High-throughput genomic research tools are becoming standard in the biologist's toolbox. After processing the genomic data with one of the many available statistical algorithms to identify statistically significant genes, these genes need to be further analyzed for biological significance in light of all the existing knowledge. Literature mining – the process of representing literature data in a fashion that is easy to relate to genomic data – is one solution to this problem.We present a web-based tool, MILANO (Microarray Literature-based Annotation), that allows annotation of lists of genes derived from microarray results by user defined terms. Our annotation strategy is based on counting the number of literature co-occurrences of each gene on the list with a user defined term. This strategy allows the customization of the annotation procedure and thus overcomes one of the major limitations of the functional annotations usually provided with microarray results. MILANO expands the gene names to include all their informative synonyms while filtering out gene symbols that are likely to be less informative as literature searching terms. MILANO supports searching two literature databases: GeneRIF and Medline (through PubMed), allowing retrieval of both quick and comprehensive results. We demonstrate MILANO's ability to improve microarray analysis by analyzing a list of 150 genes that were affected by p53 overproduction. This analysis reveals that MILANO enables immediate identification of known p53 target genes on this list and assists in sorting the list into genes known to be involved in p53 related pathways, apoptosis and cell cycle arrest..MILANO provides a useful tool for the automatic custom annotation of microarray results which is based on all the available literature. MILANO has two major advances over similar tools: the ability to expand gene names to include all their informative synonyms while removing synonyms that are not informative and access to the GeneRIF database which provides short summaries of curated articles relevant to known genes. MILANO is available at  Saccharomyces cerevisiae, there are excellent curated databases, such as the Yeast Proteome Database [In the post-genomic era, biologists encounter a flood of information derived mainly from microarray experiments. The blessing of this wealth of information is accompanied by a great difficulty in identifying the biologically significant findings, which are often embedded in irrelevant information. Currently, there are several approaches to deal with this problem. One approach is to identify a category of genes which is overrepresented in the microarray output. This approach can be carried out using the Gene Ontology project (GO) which describes gene products in terms of their associated biological processes, cellular components and molecular functions . The advDatabase  and the Database , which cWe have developed an analysis tool that combines the advantages of all the mentioned approaches and overcomes some of the disadvantages. Our tool (MILANO – Microarray Literature-based Annotation) uses an automatic search of literature databases for performing custom annotation of the list of genes obtained from a microarray output. This is done by generating dynamic annotations for genes, built according to terms provided by the researcher. The program receives as input a list of gene identifiers obtained from any microarray experiment and a set of custom search terms. The program expands each gene identifier to its informative synonyms and searches literature databases for co- occurrences of every gene on the list with each of the custom terms. The program's output is an annotation table with the numbers of publications for each gene-term combination (hit-counts). This novel annotation format can be easily used within a web browser or a spreadsheet program to quickly identify genes within the list that are related to the terms provided by the researcher, and may be easily extended, as every hit-count in the annotation is a hyperlink to the query's results. The great advantage achieved by this method over standard static annotations, such as Gene Ontology (GO) annotations, is that the annotations are generated based on terms provided by the researcher, and therefore help in addressing the specific scientific question the researcher is pursuing.The program is able to search two literature databases, GeneRIF  and MedlTo demonstrate the power of this strategy, we have analyzed a list of 148 genes affected by over-expression of p53 . Our anaMILANO is accessed through a familiar web form Fig . A CGI . Although expansion of the search terms is a useful tool to increase the number of articles retrieved for each gene it also adds many irrelevant articles due to the fact that some of the gene symbols are not informative as Medline query terms. For example one of the aliases of the gene aquaporin_1 is CO, a term that is mostly mentioned as an abbreviation for Carbon mono-oxide, and one of the aliases of the gene CD36_antigen is FAT, which is found in over 100,000 articles, unrelated to CD36. In order to diminish this problem we filtered out from the list of gene symbols any term that was shorter than three characters and any term that is an English word. In order to check our name expansion strategy we conducted a Medline search for 16862 well-known human genes , using three search strategies: using only the official symbol for each gene (Symbol), using the official symbol together with all its aliases and the gene product (Expanded) and using only the informative terms (Filtered). Using the Expanded search allowed the identification of literature information on about ~1900 additional genes over a query using the official symbol only  with user defined secondary terms Figure . The proIn order to assist in further evaluation of the results, we have built the annotation table such that each number in the table is a hyper-link to the literature database and thus clicking on it will perform this specific search again and will open a window containing the actual abstracts found by this combination of search terms.The MILANO program can search two databases Figure  – the fuTo demonstrate the power of our literature-based annotation strategy, we analyzed a list of 148 genes affected by over-expression of p53 . This liOur first aim was to identify the known p53 target genes that were affected by p53 overproduction in this experiment. By using specific secondary terms, we were able to trim down the list of 148 genes to a much shorter list of genes highly enriched for known p53 target genes Figure . In ordeP53 is involved in apoptosis, cell cycle arrest and cancer. It is interesting to find out which of the genes affected by p53 is involved in these processes. Using MILANO we easily identified genes known to be involved in these processes Figure , which hRecently, few literature mining tools has been developed, using a similar approach to the one presented here -17, howest and the 100th query in an unbiased manner. The MILANO program uses all the published information for the annotation of each gene according to its co-occurrence in the literature with a user defined secondary search term. These features of MILANO makes it especially suitable for analyzing microarray results, since it can be used to annotate the results with terms defined by the user and not limited by preset terms such as the GO terms based annotation.MILANO is a simple and intuitive literature search tool. It allows automatic Medline and GeneRIF searches followed by a quick survey of the results. Using this tool dramatically reduces the time needed to query literature databases. Moreover, due to its systematic nature, it assists in treating the 1We have demonstrated the power of our program by the analysis of a list of 148 genes that were deregulated in cells that overproduced the p53 tumor suppressor gene . FrequenSeveral literature mining approaches have been developed to integrate multiplex biological datasets into the context of published medical literature. A good example of such an approach is the PubGene program , which sMILANO's aim is to further improve the literature based automatic annotation approach by adding two essential features that address these limitations:Each gene symbol is expanded to all its aliases, while removing non-informative terms, and the gene product name is added to the query. This addresses the synonym problem, while omitting many of the irrelevant results, thus reducing the polysemy problem (words with multiple meanings). The advantage of our synonym expansion scheme over the existing tools is demonstrated by the comparison presented in table In contrast to the existing tools, MILANO is able to search not only the Medline database, but also the GeneRIF database, which contains short summaries of articles relevant to known genes. The curation of GeneRIF is done by the National Library of Medicine's MeSH indexing staff, who have advanced degrees in the life sciences and use the full text of articles for the indexing process . Using t, a literature mining tool that can help in annotating microarray results in light of all available literature using experiment-specific terms. In designing MILANO we focused on the accuracy of the search results by providing two novel features: i) Expansion of gene names to include in the literature searches all their informative synonyms, while removing non-informative synonyms; ii) Searching two literature databases – Medline and GeneRIF. While Medline encompasses all the literature and provides the most comprehensive results, it also contains many irrelevant articles. GeneRIF provides a subset of Medline articles that are relevant to known genes and thus avoids most of the irrelevant results often found in Medline searches.We present MILANO The usefulness of MILANO is demonstrated by the automatic analysis of a list of 148 p53 target genes. The use of literature mining dramatically reduced the time and effort required for a task such as identifying the known p53 target genes within this list. A search in GeneRIF immediately discovered the full list of target genes, with no false hits.. The author will provide data, scripts and programs used on demand. We encourage users to install the software on their own servers, as we provide no assurance to the privacy or accuracy of the results.All software and databases are freely available and may be executed online at our web site: RR designed and programmed the software. IS managed the project and drafted the manuscript.


