Circular dichroism of DNA frayed wires.

Ultraviolet circular dichroism spectra are reported for the oligonucleotide d(A15G15) in aqueous solutions containing 5 mM MgCl2 at several temperatures and in the presence of partially complementary oligonucleotides. Oligonucleotides with several consecutive terminal guanine residues self-associate to form aggregates, called frayed wires, that consist of integer numbers of strands. A "stem" is formed through interactions between the guanine residues of the associated oligonucleotides, whereas the adenine "arms" remain single stranded. Upon subtracting the circular dichroism spectrum of d(A15) from that of d(A15G15), one obtains a spectrum that closely resembles previously published spectra of poly(G). Subtracting spectra measured at temperatures between 10 degrees C and 60 degrees C reveals the resultant spectra to be independent of temperature, consistent with the extreme thermal stability observed for the aggregated structures. Upon the addition of d(T15) to the solution, complexes with the adenine portion of the d(A15G15) frayed wires are formed. Subtraction of d(A15):d(T15) spectra measured at several temperatures from those of the d(A15G15):d(T15) does not significantly alter the spectrum of the guanines. The helix-coil transition temperature of d(A15):d(T15) duplex is identical to that of the unbinding of d(T15) from d(A15G15):d(T15) complexes. Experiments using oligonucleotides in which the adenines were replaced with sequences of bases yielded similar results. By varying the length of the nonguanine tract, it is shown that the solubility of the complexes increases with the length of the nonguanine region of the oligonucleotide.     

Efficacy of Euphorbia splendens and Leonotis nepetaefolia on aflatoxin producing fungi Aspergillus flavus and Aspergillus parasiticus

Efficacy of three different concentrations (5, 10 and 15 mg/ml) of dry flower powder of E. splendens and L. nepetaefolia was tested on the growth of aflatoxin-producing toxigenic strains of fungi A. flavus (NCBT 101) and A. parasiticus (NCBT 128) in Sabouraud dextrose agar medium (SDA). Maximum (75%) inhibition of growth of A. flavus was seen at 15 mg/ml concentration of E. splendens flower dry powder, while A. parasiticus showed 50% inhibition of growth at 10 and 15 mg/ml concentrations. Total inhibition (100%) of growth of A. flavus was seen at 10 and 15 mg/ml for L. nepetaefolia and maximum (75%) inhibition of growth was seen for A. parasiticus at 15 mg/ml concentration. Bioassay with groundnut seeds soaked with different concentrations of flower extract proved that both fungi were incapable of infecting the seeds at 10 and 15 mg/ml of L. nepetaefolia flower extracts.     

Proteomic identification of protein targets for 15-deoxy-Δ(12,14)-prostaglandin J2 in neuronal plasma membrane

15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is one of factors contributed to the neurotoxicity of amyloid β (Aβ), a causative protein of Alzheimer's disease. Type 2 receptor for prostaglandin D(2) (DP2) and peroxysome-proliferator activated receptorγ (PPARγ) are identified as the membrane receptor and the nuclear receptor for 15d-PGJ(2), respectively. Previously, we reported that the cytotoxicity of 15d-PGJ(2) was independent of DP2 and PPARγ, and suggested that 15d-PGJ(2) induced apoptosis through the novel specific binding sites of 15d-PGJ(2) different from DP2 and PPARγ. To relate the cytotoxicity of 15d-PGJ(2) to amyloidoses, we performed binding assay [(3)H]15d-PGJ(2) and specified targets for 15d-PGJ(2) associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ(2) and fibrillar Aβ. Specific binding sites of [(3)H]15d-PGJ(2) were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [(3)H]15d-PGJ(2) were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ(2) in the plasma membrane. By using biotinylated 15d-PGJ(2), eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α). GAPDH, PKM1 and Tubulin β are Aβ-interacting proteins. Thus, the present study suggested that 15d-PGJ(2) plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues.     

