Stefan Andersson

Bio: Stefan Andersson is an academic researcher from University of Texas Southwestern Medical Center. The author has contributed to research in topics: Testosterone & Isozyme. The author has an hindex of 35, co-authored 47 publications receiving 7890 citations. Previous affiliations of Stefan Andersson include Merck & Co. & University of Texas System.

Cloning, structure and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase

Abstract: The conversion of cholesterol into bile acids in the liver represents the major catabolic pathway for the removal of cholesterol from the body. In this complex biosynthetic pathway, at least 10 enzymes modify both the ring structure and side chain of cholesterol, resulting in the formation of the primary bile acids, cholic acid, and chenodeoxycholic acid. To gain insight into the details and regulation of this pathway, we have used protein sequencing and molecular cloning techniques to isolate and characterize a cDNA encoding the rabbit mitochondrial sterol 26-hydroxylase. This enzyme catalyzes the first step in the oxidation of the side chain of sterol intermediates in the biosynthesis of bile acids. The structure of the sterol 26-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, reveals it to be a mitochondrial cytochrome P-450. A signal sequence of 36 residues precedes a coding region of 499 amino acids, predicting a molecular weight of 56,657 for the mature protein. The identity of the 26-hydroxylase cDNA was further confirmed by expression in monkey COS cells employing a versatile eukaryotic expression vector. Blotting experiments revealed that the mRNA for this enzyme is expressed in many tissues and that it is encoded by a low copy number gene in the rabbit genome.

Deletion of steroid 5α-reductase 2 gene in male pseudohermaphroditism

Abstract: THE conversion of testosterone into dihydrotestosterone by steroid 5α-reductase is a key reaction in androgen action, and is essential both for the formation of the male phenotype during embryogenesis and for androgen-mediated growth of tissues such as the prostate1,2. Single gene defects that impair this conversion lead to pseudohermaphroditism in which 46 X, Y males have male internal urogenital tracts, but female external genitalia3. We have described the isolation of a human 5α-reductase complementary DNA from prostate4. Subsequent cloning and genetic studies showed that this gene (designated 5α-reductase 1) was normal in patients with Sor-reductase deficiency26. We report here the isolation of a second 5α-reductase cDNA by expression cloning and the polymerase chain reaction. The biochemical and pharmacological properties of this cDNA-encoded enzyme (designated 5α-reductase 2) are consistent with it being the major isozyme in genital tissue. A deletion in this gene is present in two related individuals with male pseudohermaphroditism caused by 5α-reductase deficiency. These results verify the existence of at least two 5α-reductases in man and provide insight into a fundamental hormone-mediated event in male sexual differentiation.

Male pseudohermaphroditism caused by mutations of testicular 17β-hydroxysteroid dehydrogenase 3

Abstract: Defects in the conversion of androstenedione to testosterone in the fetal testes by the enzyme 17β–hydroxysteroid dehydrogenase (17β–HSD) give rise to genetic males with female external genitalia. We have used expression cloning to isolate cDNAs encoding a microsomal 17β–HSD type 3 isozyme that shares 23% sequence identity with other 1 7β–HSD enzymes, uses NADPH as a cofactor, and is expressed predominantly in the testes. The 17βHSD3 gene on chromosome 9q22 contains 11 exons. Four substitution and two splice junction mutations were identified in the 17βHSD3 genes of five unrelated male pseudohermaphrodites. The substitution mutations severely compromised the activity of the 17β–HSD type 3 isozyme.

Structural and biochemical properties of cloned and expressed human and rat steroid 5 alpha-reductases.

Abstract: The microsomal enzyme steroid 5 alpha-reductase is responsible for the conversion of testosterone into the more potent androgen dihydrotestosterone. In man, this steroid acts on a variety of androgen-responsive target tissues to mediate such diverse endocrine processes as male sexual differentiation in the fetus and prostatic growth in men. Here we describe the isolation, structure, and expression of a cDNA encoding the human steroid 5 alpha-reductase. A rat cDNA was used as a hybridization probe to screen a human prostate cDNA library. A 2.1-kilobase cDNA was identified and DNA sequence analysis indicated that the human steroid 5 alpha-reductase was a hydrophobic protein of 259 amino acids with a predicted molecular weight of 29,462. A comparison of the human and rat protein sequences revealed a 60% identity. Transfection of expression vectors containing the human and rat cDNAs into simian COS cells resulted in the synthesis of high levels of steroid 5 alpha-reductase enzyme activity. Both enzymes expressed in COS cells showed similar substrate specificities for naturally occurring steroid hormones. However, synthetic 4-azasteroids demonstrated marked differences in their abilities to inhibit the human and rat steroid 5 alpha-reductases.

Cloning of a novel receptor expressed in rat prostate and ovary.

Abstract: We have cloned a novel member of the nuclear receptor superfamily. The cDNA of clone 29 was isolated from a rat prostate cDNA library and it encodes a protein of 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Clone 29 protein is unique in that it is highly homologous to the rat estrogen receptor (ER) protein, particularly in the DNA-binding domain (95%) and in the C-terminal ligand-binding domain (55%). Expression of clone 29 in rat tissues was investigated by in situ hybridization and prominent expression was found in prostate and ovary. In the prostate clone 29 is expressed in the epithelial cells of the secretory alveoli, whereas in the ovary the granuloma cells in primary, secondary, and mature follicles showed expression of clone 29. Saturation ligand-binding analysis of in vitro synthesized clone 29 protein revealed a single binding component for 17beta-estradiol (E2) with high affinity (Kd= 0.6 nM). In ligand-competition experiments the binding affinity decreased in the order E2 > diethylstilbestrol > estriol > estrone > 5alpha-androstane-3beta,17beta-diol >> testosterone = progesterone = corticosterone = 5alpha-androstane-3alpha,17beta-diol. In cotransfection experiments of Chinese hamster ovary cells with a clone 29 expression vector and an estrogen-regulated reporter gene, maximal stimulation (about 3-fold) of reporter gene activity was found during incubation with 10 nM of E2. Neither progesterone, testosterone, dexamethasone, thyroid hormone, all-trans-retinoic acid, nor 5alpha-androstane-3alpha,I7beta-diol could stimulate reporter gene activity, whereas estrone and 5alpha-androstane-3beta,17beta-diol did. We conclude that clone 29 cDNA encodes a novel rat ER, which we suggest be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.

Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15 (23):6541-6551

Abstract: Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12‐fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20‐ and 50‐fold in response to insulin and IGF‐1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3‐kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild‐type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase‐2. These experiments demonstrated that activation of PKBalpha by insulin or insulin‐like growth factor‐1 (IGF‐1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF‐1‐stimulated cells by an upstream kinase(s).

Mechanism of activation of protein kinase B by insulin and IGF-1.

Abstract: Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).

Neuroscience 細胞死：最近の知見

Abstract: 中枢神経系疾患の治療は正常細胞（ニューロン）の機能維持を目的とするが，脳血管障害のように機能障害の原因が細胞の死滅に基づくことは多い．一方，脳腫瘍の治療においては薬物療法や放射線療法といった腫瘍細胞の死滅を目標とするものが大きな位置を占める．いずれの場合にも，細胞死の機序を理解することは各種病態や治療法の理解のうえで重要である．現在のところ最も研究の進んでいる細胞死の型はアポトーシスである．そのなかで重要な位置を占めるミトコンドリアにおける反応および抗アポトーシス因子について概要を紹介する．