BMP7 acts in murine lens placode development.
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Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation. (+info)
Selective effects of a 4-oxystilbene derivative on wild and mutant neuronal chick alpha7 nicotinic receptor.
(2/1448)
1. We assessed the pharmacological activity of triethyl-(beta-4-stilbenoxy-ethyl) ammonium (MG624), a drug that is active on neuronal nicotinic receptors (nicotinic AChR). Experiments on the major nicotinic AChR subtypes present in chick brain, showed that it inhibits the binding of [125I]-alphaBungarotoxin (alphaBgtx) to the alpha7 subtype, and that of [3H]-epibatidine (Epi) to the alpha4beta2 subtype, with Ki values of respectively 106 nM and 84 microM. 2. MG624 also inhibited ACh elicited currents (I(ACh)) in the oocyte-expressed alpha7 and alpha4beta2 chick subtypes with half-inhibitory concentrations (IC50) of respectively 109 nM and 3.2 microM. 3. When tested on muscle-type AChR, it inhibited [125I]-alphaBgtx binding with a Ki of 32 microM and ACh elicited currents (I(ACh)) in the oocyte-expressed alpha1beta1gammadelta chick subtype with an IC50 of 2.9 microM. 4. The interaction of MG624 with the alpha7 subtype was investigated using an alpha7 homomeric mutant receptor with a threonine-for-leucine 247 substitution (L247T alpha7). MG624 did not induce any current in oocytes expressing the wild type alpha7 receptor, but did induce large currents in the oocyte-expressed L247T alpha7 receptor. The MG624 elicited current (I(MG62)) has an EC50 of 0.2 nM and a Hill coefficient nH of 1.9, and is blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). 5. These binding and electrophysiological studies show that MG624 is a potent antagonist of neuronal chick alpha7 nicotinic AChR, and becomes a competitive agonist following the mutation of the highly conserved leucine residue 247 located in the M2 channel domain. (+info)
Evaluation of ZP2 domains of functional importance with antisera against synthetic ZP2 peptides.
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The mouse zona pellucida protein ZP2 plays an important role in the process of fertilization by mediating secondary sperm binding to mammalian oocytes. ZP2 primary structures are highly conserved as revealed by cDNA cloning. The aim of the study was to identify ZP2 domains of functional relevance. Antisera were raised against synthetic peptides that are either conserved in the structure of ZP2 from different mammalian species (AS ZP2-20) or present in the human ZP2 but not in the mouse ZP2 amino acid sequence (AS ZP2-26). Antibody binding to zona pellucida proteins was assessed by assaying the antisera with human hemizonae. Using human zonae pellucidae, we demonstrated that anti-ZP2 common antibodies and anti-ZP2 human peptide antibodies react with human zona pellucida antigens. For the first time, ZP2 domains of functional relevance for human sperm-oocyte interaction could be identified applying the competitive hemizona assay. Antiserum AS ZP2-20 significantly inhibited binding of spermatozoa to test hemizonae, whereas treatment of hemizonae with AS ZP2-26 did not influence sperm-oocyte interaction. These results show that antibodies against synthetic ZP2 peptides react with ZP2 protein and that AS ZP2-20 identifies a linear ZP2 epitope that is of possible functional importance for sperm-oocyte interaction. (+info)
Cloning of mnuA, a membrane nuclease gene of Mycoplasma pulmonis, and analysis of its expression in Escherichia coli.
(4/1448)
Membrane nucleases of mycoplasmas are believed to play important roles in growth and pathogenesis, although no clear evidence for their importance has yet been obtained. As a first step in defining the function of this unusual membrane activity, studies were undertaken to clone and analyze one of the membrane nuclease genes from Mycoplasma pulmonis. A novel screening strategy was used to identify a recombinant lambda phage expressing nuclease activity, and its cloned fragment was analyzed. Transposon mutagenesis was used to identify an open reading frame of 1,410 bp, which coded for nuclease activity in Escherichia coli. This gene coded for a 470-amino-acid polypeptide of 53,739 Da and was designated mnuA (for "membrane nuclease"). The MnuA protein contained a prolipoprotein signal peptidase II recognition sequence along with an extensive hydrophobic region near the amino terminus, suggesting that the protein may be lipid modified or that it is anchored in the membrane by this membrane-spanning region. Antisera raised against two MnuA peptide sequences identified an M. pulmonis membrane protein of approximately 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nucleolytic band of the same size. Maxicell experiments with E. coli confirmed that mnuA coded for a nuclease of unknown specificity. Hybridization studies showed that mnuA sequences are found in few Mycoplasma species, suggesting that mycoplasma membrane nucleases display significant sequence variation within the genus Mycoplasma. (+info)
In vitro metabolism of quinidine: the (3S)-3-hydroxylation of quinidine is a specific marker reaction for cytochrome P-4503A4 activity in human liver microsomes.
