Theca interna: the other side of bovine follicular atresia

Currently, histological classifications of ovarian follicular atresia are almost exclusively based on the morphology of the membrana granulosa without reference to the theca interna. Atresia in the bovine small antral ovarian follicle has been redefined into antral or basal atresia where cell death commences initially within antral or basal regions of the membrana granulosa, respectively. To examine cell death in the theca interna in the two types of atretic follicles, bovine ovaries were collected and processed for immunohistochemistry and light microscopy. Follicles were classified as healthy, antral atretic, or basal atretic. Follicle diameter was recorded and sections stained with lectin from Bandeiraea simplicifolia to identify endothelial cells or with an antibody to cytochrome P450 cholesterol side-chain cleavage to identify steroidogenic cells and combined with TUNEL labeling to identify dead cells. The numerical density of steroidogenic cells within the theca interna was significantly reduced (P < 0.001) in basal atretic follicles in comparison with other follicles. Cell death was greater in both endothelial cells (P < 0.05) and steroidogenic cells (P < 0.01) of the theca interna of basal atretic follicles compared with healthy and antral atretic follicles. Thus, we conclude that the theca interna is susceptible to cell death early in atresia, particularly in basal atretic follicles.     

Sialic acid recognition by Vibrio cholerae neuraminidase

Vibrio cholerae neuraminidase (VCNA) plays a significant role in the pathogenesis of cholera by removing sialic acid from higher order gangliosides to unmask GM1, the receptor for cholera toxin. We previously showed that the structure of VCNA is composed of a central beta-propeller catalytic domain flanked by two lectin-like domains; however the nature of the carbohydrates recognized by these lectin domains has remained unknown. We present here structures of the enzyme in complex with two substrates, alpha-2,3-sialyllactose and alpha-2,6-sialyllactose. Both substrate complexes reveal the alpha-anomer of N-acetylneuraminic acid (Neu5Ac) bound to the N-terminal lectin domain, thereby revealing the role of this domain. The large number of interactions suggest a relatively high binding affinity for sialic acid, which was confirmed by calorimetry, which gave a Kd approximately 30 microm. Saturation transfer difference NMR using a non-hydrolyzable substrate, Neu5,9Ac2-2-S-(alpha-2,6)-GlcNAcbeta1Me, was also used to map the ligand interactions at the VCNA lectin binding site. It is well known that VCNA can hydrolyze both alpha-2,3- and alpha-2,6-linked sialic acid substrates. In this study using alpha-2,3-sialyllactose co-crystallized with VCNA it was revealed that the inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) was bound at the catalytic site. This observation supports the notion that VCNA can produce its own inhibitor and has been further confirmed by 1H NMR analysis. The discovery of the sialic acid binding site in the N-lectin-like domain suggests that this might help target VCNA to sialic acid-rich environments, thereby enhancing the catalytic efficiency of the enzyme.     

Mutations in LRP5 or FZD4 underlie the common familial exudative vitreoretinopathy locus on chromosome 11q

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder of the retinal vascular system. Autosomal dominant FEVR is genetically heterogeneous, but its principal locus, EVR1, is on chromosome 11q13-q23. The gene encoding the Wnt receptor frizzled-4 (FZD4) was recently reported to be the EVR1 gene, but our mutation screen revealed fewer patients harboring mutations than expected. Here, we describe mutations in a second gene at the EVR1 locus, low-density-lipoprotein receptor-related protein 5 (LRP5), a Wnt coreceptor. This finding further underlines the significance of Wnt signaling in the vascularization of the eye and highlights the potential dangers of using multiple families to refine genetic intervals in gene-identification studies.     

Nuclear overexpression of NAD(P)H:quinone oxidoreductase 1 in Chinese hamster ovary cells increases the cytotoxicity of mitomycin C under aerobic and hypoxic conditions

The effects of the subcellular localization of overexpressed bioreductive enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) on the activity of the antineoplastic agent mitomycin C (MC) under aerobic and hypoxic conditions were examined. Chinese hamster ovary (CHO-K1/dhfr(-)) cells were transfected with NQO1 cDNA to produce cells that overexpressed NQO1 activity in the nucleus (148-fold) or the cytosol (163-fold) over the constitutive level of the enzyme in parental cells. Subcellular localization of the enzyme was confirmed using antibody-assisted immunofluorescence. Nuclear localization of transfected NQO1 activity increased the cytotoxicity of MC over that produced by overexpression in the cytosol under both aerobic and hypoxic conditions, with greater cytotoxicity being produced under hypoxia. The greater cytotoxicity of nuclear localized NQO1 was not attributable to greater metabolic activation of MC but instead was the result of activation of the drug in close proximity to its target, nuclear DNA. A positive relationship existed between the degree of MC-induced cytotoxicity and the number of MC-DNA adducts produced. The findings indicate that activation of MC proximal to nuclear DNA by the nuclear localization of transfected NQO1 increases the cytotoxic effects of MC regardless of the degree of oxygenation and support the concept that the mechanism of action of MC involves alkylation of DNA.     

Missense mutations in ABCG5 and ABCG8 disrupt heterodimerization and trafficking

Mutations in ABCG5 (G5) or ABCG8 (G8) cause sitosterolemia, an autosomal recessive disease characterized by sterol accumulation and premature atherosclerosis. G5 and G8 are ATP-binding cassette (ABC) half-transporters that must heterodimerize to move to the apical surface of cells. We examined the role of N-linked glycans in the formation of the G5/G8 heterodimer to gain insight into the determinants of folding and trafficking of these proteins. Site-directed mutagenesis revealed that two asparagine residues (Asn(585) and Asn(592)) are glycosylated in G5 and that G8 has a single N-linked glycan attached to Asn(619). N-Linked glycosylation of G8 was required for efficient trafficking of the G5/G8 heterodimer, but mutations that abolished glycosylation of G5 did not prevent trafficking of the heterodimer. Both G5 and G8 are bound by the lectin chaperone, calnexin, suggesting that the calnexin cycle may facilitate folding of the G5/G8 heterodimer. To determine the effects of 13 disease-causing missense mutations in G5 and G8 on formation and trafficking of the G5/G8 heterodimer, mutant forms of the half-transporters were expressed in CHO-K1 cells. All 13 mutations reduced trafficking of the G5/G8 heterodimer from the endoplasmic reticulum to the Golgi complex, and most prevented the formation of stable heterodimers between G5 and G8. We conclude that the majority of the molecular defects in G5 and G8 that cause sitosterolemia impair transport of the sterol transporter to the cell surface.