β-Ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

Regionally selective alterations in enzymatic activities and metabolic fluxes during thiamin deficiency

To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.¹⁴CO₂ production from¹⁴C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO₂ production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC 2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3. - PDHC pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3 - TK transketolase (EC 2.2.1.1) - TPP thiamin pyrophosphate     

Selective inhibition of 11β-hydroxysteroid dehydrogenase 1 by 18α-glycyrrhetinic acid but not 18β-glycyrrhetinic acid

Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11β-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11β-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18β-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11β-HSD1 and 11β-HSD2. Here, we compare the biological activity of 18β-GA and its diastereomer 18α-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18α-GA selectively inhibits 11β-HSD1 but not 11β-HSD2. This is in contrast to 18β-GA, which preferentially inhibits 11β-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11β-HSD1, we provide an explanation for the differences in the activities of 18α-GA and 18β-GA. This model will be used to design novel selective derivatives of GA.     

Mitochondrial dihydrolipoyl succinyltransferase deficiency accelerates amyloid pathology and memory deficit in a transgenic mouse model of amyloid deposition

Mitochondrial dysfunction and oxidative stress are involved in Alzheimer disease (AD) pathogenesis. In human AD brains, the activity of the α-ketoglutarate dehydrogenase enzyme complex (α-KGDHC) is reduced. KGDHC is mostly involved in NADH production. It can also participate in oxidative stress and reactive oxygen species (ROS) production. The mitochondrial dihydrolipoyl succinyltransferase enzyme (DLST) is a key subunit specific to the α-KGDHC. In cultured cells, reduction of DLST increased H₂O₂-induced ROS generation and cell death. Thus, we asked whether partial genetic deletion of DLST could accelerate the onset of AD pathogenesis, using a transgenic mouse model of amyloid deposition crossed with DLST^+/− mice. Tg19959 mice, which carry the human amyloid precursor protein with two mutations, develop amyloid deposits and progressive behavioral abnormalities. We compared Tg19959 mice to Tg19959-DLST^+/− littermates at 2–3 months of age and studied the effects of DLST deficiency on amyloid deposition, spatial learning and memory, and oxidative stress. We found that α-KGDHC activity was reduced in DLST^+/− mice. We also found that DLST deficiency increased amyloid plaque burden, Aβ oligomers, and nitrotyrosine levels and accelerated the occurrence of spatial learning and memory deficits in female Tg19959 mice. Our data suggest that α-KGDHC may be involved in AD pathogenesis through increased mitochondrial oxidative stress.     

On-line deconjugation of glucuronides using an immobilized enzyme reactor based upon β-glucuronidase

An immobilized enzyme reactor based upon β-glucuronidase (BG–IMER) has been developed for the on-line deconjugation of substrates. The activity of the BG–IMER and its applicability to on-line deconjugation was investigated. The BG–IMER was coupled to a reversed-phase column (C₈ or C₁₈) and the latter column was used to separate substrates and products eluted from the β-glucuronidase reactor. The activity of the BG–IMER was followed by measurement of percent deconjugation and the parameters investigated were: substrate concentration, pH (4 to 6), temperature (r.t., 37°C), enzyme–substrate contact time using flow-rates of 0.1 to 1.0 ml/min (15–1.5 min). The glucuronides used in the evaluation of the BG–IMER were: 4-methylumbelliferyl-β- -glucuronide, p-acetaminophen-β- -glucuronide, 3′-azido-3′-deoxythymidine-β- -glucuronide, phenyl-β- -glucuronide, chloramphenicol-β- -glucuronide, estradiol-17-β- -glucuronide and morphine-β- -glucuronide. The development of on-line HPLC deconjugation of glucuronide substrates using the BG–IMER will facilitate the identification of metabolites and quantification of aglycones in metabolic and pharmacokinetic studies.     

Beta-ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.     

