Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles

Background  

Bovine follicular development is regulated by numerous molecular mechanisms and biological pathways. In this study, we tried to identify differentially expressed genes between largest (F1) and second-largest follicles (F2), and classify them by global gene expression profiling using a combination of microarray and quantitative real-time PCR (QPCR) analysis. The follicular status of F1 and F2 were further evaluated in terms of healthy and atretic conditions by investigating mRNA localization of identified genes.     

Interactions of STAP-2 with Brk and STAT3 participate in cell growth of human breast cancer cells

STAP-2 (signal transducing adaptor protein-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) and Src homology 2-like domains, as well as a STAT3-binding motif in its C-terminal region. STAP-2 is also a substrate of breast tumor kinase (Brk). In breast cancers, Brk expression is deregulated and promotes STAT3-dependent cell proliferation. In the present study, manipulated STAP-2 expression demonstrated essential roles of STAP-2 in Brk-mediated STAT3 activation. STAP-2 interacts with both Brk and STAT3. In addition, small interfering RNA-mediated reduction of endogenous STAP-2 expression strongly decreased Brk-mediated STAT3 activation in T47D breast cancer cells. The PH domain of STAP-2 is involved in multiple steps: the binding between Brk and STAP-2, the activation and tyrosine phosphorylation of STAT3, and the activation of Brk. Notably, a STAP-2 PH-Brk fusion protein exhibited robust kinase activity and increased activation and tyrosine phosphorylation of STAT3. Finally, STAP-2 knockdown in T47D cells induced a significant decrease of proliferation, as strong as that of Brk or STAT3 knockdown. Taken together, our findings are likely to inform the development of a novel therapeutic strategy, as well as the determination of novel prognostic values, in breast carcinomas.     

Differential mechanisms for the inhibition of human cytochrome P450 1A2 by apigenin and genistein

The inhibitory effects of flavonoids on the human cytochrome P450 1A2 (CYP1A2) were examined. Among flavonoids tested, galangin, kaempferol, chrysin, and apigenin were potent inhibitors. Although apigenin belonging to flavones and genistein belonging to isoflavones are similar in the chemical structures, the inhibitory potencies for CYP1A2 were distinguished markedly between these two flavonoids. In computer‐docking simulation, apigenin interacted with the same mode of cocrystallized α‐naphthoflavone in the active site of CYP1A2, and then the B ring of apigenin was placed close to the heme iron of the enzyme with a single orientation. In contrast, the docked genistein conformation showed two different binding modes, and the A ring of genistein was oriented to the heme iron of CYP1A2. Furthermore, the binding free energy of apigenin was lower than that of genistein. These results demonstrate a possible mechanism that causes the differential inhibitory potencies of apigenin and genistein for CYP1A2. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:230–234, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20328     

Ceramide in lipid particles enhances heparan sulfate proteoglycan and low density lipoprotein receptor-related protein-mediated uptake by macrophages

Arterial wall sphingomyelinase (SMase) has been proposed to be involved in atherogenesis. SMase modification of lipoproteins has been shown to occur in atherosclerotic lesions and to facilitate their uptake by macrophages and foam cell formation. To investigate the mechanism of macrophage uptake enhanced by SMase, we prepared lipid emulsions containing sphingomyelin (SM) or ceramide (CER) as model particles of lipoproteins. SMase remarkably increased the uptake of SM-containing emulsions by J774 macrophages without apolipoproteins. The emulsion uptake was negatively correlated with the degree of particle aggregation by pretreatment with SMase, whereas the uptake of CER-containing emulsions was significantly larger than SM-containing emulsions, indicating that enhancement of uptake is due to the generation of CER molecules in particles but not to the aggregation by SMase. Heparan sulfate proteoglycans (HSPGs) and low density lipoprotein receptor-related protein (LRP) were crucial for CER-enhanced emulsion uptake, because heparin or lactoferrin inhibited the emulsion uptake. Confocal microscopy also showed that SMase promoted both binding and internalization of emulsions by J774 macrophages, which were almost abolished by lactoferrin. Apolipoprotein E further increased the uptake of CER-containing emulsions compared with SM-containing emulsions. These findings suggest the generation of CER in lipoproteins by SMase facilitates the macrophage uptake via HSPG and LRP pathways and plays a crucial role in foam cell formation. Thus, CER may act as an important atherogenic molecule.     

