Isoform-specific activation of protein kinase c in irradiated human fibroblasts and their bystander cells

Studies over the last several years have revealed the existence of a biological phenomenon known as "bystander effect", wherein cells that are not exposed to radiation elicit a similar response to that of irradiated cells. Understanding the mechanism(s) underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for cancer radiotherapy. Evaluation of signaling pathways in bystander cells may provide an insight to understand the molecular mechanisms(s) responsible for this complex phenomenon. With this objective, the time course kinetics of intracellular distribution of protein kinase C (PKC isoforms PKC-betaII, PKC-alpha/beta, PKC-theta) was investigated in total and subcellular (cytosolic and nuclear) fractions of human lung fibroblast (MRC-5) cells. MRC-5 cells were either irradiated or treated with the irradiated conditioned medium collected 1h after 1 or 10 Gy of gamma-irradiation. The radiation dose selected was in the range of therapeutic usage of radiation for the human cancer treatment. Unexpectedly, bystander cells showed higher activation of protein kinase C isoforms as compared to irradiated and sham-treated control cells. Protein kinase C isoforms were more enriched in the nuclear fraction than the cytosolic fraction proteins. Induction of PKC isoforms in bystander cells are due to post-translational modifications as shown by the non-phosphorylated protein kinase C level in both irradiated and bystander cells did not differ from the sham-treated control cells. The specific activation of protein kinase C isoforms in bystander cells as demonstrated for the first time in this study may help to identify the effect of therapeutically used radiation exposure for the tumor destructions along with its implications for adjacent non-irradiated cells and organs.     

Ionic polymeric amphiphiles with cholesterol mesogen: adsorption and organization characteristics at the air/water interface from Langmuir film balance studies

Ionic polymeric amphiphiles consisting of cholesterol mesogen were investigated for the interfacial adsorption characteristics at the air/water interface using a Langmuir film balance with an aim to understand the influence of ionic segment from 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS) on the packing behavior of cholesterol at the interface. From surface pressure (pi)-area (A) isotherm characteristics, it is demonstrated that the homopolymer and the copolymer C consisting of 0.15 mol fraction CAB segments exhibit the most expanded structures contributing to surface area of about 84 A(2)/molecule. It is shown that the copolymer B with 0.1 mol fraction CAB provides optimum hydrophilic liphophilic balance to form the most compact structures contributing to a surface area of 35.75 A(2)/molecule. The high surface pressure, >40 mN/m, in contrast to that of PAMPS demonstrates significant adsorption of the copolymers at the interface. An interesting correlation among interfacial packing characteristics, thermal behavior, and solution structures is demonstrated. From molecular models developed for CAB, it is shown that the horizontal orientation of the linker group with respect to cholesterol chain in CAB underlies the expanded structures observed in PCAB and copolymer C.     

Sequence and stability of the goat cytochrome c

We have determined the sequence of mitochondrial cytochrome c (cyt-c) from the goat heart, and it was found to have a unique amino acid sequence among all amino acid sequences of cyt-c reported till date. Its sequence alignment with the bovine cytochrome c (b-cyt-c) led us to conclude that the goat cytochrome c (g-cyt-c) differs in amino acid sequence from b-cyt-c at only one position, i.e., Pro44(bovine) --> Ala44(goat). It has been observed that guanidinium chloride (GdmCl) induces a two-state transition between the native (N) and denatured (D) states of g-cyt-c. This conclusion is reached from the coincidence of GdmCl-induced transition curves monitored by measurements of absorbance at 405, 530 and 695 nm and circular dichroism (CD) at 222, 416 and 405 nm. Analysis of denaturation curves for the Gibbs energy of stabilization suggests that the stability of g-cyt-c is, within experimental errors, identical to that of b-cyt-c. We have also measured the effect of temperature on the equilibrium, N state <--> D state of g-cyt-c in the presence of different GdmCl concentrations. These measurements gave values of transition temperature (T(m)), changes in enthalpy (DeltaH(m)) and heat capacity (DeltaC(p)) of g-cyt-c in the absence of GdmCl, which are compared with those of b-cyt-c. We have used crystal structure coordinates of b-cyt-c to predict the structure and stability of g-cyt-c, which are compared with those of the bovine protein.     

