Annika Lindqvist

Biochemical Properties of Purified Recombinant Human β-Carotene 15,15′-Monooxygenase

β-Carotene 15,15′-monooxygenase (BCO), formerly known as β-carotene 15,15′-dioxygenase, catalyzes the first step in the synthesis of vitamin A from dietary carotenoids. We have biochemically and enzymologically characterized the purified recombinant human BCO enzyme. A highly active BCO enzyme was expressed and purified to homogeneity from baculovirus-infectedSpodoptera frugiperda 9 insect cells. TheK m and V max of the enzyme for β-carotene were 7 μm and 10 nmol retinal/mg × min, respectively, values that corresponded to a turnover number (k cat) of 0.66 min−1 and a catalytic efficiency (k cat/K m ) of ∼105 m −1·min−1. The enzyme existed as a tetramer in solution, and substrate specificity analyses suggested that at least one unsubstituted β-ionone ring half-site was imperative for efficient cleavage of the carbon 15,15′-double bond in carotenoid substrates. High levels of BCO mRNA were observed along the whole intestinal tract, in the liver, and in the kidney, whereas lower levels were present in the prostate, testis, ovary, and skeletal muscle. The current data suggest that the human BCO enzyme may, in addition to its well established role in the digestive system, also play a role in peripheral vitamin A synthesis from plasma-borne provitamin A carotenoids.

alpha(1)-Microglobulin: a yellow-brown lipocalin

alpha(1)-Microglobulin, also called protein HC, is a lipocalin with immunosuppressive properties. The protein has been found in a number of vertebrate species including frogs and fish. This review summarizes the present knowledge of its structure, biosynthesis, tissue distribution and immunoregulatory properties. alpha(1)-Microglobulin has a yellow-brown color and is size and charge heterogeneous. This is caused by an array of small chromophore prosthetic groups, attached to amino acid residues at the entrance of the lipocalin pocket. A gene in the lipocalin cluster encodes alpha(1)-microglobulin together with a Kunitz-type proteinase inhibitor, bikunin. The gene is translated into the alpha(1)-microglobulin-bikunin precursor, which is subsequently cleaved and the two proteins secreted to the blood separately. alpha(1)-Microglobulin is found in blood and in connective tissue in most organs. It is most abundant at interfaces between the cells of the body and the environment, such as in lungs, intestine, kidneys and placenta. alpha(1)-Microglobulin inhibits immunological functions of white blood cells in vitro, and its distribution is consistent with an anti-inflammatory and protective role in vivo.

Male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase 3 deficiency. Diagnosis, psychological evaluation, and management.

Ten male pseudohermaphrodites with 17 beta-hydroxysteroid dehydrogenase 3 (17 beta-HSD3) deficiency were evaluated in 1 clinic with an average follow-up of 10.1 years. The diagnoses were made by demonstrating low to normal serum testosterone levels, high androstenedione levels, and high ratios of serum androstenedione to testosterone in the basal state or after treatment with human chorionic gonadotropin. The molecular features of the underlying mutations were identified in all 7 families. Two additional males in the same families are believed to be affected on the basis of history obtained from family members. All of the 46,XY individuals in these families were registered at birth and raised as females (despite the presence of ambiguous genitalia in all or most), and all virilized after the time of expected puberty due to a rise in serum testosterone to or toward the normal male range. The age at diagnosis varied from 4 to 37 years. Ten individuals were studied by the same psychologist, and change of gender role (social sex) from female to male occurred in 3 subjects and in the 2 presumed affected subjects not studied. The individual with the highest serum testosterone level maintained female sexual identity, and in 2 families some of the affected males changed gender role and others did not. Thus, while androgen action plays a role in the process, additional undefined psychological, social, and/or biologic factors must be determinants of gender identity/role behavior. Management of the 7 individuals who chose to maintain female sex roles included castration, clitoroplasty, vaginal enlargement procedures when appropriate, treatment of hirsutism, cricoid cartilage reduction, and estrogen replacement. Three of the 7 are married (2 twice), 1 is involved in a long-term heterosexual relationship, 1 is engaged to be married, and the other 2 are not married and not believed to be sexually active. The 3 subjects who changed gender role behavior to male underwent hypospadias repair, and 1 was given supplemental testosterone therapy. One of these men is divorced, and the other 2 (aged 29 and 35 years) are unmarried. The diagnosis in 8 of these subjects was made after the time of expected puberty; it is unclear whether the functional and social outcomes would have been different if the diagnosis had been made and therapy begun earlier in life.

