Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.     

The parturition defect in steroid 5alpha-reductase type 1 knockout mice is due to impaired cervical ripening.

Successful delivery of the fetus (parturition) depends on coordinate interactions between the uterus and cervix. A majority (70%) of mice deficient in the type 1 isozyme of steroid 5alpha-reductase fail to deliver their young at term and thus manifest a parturition defect. Using in vitro and in vivo measurements we show here that rhythmic contractions of the uterus occur normally in these mutant mice at the end of gestation. In contrast, the cervix of the mutant animal fails to ripen at term as judged by biomechanical, histological, and endocrinological assays. Impaired metabolism of progesterone in the cervix of the mutant mice in late gestation leads to an accumulation of this steroid in the tissue. We conclude that a failure of cervical ripening underlies the parturition defect in mice lacking steroid 5alpha-reductase type 1 and that this enzyme normally plays an essential role in cervical progesterone catabolism at the end of pregnancy.     

Human aldehyde dehydrogenase. Activity with aldehyde metabolites of monoamines, diamines, and polyamines

Two isozymes (E1 and E2) of human aldehyde dehydrogenase (EC 1.2.1.3) were purified to homogeneity 13 years ago and a third isozyme (E3) with a low Km for gamma-aminobutyraldehyde only recently. Comparison with a variety of substrates demonstrates that substrate specificity of all three isozymes is broad and similar. With straight chain aliphatic aldehydes (C1-C6) the Km values of the E3 isozyme are identical with those of the E1 isozyme. All isozymes dehydrogenate naturally occurring aldehydes, 5-imidazoleacetaldehyde (histamine metabolite) and acrolein (product of beta-elimination of oxidized polyamines) with similar catalytic efficiency. Differences between the isozymes are in the Km values for aminoaldehydes. Although all isozymes can dehydrogenate gamma-aminobutyraldehyde, the Km value of the E3 isozyme is much lower: the same appears to apply to aldehyde metabolites of cadaverine, agmatine, spermidine, and spermine for which Km values range between 2-18 microM and kcat values between 0.8-1.9 mumol/min/mg. Thus, the E3 isozyme has properties which make it suitable for the metabolism of aminoaldehydes. The physiological role of E1 and E2 isozymes could be in dehydrogenation of aldehyde metabolites of monoamines such as 3,4-dihydroxyphenylacetaldehyde or 5-hydroxyindoleacetaldehyde; the catalytic efficiency with these substrates is better with E1 and E2 isozymes than with E3 isozyme. Isoelectric focusing of liver homogenates followed by development with various physiological substrates together with substrate specificity data suggest that aldehyde dehydrogenase (EC 1.2.1.3) is the only enzyme in the human liver capable of catalyzing dehydrogenation of aldehydes arising via monoamine, diamine, and plasma amine oxidases. Although the enzyme is generally considered to function in detoxication, our data suggest an additional function in metabolism of biogenic amines.     

Steroid metabolism and profile of steroidogenic gene expression in Episkin: high similarity with human epidermis

The skin is a well-recognized site of steroid formation and metabolism. Episkin is a cultured human epidermis. In this report, we investigate whether Episkin possesses a steroidogenic machinery able to metabolize adrenal steroid precursors into active steroids. Episkin was incubated with [14C]-dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) and their metabolites were analyzed by liquid chromatography/mass spectrometry (LC/MS/MS). The results show that the major product of DHEA metabolism in Episkin is DHEA sulfate (DHEAS) (88% of the metabolites) while the other metabolites are 7alpha-OH-DHEA (8.2%), 4-dione (1.3%), 5-androstenediol (1.3%), dihydrotestosterone (DHT) (1.4%) and androsterone (ADT) (2.3%). When 4-dione is used as substrate, much higher levels of C19-steroids are produced with ADT representing 77% of the metabolites. These data indicate that 5alpha-reductase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 3alpha-hydroxysteroid dehdyrogenase (3alpha-HSD) activities are present at moderate levels in Episkin, while 3beta-HSD activity is low and represents a rate-limiting step in the conversion of DHEA into C19-steroids. Using realtime PCR, we have measured the level of mRNAs encoding the steroidogenic enzymes in Episkin. A good agreement is found between the mRNAs expression in Episkin and the metabolic profile. High expression levels of steroid sulfotransferase SULT2B1B and type 3 3alpha-HSD (AKR1C2) correspond to the high levels of DHEA sulfate (DHEAS) and ADT formed from DHEA and 4-dione, respectively. 3beta-HSD is almost undetectable while the other enzymes such as type 1 5alpha-reductase, types 2, 4, 5, 7, 8, and 10 17beta-HSD and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) (AKR1C1) are highly expressed. Except for UGT-glucuronosyl transferase, similar mRNA expression profiles between Episkin and human epidermis are observed.     

