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J Adamski

Publications and source records attributed to J Adamski.

At least 73 records · Page 4Linked to original sources
PubMed1995

Molecular characterization of mouse 17 beta-hydroxysteroid dehydrogenase IV.

17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) catalyze the conversion of estrogens and androgens at the C17 position. The 17 beta-HSD type I, II, III and IV share less than 25% amino acid similarity. The human and porcine 17 beta-HSD IV reveal a three-domain structure unknown among other dehydrogenases. The N-terminal domains resemble the short chain alcohol dehydrogenase family while the central parts are related to the C-terminal parts of enzymes involved in peroxisomal beta-oxidation of fatty acids and the C-terminal domains are similar to sterol carrier protein 2. We describe the cloning of the mouse 17 beta-HSD IV cDNA and the expression of its mRNA. A probe derived from the human 17 beta-HSD IV was used to isolate a 2.5 kb mouse cDNA encoding for a protein of 735 amino acids showing 85 and 81% similarity with human and porcine 17 beta-HSD IV, respectively. The calculated molecular mass of the mouse enzyme amounts to 79,524 Da. The mRNA for 17 beta-HSD IV is a single species of about 3 kb, present in a multitude of tissues and expressed at high levels in liver and kidney, and at low levels in brain and spleen. The cloning and molecular characterization of murine, human and porcine 17 beta-HSD IV adds to the complexity of steroid synthesis and metabolism. The multitude of enzymes acting at C17 might be necessary for a precise control of hormone levels.

17-Hydroxysteroid Dehydrogenases↗
PubMed1995

Intrinsic sterol- and phosphatidylcholine transfer activities of 17 beta-hydroxysteroid dehydrogenase type IV.

Previous studies have shown that the 80 kDa 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) type IV comprises distinct domains, including an N-terminal region related to the short chain alcohol dehydrogenase multigene family and a C-terminal part related to the lipid transfer protein sterol carrier protein 2 (SCP2). In this study, we have investigated whether the SCP2-related part of the 80 kDa protein leads to an intrinsic sterol and phospholipid transfer activity, as shown earlier for the 60 kDa SCP2-related peroxisomal 3-ketoacyl CoA thiolase with intrinsic sterol and phospholipid transfer activity called sterol carrier protein x (SCPx). Our results indicate that a fraction rich in the 80 kDa form of 17 beta-HSD type IV exhibits high transfer activities for 7-dehydrocholesterol and phosphatidylcholine. In addition, a purified recombinant peptide derived from the SCP2-related domain of the 17 beta-HSD type IV has about 30% of the transfer activities for 7-dehydrocholesterol and phosphatidylcholine seen with purified recombinant human SCP2. We conclude that the 80 kDa type IV 17 beta-HSD represents a potentially multifunctional protein with intrinsic in vitro sterol and phospholipid transfer activity in addition to its enzymatic activity.

17-Hydroxysteroid Dehydrogenases↗
PubMed1995

Expression and regulation of aromatase and 17 beta-hydroxysteroid dehydrogenase type 4 in human THP 1 leukemia cells.

Estradiol is active in proliferation and differentiation of sex-related tissues like ovary and breast. Glandular steroid metabolism was for a long time believed to dominate the estrogenic milieu around any cell of the organism. Recent reports verified the expression of estrogen receptors in "non-target" tissues as well as the extraglandular expression of steroid metabolizing enzymes. Extraglandular steroid metabolism proved to be important in the brain, skin and in stromal cells of hormone responsive tumors. Aromatase converts testosterone into estradiol and androstenedione into estrone, thereby activating estrogen precursors. The group of 17 beta-hydroxysteroid dehydrogenases catalyzes the oxidation and/or reduction of the forementioned compounds, e.g. estradiol/estrone, thereby either activating or inactivating estradiol. Aromatase is expressed and regulated in the human THP 1 myeloid leukemia cell line after vitamin D/GMCSF-propagated differentiation. Aromatase expression is stimulated by dexamethasone, phorbolesters and granulocyte/macrophage stimulating factor (GMCSF). Exons I.2 and I.4 are expressed in PMA-stimulated cells only, exon I.3 in both PMA- and dexamethasone-stimulated cells. Vitamin D-differentiated THP 1 cells produce a net excess of estradiol in culture supernatants, if testosterone is given as aromatase substrate. In contrast, the 17 beta-hydroxysteroid dehydrogenase type 4 (17 beta-HSD 4) is abundantly expressed in unstimulated THP 1 cells and is further stimulated by glucocorticoids (2-fold). The expression is unchanged after vitamin D/GMCSF-propagated differentiation. 17 beta-HSD 4 expression is not altered by phorbolester treatment in undifferentiated cells but is abolished after vitamin D-propagated differentiation along with downregulation of beta-actin. Protein kinase C activation therefore appears to dissociate the expression of aromatase and 17 beta-HSD 4 in this differentiation stage along the monocyte/phagocyte pathway of THP 1 myeloid cells. The expression of steroid metabolizing enzymes in myeloid cells is able to create a microenvironment which is uncoupled from dominating systemic estrogens. These findings may be relevant in the autocrine, paracrine or iuxtacrine cellular crosstalk of myeloid cells in their respective states of terminal differentiation, e.g. in bone metabolism and inflammation.

