No abstract

Inhibitors of heat-induced heat shock protein 70 (HSP70)a expression have the potential to enhance the therapeutic effectiveness of heat induced radiosensitization of tumors. Among known small molecule inhibitors, quercetin has the advantage of being easily modified for structure-activity studies. Herein, we report the ability of five mono-methyl and five carbomethoxymethyl derivatives of quercetin to inhibit heat-induced HSP70 expression and enhance HSP27 phosphorylation in human cells. While quercetin and several derivatives inhibit HSP70 induction and enhance HSP27 phosphorylation at Ser78, other analogs selectively inhibit HSP70 induction without enhancing HSP27 phosphorylation that would otherwise aid in cell survival. We also show that good inhibitors of HSP70 induction are also good inhibitors of both CK2 and CamKII, kinases that are known to activate HSP70 expression by phosphorylation of heat shock transcription factor 1. Derivatives that show poor inhibition of either or both kinases are not good inhibitors of HSP70 induction, suggesting that quercetin's effectiveness is due to its ability to inhibit both kinases.

Irradiation of the dinucleotide TpdA and TA-containing oligonucleotides and DNA produces the TA* photoproduct which was proposed to be the [2+2] cyclo-addition adduct between the C5-C6 double bonds of the T and the A [Bose,S.N., Kumar,S., Davies,R.J.H., Sethi,S.K. and McCloskey,J.A. (1984) Nucleic Acids Res. 12, 7929-7947]. The proposed structure was based on a variety of spectroscopic and chemical degradation studies, and the assignment of a trans-syn-I stereochemistry was based on an extensive 1H-NMR and molecular modeling study of the dinucleotide adduct [Koning,T.M.G., Davies,R.J.H. and Kaptein,R. (1990) Nucleic Acids Res. 18, 277-284]. However, a number of properties of TA* are not in accord with the originally proposed structure, and prompted a re-evaluation of the structure. To assign the 13C spectrum and establish the bond connectivities of the TA* photoproduct of TpdA [d(TpA)*], 1H-13C heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) spectra were obtained. The 13C shifts and connectivities were found to be inconsistent with the originally proposed cyclobutane ring fusion between the thymine and adenine, but could be explained by a subsequent ring-expansion reaction to give an eight-membered ring valence isomer. The new structure for the d(TpA)* resolves the inconsistencies with the originally proposed structure, and could have a stereochemistry that arises from the anti, anti glycosyl conformation found in B form DNA.

UVB irradiation of DNA produces photodimers in adjacent DNA bases and on rare occasions in nonadjacent bases. UVB irradiation (312 nm) of d(GTATCATGAGGTGC) gave rise to an unknown DNA photoproduct in approximately 40% yield at acidic pH of about 5. This product has a much shorter retention time in reverse phase HPLC compared to known dipyrimidine photoproducts of this sequence. A large upfield shift of two thymine H6 NMR signals and photoreversion to the parent ODN upon irradiation with 254 nm light indicates that the photoproduct is a cyclobutane thymine dimer. Exonuclease-coupled MS assay establishes that the photodimer forms between T2 and T7, which was confirmed by tandem mass spectrometric MS/MS identification of the endonuclease P1 digestion product d(T2[A3])=pd(T7[G8]). Acidic hydrolysis of the photoproduct gave a product with the same retention time on reverse phase HPLC and the same MS/MS fragmentation pattern as authentic Thy[c,a]Thy. 2D NOE NMR data are consistent with a cis-anti cyclobutane dimer between the 3′-sides of T2 and T7 in anti glycosyl conformations that had to have arisen from an inter-stand type reaction. In addition to pH-dependent, the photoproduct yield is highly sequence specific and concentration dependent, indicating that it results from a higher order folded structure. The efficient formation of this inter-strand-type photoproduct suggests the existence of a new type of folding motif and the possibility that this type of photoproduct might also form in other folded structures, such as G-quadruplexes and i-motif structures which can be now studied by the methods described.

Photolysis of thymidylyl-(3'-->5')-thymidine (TpT) has been previously reported by many workers to lead to only two cyclobutane dimers, the cis-syn and trans-syn-I dimers. This is curious in light of the fact that photolysis of thymidylyl-(3'-->5')-deoxyuridine (TpdU), which has a hydrogen in place of a methyl group at C6 of the 3'-thymidine, produces two trans-syn diastereomers. Recently, we discovered by way of X-ray crystallography that photolysis of the (Rp)-methyl phosphate ester of TpT leads to two trans-syn diastereomers, prompting us to reexamine the photochemistry of TpT. In this paper we show that sensitized photolysis of TpT also leads to the hitherto unknown trans-syn-II diastereomer in 2% yield. We also report the solution-state 1H NMR assignment of the trans-syn-II photodimer of TpT and its (Rp)-methyl phosphate ester by way of 2D homonuclear Hartmann--Hahn experiment, rotating frame nuclear Overhauser effect spectroscopy, and proton-detected 1H-31P correlation spectroscopy. Conformational analysis of three-bond 1H-1H, 1H-31P, and 13C-31P coupling constant data established a close similarity between the solution-state structures of the trans-syn-II photodimer of TpT and its (Rp)-methyl phosphate ester, with the crystal structure of the methyl phosphate ester.

