Cooperative effects in the peptidyltransferase center of Escherichia coli ribosomes

We have measured the binding isotherms of C--A--C--C--A(3'NH)-[14C]Phe to the 70S ribosomes and 50S subunits of Escherichia coli and proposed a theoretical model for adsorption when cooperative interaction occurs between ligands that are adsorbed on ribosomes. Analysis of the experimental binding isotherms leads to the following conclusions. A ribosome (or subunit) binds two C--A--C--C--A(3'NH)-Phe molecules. The binding of C--A--C--C--A(3'NH)-Phe to a ribosome (or subunit) is a cooperative process, characterized by a cooperativity coefficient tau = 40 +/- 5 or more. The binding of C--A--C--C--A(3'NH)-AcPhe at the donor site of the peptidyltransferase center (association binding constant 1.5 X 10(6) M-1) and the binding of puromycin at the acceptor site also occur cooperatively with a coefficient of 10-25, the association binding constant of puromycin at the acceptor site being (1-2) X 10(4) M-1. The puromycin association binding constant at the donor site multiplied by the cooperativity coefficient of two interacting puromycin molecules absorbed on a ribosome equals 100-200 M-1.     

New non-nucleoside substrates for terminal deoxynucleotidyl transferase: the synthesis and the dependence of substrate properties on structure

N-(9-Fluorenylmethoxycarbonyl)-omega-aminoalkyl, N-(9-fluorenylmethoxycarbonyl)-8-amino-3,6-dioxaoctyl, and N-(9-fluorenylmethoxycarbonyl)-6-aminohexanoyl]-2-aminoethyl triphosphates were synthesized. All of them were shown to be the substrates of the calf thymus terminal deoxynucleotidyl transferase. Their substrate properties depend on the length and structure of linker between the 9-fluorenylmethoxycarbonyl and triphosphate moieties.     

Possible role of eclosion rhythm in mediating the effects of light-dark environments on pre-adult development in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background  

In insects, circadian clocks have been implicated in affecting life history traits such as pre-adult development time and adult lifespan. Studies on the period (per) mutants of Drosophila melanogaster, and laboratory-selected lines of Bactrocera cucurbitae suggested a close link between circadian clocks and development time. There is a possibility of clock genes having pleiotropic effects on clock period and pre-adult development time. In order to avoid such pleiotropic effects we have used wild type flies of same genotype under environments of different periodicities, which phenotypically either speeded up or slowed down the eclosion clock of D. melanogaster.     

Synthesis and Substrate Properties of Modified Nucleoside 5"-Triphosphates Mimicking dATP in the Reactions of DNA Synthesis Catalyzed by DNA Polymerases

Several modified nucleoside 5"-triphosphates containing adenine-mimicking pyrimidine derivatives as an aglycone were synthesized. The study of substrate properties of these compounds towards DNA-synthesizing enzymes showed their ability of being incorporated into the growing DNA chain in place of dATP.     

Synthesis and substrate properties of modified nucleoside 5'-triphosphates mimicking dATP in the reactions of DNA synthesis catalyzed by DNA polymerases

Several modified nucleoside 5'-triphosphates were synthesized containing adenine-mimicking pyrimidine derivatives as an aglycone. The study of substrate properties of these compounds towards DNA-synthesizing enzymes showed their ability of being incorporated into the growing DNA chain in place of dATP.     

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERβ mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class “S1.2 Other Anabolic Agents” of the list of prohibited substances of the World Anti-Doping Agency.     

Excision of β-L- and β-D-Nucleotide Analogs From DNA by p53 Protein

Abstract

The tumor suppressor p₅₃ protein plays a critical role in the cell-cycle progression. The role of the 3′-to-5′ exonuclease activity of p53 protein in the DNA repair process remains elusive. Using an in vitro exonuclease assay and defined oligonucleotides terminated with β-D- and β-L-nucleoside analogs at the 3′-terminus, we studied the ability of p53 protein to excise β-L- and β-D-nucleoside analogs which have anticancer or antiviral potential. p53 protein removes β-D-nucleoside analogs more efficiently compared to that of β-L-nucleoside analogs. The affinity of p53 protein for an β-L-nucleotide terminated primer was 5 fold lower compared to non-modified primer. The hypothesis on an important role of the 3′-to-5′ exonuclease activity of p53 protein in the action of nucleoside analogs was proposed.     

Acyclic 2′, 3′-Dideoxy-2′,3′-didehydronucleoside 5′-Triphosphates as Termination Substrates of Broad Set of DNA Polymerases

Abstract

O4′-Nor-2′,3′-dideoxy-2′,3′-didehydronucleoside 5′-triphosphates (acyclo-d₄NTP) have been shown to have the properties of effective termination substrates for DNA biosynthesis, catalyzed by several different DNA polymerases.     

In silico characterization of a novel pathogenic deletion mutation identified in XPA gene in a Pakistani family with severe xeroderma pigmentosum

Background

Xeroderma Pigmentosum (XP) is a rare skin disorder characterized by skin hypersensitivity to sunlight and abnormal pigmentation. The aim of this study was to investigate the genetic cause of a severe XP phenotype in a consanguineous Pakistani family and in silico characterization of any identified disease-associated mutation.

Results

The XP complementation group was assigned by genotyping of family for known XP loci. Genotyping data mapped the family to complementation group A locus, involving XPA gene. Mutation analysis of the candidate XP gene by DNA sequencing revealed a novel deletion mutation (c.654del A) in exon 5 of XPA gene. The c.654del A, causes frameshift, which pre-maturely terminates protein and result into a truncated product of 222 amino acid (aa) residues instead of 273 (p.Lys218AsnfsX5). In silico tools were applied to study the likelihood of changes in structural motifs and thus interaction of mutated protein with binding partners. In silico analysis of mutant protein sequence, predicted to affect the aa residue which attains coiled coil structure. The coiled coil structure has an important role in key cellular interactions, especially with DNA damage-binding protein 2 (DDB2), which has important role in DDB-mediated nucleotide excision repair (NER) system.

Conclusions

Our findings support the fact of genetic and clinical heterogeneity in XP. The study also predicts the critical role of DDB2 binding region of XPA protein in NER pathway and opens an avenue for further research to study the functional role of the mutated protein domain.     

Quantitative,high-resolution epigenetic profiling of CpG loci identifies associations with cord blood plasma homocysteine and birth weight in humans

Supplementation with folic acid during pregnancy is known to reduce the risk of neural tube defects and low birth weight. It is thought that folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. We examined the effects of folate on the human methylome using quantitative interrogation of 27,578 CpG loci associated with 14,496 genes at single-nucleotide resolution across 12 fetal cord blood samples. Consistent with previous studies, the majority of CpG dinucleotides located within CpG islands exhibited hypomethylation while those outside CpG islands showed mid-high methylation. However, for the first time in human samples, unbiased analysis of methylation across samples revealed a significant correlation of methylation patterns with plasma homocysteine, LINE-1 methylation and birth weight centile. Additionally, CpG methylation significantly correlated with either birth weight or LINE-1 methylation were predominantly located in CpG islands. These data indicate that levels of folate-associated intermediates in cord blood reflect their influence and consequences for the fetal epigenome and potentially on pregnancy outcome. In these cases, their influence might be exerted during late gestation or reflect those present during the peri-conceptual period.Key words: cord blood, birth weight, folic acid, homocysteine, BeadArray, hierarchical clustering, Illumina