Actins↗
PubMed1995

Modulation of apoptosis by starvation: morphological and biochemical study of rat intestinal mucosa.

Morphology at light and electron microscopic levels, expression and activation of transglutaminase and DNA fragmentation at internucleosomal sites were used as markers to study the effect of starvation on the apoptosis of small intestinal epithelial cells. The cells entering apoptotic programme in well-fed animals undergo many morphological changes in apical cytoplasm involving alterations in actin cytoskeleton organisation which may cause a discharge of microvilli. Some free floating cells in the intestinal lumen show characteristics of apoptotic cell death, e.g. shrinkage of cell and peripheral condensation of chromatin, while mitochondria and lysosomes remain unchanged. Apoptotic bodies are also seen in scanning electron micrographs. During progressive starvation, epithelial cells do not enter the apoptotic cell death programme. Biochemical markers for apoptosis such as increased transglutaminase activity and DNA fragmentation are clearly discernible in normally fed animals. The percentage of cells labelled immunohistochemically by antibody against transglutaminase decreased during starvation while DNA fragmentation was absent. The exact mechanism for suppressing apoptosis in intestinal cells under starvation is not known. However, the data presented here support the existence of such a regulatory process.

Journal Article↗
PubMed1995

The organelles containing porcine 17 beta-estradiol dehydrogenase are peroxisomes.

Porcine 17 beta-estradiol dehydrogenase was recently purified and cloned. It catalyzes the NAD(+)-dependent oxidation of estradiol to estrone 360-fold more efficiently than the reverse reaction with NADPH. Immunogold electron microscopy localizes 17 beta-estradiol dehydrogenase in organelles of 120 to 500 nm with moderate electron-dense matrices bounded by single membranes. Antibodies against the peroxisomal markers catalase and acyl-CoA oxidase recognize the same organelles in double-labeling studies. This is the first report on the participation of peroxisomes in the metabolism of estradiol.

Acyl-CoA Oxidase↗
PubMed1994

The effect of antisense inhibition of urokinase receptor in human squamous cell carcinoma on malignancy.

Concomitant expression of urokinase type plasminogen activator (uPA) and its surface receptor (uPAR) has been shown to correlate strongly with a more invasive tumor cell phenotype. A highly malignant human epidermoid carcinoma cell line (HEp3) was transfected with a vector capable of expressing an antisense transcript complementary to 300 bases of the 5' end of uPAR, including the ATG codon. Six stably transfected antisense (AS-2, 3, 5, 9, 10, 12) and eight control clones were characterized. All clones produced high levels of uPA activity. Examination of collagenase production and doubling time showed that all of the clones tested produced similar activities. The antisense clones showed a 20-74% reduction in the uPAR sites; the uPAR mRNA level was also reduced. A test of the invasive ability of all clones in a modified chorioallantoic membrane (CAM) showed that invasiveness of the antisense-inhibited clones was directly proportional to the density of surface uPAR. The AS-2 clone, which expressed the lowest number of uPARs showed a significantly reduced level of invasion. The invasiveness of additional AS-inhibited clones was also reduced. Seven control and four AS-inhibited clones were tested for tumorigenicity on CAMs of chick embryos. Inoculation of control cells produced large tumors, while the As clones were non-tumorigenic. AS-2 did not produce tumors even if kept in vivo for up to 10 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Allantois↗
PubMed1994

Molecular cloning and amino acid sequence of the porcine 17 beta-estradiol dehydrogenase.