Irradiation of d(GTATTATG) with 254 nm light gave rise to four major photoproducts, two of which were readily identified by NMR as the cis-syn cyclobutane dimer and the (6-4) photoproduct of the central TT site. Analysis of the NMR data for the other two photoproducts indicated that they were not any of the other known photoproducts of a TT site and might be TA* photoproducts [Bose, S. N., et al. (1983) Science 220, 723-725]. In support of this possibility, the fluorescence spectra of the products of acid hydrolysis of the two photoproducts were very similar to that reported for the hydrolysis product of the TA* photoproduct of TpdA. Only one of the two TA*-containing octamers could be ligated at both ends to form a 49-mer oligonucleotide in the presence of a complementary oligonucleotide scaffold, suggesting that the TA* photoproduct had formed between T5 and A6. The position of the TA* photoproduct was confirmed by mapping the arrest sites for 3'-->5' exonucleolytic degradation of the 49-mer by T4 DNA polymerase and for primer extension opposite the 49-mer by exonuclease deficient Klenow fragment (KF) and Sequenase Version 2.0. The TA* product could also be bypassed by both polymerases, but it was less of a block to KF. Treatment with 1 M aqueous piperidine at 100 degrees C led to a maximum of about 34% cleavage of the DNA at the site of the TA* product.(ABSTRACT TRUNCATED AT 250 WORDS)

The N-terminal fragment of pro B-type natriuretic peptide (NT-proBNP) and proBNP are used as gold standard clinical markers of myocardial dysfunction such as cardiac hypertrophy and left ventricle heart failure. The actual circulating molecular forms of these peptides have been the subject of intense investigation particularly since these analytes are measured in clinical assays. Conflicting data has been reported and no firm consensus on the exact nature of the molecular species exists. Because these clinical assays are immunoassay-based, specific epitopes are detected. It is conceivable then that certain epitopes may be masked and therefore unavailable for antibody binding, thus the importance of determining the nature of the circulating molecular forms of these analytes. This situation is an unavoidable Achilles' heel of immunoassays in general.A recombinant O-linked glycosylated form of proBNP has been show to mimic some of the properties of extracted plasma from a heart failure patient. In particular the recombinant and native material co-migrated as diffuse Western-immunostained bands on SDS-PAGE and each band collapsed to an apparent homogeneous band following deglycosylation. Thus, glycosylated-proBNP may be one such circulating form. Here we provide extensive physiochemical characterization for this O-linked protein and compare these results to other described circulating species, non-glycosylated-proBNP and NT-proBNP. It will be shown that glycosylation has no influence on the secondary and quaternary structure of proBNP. In fact, at moderate concentration in benign physiological neutral pH buffer, all three likely circulating species are essentially devoid of major secondary structure, i.e., are intrinsically unstructured proteins (IUPs). Furthermore, all three proteins exist as monomers in solution. These results may have important implications in the design of NT-proBNP/BNP immunoassays. Immunoassays utilize antibodies that react with specific analyte (usually protein) epitope(s). If these epitopes become unreactive due to say proteolytic processing, or masked by interaction with other molecule(s), or are post-or co-translationally modified, then the immunoassay can generate incorrect results. It becomes important then to identify the circulating forms of the analytes. This analysis can include primary sequence, post-translational modifications, and quaternary structure as homo-and hetero-interaction partners. Knowledge of such can aid in the development of immunoassays that accurately detect the circulating species. Keywords GlycosylatedPrevious characterization of human NT-proBNP whether as a recombinant product or synthetic peptide showed this N-terminal fragment to be a largely unfolded, flexible protein, termed an IUP, which exists as a monomer in solution [7,8]. No comparable data were hitherto available for proBNP and a recently discovered [9] glycosylated form of proBNP. Here we use a combination of CD to assess the global average secondary structure of proBNP and glycosylated-proBNP...

The incorporation of the 2,2′:6′,2′′-terpyridyl (terpy) complex of Cu(II) into a deoxyoligonucleotide has led to a functional mimic of ribozymes. The resulting mimic can cleave target RNA in a sequence-directed manner by a hydrolytic mechanism. Here we describe the synthesis and characterization of four modified nucleoside phosphoramidite reagents (7, 10, 14, and 18) that contain pendant 2,2′-bipyridine or terpy ligands. The ligands are attached either to the nucleobase (7, 10) or to the sugar (14, 18). Nucleobase modification was carried out at the C-5 position of 2′-deoxyuridine (dU). One route to sugar modification was performed by synthesis of 18, a 1′functionalized analog of dU based on 1-[3-deoxy-β-D-psicofuranosyl]uracil. Another route to sugar functionaliztion resulted in 14, a 2′-O-alkyl derivative of adenosine. These modified nucleosides are building blocks for ribozyme mimics. They are designed to deliver hydrolytically active metal complexes across either the major groove (7, 10) or the minor groove (14, 18) of an RNA/DNA duplex.