Variation of monoterpenoids inartemisia feddei andArtemisia scoparia

The composition and concentration of monoterpenoids in the leaves and stems ofArtemisia feddei andArtemisia scoparia were determined, and seasonal variation in the monoterpenoids ofArtemisia species were investigated. The two species possessed different compositions and concentrations of monoterpenoids. The total amount of monoterpenoid inA. scoparia was always higher than that of A.feddei, and the monoterpenoid yields of leaves were higher than stem yields in both species as compounds formed. The major constituents of A.scoparia were 25 while A.feddei consisted of 26 compounds. Sixteen common monoterpenoid compounds were found in both plants. Large differences in the relative amounts of the monoterpenoids were found between species and seasons. Extremely large differences in the relative amounts of naphtalene, sabinene, β-pinene, cyclohexene, and octatrine were found in the leaf monoterpenoids of the two species. The largest differences in relative amounts of stem monoterpenoids were in s abinene and β-pinene levels.     

Monoterpenoid indole alkaloids from Palicourea crocea

A new monoterpenoid indole alkaloid N-oxide, 3,4-dihydro-1-(1-β-d-glucopyranosyloxy-1,4a,5,7a-tetrahydro-4-methoxycarbonylcyclopenta[c]pyran-7-yl)-β-carboline-N²-oxide (3) and two known monoterpenoid indole alkaloids, croceaine A (1) and psychollatine (2), were isolated from Palicourea crocea (Rubiaceae) from Trinidad. The structures of 1–3 were determined on the basis of spectral and other physical data. Compounds 1 and 2 were previously isolated from plants of different genera viz. Palicourea crocea and Psychotria umbellata, respectively, both collected in Brazil. The results support the proposal that the genus Palicourea and the subgenus Heteropsychotria should be combined into a single genus.     

Pleurotus species convert monoterpenes to furanoterpenoids through 1,4-endoperoxides

Enzymatic synthesis of furanoterpenoids from β-myrcene and related monoterpenes was observed using a solubilised enzyme fraction of mycelium lyophilisates of several Pleurotus species. The initial enzymatic step, the incorporation of molecular oxygen into the conjugated 1,3-diene moiety, was similar to a 2 + 4 cycloaddition of 1,3-dienes with dienophilic ¹O₂, and was followed by a non-catalysed degradation sequence leading to the furans. The cyclic peroxides 3,6-dihydro-4-(2-(3,3-dimethyloxiran-2-yl)ethyl)-1,2-dioxine and 5-(3,6-dihydro-1,2-dioxin-4-yl)-2-methylpentan-2-ol were identified as key intermediates. Biotransformation of β-myrcene in ¹⁸O-labelled HEPES-buffer did not yield a detectable label in perillene, so a water addition to 3,10-epoxy-β-myrcenes as an alternative was ruled out. The pathway suggested presents a substantiated biogenetic scheme for the formation of monoterpenoid furans and opens biotechnological access to valuable natural flavour compounds, such as perillene and rosefurane. Only one metabolite, identified as the new natural compound 6-methyl-2-methylene-hept-5-enal, carried the ¹⁸O-label. The enzymatic formation of this compound through a 1,2-endoperoxide (3-(5-methyl-1-methylene-hex-4-enyl)-[1,2]-dioxetane) is suggested. The label may simply result from a chemical oxygen exchange between the carbonyl group and the ¹⁸O-labelled water.     

Biosynthesis of triterpenoids from amino acids in Pisum sativum. The distribution of the radioactivity in squalene biosynthesized from radioisotopically labelled l-leucine and l-valine

Radioisotopically labelled l-leucine and l-valine were fed to Pisum sativum and incorporated into squalene and β-amyrin. Chemical degradation of the radioactive squalene revealed an equal distribution of the radioactivity in the isopentenyl pyrophosphate(IPP)-derived and the 3,3-dimethylallyl pyrophosphate(DMAPP)-derived moieties of the squalene molecule, unlike the unbalanced distribution in favour of the DMAPP-derived moiety of a monoterpenoid molecule biosynthesized from these amino acids by higher plants.     

γ-N-Methylasparagine in phycobiliproteins from the cyanobacteria Mastigocladus laminosus and Calothrix

Reinvestigation of the amino acid sequences of all phycobiliproteins from Mastigocladus laminosus showed that there is a post-translationally modified asparagine residue at position 72 of the phycobiliprotein subunits β^PC, β^AP and β^16.2. This residue was identified as γ-N-methylasparagine and it was also found in β^PE of Calothrix. This study also revealed some differences in the amino acid sequences β^AP and β^PC compared to the published data.     