We describe the cloning and sequencing of porcine 17 beta-estradiol dehydrogenase. The enzyme performs oxidation 360-fold more efficiently than reduction, both measured under optimal conditions. It is localized in specialized vesicles of epithelial cells. The cDNA clones were isolated from a lambda UNI ZAP XR library of porcine kidney and polymerase-chain-reaction-amplified from templates of uterus epithelium. In both tissues, the same enzyme is coded by a transcript of 2.9 kb. It contains a 69-b 5'-noncoding region, an open reading frame of 2211 b and a 3'-noncoding region of 624 b. The open reading frame of 737 amino acids with a predicted molecular mass 79,973 Da was confirmed by amino acid sequencing of peptides. The 80-kDa translation product is processed to the N-terminal 32-kDa enzyme, part of which is then covalently linked to actin. The estradiol dehydrogenase/actin complex and the 80-kDa translation product comigrate in SDS/PAGE.

Amino Acid Sequence↗
PubMed1994

Alterations in the subcellular distribution of 17 beta-estradiol dehydrogenase in porcine endometrial cells over the course of the estrous cycle.

The uteri of German landrace gilts slaughtered at different days of the cycle were processed for immunocytochemistry and biochemical analyses. Plasma was collected for hormone assays. The monoclonal antibody F1 against the structure-bound 17 beta-estradiol dehydrogenase of porcine endometrial epithelium was applied to rehydrated paraffin sections either as a direct, peroxidase-linked probe or in combination with a fluorescing secondary antibody. The oxidation of estradiol was measured in homogenates of tissue powdered in liquid nitrogen. Immunoreactivity was restricted to endometrial epithelium. In the glandular epithelium, faint dots of fluorescence became visible at day 4, which apparently coalesced to spherical structures of 2-4 microns diameter at the cell basis between days 11 through 17 before disappearing by day 18. A similar distribution was observed for the oxidation products of diaminobenzidine beginning with a faint uniform staining and followed by the appearance of intensely stained basal bodies persisting until day 17. Essentially the same time course was seen in the luminal epithelium but with a different distribution. Immunoreactive material amassed in the apical region of the cells, but the conspicuous aggregations were absent. Time course and intensities of the immunological responses are matched by the enzymatic activity measured in parallel. Both correlate with the plasma progesterone levels, suggesting an induction of the enzyme by the hormone. An involvement of the cytoskeleton in the sequence of subcellular distribution patterns is discussed.

Animals↗
PubMed1994

The sequence of porcine 80 kDa 17 beta-estradiol dehydrogenase reveals similarities to the short chain alcohol dehydrogenase family, to actin binding motifs and to sterol carrier protein 2.

The cDNA of porcine 17 beta-estradiol dehydrogenase codes for a polypeptide of 737 amino acids. The dehydrogenase activity of the 80 kDa translation product is located in its N-terminal 32 kDa fragment, which is the major form isolated from endometrial epithelium. beta-Actin co-purifies with some of the 32 kDa enzyme, which contains actin-binding motifs and is homologous to hydratase-dehydrogenase-epimerase of Candida tropicalis. The microbody-targeting signal AKI and sequences resembling sterol carrier protein 2 are present in the C-terminal part of the 80 kDa protein. The N- and C-terminal parts are connected by a sequence containing the putative protease recognition signal AAP.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1994

Characterization of estrone hydroxylase activities in porcine endometrial cells.

The oxidation of estradiol to estrone in porcine endometrial cells is succeeded by hydroxylation at either 6 alpha- or 7 alpha-. The products are devoid of receptor affinity. Their formation is inhibited by cytochrome P450 blockers like ketoconazol but not by chloroquine and analogues. The hydroxylation at 6 alpha- proceeds with KM = 1.9 x 10(-7) M, that at 7 alpha- with KM = 3.6 x 10(-7) M. The respective values for the cytochrome P450-reductase cosubstrate NADPH are 1.7 x 10(-5) M and 1.9 x 10(-5) M. The kinetic parameters of the enzymes are compatible with a metabolic sequence: estradiol-->estrone--> 6 alpha/17 alpha-estrone.

Alcohol Oxidoreductases↗
PubMed1994

Postoperative pain relief in children. A comparison between caudal bupivacaine and intramuscular diclofenac sodium.