Cell Type-specific Expression of β-Carotene 15,15′-Mono-oxygenase in Human Tissues

We studied the cell type-specific expression of human β-carotene 15,15′-mono-oxygenase (BCO1), an enzyme that catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A. Immunohistochemical analysis using two monoclonal antibodies against different epitopes of the protein revealed that BCO1 is expressed in epithelial cells in a variety of human tissues, including mucosa and glandular cells of stomach, small intestine, and colon, parenchymal cells in liver, cells that make up the exocrine glands in pancreas, glandular cells in prostate, endometrium, and mammary tissue, kidney tubules, and in keratinocytes of the squamous epithelium of skin. Furthermore, BCO1 is detected in steroidogenic cells in testis, ovary, and adrenal gland, as well as skeletal muscle cells. Epithelia in general are structures that are very sensitive to vitamin A deficiency, and although the extraintestinal function of BCO1 is unclear, the finding that the enzyme is expressed in all epithelia examined thus far leads us to suggest that BCO1 may be important for local synthesis of vitamin A, constituting a back-up pathway of vitamin A synthesis during times of insufficient dietary intake of vitamin A.

Deoxycorticosterone inactivation by AKR1C3 in human mineralocorticoid target tissues

Aldosterone is the principal endogenous mineralocorticoid in humans and regulates salt and water homeostasis. Cortisol, the major glucocorticoid, has high affinity for the mineralocorticoid receptor; however, 11beta-hydroxysteroid dehydrogenase type 2 converts cortisol to the inactive steroid cortisone in aldosterone target cells of the kidney, thus limiting the mineralocorticoid action of cortisol. Deoxycorticosterone (DOC) binds to the mineralocorticocoid receptor with high affinity and circulates at concentrations comparable to aldosterone. Severe DOC excess as is seen in 17alpha- and 11beta-hydroxylase deficiencies causes hypertension, and moderate DOC overproduction in late pregnancy is associated with hypertension. Here, we demonstrate that DOC is inactivated by the 20-ketosteroid reductase activity of the human AKR1C3 isozyme. Immunohistochemical analyses demonstrate that AKR1C3 is expressed in the mineralocorticoid-responsive epithelial cells of the renal cortical and medullary collecting ducts, as well as the colon. Our findings suggest that AKR1C3 protects the mineralocorticoid receptor from activation by DOC in mineralocorticoid target cells of the kidney and colon, analogous to cortisol inactivation by 11beta-hydroxysteroid dehydrogenase type 2.

Transforming Growth Factor β1 (TGFβ1) and Progesterone Regulate Matrix Metalloproteinases (MMP) in Human Endometrial Stromal Cells

CONTEXT Menstruation is preceded by progesterone withdrawal and endometrial matrix remodeling predominantly through induction of matrix metalloproteinases (MMP) and recruitment of invading neutrophils. DESIGN Using endometrial tissues from women during various phases of the menstrual cycle, we found that MMP2, MMP9, and MMP11 were up-regulated in the late secretory phase/premenstrual phase. Because TGFβ-responsive genes were also up-regulated in endometrium during this time, we tested the hypothesis that TGFβ1 and progesterone regulate expression of MMP in human endometrial stromal cells (HESC). RESULTS Treatment of HESC with TGFβ1 resulted in marked increases in MMP2 and MMP11 mRNA and pro- and active MMP2 activity. Progesterone inhibited TGFβ1-induced stimulation of MMP2 and MMP11 through its nuclear hormone receptors. Interestingly, TGFβ1 also decreased progesterone receptor (PR)-A and PR-B in HESC with a more pronounced effect on PR-A. CONCLUSIONS These data support the hypothesis that TGFβ1 has endogenous anti-progestational effects in HESC and that the opposing effects of progesterone and TGFβ1 are important in regulation of matrix integrity in human endometrium.