Fate and function of anti-CD3/CD28-activated T cells following adoptive transfer: IL-2 promotes development of anti-tumor memory T cells in vivo

BACKGROUND: Adoptive immunotherapy with T cells activated through CD3 alone requires exogenous IL-2 for T-cell function and survival after transfer, but the in vivo cytokine requirement of T cells activated through CD3 and CD28 is unknown. We hypothesized that CD3/CD28-activated T cells, unlike those activated through CD3 alone, might develop into long-lived memory T cells, either with or without systemic IL-2. METHODS: We used MHC class I-restricted TCR transgenic T cells from the OT-1 mouse, specific for the surrogate tumor Ag ovalbumin (OVA), to assess the trafficking kinetics, antigenic responsiveness and anti-tumor efficacy of dual-activated T cells in vivo as a function of IL-2 administration. At days 7, 14, and 28 after transfer, lymph node cells and splenocytes were examined for donor cell persistence and antigenic responsiveness by FACS and ELISA, respectively. RESULTS: In IL-2-treated mice, donor CD8+ T cells persisted and developed a memory phenotype, based on CD44 and Ly6c expression at day 28, while mice given no IL-2 had fewer donor cells at all time points. OVA-specific release of IFN-gamma was higher from lymphocytes of IL-2-treated mice compared with no-IL-2 mice (P<0.02 at all time points). In mice challenged with an OVA-bearing subline of the AML leukemia model C1498, IL-2 did not confer added protection from tumor challenge at 1 or 2 weeks after adoptive transfer, but gave improved survival at 4 weeks post-transfer. DISCUSSION: We conclude that exogenous IL-2 is not required for anti-tumor activity of CD3/CD28-activated CD8+ cells early after adoptive transfer, but promotes T-cell persistence that confers disease protection at more remote times.     

Bioefficacy of Ceasalpinea bonduc (L.) Roxb. against Spodoptera litura Fab. (Lepidoptera: Noctuidae)

Bioefficacy of hexane, chloroform and ethyl acetate extracts of the seeds of Ceasalpinea bonduc (L.) Roxb. was studied against third instar larvae of Spodoptera litura at 0.25, 0.5, 1, 2.5 and 5.0% concentrations. Significant antifeedant, larvicidal and pupicidal activities and least LC₅₀ values were observed in chloroform extract. The chloroform extract was subjected to fractionation using silica gel column chromatography. Six fractions were obtained; among these, the third fraction showed high antifeedant, larvicidal and pupicidal activities at 1000 ppm concentration. Abnormalities in adults were also observed. All the activities were concentration dependent. C. bonduc could be useful in integrated pest management programme.     

Short dysfunctional telomeres impair the repair of arsenite-induced oxidative damage in mouse cells

Telomeres and telomerase appear to participate in the repair of broken DNA ends produced by oxidative damage. Arsenite is an environmental contaminant and a potent human carcinogen, which induces oxidative stress on cells via the generation of reactive oxygen species affecting cell viability and chromosome stability. It promotes telomere attrition and reduces cell survival by apoptosis. In this study, we used mouse embryonic fibroblasts (MEFs) from mice lacking telomerase RNA component (mTERC(-/-) mice) with long (early passage or EP) and short (late passage or LP) telomeres to investigate the extent of oxidative damage by comparing the differences in DNA damage, chromosome instability, and cell survival at 24 and 48 h of exposure to sodium arsenite (As3+; NaAsO2). There was significantly high level of DNA damage in mTERC(-/-) cells with short telomeres as determined by alkaline comet assay. Consistent with elevated DNA damage, increased micronuclei (MN) induction reflecting gross genomic instability was also observed. Fluorescence in situ hybridization (FISH) analysis revealed that increasing doses of arsenite augmented the chromosome aberrations, which contributes to genomic instability leading to possibly apoptotic cell death and cell cycle arrest. Microarray analysis has revealed that As3+ treatment altered the expression of 456 genes of which 20% of them have known functions in cell cycle and DNA damage signaling and response, cell growth, and/or maintenance. Results from our studies imply that short dysfunctional telomeres impair the repair of oxidative damage caused by arsenite. The results will have implications in risk estimation as well as cancer chemotherapy.