Effects of testosterone on brain mRNA levels of steroid 5alpha-reductase isozymes in early postnatal life of rat

The enzyme 5alpha-reductase (5alpha-R) (EC 1.3.99.5) exists as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2), and both are present in the brain. 5alpha-R1 has been proposed as a constitutive enzyme that essentially plays a catabolic and neuron protective role whereas 5alpha-R2 has been associated with sexually dimorphic functions of the male. In this work, we studied the effects of testosterone (T), the masculinizing hormone of the central nervous system (CNS), on mRNA levels of both 5alpha-R isoforms in the prefrontal cortex of male and female rats during the postnatal sexual differentiation of the CNS in the rat, using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis (LIF-CE). We found an increase in 5alpha-R2 mRNA levels in both male and female rats after T treatment, while 5alpha-R1 mRNA levels were decreased in the same experimental conditions. Our results clearly indicated that T regulates the expression of both 5alpha-R1 and 5alpha-R2 genes in an opposite manner and independently of the sex. This could point to a crucial role of T in the sexual dimorphism for both 5alpha-R isozymes in the neonatal brain. These results open up a new research line that could improve understanding of the role of 5alpha-R isozymes in the physiology of the CNS.     

Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver

The regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by five isozymes of cytochrome P-450 purified from phenobarbital-induced rat liver were studied in a reconstituted monooxygenase system using testosterone (T) and androst-4-ene-3,17-dione (delta 4-A) as substrates. P-450 PB-3, an isozyme exhibiting low catalytic activity with many xenobiotic substrates, catalyzed efficient (turnover = 15.7 to 18.5 min-1 P-450-1 at 25 microM substrate) and highly stereoselective B-ring hydroxylations of both steroid substrates, with the corresponding 7 alpha- and 6 alpha-hydroxy alcohols formed in ratios of approximately 20 to 30:1, respectively. P-450 PB-2c metabolized testosterone to a mixture of 16 alpha OH-T, 2 alpha OH-T, and delta 4-A (product ratio = 1.0/0.78/0.33; turnover = 10.2 min-1 P-450-1). PB-2c is present in significantly larger amounts in mature male rats as compared to immature males, and probably catalyzes the male-specific testosterone 16 alpha-hydroxylase activity known to be induced at puberty and subject to endocrine control. P-450 PB-4, the major phenobarbital-induced isozyme in rat liver, catalyzed efficient D-ring hydroxylations, yielding 16 beta OH- delta 4-A as the predominant product with delta 4-A as substrate (turnover = 12.0 min-1 P-450-1) and a mixture of 16 beta OH-T, 16 alpha OH-T, and delta 4-A (the latter compound presumably formed via 17 alpha hydroxylation) with testosterone as substrate (turnover = 5.2 min-1 P-450-1). P-450 isozymes PB-1 and PB-5 hydroxylated both steroids with essentially the same regioselectivity as PB-4 but at only 5 to 10% the catalytic rate. Cytochrome b5 stimulated most of these steroid hydroxylations up to 2-fold with no change in regio- or stereoselectivity. The identification of specific steroid metabolites as diagnostic of particular P-450 isozymes should be useful for the assessment of isozymic contributions to microsomal activities and, in addition, facilitate comparisons of P-450 isozymes isolated in different laboratories.     

Selective non-steroidal inhibitors of 5 alpha-reductase type 1

The enzyme 5 alpha-reductase (5 alpha R) catalyses the reduction of testosterone (T) into the more potent androgen dihydrotestosterone (DHT). The abnormal production of DHT is associated to pathologies of the main target organs of this hormone: the prostate and the skin. Benign prostatic hyperplasia (BPH), prostate cancer, acne, androgenetic alopecia in men, and hirsutism in women appear related to the DHT production. Two isozymes of 5 alpha-reductase have been cloned, expressed and characterized (5 alpha R-1 and 5 alpha R-2). They share a poor homology, have different chromosomal localization, enzyme kinetic parameters, and tissue expression patterns. Since 5 alpha R-1 and 5 alpha R-2 are differently distributed in the androgen target organs, a different involvement of the two isozymes in the pathogenesis of prostate and skin disorders can be hypothesized. High interest has been paid to the synthesis of inhibitors of 5 alpha-reductase for the treatment of DHT related pathologies, and the selective inhibition of any single isozyme represents a great challenge for medical and pharmaceutical research in order to have more specific drugs. At present, no 5 alpha R-1 inhibitor is marketed for the treatment of 5 alpha R-1 related pathologies but pharmaceutical research is very active in this field. This paper will review the major classes of 5 alpha R inhibitors focusing in particular on non-steroidal inhibitors and on structural features that enhance the selectivity versus the type 1 isozyme. Biological tests to assess the inhibitory activity towards the two 5 alpha R isozymes will be also discussed.     

