Prognostic significance of DNA aneuploidy and p21 ras oncoprotein expression in colorectal cancer and their role in the determination of treatment modalities

The purpose of the present study was to investigate the prognostic significance of DNA ploidy, S-phase fraction and p21 ras oncoprotein expression in patients with colorectal cancer and to correlate these factors with the clinical behavior of the tumors and their response to therapy. Of 79 patients with colorectal cancer 57% (45/79) had early stage disease. Forty-one percent (32/79) had aneuploid tumors while 30% (24/79) of the tumors had a high (>10%) S-phase fraction. p21ras oncoprotein expression was detected in 38% (30/79) of tumors. Patients with aneuploid tumors had a worse prognosis than patients with diploid tumors (p=0.0002). Similarly, patients with high S-phase fraction tumors had a shorter survival than those with low S-phase fraction tumors (p=0.005). No such difference was found between p21 raspositive and p21 ras-negative tumor subgroups. In early stage colorectal cancer, aneuploidy was closely correlated with disease outcome (p=0.029). Early stage patients with diploid tumors who received radiotherapy and chemotherapy had a better prognosis than patients with aneuploid tumors. In conclusion, DNA ploidy is a significant and independent prognostic factor in colorectal cancer. Aneuploidy and genetic alteration of the p21 ras oncoprotein are important in determining the biological aggressiveness of colorectal cancer. Furthermore, DNA ploidy may identify those subgroups of patients with early stage disease who may benefit from more aggressive treatment.     

Immunization with Brugia malayi Myosin as Heterologous DNA Prime Protein Boost Induces Protective Immunity against B. malayi Infection in Mastomys coucha

The current control strategies employing chemotherapy with diethylcarbamazine, ivermectin and albendazole have reduced transmission in some filaria-endemic areas, there is growing interest for complementary approaches, such as vaccines especially in light of threat of parasite developing resistance to mainstay drugs. We earlier demonstrated recombinant heavy chain myosin of B. malayi (Bm-Myo) as a potent vaccine candidate whose efficacy was enhanced by heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) vaccination in BALB/c mice. BALB/c mouse though does not support the full developmental cycle of B. malayi, however, the degree of protection may be studied in terms of transformation of challenged infective larvae (L3) to next stage (L4) with an ease of delineating the generated immunological response of host. In the current investigation, DNA vaccination with Bm-Myo was therefore undertaken in susceptible rodent host, Mastomys coucha (M. coucha) which sustains the challenged L3 and facilitates their further development to sexually mature adult parasites with patent microfilaraemia. Immunization schedule consisted of Myo-pcD and Myo-pcD+Bm-Myo followed by B. malayi L3 challenge and the degree of protection was evaluated by observing microfilaraemia as well as adult worm establishment. Myo-pcD+Bm-Myo immunized animals not only developed 78.5% reduced blood microfilarial density but also decreased adult worm establishment by 75.3%. In addition, 75.4% of the recovered live females revealed sterilization over those of respective control animals. Myo-pcD+Bm-Myo triggered higher production of specific IgG and its isotypes which induced marked cellular adhesion and cytotoxicity (ADCC) to microfilariae (mf) and L3 in vitro. Both Th1 and Th2 cytokines were significantly up-regulated displaying a mixed immune response conferring considerable protection against B. malayi establishment by engendering a long-lasting effective immune response and therefore emerges as a potential vaccination method against LF.     

Measurement and comparison of head scatter factor for 7 MV unflattened (FFF) and 6 MV flattened photon beam using indigenously designed columnar mini phantom

Aim

To measure and compare the head scatter factor for 7 MV unflattened and 6 MV flattened photon beam using a home-made designed mini phantom.

Background

The head scatter factor (Sc) is one of the important parameters for MU calculation. There are multiple factors that influence the Sc values, like accelerator head, flattening filter, primary and secondary collimators.

Materials and methods

A columnar mini phantom was designed as recommended by AAPM Task Group 74 with high and low atomic number material for measurement of head scatter factors at 10 cm and d_max dose water equivalent thickness.

Results

The Sc values measured with high-Z are higher than the low-Z mini phantoms observed for both 6MV-FB and 7MV-UFB photon energies. Sc values of 7MV-UFB photon beams were smaller than those of the 6MV-FB photon beams (0.6–2.2% (Primus), 0.2–1.4% (Artiste) and 0.6–3.7% (Clinac iX (2300CD))) for field sizes ranging from 10 cm × 10 cm to 40 cm × 40 cm. The SSD had no influence on head scatter for both flattened and unflattened beams. The presence of wedge filters influences the Sc values. The collimator exchange effects showed that the opening of the upper jaw increases Sc irrespective of FF and FFF.

Conclusions

There were significant differences in Sc values measured for 6MV-FB and unflattened 7MV-UFB photon beams over the range of field sizes from 10 cm × 10 cm to 40 cm × 04 cm. Different results were obtained for measurements performed with low-Z and high-Z mini phantoms.     

Overexpression of <Emphasis Type="Italic">MuHSP70</Emphasis> gene from <Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> confers multiple abiotic stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H₂O₂, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.     

Tamoxifen,protein kinase C and rat sperm mitochondria

Tamoxifen at a dose of 400 microg/kg/day has been reported to reduce the fertility of adult male rats and alter the pattern of cauda sperm motility from forward progressive to circular yawing type. Since sperm motility is powered by mitochondria, the effect of tamoxifen on mitochondrial function was studied. Tamoxifen treatment significantly increased rhodamine 123 fluorescent dye uptake by sperm mitochondria, reflecting an altered mitochondrial membrane potential. ATP and DAG levels, activities of glycolytic enzymes, creatine kinase and PKC all remained unaffected by tamoxifen. This is also the first report describing the presence of PKC alpha and beta in rat sperm. Morphological and biochemical integrity of sperm membranes was determined by electron microscopy and malondialdehyde levels, which were unaltered after tamoxifen treatment. This study indicates that the altered sperm motility induced by tamoxifen is accompanied by changes in mitochondrial membrane potential, but in the absence of any detectable change in membrane integrity, lipid peroxidation, ATP levels and activities of glycolytic enzymes, creatine kinase and PKC.     

