10.G4.1 cells), respectively. Am-80 selectively inhibited bacterial lipopolysaccharide (LPS)-induced IL-6, but not TNF-alpha and IL-1beta, production in mice. Moreover Am-80 inhibited IL-1beta induced IL-6 production and IL-6 mRNA expression in human osteoblast-like cells (MG-63). The inhibition of IL-6 production by Am-80 was due to downregulation of the pretranscription or the transcription of IL-6 in MG 63 cells. These findings suggest that the inhibitory effect of Am-80 on CIA is partially by modulating the production of the proinflammatory cytokine, IL-6.

Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata).

Synthetic urea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin-specific binding protein (CSBP) of high affinity (Ka for 4PU30 = 4x10(10) M(-1)) in the soluble fraction of etiolated mung bean seedlings [Nagata, R., Kawachi, E., Hashimoto, Y. & Shudo, K. (1993) Biochem. Biophys. Res. Commun. 191, 543-549]. In this report, we purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel liganded with 4PU. We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse-transcription (RT) PCR. The cDNA encoded a protein with a calculated molecular mass of 17 kDa. A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis-related protein family. Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins.

[Chemistry of benzoxazinoids produced by plants as phytoalexin].

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one and its desmethoxy derivative (DIMBOA and DIBOA) are major phytoalexins produced by rye, wheat, zea maize and related monocotyledoneae plants. These compounds elicit a wide variety of biological activities including antifungal and mutagenic activities. Structure-activity relationships of these compounds and their derivatives (benzoxazinoids), and the reactivity of benzoxazinoids with nucleophiles are discussed in relation to the molecular mechanism of their biological activity. The electrophilic reaction mechanism of benzoxazinoids and substituent effects of namely 7-methoxy and 2-hydroxy groups are also discussed.

Cytokinin-specific binding protein in etiolated mung bean seedlings.

We have found that a synthetic urea derivative, N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30, forchlorfenuron), has even higher cytokinin activity than N6-benzyladenine (BA). Using [3H]4PU30 (2.83 TBq/mmol) as a probe, we confirmed chromatographically the presence of a high-affinity cytokinin-specific binding protein (CSBP) in whole-cell extract from etiolated mung bean seedlings. The apparent molecular weight of CSBP was estimated to be 21 kDa. The association constants (Ka's) of CSBP for 4PU30 and BA were calculated to be 4 x 10(10) M-1 and 3 x 10(9) M-1, respectively. Various active cytokinins showed mutually competitive binding to CSBP, and their affinities for CSBP corresponded well to their cytokinin activities at the tissue level.

Meso-oligodeoxyribonucleotides.

Meso-DNAs, containing L-sugar and D-sugar in an alternate sequence as the backbone [Fig. 1, designated as LD-(dN)n], were prepared as a modificated oligonucleotides of natural- and enantio-DNA. The characteristics of meso-DNAs were analyzed, focusing on LD-(dA)n and LD-(dT)n. These L-sugar containing oligomers show resistancy to phosphodiesterases. Though both LD-(dA)n and LD-(dT)n bound their corresponding complementary homopolymers, apparent differences of the interaction mode between these two meso-DNAs were observed: [1] Concerning the complex formation with the complementary homopolymers, LD-(dA)n favors a triplex formation while LD-(dT)n forms only duplex, and [2] Concerning DNA/RNA selectivity for the complex formation, LD-(dA)n interacted with poly U more strongly than poly dT (RNA-selective), while LD-(dT)n showed stronger interaction with poly dA than poly A (DNA-selective). The similarities in CD-spectra of LD-oligomer/natural polymer complexes to natural complexes suggest that the conformation possesses a structure close to a natural right-handed helix conformation.

Expression of nuclear retinoic acid receptors in wild-type and mutant embryonal carcinoma PCC4.aza1R cells.

Retinoic acid (RA) induces differentiation of murine embryonal carcinoma PCC4.aza1R cells. In this study, the expression of nuclear retinoic acid receptors (RARs) in PCC4.aza1R cells is examined. Analyses of [3H]RA-labeled nuclear extracts prepared from PCC4.aza1R cells by size-exclusion high-performance liquid chromatography demonstrated the presence of a specific RA-binding activity that migrated with a molecular weight of approximately 50,000. More than 95% of this binding activity was associated with the nuclear fraction. In contrast to cytosolic retinoic acid-binding protein, the RARs bound RA analogues of the Ch-series very effectively. Northern blot analyses of total RNA with complementary DNA probes specific for RAR alpha, RAR beta, and RAR gamma showed that PCC4.aza1R cells contain predominantly transcripts encoding RAR alpha and RAR gamma; RAR beta transcripts were undetectable. Treatment of PCC4.aza1R cells with RA increased the levels of RAR beta mRNA in a dose- and time-dependent manner. The RA concentration for half-maximum induction of RAR beta mRNA was 1 nM. An increase in RAR beta mRNA was detectable as early as 2 h after the addition of RA. This increase was not abrogated by cycloheximide, suggesting that protein synthesis is not required for this response. The ability of several retinoids to increase RAR beta mRNA levels in PCC4.aza1R cells correlated well with their binding affinity to the RARs but not with their binding affinity to cytosolic retinoic acid-binding protein. Two mutant cell lines, PCC4(RA)-1 and (RA)-2, which do not undergo differentiation after RA treatment, contained levels of RAR-binding activity very similar to those of the parental cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Zinc ion enhances the blocking potency of synthetic analogs of spider toxin (JSTX) on the glutamate receptor.

