Antitumor promoting activities of 3-O-acyl-(-)epigallocatechins

As an exploratory investigation of antitumor promoting compounds, 3-O-acyl-(-)-epigallocatechins possessing a straight-, branched-, phenyl-inserted- or 1,4-phenylene-inserted-acyl chain of varying length from C4 to C18 were synthesized and evaluated their inhibitory effects against the activation of the Epstein-Barr virus early antigen (EBV-EA). It was indicated that the epigallocatechin derivatives having the straight- or branched-acyl chain of C8 to C11 carbon atoms achieve marked effects.     

Synthesis of covalently immobilized phospholipid analogues

Cyclic 1-O-acyl-2-O-alkyl-glycero-3-phosphotriesters and 1-O-acyl-2-O-alkyl-glycero-3-bromoethylphosphate with a free acyl moiety in position 1 of the glycerol backbone were synthesized. These phospholipid intermediates were covalently bound to AH-Sepharose via the carbodiimide method. After immobilization the corresponding phosphatidylethanolamine analogues were obtained by acid hydrolysis of the cyclic phosphotriesters and by direct amination of the bromoethylphosphate. Thus, in a short, stepwise synthesis including minimum use of protecting groups, a variety of immobilized phospholipid analogues are available as affinity adsorbents for the purification of enzymes related to phospholipid metabolism.     

The chemical synthesis of metabolically stabilized 2-OMe-LPA analogues and preliminary studies of their inhibitory activity toward autotaxin

The chemical synthesis of five new metabolically stabilized 2-OMe-LPA analogues (1a-e) possessing different fatty acid residues has been performed by phosphorylation of corresponding 1-O-acyl-2-OMe-glycerols which were prepared by multistep process from racemic glycidol. The now analogues were subjected to biological characterization as autotaxin inhibitors using the FRET-based, synthetic ATX substrate FS-3. Among tested compounds 1-O-oleoyl-2-OMe-LPA (1e) appeared to be the most potent, showing ATX inhibitory activity similar to that of unmodified 1-O-oleoyl-LPA. Parallel testing showed, that similar trend was also observed for corresponding 1-O-acyl-2-OMe-phosphorothioates (2a-e, synthesized as described by us previously). 1-O-oleoyl-2-OMe-LPA (1e) was found to be resistant toward alkaline phosphatase as opposed to unmodified 1-O-oleoyl-LPA.     

Phorbol diester enhances calcium ionophore A23187-induced [3H]acetate incorporation into platelet-activating factor in murine macrophages: predominant incorporation into 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine

Pretreatment of macrophages with 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to enhance the release of arachidonic acid from cell phospholipids in response to agonist stimulation. This study describes the ability of TPA to also alter calcium ionophore A23187-induced incorporation of [3H]acetate into platelet activating factor (PAF). Cultured murine peritoneal macrophages were preincubated with [3H]acetate (25 muCi) and TPA (10 ng/ml) for 10 min, and subsequently incubated with 0.1 microM A23187 for 0.5-10 min. Buffer and cells were then extracted and PAF resolved by normal-phase HPLC. Sequential exposure to TPA and A23187 resulted in a greatly enhanced incorporation (11,861 dpm/10(6) cells) of [3H]acetate into PAF compared to TPA alone, which did not significantly influence [3H]acetate incorporation into PAF, and 0.1 microM A23187, which induced minimal incorporation (688 dpm/10(6) cells). Macrophage-produced [3H]PAF was resolved by HPLC, extracted, treated with phospholipase-C, and acetylated to facilitate quantitation of 1-O-alkyl-2-acetyl-GPC (PAF) from 1-O-acyl-2-acetyl-GPC (acylPAF). A23187 alone (1 microM) produced 72% 1-O-acyl-2-[3H]acetyl-GPC, and A23187 (0.1 microM) following TPA pretreatment produced 81% 1-O-acyl-2-[3H]acetyl-GPC. Less than 2% of the radioactivity of acylPAF was in the acyl moiety. These data support a role for protein kinase C in modulating agonist-induced PAF synthesis. The results also suggest that acetyltransferase of murine macrophages does not possess specificity for 1-O-alkyl-2-lyso-GPC, and that availability of specific species of lyso-phospholipid may determine the type of PAF produced.     