Fatty Acid Amide Hydrolase-Dependent Generation of Antinociceptive Drug Metabolites Acting on TRPV1 in the Brain

The discovery that paracetamol is metabolized to the potent TRPV1 activator N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) and that this metabolite contributes to paracetamol’s antinociceptive effect in rodents via activation of TRPV1 in the central nervous system (CNS) has provided a potential strategy for developing novel analgesics. Here we validated this strategy by examining the metabolism and antinociceptive activity of the de-acetylated paracetamol metabolite 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA), both of which may undergo a fatty acid amide hydrolase (FAAH)-dependent biotransformation to potent TRPV1 activators in the brain. Systemic administration of 4-aminophenol and HMBA led to a dose-dependent formation of AM404 plus N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) and arvanil plus olvanil in the mouse brain, respectively. The order of potency of these lipid metabolites as TRPV1 activators was arvanil = olvanil>>AM404> HPODA. Both 4-aminophenol and HMBA displayed antinociceptive activity in various rodent pain tests. The formation of AM404, arvanil and olvanil, but not HPODA, and the antinociceptive effects of 4-aminophenol and HMBA were substantially reduced or disappeared in FAAH null mice. The activity of 4-aminophenol in the mouse formalin, von Frey and tail immersion tests was also lost in TRPV1 null mice. Intracerebroventricular injection of the TRPV1 blocker capsazepine eliminated the antinociceptive effects of 4-aminophenol and HMBA in the mouse formalin test. In the rat, pharmacological inhibition of FAAH, TRPV1, cannabinoid CB1 receptors and spinal 5-HT₃ or 5-HT_1A receptors, and chemical deletion of bulbospinal serotonergic pathways prevented the antinociceptive action of 4-aminophenol. Thus, the pharmacological profile of 4-aminophenol was identical to that previously reported for paracetamol, supporting our suggestion that this drug metabolite contributes to paracetamol’s analgesic activity via activation of bulbospinal pathways. Our findings demonstrate that it is possible to construct novel antinociceptive drugs based on fatty acid conjugation as a metabolic pathway for the generation of TRPV1 modulators in the CNS.     

Impaired oxidoreduction by 11β-hydroxysteroid dehydrogenase 1 results in the accumulation of 7-oxolithocholic acid

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) mediates glucocorticoid activation and is currently considered as therapeutic target to treat metabolic diseases; however, biomarkers to assess its activity in vivo are still lacking. Recent in vitro experiments suggested that human 11β-HSD1 metabolizes the secondary bile acid 7-oxolithocholic acid (7-oxoLCA) to chenodeoxycholic acid (CDCA) and minor amounts of ursodeoxycholic acid (UDCA). Here, we provide evidence from in vitro and in vivo studies for a major role of 11β-HSD1 in the oxidoreduction of 7-oxoLCA and compare its level and metabolism in several species. Hepatic microsomes from liver-specific 11β-HSD1-deficient mice were devoid of 7-oxoLCA oxidoreductase activity. Importantly, circulating and intrahepatic levels of 7-oxoLCA and its taurine conjugate were significantly elevated in mouse models of 11β-HSD1 deficiency. Moreover, comparative enzymology of 11β-HSD1-dependent oxidoreduction of 7-oxoLCA revealed that the guinea-pig enzyme is devoid of 7-oxoLCA oxidoreductase activity. Unlike in other species, 7-oxoLCA and its glycine conjugate are major bile acids in guinea-pigs. In conclusion, the oxidoreduction of 7-oxoLCA and its conjugated metabolites are catalyzed by 11β-HSD1, and the lack of this activity leads to the accumulation of these bile acids in guinea-pigs and 11β-HSD1-deficient mice. Thus, 7-oxoLCA and its conjugates may serve as biomarkers of impaired 11β-HSD1 activity.     

Enzymatic synthesis of trisaccharides and alkyl β-d-glucosides by the transglycosylation reaction of β-glucosidase from Fusarium oxysporum

Purified β-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl β-d-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl β-d-glucosides were 22–37% and 10–33% of the total sugar, respectively. The enzyme retained 70–80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, β-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl β-d-glucosides.     

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC–MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1000 μg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1±13.5 μg/l for 2-ethylhexanoic acid, 104.8± 80.6 μg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2± 389.5 μg/l for 2-ethyl-3-oxohexanoic acid.     

Selective separation process of proteins based on the heat stress-induced translocation across phospholipid membranes

Heating of several protein solutions at 40–47°C for 5–60 min in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of β-galactosidase (β-gal), α-glucosidase (α-glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of β-gal at various temperatures were maximized under suitable heating conditions (45°C, 30 min). Those of α-glu and CAB were maximized at 40–45 and 60°C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of β-gal, α-glu and CAB.     