The effect of synthetic spider toxin analogs containing aza-crown in combination with zinc and copper was studied on the lobster neuromuscular synapse. The suppressive action of N4-type spider toxin analog (N4) on excitatory postsynaptic potentials (EPSPs) was markedly enhanced in the presence of 10(-5)-10(-4) M Zn2+. Cu2+ (10(-4) M) also had a potentiating effect on the suppression of EPSPs by N4 but to a lesser degree than Zn2+. While N6-type spider toxin analog (N6) suppressed EPSPs more effectively than N4, additional suppression was not pronounced in the presence of Zn2+ or Cu2+. The results suggest that chelatable divalent metal ions play an important role in the blocking mechanism of glutamate receptor by the spider toxin.

A new tumor promoter from the seed oil of Jatropha curcas L., an intramolecular diester of 12-deoxy-16-hydroxyphorbol.

A new type of phorbol ester, which has a macrocyclic dicarboxylic acid diester structure, was isolated from the seed oil of Jatropha curcas L. (Euphorbiaceae). Based on the results of spectroscopic analyses of the compound and its chemical degradation products, its structure is proposed to be an intramolecular 13,16-diester of 12-deoxy-16-hydroxyphorbol, 12-deoxy-16-hydroxyphorbol-4'-[12',14'-butadienyl]-6'-[16',18',20' - nonatrienyl]-bicyclo[3.1.0]hexane-(13-O)-2'-[carboxylate]-(16-O)-3 '- [8'-butenoic-10']ate (DHPB). DHPB showed slightly weaker biological and biochemical activities than 12-O-tetradecanoylphorbol-13-acetate (TPA). DHPB induced ornithine decarboxylase in mouse skin (2.8 nmol CO2/30 min/mg protein/34 nmol application), inhibited the specific binding of [3H]-12-O-tetradecanoylphorbol-13-acetate to phorbol ester receptors (50% effective dose, 17.0 nM), and activated protein kinase C in vitro (50% effective dose, 36.0 nM). Also, a weak tumor-promoting activity of DHPB was found in a two-stage carcinogenesis experiment on mouse skin. One week after initiation of mice with 100 micrograms of 7,12-dimethyl-benz(a)anthracene, topical application, twice a week, of 2 micrograms of DHPB until week 17, followed by application of 5 microgram of DHPB until week 30 at the same rate, resulted in 46.7% incidence of tumors by week 30. The groups treated with 7,12-dimethylbenz(a)anthracene alone or DHPB alone did not produce significant numbers of tumors. These results indicate that the new phorbol ester, DHPB, is a tumor promoter with weaker activity than 12-O-tetradecanoylphorbol-13-acetate.

Newly synthesized analogues of the spider toxin block the crustacean glutamate receptor.

Effects of synthetic compounds similar to the structure of a spider toxin were studied on the glutamate receptors in crustacean neuromuscular synapses. Two kinds of analogues, 2,4-dihydroxyphenylacetyl-asparaginyl cadaverine (C-1) and 2,4-dihydroxyphenylacetyl-asparaginyl spermine (C-2), suppressed the excitatory postsynaptic potentials in a manner similar to natural spider toxin (JSTX). The dose-response relationship showed that the relative potency of the compounds is C-1 less than C-2 less than JSTX. While the effect of JSTX was irreversible, those of C-1 and C-2 were reversible. These synthetic compounds may serve as important tools in studying the structure and function of glutamate receptors.

Clastogenic potential of heavy oil extracts and some aza-arenes in Chinese hamster cells in culture.

Chromosomal aberration tests in vitro, with a Chinese hamster fibroblast cell line, CHL, were carried out on B class heavy oil fractions, obtained by silica-gel column chromatography and a liquid-liquid extraction method. The original oil and basic nitrogen-containing fractions induced structural chromosomal aberrations in the CHL cells in the presence of rat-liver microsome fraction (S9 mix). 8 tricyclic or pentacyclic aza-arenes, which possibly exist in the positive fractions, were also examined for their clastogenic activities in the system with or without S9 mix. Acridine, benzo[f]quinoline, pyrenoline and pyrenoline 4,5-oxide induced chromosomal aberrations both with and without S9 mix. Benzo[h]quinoline was positive without S9 mix, whereas dibenz[c,h]acridine and dibenz[a,j]acridine were only positive with S9 mix. The results suggest that the clastogenic effects of the heavy oil may in part be due to the presence of nitrogen-containing polycyclic hydrocarbons such as aza-arenes.