If there is life on distant worlds, how would we go about finding it? Star Trek that can detect life forms even from orbit. Unfortunately, we don't have a clue how a tricorder might work, since life forms don't seem to have any observable property that distinguishes them from inanimate matter. Furthermore, we lack a definition of life that can guide a search for life outside Earth. How can we find what we can't define? An answer may lie in the observation that life uses a small, discrete set of organic molecules as basic building blocks. On the surface of Europa and in the subsurface of Mars, we can search for alien but analogous patterns in the organics.I need a “tricorder”—the convenient, hand-held device featured on The obvious diversity of life on Earth overlies a fundamental biochemical and genetic similarity. The three main polymers of biology—the nucleic acids, the proteins, and the polysaccarides—are built from 20 amino acids, five nucleotide bases, and a few sugars, respectively. Together with lipids and fatty acids, these are the main constituents of biomass: the hardware of life , p 21. TOur lack of data is reflected in our attempts to define life. The simplest, but not the only, proof of life is to find something that is alive. There are only two properties that can determine if an object is alive: metabolism and motion.  All living things require some level of metabolism to remain viable against entropy. Movement (either microscopic or macroscopic) in response to stimuli or in the presence of food can be a convincing indicator of a living thing. But both metabolism (fire) and motion (wind) occur in nature in the absence of biology.The practical approach to the search for life is to determine what life needs. The simplest list is probably: energy, carbon, liquid water, and a few other elements such as nitrogen, sulfur, and phosphorus . Life reOrbital images, such as the canyon in On Jupiter's moon Europa, the cracks and icebergs on the surface of the ice indicate water beneath the ice, but not necessarily at the present time. Present water on Europa is indicated by the magnetic disturbance Europa makes as it moves through Jupiter's magnetic field, not unlike the way coins in the pocket of a passenger will set off an airport metal detector. Europa has a large conductor, and this is most likely a global, salty layer of water.The Viking missions to Mars in the late 1970s were the first  search for life outside Earth. Each Viking conducted three incubation experiments to detect the presence of metabolism in the Martian soil. Each lander also carried a sophisticated Gas Chromatograph Mass Spectrometer for characterizing organic molecules. The results were unexpected , 1999. TIf considered alone, the Labeled Release results would be a plausible indication for life on Mars. However, the Gas Chromatograph Mass Spectrometer did not detect the presence of any organic molecules in the soil at level of one part per billion . It is dLife on Mars is also likely to be the same at the top of the table: at the ecological level. Primary production in a Martian ecosystem is likely to be phototrophic, using carbon dioxide and water. Heterotrophs are likely to be present to consume the phototrophs and in turn to be consumed by predators. Darwinian evolution would result in many of the same patterns we see in ecosystems on Earth. While it may be similar at the top  and bottom  levels, life on Mars could be quite alien in the middle, in the realm of biochemistry. Future missions to Mars might find microfossils in sedimentary rocks such as those at Meridiani Planum. Microbes don't readily form convincing fossils; the one exception may be the strings of magnetite formed by magnetotactic bacteria . As inteIf we were to find organic material in the subsurface of Mars or on the ice of Europa, how could we determine whether it was the product of a system of biology or merely abiotic, organic material from meteorites or photochemistry? If this life were in fact related to Earth life, this should be easy to determine. We now have very sensitive methods, such as PCR and fluorescent antibody markers, for detecting life like us. This case would be the simplest to determine, but it would also be the least interesting. If the life turned out to be truly alien, then the probes specific to our biology would be unlikely to work. What, then, could we do to determine a biological origin?The question is open and possibly urgent. On space missions already being planned, we may have the opportunity to analyze the remains of alien organics on the surface of Europa or frozen below ground on Mars. The instruments that will make this analysis must be designed in the next couple of years.d- instead of l-amino acids.One approach appears promising. I call it the “Lego Principle.” It is based on the patterns of the molecules of life. Biological processes, in contrast to abiotic mechanisms, do not make use of the range of possible organic molecules. Instead, biology is built from a selected set. Thus, organic molecules that are chemically very similar to each other may have widely different concentrations in a sample of biological organics. An example of this on Earth is the 20 amino acids used in proteins and the use of the left-handed version of these amino acids. The selectivity of biological processes is shown schematically in This approach has immediate practical benefit in the search for biochemistry in the Solar System. Samples of organic material collected from Mars and Europa can be easily tested for the prevalence of one chirality of amino acid over the other. More generally, a complete analysis of the relative concentration of different types of organic molecules might reveal a pattern that is biological even if that pattern does not involve any of the familiar biomolecules. Interestingly, if a sample of organics from Mars or Europa shows a preponderance of D-amino acids, this would be evidence of life, and at the same time would show that this life was distinct from Earth life. This same conclusion would apply to any clearly biological pattern that was distinct from that of Earth life.Organic material of biological origin will eventually lose its distinctive pattern when exposed to heat and other types of radiation, , but at the low temperatures in the Martian permafrost, calculations suggest that there has been no thermal alteration . An inteOn Europa, the organic material for our tests might be collected right from the dark regions on the surface. On Mars, there is ice-rich ground in the cratered southern polar regions , which p


Cercopithecus mitis) make up an abundant, widespread and morphologically diverse species complex in eastern Africa that naturally harbors a distinct simian immunodeficiency virus (SIVsyk). We carried out a retrospective serological survey of SIV infection from both wild and captive Sykes' monkeys from Kenya. We compared two commercially available, cross-reactive ELISA tests using HIV antigens with a novel SIVsyk antigen-specific Western blot assay and analyzed the data by origin, subspecies, age and sex.The Sykes' monkey and related forms . Females were infected more often than males in both wild and captive populations. Seropositive infants were common. However, no seropositive juveniles were identified.We have developed a specific and sensitive Western blot assay for anti-SIVsyk antibody detection. Sykes' monkeys are commonly infected with SIVsyk, but with extremely variable prevalence in the wild. Higher infection prevalence in females suggests predominantly sexual transmission. High infection prevalence in infants, but none in juveniles, suggests maternal antibodies, but little or no vertical transmission. Pan troglodytes troglodytes) and West African sooty mangabey monkeys (Cercocebus atys), respectively [Macaca mulatta) and is pathogenic in that species, producing an acquired immunodeficiency syndrome-like condition [Both human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) have been evolutionarily linked to zoonotic transmissions of naturally occurring simian immunodeficiency viruses (SIVs) from Central African chimpanzees  belongs to an abundant, widespread and morphologically diverse species complex found throughout East Africa [C. mitis have previously been shown to have antibodies that cross-reacted with SIVsmm and SIVagm [The Sykes' monkey  serum samples from the same animals taken at different times. From these sera, the Innotest HIV-1/HIV-2 antibodies test  detected a total of 132 positive samples and 144 negative samples. The Genescreen HIV-1/2 version 2 test . detected 129 positive samples and 147 negative samples. Five samples gave disparate results using these two tests, four of these samples were positive using the Innotest kit, but negative using the Genscreen test and one sample was positive using the Genscreen test, but negative using the Innotest kit.Two hundred and seventy six serum samples from both wild and captive To confirm these results and compare the sensitivity and specificity of these assays, all 276 samples were screened by an SIVsyk viral antigen-specific Western blot assay. Serum samples were considered positive by SIVsyk Western blot assay if they showed reactivity to ENV, GAG or CA proteins. The SIVsyk Western assay identified a total of 138 samples as positive and 138 as negative. Ten samples that were negative by the Genscreen assay and seven samples that were negative by the Innotest assay were identified as positive by the SIVsyk Western assay . This includes both sexes and all age classes. Ten of 17 captive adult highland Sykes' and 3 of 4 captive adult lowland Sykes' were positive, yielding prevalences of 59.0% and 75.0%, respectively for these two subspecies. No known captive Blue Sykes' were available for testing. Among captive