Identification of a conserved sequence motif that promotes Cdc37 and cyclin D1 binding to Cdk4

Cdc37 is a molecular chaperone that is important for the stability and activity of several protein kinases, including Cdk4 and Raf1. We first determined, using in vitro assays, that Cdc37 binds to the amino-terminal lobe of Cdk4. Subsequent mutagenesis revealed that Gly-15 (G15A) and Gly-18 (G18A) were critical for Cdc37-Cdk4 complex formation. Gly-15 and Gly-18 of Cdk4 are within the conserved Gly-X-Gly-X-X-Gly motif that is required for ATP binding to the kinase. Mutation of either glycine at the equivalent positions of Raf1 (G358A and G361A) also inhibited Cdc37 binding to Raf1. Replacing another conserved residue critical for ATP binding and kinase activity, Lys-35 (K35A), reduced Cdc37-Cdk4 complex formation but to a lesser extent. The interaction of Cdk4 with Cdc37 in vitro was not sensitive to changes in ATP levels. Cell-based assays indicated that Cdk4(G15A) and Cdk4(G18A) were present at the same level as wild type Cdk4. Equivalent amounts of p16 bound to Cdk4(G15A) and Cdk4(G18A) relative to wild type Cdk4, suggesting that Cdk4(G15A) and Cdk4(G18A) adopt significant tertiary structure. However, in contrast to wild type Cdk4, Cdk4(G15A), and Cdk4(G18A) had greatly reduced binding of cyclin D1, Cdc37, and Hsp90. Importantly, overexpression of Cdc37 not only stimulated cyclin D1 binding to wild type Cdk4 but also restored its binding to Cdk4(G15A). Under the same conditions, p16 binding to wild type Cdk4 was suppressed. Our findings show that the interaction of Cdc37 with its client protein kinases requires amino acid residues within a motif that is present in many protein kinases.     

Functional annotation of the putative orphan Caenorhabditis elegans G-protein-coupled receptor C10C6.2 as a FLP15 peptide receptor

This report describes the cloning and functional annotation of a Caenorhabditis elegans orphan G-protein-coupled receptor (GPCR) (C10C6.2) as a receptor for the FMRFamide-related peptides (FaRPs) encoded on the flp15 precursor gene, leading to the receptor designation FLP15-R. A cDNA encoding C10C6.2 was obtained using PCR techniques, confirmed identical to the Worm-pep-predicted sequence, and cloned into a vector appropriate for eucaryotic expression. A [35S]guanosine 5'-O-(thiotriphosphate) (GTPgammaS) assay with membranes prepared from Chinese hamster ovary (CHO) cells transiently transfected with FLP15-R was used as a read-out for receptor activation. FLP15-R was activated by putative FLP15 peptides, GGPQGPLRF-NH2 (FLP15-1), RGPSGPLRF-NH2 (FLP15-2A), its des-Arg1 counterpart, GPSGPLRF-NH2 (FLP15-2B), and to a lesser extent, by a tobacco hornworm Manduca sexta FaRP, GNSFLRFNH2 (F7G) (potency ranking FLP15-2A > FLP15-1 > FLP15-2B > F7G). FLP15-R activation was abolished in the transfected cells pretreated with pertussis toxin, suggesting a preferential receptor coupling to Gi/Go proteins. The functional expression of FLP15-R in mammalian cells was temperature-dependent. Either no stimulation or significantly lower ligand-evoked [35S]GTPgammaS binding was observed in membranes prepared from transfected FLP15-R/CHO cells cultured at 37 degrees C. However, a 37 to 28 degrees C temperature shift implemented 24 h post-transfection consistently resulted in an improved activation signal and was essential for detectable functional expression of FLP15-R in CHO cells. To our knowledge, the FLP15 receptor is only the second deorphanized C. elegans neuropeptide GPCR reported to date.     

