2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)     

2- and 8-azido photoaffinity probes. 2. Studies on the binding process of 2-5A synthetase by photosensitive ATP analogues

The photoaffinity probes [gamma-32P]2-azidoATP (2-N3ATP) and [alpha-32P]8-azido-ATP (8-N3ATP) were used to investigate the binding of ATP to highly purified 2-5A synthetase. 2-N3ATP and 8-N3ATP are substrates for 2-5A synthetase [Suhadolnik, R.J., Karikó, K., Sobol, R.W., Jr., Li, S.W., Reichenbach, N.L., & Haley, B.E., preceding paper]. In this study we show that 2- and 8-N3ATP are competitive inhibitors of the enzymatic conversion of ATP to 2-5A. Ultraviolet irradiation results in the photoinsertion of 2-N3ATP and 8-N3ATP into the enzyme. The covalent photoinsertion of [alpha-32P]8-N3ATP into the 2-5A synthetase is proportional to the inactivation of the enzyme as UV irradiation is increased. Photolabeling of 2-5A synthetase is saturated at 1.5 mM 2-N3ATP and 2.0 mM 8-N3ATP. Computer analysis of the curvilinear Scatchard plots of the 2-5A synthetase suggests the presence of high-affinity and low-affinity binding sites that may correspond to the acceptor and the 2'-adenylation sites of the enzyme. The competition of nucleotides for the covalent photoinsertion of 8-N3ATP into the binding site(s) of the synthetase was as follows: ATP greater than 2'dATP = 3'dATP greater than CTP greater than ITP greater than AMP greater than NAD+ greater than UTP greater than UMP greater than CMP. Photoinsertion of 8-N3ATP into 2-5A synthetase increases with the addition of poly(rI).poly(rC).(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic analysis of 24 French families with multiple endocrine neoplasia type 2A.

The gene for multiple endocrine neoplasia type 2A (MEN2A) has been mapped to the pericentromeric region of chromosome 10 by linkage analysis. Thirty-four families with multiple cases of medullary carcinoma of the thyroid (MTC), including 24 families with origins in France, have been typed with nine polymorphic markers spanning the centromere of chromosome 10. No recombination was observed between the MEN2A locus and either of the four loci D10Z1 (lod score 12.79), D10S102 (lod score 6.38), D10S94 (lod score 7.76), and D10S34 (lod score 5.94). There was no evidence for genetic linkage heterogeneity in the panel of 34 families. Haplotypes were constructed for a total of 11 polymorphisms in the MEN2A region, for mutation-bearing chromosomes in 24 French families and for 100 spouse controls. One haplotype was present in four MEN2A families but was not observed in any control (P less than .01). Two additional families share a core segment of this haplotype near the MEN2A gene. It is likely that these six families have a common affected ancestor. Because the incidence of pheochromocytoma among carriers varies from 0% to 74% within these six families, it is probable that additional factors modify the expression of the MEN2A gene.     

Familial Site-specific Ovarian Cancer Is Linked to BRCA1 on 17q12-21

In a study of nine families with “site-specific” ovarian cancer (criterion: three or more cases of epithelial ovarian cancer and no cases of breast cancer diagnosed at age <50 years) we have obtained evidence of linkage to the breast-ovarian cancer susceptibility gene, BRCA1 on 17q12-21. If the risk of cancer in these families is assumed to be restricted to the ovary, the best estimate of the proportion of families linked to BRCA1 is .78 (95% confidence interval .32–1.0). If predisposition to both breast and ovarian cancer is assumed, the proportion linked is 1.0 (95% confidence interval .46–1.0). The linkage of familial site-specific ovarian cancer to BRCA1 indicates the possibility of predictive testing in such families; however, this is only appropriate in families where the evidence for linkage to BRCA1 is conclusive.     

Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom

Summary Two-dimensional ¹H NMR techniques were used to determine the spatial structure of ectatomin, a toxin from the venom of the ant Ectatomma tuberculatum. Nearly complete proton resonance assignments for two chains of ectatomin (37 and 34 amino acid residues, respectively) were obtained using 2D TOCSY, DQF-COSY and NOESY experiments. The cross-peak volumes in NOESY spectra were used to define the local structure of the protein and generate accurate proton-proton distance constraints employing the MARDIGRAS program. Disulfide bonds were located by analyzing the global fold of ectatomin, calculated with the distance geometry program DIANA. These data, combined with data on the rate of exchange of amide protons with deuterium, were used to obtain a final set of 20 structures by DIANA. These structures were refined by unrestrained energy minimization using the CHARMm program. The resulting rms deviations over 20 structures (excluding the mobile N- and C-termini of each chain) are 0.75 ? for backbone heavy atoms, and 1.25 ? for all heavy atoms. The conformations of the two chains are similar. Each chain consists of two α-helices and a hinge region of four residues; this forms a hairpin structure which is stabilized by disulfide bridges. The hinge regions of the two chains are connected together by a third disulfide bridge. Thus, ectatomin forms a four-α-helical bundle structure.     

Role of Microbiota in Neurodegenerative Diseases

Russian Journal of Developmental Biology - Functional interaction of the gastrointestinal tract (GI) and the central nervous system (CNS) is due to various relationships, which includes autonomic...     

Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity

Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.     

A comparative study of the effects of Pr<Superscript>3+</Superscript> and La<Superscript>3+</Superscript> ions on calcium dependent processes in frog cardiac muscle and rat heart mitochondria

The inotropic effect of Pr³⁺ and La³⁺ ions on the heart muscle of frog Rana ridibunda, as well as the influence of the ions on respiration, swelling, and the potential (ΔΨ_mito) on the inner membrane of Ca²⁺- loaded rat heart mitochondria, energized by glutamate and malate or succinate in the presence of rotenone were studied. It was found that 2 mM Pr³⁺ in Ringer’s solution reduces the force of spontaneous contractions and those induced by electrical stimulation in the heart; it had a negative chronotropic effect, decreasing the frequency of spontaneous contractions. Pr³⁺ and La³⁺ prevented a decrease in the 2,4-dinitrophenol (DNP)- uncoupled respiration of energized rat heart mitochondria, swelling of these organelles in salt media, and a reduction in ΔΨ_mito on the inner mitochondrial membrane that were induced by Ca²⁺ ions. Retardation by Pr³⁺ and La³⁺ ions of these calcium-induced effects may suggest that in the inner mitochondrial membrane these metals inhibit the opening of the mitochondrial permeability transition pore caused by Ca²⁺ overload of mitochondria. The data we obtained are important for a better understanding of the mechanisms of the damaging action of rare-earth elements on Ca²⁺-dependent processes in the vertebrate myocardium.