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Fractionation guided by immunomodulatory activity of the EtOAc extract of the Ascomycete Eupenicillium crustaceum has afforded two new naturally occurring products, 4‘-oxomacrophorin D (1) and 4‘-oxomacrophorin A (2), as the immunosuppressive components of this fungus [1:  3-hydroxy-3-methylglutaryl (HMG) conjugate of 2]. The structures including the absolute configurations of 1 and 2 have been determined on the basis of chemical correlation of 1 with macrophorin D (3). The absolute configuration of the HMG moiety in 3 has been revised from 3R to 3S.

We present a new analytical method of molecular orientation in films using a phase measurement of second harmonic generation (SHG) with a theoretical curve fitting. The new analytical method provides the average direction of molecules and the absolute value of the effective second-order nonlinear optical constant (|d ^eff|) simultaneously. In conventional methods, the average direction of molecules in a sample has been relatively determined with that in a standard sample having preknown molecular direction. The second harmonic (SH) interference fringe obtained between a reference having preknown molecular direction and the sample has been compared with that between the reference and the standard sample. In the new method, however, the average direction of molecules can be obtained without the standard sample. The value of |d^eff| is calculated through the fitting process, the average direction of molecules is determined by the sign or the phase of d^eff, which is introduced by a reference. Complete expression with physical meanings is given for the valley points and the contrast of SH interference fringe. There, effect of a lens to focus a fundamental wave to a sample is also taken into account. The new phase measurement of SHG with the curve fitting has been carried out with poled poly(methyl methacrylate) films doped with 2 wt % of p-nitroaniline (PNA/PMMA), which were used as a reference and also as a sample. The accuracy of the new method has been demonstrated in comparison of the value |d^eff| of the sample with that determined by Maker fringe measurement. The determined average direction of molecules for the sample was confirmed by experimental geometry of the reference and the sample and also from the poling geometry for the sample. We have shown that the phase measurement of SHG and the curve fitting are useful tools to investigate the molecular orientation.

We report here kinetic analysis and identification of the two cyclase domains in a bifunctional diterpene cyclase, Phaeosphaeria ent-kaurene synthase (FCPS/KS). Kinetics of a recombinant FCPS/KS protein indicated that the affinity for copalyl diphosphate is higher than that for geranylgeranyl diphosphate (GGDP). ent-Kaurene production from GGDP by FCPS/KS was enhanced by the addition of a plant ent-kaurene synthase (KS) but not by plant CDP synthase (CPS), suggesting that the rate of ent-kaurene production of FCPS/KS may be limited by the KS activity. Site-directed mutagenesis of aspartate-rich motifs in FCPS/KS indicated that the (318)DVDD motif near the N terminus and the (656)DEFFE motif near the C terminus may be part of the active site for the CPS and KS reactions, respectively. The other aspartate-rich (132)DDVLD motif near the N terminus is thought to be involved in both reactions. Functional analysis of the N- and C-terminal truncated mutants revealed that a N-terminal 59-kDa polypeptide catalyzed the CPS reaction and a C-terminal 66-kDa polypeptide showed KS activity. A 101-kDa polypeptide lacking the first 43 amino acids of the N terminus reduced KS activity severely without CPS activity. These results indicate that there are two separate interacting domains in the 106-kDa polypeptide of FCPS/KS.

ent-Kaurene is the first cyclic diterpene intermediate of gibberellin biosynthesis in both plants and fungi. In plants, ent-kaurene is synthesized from geranylgeranyl diphosphate via copalyl diphosphate in a two-step cyclization catalyzed by copalyl diphosphate synthase and ent-kaurene synthase. A cell-free system of the fungus Phaeosphaeria sp. L487 converted labeled geranylgeranyl diphosphate to ent-kaurene. A cDNA fragment, which possibly encodes copalyl diphosphate synthase, was isolated by reverse transcription-polymerase chain reaction using degenerate primers based on the consensus motifs of plant enzymes. Translation of a full-length cDNA sequence isolated from the fungal cDNA library revealed an open reading frame for a 106-kDa polypeptide. The deduced amino acid sequence shared 24 and 21% identity with maize copalyl diphosphate synthase and pumpkin ent-kaurene synthase, respectively. A fusion protein produced by expression of the cDNA in Escherichia coli catalyzed the two-step cyclization of geranylgeranyl diphosphate to ent-kaurene. Amo-1618 completely inhibited the copalyl diphosphate synthase activity of the enzyme at 10(-6) M, whereas it did not inhibit the ent-kaurene synthase activity even at 10(-4) M. These results indicate that the fungus has a bifunctional diterpene cyclase that can convert geranylgeranyl diphosphate into ent-kaurene. They may be separate catalytic sites for the two cyclization reactions.

Phenotypes of Caenorhabditis elegans unc-18 and unc-64 gene mutations are similar. While unc-18 is known to be essential for normal synaptic transmission (Hosono, R., Hekimi, S., Kamiya, Y., Sassa, T., Murakami, S., Nishiwaki, S., Miwa, J., Taketo, A., and Kodaira, K.-I. (1992) J. Neurochem. 58, 1517-1525), the function of unc-64 remains unclear. Here we describe the cloning, and the molecular and genetic characterization of the unc-64 gene, especially in relation to unc-18. unc-64 encodes a protein (C. elegans syntaxin) showing sequence and structural similarities to mammalian syntaxin 1A. From unc-64, at least three types of poly(A)+ RNA are transcribed, which encode two types of syntaxin that differ in the deduced transmembrane domain. In gene expression, unc-64 closely resembles unc-18, that is, both are expressed in neural cells, especially in motor neurons and neurons constituting head ganglions. C. elegans syntaxin binds to UNC-18 with high affinity. The unc-64 (e246) mutation producing a mild phenotype causes an Ala-->Val conversion in the conserved COOH-terminal region in mammalian syntaxin 1A or Drosophila syntaxin-1A whose site is included in three types of transcripts. The binding of the mutant C. elegans syntaxin to UNC-18 is greatly reduced, indicating the mutation site contributes to the binding.

Protein kinase C (PKC) of Caenorhabditis elegans was identified by enzymatic activity and [3H]phorbol 12,13-dibutyrate binding after DEAE-Sephacel column chromatography of a crude cytosolic extract. Ca(2+)-dependent activation of nematode PKC was observed in the presence of phosphatidylserine. The enzyme was maximally activated by 1,2-dioleoylglycerol or phorbol 12-myristate 13-acetate in the presence of phosphatidylserine and Ca2+. Hydroxyapatite column chromatography showed only one peak of PKC activity with histone H1 and myelin basic protein as substrates. The enzyme was purified to near homogeneity by sequential chromatography on polylysine-agarose and phosphatidylserine affinity columns. The purified protein showed a molecular mass of 79 kDa on SDS/PAGE. The substrate specificity of the C. elegans enzyme was shown to be different from that of mammalian PKCs. Here we describe some of the properties of the nematode enzyme.

Xylem cell radial growth of 1-year-old seedlings of P. radiata was significantly increased by application of gibberellin A4 (GA4), or of abscisic acid (ABA) plus a smaller, and non-significant, dosage of GA4. In 10 out of 15 additional tests throughout the growing season, ABA tended to increase radial growth, and only once (in mid-summer) did ABA significantly reduce xylem cell radial growth. Seedlings were grown under well watered, warm, long-day conditions. Where GA4 (22 g) or ABA (20 g) plus GA4 (2 g) increased xylem cell radial growth, the metabolism of [3H]GA4 was followed. Although not characterized from P. radiata, GA4 is native to P. attenuata, a species which will hybridize with P. radiata. At low levels (2 g) of carrier GA4, [3H]GA4 metabolism was rapid, and ABA (20 g) enhanced metabolism, especially into acidic metabolites. One metabolite was definitively characterized as GA34 and other metabolites eluted in regions of a SiO2 partition column coincidental with GA1, GA2, GAB and GA4 glucosyl ester. At high levels (22 g) of carrier GA4, [3H]GA4 metabolism was dampened, and ABA reduced the metabolism of [3H]GA4 even further. Enhanced metabolism of [3H]GA4 in P. radiata by ABA is analogous to an earlier report [Nadeau et al. (1972). Planta 107, 315-24] where ABA enhanced the metabolism of [3H]GA1 in barley aleurone layers while reducing their GA1-stimulated amylase production. However, in P. radiata, ABA increased xylem cell radial growth while enhancing [3H]GA4 metabolism. If the metabolites of GA4 that were enhanced by ABA were highly active, a similar effect might also be exerted on endogenous GAs, thus explaining the modest tendency of ABA to promote radial growth through much of the year.

Ethylene-propylene rubber is particularly excellent in heat resistance, weather resistance and ozone resistance. It is widely used in automotive rubber parts, rubber sheets, etc. This report describes the research on electron beam crosslinking of ethylene-propylene rubber and its application in rubber products.

We investigated how partial fluorination of a phenyl group affects the CH stretching vibrational absorptions of polystyrene-based optical fiber materials. We confirmed that the fluorination can decrease not only number density of CH bonds but also the CH absorption per bond. Moreover, by quantitatively correlating the CH stretching vibration absorption spectra with anharmonic vibrational potential, we suggested that the decrease efficiency of attenuation depends on the substituted position in the benzene ring and the chemical structure itself. [ABSTRACT FROM AUTHOR]