Low pKa lysine residues at the active site of sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is inactivated by iodoacetamide with the modification of two specific residues. Comparing the amino acid sequence and mass spectra of the peptide fragments containing the modified residues with those from the native enzyme, the modified residues were identified to be lysine. The pKa of these residues were estimated to be 8.5 and 6.7 from the pH dependence of inactivation in the presence and absence of the competitive inhibitor, acetate. These estimated pKa values are much lower than that of the epsilon-amino group of lysine residue. There may be unique microenvironments around these residues that activate their -amino groups to be susceptible to iodoacetamide. A possible role of the lysine residue with pKa 6.7 is discussed.     

3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factor II receptor from the early endosome to the TGN

3-Methyladenine (3-MA), a well-known inhibitor of autophagic sequestration, can also prevent class III phosphatidylinositide (PI) 3-kinase activity, which is required for many processes in endosomal membrane trafficking. Although much is known about the effects of other PI 3-kinase inhibitors, such as wortmannin and LY294002, on endosomal membrane trafficking, little is known about those of 3-MA. Here we show that the treatment of cells with 3-MA results in a specific redistribution of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR300) from the trans-Golgi network (TGN) to early/recycling endosomal compartments containing internalized transferrin. Importantly, in contrast to wortmannin and LY294002, 3-MA did not cause the enlargement of late endosomal/lysosomal compartments. The results suggest that the effect of 3-MA is restricted to the retrieval of MPR300 from early/recycling endosomes.     

Deciphering the molecular basis of the broad substrate specificity of alpha-glucosidase from Bacillus sp. SAM1606

The alpha-glucosidase of Bacillus sp. strain SAM1606 is a member of glycosyl hydrolase family 13, and shows an extraordinarily broad substrate specificity and is one of very few alpha-glucosidases that can efficiently hydrolyze the alpha-1,1-glucosidic linkage of alpha,alpha'-trehalose (trehalose). Phylogenetic analysis of family-13 enzymes suggests that SAM1606 alpha-glucosidase may be evolutionally derived from an alpha-1,6-specific ancestor, oligo-1,6-glucosidase (O16G). Indeed, replacement of Pro(273*) and Thr(342*) of B. cereus O16G by glycine and asparagine (the corresponding residues in the SAM1606 enzyme), respectively, was found to cause 192-fold enhancement of the relative catalytic efficiency for trehalose, suggesting that O16G may easily "evolved" into an enzyme with an extended substrate specificity by substitution of a limited number of amino acids, including that at position 273* (an asterisk indicates the amino-acid numbering of the SAM1606 sequence). To probe the role of the amino acid at position 273* of alpha-glucosidase in determination of the substrate specificity, the amino acid at position 273 of SAM1606 alpha-glucosidase was replaced by all other naturally occurring amino acids, and the resultant mutants were kinetically characterized. The results showed that substitution of bulky residues (e.g., isoleucine and methionine) for glycine at this position resulted in large increases in the K(m) values for trehalose and maltose, whereas the affinity to isomaltose was only minimally affected by such an amino-acid substitution at this position. Three-dimensional structural models of the enzyme-substrate complexes of the wild-type and mutant SAM1606 alpha-glucosidases were built to explore the mechanism responsible for these observations. It is proposed that substitution by glycine at position 273* could eliminate steric hindrance around subsite +1 that originally occurred in parental O16G and is, at least in part, responsible for the acquired broad substrate specificity of SAM1606 alpha-glucosidase.     

Functional complementation in yeast reveals a protective role of chloroplast 2-Cys peroxiredoxin against reactive nitrogen species

The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism.