Membrane dopamine β-hydroxylase: a precursor for the soluble enzyme in the bovine adrenal medulla

• 1.1. Searching for endogenous proteolytic activities converting the membrane form of dopamine β-hydroxylase (dopamine β-monooxygenase, DBH) into the soluble and releasable form, DBH was monitored enzymatically and immunologically in aqueous and detergent-solubilized extracts of the adrenomedullary fractions.
• 2.2. Degradation of the soluble DBH and acidic chromogranins by activation of endogenous proteases occurred during lysis in H₂O.
• 3.3. Shifts in the hydrophobicity of the membrane DBH were also apparent. Loss in enzyme protein or activity was, on the other hand, not observed for bufier-dialysed CG (pH 5–6).
• 4.4. Limited proteolysis within the membrane phase was, however, indicated by the shift towards dominance of the intermediate hydrophobic DBH in the buffer-dialysed CG.
• 5.5. By two-dimensional, crossed immunoelectrophoresis with cationic detergent the microsomal DBH was immunologically identical to the granule-bound enzyme but differed from the latter in molecular heterogeneity and in susceptibility to proteolytic solubilization by endogenous protease activities.
• 6.6. DBH in the membranes of the chromaffin granules was proteolytically solubilized at pH 6–8 and the soluble DBH further degraded at pH 5.
• 7.7. The results indicate that a post-translational conversion of the amphiphilic DBH into the soluble form, initiated at the level of the microsomes, may continue within the light and the heavy granule fractions which contain several DBH-converting and degrading proteolytic activities with acid optima.

     

Immunocytochemical and biochemical studies on the localization and changes of 17 α-hydroxylase/C17–C20 lyase activity in immature rat ovary treated with PMSG and hCG

Summary The localization of cytochrome P-450 of 17 -hydroxylase/C17–C20 lyase (P-450_{17 , lyase}) and the changes of the enzyme activity were studied immunocytochemically and biochemically in the ovaries of immature rats treated with PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin). Immunocytochemically, P-450_{17 , lyase} was localized in both the theca interna cells and interstitial gland cells of the ovaries of immature rats treated with PMSG for 48 h. After hCG administration, the immunoreactive cells rapidly decreased in number in the PMSG-pretreated rat ovary. Namely, 6 h after the hCG injection, positive staining for P-450_{17 , lyase} was recognized only in a few theca interna cells, while 12 h after the injection to immunostained cells were detected in the ovary. Forty-eight hours after the hGC treatment (96 h after the PMSG injection), most of the theca interna cells and the interstitial gland cells became immunopositive for P-450_{17 , lyase} again. The 17 -hydroxylating activity of P-450_{17 , lyase} was 0.5, 0.22 and 0.03 nmol/min/mg protein in the ovarian microsomes of PMSG-treated, PMSG+hCG(3 h)-treated and PMSG+hCG(6 h)-treated rats, respectively. Changes of the hydroxylase activities in all the experimental groups are almost parallel to those of P-450 contents in the microsomes. These immunocytochemical and biochemical findings suggest that 1) P-450_{17 , lyase} is localized in both the theca interna cell and interstitial gland cell, and these cells are the main site of the androstenedione production in the ovary, and that 2) the decreased production of estrogen occurring just before ovulation is not brought about by the decreased activity of P-450_{17 , lyase}, but done by the loss of the enzyme.Supported by grants from the Ministry of Education, Science and Culture, Japan     

Patterns of 3β-hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ activity (3Δ-HSDH) was examined in the rhesus monkey ($\text{Macaca mulatta}$) placenta and fetal adrenal at 135 and 155–162 days of gestation. Activity was evaluated in microsomes by the conversion of [³H]pregnenolone to [³H]progesterone. There was a 7-fold increase in enzyme activity in the whole adrenal (minus medulla) between the two stages of development. Combining data from both periods, enzyme activity was greater in the outer than in the inner region of the adrenal. No stage-dependent change in placental activity was evident. The temporal patterns in 3β-HSDH activity are consistent with corticoid and progesterone patterns in the circulation. Thus, the level of 3β-HSDH activity may be rate limiting in both the fetal adrenal and placenta.Enzyme activity was assessed in incubations which included unex-tracted, heat-treated, 100,000 g tissue supernatants. In both placental and adrenal incubations, competitive inhibition was noted. Ethyl ether extracts of 100,000 g tissue supernatants also inhibited 3β-HSDH in the respective tissues. GLC analysis of these extracts revealed the presence of putative dehydroepiandrosterone. Hormone levels and the nature of the inhibition that were observed are compatible with the conclusion that dehydroepiandrosterone can inhibit the conversion of pregnenolone to progesterone $\text{in vivo}$. The physiological importance of this remains to be determined.     