Adipose tissue intracrinology: potential importance of local androgen/estrogen metabolism in the regulation of adiposity.

The present article summarizes some of the studies available on steroid hormone conversion through the specific expression of steroidogenic enzymes in adipose tissue (adipose tissue intracrinology) and discusses the potential impact of local adipose tissue steroid metabolism on the regulation of adipocyte function and other metabolic parameters. Several studies have demonstrated significant steroid hormone uptake and conversion by adipose tissues from various body sites and in various cell fractions. Activities and/or mRNAs of aromatase, 3beta-hydroxysteroid dehydrogenase (HSD), 3alpha-HSD, 11beta-HSD, 17beta-HSD, 7alpha-hydroxylase, 17alpha-hydroxylase, 5alpha-reductase and UDP-glucuronosyltransferase 2B15 have been detected in adipose tissue or adipose cells. These studies have demonstrated potentially important roles for these enzymes in obesity, central fat accumulation, and the metabolic syndrome. Future studies on adipose tissue intracrinology will contribute further to our understanding of steroid action in adipocytes.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulatory effects of indomethacin on androgen metabolism in human gingival and oral periosteal fibroblasts.

The non-steroidal anti-inflammatory agent indomethacin (I) suppresses gingival inflammation and alveolar bone resorption. Androgens particularly 5 alpha-dihydrotestosterone (DHT) have anabolic effects on connective tissue and bone matrices. Human oral periosteal fibroblasts (HPF) and gingival fibroblasts (HGF) instigate healing in inflammatory periodontal lesions. The aim of this investigation was to compare the modulatory effects of I on the metabolism of two androgen substrates in human oral periosteal and gingival fibroblasts in culture. Monolayer cultures of both cell types (5(th)-9(th) passage) were established in Eagle's MEM and incubated with 14C-testosterone/14C-4-androstenedione and serial concentrations of I (0.5-50 microg/ml) for 24 h. The steroid metabolites were solvent extracted from the medium, separated by TLC and quantified using a radioisotope scanner. Both androgen substrates were metabolized mainly to DHT and 4-androstenedione/testosterone respectively, expressing 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity in both HPF and HGF. There were 51% and 73% increases in the levels of DHT over controls, with HGF and HPF respectively (n = 6; n = 4, P < 0.01) in response to I at 1-5 microg/ml, often reaching control values at 50 microg/ml. The expression of 17 beta-HSD activity showed less stimulation than the levels of DHT. Both androgen substrates were effective in this metabolic conversion, which is applicable to healing responses in both males and females in vivo. There were 57% increases (n = 4; P < 0.01) over controls, in the formation of androstanediol from 14C-4-androstenedione at 10 microg of I, in HPF. This transformation may regulate androgen action in androgen-dependent tissue. In addition to its anti-inflammatory properties, indomethacin can contribute to anabolic reparatory responses, by increasing the expression of steroid metabolizing enzymes in gingival and periosteal fibroblasts, in the inflammatory periodontal lesion.     

Glycolate oxidase isozymes are coordinately controlled by GLO1 and GLO4 in rice

Glycolate oxidase (GLO) is a key enzyme in photorespiratory metabolism. Four putative GLO genes were identified in the rice genome, but how each gene member contributes to GLO activities, particularly to its isozyme profile, is not well understood. In this study, we analyzed how each gene plays a role in isozyme formation and enzymatic activities in both yeast cells and rice tissues. Five GLO isozymes were detected in rice leaves. GLO1 and GLO4 are predominately expressed in rice leaves, while GLO3 and GLO5 are mainly expressed in the root. Enzymatic assays showed that all yeast-expressed GLO members except GLO5 have enzymatic activities. Further analyses suggested that GLO1, GLO3 and GLO4 interacted with each other, but no interactions were observed for GLO5. GLO1/GLO4 co-expressed in yeast exhibited the same isozyme pattern as that from rice leaves. When either GLO1 or GLO4 was silenced, expressions of both genes were simultaneously suppressed and most of the GLO activities were lost, and consistent with this observation, little GLO isozyme protein was detected in the silenced plants. In contrast, no observable effect was detected when GLO3 was suppressed. Comparative analyses between the GLO isoforms expressed in yeast and the isozymes from rice leaves indicated that two of the five isozymes are homo-oligomers composed of either GLO1 or GLO4, and the other three are hetero-oligomers composed of both GLO1 and GLO4. Our current data suggest that GLO isozymes are coordinately controlled by GLO1 and GLO4 in rice, and the existence of GLO isozymes and GLO molecular and compositional complexities implicate potential novel roles for GLO in plants.     