adult animals, 4 of 8 (50.0%) males and 9 of 13 (69.2%) females were positive. Four of 16 (25.0%) captive infant males and 7 of 19 (36.8%) captive infant females were positive. However, no captive juveniles (0 of 6 (0.0%) males and 0 of 5 (0.0%) females) were positive. Tables 4/67. ThiThe overall captive seroprevalence was 35.8%, which is slightly lower than the overall wild seroprevalence of 46.8%. Infection prevalence was highest in adults at 69.8% for wild populations and 61.9% in captive populations. Adult females were infected at a higher prevalence than adult males in both the wild (74.1% (20/27) for wild adult females and 65.4% (17/26) for wild adult males) and captive populations (69.2% (9/13) for captive adult females and 50.0% (4/8) for captive adult males). Infants were also found to have a high seroprevalence at 66.7% (2/3) from wild populations and 31.4% (11/35) from captivity, although few wild infants were surveyed. No seropositive juveniles were identified from either wild (0/3) or captive (0/11) populations. A Fisher's exact (2 × 2) contingency test for sex-related differences in the combined wild and captive populations yielded a p value of 0.455. This indicates that while the there is a clear trend in each data set showing higher prevalence among females, the small numbers of samples do not show a statistically significant difference between infection in males and females. An exact 3 × 2 contingency test for age-related differences in the combined wild and captive populations yielded a p value of 0.00001, indicating that age-related differences in the combined populations are highly statistically significant.Cercopithecus mitis have used assays relying on cross-reactivity between antibodies against SIVsyk and other HIVs or SIVs. To assess the relative effectiveness of the use of cross-reactive tests, we compared serological results using two commercially available HIV-1/HIV-2 ELISA kits with an authentic SIVsyk antigen Western blot assay. The cross-reactive ELISA assays were very specific and in only one out of 276 samples gave a false positive result compared to the SIVsyk antigen Western assay. Curiously, this one particular captive juvenile female was repeatedly positive by both ELISAs but repeatedly negative by Western. This animal may possibly have been infected in captivity with a different SIV that produces cross-reactive antibodies to HIV-1 and HIV-2, but not to SIVsyk. On the other hand, the cross-reactive ELISAs failed to detect between 4.2 to 6.1% of the animals that were positive by the SIVsyk antigen Western assay. This result may be due to increased sensitivity using a Western blot format, but is also possibly due to increased sensitivity to the authentic SIVsyk viral antigens.Previous SIV serosurveys of Variable SIV infection prevalences have previously been reported in this species. However, information on age, sex and captive or wild status was not provided in any previous survey. Lowenstine et al, 1986  found noTo avoid age-related bias, a comparison between captive and wild populations should be made using only adult animals because the captive population contained many more samples from infants and juveniles than did the wild population  includes coastal and eastern regions of Kenya [C. mitis kolbi Neumann) includes the upland central portion of the country [C. mitis stuhlmannii Matschie) includes western Kenya and the border with Uganda [The sera used in this study were obtained between 1981 and 1996 from animals that had been previously trapped in the wild or from previously sampled captive animals housed at the Institute of Primate Research in Nairobi, Kenya. Serum samples were stored continuously at -20°C. All animals were captured, housed and sampled in accordance with humane animal use guidelines established by the Kenya Institute of Primate Research. Wild born monkey samples used in this study were derived from eight locations. In general, these sites were rural or suburban areas that bordered forested areas with natural flowing water. These sites are typical of areas in which humans have encroached on natural habitats and now may come in contact with these monkeys as they forage for food. The geography of the sites varied considerably from heavily forested areas in the highlands of Kenya to drier forests along the lowland coast of Kenya. The range of the lowland Sykes'  and Genscreen HIV1/2 version 2 . The Innotest ELISA kit uses synthetic peptides representing HIV-1 subtype M envelope, HIV-2 envelope, and HIV-1 group O envelope antigens. The Genscreen uses purified gp160 envelope and p25 nucleocapsid recombinant protein of HIV-1 and a peptide that mimics the immunodominant epitope of HIV-2 envelope. Each kit was used according to the manufacturers' recommendations. Photometric measurements were performed with a Dynatech MRX plate reader at 450 and 650 nm wavelengths. Positive and negative determinations were based upon cutoff values as determined by the manufacturers' protocols.CD4+ CEMx174 cells were transduced with a CCR5-encoding vector  as previously described . The resSerum samples were screened by Western blot using SIVsyk-specific viral antigens. SIVsyk-1.2 infected cells were centrifuged for 5 min at 1,000 × g and resuspended in lysis buffer containing 1% triton, 50 mM tris pH 7.5, 2 mM EDTA. Cell debris was pelleted for 2 min. at 12,000 × g and the supernatant was added to loading buffer containing 50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% SDS, 10% glycerol, 0.1% bromophenol blue. Viral antigens were separated on a 12% polyacrylamide gel and subsequently transferred to a PVDF membrane. Membrane strips were blocked with a solution of PBS-T containing 5% dry milk. Serum samples were diluted 1:500 and incubated for one hour at room temperature with individual membrane strips. Primary serum antibodies were detected with a 1:4,000 dilution of goat-anti-rhesus HRP conjugated secondary antibody . Colorimetric detection was performed using the Immunopure Metal Enhanced DAB Substrate Kit .The author(s) declare that the have no competing interests.BRE and EM conducted the ELISA and Western blot experiments and BRE wrote the draft manuscript. DE and JR cultured the SIVsyk virus. MGO provided serum samples and oversaw the ELISA experiments. SFM assisted with and oversaw the Western blot experiments and prepared the final manuscript. All authors read and approved the final manuscript.


The Darlington and Northallerton Asthma Study is an observational cohort study started in 1983. At that time little was published about long term outcome in asthma and the contribution of change in reversible disease or airway remodelling to any excess deterioration in function. The study design included regular review of overall and fixed function lung. We report the trends over fifteen years.All asthmatics attending secondary care in 1983, 1988 and 1993 were recruited. Pulmonary function was recorded at attendance and potential best function estimated according to protocol. Rate of decline was calculated over each 5-year period and by linear regression analysis in those seen every time. The influence of potential explanatory variables on this decline was explored.1724 satisfactory 5-year measurements were obtained in 912 subjects and in 200 subjects on all occasions. Overall rate of decline (ml/year (95%CI)) calculated from 5-year periods was FEV1 ♂41.0 (34.7–47.3), ♀28.9 (23.2–34.6) and best FVC ♂63.1 (55.1–71.2)ml/year, ♀45.8 (40.0–51.6).The principal association was with age. A dominant cubic factor suggested fluctuations in the rate of change in middle life with less rapid decline in youth and more rapid decline in the elderly. Rapid decline was possibly associated with short duration. Treatment step did not predict rate of deterioration.Function declined non-linearly and more rapidly than predicted from normal subjects. It reports for the first time a cubic relationship between age and pulmonary function. This should be taken into account when interpreting other articles reporting change in function over time. It is recognised that the average decline in pulmonary function is greater in asthmatic subjects than in the general population ,2. This We wished this to be a population study as far as possible, so we invited all subjects satisfying a broad definition of asthma referred to a single-handed respiratory physician in a well defined geographical area to participate. Very few refused, but patients managed entirely in general practice were necessarily excluded. Best function, assessed according to a defined protocol  implicitThese, described in detail elsewhere -11 are sst April and the 31st March in subsequent 12-month periods[All asthmatics currently attending secondary care clinics in the Darlington and Northallerton Health Districts were eligible for recruitment in 1983, 1988 and 1993, and reviewed in 1988, 1993 and 1998. Asthma was diagnosed clinically and confirmed by reversibility of FEV1 or peak flow of at least 15% on more than one occasion, either spontaneously, or in response to bronchodilator at any time since referral . The firh periods. AllowanThe following variables recorded included: age, gender, height, duration of asthma, atopy, childhood asthma, smoking habit and lifetime amount smoked, social class. Duration of asthma was determined from the first onset or from relapse after a symptom free interval of at least five years, if this was applicable. Atopy was determined by at least one of the following skin tests resulting in a diameter at least 3 mm greater than control: D Pterynissimus, cat, grass pollen, A fumigatus. Childhood asthma was defined as a childhood history of recurrent lower respiratory tract symptoms with wheeze, in the absence of a localised structural damage such as bronchiectasis. It was sub-divided