Synthetic deoxy-oligo duplexes containing short gaps of 1 and 4 nucleotides were used as model substrates to assess the DNA gap repair ability of the neuronal extracts prepared from cerebral cortex of rats of different ages. Our results demonstrate that gap repair activity in neurons decreases markedly with age. The decreased activity could be restored by supplementing the neuronal extracts with pure recombinant rat liver DNA polymerase b. High levels of DNA polymerase b supplementation resulted in gap-filling activity that proceeded essentially through addition of nucleotides through a slow distributive strand displacement mode to achieve full template length (32-mer). However, at lower concentrations of DNA polymerase b, the gap repair takes place quickly through gap filling followed by ligation to downstream primer, in an energy efficient manner. For this to happen, the conditions required are the presence of 5¢-PO 4 on the downstream primer and supplementation of aging neuronal extracts with DNA-ligase in addition to recombinant DNA polymerase b. These results demonstrate that aging neurons are unable to affect base excision repair (BER) due to deficiency of DNA polymerase b and DNA-ligase and fortifying aged neuronal extracts with these two factors can restore the lost BER activity. Recent studies have revealed that the most predominant DNA polymerase present in adult rat brain is DNA polymerase b (pol b; Waser et al. 1979;Prapurna and Rao 1997;Raji et al. 2002). It is suggested that the activity of pol b is markedly reduced in whole brain extracts, as well as in isolated neuronal and glial cell fractions with age (Prapurna and Rao 1997;Rao et al. 2000;Cabelof et al. 2002;Raji et al. 2002). On a different note, it is now well established that pol b is an important component of DNA base excision repair (BER) pathway (Wilson 1998;Idriss et al. 2002) and pol b is considered as the key polymerase for both the short and long patch repair modes of BER (Dianov et al. 1999;Podlutsky et al. 2001). Such unconnected observations led to the hypothesis that BER might be adversely affected in the aging brain and reflect its diminished function with age.Our earlier studies to examine the pol b-dependent repair activity in extracts of neurons isolated from cerebral cortices of rats of varying ages used primer extension assays (Rao et al. 2000). It was observed that primer extension activity was drastically reduced, particularly if the 3¢-end of the primer had contained a mismatched base, in the aging neurons and that addition of pure recombinant rat liver pol b to the reaction mixture restored the lost activity. However, it is now well established that the most preferred and relevant substrate for pol b (therefore BER) is a duplex DNA with a gap of 1-6 nucleotides (Singhal and Wilson 1993;Chagovetz et al. 1997). Hence, in the present study we used relevant substrates, oligo duplexes with a gap of 1-4 nucleotides in one of the strands, for measuring the BER activity in Received July 12, 2004; revised manuscript rece...

The relative proportions of DNA-polymerases alpha, beta, delta and epsilon (pols alpha, beta, delta and epsilon ) activities in isolated neuronal and astroglial cell fractions from developing, adult and aging rat brain cerebral cortex, were examined. This was achieved through a protocol that takes advantage of the reported differential sensitivities of different DNA-polymerases towards certain inhibitors like butylphenyl and butylanilino nucleotide analogs, 2',3'-dideoxythymidine triphosphate (ddTTP), monoclonal antibody of human alpha polymerase and the use of two template primers as substrates. The results indicate that while DNA-polymerase beta (pol beta) is the predominant enzyme, significant levels of DNA-polymerases alpha and delta/epsilon (pols alpha and delta/epsilon ) are also present in both cell types at all the post-natal ages studied. A notable difference regarding the relative abundance of DNA-polymerases other than beta is the higher percentage of pol delta/epsilon in neurons and a more sustained pol alpha activity through the life span in astroglia. The presence of detectable proportion of DNA-polymerases other than beta (particularly the delta/epsilon type) may be taken to indicate their role in long patch base excision repair as well as in other modes of DNA repair.

The corrosion behaviour of the hard anodic coatings prepared by three different methods viz., conventional hard anodizing (C-HA), pulse hard anodizing (P-HA) and low voltage room temperature pulse hard anodizing (LVP-HA) on AA 6061 was compared using electrochemical impedance spectroscopy (EIS) and linear polarisation. Scanning electron microscope (SEM) was used to study the surface morphology before and after corrosion. EIS data revealed that no significant difference in corrosion resistance was observed among three types. R p , I corr and E corr were found to be highly stable over the exposure period up to 72 h which confirms the excellent corrosion resistance. The studies indicate that the corrosion resistance of LVP-HA and P-HA, which are processed at lower voltage and at relatively higher temperatures, are comparable to that of C-HA which is processed at sub-zero temperatures and high operating voltages. The corrosion resistance of all coatings was found to be improved after hydrothermal sealing.

This study investigated the hot corrosion performance of a dissimilar weldment of Ni-based superalloy and stainless steel joined by CO2 laser welding and improved by high-velocity oxy-fuel (HVOF) coating in a Na2SO4−60wt%V2O5 atmosphere at 900°C. A dissimilar butt joint of AISI 321 and alloy 825 was fabricated by CO2 laser welding with low heat input after obtaining the optimum welding parameters by bead-on-plate trials. The metallurgical and mechanical properties of the laser weldment were evaluated. The tensile test results indicated the occurrence of fracture in the base metal AISI 321 side. The HVOF process was employed to coat Ni−20wt%Cr on the weldment. To evaluate the surface morphology of the corrosion products formed on the uncoated and HVOF-coated weldments, scanning electron microscopy (SEM) analysis was performed. Energy-dispersive spectroscopy (EDS) was used to determine the different elements present on the surface scales. The existence of oxide phases on the weldments was determined by X-ray diffraction (XRD). The cross sections of the weldments were characterized by SEM with EDS line mapping analysis. The results indicated that the HVOF-coated weldment exhibited superior hot corrosion resistance due to the development of Cr2O3 and NiCr2O4 protective oxide scales.

A three-step reaction strategy has been developed to examine the mechanism of extension of a mismatched primer in an oligoduplex substrate by rat neuronal extracts and DNA polymerase beta. The results revealed that in the case of duplexes with a mismatch at 3'-end of primer, significant extension by DNA polymerase beta has taken place only after the removal of the mismatched base, thus indicating the presence of a proof reading 3'-5' exonuclease activity in neuronal extracts of all ages. A closer examination of the neuronal exonuclease activity revealed that bases are excised from the 3' end in a sequential and nonspecific manner, although initial excision of a mismatched base was slightly faster. Further, the excision efficiency is seen to decrease with the age of the animal but apparently does not go below a critical level so as to become a rate-limiting factor for the DNA-repair activity.