Evaluation of K-RAS gene in colorectal cancer

The aim of the study was to assess the prevalence of K-RAS gene mutations in colorectal cancer and their role in diagnosis and prognosis. The study involved 36 patients with colorectal cancer at different stages of the disease progression and with different histopathologic grading. Mutations of codon 12 of K-RAS gene investigated using PCR-RFLP technique were found in 15 patients (41.67%). Although no statistically significant correlation was observed between the disease progression, histopathologic findings, gender and age, we suppose that assessment of K-RAS gene mutations might be of clinical value in the prognosis of colorectal cancer.     

Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians

Batrachochytrium dendrobatidis is a pathogen of amphibians that has been implicated in severe population declines on several continents. We investigated the zoospore activity, physiology and protease production of B. dendrobatidis to help understand the epidemiology of this pathogen. More than 95% of zoospores stopped moving within 24 h and swam less than 2 cm before encysting. Isolates of B. dendrobatidis grew and reproduced at temperatures of 4-25 C and at pH 4-8. Growth was maximal at 17-25 C and at pH 6-7. Exposure of cultures to 30 C for 8 d killed 50% of the replicates. B. dendrobatidis cultures grew on autoclaved snakeskin and 1% keratin agar, but they grew best in tryptone or peptonized milk and did not require additional sugars when grown in tryptone. B. dendrobatidis produced extracellular proteases that degraded casein and gelatin but had no measurable activity against keratin azure. The proteases were active against azocasein at temperatures of 6-37 C and in a pH range of 6-8, with the highest activity at temperatures of 23-30 C and at pH 8. The implications of these observations on disease transmission and development are discussed.     

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.     

Physiological and biochemical characterization of ethylene-generated gravicompetence in primary shoots of coleoptile-less gravi-incompetent rye seedlings

A recent study demonstrated that gravi-incompetent coleoptile-less seedlings of rye exhibit gravi-competence after exogenous application of ethylene. Treatments and conditions which induce and interfere with this phenomenon were analysed in more detail. Aminocyclopropane-1-carboxylic acid (ACC) as a precursor of ethylene has similar gravicompetence-inducing effects and also appropriate conditions of light, which strongly enhances ethylene synthesis. Both effects can be inhibited by the ethylene-perception blocking agent methylcyclopropene (MCP) or inhibitors of ethylene synthesis such as aminovinylglycine (AVG), indicating that light exerts its gravicompetence-generating effect via induced/enhanced ethylene synthesis. Gain in gravicompetence is accompanied by the induced/enhanced occurrence of calreticulin and lipoxygenase as detected by 2D-gels and Q-TOFF-analyses. Previously gravicompetent, light-grown coleoptile-less seedlings are characterized by gravi-incompetent growth during subsequent horizontal gravistimulation when perception of ethylene is inhibited by MCP. The results demonstrate that continuous perception of ethylene is an indispensable step, permanently required for the regulation of gravitropic growth in germinating primary shoots of rye, either within the process of graviperception and/or of the transduction of the gravi-signal.     

Optimization of naked DNA delivery for interferon subtype immunotherapy in cytomegalovirus infection

Type I interferon (IFN) gene therapy modulates the immune response leading to inflammatory heart disease following cytomegalovirus (CMV) infection in a murine model of post-viral myocarditis. Efficacy of different immunisation protocols for the IFN constructs was influenced by the dose of DNA, subtype choice, combination use, pre-medication, and timing of DNA administration. Optimal efficacy was found with bupivacaine treatment prior to DNA inoculation of 200μgIFN DNA 14 days prior to virus challenge. Maximal antiviral and antimyocarditic effects were achieved with this vaccination schedule. Furthermore, inoculation of synergistic IFN subtypes demonstrated enhanced efficacy when delivered either alone or with CMVgB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced diseases. Published: February 17, 2003     

Sepsis induces DNA fragmentation in rat skeletal muscle

Recent studies have demonstrated the activation of skeletal muscle DNA fragmentation in some catabolic conditions. In an attempt to elucidate if sepsis (a catabolic state) was also associated with muscle apoptosis, sepsis was induced by cecal ligation and puncture, and the results clearly show an induction of DNA fragmentation in gastrocnemius muscle following the induction of the septic state. Administration of rolipram (an inhibitor of tumour necrosis factor-a (TNF-alpha) synthesis) to septic rats clearly prevented the increased DNA fragmentation, suggesting that TNF-alpha is involved in the activation of the apoptotic events in septic rat skeletal muscle.     