3β-Hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal,testis and ovary during late gestation

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ (3β-HSDH) was measured in the rhesus monkey ($\text{Macaca mulatta}$) placenta, fetal adrenal (whole organ minus medulla), testis and ovary during late gestation (Days 145–162). Activities were evaluated from the conversion of [³H]-pregnenolone to [³H]progesterone. The maximum enzyme velocity (V_m) in adrenal microsomes (100,000 g pellet) was significantly higher (146 nmoles progesterone/h x mg^?1protein) than in microsomes from the other tissues. Testicular V_m was greater than either ovarian or placental V_m which were not different from one another (11.5 versus 1.9, 1.2 nmoles progesterone/h x mg^?1protein, respectively). Apparent Michaelis-Menten constants in the adrenal, placenta, testis and ovary averaged 1.8,2.5,0.27 and 0.16 μM, respectively. In some cases, substrate inhibition was noted. Estimated dissociation constants for pregnenolone were 2.3 μM (adrenal), 2.1 μM (placenta), 0.74 μM (testis) and 0.13 μM (ovary). 3β-HSDH was less active in a crude mitochondrial preparation from the fetal adrenal (10,000 g pellet) than in microsomes, whereas activity in the placenta and testis appeared to be equally distributed between mitochrondria and microsomes.Rate measurements were consistent with the apparent potentials of these organs to synthesize their characteristic hormones. Thus, 3β-HSDH activity may be an important rate determining step in hormone synthesis. The importance of substrate inhibition in progesterone formation remains to be assessed.     

17 β-hydroxysteroid dehydrogenase in monkey endometrium during the menstrual cycle and at the time of implantation

In order to further identify physiological similarities between 17β-hydroxysteroid dehydrogenase (HSD) in human and monkey endometrium, and to evaluate the role of estradiol-17β (E₂) oxidation to estrone (E₁) during periimplantation events, 30 rhesus monkeys were studied at different intervals of the nonfertile menstrual cycle (days 8, 12, 15, 18 and 24). Also, five pregnant monkeys provided endometrial tissue on day 24 of the fertile menstrual cycle, near the expected time of implantation. HSD activity in endometrium was low at midfollicular phase (day 8), increased to maximal levels (8-fold) during the periovulatory span (days 12 and 15),and was intermediate in mid to late luteal phase (days 18 and 24) in non-fertile menstrual cycles. In the absence of ovulation, HSD was low throughout. These enzyme data fit with a pattern of daily peripheral serum levels of E₂ and progesterone (P) and suggest that when the normal sequence of P follows elevated estrogens in late follicular phase, HSD activity is markedly enhanced in the early luteal phase. However, HSD activity in endometrium did not increase more in the fertile menstrual cycle, despite further elevations of serum P during rescue of the corpus luteum.     

Short-term bioaccumulation, circulation and metabolism of estradiol-17β in the oyster Crassostrea gigas

Steroids are active signal transmitters in Vertebrates. These roles have also been hypothesized in other Phyla and endocrine disrupting effects have been reported for different estrogen-like compounds in fishes and some marine invertebrates. As estradiol-17β has shown some physiological activities in the oyster and as estrogens or estrogen-like molecules can be present in water, we have investigated the bioaccumulation and metabolism of this estrogen in vivo in the oyster Crassostrea gigas. When dissolved in seawater, in less than 48 h estradiol-17β concentrated up to 31 times in the soft tissues of the suspension-feeder mollusc. Injected in the adductor muscle, estradiol-17β circulated from muscle to the gonad, the gills, the mantle, the labial palps, and to a lesser extent to the digestive gland. After 2 h, estradiol flow increased specifically towards this gland. Different hypotheses were raised concerning the circulation paths. However, in all cases estradiol metabolism primarily evidenced an in vivo transformation into estrone in the whole oyster and in its digestive gland. This strong 17β-hydroxysteroid-dehydrogenase activity confirms our previous in vitro results. In conclusion, it is proposed that oyster is able to take in charge estradiol as a potential contaminant in seawater. Therefore, its bioaccumulation and transformation into estrone could be studied as potential biomarkers of endocrine disruption. Furthermore, the experimental approach with dissolved steroids in the seawater combined to an anatomical screening appears as an interesting tool to investigate the bivalve endocrinology.     