Evaluation of 4'-substituted bicyclic pyridones as non-steroidal inhibitors of steroid 5alpha-reductase

4'-Substituted bicyclic pyridones were prepared and evaluated as non-steroidal inhibitors of type 1 and 2 steroid 5alpha-reductase (SR). A range of 4'-substituents were incorporated into the bicyclic scaffold to investigate SAR within and across different classes of non-steroidal inhibitors of SR. Bicyclic pyridones containing a 4'-benzoyl or long carbon chain tether showed more potent inhibition against type 1 SR than inhibitors with N-substituted acetamide groups in the 4'-position. SAR derived from 4'-substituted bicyclic pyridones reported here do not correlate with SAR derived from known potent 4'-substituted biaryl acid SR inhibitors. A 4'-benzoyl group is favoured by the active site in both isozymes.     

Enzymes as modulators in malignant transformation

Experimental data suggest that sex steroids have a role in the development of breast and prostate cancers. The biological activity of sex steroid hormones in target tissues is regulated by several enzymes, including 17β-hydroxysteroid dehydrogenases (17HSD). Changes in the expression patterns of these enzymes may significantly modulate the intracellular steroid content and play a pathophysiological role in malignant transformation. To further clarify the role of 17HSDs in breast cancer, we analyzed the mRNA expressions of the 17HSD type 1, 2, and 5 enzymes in 794 breast carcinoma specimens. Both 17HSD type 1 and 2 mRNAs were detected in normal breast tissue from premenopausal women but not in specimens from postmenopausal women. Of the breast cancer specimens, 16% showed signals for 17HSD type 1 mRNA, 25% for type 2, and 65% for type 5. No association between the 17HSD type 1, 2, and 5 expressions was detected. The patients with tumors expressing 17HSD type 1 mRNA or protein had significantly shorter overall and disease-free survival than the other patients. The expression of 17HSD type 5 was significantly higher in breast tumor specimens than in normal tissue. The group with 17HSD type 5 overexpression had a worse prognosis than the other patients. Cox multivariate analyses showed that 17HSD type 1 mRNA, tumor size, and ER had independent prognostic significance.

Using an LNCaP prostate cancer cell line, we developed a cell model to study the progression of prostate cancer. In this model, androgen-sensitive LNCaP cells are transformed in culture conditions into more aggressive, androgen-independent cells. The model was used to study androgen and estrogen metabolism during the transformation process. Our results indicate that substantial changes in androgen and estrogen metabolism occur in the cells during the process. A remarkable decrease in oxidative 17HSD activity was seen, whereas reductive activity seemed to increase. Since local steroid metabolism controls the bioavailability of active steroid hormones of target tissues, the variations in steroid-metabolizing enzymes during cancer progression may be crucial in the regulation of the growth and function of organs.     

Sexual dimorphism of growth plate prehypertrophic and hypertrophic chondrocytes in response to testosterone requires metabolism to dihydrotestosterone (DHT) by steroid 5-alpha reductase type 1

Rat costochondral growth plate chondrocytes exhibit sex-specific and cell maturation dependent responses to testosterone. Only male cells respond to testosterone, although testosterone receptors are present in both male and female cells, suggesting other mechanisms are involved. We examined the hypothesis that the sex-specific response of rat costochondral cartilage cells to testosterone requires further metabolism of the hormone to dihydrotestosterone (DHT). Resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) chondrocytes from male and female Sabra strain rats exhibited sex-specific responses to testosterone and DHT: only male cells were responsive. Testosterone and DHT treatment for 24 h caused a comparable dose-dependent increase in [3H]-thymidine incorporation in quiescent preconfluent cultures of male GC cells, and a comparable increase in alkaline phosphatase specific activity in confluent cultures. RC cells responded in a differential manner to testosterone and DHT. Testosterone decreased DNA synthesis in male RC cells but DHT had no effect and alkaline phosphatase specific activity of male RC cells was unaffected by either hormone. Inhibition of steroid 5alpha-reductase activity with finasteride (1, 5, or 10 microg/ml), reduced the response of male GC cells to testosterone in a dose-dependent manner, indicating that metabolism to DHT was required. RT-PCR showed that both male and female cells expressed mRNAs for steroid 5alpha-reductase type 1 but lacked mRNAs for the type 2 form of the enzyme. Male cells also exhibited 5alpha-reductase activity but activity of this enzyme was undetectable in female cells. These observations show that sex-specific responses of rat growth zone chondrocytes to testosterone requires the further metabolism of the hormone to DHT and that the effect of DHT in the male growth plate is maturation-state dependent. Failure of female chondrocytes to respond to testosterone may reflect differences in testosterone metabolism, since these cells possess greater ability to aromatize the hormone to estradiol.     

