Magnesium is required for specific DNA binding of the CREB B-ZIP domain

We have examined binding of the CREB B-ZIP protein domain to double-stranded DNA containing a consensus CRE sequence (5′-TGACGTCA-3′), the related PAR, C/EBP and AP-1 sequences and the unrelated SP1 sequence. DNA binding was assayed in the presence or absence of MgCl₂ and/or KCl using two methods: circular dichroism (CD) spectroscopy and electrophoretic mobility shift assay (EMSA). The CD assay allows us to measure equilibrium binding in solution. Thermal denaturation in 150 mM KCl indicates that the CREB B-ZIP domain binds all the DNA sequences, with highest affinity for the CRE site, followed by the PAR (5′-TAACGTTA-3′), C/EBP (5′-TTGCGCAA-3′) and AP-1 (5′-TGAGTCA-3′) sites. The addition of 10 mM MgCl₂ diminished DNA binding to the CRE and PAR DNA sequences and abolished binding to the C/EBP and AP-1 DNA sequences, resulting in more sequence-specific DNA binding. Using ‘standard’ EMSA conditions (0.25× TBE), CREB bound all the DNA sequences examined. The CREB–CRE complex had an apparent K_d of ~300 pM, PAR of ~1 nM, C/EBP and AP-1 of ~3 nM and SP1 of ~30 nM. The addition of 10 mM MgCl₂ to the polyacrylamide gel dramatically altered sequence-specific DNA binding. CREB binding affinity for CRE DNA decreased 3-fold, but binding to the other DNA sequences decreased >1000-fold. In the EMSA, addition of 150 mM KCl to the gels had an effect similar to MgCl₂. The magnesium concentration needed to prevent non-specific electrostatic interactions between CREB and DNA in solution is in the physiological range and thus changes in magnesium concentration may be a cellular signal that regulates gene expression.     

Combining mutations in HIV-1 protease to understand mechanisms of resistance

HIV-1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV-1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2-1.2 A to determine the associated molecular changes. Sequence-dependent changes in protease-inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2', P3/P3'/P4/P4', and P1/P1', respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6(pol)-PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag-Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2-P2' and S2-S2' sites. The complex effects of combining mutations are discussed.     

Marathon running alters the DNA base excision repair in human skeletal muscle

Reactive oxygen and nitrogen species generated either as products of aerobic metabolism or as a consequence of environmental mutagens, oxidatively modify DNA. Formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (endo III) or their functional mammalian homologues repair 7,8-dihydro-8-oxoguanine (8-oxoG) and damaged pyrimidines, respectively, to curb the deleterious effects of oxidative DNA alterations. A single bout of physical exercise can induce oxidative DNA damage. However, its effect on the activity of repair enzymes is not known. Here we report that the activity of a functional homolog of Fpg, human 8-oxoG DNA glycosylase (hOGG1), is increased significantly, as measured by the excision of 32P labeled damaged oligonucleotide, in human skeletal muscle after a marathon race. The AP site repair enzyme did not change significantly. Despite the large individual differences among the six subjects measured, data suggest that a single-bout of aerobic exercise increases the activity of hOGG1 which is responsible for the excision of 8-oxoG. The up-regulation of DNA repair enzymes might be an important part of the regular exercise induced adaptation process.     

Conservation of biodiversity in Romania

This paper briefly discusses the history and development of nature protection in Romania. It summarises the current situation of protected areas, and discusses the ecological, ethical and philosophical ideas concerning biodiversity conservation in the country.     