Development of streptococcal pyrogenic exotoxin C vaccine toxoids that are protective in the rabbit model of toxic shock syndrome

Streptococcal pyrogenic exotoxin C (SPE C) is a superantigen produced by many strains of Streptococcus pyogenes that (along with streptococcal pyrogenic exotoxin A) is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Based on the three-dimensional structure of SPE C, solvent-exposed residues predicted to be important for binding to the TCR or the MHC class II molecule, or important for dimerization, were generated. Based on decreased mitogenic activity of various single-site mutants, the double-site mutant Y15A/N38D and the triple-site mutant Y15A/H35A/N38D were constructed and analyzed for superantigenicity, toxicity (lethality), immunogenicity, and the ability to protect against wild-type SPE C-induced STSS. The Y15A/N38D and Y15A/H35A/N38D mutants were nonmitogenic for rabbit splenocytes and human PBMCs and nonlethal in two rabbit models of STSS, yet both mutants were highly immunogenic. Animals vaccinated with the Y15A/N38D or Y15A/H35A/N38D toxoids were protected from challenge with wild-type SPE C. Collectively, these data indicate that the Y15A/N38D and Y15A/H35A/N38D mutants may be useful as toxoid vaccine candidates.     

Novel S100A7 (psoriasin)/S100A15 (koebnerisin) subfamily: highly homologous but distinct in regulation and function

S100A7 (psoriasin) and S100A15 (koebnerisin) were first identified in inflamed psoriatic skin. They are of major interest because of their putative functional roles in innate immunity, epidermal cell maturation, and epithelial tumorigenesis. Human S100A7 and S100A15 have lately evolved by gene duplications within the epidermal differentiation complex (chromosome 1q21) during primate evolution forming a novel S100 subfamily. Therefore, S100A7 and S100A15 are almost identical in sequence (>90%) and are difficult to discriminate. Despite their high homology, S100A7 and S100A15 are distinct in tissue distribution, regulation, and function, and thus, exemplary for the diversity within the S100 family. Their different properties are compelling reasons to discriminate S100A7 (psoriasin) and S100A15 (koebnerisin) in epithelial homeostasis, inflammation, and cancer.     

Biochemical and genetic analyses of murine hepatitis virus Nsp15 endoribonuclease

The goal of this project was to better define the relationship between the endoribonuclease activity of murine hepatitis virus (MHV) Nsp15 (mNsp15) and its role in virus infection. Molecular modeling demonstrated that the catalytic residues of mNsp15 are superimposable with its severe acute respiratory syndrome coronavirus ortholog. Alanine substitutions at three key residues in the mNsp15 catalytic pocket (H262, H277, and G275) and a double-mutant version (H262P and H277A) generated proteins with greatly reduced but detectable endoribonuclease activities. Furthermore, these mutant proteins demonstrated lower cleavage specificities for uridylate than wild-type (WT) mNsp15. These mutations were successfully incorporated into viruses named vH262A, vH277A, vG275A, and vH262P+H277A. All four mutant viruses formed plaques with diameters similar to that of MHV-A59 1000 (WT virus) on several different cell lines. Interestingly, viruses with a mutation at a noncatalytic residue, D324A, could not be recovered despite repeated attempts, and expression of mNsp15 containing the D324A mutation in Escherichia coli resulted in an insoluble protein. Plaques derived from vH262A produced approximately 6- to 13-fold fewer PFU than those from WT virus. Cells infected with mNsp15 mutant viruses accumulated lesser amounts of plus- and minus-sense subgenomic RNAs and spike protein than WT virus. The expression of mNsp15 in trans by transient transfection partially restored RNA synthesis by vH262A. These results demonstrate that mNsp15 is required for optimal infection by MHV.     

