The metabolism of 1-methylhydantoin via 5-hydroxy-1-methylhydation in mammals

The metabolic pathway of 1-methylhydantoin (2) via 5-hydroxy-1-methylhydantoin (3), methylparabanic acid (4) and N⁵-methyloxaluric acid (5) proved to be a major and general one in mammals. Hence the formation of (3), which has not been detected in normal tissue, is likely to be indirect in inflamed tissue, probably depending on the arising formation of (2) from creatinine (1).     

Creatinine metabolite,HMH (5-hydroxy-1-methylhydantoin; NZ-419), modulates bradykinin-induced changes in vascular smooth muscle cells

Abstract

A creatinine metabolite, 5-hydroxy-1-methylhydantoin (HMH: NZ-419), a hydroxyl radical scavenger, has previously been shown to confer renoprotection by inhibiting the progression of chronic kidney disease in rats. In the current study, we demonstrate that HMH modulates the effects of glucose and bradykinin (BK) in vascular smooth muscle cell (VSMC). HMH a novel anti-oxidant drug completely suppressed the expression of B2-kinin receptors (B2KR) in response to high glucose (25?mM) stimulation in VSMC and was also shown to attenuate the effects of BK on VSMC remodeling. HMH inhibited the BK-induced increase in MAPK phosphorylation and attenuated the increase in connective tissue growth factor (CTGF) protein levels in VSMC. These findings suggest that HMH may confer vascular protection against high glucose concentrations and BK-stimulation to ameliorate vascular injury and remodeling through its anti-oxidant properties.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases

DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.     

Activation of phospholipase D by endothelin-1 and other pharmacological agents in rabbit iris sphincter smooth muscle

The stimulation of phospholipase D (PLD) activity by endothelin-1 (ET1) was investigated in rabbit iris sphincter perlabelled with [³H]myristic acid. In the presence of 0.5% ethanol, ET1 caused a time- and dose-dependent increase in the production of [³H]phophatidylethanol ([³H]PEt). Within 30 s the peptide increased PEt formation by 30% and after 5 min increased it by 140%. The ₅₀ value for ET1-stimulated PEt formation was found to be 30 nM. This value is appreciably lower than the ₅₀ we previously obtained for ET1-induced inositol triphosphate production (45 nM), but considerably higher than that for arachidonic acid release (1 nM). PEt formation was significantly stimulated by prostaglandin F₂₀, phorbol 12,13-dibutyrate (PDBu), chloroform, A23187 and A1F₄⁻, but it was not affected by carbachol or the platelet-activating factor. PDBu-stimulated PEt formation was blocked by staurosporine and it was not potentiated by A23187. Staurosporine had no effect on ET1-stimulated PEt formation. Our data indicate that ET1 stimulation of PLD occurs independently of protein kinase C activation, phospholipase C activation and intracellular Ca²⁺ mobilization, and phospholipase A₂ activation. In this tissue the ET1 receptor is probably coupled to the three phospholipases through several G-proteins, and this appears to be species and receptor type specific.     

The Effects of Auxins, Amino Acids, and Catechols on the Formation of Red Pigment in Helianthus tuber slices

The mechanism of red pigment formation in tuber slices of Helianthus tuberosus during incubation with auxins has been studied and the following results were obtained. 1. The degree of expansion of the tissue slices in the presence of indole-acetic acid (IAA), 2,4-dichloropbenoxyacetic acid (2,4-D), or α-naphthaleneacetic acid (NAA) was not parallel to the colour intensity which developed during incubation. 2. The pigment solutions which were extracted by pyridine. ethanol, or dioxane commonly showed a peak at around 515 nm in the absorption spectrum. 3. The o-diphenol oxidase activity in the tissue slices was increased by incubation with 2.4-D. 4. I.-Proline or hydroxy-L-proline as reactants in the formation of red pigment by the o-diphenol oxidase system could not be replaced by L-arginine, L-histidine and L-tryptophan. 5. The red pigment may resemble 4-(4′-hydroxy-2′-carbethoxy -1′-pyrrolidyl)-5-methyl-o-benzoquinone, formed by the o-diphenol oxidase system in the presence of homocatechol and hydroxy-L-proline.     