Influence of hyperthyroidism on the effect of adenosine transport blockade assessed by a novel method in guinea pig atria

The aim of the present study was to investigate the effect of hyperthyroidism on the trans-sarcolemmal adenosine (Ado) flux via equilibrative and nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporters (ENT1) in guinea pig atria, by assessing the change in the Ado concentration of the interstitial fluid ([Ado]_ISF) under nucleoside transport blockade with NBTI. For the assessment, we applied our novel method, which estimates the change in [Ado]_ISF utilizing the altered inotropic response to N⁶-cyclopentyladenosine (CPA), a relative stable selective agonist of A₁ Ado receptors, by providing a relative index, the equivalent concentration of CPA. Our results show an interstitial A do accumulation upon ENT1 blockade, which was more extensive in the hyperthyroid samples (CPA concentrations equieffective with the surplus [Ado]_ISF were two to three times higher in hyperthyroid atria than in euthyroid ones, with regard to the negative inotropic effect of CPA and Ado). This suggests an enhanced Ado influx via ENT1 in hyperthyroid atria. It is concluded that hyperthyroidism does not alter the prevailing direction of the Ado transport, moreover intensifies the Ado influx in the guinea pig atrium.     

Discovery, synthesis, and structure activity of a highly selective alpha7 nicotinic acetylcholine receptor antagonist

Nicotinic acetylcholine receptors (nAChRs) that contain an alpha7 subunit are widely distributed in neuronal and nonneuronal tissue. These receptors are implicated in the release of neurotransmitters such as glutamate and in functions ranging from thought processing to inflammation. Currently available ligands for alpha7 nAChRs have substantial affinity for one or more other nAChR subtypes, including those with an alpha1, alpha3, alpha6, and/or alpha9 subunit. An alpha-conotoxin gene was cloned from Conus arenatus. Predicted peptides were synthesized and found to potently block alpha3-, alpha6-, and alpha7-containing nAChRs. Structure-activity information regarding conotoxins from distantly related Conus species was employed to modify the C. arenatus derived toxin into a novel, highly selective alpha7 nAChR antagonist. This ligand, alpha-CtxArIB[V11L,V16D], has low nanomolar affinity for rat alpha7 homomers expressed in Xenopus laevis oocytes, and antagonism is slowly reversible. Kinetic analysis provided insight into the mechanism of antagonism. alpha-CtxArIB interacts with five ligand binding sites per alpha7 receptor, and occupation of a single site is sufficient to block function. The peptide was also shown to be highly selective in competition binding assays in rat brain membranes. alpha-CtxArIB[V11L,V16D] is the most selective ligand yet reported for alpha7 nAChRs.     

Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis

We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5′ fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30°C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.     

Protein kinase C inhibitor and irradiation-induced apoptosis: relevance of the cytochrome c-mediated caspase-9 death pathway.

Caspases are a family of cysteine proteases that constitute the apoptotic cell death machinery. We report the importance of the cytochrome c-mediated caspase-9 death pathway for radiosensitization by the protein kinase C (PKC) inhibitors staurosporine (STP) and PKC-412. In our genetically defined tumor cells, treatment with low doses of STP or the conventional PKC-specific inhibitor PKC-412 in combination with irradiation (5 Gy) potently reduced viability, enhanced mitochondrial cytochrome c release into the cytosol, and specifically stimulated the initiator caspase-9. Whereas treatment with each agent alone had a minimal effect, combined treatment resulted in enhanced caspase-3 activation. This was prevented by broad-range and specific caspase-9 inhibitors and absent in caspase-9-deficient cells. The tumor suppressor p53 was required for apoptosis induction by combined treatment but was dispensable for dose-dependent STP-induced caspase activation. These results demonstrate the requirement for an intact caspase-9 pathway for apoptosis-based radiosensitization by PKC inhibitors and show that STP induces apoptosis independent of p53.     

Accumulation of red elemental selenium nanoparticles and their biological effects in Nicotinia tabacum

The uptake, accumulation and biological effects of red nano-sized elemental selenium (nanoSe) in comparison to selenate were investigated in plant system at the first time. The data clearly indicated that red nanoSe was taken up by tobacco callus cultures and rooted tobacco plantlets. The roots of regenerated plantlets accumulated selenium in very high concentrations, 2,947 ± 99 mg/kg DW, from the medium containing 530 μM nanoSe. The biological effects of nanoSe were different from the selenate ion in plant tissue culture. NanoSe (265–530 μM concentration range) stimulated the organogenesis and the growth of root system significantly (~40 %) while selenate did not show these effects at any concentration moreover inhibited both callus growth and root regeneration totally in 265–530 μM concentrations.