Interaction between streptococcal protein Arp and different molecular forms of human immunoglobulin A

Protein Arp, the IgA-binding protein of the group A Streptococcus, has affinity for the Fc-part of IgA. The binding between protein Arp and several different molecular forms of human IgA was characterized. It was found that protein Arp bound with higher affinity to uncomplexed forms of IgA than to complexed forms (secretory IgA, alpha 1-antitrypsin-IgA and alpha 1-microglobulin-IgA). Thus, the affinity constant was 2.0-5.9 x 10(8) M-1 for the binding to monomeric, dimeric, trimeric, and quadrimeric IgA, and 4.5-5.0 x 10(7) M-1 for binding to the complexed forms. Among the uncomplexed IgA-molecules, the affinity constant was in the same range for J chain-containing forms (dimeric, trimeric and quadrimeric IgA) as for forms without J chain (monomeric and a particular quadrimeric IgA devoid of J chain). Western blotting demonstrated that protein Arp bound exclusively to the alpha-chain of all IgA-forms. Several lines of evidence pointed to a localization of the binding site to the C alpha 3-domain. First, protein Arp did not bind to three N-terminal alpha-chain fragments which lacked a region corresponding to the C alpha 3-domain, including that form a four-chain myeloma IgA, naturally occurring in plasma. Second, the binding to dimeric and tri/quadrimeric IgA was partially blocked by an added secretory component, which has been suggested to bind to the C alpha 2- and C alpha 3-domains of the alpha-chain. Finally, alpha 1-antitrypsin and alpha 1-microglobulin, in the weakly binding IgA-complexes, have been shown to be linked to the C alpha 3-domain via the penultimate amino acid residue of the alpha-chain peptide, supporting the hypothesis of a localization of the binding site of protein Arp to the C alpha 3-domain.

The impact of reference gene selection in quantification of gene expression levels in guinea pig cervical tissues and cells

BackgroundAccurate measurements of mRNA expression levels in tissues or cells are crucially dependent on the use of relevant reference genes for normalization of data. In this study we used quantitative real-time PCR and two Excel-based applets (geNorm and BestKeeper) to determine the best reference genes for quantification of target gene mRNA in a complex tissue organ such as the guinea pig cervix.ResultsGene expression studies were conducted in cervical epithelium and stroma during pregnancy and parturition and in cultures of primary cells from this tissue. Among 15 reference gene candidates examined, both geNorm and BestKeeper found CLF1 and CLTC to be the most stable in cervical stroma and cervical epithelium, ACTB and PPIB in primary stroma cells, and CLTC and PPIB in primary epithelial cells. The order of stability among the remaining candidate genes was not in such an agreement. Commonly used reference such as GAPDH and B2M demonstrated lower stability. Determination of pairwise variation values for reference gene combinations using geNorm revealed that the geometric mean of the two most stable genes provides sufficient normalization in most cases. However, for cervical stroma tissue in which many reference gene candidates displayed low stability, inclusion of three reference genes in the geometric mean may improve accuracy of target gene expression level analyses. Using the top ranked reference genes we examined the expression levels of target gene PTGS2 in cervical tissue and cultured cervical cells. We compared the results with PTGS2 expression normalized to the least stable gene and found significant differences in gene expression, up to 10-fold in some samples, emphasizing the importance of appropriately selecting reference genes.ConclusionsWe recommend using the geometric mean of CFL1 and CLTC for normalization of qPCR studies in guinea pig cervical tissue studies, ACTB and PPIB in primary stroma cells and CLTC and PPIB in primary epithelial cells from guinea pig.