into those with (Gap Asthmatics) and without (Continuous since Childhood) a symptom free gap of at least five years. Never smokers were those smoking less than one cigarette a day for one year. Ex-smokers had been abstinent for at least three months at the time of examination. The lifetime amount smoked was determined from the average consumption (expressed in packs of 20 cigarettes) multiplied by the duration of smoking to give a figure in pack-years.This was characterised by the use of long acting beta agonists and the corticosteroid step: none, low dose inhaled (<800 micrograms per day), intermediate dose inhaled (800–1000 micrograms per day), high dose inhaled (>1000 micrograms per day), oral 1–9 mgs per day, oral ≥ 10 mgs per day, unstable .Actual function was that recorded at attendance.st of the previous year, and the highest value, including the after-bronchodilator reading at attendance, was accepted as best subject to the following.This was estimated according to the published protocol . The not(a) If >80% predicted (Cotes ); after-(b) If 70–80% predicted; after-bronchodilator and stable on mutually agreed satisfactory preventative treatment with twice daily recording of peak flow for one week(c) If <70% of predicted; after-bronchodilator with formal trial of corticosteroids (prednisolone 30 mgs for at least five days with stability of peak flow for at least 48 hours)If the above was not already satisfied, best function was immediately established according to the protocol.Spirometry was performed using a rolling wedge spirometer  and peak flow with the mini peak flow meter (Clement Clark Limited). Actual function was measured opportunistically at the clinic in current attenders and at a special clinic for those who had been discharged. One of three research fellows, one of two research nurses or CKC were responsible for checking current pulmonary tests in the clinical records and performing further tests when required by protocol for best function.This was obtained from the Darlington, Northallerton and South Durham Ethics Committees at various stages of the study.The principal independent variable was change during each 5-year period so each individual contributed up to 3 observations to the analysis. For the 200 subjects with observations on all four occasions a secondary analysis could be based on decline over 15 years, and for each of these subjects the mean rate of decline was estimated from the four observations using linear regression analysis.Best and actual/best peak flow measured independently of the spirometric outcome variables were chosen as the functional potential predictors of outcome. This approach was taken to avoid the mathematical relationship between baseline level and change that applies if the spirometric variables are used as predictors of outcome. Many of these potential predictive variables are correlated with each other and there are arguments for and against models examining the effect of socio-economic variables with or without allowance for the respiratory function of the patients and their age. We present a relevant selection from the large range of unrestricted and hierarchical models that we constructed.We allowed for the inevitable regression to the mean associated with outcome measures subject to appreciable variability. Initially the starting value of an outcome functional variable was regressed against a full set of potential predictors of this function in order to generate a set of residuals indicating the extent to which an individual starting measurement is higher or lower than would be expected. Then the change in function was regressed against the residual. The new set of residuals indicates the extent to which the apparent rate of change is affected by regression to the mean enabling an adjusted rate of change to be calculated. This adjusted change in function was then taken as the dependent variable in subsequent analyses. These included the univariate effect of potential risk factors on the adjusted change in function. In the multivariate analyses we omitted variables which were consistently non-significant, but all others are retained in the presentation. Models are shown with and without the inclusion of age. Univariate analysis showed that there were cubic relationships between change in function and age and duration. The cubic terms are retained in the multivariate presentation. We also examined interactions between predictor variables where there was some a priori reason for believing that such an interaction was plausible but none was demonstrated.Of 1138 subjects recruited, 155 died before the first review, 49 were lost to follow up and 22 did not have satisfactory spirometric tests. The remaining 912 had at least one paired result of actual or best FEV1 or best FVC and 200 had satisfactory assessments of best FVC on all four occasions. Demographic details at entry are given in Table 2 for trend, 59.0 (p < 0.001)).The mean annual decline in function calculated using all five-year intervals Table  was greaFor the 200 subjects with complete observations over 15 years the mean rate of decline was similar to that observed over five year periods, but the standard deviation of the rates of decline was half that of the 5-year estimates, reflecting greater accuracy from multiple measurements. Even when calculating change this way several individuals improved or showed excessive loss in function against predicted .As there was no secular trend in the change of function over the five year periods, the date of observation is not considered in the analyses below.The univariate relationships between the potential explanatory variables and change in function after allowance for regression to the mean are summarised in Table There were no statistically significant relationships with atopy. Longer duration of asthma was significantly associated with favourable outcome for all variables. In all cases a cubic relationship between loss of function and duration suggests high initial rates of loss , with improvement to a plateau at around 20 years duration . Childhood asthma was significantly associated with a relatively favourable outcome, though this benefit was only seen in those for whom asthma had been continuous. The outcome of the 'gap asthmatics' was similar to those with adult onset. Higher social class was associated with worse outcome, significantly so for best FEV1. Low initial function and worse control as assessed by actual/best PEF both predicted less loss in actual FEV1. Although there were no significant associations with current smoking status, there was significantly greater loss in best FVC with amount smoked.As childhood asthma, duration of asthma and age at entry are potentially confounded and were significant in some of the univariate analyses, we performed a series of multivariate analyses progressively including each in turn. Tables Height (not tabulated) was consistently directly associated with decline at a significance level of the order of 15% in univariate and multivariate analyses. In the latter analyses, height suppressed the associations with gender and social class. There were no significant associations with instability of regimen, nor with steroid step or the regular prescription of bronchodilators. None of the models considered made any relevant difference to the shape or gradients of the curves shown in fig Generally similar univariate associations were demonstrated. However on multivariate analysis, amount smoked remained in the FVC model (coefficient 13.3 ml/year per 10 pack years P = 0.020), and the effect of gender was stronger in both models . Height was again consistently directly associated with decrease in function at a significance level of the order of 15%. When height was included to allow for its association with gender, the difference in rate of decline in FEV1 between males and females was substantially reduced to 12.5 ml per year (p = 0.010) and was no longer significant for best FVC (p = 0.14).The hypothesis that persistent airway obstruction may develop in asthmatics implies that persistent airway obstruction and reversible obstruction associated with asthma are not mutually exclusive diagnoses However if COPD is regarded as a syndrome; this implies a non-asthmatic inflammatory process . As the We are satisfied that our subjects satisfied the clinical criteria for asthma and had the relevant inflammatory process. Of course, the study is not of the entire asthmatic population as some individuals will remain undiagnosed while others will have been managed entirely in general practice, but all those referred to hospital and willing were entered into the study. We believe that the referral threshold was relatively low, and very few patients were referred outwith the local area. The subjects were recruited by a single handed hospital physician, with no other selection. There were no differences in the demography or function of those entering at different times . Most weThe rate of decline of actual FEV1 was similar to that reported by Ulrik   declare that they have no competing interests.CKC conceived the study and was responsible for design of data forms, recruitment and all the clinical aspects. RJP undertook the analysis of data. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Oxidative stress is involved in cisplatin-nephrotoxicity. However, it has not completely established if reactive nitrogen species and nitrosative stress are involved in this experimental model. The purpose of this work was to study the role of peroxynitrite, a reactive nitrogen specie, in cisplatin-nephrotoxicity using the compound 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) (FeTPPS), a soluble complex able to metabolize peroxynitrite.In rats treated with cisplatin , renal nitrosative stress was made evident by the increase in 3-nitrotyrosine on day 3. In addition, cisplatin-induced nephrotoxicity was evident by the histological damage of proximal tubular cells and by the increase in (a) serum creatinine, (b) blood urea nitrogen, and (c) urinary excretion of N-acetyl-β-D-glucosaminidase and total protein. Cisplatin-induced nitrosative stress and nephrotoxicity were attenuated by FeTPPS-treatment .Nitrosative stress is involved in cisplatin-induced nephrotoxicity in rats. Our data suggest that peroxynitrite is involved, at least in part, in cisplatin-induced nephrotoxicity and protein nitration. Furthermore, the inhibition of NOS by L-NAME [•NO is playing a toxic role in this experimental model. However, it is unknown if peroxynitrite (ONOO-), a RNS that is generated by the reaction of •NO and O2•-, is involved in the renal damage induced by cisplatin. It has been shown that ONOO-, which is not a free radical, is involved in the pathogenesis of many diseases [- can react with different biomolecules including amino acids such as cysteine, methionine, tryptophan, and tyrosine leading to changes in protein structure and function [- has been shown to cause lipid peroxidation, chemical cleavage of DNA, and reduction in cellular defenses by oxidation of thiol pools [On the other hand, the role of reactive nitrogen species (RNS) and nitrosative stress has been less explored in cisplatin-induced nephrotoxicity. In this context, it has been studied the role of nitric oxide (se (NOS) -19. It hse (NOS) ,19 suggey L-NAME  or by amy L-NAME  decreasediseases -25. ONOOfunction . ONOO- hol pools .- is involved in the nephrotoxicity induced by cisplatin by using 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) (FeTPPS). This compound is a water-soluble Fe (III) porphyrin complex that catalyzes rapid isomerization of ONOO- to nitrate (NO3-) under physiologically relevant conditions  [In this work, we studied if ONOO4, 37°C) . The cyt4, 37°C) .Body weight decreased 8.5% in cisplatin (Cis) group on day 3 and FeTPPS tended to prevent this decrease in Cis+FeTPPS group, however there was no significative difference between Cis and Cis+FeTPPS groups. Body weight was similar in control (Ct), FeTPPS, and Cis+FeTPPS groups. Urinary volume was not significative difference among the four groups along the study and on day of sacrifice Table .Serum creatinine increased 4.9 times and blood urea nitrogen (BUN) increased 5.5 times in Cis group compared to control one Fig . FeTPPS After three days of cisplatin-treatment, the epithelium from proximal convoluted tubules  showed cytoplasmic vacuolization, intracellular edema and extensive damage which affected 87 ± 4% of their surface area Fig . The cisA strong 3-NT immunostaining was observed in the straight portion of the proximal convoluted tubules located in the inner area of the kidney cortex of cisplatin-treated rats Fig . Interesin vivo administration of some antioxidants such as vitamins C and E [2•- scavenger), pyruvate and catalase (H2O2 scavengers), and dimethylthiourea and thiourea (•OH scavengers) in renal proximal tubular epithelial cells (LLC-PK1 cells) treated with cisplatin also strongly support the role of ROS in cisplatin renal toxicity [Cisplatin in an effective chemotherapeutic agent for a wide variety of tumors, nevertheless, nephrotoxicity is the major complication of this antineoplasic treatment . The mec C and E ,10, mela C and E , or sele C and E  in cispltoxicity .•NO and RNS in cisplatin-induced nephrotoxicity has not been completely established. It has been shown that the renal content of nitrate/nitrite is increased in cisplatin-treated rats suggesting that •NO is increased in these animals [•NO on cisplatin-induced renal toxicity: (a) aminoguanidine, an inhibitor of inducible NOS, decreased nephrotoxicity and prevented kidney lipid peroxidation and reduction of antioxidant enzymes induced by cisplatin [et al. [et al. [et al. [•NO is not playing a toxic role in cisplatin-induced nephrotoxicity. The above mentioned disagreement justify the performance of additional experiments to clarify the role of NOS and •NO in cisplatin-induced nephrotoxicity.In contrast the role of  animals ,19. In f animals . In addiisplatin , and (b)isplatin . In cont [et al.  and Li e [et al.  have fou [et al.  found th•NO is able to react with O2 •- to produce ONOO-, which is a powerful oxidant more, reactive than its precursors, and has been implicated in an increasing list of diseases: hyperlipidemia [- decomposition catalyst FeTPPS is a water-soluble Fe (III) porphyrin complex able to block ONOO- toxicity [•NO and O2•- results in continuous formation of ONOO- even several hours after ischemia-reperfusion insult [- as measured by plasma rhodamine fluorescence and heart 3-NT staining [On the other hand, ipidemia , Alzheimipidemia , acute ripidemia , neurotoipidemia , and diaipidemia . The ONOtoxicity ,29 and tn insult . Signifin insult . In a mostaining . Interesstaining .- which could be responsible, at least in part, of the alterations induced by cisplatin. This ameliorative effect of FeTPPS was associated with the decrease in 3-NT staining suggesting that ONOO- is involved in protein nitration in cisplatin-nephrotoxicity. It is known that another RNS such as N2O4, HONOO, •NO2 [2Cl) [2- and HOCl-derived myeloperoxidase [In this work it was found that FeTPPS partially prevented the increase in BUN and serum creatinine (markers of glomerular damage) and urinary excretion of NAG and total protein (markers of tubular damage) induced by cisplatin-treatment. The increase in urinary NAG and total protein excretion could be associated with necrosis of the proximal convoluted tubules, the primary site of drug accumulation . FeTPPS OO, •NO2 , and nitO2 [2Cl) , are invroxidase .- may also be involved in the glomerular alterations in cisplatin-treated rats.Studies in animals have established that tubular injury plays a central role in the reduction of glomerular filtration rate in acute tubular necrosis. Two major tubular abnormalities could be involved in the decrease in glomerular fucntion in cisplatin-treated rats: obstruction and backleak of glomerular filtrate. The alteration in glomerular function can not be attributed to structural damage since glomeruli structure is normal in cisplatin-treated rats. The alterations in glomerular function in cisplatin-treated rats may also be secondary to ROS  which in- induced by cisplatin may be secondary to the increase in •NO and O2•- production. In fact, there are evidences of the renal increase in •NO production in cisplatin nephrotoxicity [2•- generation in cisplatin-treated LLC-PK1 cells [2•- increase in cisplatin-nephrotoxicity may be simply consequence of the mitochondrial dysfunction [The increase in renal ONOOtoxicity ,19 and OK1 cells . The O2•function  and the function .- is involved in both protein nitration and nephrotoxicity in these animals.Nitrosative stress is involved in cisplatin-induced nephrotoxicity in rats. The ameliorative effect of FeTPPS on cisplatin-induced nephrotoxicity in rats was associated with the decrease in protein nitration suggesting that ONOOCisplatin  was from Sigma-Aldrich . FeTPPS  was from . Rabbit anti-3-NT polyclonal antibodies  were from Upstate . Anti-rabbit Ig horseradish peroxidase antibodies  were purchased from Stressgen . Commercial kits to measure creatinine and urea were from Spinreact . All other chemicals were reagent grade and commercially available.Male Wistar rats  initially weighing 200–250 g were used. Experimental work was approved by DGAPA (IN227103) and followed the guidelines of Norma Oficial Mexicana (NOM-ECOL-087-1995). All animals had free access to water and commercial rodent diet , and were randomly divided in four groups (n = 6 rats/group) as follows: (1) CT, injected intraperitoneally (i.p.) with isotonic saline solution; (2) Cis, treated with a single dose of cisplatin (7.5 mg/Kg b.w./i.p.) ; (3) FeTThe markers of glomerular damage, creatinine and urea, were measured using commercial kits. BUN was obtained by correcting the urea value by a 2.14 factor . As markThin slices of kidney tissue with cortex and medulla were fixed by immersion in buffered formalin (pH 7.4), dehydrated and embedded in paraffin . Section2O2 in methanol by incubation for 1.5 h at room temperature. Nonspecific adsorption was minimized by leaving the sections in 3% bovine serum albumin in phosphate buffer saline for 30 min. Sections were incubated overnight with a 1:700 dilution of anti-3-NT antibody. After extensive washing with phosphate buffer saline, the sections were incubated with a 1:1000 dilution of a peroxidase conjugated anti-rabbit Ig antibody for 1 h, and finally incubated with hydrogen peroxide-diaminobenzidine for 10 s. Sections were counterstained with hematoxilin and observed under light microscopy. All the sections from the four studied groups were incubated under the same conditions with the same antibodies concentration, and in the same running, so the immunostaining was comparable among the different experimental groups [For immunohistochemistry, 3 μm sections were deparaffined with xylol and rehydrated with ethanol. Endogenous peroxidase was quenched/inhibited with 4.5% Hl groups .Results are expressed as the mean ± SEM. Data were analyzed by one-way ANOVA followed by Bonferroni's multiple comparisons. Non-paired t-test was used to compare the quantitative histological damage data using the software Prism 3.02 . P ≤ 0.05 was considered statistically significant.YICH performed animal experimentation, biochemical determinations, statistical analyses, light microscopy and immunohistochemical studies. RHP supported the light microscopy and immunohistochemical studies. JPCH conceived, designed and coordinated the study. All authors read and approved the final manuscript.