Characterization of β-endorphin- and α-MSH-related peptides in rat heart

POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that β-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to β-endorphin- and α-MSH-related peptides. Ion exchange HPLC analysis revealed that β-endorphin(1–31) was further processed to α-N-acetyl-β-endorphin(1–31), which comprised 35.9 ± 0.1% of total immunoreactivity, and smaller amounts of β-endorphin(1–27), β-endorphin(1–26), and their α-N-acetylated derivatives. The predominant α-MSH immunoreactive peptides coeluted with α-MSH and N,O-diacetyl-α-MSH by reverse-phase HPLC, although small amounts of ACTH(1–13)-NH₂ were also present. Thus, multiple forms of β-endorphin and α-MSH are localized in rat heart. β-Endorphin(1–31) is a minor constituent, however, indicating that nonopioid β-endorphin peptides predominate.     

Analysis of some metabolites of organic solvents in urine by high-performance liquid chromatography with β-cyclodextrin

Chromatographic separation of the metabolites derived from toluene, ethylbenzene, styrene and xylene was carried out on untreated urine samples from factory workers. The elution sequence was as follows: phenylglyoxilic acid, 3-hydroxy-2-butanone, hippuric acid, o-methylhippuric acid, p-methylhippuric acid, m-methylhippuric acid, p-cresol, m-cresol and o-cresol. The stability constants (K_G) of cresol and methylhippuric acid derivatives were evaluated. The capacity factor (k′), selectivity factor (α) and resolution (R_s) are described with a variety of mobile phases containing β-cyclodextrin (β-CD). The optimum concentration ratio of ethanol–water–acetic acid–β-CD was determined to be 20:80:0.3:1.4%. Under these conditions, k′ values of the five metabolites were 2<k′<6, and all α values were greater than 1.5. Simultaneous determinations of the metabolites were carried out in real urine samples from factory workers using the standard addition method. Validation of the method and the detection limit are described under the optimum conditions attained in this experiment.     

Radioimmunoassay of main urinary metabolite of prostaglandin F2α

Radioimmunoassay of 5α,7α-dihydroxy-11-keto-tetranorprosta-1,16-dioic acid, main urinary metabolite of prostaglandin F₂α (PGF₂α), was performed using an antiserum produced in the rabbit.The antibody in 100 μ1 of 1,600-fold diluted antiserum binds with 60 picograms of metabolite.The main urinary metabolite level fell when flufenamic acid, a prostaglandin synthetase inhibitor, was given to rats. In contrast, it was significantly elevated when PGF₂α was administered.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Distinct effect of a cationic surfactant on the transient and steady state phases of 2-naphthyl acetate hydrolysis catalyzed by α-chymotrypsin

Results obtained on the effect of addition of dodecyltrimethylammonium bromide (DTAB) upon the α-chymotrypsin (α-CT) catalyzed hydrolysis of 2-naphthyl acetate (2-NA) under steady state conditions for the acyl–enzyme intermediate are compared with those previously obtained in the transient (pre-steady state or “burst”) phase. It is found that, while in the transient phase there is no effect of DTAB addition on the kinetic parameters at concentrations below the critical micelle concentration (CMC) of the surfactant, super-activity is observed when the acyl–enzyme intermediate reaches the steady state condition. This difference implies that the surfactant does not modify either the formation or the decomposition of the enzyme–substrate complex (transient phase) but notably increases the rate of disruption of the acyl–enzyme intermediate.     

The interaction of estrogen receptor α and caveolin-3 regulates connexin43 phosphorylation in metabolic inhibition-treated rat cardiomyocytes

Caveolin-3, the major caveolin isoform in cardiomyocytes, plays an important role in the rapid signaling pathways initiated by stimulation of the membrane-associated molecules. To examine the role of caveolin-3 in regulating estrogen receptor α in cardiomyocytes, we investigate whether the membrane estrogen receptor α associates with caveolin-3 and whether this association is linked to the 17β-estradiol-mediated signals. In control cardiomyocytes, following discontinuous sucrose gradient centrifugation, caveolin-3 was found predominantly in the lipid raft buoyant fractions, whereas it was distributed to both the buoyant and non-lipid raft heavy fractions following metabolic inhibition treatment. Confocal microscopy showed that estrogen receptor α co-localized with caveolin-3 on the plasma membrane of neonatal and adult rat cardiomyocytes. This membrane labeling of estrogen receptor α was not seen following treatment with the cholesterol-depleting agent methyl-β-cyclodextrin (5 mM), whereas metabolic inhibition had little effect on the membrane distribution of estrogen receptor α. Metabolic inhibition induced tyrosine phosphorylation of caveolin-3 and decreased its association with estrogen receptor α, both effects being mediated via a Src activation mechanism, since they were inhibited by the selective tyrosine kinase inhibitor PP2. Metabolic inhibition also induced tyrosine phosphorylation of connexin43 and increased its association with c-Src, both effects being prevented by 17β-estradiol (200 nM). The effect of 17β-estradiol on metabolic inhibition-induced tyrosine phosphorylation of connexin43 was inhibited by the specific estrogen receptor antagonist ICI182780. These data identify cardiac caveolin-3 as juxtamembrane scaffolding for estrogen receptor α docking at caveolae, which provide a unique compartment for conveying 17β-estradiol-elicited, rapid signaling to regulate connexin43 phosphorylation during ischemia.     

