Cytochrome P450/NADPH-dependent formation of trans epoxides from trans-arachidonic acids

Trans-arachidonic acids (trans-AA) are products of cis-trans isomerization of arachidonic acid by nitrogen dioxide radical (NO(2)), and occur in vivo, but their metabolism is unknown. We found that hepatic microsomes oxidized trans-AA via cytochrome P450/NADPH system to epoxides, which were hydrolyzed by epoxide hydrolase to diols (DiHETEs). 14,15-trans-AA produced one erythro diol and three threo diols each having one trans double bond.     

Guanaconetins, new antitumoral acetogenins, mitochondrial complex I and tumor cell growth inhibitors

The antitumoral activity of a series of acetylated bis-tetrahydrofuranic acetogenins with a threo/trans/threo/trans/erythro relative configuration was characterized by four new natural and two semisynthetic, 15,24,30-trioxygenated acetogenins that were found to inhibit mitochondrial complex I enzyme as well as growth of several tumor cell lines. Placement of acetyl groups along the alkyl chain modulated the potency of the bis-tetrahydrofuranic acetogenins and could be important for future utilization of these compounds as chemotherapeutic agents.     

Structure–activity relationships of diverse annonaceous acetogenins against human tumor cells

Twelve annonaceous acetogenins (ACGs) with different stereochemical structures and configuration were selected to test for their inhibitions on the growth of Hela, SMMC-7541, SGC-7901, MCF-7 and A-5408 tumor cell lines using MTT method. This was the first to simultaneously investigate effects of structural factors of stereochemical structures and configuration on cytotoxicities with structure–activity relationship. The present study showed that cytotoxic selectivities of ACGs with threo/trans/threo/trans/erythro stereochemical arrangement were gently more active than those with threo/trans/threo/trans/threo stereochemical arrangement, and ACGs with cis THF ring partly produced notable cytotoxic selectivities. Furthermore, ACGs with S configuration at C-24 exhibited gently more cytotoxic selectivities potency than those with R configuration at C-24.     

Ratio of erythro and threo forms of beta-O-4 structures in tension wood lignin

The ratio of erythro and threo forms of beta-O-4 structures in tension wood lignin was investigated by ozonation analysis of wood meal taken from various positions in the stem of yellow poplar (Liriodendron tulipifera). The proportion of the erythro form was higher in tension wood than in opposite wood, and the methoxyl group content showed a similar trend. The proportion of the erythro form and the methoxyl group content in the 7 positions in the stem lignin was correlated (correlation coefficient R=0.98), suggesting that the type of aromatic ring, syringyl or guaiacyl, is one of the factors which stereochemically controls the ratio of erythro and threo forms of beta-O-4 structures during lignin formation.     

Synthesis of erythro and threo furanoid glycals from 1- and 2-phenylselenenyl-carbohydrate derivatives.

Differently protected erythro and threo furanoid glycals were synthesized by selenoxide elimination when phenyl 1-selenoglycosides were treated in oxidizing conditions (tBuOOH, Ti(O(i)Pr)(4), Et(2)(i)PrN). The phenyl 1-selenoglycosides were obtained from methyl 2-deoxy-D-erythro-pentofuranoside by protection of the primary hydroxyl or both hydroxyls and further reaction with PhSeH in the presence of BF(3).Et(2)O. Erythro and threo furanoid glycals were also prepared by treating 2-deoxy-2-phenylselenenyl-1,4-anhydrocyclitols under similar conditions. The 2-deoxy-2-phenylselenenyl-1,4-anhydrocyclitols were obtained from 4-pentene-1,2,3-triols by a 5-endo selenium electrophilic induced cyclization.     

Biosynthesis of trans,trans,trans-geranylgeranyl diphosphate by the cytosolic fraction from rat tissues.

The cytosolic fractions from rat liver, brain, kidney, spleen and testis demonstrate the capacity to synthesize two products from [3H]isopentenyl diphosphate, i.e., farnesyl diphosphate and geranylgeranyl diphosphate. The highest rate of geranylgeranyl diphosphate synthesis was found in brain, testis and spleen, accounting for up to 30% of the total incorporation of radioactivity under optimal conditions. In all tissues examined the geranylgeranyl diphosphate formed was identified as the trans,trans,trans-isomer. The ratio of geranylgeranyl diphosphate to farnesyl diphosphate produced was specific for the tissue investigated and could be altered by the addition of divalent cations. The results in this study demonstrate the presence of a specific trans,trans,trans-geranylgeranyl diphosphate synthetase showing high affinity for farnesyl diphosphate.     