Generation of anti-idiotypic reagents in the EGFRvIII tumor-associated antigen system

The use of anti-idiotype (anti-id) vaccines for immunotherapy of human cancers is attractive, as immunization with true anti-id reagents (Ab2 beta) has been shown to induce both cellular and humoral immunity, frequently when the original antigen does not, or when a state of anergy to the self-expressed tumor-associated antigen exists. The aim of this study was to investigate the potential of an anti-id vaccine approach to the glioma-associated antigen epidermal growth factor receptor variant III (EGFRvIII) for human clinical trials. By using conventional methodology, seven rat mAbs specific for the binding site of the murine anti-EGFRvIII-specific mAb Y10, as defined by the ability to inhibit the binding of mAb Y10 to EGFRvIII expressed on cells or as purified protein, were generated, and a subset (3/7) was found to be true Ab2 beta, as defined by the ability to induce the formation of antibody directed against EGFRvIII in two species (mouse and rabbit) when used as immunogen. The ability of these three Ab2 beta to elicit a protective anti-tumor response when used as a vaccine in the syngeneic, subcutaneous C57Bl/6-B16mseEGFRvIII tumor model was investigated. Following vaccination with one Ab2 beta mAb (2C7), 6/20 mice failed to develop tumor upon challenge, and 3/20 mice with outgrowing tumors exhibited dramatic regression of incipient tumors. Vaccination with a second mAb (5G8) resulted in one tumor-free survivor and one tumor regressor; vaccination with the third Ab2 beta mAb (7D3) did not confer protection, but did significantly increase the latency period until tumor outgrowth in all vaccinated recipients. The ability of Ab2 beta mAb 2C7 to induce an anti-EGFRvIII response in non-human primates was investigated by using the saponin adjuvant approved for human clinical trial, QS-21. Three of three macaques produced anti-EGFRvIII titers, as detected on EGFRvIII-expressing cells by both ELISA and fluorescence-activated cytometric analysis, following six immunizations with Ab2 beta mAb 2C7 and QS-21. The results obtained confirm that an anti-id response in the EGFRvIII antigen system can be induced in rodents, rabbits, and non-human primates, and it may prove a useful adjunct to immunotherapeutic approaches to EGFRvIII-positive gliomas, breast carcinomas, and non-small-cell lung tumors.     

Interactions of inositol 1,4,5-trisphosphate (IP(3)) receptors with synthetic poly(ethylene glycol)-linked dimers of IP(3) suggest close spacing of the IP(3)-binding sites

The distances between the inositol 1,4,5-trisphosphate (IP(3))-binding sites of tetrameric IP(3) receptors were probed using dimers of IP(3) linked by poly(ethylene glycol) (PEG) molecules of differing lengths (1-8 nm). Each of the dimers potently stimulated (45)Ca(2+) release from permeabilized cells expressing predominantly type 1 (SH-SY5Y cells) or type 2 (hepatocytes) IP(3) receptors. The shortest dimers, with PEG linkers of an effective length of 1.5 nm or less, were the most potent, being 3-4-fold more potent than IP(3). In radioligand binding experiments using cerebellar membranes, the shortest dimers bound with highest affinity, although the longest dimer (8 nm) also bound with almost 4-fold greater affinity than IP(3). The affinity of monomeric IP(3) with only the PEG attached was 2-fold weaker than IP(3), confirming that the increased affinity of the dimers requires the presence of both IP(3) motifs. The increased affinity of the long dimer probably results from the linked IP(3) molecules binding to sites on different receptors, because the dimer bound with greater affinity than IP(3) to cerebellar membranes, where receptors are densely packed, but with the same affinity as IP(3) to purified receptors. IP(3) and the IP(3) dimers, irrespective of their length, bound with similar affinity to a monomeric IP(3)-binding domain of the type 1 IP(3) receptor expressed in bacteria. Short dimers therefore bind with increased affinity only when the receptor is tetrameric. We conclude that the four IP(3)-binding sites of an IP(3) receptor may be separated by as little as 1.5 nm and are therefore likely to be placed centrally in this large (25 x 25 nm) structure, consistent with previous work indicating a close association between the central pore and the IP(3)-binding sites of the IP(3) receptor.     

DNA damage in female workers exposed to pesticides in banana plantations at Limón,Costa Rica

Pesticide use in Costa Rica is very high and all year round. A high percentage of what is sprayed remains in the environment and in the living organisms around. This situation brings contamination and health problems to people in contact with them. The onset of adverse effects may be in the short or the long term, and symptoms vary widely, from headaches to cancer. Much research in this area has been devoted to acute or chronic effects, and not until recently to the genotoxic effect of pesticides. This study evaluated the genotoxic effect of pesticides used in banana packing activities, using the comet assay (single cell electrophoresis) as the biological marker in lymphocytes. This was a case-control double blind study of 30 exposed women from 15 banana farms and 28 women not occupationally exposed to pesticides from the same geographic area. Results show damage to single stranded DNA after working from 5 to 15 years (R2 = 0.12). In Costa Rica we do not have an historical record of the kind of pesticides used in banana farms, the period of time and for how long were they used. This prevented further analysis concerning dose, frequency of exposure and use of new or old kind of pesticides in the farms in relation to DNA damage. The comet assay is of value in the genetic monitoring of pesticide exposed populations.