d-p-Hydroxyphenylglycine production from dl-5-p-hydroxyphenylhydantoin by Agrobacterium sp.

Summary A bacterium that stereospecifically produces D-p-hydroxyphenylglycine (D-PHPG) from DL-5-p-hydroxyphenylhydantoin (DL-5-PHPH) was isolated from soil and identified as Agrobacterium sp. IP-I 671. The hydantoinase and the N-carbamyl-amino acid amido-hydrolase involved in this biotransformation process were both strictly D-stereospecific. Their biosynthesis was found to be inducible by addition of 2-thiouracil to the cultivation media, or to a lesser extent by uracil. The amidohydrolase activity of Agrobacterium sp. was strongly inhibited by ammonium ions co-produced with D-PHPG, whereas the hydantoinase activity under the same conditions was unaffected. Optimum temperature and pH were respectively 55° C and 10 for the partially purified hydantoinase, 45° and 6.75 when resting cells were used. Biotransformation under these slightly acidic conditions allowed to complete conversion of 30 g/1 DL-5-PHPH into 25 g/l of D-PHPG (molar yield 96%) and involved enzymatic racemization of DL-5-PHPH. Offprint requests to: S. Runser     

Enzymatic production of l-tryptophan from dl-5-indolylmethylhydantoin by Flavobacterium sp.

An enzymatic production of l-tryptophan from dl-5-indolylmethylhydantoin by the action of hydantoinase and carbamoylase has been investigated. A strain identified as (Flavobacterium) sp. I-3 isolated from soil was found to form l-tryptophan from dl-5-indolylmethylhydantoin. Cultural conditions for the formation of the l-tryptophan-forming activity were investigated, and the highest activity of 0.81 μmol min⁻¹of l-tryptophan formed per 1 ml of culture broth (hydantoinase, 3.6 μmol min⁻¹of N-carbamoyl-l-tryptophan formed per 1 ml of culture broth; carbamoylase, 0.92 μmol min⁻¹of l-tryptophan formed per 1 ml of culture broth) was obtained. These activities were found to be inducible and intracellular. Optimization of the parameters of the conversion reaction resulted in accumulation of 50 mg of l-tryptophan per 1 ml of cultural broth per day. The conversion yield from dl-5-indolylmethylhydantoin was about 100%. Accumulated l-tryptophan was readily isolated in pure form by ordinary procedures.     

Synthesis of dl-4-Amino-2-hydroxybutyric Acid

Male Wistar rats were given purified diets containing safflower (SAF), perilla (PER), or palm (PAL) oils with or without 1% tea polyphenols (TP) for 3 weeks, and chemical mediator releasing activity from rat peritoneal exudate cells (PEC) was measured. Histamine releasing activity was not influenced by TP, while histamine release and intracellular histamine content were significantly increased in the PAL-fed group. On the contrary, leukotriene B₄ (LTB₄) release was significantly lower in rats fed PER than in those fed SAF and PAL, and TP significantly decreased the release in all fat groups. TP also significantly inhibited the release of LTB₅, which was generated only in rats fed PER. TP significantly decreased the proportion of arachidonic acid (AA) in PEC in the SAF-fed group and that of eicosapentaenoic acid (EPA), the precursor of LTB₅ in the PER-fed group, but did not influence that of AA in the PAL- and PER-fed group. These results suggest that ingestion of TP improves type I allergic symptom through the inhibition of LT release though the inhibition by TP could not be totally explained by the reduction of substrate fatty acid.     