Two hundred and fifty children undergoing herniotomy or orchidopexy under general anaesthesia were randomly allocated to receive pre-operatively either diclofenac sodium 1 mg.kg-1 given intramuscularly or a caudal injection of bupivacaine 0.25% 1 ml.kg-1 with or without adrenaline or no analgesia. Plasma diclofenac and beta-endorphin concentrations were determined in eight and 21 patients respectively. Postoperative pain was assessed by ward nurses who were blinded to the group allocation. Comparison with the control group showed diclofenac to be an effective analgesic. Caudal bupivacaine provided more pain-free children during the early postoperative hours, but later the need for pethidine as rescue analgesic was lower among the children who had received intramuscular diclofenac. Caudal analgesia abolished the stress-induced increase in plasma beta-endorphin level which was found in the children given diclofenac and in those who served as controls. Total plasma clearance of intramuscular diclofenac sodium appears to be higher in children than in adults. A single intramuscular dose of diclofenac significantly reduces the need for an opioid analgesic in children after inguinal herniotomy or orchidopexy, and owing to its long duration of action, it offers an alternative or complementary method of pain relief to caudal analgesia.

Analgesia, Epidural↗
PubMed1993

Linkage of 17 beta-oestradiol dehydrogenase to actin by epsilon-(gamma-glutamyl)-lysine in porcine endometrial cells.

We report on the discovery of interactions of porcine endometrial 17 beta-oestradiol dehydrogenase with actin. The 17 beta-oestradiol dehydrogenase of porcine uteri is an essentially unidirectional enzyme compounded in specialized organelles. The enzyme activity in Brij 35 extracts of the particulate fraction of epithelial cells sedimenting between 1800 and 11,000 g(av). was collected by immunoadsorption and eluted at low pH. The eluate contained three proteins of 32, 45 and 80 kDa as shown by SDS/PAGE and silver staining. They were identified by amino acid sequencing and immunotyping as oestradiol dehydrogenase (32 kDa), actin (45 kDa) and a covalent dehydrogenase-actin complex (80 kDa). Disulphides, aldimines, periodate-degradable bonds and hydrophobic interactions were excluded as linkages in the 80 kDa protein. The epsilon-(gamma-glutamyl)-lysine nature of the covalent cross-link was recognized by narrow-bore h.p.l.c. analysis of enzymic digests of electro-eluted 80 kDa material. An involvement of the actin anchor in positioning of the oestradiol dehydrogenase-containing organelles according to metabolic requirements is discussed.

Actins↗
PubMed1993

The 17 beta-oestradiol dehydrogenase of pig endometrial cells is localized in specialized vesicles.

Two monoclonal antibodies against the 17 beta-oestradiol dehydrogenase of pig endometrial cells have been used in localization studies with immunogold electron microscopy. The antibodies attach both to a fraction of dehydrogenase-rich cytoplasmic vesicles isolated from homogenates and to vesicles of similar appearance in cells. The vesicles are filled with electron-dense material. Their tagging intensity indicates a high degree of specialization. Endometrial cells from mature animals contain a host of dehydrogenase vesicles, and cells from prepubertal animals only a few. Functional aspects of the novel organelle are discussed.

Animals↗
PubMed1992

Purification and properties of oestradiol 17 beta-dehydrogenase extracted from cytoplasmic vesicles of porcine endometrial cells.

Porcine endometrial oestradiol-17 beta dehydrogenase was solubilized from the particulate fraction of homogenates sedimenting between 1200 g and 10,000 g by treatment with 0.4% Brij 35 in neutral buffers. The extracts were processed by successive passage through DEAE-Sepharose, Amberlyte XAD-2 and Blue-Sepharose, and the enzyme was collected from the washed affinity matrix at 0.8 M of a 0-2 M-KCl gradient. A genuine oestrone reductase was eluted at 1.9 M-KCl. The dehydrogenase pool was resolved by butyl-Sepharose chromatography into a major (80%) peak (EDHM) eluted at 0.8 M-(NH4)2SO4 and a very hydrophobic fraction (VHF) recovered at 0.1 M. EDHM was further purified by filtration through Sephadex G-200 and cation-exchange chromatography on Mono S. Sephacryl 300 was used for VHF followed by Mono S. Enrichments from the homogenate amounted to 1074-fold for EDHM and 632-fold for VHF. A single silver-stained band at 32 kDa is seen on SDS/PAGE of EDHM, and VHF contains additional bands at 45 and 80 kDa. Polyclonal antibodies (G436) raised against EDHM and the monoclonal antibody F1 raised against VHF recognize the single 32 kDa band in EDHM and both the 32 kDa and 80 kDa bands in composite VHF. The 45 kDa band of VHF reacts with neither. Monoclonal antibody W1 raised against EDHM only recognizes the 32 kDa peptide of EDHM and VHF. The specific activity for oestradiol oxidation amounts to 4081 mu-units/mg for EDHM and to 2402 mu-units/mg for VHF. Both possess a minimal (1/260) endogenous reductase activity and are devoid of 3 beta, 3 alpha- and 20 alpha-dehydrogenases. We consider EDHM to be authentic oestradiol-17 beta dehydrogenase of porcine endometrium. The composite VHF could reflect the situation of the enzyme in vivo or result from aggregations occurring during processing.