Genotoxic and cell-transforming properties of (trans,trans)-muconaldehyde

(trans,trans)-Muconaldehyde, a putative metabolite of benzene, should be expected to have mutagenic properties by virtue of its twin alpha,beta-unsaturated carbonylic function. It displayed definitely mutagenic properties in S. typhimurium TA100 without metabolic activation and with a 5-fold concentration of tester organisms in the preincubation assay and induced SOS response in E. coli. It induced micronucleus formation and morphological transformation in a dose-dependent manner in Syrian hamster embryofibroblasts. No DNA single-strand breaks or interstrand cross-links could be detected using the alkaline elution technique; however, strand-break generation by subsequent gamma-irradiation was found to be increased.     

Asitrilobins C and D: two new cytotoxic mono-tetrahydrofuran annonaceous acetogenins from Asimina triloba seeds

Two new bioactive mono-tetrahydrofuran (THF) gamma-lactone acetogenins, asitrilobins C (1) and D (2), were isolated from the seeds of Asimina triloba (Annonaceae) by directing the fractionation with brine shrimp lethality. Compounds 1 and 2 have a relative stereochemical relationship of threo/trans/threo across the mono-THF ring with its two flanking hydroxyls. Their structures were established on the basis of chemical and spectral evidence. Compounds 1 and 2 showed selective cytotoxicity comparable with adriamycin for the breast carcinoma (MCF-7) and the colon adenocarcinoma (HT-29) cell lines.     

Hydrolysis and isomerization of trans,trans-farnesyl diphosphate by Andrographis tissue-culture enzymes.

Incubation of (3R,5S)-[5-3H1]mevalonate + (3RS)-[2-14C]mevalonate with Andrographis cell-free extract leads to trans,trans-farnesol and cis,trans-farnesol which both totally retain tritium. 2. This conflicts with our previous results which predict one third tritium loss in the cis,trans-farnesol. Inversion at C-1 during hydrolysis of trans,trans-farnesyl diphosphate to trans,trans-farnesol could explain this anomaly. 3. (1s)-trans,trans-[1-3H1]Farnesyl diphosphate and phosphate and (1R)-trans,trans-[1-3H1]-farnesyl diphosphate and phosphate, all prepared chemically, were hydrolysed with Andrographis phosphatase, and alkaline phosphatase and hydrogenolysed with lithium aluminium hydride and the product alcohols exchanged with liver alcohol hydrogenase. 4. Both Andrographis phosphatase and alkaline phosphatase hydrolyse trans,trans-farnesyl diphosphate and trans,trans-farnesyl phosphate with retention. 5. Hydrolysis of trans,trans-[1-18O]farnesyl diphosphate in H2(18O with both phosphatases supports P-O fission. 6. The C-1 configuration in (1S)-TRANS,TRANS-[1-3H1]farnesyl diphosphate and phosphate and (1R)-trans,trans-[1-3H1]farnesyl diphosphate and phosphate is progressively racemised in 0.01 M NH4OH/MeOH (1/9) AT - 20 degrees C.     

Biosynthesis of trans,trans- and cis,trans-farnesols by soluble enzymes from tissue cultures of Andrographis paniculata

A cell-free system obtained from tissue cultures of Andrographis paniculata produces 2-trans,6-trans-farnesol (trans,trans-farnesol) and 2-cis,6-trans-farnesol (cis,trans-farnesol) (5:1), incorporating 10% of the radioactivity from 3R-[2-(14)C]mevalonate. There is total loss of (3)H from 3RS-[2-(14)C,(4S)-4-(3)H(1)]mevalonate and total retention from the (4R) isomer in both the trans,trans-farnesol and cis,trans-farnesol formed. When 3RS-[2-(14)C,5-(3)H(2)]mevalonate is used as substrate, there is total retention of (3)H in the trans,trans-farnesol, but loss of one-sixth of the (3)H in the cis,trans-farnesol. With (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-trans,trans -farnesol and (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-cis, trans-farnesol as substrates, the label is lost from the (1R)-cis,trans and (1S)-trans,trans isomers but retained in the (1R)-trans,trans and (1S)-cis,trans isomers; this shows that the pro-1S hydrogen is exchanged in the conversion of trans,trans-farnesol into cis,trans-farnesol and the pro-1R hydrogen in the conversion of cis,trans-farnesol into trans,trans-farnesol. (1R)-[1-(3)H(1)]-trans,trans-Farnesol and (1R)-[1-(3)H(1)]-cis,trans-farnesol have been synthesized by asymmetric chemical synthesis and exchanged with liver alcohol dehydrogenase. Both the trans- and the cis-alcohol exchange the pro-1R hydrogen atom.     