Determination of genetic relatedness among microorganisms provides information necessary for making inferences regarding phylogeny. However, there is little information available on how well the genetic relationships inferred from different genotyping methods agree with true genetic relationships. In this report, two genotyping methods – restriction fragment analysis (RFA) and partial genome DNA sequencing – were each compared to complete DNA sequencing as the definitive standard for classification.Using the Genbank database, 16 different types or subtypes of papillomavirus were selected as study samples, because numerous complete genome sequences were available. RFA was achieved by computer-simulated digestion. The genetic similarity of samples, based on RFA, was determined from the proportion of fragments that matched in size. DNA sequences of four specific genes , representing partial genome sequencing, were also selected for comparison to complete genome sequencing. Laboratory error was not taken into account. Evaluation of the correlation between genetic similarity matrices (Mantel's r) and comparisons of the structure of the derived dendrograms (partition metric) indicated that partial genome sequencing (for single genes) had higher agreement with complete genome sequencing, achieving a maximum Mantel's r = 0.97 and a minimum partition metric = 10. RFA had lower agreement, with a maximum Mantel's r = 0.60 and a minimum partition metric = 18.This simulation indicated that for smaller genomes, such as papillomavirus, partial genome sequencing is superior to restriction fragment analysis in representing genetic relatedness among isolates. The generalizability of these results to larger genomes, as well as the impact of laboratory error, remains to be demonstrated. Precise estimation of genetic relatedness between isolates of a microorganism is important for determination of phylogenetic relationships, which has important applications in studies of disease transmission ,2. The dCurrently, one genotyping technique used frequently as an alternative to complete genome sequencing is restriction fragment analysis (RFA), in which restriction endonuclease enzymes cleave the genome at specific sites, producing DNA fragments that are then separated by size using electrophoresis . The perAnother common alternative to complete genome sequencing is partial genome sequencing, i.e., the nucleotide sequencing of a particular gene or segment of the genome ,10. The In a comparison of RFA and partial genome sequencing with respect to their similarity in interpreting a disease outbreak caused by pseudorbabies virus in a swine producing region in Illinois, USA, both genotyping methods generated similar conclusions about patterns of spread of the virus . HoweverRestriction fragment analysis detects genetic variation by surveying specific endonuclease restriction sites over the entire genome; in contrast, partial genome sequencing detects genetic variation by comparing nucleotide bases from a specific region of the genome. Each method detects a different dimension of genetic variation, and each can detect only a proportion of the genetic variation present in the entire genome. Therefore, it is important to determine which method, using partial information, provides a more accurate estimation of genetic relatedness.The primary purpose of this study was to compare both restriction fragment analysis and partial genome sequencing to complete genome sequencing, with regard to their agreement in estimating genetic relationships and in reconstructing phylogenies under the ideal conditions of absence of laboratory error. Computer simulation of the genotyping analysis was conducted, using completely sequenced papillomavirus isolates obtained from Genbank.Table Sequencing entire genomes is impractical in most investigations of genetic relationships. The computer simulation conducted here determined that compared to restriction fragment analysis, partial genome sequencing had higher agreement with complete genome sequencing in estimating genetic relatedness and greater similarity in the topology of the dendrograms of phylogenetic relationships derived from these estimates. These results using papallomavirus sequences with a genome length averaging less than 8 kb, indicate that for microorganisms with small genomes, partial genome sequencing targeting genes comprising approximately 20–25% of the total genome length can provide a very good estimate of genetic relatedness. The topological structure of phylogenetic trees was also stable for partial genome sequencing, particularly for the most closely related samples. The degree to which these results generalize to larger genomes is unknown, in part because microorganisms with large genomes are rarely, if ever, sequenced in their entirety. There are also other considerations in selecting partial genome sequencing as a genotyping method, such as presence of the gene in all isolates, and sufficient variability to differentiate isolates . In addiOne might expect that increasing genome size would diminish the advantage of partial genome sequencing compared to restriction fragment analysis. As total genome size increases, the number of restriction sites cut by restriction enzymes is expected to increase, providing more fragments and more genetic information for estimating genetic relatedness at no increased cost. This also needs to be taken into account in the selection of a genotyping method. However, it has been argued that if a gene is selectively neutral , it is only the length of the gene sequenced, not the ratio of sequenced gene length to genome size, that is important for determining the degree of divergence from a common ancestor . To the The general conditions under which partial genome sequencing is more accurate than restriction fragment analysis in representing true genetic relatedness have not been addressed in the analysis conducted here. However, another study from our laboratory , using sThe simulated genotyping conducted here assumed no error of measurement. The sources of error in restriction fragment analysis are well known -24. FragIn practical terms, laboratory error and cost need to be taken into account in the selection of a genotyping method. However, when the impact of these factors is minimized, the computer simulation analysis conducted here indicates that partial genome sequence becomes the preferred alternative for representing genetic relationships.For small genomes, partial genome sequencing of target genes comprising 20–25% of the total genome provides a more accurate estimate of genetic relatedness and more accurate representation of evolutionary and transmission histories than does restriction fragment analysis and thus is indicated to be the preferred genotyping method for phylogenetic reconstruction under these conditions. The degree to which these results are generalizable to larger genomes and conditions of laboratory error remains to be determined.The source of information on nucleotide sequences was the Genbank database . The orgThe complete DNA sequences of the 16 papillomavirus samples were aligned using ClustalW software . The genCommonly used restriction endonuclease enzymes were selected , based oSimulated digestion of each papillomavirus DNA sample by each restriction enzyme was conducted using the DIGEST program . The resxy = 2nxy/(nx+ny), where nxy is the number of fragments matching in size for samples x and y, and nx and ny are the number of fragments in samples x and y, respectively. Then, Dxy = 1-Sxy was calculated as a distance measure. Pairwise distances between samples were computed for each individual enzyme. Also, pairwise distances were obtained for up to 4 enzymes, by using for each condition  the fragment size distributions for 30 randomly selected combinations of enzymes, and calculating the composite distance [Based on the distribution of restriction fragment sizes, the genetic similarity between any two papillomavirus samples was calculated for each restriction enzyme using the Dice coefficient ,6: Sxy =distance .The E1, E6, E7, and L1 genes, which have been of interest in studies of papillomavirus, were used for estimating genetic relatedness. The ClustalW program  was usedThe matrix of genetic distances based on complete DNA sequences was considered the definitive standard. The genetic distance matrices based on RFA and partial genome sequencing were compared to complete genome sequencing by calculating Mantel's coefficient of correlation between matrices (Mantel's r) .The genetic distance matrices for RFA, partial genome sequencing, and complete genome sequencing were used to construct phylogenetic trees, using the Neighboring-joining algorithm , as implbps: base pairsBPV: bovine papillomaviruscaninePV: canine papillomaviruschimpPV: chimpanzee papillomavirusHPV: human papillomaviruskb: kilobaseMantel's r: Mantel's coefficient of correlation between matricesPCR: polymerase chain reactionRFA: restriction fragment analysisBQ designed the investigation, collected the data, conducted the data analysis, and wrote the manuscript. RW identified the problem to be investigated, provided statistical guidance, assisted in interpretation of results, and edited the final drafts of the manuscript. Both authors read and approved the final manuscript.