Synthesis of methyl dl-(1,3/2,4,5)- and dl-(1,3,4/2,5)-2,3,4,5-tetrahydroxycyclohexane-1-carboxylates

Two new diastereoisomers of the pseudo-hexuronate methyl 2,3,4,5-tetrahydroxycyclohexane-1-carboxylate, having (1,3/2,4,5)- (8) and (1,3,4/2,5)-configurations (16), have been synthesised from the readily available bromo-lactone (1) of the endo-adduct of furan and acrylic acid. Treatment of 1 with 20% hydrogen bromide in acetic acid at 80° resulted in regioselective cleavage of the 1,4-anhydro ring to give dl-(1,3,5/2,4)-2,3-diacetoxy-4,5-dibromocyclohexane-1-carboxylic acid (2). Debromination of 2 with zinc dust gave dl-(1,3/2)-2,3-diacetoxycyclohex-4-ene-1-carboxylic acid (5). The methyl ester (6) of 5 was oxidised with osmium tetraoxide and hydrogen peroxide, followed by acetylation, to give the tetra-acetate of 8. Epoxidation of 6 gave two isomeric epoxides, each of which gave 16 on hydrolysis followed by O-deacetylation.     

Interleukin-18.

Interleukin (IL)-18 is a newly discovered cytokine, structurally similar to IL-1, with profound effects on T-cell activation. This short review summarizes the present knowledge on IL-18, to give an insight into the future perspectives for its possible use as vaccine adjuvant. Formerly called interferon (IFN) gamma inducing factor (IGIF), IL-18 is the new name of a novel cytokine that plays an important role in the T-cell-helper type 1 (Th1) response, primarily by its ability to induce IFNgamma production in T cells and natural killer (NK) cells. Mice deficient in IL-18 have suppressed IFNgamma production despite the presence of IL-12 IL-18 is related to the IL-1 family in terms of structure, receptor family, and function. In terms of structure, IL-18 and IL-1beta share primary amino acid sequences of the so-called "signature sequence" motif and are similarly folded as all-beta pleated sheet molecules. Also similar to IL-1beta, IL-18 is synthesized as a biologically inactive precursor molecule lacking a signal peptide which requires cleavage into an active, mature molecule by the intracellular cysteine protease called IL-1beta-converting enzyme (ICE, also called caspase-1). The activity of mature IL-18 is closely related to that of IL-1. IL-18 induces gene expression and synthesis of tumor necrosis factor (TNF), IL-1, Fas ligand, and several chemokines. The activity of IL-18 is via an IL-18 receptor (IL-18R) complex. This IL-18R complex is made up of a binding chain termed IL-18Ralpha, a member of the IL-1 receptor family previously identified as the IL-1 receptor-related protein (IL-1Rrp), and a signaling chain, also a member of the IL-1R family. The IL-18R complex recruits the IL-1R-activating kinase (IRAK) and TNFR-associated factor-6 (TRAF-6) which phosphorylates nuclear factor kappaB (NFkappaB)-inducing kinase (NIK) with subsequent activation of NFkappaB. Thus on the basis of primary structure, three-dimensional structure, receptor family, signal transduction pathways and biological effects, IL-18 appears to be a new member of the IL-1 family. Similar to IL-1, IL-18 participates in both innate and acquired immunity.     

Synthesis of dl-4,5,6-trinor-3,7-inter-m-phenylene-3oxaprostaglandins including one which inhibits platelet aggregation.

The synthesis of dl-4,5,6-trinor-3,7-inter-m-phenylene-3-oxaprostaglandins of the E1 and F1alpha series from 6-endo-(1-heptenyl)-bicyclo[3:1:0]hexan-3-one (III), is described. Preliminary biological screening data for gerbil colon smooth muscle stimulation, rat blood pressure and substrate specificity toward 15-hydroxyprostaglandin dehydrogenase is presented. Platelet function studies, both in vitro and in vivo of dl-4,5,6-trinor-3,7-inter-m-phenylene-3-oxa-PGE1, methyl ester (VIII) are presented.     

Stereospecificity and mechanism of adenosylcobalamin-dependent diol dehydratase. Catalysis and inactivation with meso- and dl-2,3-butanediols as substrates.