Tubules of the trans Golgi apparatus visualized by immunoelectron microscopy

 Tubules constitute an integral part of the Golgi apparatus and have been shown to form a complex and dynamic network at its trans side. We have studied in detail structural features of the trans Golgi network and its relationship with the cisternal stack in thin sections of Lowicryl K4M embedded human absorptive enterocytes by immunolectron microscopy. Immunoreactive sites for α1,3 N-acetylgalactosaminyltransferase and blood group A substance were detectable troughout the cisternal stack and the entire trans Golgi network. Furthermore, the entire trans Golgi network was reactive for CMPase activity. Evidence for two kinds of tubules at the trans side of the Golgi apparatus was found: tubules that laterally connect adjacent and distant cisternal stacks, and others extending from central and lateral portions of trans cisternae to form the complex and extensive trans Golgi network. Trans cisternae showed often the peeling-off phenomenon and were continuous with the trans Golgi network. Both, trans cisternae and tubules of the trans Golgi network exhibited regionally buds and vesicles with a lace-like, non clathrin coat, previously reported by others in NRK cells, which contained glycoproteins with terminal N-acetylgalactosamine residues. These buds and vesicle are therefore involved in constitutive exocytosis. Accepted: 12 January 1998     

A note on urine trans, trans muconic acid level among a sample of Thai police: implication for an occupational health issue

Benzene exposure is of particular concern because recent research indicating that benzene exposure can result in chronic toxicity, therefore, monitoring for benzene exposure among at-risk workers is recommended. In exposure- and risk-evaluation, the monitoring of benzene by peripheral biomarker has several advantages over technical assessment of exposure. For this purpose, the urine trans, trans muconic acid (ttMA)e level is accepted as a useful monitoring tool for early diagnosis of dangerous exposure. Apart from the industrial workers, there are other occupations with high risk for benzene exposure. In this study, we study another at-risk occupation, the police. Thirty-nine urine samples were obtained from 39 Thai police working close to traffic in an urban area. All 39 samples were analyzed for ttMA level and compared to 10 other controls. The average urine ttMA level for the control and exposed group were < 0.05 mg/gCr and was 0.79 +/- 1.43 mg/gCr, respectively. Significant higher urine ttMA acid level among the police were observed (p <.05). Working in the air pollution in the urban area can be health hazard for the police. Exposure to the benzene from automobile exhaust can be an important occupational problem for these police.     

Novel proline hydroxylase activities in the pneumocandin-producing fungus Glarea lozoyensis responsible for the formation of trans 3- and trans 4-hydroxyproline

Novel proline 3-hydroxylase (P3H) and proline 4-hydroxylase (P4H) activities that convert free l-proline to both trans 3- and trans 4-hydroxy- l-proline were detected in protein extracts of the anamorphic fungus Glarea lozoyensis. The enzymatic conversion of l-proline to trans 3- and trans 4-hydroxy- l-proline was strictly dependent on alpha-ketoglutarate, ascorbate, and dithiothreitol. Ferrous iron was required for optimal P3H and P4H activity. These substrate and co-factor requirements indicate these enzyme activities belong to the class of 2-oxoglutarate-dependent dioxygenases. Both P3H and P4H were inhibited by zinc and other trace metals. The addition of proline to the fermentation medium resulted in an increase in the specific activity of P4H and a decrease in the synthesis of pneumocandin C(0). Additionally, the synthesis of trans 3- and trans 4-hydroxy- l-proline in vivo was affected differently by the proline concentration in the medium. This result suggested that two enzymes may be responsible for the regio- and stereospecific hydroxylation of l-proline.     

Sequence elements in cis influence heterochromatic silencing in trans

The brown(Dominant) (bw(D)) allele contains a large insertion of heterochromatin, which causes the locus to aberrantly associate with heterochromatin in interphase nuclei and silences the wild-type allele in heterozygotes. Transgenes placed near the bw(+) locus, in trans to bw(D), can also be silenced. The strength of silencing (called trans inactivation) varies with the regulatory sequences of the transgene and its distance away from the bw(D) insertion site in trans. In this study, we examine endogenous sequences in cis that influence susceptibility of a reporter gene to trans inactivation. Flanking deletions were induced in two parental lines containing P-element transgenes showing trans inactivation of the mini-white reporter. These new lines, which have mini-white under the influence of different endogenous sequence elements, now show varied ability to be silenced by bw(D). Determination of the deleted regions and the levels of mini-white expression and trans inactivation has allowed us to explore the correlation between cis sequence elements and susceptibility to trans inactivation and to identify a 301-bp sequence that acts as an enhancer of trans inactivation. Intriguingly, this region encompasses the upstream regions of two divergently transcribed genes and contains a sequence motif that may bind BEAF, a protein involved in delimiting chromatin boundaries.     