Influences of genetic variants in interleukin-15 gene and serum interleukin-15 levels on coronary heart disease

Interleukin-15 (IL-15) is a potent proinflammatory cytokine that is now considered a key component of atherosclerosis. Proinflammatory gene polymorphisms lead to variations in the production and level of the proteins. In light of these findings, we hypothesized that variations in the gene coding for IL-15 influence the risk of coronary heart disease (CHD) by modulating the IL-15 levels. To test this hypothesis, we examined 5 single nucleotide polymorphisms (SNPs) in IL-15 gene and IL-15 levels in 102 patients with acute coronary syndrome (ACS), 102 patients with chronic ischemic stable CHD and 162 healthy control subjects. This study is the first report showing the influences of IL-15 gene variants and IL-15 levels on CHD. The five single nucleotide polymorphisms (SNPs) within the IL-15 gene, G367A, C267T, A14035T, C13687A, and A10504G were carried out by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Serum IL-15 levels were significantly higher in both acute and chronic patients than in controls. Genetic variants of IL-15 gene and IL-15 levels were associated with CHD. In conclusion, our study supports the hypothesis that genetic variation in IL-15 gene and IL-15 levels influence the risk of CHD. Further studies are needed to confirm our hypothesis.     

细胞酶联反应法 (cell-ELA) 测定特异结合U87-EGFRvⅢ细胞的适配子的亲和力

通过细胞-指数级富集的配基系统进化(Cell based systematic evolution of ligands by exponentialenrichment,cell-SELEX)方法从随机文库中筛选得到与稳定过表达人表皮生长因子受体Ⅲ型突变体(Epidermal growth factor receptor variantⅢ,EGFRvⅢ)的胶质瘤细胞U87细胞株(U87-EGFRvⅢ)特异结合的DNA适配子。以U87-EGFRvⅢ细胞作为检测对象,筛选到的适配子A15作为检测分子,建立一种细胞酶联反应(Cell enzyme-linked assay,cell-ELA)方法测定适配子的亲和力,并用EGFR抗体作为对照来比较此种DNA适配子的亲和力。结果显示,所测定的DNA适配子A15的解离平衡常数(Equilibrium dissociation constants,Kd)小于100 nmol/L,对U87-EGFRvⅢ细胞的结合能力与抗体相似。Cell-ELA法可较方便地用于cell-SELEX中适配子亲和力的测定。     

Labdane diterpenes from Marrubium velutinum and Marrubium cylleneum

From the aerial parts of Marrubium velutinum and Marrubium cylleneum, seven labdane diterpenes, velutine A, 15-epi-velutine A, velutine B, 15-epi-velutine B, velutine C, cyllenine A and 15-epi-cyllenine A, have been isolated together with five known diterpenes and four known flavones. The structures of the isolated compounds were established by means of NMR [(1)H-(1)H-COSY, (1)H-(13)C-HMQC, HMBC, HMQC-TOCSY, NOESY] and MS spectral analyses. Complete NMR assignments are reported for known compounds.     

15-hydroxyprostaglandin dehydrogenase (15-PGDH) and lung cancer

15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes NAD(+)-linked oxidation of 15 (S)-hydroxyl group of prostaglandins and lipoxins and is the key enzyme responsible for the biological inactivation of these eicosanoids. The enzyme was found to be under-expressed as opposed to cyclooxygenase-2 (COX-2) being over-expressed in lung and other tumors. A549 human lung adenocarcinoma cells were used as a model system to study the role of 15-PGDH in lung tumorigenesis. Up-regulation of COX-2 expression by pro-inflammatory cytokines in A549 cells was accompanied by a down-regulation of 15-PGDH expression. Over-expression of COX-2 but not COX-1 by adenoviral-mediated approach also attenuated 15-PGDH expression. Similarly, over-expression of 15-PGDH by the same strategy inhibited IL-1beta-induced COX-2 expression. It appears that the expression of COX-2 and 15-PGDH is regulated reciprocally. Adenoviral-mediated transient over-expression of 15-PGDH in A549 cells resulted in apoptosis. Xenograft studies in nude mice also showed tumor suppression with cells transiently over-expressing 15-PGDH. However, cells stably over-expressing 15-PGDH generated tumors faster than those control cells. Examination of different clones of A549 cells stably expressing different levels of 15-PGDH indicated that the levels of 15-PGDH expression correlated positively with those of mesenchymal markers, and negatively with those of epithelial markers. It appears that the stable expression of 15-PGDH induces epithelial-mesenchymal transition (EMT) which may account for the tumor promotion in xenograft studies. A number of anti-cancer agents, such as transforming growth factor-beta1 (TGF-beta1), glucocorticoids and some histone deacetylase inhibitors were found to induce 15-PGDH expression. These results suggest that tumor suppressive action of these agents may, in part, be related to their ability to induce 15-PGDH expression.     