A comparative study of the reaction of meso-, d-, and dl-2,3-butanediols with adenosylcobalamin-dependent dioldehydratase was carried out. While the meso isomer is both a substrate and inactivator of holoenzyme, the d and dl compounds act as purely competitive inhibitors, neither undergoing catalysis nor inactivating holoenzyme. Furthermore, d- and dl-2,3-butanediols protect holoenzyme from oxygen inactivation and enzyme-bound cofactor from photolysis, and do not induce detectable cleavage of the carbon-cobalt bond of cofactor. These results show that the stereospecificity of the inactivation reaction is the same as that of catalysis, suggest that hydrogen abstraction from C-1 of substrate may be concerted with cleavage of the carbon-cobalt bond of adenosylcobalamin, and further suggest that formation of a carbon-cobalt bond between coenzyme and substrate is not obligatory for catalysis.     

Synthesis of dl-3-Hydroxydihydro-β-damascone and Dihydro-β-damascone

The Rupe rearrangement of the appropriate acetylenic alcohols afforded dihydro-β-damascone (XII) and its 3-hydroxy derivative (I). The latter is an aroma component of Manila leaf tobacco.     

A circular dichroism study of the binding of CC-1065 to B and Z form poly(dl-5BrdC).poly(dl-5BrdC)

CC-1065, Benzo[1,2-b:4,3-b']dipyrrole-3(2H)-carboxamide, 7-[[1,6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8,8a-tetrahydro-7-methyl-4- oxocyclopropa[c]pyrrolo[3,2-e]indol-2(1H)-yl)carbonyl]benzo [1,2-b:4,3-b']dipyrrol-3(2H)-yl]carbonyl]-1,6-dihydro-4-hydroxy- 5-methoxy-, (7bR,8aS), binds to the B form of poly(dl-5BrdC).poly(dl-5BrdC) to yield a reversibly bound species whose stability with respect to an irreversibly bound species (presumably the inosine N-3 adduct) is much greater than it is for other DNA polymers. Competitive binding experiments with netropsin, show that this reversibly bound species of CC-1065 contains CC-1065 in the minor groove of the double helix. A review of the CC-1065 binding data obtained on other synthetic DNA polymers suggests that the widely different rates of species conversion shown by these polymers may result from small differences in DNA secondary structure rather than from different alkylating abilities of the adenine or inosine N-3 active site. CC-1065 converts the Z-form of poly(dl-5BrdC).poly(dl-5BrdC) in 3.5 M sodium chloride to the B form and does not bind to the Z form in this solvent system. CC-1065 bound to the B form polymer inhibits the formation of the Z form if the helix is saturated with CC-1065. Regions of the polymer without bound CC-1065 can convert to the Z form with added salt, producing a situation where the polymer contains both the B and Z conformations. In 4.0 M sodium chloride, where the Z conformation is also predominate, the addition of CC-1065 causes chiral aggregates to form, and CC-1065 binds to the aggregates. The addition of dimethylformamide in the absence of CC-1065 or a simple dilution of the 4.0 M sodium chloride polymer solution with water also causes aggregation, indicating that the Z form of this polymer in 4.0 M sodium chloride is unstable with respect to an aggregated form.     

Incorporation of dl-[2-14C]ornithine and dl-[5-14C]arginine in milk constituents by the isolated lactating sheep udder

1. Lactating mammary glands of sheep were perfused for several hours in the presence of dl-[2-(14)C]ornithine or dl-[5-(14)C]arginine and received adequate quantities of acetate, glucose and amino acids. 2. In the [(14)C]ornithine experiment 1.4% of the casein and 1% of the expired carbon dioxide came from added ornithine; 96% of the total radioactivity in casein was recovered in proline; 13% of the proline of casein originated from plasma ornithine. 3. In this experiment the results of chemical degradation of proline of casein as well as relative specific activities in the isolated products are consistent with the view that ornithine is metabolized, by way of glutamic gamma-semialdehyde, to proline or glutamic acid. 4. In the [(14)C]arginine experiments 3% of the casein and 1% of the expired carbon dioxide came from arginine; 84% of the arginine and 9% of the proline of casein originated from plasma arginine. 5. In these experiments the relative specific activities of arginine, ornithine and proline in plasma are in agreement with the view that arginine is metabolized by way of ornithine to proline. The conversion of arginine into ornithine is probably catalysed by arginase, so that arginase in mammary tissue may be involved in the process of milk synthesis.