Animals↗
PubMed1991

Isolation of vesicles mediating the conversion of 17 beta-estradiol to estrone.

The 17 beta-estradiol dehydrogenase of porcine endometrium was found to be localized in specialized particles. Their isolation was achieved by successive partitioning of postmitochondrial supernatants in isopycnic Percoll gradients and on sucrose gradients in a vertical rotor. The purified structures, rich in estradiol dehydrogenase, equilibrate at 1.180 g/ml in sucrose gradients, are on average 150 nm in diameter, have electron-dense cores and are studded with particles of ribosome-like appearance. They do not coequilibrate with enzymic markers for the endoplasmic reticulum (P450 enzymes and RNA), mitochondria, the Golgi apparatus, and lysosomes. The estradiol dehydrogenase activity in the isolated particles was enriched 20-fold from the homogenate.

Animals↗
PubMed1989

Assignment of estradiol-17 beta dehydrogenase and of estrone reductase to cytoplasmic structures of porcine endometrium cells.

Homogenates of porcine endometrium contain substantial activity for the dehydrogenation of estradiol-17 beta but little for estrone reduction. Both activities are associated with cytoplasmic structures. The dehydrogenase is characterized by a pH 7.7 optimum, Km 2.2 x 10(-7) mol/l for estradiol and Km 4.4 x 10(-5) mol/l for the cosubstrate NAD+. The corresponding figures for the reductase are pH 6.6, Km 1.1 x 10(-6) mol/l for estrone and Km 2.1 x 10(-5) mol/l for the cosubstrate NADPH. The (mitochondrial/lysosomal) 17,000 x g particulate fraction contains a 52-fold higher dehydrogenase than reductase activity. The (microsomal) 200,000 x g particulate fraction is only 16-fold richer in dehydrogenase. Isopycnic centrifugations of the two fractions in Percoll gradients reveal that estrone reductase and the coequilibrating marker enzyme cytochrome c reductase occur in constant proportions, whereas the dehydrogenase/cytochrome c reductase ratios are different. Both, the kinetic data and the structural assignments speak in favour of individual enzymes catalyzing the dehydrogenation of estradiol and the reduction of estrone. All gradient fractions exhibiting dehydrogenase activity feature small, electrodense vesicles of 0.15-0.20 microns in diameter as a common structural element which might harbour the dehydrogenase.

17-Hydroxysteroid Dehydrogenases↗
PubMed1988

Harvesting and separation of two populations of lysosomes from porcine endometrium.

The lysosomes present in homogenates of porcine endometrium epithelium equilibrate in two density regions of Percoll gradients. Patterns with varying proportions between high and low density peaks are observed, when aliquots of a tissue sample are processed with different all-glass Potter-Elvejhem homogenizers. The described constant-tolerance shearing device (CTSD), in contrast, provides homogenate fractions with higher latencies and steady distribution patterns. They are characteristic for each of the six lysosomal markers and the six other structure-bound enzymes measured in gradient fractions of the particulate matter harvested between 600g and 17,000g. The 17,000g sediments of CTSD homogenates contain more than 40% of the total lysosomal enzymatic activities. Recoveries from Percoll gradients are between 93 and 101%. Enrichments in the high density region range from 35-fold (beta-glucosidase) to 82-fold (acid ribonuclease). Both lysosomal populations exhibit latencies between 89 and 94%. Our results indicate that light lysosomes can be artificially generated by inappropriate homogenization, which should be considered in experiments on the formation and maturation of lysosomes.

Animals↗