Immobilized 4-aminophenyl 1-thio-β- -galactofuranoside as a matrix for affinity purification of an exo-β- -galactofuranosidase

An alternative and fast method for the purification of an exo-β- -galactofuranosidase has been developed using a 4-aminophenyl 1-thio-β- -galactofuranoside affinity chromatography system and specific elution with 10 mM -galactono-1,4-lactone in a salt gradient. A concentrated culture medium from Penicillium fellutanum was chromatographed on DEAE–Sepharose CL 6B followed by chromatography on the affinity column, yielding two separate peaks of enzyme activity when elution was performed with 10 mM -galactono-1,4-lactone in a 100–500 mM NaCl salt gradient. Both peaks behaved as a single 70 kDa protein, as detected by SDS-PAGE. Antibodies elicited against a mixture of the single bands excised from the gel were capable of immunoprecipitating 0.2 units out of 0.26 total units of the enzyme from a crude extract. The glycoprotein nature of the exo-β- -galactofuranosidase was ascertained through binding to Concanavalin A–Sepharose as well as by specific reaction with Schiff reagent in Western blots. The purified enzyme has an optimum acidic pH (between 3 and 6), and K_m and V_max values of 0.311 mM and 17 μmol h⁻¹ μg⁻¹ respectively, when 4-nitrophenyl β- -galactofuranoside was employed as the substrate.     

Androgen metabolism and biotransformation in nontumoral and malignant human liver tissues and cells

There is indirect multiple evidence that hints at a potential role of sex steroids in development and progression of human hepatocellular carcinoma (HCC). In the present study, we have investigated androgen metabolism in a panel of human liver cancer cell lines (HA22T, Huh7, HepG2) and in normal, cirrhotic and malignant human liver tissues aiming to dissect the potential impact of individual enzyme activities and their products in normal and diseased human liver, both in vivo and in vitro. Using our intact cell analysis we were able to assess rates and pathways of androgen metabolism in living conditions. Overall, incubation of cultured cells or tissue minces with either testosterone (T) or androstenedione (Ad) used as precursor resulted in a large extent of 17βoxidation of T to Ad (cells: 28–77%; tissues: 35–50%). In malignant liver cell lines, both HA22T and Huh7 cells showed consistent amounts of the 5α-reductase enzyme products (18% and 15%, respectively), while 5β-reductase activity was more pronounced in Huh7 cells (18%) than in HA22T cells (1.8%). Interestingly, a significant extent of estrogen formation could be observed in Huh7 cells (5.4–11.5%), while no aromatase activity could be detected in HA22T cells. In HepG2 cells, along with a relatively high proportion of Ad, estrogens represented the most prominent (50–55%) end product of androgen metabolism, regardless of the precursor used. In liver tissues, equivalent results could be obtained, with a consistent proportion of 17βoxidation of T to Ad (35–50%) being observed in the majority of samples. However, while normal liver tissue samples exhibited a minor proportion of bioactive androgens (3.4%) with no aromatase products, HCC tissues showed a significant extent of aromatase activity (nearly 20%) with estrogen representing the most prominent metabolic product after 24 h incubation with either T or Ad. HCV and alcoholic cirrhotic tissues displayed different patterns of androgen metabolism. The former produced limited amounts of bioactive androgens (5.3%) and considerable levels of the intermediate aromatase product 19OH-Ad (up to 28%), the latter exhibited a prevalence of androgen degradation through the 5β-reductase pathway (9.8%) and a significant extent of aromatase activity (16% as a whole). In conclusion, three major metabolic states could be depicted, depending on prevalent pathways of androgen metabolism and steroid receptor status: estrogenic, androgenic, and mixed. This model supports the idea that local estrogen biosynthesis may be implicated in human HCC and provides a basis for the exploitation of aromatase inhibitors and/or ER antagonists or selective estrogen receptor modulators (SERMs) as a new therapeutic strategy in HCC patients.