V12/E1A-transformed mouse embryo fibroblasts in order to identify the genes that could be involved in tumour development.In cancer, cellular transformation is followed by tumour development. Knowledge on the mechanisms of transformation, involving activation of proto-oncogenes and inactivation of tumour-suppressor genes has considerably improved whereas tumour development remains poorly understood. An interesting way of gaining information on tumour progression mechanisms would be to identify genes whose expression is altered during tumour formation. We used the Affymetrix-based DNA microarray technology to analyze gene expression profiles of tumours derived from rasAmong the 12,000 genes analyzed in this study, only 489 showed altered expression during tumour development, 213 being up-regulated and 276 down-regulated. The genes differentially expressed are involved in a variety of cellular functions, including control of transcription, regulation of mRNA maturation and processing, regulation of protein translation, activation of interferon-induced genes, intracellular signalling, apoptosis, cell growth, angiogenesis, cytoskeleton, cell-to-cell interaction, extracellular matrix formation, metabolism and production of secretory factors.V12/E1A transformation of MEFs, could be new cancer therapeutic targets.Some of the genes identified in this work, whose expression is altered upon ras Therefras oncogene can transform most immortalized rodent cells to generate tumour cells, whereas transformation of primary cells requires either a cooperating oncogene or the inactivation of a tumour suppressor gene. The adenovirus E1A oncogene cooperates with ras to transform primary rodent fibroblasts [V12/E1A model of tumour formation was used in this work to analyze genes necessary for tumour progression. This model was chosen because transformation is induced in a simple and controlled way, avoiding the difficulties of analyzing the multiple and complex transformation mechanisms observed in cellular models derived from human tumours. Mouse embryo fibroblasts (MEFs) were chosen for transformation by rasV12/E1A to keep the model homogeneous, the host being the athymic mouse. Because non-transformed MEFs are unable to induce tumour when injected into athymic mice, we previously analyzed the change in gene expression profile induced in MEFs by rasV12/E1A-transformation [V12/E1A-transformed MEFs and in the tumours formed after their injection into nu/nu mice. With Affymetrix microarray technology, differential expression values greater than 1.7 are likely to be significant, based on internal quality control data. We present data that use a more stringent ratio, restricting our analysis to genes overexpressed or under-expressed at least 2.0 fold in tumours, compared to the parent rasV12/E1A-transformed fibroblasts. Most striking findings are summarized below while complete data are presented in Tables 1 and 2  will very probably originate from erythrocytes, endothelium and leucocytes respectively.Down-regulation was observed for some DNA-binding proteins and transcriptional factors such as zinc finger protein 36, C3H type-like 2, forkhead box M1, T-box 14, SET and MYND domain containing 2, sine oculis-related homeobox 1 homolog (Drosophila), liver-specific bHLH-Zip transcription factor, deformed epidermal autoregulatory factor 1 homolog (Drosophila), HMG box Bromodomain (5 domains) Zinc finger C2H2 type, BTB (POZ) domain containing 14A, Jun oncogene, general transcription factor III A, MYB binding protein (P160) 1a, general transcription factor II H polypeptide 1, transcription factor Dp 1, myeloblastosis oncogene-like 2, general transcription factor IIF polypeptide 1, v-ets erythroblastosis virus E26 oncogene homolog 1 (avian), transcriptional regulator SIN3B homolog (yeast), RNA polymerase I transcription factor RRN3, Kruppel-like factor 5, general transcription factor III C 1, nucleosome assembly protein 1-like 1, general transcription factor IIB and transformation related protein 53 (p53) whereas CCR4-NOT transcription complex subunit 7, transcription factor 15, dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1), chromobox homolog 3 (Drosophila HP1 gamma), ets variant gene 1, basic helix-loop-helix domain containing class B2, pre-B-cell colony-enhancing factor 1, homeo box C6, BTB (POZ) domain containing 1, zinc finger protein 37, enhancer of zeste homolog 1 (Drosophila) and AT rich interactive domain 3B (Bright like) were up-regulated.A number of genes involved in RNA maturation, protein translation, processing and secretion were down-regulated in tumours, such as eukaryotic translation initiation factor 4 gamma 1, ER degradation enhancer mannosidase alpha-like 1, paraspeckle protein 1, CUG triplet repeat, RNA binding protein 1, copper chaperone for superoxide dismutase, splicing factor arginine/serine-rich 2 interacting protein, ribosomal protein S6 kinase polypeptide 4, sorting nexin 14, sorting nexin 17, elongation protein 3 homolog (S. cerevisiae), eukaryotic translation initiation factor 5B, peptidylprolyl isomerase F (cyclophilin F), ubiquitin-conjugating enzyme E2S, DnaJ homolog subfamily C member 3, SEC14-like 1 (S. cerevisiae), mitochondrial ribosomal protein L18, mitochondrial isoleucine tRNA synthetase, brix domain-containing protein, proteasome 26S subunit non-ATPase 1, COP9 subunit 5, small nuclear ribonucleoprotein polypeptide A, heat shock protein 105, heat shock protein 1, ribosome-binding protein p34, splicing factor 3b subunit 1, proteasome 26S subunit non-ATPase 7, mitochondrial processing peptidase beta, translocating chain-associating membrane protein 1, mitochondrial ribosomal protein L44, mitochondrial ribosomal protein S25, mitochondrial ribosomal protein S10, eukaryotic translation initiation factor 3 subunit 1 alpha, eukaryotic translation initiation factor 1A, COP9 subunit 2, RNA-binding region  containing 1, DNAJ domain-containing and Der1-like family protein, whereas only 7 genes from this group were up-regulated including ubiquitin specific protease 18, proteosome subunit beta type 8, heterogeneous nuclear ribonucleoprotein U, ERO1-like (S. cerevisiae), proteasome subunit beta type 10, hnRNP-associated with lethal yellow and peptidylprolyl isomerase (cyclophilin)-like 2.in vitro.Altogether these results show that transcriptional factors and DNA-binding proteins involved in transcription, as well as proteins involved in RNA maturation, protein translation, processing and secretion are preferentially down regulated during tumour development, suggesting that protein synthesis is less active in tumours than in transformed cells It is interesting to note that 11 genes activated by interferon were found up-regulated in tumours from 3.2 to 44.6 folds. They comprise the interferon stimulated gene 12 and genes encoding the interferon-induced protein with tetratricopeptide repeats 1, the interferon-induced protein with tetratricopeptide repeats 3, the interferon regulatory factor 7, the interferon alpha-inducible protein, the interferon consensus sequence binding protein 1, the interferon-inducible GTPase, the interferon induced transmembrane protein 2, the interferon gamma induced GTPase, the interferon-g induced GTPase and the interferon gamma-inducible protein 16Expression of several genes involved in signalling was modified in tumours. Among up-regulated were genes encoding the TYRO protein tyrosine kinase binding protein, ADP-ribosylation factor-like 4, ARF-GAP RHO-GAP ankyrin repeat and pleckstrin homology domains-containing protein 3, guanosine diphosphate dissociation inhibitor 2, SET domain bifurcated 1, acid phosphatase 5, guanylate nucleotide binding protein 3, SH3-domain GRB2-like 2, rap2 interacting protein x, TGFB inducible early growth response 1, pleckstrin homology domain containing family A (phosphoinositide binding specific) member 1, A kinase (PRKA) anchor protein 8, PDZ and LIM domain 4, protein tyrosine phosphatase non-receptor type 13, calcium and integrin binding family member 2, diaphanous homolog 2 (Drosophila), SH3-domain GRB2-like 3, Janus kinase 3, dual specificity phosphatase 9, Nik related kinase, synaptojanin 2, zinc finger RAN-binding domain containing 1, inositol hexaphosphate kinase 1, spleen tyrosine kinase, ribitol kinase putative, B lymphoid kinase, DEAD box polypeptide 27, mitogen activated protein kinase kinase kinase kinase 1, tumor-associated calcium signal transducer 1 and ral guanine nucleotide dissociation stimulator. Among down-regulated during tumour development where genes encoding adenylate kinase 1, serum deprivation response, ATP-binding cassette sub-family F member 2, protein kinase C alpha, RIO kinase 1 homolog (yeast), protein phosphatase 1 regulatory (inhibitor) subunit 2, phosphotidylinositol transfer protein beta, RAN GTPase activating protein 1, v-crk sarcoma virus CT10 oncogene homolog (avian)-like, serum/glucocorticoid regulated kinase, DEAD box polypeptide 48, nucleolar GTPase, calcium/calmodulin-dependent protein kinase II delta, cellular retinoic acid binding protein I, AFG3(ATPase family gene 3)-like 1 homolog (yeast), transforming growth factor beta regulated gene 4, enabled homolog (Drosophila), Rho GDP dissociation inhibitor gamma, S100 calcium binding protein A11 , cornichon homolog (Drosophila), butyrate-induced transcript 1, phosphatidylinositol 3-kinase regulatory subunit polypeptide 1 , mitogen activated protein kinase 1, IQ motif containing GTPase activating protein 1, protein kinase C epsilon, transducin (beta)-like 3, serine/threonine kinase 16, thymoma viral proto-oncogene 1, ATP-binding cassette sub-family B (MDR/TAP) member 7, protein phosphatase 1 regulatory (inhibitor) subunit 7, dual specificity phosphatase 1, Rho-associated coiled-coil forming kinase 2, acid phosphatase 1 soluble, PAK1 interacting protein 1, RAN binding protein 1, dual specificity phosphatase 16, RAB23 member RAS oncogene family, WD repeat domain 26, PDZ and LIM domain 1 (elfin) and mitogen activated protein kinase kinase 4.V12/E1A-transformed MEFs are very sensitive to apoptosis in vitro [The rasin vitro  whereas,in vitro than during tumour development.Another interesting point to be underscored is that expression of many cell growth-related genes was found decreased in tumours, whereas none of them was up-regulated. Among up-regulated genes were those coding for he bone morphogenetic protein 10, cytokine receptor-like factor 1, insulin-like growth factor binding protein 4, ephrin A2, schlafen 4, early growth response 2, retinoblastoma-associated factor 600, receptor tyrosine kinase-like orphan receptor 2, cyclin-dependent kinase 7 (homolog of Xenopus MO15 cdk-activating kinase), inhibitor of growth family member 4 and neoplastic progression 1 were up-regulated and connective tissue growth factor, ephrin B2, neural proliferation differentiation and control gene 1, nerve growth factor beta, Eph receptor A2, neoplastic progression 3, cyclin G1, bone morphogenetic protein receptor type 1A, cell division cycle 34 homolog (S. cerevisiae), cyclin-dependent kinase inhibitor 1A (p21), SGT1 suppressor of