A single amino acid mutation at position 170 of human parainfluenza virus type 1 fusion glycoprotein induces obvious syncytium formation and caspase-3-dependent cell death

An escape mutant of human parainfluenza virus type 1 (hPIV1), which was selected by serial passage in the presence of a sialidase inhibitor, 4-O-thiocarbamoylmethyl-2-deoxy-2,3-didehydro-N-acetylneur-aminic acid (TCM-Neu5Ac2en), exhibited remarkable syncytium formation and virus-induced cell death in LLC-MK2 cells but no difference in susceptibility for the sialidase inhibitor TCM-Neu5Ac2en from that of wild-type hPIV1 strain C35 (WT). The mutant virus also had higher replication and plaque formation abilities. The mutant virus acquired two amino acid mutations, Glu to Gly at position 170 and Ala to Glu 442 in fusion (F) glycoprotein, but no mutations in haemaggulutinin-neuraminidase (HN) glycoprotein. Using cells co-expressing F and HN genes with site-specific mutagenesis, we demonstrated that a point mutation of Glu to Gly at position 170, which was estimated to be located in hPIV1 F glycoprotein heptad repeat 1, was required for obvious syncytium formation and caspase-3-dependent cell death. In contrast, wild-type F glycoprotein induced no synctium formation or cell death. The findings suggest that a single amino acid mutation of hPIV1 F glycoprotein promotes syncytium formation that is followed by caspase-3-dependent cell death.     

Biosynthesis of 5,6-dihydroxyprostaglandin E1 and F1 alpha from 5,6-dihydroxyeicosatrienoic acid by ram seminal vesicles

cis-5(6)Epoxy-cis-8,11,14-eicosatrienoic acid was recently found to be metabolized by ram seminal vesicles to 5-hydroxyprostaglandin I 1 alpha and 5-hydroxyprostaglandin I 1 beta, 5(6)epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1. The epoxide can be hydrolyzed by epoxide hydrolases to 5,6-dihydroxy-8,11,14-eicosatrienoic acid. The latter was incubated with microsomes of ram seminal vesicles for 2 min at 37 degrees C and the polar metabolites were purified by reversed phase HPLC and analyzed by capillary column gas chromatography-mass spectrometry. The major metabolite was identified as 5,6-dihydroxyprostaglandin F 1 alpha. In the presence of glutathione (1 mM), 5,6-dihydroxyprostaglandin E1 was also formed. The 3H-labelled vicinal diol and the 3H-labelled epoxide were metabolized to polar products to a similar extent, but the formation of prostaglandin E compounds in the presence of glutathione was lower from the diol than from the epoxide or from arachidonic acid. The likely prostaglandin endoperoxide intermediates in the metabolism of the diol (5,6-dihydroxyprostaglandin G1 and 5,6-dihydroxyprostaglandin H1) thus appear to be less prone to be isomerized to prostaglandin E compounds than prostaglandins G2 and H2 and their 5(6)epoxy counterparts. 5(6)Epoxyprostaglandin E1 and 5,6-dihydroxyprostaglandin E1 can be chemically transformed into 5,6-dihydroxyprostaglandin B1. The latter can be analyzed by HPLC or by mass fragmentography, and a simple chemical synthesis of 5,6-dihydroxyprostaglandin B1 from prostaglandin E2 is described.     

Thymine lesions produced by ionizing radiation in double-stranded DNA

A DNA glycosylase which catalyzes the release of thymine residues damaged by ring saturation, fragmentation, or ring contraction from double-stranded DNA has been used to characterize such base derivatives in gamma-irradiated DNA. It is shown by chromatographic analysis that irradiation of DNA in neutral solution generates the ring-saturated forms cis-thymine glycol, trans-thymine glycol, and a monohydroxydihydrothymine, probably 6-hydroxy-5,6-dihydrothymine. The latter compound is only observed after irradiation under hypoxic conditions. The ring-contracted thymine derivative 5-hydroxy-5-methylhydantoin is also formed, and it is the major lesion after irradiation of DNA under O2. Ring-fragmented products such as methyltartronylurea were only generated in small quantities. Isolation and analysis of the DNA from gamma-irradiated human cells also revealed the formation of ring-saturated thymine derivatives, but 5-hydroxy-5-methylhydantoin was not found in this case.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

The role of methylglyoxal in the non-enzymatic conversion of tryptophan, its methyl ester and tryptamine to 1-acetyl-beta-carbolines

Non-enzymatic modification of l-tryptophan (1) and its metabolites and derivatives with aldehydes, via the Pictet-Spengler reaction, affords beta-carbolines. Here we demonstrate that methylglyoxal (2) generates 1-acetyl-beta-carbolines from tryptophan (1), from its methyl ester (6) and from tryptamine (4); however, 2 did not generate 1-(1-hydroxyethyl)-beta-carboline derivates. HPLC analysis of model reaction systems showed formation of 1-acetyl-beta-carboline (3) and 1-acetyl-beta-carboline-3-carboxylic acid (5) during incubation of 1 and 2, at pH 5.7 and 7.4, at 100 degrees C, and only 5 at 37 degrees C, at the same pH values, with limited access of oxygen. Aerobic conditions caused higher formation of 3 at 37 degrees C at both pH values, while, at higher temperature, the same effect was only observed at pH 5.7. Lack of oxygen did not much influence the formation of 3 or 5 at both pH and temperature values, in comparison with the formation at limited access of oxygen. Incubation of 2 and 6 generated methyl-1-acetyl-beta-carboline-3-carboxylate (7) together with 3 and 5 as a result of hydrolysis of 6 into 1 and, partially, 7 into 5, while in incubation mixtures of 2 and 4 only unstable 1-acetyl-3,4-dihydro-beta-carboline (8) was observed. Incubation of 1 with d-glucose as well as incubation of tryptophan with Amadori product 18 under similar conditions did not generate carbolines 3 or 5. For the first time, we were able to demonstrate the presence of 1-acetyl-beta-carboline-3-carboxylic acid (5) in some commercially available ketchups and in previously heated tomato concentrate.     