Synthesis and antibacterial activity of 3-O-acyl-6-O-carbamoyl erythromycin A derivatives

A series of 3-O-acyl-6-O-carbamoyl erythromycin A derivatives has been synthesized. Several functional groups were identified as the optimal C3-substituents, and the best compounds in this series possess potent in vitro antibacterial activity against erythromycin-susceptible and erythromycin-resistant bacteria.     

Structure determination of the polymorphism of acylgalactosylceramide in rat brain by gas chromatography/mass spectrometry and proton magnetic resonance

This paper describes the structure of acylcerebrosides isolated from rat brains. Three fractions (acylglycosylceramides I, II, III) were resolved according to their decreasing RF values on TLC. GLC analysis of acylglycosylceramides II and III indicates that their ester-linked fatty acids are short and rather unsaturated, while amide-linked fatty acids are longer and hydroxylated. Sugar GLC analysis indicates that acylglycosylceramides II and III contain only galactose. To determine the substitution position of the acyl group on the galactose moiety, the free hydroxyl groups of acylglycosylceramide were protected with dihydropyran, deacylated and subjected to permethylation. The methylated galactoside acetates obtained after hydrolysis and reduction were then analyzed by gas chromatography/mass spectrometry. Acylglycosylceramides II and III turned out to be complex mixtures of 2-O-acyl-, 3-O-acyl-, 4-O-acyl- and 6-O-acylgalactosylceramides. Moreover, the abundance of alpha-methylgalactoside reveals the existence of unsubstituted galactose, suggesting that some ester-linked fatty acids could be esterified to the hydroxyl group of hydroxy fatty acids linked to sphingosine. NMR spectrometry was used to confirm this ester linkage. The key spectral feature of the fatty acid-galactose linkage (4.45 ppm) did move to 4.15 ppm after saponification of acylglycosylceramide II; on the other hand, acylglycosylceramide III contained only the spectral feature 4.15 ppm, corresponding to a high percentage of unsubstituted galactose and consistent with the presence in the molecule of a fatty acid esterified by the omega-OH group of the hydroxy fatty acid (3.95 ppm).     

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.     

Opiate receptor binding properties of morphine-, dihydromorphine-, and codeine 6-O-sulfate ester congeners

A series of 3-O-acyl-6-O-sulfate esters of morphine, dihydromorphine, N-methylmorphinium iodide, codeine, and dihydrocodeine were prepared and evaluated for their ability to bind to mu-, delta-, kappa(1)-, kappa(2)-, and kappa(3)-opiate receptors. Several compounds exhibited good affinity for the mu-opiate receptor. Morphine-3-O-propionyl-6-O-sulfate had four times greater affinity than morphine at the mu-opiate receptor and was the most selective compound at this receptor subtype.     

N,N-dimethylcarbamyl derivatives of oxazepam

Three N,N-dimethylcarbamyl derivatives of oxazepam (1-(N,N-dimethylcarbamyl)oxazepam, 3-O-(N,N-dimethylcarbamyl)oxazepam, and 1,3-O-bis(N,N-dimethylcarbamyl) oxazepam) and a 3-O-acyl-1-(N,N-dimethylcarbamyl)-oxazepam were synthesized from either oxazepam or demoxepam. Enantiomeric pairs of these derivatives and of camazepam were resolved by high-performance liquid chromatography on at least two of three commercially available chiral stationary phase columns employed. Absolute configurations of resolved enantiomers were established by comparing their circular dichroism spectra to those of enantiomeric oxazepams with known absolute stereochemistry. Similar to those of oxazepam, enantiomers of 1-(N,N-dimethylcarbamyl)oxazepam undergo rapid racemization (t1/2 1.9 min at 23 degrees C and 0.9 min at 37 degrees C) in an aqueous solution at pH 7.5. The (R)-enantiomer of rac-3-O-acyl-1-(N,N-dimethylcarbamyl)oxazepam was hydrolyzed approximately 4.6-fold faster than the (S)-enantiomer by esterases in rat liver microsomes, whereas the (S)-enantiomer was hydrolyzed approximately 43-fold faster than the (R)-enantiomer by esterases in rat brain S9 fraction.     

Induction of erythroid differentiation of human K562 cells by 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives

In this paper we report the synthesis of twelve 3-O-acyl-1,2-O-isopropylidene-D-glucofuranose derivatives and the results obtained on their effects in inducing erythroid differentiation of human leukemic K562 cells. The data obtained demonstrate that two of the newly synthetized compounds are able to induce erythroid differentiation of K562 cells. In addition, these same compounds potentiate K562 erythroid differentiation induced by cytosine arabinoside, retinoic acid and mithramycin. Inducers of erythroid differentiation stimulating fetal gamma-globin synthesis could be considered for possible use in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.     