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The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-oxidation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity in human liver microsome preparations. An HPLC method was developed to assay the metabolites (3S)-3-hydroxyquinidine (3-OH-Q) and quinidine N-oxide (Q-N-OX) formed during incubation with microsomes from human liver and from Saccharomyces cerevisiae strains expressing 10 human CYPs. 3-OH-Q formation complied with Michaelis-Menten kinetics (mean values of Vmax and Km: 74.4 nmol/mg/h and 74.2 microM, respectively). Q-N-OX formation followed two-site kinetics with mean values of Vmax, Km and Vmax/Km for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 microM and 0.03 ml/mg/h, respectively. 3-OH-Q and Q-N-OX formations were potently inhibited by ketoconazole, itraconazole, and triacetyloleandomycin. Isozyme specific inhibitors of CYP1A2, -2C9, -2C19, -2D6, and -2E1 did not inhibit 3-OH-Q or Q-N-OX formation, with Ki values comparable with previously reported values. Statistically significant correlations were observed between CYP3A4 content and formations of 3-OH-Q and Q-N-OX in 12 human liver microsome preparations. Studies with yeast-expressed isozymes revealed that only CYP3A4 actively catalyzed the (3S)-3-hydroxylation. CYP3A4 was the most active enzyme in Q-N-OX formation, but CYP2C9 and 2E1 also catalyzed minor proportions of the N-oxidation. In conclusion, our studies demonstrate that only CYP3A4 is actively involved in the formation of 3-OH-Q. Hence, the (3S)-3-hydroxylation of quinidine is a specific probe for CYP3A4 activity in human liver microsome preparations, whereas the N-oxidation of quinidine is a somewhat less specific marker reaction for CYP3A4 activity, because the presence of a low affinity enzyme is demonstrated by different approaches. (+info)
The expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its regulation by ovarian steroids in rat uterine stromal cells.
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The effects of ovarian steroids on the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in rat uterus were examined. Intense expression of GM-CSF mRNA was dispersedly located in the endometrial-myometrial junction and stroma of the uterus. GM-CSF was immunohistochemically localized in stromal cells and luminal epithelium. Ovariectomy significantly reduced the appearance of GM-CSF-mRNA-positive cells and the levels of expression for GM-CSF mRNA in the whole uterus, whereas treatment with 17beta-estradiol (E2) or a combination of E2 and progesterone (P4) for 5 days on ovariectomized animals recruited GM-CSF-mRNA-positive cells and stimulated its expression. The combined treatment with E2 and P4 also stimulates the expression for GM-CSF mRNA and the production of immunoreactive GM-CSF in the stromal tissues. These data suggest that the expression of GM-CSF in uterine stromal cells is partially regulated by ovarian steroids. (+info)
Identification of megalin as the sole rat kidney sialoglycoprotein containing poly alpha2,8 deaminoneuraminic acid.
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Recently, poly alpha2,8 deaminoneuraminic acid (poly alpha2,8 KDN) was demonstrated in various embryonic and adult mammalian tissues. This study reports the purification and characterization of the single poly alpha2,8 KDN-bearing glycoprotein from rat kidney. Amino acid sequences of proteolytic fragments shared homology with megalin, a member of the LDL receptor family. Immunochemical analysis supported this finding, since immunoprecipitated poly alpha2,8 KDN-bearing glycoprotein was immunoreactive with anti-megalin antibodies in Western blotting and conversely immunoprecipitated megalin was immunoreactive with the monoclonal anti-poly alpha2,8 KDN antibody. Furthermore, receptor-associated protein affinity-purified megalin reacted with the anti-poly alpha2,8 KDN antibody. By immunoelectron microscopy, labeling for both poly alpha2,8 KDN and megalin coincided in the brush border, endocytic invaginations and vesicles, and apical dense tubules of proximal convoluted tubules. Immunoreactivity for poly alpha2,8 KDN on purified megalin was abolished by beta-elimination reaction but not by N-glycosidase F treatment. These data identified megalin as the sole glycoprotein of rat kidney, which contains poly alpha2,8 KDN present on O-glycosidically linked oligosaccharides. Furthermore, this study shows that megalin carries N-glycosidically linked hybrid and complex-type oligosaccharides terminating with sialic acid. Both poly alpha2,8 KDN and sialic acids on megalin may contribute to the binding of Ca2+ and cationic ligands. (+info)
pICln inhibits snRNP biogenesis by binding core spliceosomal proteins.
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The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) form essential components of spliceosomes, the machinery that removes introns from pre-mRNAs in eukaryotic cells. A critical initial step in the complex process of snRNP biogenesis is the assembly of a group of common core proteins (Sm proteins) on spliceosomal snRNA. In this study we show by multiple independent methods that the protein pICln associates with Sm proteins in vivo and in vitro. The binding of pICln to Sm proteins interferes with Sm protein assembly on spliceosomal snRNAs and inhibits import of snRNAs into the nucleus. Furthermore, pICln prevents the interaction of Sm proteins with the survival of motor neurons (SMN) protein, an interaction that has been shown to be critical for snRNP biogenesis. These findings lead us to propose a model in which pICln participates in the regulation of snRNP biogenesis, at least in part by interfering with Sm protein interaction with SMN protein. (+info)