Evidence for 6-keto-PGF1α in adrenal cortex of the rat and effects of 6-keto-PGF1α and PGI2 on adrenal cAMP levels and steroidogenesis

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF_1α, the hydrolysis metabolite of PGI₂, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF_1α was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF_2α or PGE₂. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI₂ metabolite. Studies $\text{in vitro}$, using isolated cortical cells exposed to 6-keto-PGF_1α (10^?6-10^?4M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI₂ (10^?6-10^?4M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering corticosterone production. ACTH (5–200 μU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI₂ produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused $\text{in situ}$ with PGI₂ showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF_1α is present in the rat adrenal cortex, its precursor, PGI₂, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Identification of a novel splicing mutation and 1-bp deletion in the 17α-hydroxylase gene of Japanese patients with 17α-hydroxylase deficiency

We report studies of two unrelated Japanese patients with 17α-hydroxylase deficiency caused by mutations of the 17α-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17α-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient’s mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17α-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation. Received: 15 November 1997 / Accepted: 15 March 1998     

Immunocytochemical localization of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase in the adrenal glands of the frog and rat by a peroxidase-antiperoxidase method

Summary The subcellular locilazations of tryrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) in the adrenal glands of the frog and rat have been examined by a peroxidase-antiperoxidase (PAP) method. TH was localized in the ground substance of the adrenaline-containing cells and noradrenaline-containing cells, but not in the nucleus or in the mitochondria. TH was also located on the outside of the membrane of the chromaffin granules. DBH was observed only inside the granules. PNMT was found not only in the ground substance but also on the membrane of some adrenaline-containing granules. Cortical lipid cells of the frog adrenals did not show TH-, DBH-, and PNMT-reactions. The negative reactions to TH-, DBH-, and PNMT-antiserum exhibited by the summer cells of the frog adrenals prove that they belong to the cortical cells.     

Steroid modification by filamentous fungus Drechslera sp.: Focus on 7-hydroxylase and 17β-hydroxysteroid dehydrogenase activities

Fungal strain Drechslera sp. Ph F-34 was shown to modify 3-oxo- and 3-hydroxy steroids of androstane series to form the corresponding allylic 7-alcohols and 17β-reduced derivatives thus evidencing the presence of 7α-, 7β-hydroxylase and 17β-hydroxysteroid dehydrogenase (17β-HSD) activities. The growing mycelium predominantly hydroxylated androsta-1,4-diene-3,17-dione (ADD) at the 7β-position, while much lower 7α-hydroxylation was observed. Along with 7β-hydroxy-ADD and its corresponding 7α-isomer, their respective 17β-alcohols were produced.In this study, transformation of ADD, androst-4-en-17β-ol-3-one (testosterone, TS) and 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone, DHEA) by resting mycelium of Drechslera sp. have been estimated in different conditions with regard to the inducibility and functionality of the 17β-HSD and 7-hydroxylase enzyme systems. Steroids of androstane, pregnane and cholane series were evaluated as inducers. The inhibitory analysis was provided using cycloheximide (CHX). Steroids were assayed using TLC and HPLC methods, and the structures were confirmed by mass-spectrometry, ¹H and ¹³C NMR spectroscopy data.17β-HSD of the mycelium constitutively reduced 17-carbonyl group of ADD and DHEA to form the corresponding 17β-alcohols, namely, androsta-1,4-diene-17β-ol-3-one (1-dehydro-TS), and androst-5-ene-3β,17β-diol. Production of the 7α- and 7β-hydroxylated derivatives depended on the induction conditions. The inducer effect relied on the steroid structure and decreased in the order: DHEA > pregnenolone > lithocholic acid. β-Sitosterol did not induce hydroxylase activity in Drechslera sp. CHX fully inhibited the synthesis of 7-hydroxylase in Drechslera mycelium thus providing selective 17-keto reduction.Results contribute to the diversity of steroid modifying enzymes in fungi and can be used at the development of novel biocatalysts for production of valuable steroid 7(α/β)- and 17β-alcohols.     

Preparturient changes in plasma concentrations of estrone,estradiol-17β and estradiol-17α in the fetal calf: Effects of dexamethasone infusion

The concentrations of total estrogens in fetal calf plasma were determined during a 6–10 day period immediately before delivery. Comparison was made between levels found in untreated calves and calves infused with dexamethasone at the rate of 0.1, 1.0 and 10 mg/24 hours. In untreated calves the plasma estrone, estradiol-17β and estradiol-17α levels remained relatively constant at 38 ± 7 ng ml^?1 (mean ± SEM n = 3), 46 ± 6 ng ml^?1 and 29 ± 5 ng ml^?1 respectively. Infusion with dexamethasone at 0.1 mg/24 hr (3 calves) and 1.0 mg/24 hr (3 calves) was without dramatic effect on plasma estrogen levels. However, in one fetus infused with 10.0 mg/24 hr the dexamethasone treatment may have caused a transitory rise in the levels of all estrogens examined.     