The effect of aldehydogenic and acyl structural analogs of platelet activating factor on the formation of superoxide radicals by human blood leukocytes]

The effects of plasmalogenic and acyl structural analogs of the platelet activation factor (PAF) on the superoxide radical production by human blood leukocytes were studied. It was found that 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine and 1-O-(1'-alkenyl)-2-acetyl-sn-glycero-3-phosphocholine stimulates the production of leukocyte superoxide radicals, their activity being comparable with that of PAF. Stimulation of superoxide radical production strongly depends on the structure of the polar heads of PAF analogs. It is supposed that choline-containing plasmalogenic and acyl PAF analogs may act as specific lipid mediators of the neutrophil function.     

4-1BBL cooperates with B7-1 and B7-2 in converting a B cell lymphoma cell line into a long-lasting antitumor vaccine

A20 is a B cell lymphoma that constitutively expresses the costimulatory molecule B7-2 yet grows readily as a tumor in syngeneic BALB/c mice. We have compared the tumorigenicity of A20 variants expressing either B7-1 (A20/B7-1) or B7-2 (A20/B7-2) with an A20 variant expressing B7-1 and B7-2 with 4-1BBL (A20/4-1BBL), a costimulatory member of the TNF family. Mice injected with tumors expressing the vector backbone (A20/CMV) or B7-1 developed tumors within 25 days of s.c. injection. In contrast, mice injected with A20/4-1BBL were tumor free for the 150-day follow-up period, while 25% of mice injected with A20/B7-2 developed tumors. Tumorigenicity experiments using nude mice indicated the requirement for T cells for variant rejection. Almost all mice that resisted the initial tumor challenge were resistant to further challenge with the parental tumor. Splenocytes from these mice showed high CTL lytic activity against the parental tumor, A20, as well as the syngeneic BALB/c lymphoma K46J, but showed background levels of lytic activity against the congenic SCID thymoma line ST-D2 or the allogeneic EL4 thymoma. In vitro blocking experiments with anti-B7-1 plus anti-B7-2 and/or soluble 4-1BB receptor showed B7-1, B7-2, and 4-1BBL all contributed to the CTL activity. Thus, the data show that neither B7-1 or B7-2 alone can confer full immunogenicity to the A20 lymphoma but that the addition of 4-1BBL results in a tumor that is highly immunogenic and can confer long-lasting protection against challenge with parental tumor in vivo.     

Calcium-ionophore-induced formation of platelet-activating factor and leukotrienes by horse eosinophils: a comparative study

Horse eosinophils preincubated with 3H-labelled acetate and stimulated with the Ca2+ ionophores ionomycin or A23187 form a radioactive compound, which we have shown to be 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine (platelet-activating factor). We could detect no 1-O-acyl-2-[3H]acetyl-glycero-3-phosphocholine in the radioactive fraction. The formation of platelet-activating factor was strongly correlated to the generation of leukotriene C4, the main arachidonate metabolite in horse eosinophils, suggesting that platelet-activating factor and leukotriene C4 have a common precursor pool (1-O-alkyl-2-arachidonyl-glycero-3-phosphocholine) and a common regulation of synthesis. Even though both ionomycin and A23187 are described as Ca2+ ionophores, they have a series of significantly different effects on the eosinophils with respect to formation of platelet-activating factor and leukotriene C4. While A23187 induces an asymptotic maximum of mediator formation at concentrations higher than 20 microM, ionomycin expressed a narrow optimum at 2 microM. The effects of exogenous pH on the release of mediators also differ strongly between the two ionophores: for A23187 the effects are the same for both mediators but when ionomycin is used as stimulant, generation of platelet-activating factor and leukotriene C4 are affected differently.     