Stress-Induced Changes in Peptidyl-Prolyl cis-trans Isomerase Activity of Sorghum bicolor Seedlings

Developmental changes and effects of various abiotic stresses on peptidyl prolyl cis-trans isomerase (PPIase) activity were studied in the seedlings of sorghum [Sorghum bicolor (L.) Moench cv. CSH-6]. The PPIase activity of sorghum seedlings markedly decreased after two days of germination. Up to 90 % of the PPIase activity was inhibited by cyclosporin-A. Maximal increase in specific PPIase activity in the 3-d-old seedlings was observed in response to osmotic stress and it was transient in nature. The stress-induced enhancement in PPIase activity, depending upon tissue and stress treatment, was due to induction of cyclophilins as well as other PPIases. Osmotic stress-induced enhancement in PPIase activity in the drought susceptible cv. SPRU-94008B was maximal in roots, as compared to shoots in the drought tolerant cv. ICSV-272. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced proteolysis leads to pre-mature cell death under the influence of elicitor like mycelial components from karnal bunt (tilletia indica) pathogen in wheat callus cultures

Calli raised from mature embryos of susceptible wheat cultivar WH 542 were used in the present study as in vitro bioassay system to study the influence of disease determinant(s) of Karnal bunt (Tilletia indica), a semi-biotrophic fungal pathogen of wheat. Influence of elicitor and conditioned medium (CM) prepared from fungal cultures of T. indica was investigated on induction of programmed cell death (PCD). Induction of PCD was observed as hypersensitive response (HR) in terms of browning at localized regions of callus cultures and induction of proteolytic enzyme(s). Elicitor treated calli showed higher induction of protease activity than untreated and CM-treated cultures, which showed not much change in the activity. It was further substantiated by gel protease assay and activation of caspase-3 like protein(s) in callus cultures that clearly suggested the presence of signaling molecule(s) in the fungal elicitor preparation rather than in conditioned medium. This study further demonstrated that only elicitor preparation possesses such molecule(s), which might be cell wall bound components, rather than secretory in nature as CM was unable to induce PCD in wheat callus cultivars.     

Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis <Emphasis Type="Italic">NPR1</Emphasis>

Cotton is an economically important crop worldwide that suffers severe losses due to a wide range of fungal/bacterial pathogens and nematodes. Given its susceptibility to various pathogens, it is important to obtain a broad-spectrum resistance in cotton. Resistance to several fungal and bacterial diseases has been obtained by overexpressing the Non-expressor of Pathogenesis-Related genes-1 (NPR1) in various plant species with apparently minimal or no pleiotropic effects. We examined the efficacy of this approach in cotton by constitutive expression of the Arabidopsis (Arabidopsis thaliana) NPR1 gene. The results show that NPR1-expressing lines exhibited significant resistance to Verticillium dahliae isolate TS2, Fusarium oxysporum f. sp. vasinfectum, Rhizoctonia solani, and Alternaria alternata. Interestingly, the transformants also showed significant resistance to reniform nematodes. Analysis of defense-related, biochemical and molecular responses suggest that when challenged with pathogens or certain systemic acquired resistance-inducing chemicals, the transgenic lines respond to a greater degree compared to the wild-type plants. Importantly, the basal activities of the defense-related genes and enzymes in uninduced transformants were no different than those in their non-transgenic counterparts. The results provide additional evidence supporting the role of NPR1 as an important part of the plant defense system and suggest a means to achieve broad-spectrum resistance to pathogens via genetic engineering.     

Role of tryptophan-208 residue in cytochrome c oxidation by ascorbate peroxidase from Leishmania major-kinetic studies on Trp208Phe mutant and wild type enzyme

Ascorbate peroxidase from L. Major (LmAPX) is a functional hybrid between cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX). We utilized point mutagenesis to investigate if a conserved proximal tryptophan residue (Trp208) among Class I peroxidase helps in controlling catalysis. The mutant W208F enzyme had no effect on both apparent dissociation constant of the enzyme-cytochrome c complex and K(m) value for cytochrome c indicating that cytochrome c binding affinity to the enzyme did not alter after mutation. Surprisingly, the mutant was 1000 times less active than the wild type in cytochrome c oxidation without affecting the second order rate constant of compound I formation. Our diode array stopped-flow spectral studies showed that the substrate unbound wild type enzyme reacts with H(2)O(2) to form compound I (compound II type spectrum), which was quite different from that of compound I in W208F mutant as well as horseradish peroxidase (HRP). The spectrum of the compound I in wild type LmAPX showed a red shift from 409 nm to 420 nm with equal intensity, which was broadly similar to those of known Trp radical. In case of compound I for W208F mutant, the peak in the Soret region was decreased in heme intensity at 409 nm and was not shifted to 420 nm suggesting this type of spectrum was similar to that of the known porphyrin pi-cation radical. In case of an enzyme-H(2)O(2)-ascorbate system, the kinetic for formation and decay of compound I and II of a mutant enzyme was almost identical to that of a wild type enzyme. Thus, the results of cytochrome c binding, compound I formation rate and activity assay suggested that Trp208 in LmAPX was essential for electron transfer from cytochrome c to heme ferryl but was not indispensable for ascorbate or guaiacol oxidation.