Rejection of mouse cardiac allografts by costimulation in trans

The activation of T cells by B7 costimulation in trans has been demonstrated in vitro, but the in vivo relevance is unknown. To study costimulation in trans of CD4(+) T cells in vivo, we performed cardiac transplants from B7-1/B7-2-deficient mice to recipients that do not express MHC class II molecules on peripheral APCs, but do have functional CD4(+) T cells (II(-)/4(+) mice). This model restricts the B7-dependent activation of CD4(+) T cells to costimulation in trans and excludes any contribution from indirect Ag presentation. We find that II(-)/4(+) recipients reject B7-deficient grafts as rapidly as wild-type grafts, suggesting that costimulation in trans can mediate rejection as potently as costimulation in cis. Treatment of II(-)/4(+) recipients of B7-deficient grafts with depleting Abs to CD4 or CD8 demonstrates that indirect Ag presentation to CD8(+) cells does not significantly contribute to rejection. This is the first demonstration that costimulation in trans can mediate an immune response in vivo and has important therapeutic implications.     

The retina is more susceptible than the brain and the liver to the incorporation of trans isomers of DHA in rats consuming trans isomers of alpha-linolenic acid

Trans polyunsaturated fatty acids are formed during heat treatments of vegetable oils from polyunsaturated fatty acids containing cis double bonds. After dietary intake, they are distributed in the body and are incorporated into nervous tissues including the retina. Since nervous tissues are known to be rich in n-3 fatty acids such as docosahexaenoic acid (DHA), we studied the ability of the retina and the brain to incorporate trans isomers of DHA formed in vivo from the dietary precursor trans alpha-linolenic acid. Wistar rats were fed with trans isomers of alpha-linolenic acid for 21 months. A linear incorporation of trans DHA and a decrease in cis DHA was observed in the retina, whereas no major changes were observed in the brain. In parallel to the modifications in retinal cis and trans DHA levels, the retinal functionality evaluated by the electroretinogram showed defects in animals that consumed trans alpha-linolenic acid. These results suggest that the mechanisms leading to the incorporation of cis and trans fatty acids are quite different in the retina when compared to the brain and the liver, the retina being more susceptible to changes in the dietary lipid contribution.     

trans rescue of a mutant poliovirus RNA polymerase function.

A series of three-nucleotide insertions was engineered into the P2 and P3 coding regions of the T7 expression plasmid pT7(tau)-PV1, which encodes a full-length copy of poliovirus type 1 (Mahoney) cDNA. When RNA derived in vitro from these mutated templates was used to transfect HeLa cells, viable virus mutants were recovered. One mutant, Sel-3D-18, which contained a single amino acid insertion in the 3Dpol coding region, was temperature sensitive for growth at 39 degrees C and showed defects in both RNA synthesis and P1 protein processing at the nonpermissive temperature. The RNA replication defect in Se1-3D-18 was identified at the level of RNA chain elongation. A highly specific and sensitive method was developed for analyzing the ability of mutant RNA templates to replicate in the presence or absence of helper functions provided in trans. This approach was used to demonstrate that RNA synthesis in Se1-3D-18 can be rescued by helper functions provided in trans.     

Efficient, pH-dependent RNA ligation by the VS ribozyme in trans

The VS ribozyme acts as a very efficient ligase in trans when the 5' cleavage product is prevented from dissociation by an extended helix Ia in the substrate. Provided that the length of this helix is >or=10 bp, the substrate becomes approximately 80% ligated by the ribozyme acting in trans. Most of the nucleotides that have been shown to be important for cleavage are similarly important for ligation, including the critical A756 of the active site. The exception to this is C755. The variant ribozyme C755A has almost normal cleavage activity, whereas the rate of ligation is reduced 70-fold. It is therefore likely that this nucleotide plays a specific role in the organization of the termini of the ligation substrates. We have found that the rate of the trans ligation reaction depends on pH, corresponding to the protonation/deprotonation of a group with a pK(A) of 5.6. A model is suggested whereby the approach to equilibrium is catalyzed by the ribozyme catalyzing the ligation reaction in its deprotonated state (rate 1.05 min(-1)) and the cleavage reaction in its protonated state (rate 0.18 min(-1)). A756 is a candidate for the nucleobase undergoing protonation/deprotonation.