Acyl and plasmalogen analogs of platelet activating factor--new lipid cellular bioregulators]

Recent data concerning two structural platelet-activating factor (PAF) analogs-1-O-acyl-2-acetyl-sn-glycero-3-phosphocholine (acyl-PAF) and 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine (vinyl-PAF) identified in some cells and tissues are reviewed. Isolation, identification, biosynthesis, and metabolism of acyl-PAF and vinyl-PAF are considered. The activity of acyl-PAF and vinyl-PAF towards platelets, leukocytes, isolated myocardium, and ileum as well as its in vivo activity are discussed. The influence of acyl-PAF and vinyl-PAF on PAF platelet interaction, Ca2+ mobilization, and platelet adenylate cyclase activity is considered. It is concluded that similar to PAF, acyl-PAF and vinyl-PAF should be regarded as a family of PAF lipid bioregulators.     

Synthesis of (20S)-[7,7,21,21-2H4]-3beta-(tert-butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol,a useful intermediate for the preparation of deuterated isotopomers of sterols

(20S)-[7,7,21,21-2H(4)]-3beta-(tert-Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, an intermediate for the preparation of deuterated isotopomers of sterols to be used as standards for biomedical studies, was prepared by reduction with dichloroaluminum deuteride of ethyl (20S)-3beta-(tert-butyldimethylsilanyloxy)-7-oxo-pregn-5-en-20-carboxylate. Using controlled experimental conditions, it has also been shown that the dichloroaluminum hydride reduction of a 7-keto steroid affords the corresponding 7beta-hydroxy derivative in a highly stereoselective manner.     

Characterization of the segmental duplication LCR7-20 in the human genome

Our previous study described the amplification of a genomic sequence containing exon 9 of CFTR in the human genome. Here we report that this CFTR sequence is part of a large duplicated sequence unit, provisionally named LCR7-20. Through successive screening of two human chromosome 7-specific cosmid libraries to construct a cosmid contig, we assembled two sequenced BAC clones into a single contig containing a prototypic LCR7-20 unit. Subsequent searches of existing human genome sequences identified additional six copies of LCR7-20-like sequences with more than 90% sequence homology. Additional genomic clones containing LCR7-20-like sequences were then isolated from total genomic BAC and PAC libraries. Restriction fragment analysis and limited sequencing data indicated that there could be around 30 copies of LCR7-20-like sequences in the human genome and that the average region of homology could extend over 120 kb. As indicated by fluorescence in situ hybridization analysis, LCR7-20-like sequences are dispersed on different chromosomes, mainly in the centromeric and pericentromeric regions, and some may exist in tandem copies. Our study also indicates that many genomic regions containing LCR7-20's either have been misassembled or are missing in current versions of the human genome sequence.     

-8p12-23 and +20q are predictors of subtypes and metastatic pathways in colorectal cancer: construction of tree models using comparative genomic hybridization data

A substantial body of evidence suggests the genetic heterogeneous pattern and multiple pathways in colorectal cancer initiation and progression. In this study, we construct a branching tree and multiple distance-based tree models to elucidate these genetic patterns and pathways in colorectal cancer by using a data set comprised of 244 cases of comparative genomic hybridization. We identify the six most common gains of chromosomal regions of 7p (37.0%), 7q11-32 (34.8%), 8q (48.3%), 13q (49.1%), 20p (36.1%), and 20q (50.4%), and the nine most common losses of 1p13-36 (30.9%), 4p15 (24.3%), 4q33-34 (24.3%), 8p12-23 (50.9%), 15q13-14 (23.5%), 15q24-25 (24.3%), 17p (34.8%), 18p (36.5%), and 18q (61.7%) in colorectal cancer. We classify colorectal cancer into two distinct groups: one preceding with -8p12-23, and the other with +20q. The sample-based classification tree also demonstrates that colorectal cancer can be classified into multiple subtypes marked by -8p12-23 and +20q. By comparing chromosomal abnormalities between primary and metastatic colorectal cancer, we identify five potential metastatic pathways: (-18q, -18p), (-8p12-23, -4p15, -4q33-34), (+20q, +20p), (+20q, +7p, +7q11-32), and +8q. -8p12-23 and +20q are inferred to be the two marker events of colorectal cancer metastasis. The current oncogenetic tree models may contribute to our understanding towards molecular genetics in colorectal cancer. Particularly, the metastatic pathways we describe may provide pivotal clues for metastatic candidate genes, and thus impact on the prediction and intervention of metastatic colorectal cancer.     

Biotransformation of pregnenolone - 7alpha-3H, progesterone - 7alpha-3H and dehydroepiandrosterone - 7alpha-3H by porcine fetal and maternal adrenal homogenate preparations.