Endogenous formation and significance of 1,N2-propanodeoxyguanosine adducts

The detection of 1,N2-propanodeoxyguanosine adducts in the DNA of rodent and human tissues as endogenous lesions has raised important questions regarding the source of their formation and their roles in carcinogenesis. Both in vitro and in vivo studies have generated substantial evidence which supports the involvement of short- and long-chain enals derived from oxidized polyunsaturated fatty acids (PUFAs) in their formation. These studies show that: (1) the cyclic propano adducts are common products from reactions of enals with DNA bases; (2) they are formed specifically from linoleic acid (LA; omega-6) and docosahexaenoic acid (omega-3) under in vitro stimulated lipid peroxidation conditions; (3) the levels of propano adducts are dramatically increased in rat liver DNA upon depletion of glutathione; (4) the adduct levels are increased in the liver DNA of the CCl4-treated rats and the mutant strain of Long Evans rats which are genetically predisposed to increased lipid peroxidation; and (5) adduct levels are significantly higher in older rats than in newborn rats. These studies collectively demonstrate that tissue lipid peroxidation is a main endogenous pathway leading to propano adduction in DNA. The possible contribution from environmental sources, however, cannot be completely excluded. The mutagenicity of enals and the mutations observed in site-specific mutagenesis studies using a model 1,N2-propanodeoxyguanosine adduct suggest that these adducts are potential promutagenic lesions. The increased levels of the propano adducts in the tissue of carcinogen-treated animals also provide suggestive evidence for their roles in carcinogenesis. The involvement of these adducts in tumor promotion is speculated on the basis that oxidative condition in tissues is believed to be associated with this process.     

An efficient synthesis of 5alpha-androst-1-ene-3,17-dione

5alpha-Androst-1-ene-3,17-dione (5) as a prodrug of 1-testosterone (4) was prepared in four steps from 17beta-Acetoxy-5alpha-androstan-3-one (stanolone acetate) (1) in high yield. Thus, stanolone acetate (1) was brominated in the presence of hydrogen chloride in acetic acid to give 17beta-acetoxy-2-bromo-5alpha-androstan-3-one (2), which underwent dehydrobromination using lithium carbonate as base with lithium bromide as an additive to give 17beta-acetoxy-5alpha-androst-1-en-3-one (3) in almost quantitative yield with 97% of purity. Compound (3) was hydrolyzed with sodium hydroxide to give 17beta-hydroxy-5alpha-androst-1-en-3-one (4,1-testosterone), which was oxidized with chromium trioxide to afford 5alpha-androst-1-ene-3,17-dione (5). The overall yield of 5 was 78.2% with purity of 99%. In this method, the formation of 4-ene was diminished when 1-ene was introduced, and its mechanism was also discussed.     

Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.     

Two amino acids in each of D1 and D2 dopamine receptor cytoplasmic regions are involved in D1-D2 heteromer formation

D(1) and D(2) dopamine receptors exist as heteromers in cells and brain tissue and are dynamically regulated and separated by agonist concentrations at the cell surface. We determined that these receptor pairs interact primarily through discrete amino acids in the cytoplasmic regions of each receptor, with no evidence of any D(1)-D(2) receptor transmembrane interaction found. Specifically involved in heteromer formation we identified, in intracellular loop 3 of the D(2) receptor, two adjacent arginine residues. Substitution of one of the arginine pair prevented heteromer formation. Also involved in heteromer formation we identified, in the carboxyl tail of the D(1) receptor, two adjacent glutamic acid residues. Substitution of one of the glutamic acid pair prevented heteromer formation. These amino acid pairs in D(1) and D(2) receptors are oppositely charged, and presumably interact directly by electrostatic interactions.     

