Mechanisms of coupling between DNA recognition specificity and catalysis in EcoRI endonuclease

Proteins that bind to specific sites on DNA often do so in order to carry out catalysis or specific protein-protein interaction while bound to the recognition site. Functional specificity is enhanced if this second function is coupled to correct DNA site recognition. To analyze the structural and energetic basis of coupling between recognition and catalysis in EcoRI endonuclease, we have studied stereospecific phosphorothioate (PS) or methylphosphonate (PMe) substitutions at the scissile phosphate GpAATTC or at the adjacent phosphate GApATTC in combination with molecular-dynamics simulations of the catalytic center with bound Mg2+. The results show the roles in catalysis of individual phosphoryl oxygens and of DNA distortion and suggest that a "crosstalk ring" in the complex couples recognition to catalysis and couples the two catalytic sites to each other.     

Lithostratigraphy and carbonate microfacies across the Permian–Triassic boundary near Julfa (NW Iran) and in the Baghuk Mountains (Central Iran)

Permian–Triassic boundary sections in the Julfa (NW Iran) and Abadeh (Central Iran) regions display a succession of three characteristic rock units, (1) the Paratirolites Limestone with the mass extinction horizon at its top, (2) the ‘Boundary Clay’, and (3) the earliest Triassic Elikah Formation with the conodont P–Tr boundary at its base. The carbonate microfacies reveals a facies change, in the sections near Julfa, within the Paratirolites Limestone with an increasing number of intraclasts, Fe–Mn crusts, and biogenic encrustation. A decline in carbonate accumulation occurs towards the top of the unit with a sponge packstone in the sections, and finally resulting in a complete demise of the carbonate factory. The succession of the ‘Boundary Clay’ differs in the two regions; thin horizons of sponge packstone are present in the Julfa region and ‘calcite fans’ of probably inorganic origin in the Abadeh Region. The skeletal carbonate factory of the Late Permian was restored with the deposition of microbial carbonates at the base of the Elikah Formation, where densely laminated bindstone, floatstone with sparry calcite spheres, and oncoid wackestone/floatstone predominate.     

PDCD10 Gene Mutations in Multiple Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.     

Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.     

Role of the exosporium in the stability of the Bacillus sphaericus binary toxin

Abstract The persistence of toxicity of the Bacillus sphaericus 1593 binary toxin was compared when produced in B. sphaericus , inside the exosporium, or in a recombinant B. thuringiensis strain, outside the exosporium. The stability of the toxin crystal was affected by temperature and quality of the water, but not by the location of the production in the bacterial cell.     

applications of electroporation of adherent cellsIn Situ,on a partly conductive slide

One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the ch0ance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too lowT _m of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.