ISG15 Arg151 and the ISG15-Conjugating Enzyme UbE1L Are Important for Innate Immune Control of Sindbis Virus

Interferon (IFN)-stimulated gene 15 (ISG15) is a ubiquitin-like molecule that conjugates to target proteins via a C-terminal LRLRGG motif and has antiviral function in vivo. We used structural modeling to predict human ISG15 (hISG15) residues important for interacting with its E1 enzyme, UbE1L. Kinetic analysis revealed that mutation of arginine 153 to alanine (R153A) ablated hISG15-hUbE1L binding and transthiolation of UbcH8. Mutation of other predicted UbE1L-interacting residues had minimal effects on the transfer of ISG15 from UbE1L to UbcH8. The capacity of hISG15 R153A to form protein conjugates in 293T cells was markedly diminished. Mutation of the homologous residue in mouse ISG15 (mISG15), arginine 151, to alanine (R151A) also attenuated protein ISGylation following transfection into 293T cells. We assessed the role of ISG15-UbE1L interactions in control of virus infection by constructing double subgenomic Sindbis viruses that expressed the mISG15 R151A mutant. While expression of mISG15 protected alpha/beta-IFN-receptor-deficient (IFN-αβR^−/−) mice from lethality following Sindbis virus infection, expression of mISG15 R151A conferred no survival benefit. The R151A mutation also attenuated ISG15's ability to decrease Sindbis virus replication in IFN-αβR^−/− mice or prolong survival of ISG15^−/− mice. The importance of UbE1L was confirmed by demonstrating that mice lacking this ISG15 E1 enzyme were highly susceptible to Sindbis virus infection. Together, these data support a role for protein conjugation in the antiviral effects of ISG15.     

Knockdown of ribosomal protein S15A inhibits proliferation of breast cancer cells through induction of apoptosis in vitro

To explore the role of ribosomal protein S15A (RPS15A) in breast cancer. The Oncomine database was used to compare the expression of RPS15A in human breast cancer tissues and normal tissues. RPS15A in breast cancer cell line ZR-75-30 and BT474 was specifically knocked down using lentivirus-mediated short hairpin RNAs (shRNAs). RPS15A knockdown efficiency was validated by quantitative polymerase chain reaction and western blot analysis. Subsequently, the functional effects of RPS15A on proliferation of breast cancer cells were investigated by MTT, colony formation and flow cytometry assays. Functional analysis indicated that RPS15A knockdown could inhibit cell proliferation, induced cell cycle arrest and apoptosis. Mechanism analysis revealed RPS15A mediated apoptosis via activating of caspase-3 and PARP cleavage, upregulating of Bad and BAX and downregulating of Bcl-2. Our preliminary study highlighted the importance of RPS15A in breast cancer growth. The inhibition of RPS15A may be a promising therapeutic target for breast cancer treatment.     

Involvement of mitochondrial ferredoxin and Cox15p in hydroxylation of heme O

Cox15p is essential for the biogenesis of cytochrome oxidase [Glerum et al., J. Biol. Chem. 272 (1997) 19088-19094]. We show here that cox15 mutants are blocked in heme A but not heme O biosynthesis. In Schizosaccharomyces pombe COX15 is fused to YAH1, the yeast gene for mitochondrial ferredoxin (adrenodoxin). A fusion of Cox15p and Yah1p in Saccharomyces cerevisiae rescued both cox15 and yah1 null mutants. This suggests that Yah1p functions in concert with Cox15p. We propose that Cox15p functions together with Yah1p and its putative reductase (Arh1p) in the hydroxylation of heme O.     