Physicochemical and biological properties of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose (6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin)

A unique multibranched cyclomaltooligosaccharide (cyclodextrin, CD) of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose [6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin, (G(2))(3)-betaCD] was prepared. The physicochemical and biological properties of (G(2))(3)-betaCD were determined together with those of monobranched CDs (6-O-alpha-D-glucopyranosyl-alpha-cyclodextrin (G(1)-alphaCD), 6-O-alpha-D-glucopyranosyl-beta-cyclodextrin (G(1)-betaCD), and 6-O-alpha-maltosyl-beta-cyclodextrin (G(2)-betaCD)). NMR spectra of (G(2))(3)-betaCD were measured using various 2D NMR techniques. The solubility of (G(2))(3)-betaCD in water and MeOH-water solutions was extremely high in comparison with nonbranched betaCD and was about the same as that of the other monobranched betaCDs. The formation of an inclusion complex of (G(2))(3)-betaCD with stereoisomers (estradiol, retinoic acid, quinine, citral, and glycyrrhetinic acid) depends on the cis-trans isomers of guest compounds. The cis isomers of estradiol, retinoic acid, and glycyrrhetinic acid were included more than their trans isomers, while the trans isomers of citral and quinine fit more tightly than their cis isomers. (G(2))(3)-betaCD was the most effective host compound in the cis-trans resolution of glycyrrhetinic acid. Among the branched betaCDs, (G(2))(3)-betaCD exhibited the weakest hemolytic activity in human erythrocytes and showed negligible cytotoxicity in Caco-2 cells up to 200 microM. These results indicate unique characteristics of (G(2))(3)-betaCD in some biological responses of cultured cells.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine     

Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/β-HSDs and 5α/β-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17β-HSD type 2 (HSD17B2). Also, 3α-HSD and 3β-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5β-reductase (AKR1D1) are responsible for the formation of 5α- and 5β-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/β-HSD activities of AKR1C1, AKR1C2 and AKR1C3.     

In vivo formation of 1-malonylaminocyclopropane-1-carboxylic acid and its relationship to ethylene production in cocklebur seed segments: A tracer study with 1-amino-2-ethylcyclopropane-1-carboxylic acid

The in vivo formation of 1-malonylaminocyclopropane-1-carboxylic acid (malonyl-ACC) and its relationship to ethylene production in the axial tissue of cocklebur (Xanthium pennsylvanicum) seeds were investigated using the stereoisomers of the 2-ethyl derivative of ACC (AEC), as tracers of ACC. Of the four AEC isomers, the (1R, 2S)-isomer was converted most effectively to a malonyl conjugate as well as to 1-butene. Malonyl-AEC, once formed, was not decomposed, supporting the view that malonyl-ACC does not liberate free ACC for ethylene production in this tissue. d-Phenylalanine inhibited the formation of malonyl-AEC and, at the same time, promoted the evolution of 1-butene, whereas l-phenylalanine did not. Possibly, the d-amino-acid-stimulated ethylene production in cocklebur seed tissues is due to an increase in the amount of ACC available for ethylene production which results from the decrease of ACC malonylation in the tissues treated with d-amino acid. 2-Aminoisobutyric acid, a competitive inhibitor of ACC-ethylene conversion, did not affect the malonylation of AEC.     

Multiple steroid metabolic pathways in ZR-75-1 human breast cancer cells

In order to characterize the main enzymatic systems involved in androgen and estrogen formation as well as metabolism in ZR-75-1 human breast cancer cells, incubation of intact cells was performed for 12 or 24 h at 37 degrees C with tritiated estradiol (E2), estrone (E1), androst-5-ene-3 beta, 17 beta-diol (5-ene-diol), dehydroepiandrosterone (DHEA), testosterone (T), androstenedione (4-ene-dione), dihydrotestosterone (DHT) or androsterone (ADT). The extra- and intracellular steroids were extracted, separated into free steroids, sulfates and non-polar derivatives (FAE) and identified by HPLC coupled to a Berthold radioactivity monitor. Following incubation with E2, 5-ene-diol or T, E1, DHEA and 4-ene-dione were the main products, respectively, thus indicating high levels of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). When 4-ene-dione was used, on the other hand, a high level of transformation into 5 alpha-androstane-3,17-dione (A-dione), Epi-ADT and ADT was found, thus indicating the presence of high levels of 5 alpha-reductase as well as 3 alpha- and 3 beta-hydroxysteroid dehydrogenase. Moreover, some T was formed, due to oxidation by 17 beta-HSD. No estrogen was detected with the androgen precursors T or 4-ene-dione, thus indicating the absence of significant aromatase activity. Moreover, significant amounts of sulfates and non-polar derivatives were found with all the above-mentioned substrates. The present study shows that ZR-75-1 human breast cancer cells possess most of the enzymatic systems involved in androgen and estrogen formation and metabolism, thus offering an excellent model for studies of the control of sex steroid formation and action in breast cancer tissue.