The biotransformation of pregnenolone-7alpha-3H and of progesterone-7alpha-3H by porcine fetal and maternal adrenal homogenates at 56 and 112 days of pregnancy and of dehydroepiandrosterone-7alpha-3H by fetal adrenal homogenates has been investigated in vitro. Both pregnenolone-7alpha-3H and progesterone-7alpha-3H were metabolized extensively by maternal adrenal preparations, the principal radioactive metabolites isolated being cortisol, corticosterone, 11-deoxycortisol, deoxycorticosterone, 11beta-hydroxyprogesterone and androstenedione. In addition, 17alpha-hydroxyprogesterone, 20alpha-dihydroprogesterone and cortisone were formed from both substrates and 17alpha-hydroxypregnenolone and progesterone were formed from pregnenolone. Although essentially the same radioactive metabolites were isolated after incubation of fetal adrenal glands with pregnenolone-7alpha-3H or progesterone-7alpha-3H, a greater proportion of the radioactivity was associated with corticosteroids at 112 days of pregnancy than at 56 days. 11beta-Hydroxyandrostenedione and androstenedione were isolated and identified together with an unknown polar metabolite, after incubation of fetal adrenal tissue with dehydroepiandrosterone-7alpha-3H. These results are discussed in relation to feto-placental steroid biosynthesis and metabolism and the role of the fetal adrenal in the initiation of parturition in the pig.     

The 10-20-30 training concept improves performance and health profile in moderately trained runners

The effect of an alteration from regular endurance to interval (10-20-30) training on the health profile, muscular adaptations, maximum oxygen uptake (Vo(2max)), and performance of runners was examined. Eighteen moderately trained individuals (6 females and 12 males; Vo(2max): 52.2 ± 1.5 ml·kg(-1)·min(-1)) (means ± SE) were divided into a high-intensity training (10-20-30; 3 women and 7 men) and a control (CON; 3 women and 5 men) group. For a 7-wk intervention period the 10-20-30 replaced all training sessions with 10-20-30 training consisting of low-, moderate-, and high-speed running (<30%, <60%, and >90% of maximal intensity) for 30, 20, and 10 s, respectively, in three or four 5-min intervals interspersed by 2 min of recovery, reducing training volume by 54% (14.0 ± 0.9 vs. 30.4 ± 2.3 km/wk) while CON continued the normal training. After the intervention period Vo(2max) in 10-20-30 was 4% higher, and performance in a 1,500-m and a 5-km run improved (P < 0.05) by 21 and 48 s, respectively. In 10-20-30, systolic blood pressure was reduced (P < 0.05) by 5 ± 2 mmHg, and total and low-density lipoprotein (LDL) cholesterol was lowered (P < 0.05) by 0.5 ± 0.2 and 0.4 ± 0.1 mmol/l, respectively. No alterations were observed in CON. Muscle membrane proteins and enzyme activity did not change in either of the groups. The present study shows that interval training with short 10-s near-maximal bouts can improve performance and Vo(2max) despite a ～50% reduction in training volume. In addition, the 10-20-30 training regime lowers resting systolic blood pressure and blood cholesterol, suggesting a beneficial effect on the health profile of already trained individuals.     

Permeation and metabolism of a series of novel lipophilic ascorbic acid derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids with a branched-acyl chain, in a human living skin equivalent model

A series of novel lipophilic vitamin C derivatives, 6-O-acyl-2-O-alpha-D-glucopyranosyl-L-ascorbic acids possessing a branched-acyl chain of varying length from C(8) to C(16) (6-bAcyl-AA-2G), were evaluated as topical prodrugs of ascorbic acid (AA) with transdermal activity in a human living skin equivalent model. The permeability of 6-bAcyl-AA-2G was compared with those of the derivatives having a straight-acyl chain (6-sAcyl-AA-2G). Out of 10 derivatives of 6-sAcyl-AA-2G and 6-bAcyl-AA-2G, 6-sDode-AA-2G and 6-bDode-AA-2G exhibited most excellent permeability in this model. Measurement of the metabolites permeated from the skin model suggested that 6-bDode-AA-2G was mainly hydrolyzed via 6-O-acyl AA to AA by tissue enzymes, while 6-sDode-AA-2G was hydrolyzed via 2-O-alpha-D-glucopyranosyl-L-ascorbic acid to AA. The former metabolic pathway seems to be advantageous for a readily available source of AA, because 6-O-acyl AA, as well as AA, is able to show vitamin C activity.