Light-induced global conformational change of photoactive yellow protein in solution

The light-induced global conformational change of photoactive yellow protein was directly observed by small-angle X-ray scattering (SAXS). The N-terminal 6, 15, or 23 amino acid residues were enzymatically truncated (T6, T15, or T23, respectively), and their near-UV intermediates were accumulated under continuous illumination for SAXS measurements. The Kratky plot demonstrated that illumination induced partial loss of globularity. The change in globularity was marked in T6 but very small in T15 and T23, suggesting that structural change in positions 7-15 mainly reduces the globularity. The radius of gyration (R(g)) estimated by Guinier plot was increased by 1.1 A for T6 and 0.7 A for T15 and T23 upon illumination. As T23 lacks most of the N-terminal loop, structural change in the main part composed of the PAS core, helical connector, and beta-scaffold caused an increase of R(g) by 0.7 A. The structural change of positions 7-15 caused an additional increase by 0.4 A. The decrease of R(g) upon truncation of positions 7-15 for dark state was 0.3 A, while that for the intermediate was 0.7 A, suggesting that this region moves outward on formation of the intermediate. These results indicate that a light-induced structural change of PYP takes place in the main part and N-terminal 15 amino acid residues. The former induces only dimensional increase, but the latter results in additional change in shape.     

Anti-inflammatory lipid mediator 15d-PGJ2 inhibits translation through inactivation of eIF4A

The signaling lipid molecule 15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) has multiple cellular functions, including anti-inflammatory and antineoplastic activities. Here, we report that 15d-PGJ2 blocks translation through inactivation of translational initiation factor eIF4A. Binding of 15d-PGJ2 to eIF4A blocks the interaction between eIF4A and eIF4G that is essential for translation of many mRNAs. Cysteine 264 in eIF4A is the target site of 15d-PGJ2. The antineoplastic activity of 15d-PGJ2 is likely attributed to inhibition of translation. Moreover, inhibition of translation by 15d-PGJ2 results in stress granule (SG) formation, into which TRAF2 is sequestered. The sequestration of TRAF2 contributes to the anti-inflammatory activity of 15d-PGJ2. These findings reveal a novel cross-talk between translation and inflammatory response, and offer new approaches to develop anticancer and anti-inflammatory drugs that target translation factors including eIF4A.     

The M type K15 protein of Kaposi's sarcoma-associated herpesvirus regulates microRNA expression via its SH2-binding motif to induce cell migration and invasion

Kaposi's sarcoma (KS) associated herpesvirus (KSHV) is the etiological agent of KS. In vivo, KS is a tumor capable of spreading throughout the body, and pulmonary metastasis is observed clinically. In vitro, KSHV induces the invasiveness of endothelial cells. The KSHV open reading frame K15 is a KSHV-specific gene encoding a transmembrane protein. Two highly divergent forms of K15, the predominant (P) and minor (M) forms (K15P and K15M, respectively), have been identified in different KSHV strains. The two K15 alleles resemble the latent membrane protein 2A (LMP2A) gene of Epstein-Barr virus (EBV) in their genomic locations and protein topology. Also, both K15 proteins have motifs similar to those found in the EBV LMP1 protein. K15 therefore appears to be a hybrid of a distant evolutionary relative of EBV LMP1 and LMP2A. Since both LMP1 and LMP2A proteins are capable of inducing cell motility, we sought to determine whether K15 has similar abilities. In this study, we show that K15M is latently expressed in KSHV-positive PEL cells and knockdown of K15M in PEL cells reduces cell motility. K15M localizes to lysosomal membranes and induces cell migration, invasion, and NF-κB (but not AP-1) activity via its conserved SH2-binding motif. K15M also induces the expression of microRNAs miR-21 and miR-31 via this conserved motif, and knocking down both these microRNAs eliminates K15M-induced cell motility. Therefore, K15M may contribute to KSHV-mediated tumor metastasis and angiogenesis via regulation of miR-21 and miR-31, which we show here for the first time to be a specific regulator of cell migration. In light of these findings, the targeting of K15 or the downstream microRNAs regulated by it may represent novel therapies for treatment of KSHV-associated neoplasia.