Monoterpenoid glycoside derivatives from Melaleuca alternifolia

The phytochemical investigation of leaves of Melaleuca alternifolia (Maiden & Betche) Cheel led to the isolation of a new monoterpenoid glycoside derivative melaleuoside (1), together with four known monoterpenoid glycoside derivatives (2–5). Structure elucidation of these compounds was performed based on NMR spectral data and MS. Compounds 2–5 were isolated from the genus of Melaleuca for the first time. Furthermore, we also discussed the chemotaxonomic significance of the isolates in this thesis.     

Six guaianolides from Stylotrichium rotundifolium

The investigation of Stylotrichium rotundifolium afforded the epoxide of β-sesquiphellandrene, a new derivative of geranylnerol and six guaianolides not isolated previously. Furthermore, two germacranolides also present in a Lasiolaena species and several known compounds were isolated. The structures were elucidated by detailed ¹H NMR investigations. The chemotaxonomic situation is discussed briefly.     

An active site-directed, irreversible inactivation of yeast hexokinase by (R,S)2,3-epoxypropyl beta-D-glucopyranoside

(1) Only (R,S)2′,3′-epoxypropyl β-d-glucopyranoside of the complete series of mono (R,S)2′.3′-epoxypropyl ethers and glycosides of d-glucopyranose significantly inactivated yeast hexokinase.(2) (R,S)2′,3′-Epoxypropyl β-d-glucopyranoside inactivates yeast hexokinase in the absence of MgATP^2?, The rate of inactivation is unaffected by MgATP^2?.(3) The rate of inactivation of hexokinase with (R,S)2′,3′-epoxypropyl β-d-ilucopyranoside was much greater when hexokinase was present in a monomeric form than when it was present in a dimeric form.(4) (R,S)2′,3′-Epoxypropyl β-d-glucopyranoside has a high K_t (0.38 M) and at a saturating concentrarion, the first order rate constant for the inactivation of monomeric hexokinase is 8.3 · 10^?4 sec.(5) d-Glucose protects against this inactivation and this was used to derive a dissocistion constant of 0.21 mM for d-glucose in the absence of MgATP^2?.(6) The alkylation of yeast hexokinase by (R,S)2′,3′-epoxypropyl β-d-gluco-pyranoside was not specific to the active site. When the concentration of (R,S)2′,3′-epoxypropyl β-d-glucopyranoside was 50 mM two thiol groups outside the active site were also alkylated.(7) The reaction between 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and yeast hexokinase was examined in detail. Two thiol groups per monomer (mol. wt. 50000) reacted with a second order rate constant of 27 1 mole^?1 sec^?1. A third thiol group reacted more slowly with a second-order rate constant of 1.6 1 mole^?1 sec^?1 and a fourth thiol group reacted very slowly with inactivation of the enzyme. Tue second-order rate constant in this case was 0.1 1 mole^?1 sec^?1.     

Trypanosoma rangeli: An alkaline ecto-phosphatase activity is involved with survival and growth of the parasite

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca²⁺ present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. β-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (P_i). Levamisole at the IC₅₀ spared significantly parasite growth when β-GP was the sole source of P_i and stopped it in the absence of β-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.     

Synthesis of β-hydroxy fatty acids and β-amino fatty acids by the strains of Bacillus subtilis producing iturinic antibiotics

The iturinic antibiotics, which contain long chain β-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing β-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its β-hydroxy fatty acids was n C₁₆, iso C₁₆, iso C₁₅ or anteiso C₁₅. The percentages of each β-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain α-amino acids to the culture medium. These results demonstrate for the first time that iso C₁₄ β-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive β-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [¹⁴C]acetate seems also characterize the different strains producing iturinic antibiotics.     

Some Characteristics of Practical Relevance of the β -Galactosidase from Potential Probiotic Strains ofPropionibacterium acidipropionici

In the present study, factors influencing the synthesis and activity of β-galactosidase of two strains of Propionibacterium acidipropionici with some probiotic properties are described for the first time. The enzyme 6-phospho-β-D-galactosidase of the PEP-PTS system was not detected, suggesting that P. acidipropionici metabolize lactose only by using β-galactosidase. The highest enzymatic activities were obtained from cultures developed in a basal broth medium containing 1.0% sodium lactate or 0.25% lactose. Maximum β-galactosidase activity from cell-free extracts of the strains was obtained at pH 7.0 and 50°C, but a high activity was even detected at 37°C. The enzyme was competitively inhibited by lactose and activated by glucose and sodium lactate. The remaining activities after heating cell-free extracts up to 20 min at 60°C were 70% and 25% of untreated control activities for P. acidipropionici Q4 and CRL 1198, respectively. Cations like Mg²⁺, Mn²⁺, Li⁺, Na⁺, and K⁺ acted as stimulators of the β-galactosidase activity whereas Ca²⁺, Co²⁺, Ni²⁺, Hg²⁺ and Cu²⁺ showed inhibitory effect in different extent. These results suggest that the environmental conditions commonly present in the human's intestine may be adequate for the synthesis and activity of β-galactosidase from these strains of Propionibacterium. The enzyme resist the cooking temperature of Swiss-type cheeses in different extent depending on the strain tested and most of the cations present in milk stimulate the enzymatic activity. Our results suggest that a cheese would be an appropriate vehicle for delivery of β-galactosidase from propionibacteria to the host and efforts to develop a Swiss-type probiotic cheese for lactose intolerant persons should be done.     

Alternative pathways of carotene cyclisation in Phycomyces blakesleeanus

Three carotene substrates, lycopene-[¹⁴C], γ-carotene-[¹⁴C] and neurosporene-[¹⁴C] were each incorporated into β-carotene using a cell extract of the C115 car-42 mad-107(?) mutant of P. blakesleeanus. Neurosporene-[¹4C] was also converted into β-zeacarotene and lycopene. Addition of either unlabelled lycopene or β-zeacarotene with neurosporene-[¹⁴C] to the cell extract caused approximately equal reductions of incorporation into β-carotene, indicating that both cyclisation pathways are of equivalent quantitative importance.     

Biotransformation of monoterpenoids by suspension cultures of Lavandula angustifolia

Suspension cultures of Lavandula angustifolia reduced monoterpenoid aldehydes and structurally related compounds to their corresponding primary alcohols. Acyclic primary monoterpenoid alcohols were further metabolized to unidentified compounds. Cyclic, secondary, tertiary and aromatic alcohols were not metabolized.     

3-Oxo-friedelan-20α-OIC acid from gymnosporia emarginata

3-Oxo-friedelan-20α-oic acid, named as maytenonic (polpunonic) acid, has been isolated along with β-amyrin and sitosterol from Gymnosporia emarginata. ¹H and ¹³C NMR signals have been assigned for the structure. X-ray analysis of the single crystal confirmed that the carboxylic acid is α-oriented at C-20 and the D/E rings of this D:A-friedo-oleanane are in chair-chair conformation. ¹³C NMR data of the present compound also enabled the assignment of the C-20α- and β-methyl carbons in friedelin.     

Alkaloids from Melodinus yunnanensis

Ten monoterpenoid indole alkaloids, namely meloyine, 19S-methoxytubotaiwine N₄-oxide, 16,19-epoxy-Δ¹⁴-vincanol, 14β-hydroxymeloyunine, meloyunine, Δ¹⁴-vincamenine N₄-oxide, 16β,21β-epoxy-vincadine, 14β,15β-20S-quebrachamine, 3-oxo-voaphylline, 2α,7α-dihydroxy-dihydrovoaphylline, and 32 known alkaloids were isolated from leaves and twigs of Melodinus yunnanensis. Their structures were elucidated based on 1- and 2-D NMR, FTIR, UV, and MS spectroscopic data. Meloyine I showed weak cytotoxic activity against four human cancer cell lines: MCF-7 breast cancer, SMMC-7721 hepatocellular carcinoma, HL-60 myeloid leukemia, and A-549 lung cancer.     

Chemical composition and genetic basis for the isopinocamphone chemotype of Mentha citrata hybrids

The dominant I gene found in Mentha citrata causes only 1.0–1.5% isopinocamphone and 0.5–2.5% β-pinene in this species that is characterized by 60–90% linalool/linalyl acetate. If the gene Is is separated from the linked I gene that causes linalool and is substituted into M. aquatica having 66.5% menthofuran and the recessive ii genotype, the resulting hybrids will have 22.3% β-pinene, 29.9% isopinocamphone and a number of related monoterpenoids seldom observed in Mentha oils. Crosses with tester strains show that the Is gene almost entirely prevents the development of monoterpenes with a p-menthane carbon skeleton substituted at the 2- and/or 3-position which commonly occur in Mentha species. A generalized biogenetic scheme is used to hypothesize the position of the action of the Is gene in biosynthesis and its interaction with other genes controlling monoterpenoid formation in Mentha species.     

Observations and models of emissions of volatile terpenoid compounds from needles of ponderosa pine trees growing in situ: control by light,temperature and stomatal conductance

Terpenoid emissions from ponderosa pine (Pinus ponderosa subsp. scopulorum) were measured in Colorado, USA over two growing seasons to evaluate the role of incident light, needle temperature, and stomatal conductance in controlling emissions of 2-methyl-3-buten-2-ol (MBO) and several monoterpenes. MBO was the dominant daylight terpenoid emission, comprising on average 87 % of the total flux, and diurnal variations were largely determined by light and temperature. During daytime, oxygenated monoterpenes (especially linalool) comprised up to 75 % of the total monoterpenoid flux from needles. A significant fraction of monoterpenoid emissions was dependent on light and ¹³CO₂ labeling studies confirmed de novo production. Thus, modeling of monoterpenoid emissions required a hybrid model in which a significant fraction of emissions was dependent on both light and temperature, while the remainder was dependent on temperature alone. Experiments in which stomata were forced to close using abscisic acid demonstrated that MBO and a large fraction of the monoterpene flux, presumably linalool, could be limited at the scale of seconds to minutes by stomatal conductance. Using a previously published model of terpenoid emissions, which explicitly accounts for the physico-chemical properties of emitted compounds, we were able to simulate these observed stomatal effects, whether induced experimentally or arising under naturally fluctuation conditions of temperature and light. This study shows unequivocally that, under naturally occurring field conditions, de novo light-dependent monoterpenes comprise a significant fraction of emissions in ponderosa pine. Differences between the monoterpene composition of ambient air and needle emissions imply a significant non-needle emission source enriched in Δ-3-carene.     

Preparation and conformational analysis of C-glycosyl β- and β/β-peptides

Ten C-glycosyl β²- and β/β²-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble β²-amino acids in which C-glycosylic substituents are attached to the C-2 position of β-amino acids. Conformational analysis of the C-glycosyl β²-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl β/β²-peptides are governed by the configuration at the α-carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl β²-amino acids.     

Sclerostin deficient mice rapidly heal bone defects by activating β-catenin and increasing intramembranous ossification

We investigated the influence of the osteocyte protein, sclerostin, on fracture healing by examining the dynamics and mechanisms of repair of single-cortex, stabilized femoral defects in sclerostin knockout (Sost^−/−; KO) and sclerostin wild-type (Sost^+/+; WT) mice. Fourteen days following generation of bone defects, Sost KO mice had significantly more bone in the healing defect than WT mice. The increase in regenerating bone was due to an increase in the thickness of trabecularized spicules, osteoblast numbers and surfaces within the defect. Enhanced healing of bone defects in Sost KO mice was associated with significantly more activated β-catenin expression than observed in WT mice. The findings were similar to those observed in Axin2^−/− mice, in which β-catenin signaling is known to be enhanced to facilitate bone regeneration. Taken together, these data indicate that enhanced β-catenin signaling is present in Sost^−/− mice that demonstrate accelerated healing of bone defects, suggesting that modulation of β-catenin signaling in bone could be used to promote fracture repair.     

Aryl hydroxylation of isopropyl-3-chlorocarbanilate by soybean plants

Isopropyl-3-chlorocarbanilate-phenyl UL-¹⁴C (CIPC-¹⁴C) is absorbed, translocated and metabolized by soybean plants. Water-soluble metabolites in root and shoot were purified and the root major metabolite characterized. The acetylated aglucones from the β-glucosidase hydrolysis and the esters from the direct acetylation of CIPC-¹⁴C polar metabolites were purified by GLC and analysed by mass spectrometry. The data showed that the phenyl riong of CIPC-¹⁴C was hydroxylated by both root and shoot tissues. Isopropyl-5-chloro-2-hydroxycarbanilate (hydroxy-CIPC) was the predominant aglucone liberated by β-glucosidase from polar metabolites in root and shoot. The o-glucoside of hydroxy-CIPC was shown to be present, by direct acetylation and characterization. In shoot tissue the major metabolites were dechlorinated hydroxy-CIPC and were not hydrolysed by β-glucosidase. These data show that soybean root or shoot tissues hydroxylate the phenyl ring of CIPC-¹⁴C but do not alter either the isopropyl alcohol moiety or the earbamate bond.