Synthesis of regioisomeric 17β-N-phenylpyrazolyl steroid derivatives and their inhibitory effect on 17α-hydroxylase/C17,20-lyase

The reaction of 3β-hydroxy-21-hydroxymethylidenepregn-5-en-3β-ol-20-one (1) with phenylhydrazine (2a) affords two regioisomers, 17β-(1-phenyl-3-pyrazolyl)androst-3-en-3β-ol (5a) and 17β-(1-phenyl-5-pyrazolyl)androst-5-en-3β-ol (6a). The direction of the ring-closure reactions of 1 with p-substituted phenylhydrazines (2b-e) depends strongly on the electronic features of the substituents. Oppenauer oxidation of 3β-hydroxy-17β-exo-heterocyclic steroids 5a-e and 6a-e yielded the corresponding Δ⁴-3-ketosteroids 9a-e and 10a-e. The inhibitory effects (IC₅₀) of these compounds on rat testicular C_17,20-lyase were investigated by means of an in vitro radioligand incubation technique.     

Transforming growth factor β3 in the fetal and neonatal rat testis: immunolocalization and effect on fetal Leydig cell function

The localization of transforming growth factor β3 (TGFβ3) in the fetal and neonatal testis (from fetal day 13.5 to postnatal day 6) was investigated by immunohistochemical staining with a specific polyclonal antibody raised against a synthetic peptide corresponding to residues 50–75 of TGFβ3. This antibody recognized 0.5 ng TGFβ3 in western blot analysis, but did not detect 25 ng TGFβ1 or TGFβ2. The immunolocalization of TGFβ3 in the fetal and neonatal testis changed throughout development. Immunostaining was present in the gonocytes by fetal day 13.5, persisted until postnatal day 3, and was heterogeneous in spermatogonia on postnatal day 6. The Sertoli cells contained no immunoreactivity at any age. The fetal-type Leydig cells were first immunostained for TGFβ3 on day 16.5 and staining became very intense from day 18.5 onward. Staining disappeared when the antibody was presaturated with the synthetic peptide, but persisted when the antibody was presaturated with a tenfold excess of the corresponding peptide from TGFβ2. Furthermore, we researched whether TGFβ3 could act as a local regulator of fetal Leydig cell function. In a dispersed fetal testicular cell system, TGFβ3 inhibited the LH-stimulated testosterone production by Leydig cells from 20.5-day-old fetuses. The inhibitory effect of TGFβ3 was equal to that observed with TGFβ1 or TGFβ2. When compared with our previous studies showing the immunolocalization of TGFβ1 and TGFβ2, the present study shows that TGFβ3 may have a specific role in the developing rat testis, but may also overlap the action of TGFβ1 and TGFβ2. Accepted: 8 June 1999     

Canadian Mennonites and individuals residing in the Friesland region of the Netherlands share the same molecular basis of 17α-hydroxylase deficiency

Summary A common mutation within the CYP17 gene that causes 17-hydroxylase deficiency, a form of congenital adrenal hyperplasia, has been found by direct sequencing of polymerase chain reaction (PCR) fragments of genomic DNA from six families residing in the Friesland region of the Netherlands. The mutation is a 4-base duplication within exon 8 of the CYP17 gene, which alters the reading frame encoding the C-terminal 26 amino acids of cytochrome P45017. This mutation has previously been found in two Canadian patients who are members of ostensibly unrelated Mennonite families. The Mennonite Churches derive their name from Menno Simons, an early leader of the sect in Friesland. Presumably this 4-base duplication appeared within the Friesian population prior to emigration of the Mennonites from the Netherlands.     

Amide analogs of antifungal dioxane–triazole derivatives: Synthesis and in vitro activities

A new series of triazole compounds possessing an amide-part were efficiently synthesized and their in vitro antifungal activities were investigated. The amide analogs showed excellent in vitro activity against Candida, Cryptococcus and Aspergillus species. The MICs of compound 23d against C. albicans ATCC24433, C. neoformans TIMM1855 and A. fumigatus ATCC26430 were ?0.008, 0.031 and 0.031 μg/mL, respectively, (MICs of fluconazole: 0.5, >4 and >4 μg/mL; MICs of itraconazole: 0.125, 0.25, 0.25 μg/mL). Furthermore, compound 23d was stable under acidic conditions.     