Cytochromes P450 involved with benzene metabolism in hepatic and pulmonary microsomes

Benzene is an occupational hazard and environmental toxicant found in cigarette smoke, gasoline, and the chemical industry. The major health concern associated with benzene exposure is leukemia. The toxic effects of benzene are dependent on its metabolism by the cytochrome P450 enzyme system. Previous research has identified CYP2E1 as the primary P450 isozyme responsible for benzene metabolism at low concentrations, whereas CYP2B1 is involved at higher concentrations. Our studies using microsomal preparations from human, mouse, and rat indicate that CYP2E1 is the P450 isozyme primarily responsible for benzene metabolism in lung and in liver. CYP2B isozymes have little involvement in benzene metabolism by either lung or liver. Our results also indicate that isozymes of the CYP2F subfamily may play a role in benzene metabolism by lung.     

Effects of swim stress on mRNA and protein levels of steroid 5alpha-reductase isozymes in prefrontal cortex of adult male rats

The metabolite of progesterone, allopregnanolone, is among the most potent known ligands of the gamma-aminobutyric acid receptor complex (GABA(A)-R) in the central nervous system. This neuroactive steroid is markedly increased in an animal model of acute stress. Allopregnanolone is synthesized from progesterone by steroidogenic enzymes 5alpha-reductase (5alpha-R) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), with the former being the rate-limiting enzyme in this reaction sequence. In this paper, a quantitative RT-PCR method coupled to laser-induced fluorescence capillary electrophoresis (LIF-CE) and Western blot were used to measure both mRNA and protein levels of 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2) isozymes in prefrontal cortex of male rats after acute swim stress situations. Our results demonstrate that both 5alpha-R isozymes are significantly higher in prefrontal cortex of male rats after acute swim stress in comparison with control rats. These data may open up a new research line that could improve our understanding of the role of 5alpha-R isozymes in processes that accompany stress situations.     

Expression cloning and regulation of steroid 5 alpha-reductase, an enzyme essential for male sexual differentiation

The conversion of testosterone into the more potent androgen, dihydrotestosterone, catalyzed by the enzyme steroid 5 alpha-reductase, is required for the differentiation of male external genitalia. Here, we report the isolation of cDNA clones encoding the rat steroid 5 alpha-reductase using expression cloning in Xenopus oocytes. DNA sequence analysis demonstrates that the liver and ventral prostate forms of steroid 5 alpha-reductases are identical hydrophobic proteins of 29 kDa. The amount of steroid 5 alpha-reductase mRNA in liver increased in response to castration, but remained unchanged in the prostate. Testosterone administration to castrates induced expression of mRNA in the prostate but had no effect on liver. The data suggest that the steroid 5 alpha-reductase gene is differentially regulated by testosterone in androgen-responsive versus non-responsive tissues.     

Characterization of two lignin peroxidase clones from Phanerochaete chrysosporium

Two cDNA clones encoding lignin peroxidase isozymes from Phanerochaete chrysosporium have been isolated and characterized. One of the clones, lambda ML-4, encodes isozyme H8 as does the previously reported clone lambda ML-1 [Tien, M. and Tu, C.-P.D. Nature 326 (1987) 520-523; 328, 742]. Our data are consistent with lambda ML-1 and lambda ML-4 being allelic variants. The other clone, lambda ML-5, encodes a homologous isozyme. We have also isolated the genomic clone corresponding to lambda ML-4 cDNA. Conserved residues thought to be essential for peroxidase function were identified in the predicted amino acid sequences of both cDNA clones. Northern blot analyses indicate that these isozymes are expressed during secondary metabolism, appearing on day 4 of growth and increasing on days 5 and 6.