Naresh C. Verma

Modulation of radiation-induced protein kinase C activity by phenolics.

Natural phenolic compounds were tested in vitro for their effect on the activity of protein kinase C (PKC) isolated from the liver cytosol and the particulate fraction of unirradiated mice and mice irradiated at 5 Gy. Following irradiation, the PKC activity was found to be increased in both cytosolic and particulate fractions. Curcumin, ellagic acid and quercetin were effective in inhibiting radiation-induced PKC activity. Curcumin and ellagic acid were found to be more inhibitory towards radiation-induced PKC activity, while quercetin was the least effective. Curcumin was found to inhibit the activated cytosolic and particulate PKC at very low concentrations. Activation of PKC is one of the means of conferring radioresistance on a tumour cell. Suppression of PKC activity by phenolics may be one of the means of preventing the development of radioresistance following radiotherapy.

Dose-Dependent Differential Expression of Protein Kinase C Isozymes in Mouse Lymphocytes after Gamma Irradiation In Vivo and Ex Vivo

Abstract Varadkar, P. A., Krishna, M. and Verma, N. C. Dose-Dependent Differential Expression of Protein Kinase C Isozymes in Mouse Lymphocytes after Gamma Irradiation In Vivo and Ex Vivo. Radiat. Res. 159, 453–457 (2003). Protein kinase C (PKC, now known as Prkc) plays an important role in the response of cells to radiation, but little is known about the specific response of each isozyme in the radiation-induced response of cells in whole animals. However, most studies are based on single cells. There is a paucity of data on signaling after whole-body irradiation. In this study, a comparison has been made between the expression of Prkc isozymes after in vivo and ex vivo irradiation. There was a significant difference in the dose response of the isozymes. In animals in which lymphocytes were irradiated ex vivo, the expression of the Prkca isozyme was found to be maximum at 3 Gy, while in vivo irradiation did not increase the expression beyond that of 1 Gy. Prkcd was marginally activated after 0.1 Gy ex vivo irradiation, whereas there was significant activation of expression after in vivo irradiation with 3 Gy. The response of Prkcz was found to be similar to that of Prkcd. Prkc is a crucial enzyme that is being used to manipulate the response of tumors to radiotherapy. Conventional radiotherapy is delivered at low doses, and hence only those isozymes that are activated at these doses should be taken into consideration. Moreover, the differences between the response of a single cell and that of the whole animal must be considered.

Alterations in Hepatic Kinase Activity Following Whole Body γ-Irradiation of Mice

The chronological activation of the signaling molecules following whole body γ-irradiation was investigated in mouse liver. The activity of two kinases, tyrosine kinase and protein kinase C (PKC), ...

An assay for DNA photolyases using non-radioactive DNA and restriction enzymes

Restriction enzymes, such as Eco RI, Hind III, etc., which have a potential pyrimidine dimer site in their recognition sequence, fail to cleave DNA if their recognition site is modified by the formation of pyrimidine dimers as a result of UV irradiation of DNA (J. E. Cleaver, J. Mol. Biol., 170 (1983) 305-317). We have made use of this functional property of restriction enzymes to develop a rapid and sensitive assay for DNA photolyases. UV-irradiated plasmid pBR322 DNA is only partially digested when incubated with a single site enzyme Hind III even at high concentration (20 units (micrograms DNA)-1). The amount of DNA not cleaved by Hind III is determined by agarose gel electrophoresis. The effect of UV irradiation is reversed by the photoreactivation of DNA. The decrease in the amount of Hind III-resistant DNA on treatment with photolyase gives a measure of the enzymatic activity of the photolyase preparation. The advantage of using non-radioactive DNA and the high speed and simplicity of this assay make it especially suitable for use in the purification of photolyases.

Activation-dependent changes in microenvironment of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase

The spinach ribulose 1,5‐bisphosphate carboxylase/oxygenase was labelled with o‐phthalaldehyde, which forms a stable fluorescent isoindole adduct at the active site. The fluorescence behaviour of the labelled enzyme after activation to different levels by Mg2+ was compared with that of a synthetic isoindole adduct of o‐phthalaldehyde, namely 1‐(hydroxyethylthio)‐2‐β hydroxyethylisoindole in solvents of different pH and polarity. The results suggest that the microenvironment at the catalytically incompetent active site of the unactivated Rubisco is highly acidic (pH < 2) in nature. The activation by Mg2+ results in the conformational change such that the effective pH at the active site increases to > 8. The polarity of the active site of the activated enzyme was found to be similar to that of a mixture of hexane and toluene.