Caffeine and reduction of fetal ossification in the rat: fact or artifact?

This study was done to determine the gestational period during which the rat fetus is susceptible to reduction of skeletal ossification by caffeine. Caffeine, 100 mg/kg/day by gavage, caused the greatest reduction in ossification, as assessed by staining with alizarin red S, in fetuses exposed between day 16 to 19 of gestation, less in those treated between day 7 to 19, and markedly less in those receiving it between day 7 to 16; a single dose on day 19 had very little effect. This indicates that the fetus is most susceptible late in pregnancy. Bones in early stages of mineralization on day 20 showed a greater reduction in staining than those in later stages. Thus, caffeine appears to lower the rate of ossification rather than reduce its final extent. In the day 7 to 19 caffeine treatment group, but not in the day 16 to 19 group, maternal and fetal body weights were reduced, and 1.6% of the fetuses had aphalangia. The day 7 to 16 caffeine treatment reduced fetal body weight. This argues against an association between reduction in fetal weight and ossification. None of the treatments affected rates of resorption or litter size. A novel and important observation made is that the different caffeine treatments affected the staining by alizarin of both claws and bones in a qualitatively and quantitatively similar manner. Since claws are devoid of osteoid, this observation questions the specificity of alizarin for the assessment of the state of fetal ossification and raises doubt as to the significance of the observed action of caffeine on ossification.     

Granger causality in integrated GC–MS and LC–MS metabolomics data reveals the interface of primary and secondary metabolism

Metabolomics has emerged as a key technique of modern life sciences in recent years. Two major techniques for metabolomics in the last 10 years are gas chromatography coupled to mass spectrometry (GC–MS) and liquid chromatography coupled to mass spectrometry (LC–MS). Each platform has a specific performance detecting subsets of metabolites. GC–MS in combination with derivatisation has a preference for small polar metabolites covering primary metabolism. In contrast, reversed phase LC–MS covers large hydrophobic metabolites predominant in secondary metabolism. Here, we present an integrative metabolomics platform providing a mean to reveal the interaction of primary and secondary metabolism in plants and other organisms. The strategy combines GC–MS and LC–MS analysis of the same sample, a novel alignment tool MetMAX and a statistical toolbox COVAIN for data integration and linkage of Granger Causality with metabolic modelling. For metabolic modelling we have implemented the combined GC–LC–MS metabolomics data covariance matrix and a stoichiometric matrix of the underlying biochemical reaction network. The changes in biochemical regulation are expressed as differential Jacobian matrices. Applying the Granger causality, a subset of secondary metabolites was detected with significant correlations to primary metabolites such as sugars and amino acids. These metabolic subsets were compiled into a stoichiometric matrix N. Using N the inverse calculation of a differential Jacobian J from metabolomics data was possible. Key points of regulation at the interface of primary and secondary metabolism were identified.     

Multiple β-galactosidase activities in petunia hybrida: Separation and characterization

Using isoelectrofocusing (IEF), multiple forms of Petunia β-galactosidase activity could be detected. The β-galactosidase pattern showed only minor tissue-specific differences. There were, however, species-specific differences. Zea mays, for instance, showed two bands which differed from the zones obtained with Petunia preparations. Petunia and corn leaves were mixed and extracted commonly. The species-specific activity patterns remained unchanged.Petunia preparations were inactivated by 8 Murea. Following dialysis, enzymatic activity and the Petunia-specific pattern were restored. The same holds true for a mixture of Petunia and E. coli β-galactosidase preparations. On refocusing isolated Petunia zones, untreated or inactivated by 8 M urea and reactivated by dialysis, the original mobilities were shown. Therefore, it seems highly improbable that the β-galactosidase pattern was due to artefacts. Using a Petunia line which was ‘pure’, also in respect to its β-galactosidase pattern, the four main bands were preparatively separated by IEF and characterized. They showed the same pH optimum (4.3), the same temperature optimum (55°), the same inactivation kinetics by urea, the same sensitivity against Cl^?, and closely related K_m. values. In sucrose gradient centrifugation they invariably showed S values of 8–10. The multiple activities could not be separated by zone electrophoresis using various carrier systems, or by gel filtration. It seems possible that they represent forms which differ only in isoelectric points, not in MW.