G2 allele of SKP1 homolog (S. cerevisiae), nuclear casein kinase and cyclin-dependent kinase substrate, G two S phase expressed protein 1, prohibitin, nucleostemin, CDK2-associated protein 1 and block of proliferation 1 This is not a surprise since transformed cells grow more rapidly Some proangiogenic genes such as angiopoietin-like 4, selectin, endothelin 1, angiopoietin 2 and endothelial PAS domain protein 1 were up-regulated during tumour growth whereas the antiangiogenic factor thrombospondin 1 was found to be down regulated. Surprisingly, the proangiogenic endothelial cell growth factor 1 (platelet-derived) and the angiomotin like 2 were found down-regulated.Expression of some genes involved in cytoskeleton and cell-to-cell contact was modified in tumours. mRNAs encoding junctophilin 3, fibrillin 2, plexin A3, ARP1 actin-related protein 1 homolog A, MYOSIN-IXA homolog, myosin IB, tubulin alpha 1, cadherin 3, integrin beta 5, dynamin and stathmin-like 4 genes were up-regulated whereas mRNAs encoding MAP/microtubule affinity-regulating kinase 2, nucleoporin 54 kDa, smooth muscle cell associated protein-1, plakophilin 2, annexin A3, myosin X, ARP1 actin-related protein 1 homolog B (yeast), follistatin, golgi associated gamma adaptin ear containing ARF binding protein 2, filamin beta, vinculin, cytoskeleton-associated protein 1, annexin A11, lamin A, cortactin, pericentrin 2, gap junction membrane channel protein alpha 1, importin 4, nucleolar protein 5, exportin 7, vinculin, actinin alpha 1, nucleoporin 88, fibulin 2 and CD44 antigen were down regulated.In cancer, transformed and mesenchymal cells synthesize and secrete several compounds which participate to tumour organization. In tumour cells, genes encoding the procollagen type XVIII alpha 1, Nice-4 protein homolog isoform 1, glycophorin A, collagen type V alpha 1 and procollagen type VI alpha 1 were up-regulated whereas, to our surprise, procollagen type V alpha 2 was found down regulated.Finally, an interesting finding is that the majority of genes encoding enzymes involved in metabolism were down regulated in tumours whereas the majority of secretory factors or their receptors were up-regulated  cells were plated in a 6-well plate, incubated for 24 hr, and then transfected with PEI with 5 μg of retroviral plasmid. After 48 hr, the medium containing the virus was filtered  to obtain the retroviral supernatant. MEFs were plated at 2 × 105 cells per 35 mm dish and incubated overnight. For infection, the culture medium was replaced by an appropriate mix of the retroviral supernatant and culture medium (V/V), supplemented with 4 μg/ml polybrene (Sigma), and cells were incubated at 37°C. Transformation of MEFs by the pBabe-rasV12/E1A retroviral vector was evaluated by examining changes in their morphological aspect, by quantifying expression of the RAS protein by western blot, by monitoring cell proliferation, colony formation in soft-agar and tumours in nude mice as previously described [Primary embryo fibroblasts were isolated from 14.5 day-old SV129J mouse embryos and grown in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum as previously described . We tranV12/E1A transformed MEFs (106/200 μl PBS) were injected subcutaneously into the flank of male 8 week-old nu/nu mice, and tumours were allowed to develop for 20 days. Tumours were removed and stored at -80°C. Microscopical analysis reveals that tumours contain about 15% of vascular and stromal cells.Suspensions of the pBabe-rasV12/E1A-transformed cells and tumours from three independent experiments was isolated by Trizol (Gibco-BRL). Twenty μg of total RNA was converted to cDNA with SuperScript reverse transcriptase (Gibco-BRL), using T7-oligo-d(T)24 as a primer. Second-strand synthesis was performed using T4 DNA polymerase and E. coli DNA ligase followed by blunt ending by T4 polynucleotide kinase. cDNA was isolated by phenol-chloroform extraction using phase lock gels (Brinkmann). cDNA was transcribed in vitro using the T7 BioArray High Yield RNA Transcript Labeling Kit  to produce biotinylated cRNA. Labelled cRNA was isolated using an RNeasy Mini Kit column (Qiagen). Purified cRNA was fragmented to 200–300 mer cRNA using a fragmentation buffer , for 35 min at 94°C. The quality of total RNA, cDNA synthesis, cRNA amplification and cRNA fragmentation was monitored by micro-capillary electrophoresis . The cRNA probes were hybridized to an MG u74Av2 Genechip . Fifteen micrograms of fragmented cRNA was hybridized for 16 h at 45°C with constant rotation (60 rpm). Microarrays were processed in an Affymetrix GeneChip Fluidic Station 400. Staining was made with streptavidin-conjugated phycoerythrin (SAPE) followed by amplification with a biotinylated anti-streptavidin antibody and a second round of SAPE, and then scanned using an Agilent GeneArray Scanner (Agilent Technologies). The signal intensities for the β-actin and GAPDH genes were used as internal quality controls. The ratio of fluorescent intensities for the 5' and 3' ends of these housekeeping genes was <2. Scanned images were analyzed with the Microarray Suite 5.0 software (Affymetrix).Total RNA from rasV12/E1A transformed MEF and its derived tumour was subjected to PCR with reverse transcription using the One Step RT-PCR kit (Promega) according to the manufacturer's protocol. Selected RNA species were amplified using the following primers: arginase 1, sense, 5'-gaaaaggccgattcacctgag-3' and antisense, 5'-atgtggcgcattcacagtcac-3'; bone morphogenetic protein 10, sense, 5'-ggatctggacctggactcaga-3' and antisense, 5'-gaagctttctgggaattcttg-3'; cathepsin S, sense, 5'-gaagggctgcgtcactgaggt-3' and antisense, 5'-acaccgcttttgtagaagaag-3'; insulin-like growth factor binding protein 4, sense, 5'-gaaggtgtagagtagaggctc-3' and antisense, 5-ggaccagaatggggccattcc-3'; interferon stimulated gene 12, sense, 5'-ctcaacatgttgggaacactg-3' and antisense, 5-catctcctgcgtagtctgtac-3'; serum deprivation response, sense, 5'-gtctagtattataacctaacc-3' and antisense, 5-aagagtagagagttcgagccc-3'; thrombomodulin, sense, 5'-cagaaatttcaggtaaccaaa-3' and antisense, 5-tcagctcggcacgaagcacac-3'; adenylate kinase 1, sense, 5'-cactgggtgccaaggagctgt-3' and antisense, 5-ggcttcctgtgtaatgagacc-3'; connective tissue growth factor, sense, 5'-ggagtcagagccttgtctgtt-3' and antisense, 5-agtcataatcaaagaagcagc-3'; and S100 calcium binding protein A11, sense, 5'-gctgttttccaaaagtacagc-3' and antisense, 5-cgcttctgggaagtttggatg-3'. Reverse transcription was carried out 45 min at 48°C followed by 25–32 cycles of PCR, each cycle consisting in a denaturing step for 1 min at 94°C, an annealing step for 2 min at 56°C, and a polymerization step for 2 min at 72°C. PCR products were separated on a 1.0% agarose gel containing ethidium bromide and photographed under ultraviolet light.One microgram of total RNA from primary embryo fibroblasts, rasSV prepared cells and retroviruses, CM carried out RNA purification and RT-PCR analysis, ELC was in charge of microarray hybridization, JCD participated in the design of the study, JLI participated in the analysis of data and wrote the manuscript.Genes up-regulated during tumour development. Genes found up-regulated by microarray analysis are listed, with their GenBank accession number, the over-expression factors (relative to rasV12/E1A transformed MEFs) observed in three separate experiments.Click here for fileGenes down-regulated during tumour development. Genes found down-regulated by microarray analysis are listed, with their GenBank accession number, the down-regulation factors (relative to rasV12/E1A transformed MEFs) observed in three separate experiments.Click here for file


Hearts from all the groups were then processed for biochemical and histopathological studies. One way ANOVA followed by Bonferroni test was applied to test for significance and values are expressed as mean ± SE (p < 0.05).Palm olein oil (PO), obtained from refining of palm oil is rich in monounsaturated fatty acid and antioxidant vitamins and is widely used as oil in diet in many parts of the world including India. Palm oil has been reported to have beneficial effects in oxidative stress associated with hypertension and arterial thrombosis. Oxidative stress plays a major role in the etiopathology of myocardial ischemic-reperfusion injury (IRI) which is a common sequel of ischemic heart disease. Antioxidants have potent therapeutic effects on both ischemic heart disease and ischemic-reperfusion injury. Information on the effect of PO on ischemic-reperfusion injury is, however, lacking. In the present study, the effect of dietary palm olein oil on oxidative stress associated with IRI was investigated in an isolated rat heart model. Wistar rats (150–200 gm) of either sex were divided into three different groups (n = 16). Rats were fed with palm olein oil supplemented commercial rat diet, in two different doses [5% v / w (PO 5) and 10% v / w (PO 10) of diet] for 30 days. Control rats (C) were fed with normal diet. After 30 days, half the rats from each group were subjected to There was a significant increase in myocardial catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities with no significant change in myocardial thiobarbituric acid reactive substances (TBARS) only in group PO 5 as compared to group C. There was no light microscopic evidence of tissue injury. A significant rise in myocardial TBARS and depletion of myocardial endogenous antioxidants  along with significant myocyte injury was observed in control rats subjected to ischemia-reperfusion (C IR). Hearts from palm olein oil fed rats subjected to ischemia-reperfusion (PO 5 IR and PO 10 IR) were protected from increase in TBARS and depletion of endogenous antioxidants as compared to C IR group. No significant myocyte injury was present in the treated groups.The present study demonstrated for the first time that dietary palm olein oil protected rat heart from oxidative stress associated with ischemic-reperfusion injury. Ischemic heart disease (IHD) is a major cause of death all over the world. Reduction in the blood flow to myocardium leads to IHD and its restitution (reperfusion), spontaneously or by drug / surgery, is essential for tissue/organ survival. However, reperfusion itself exacerbates myocardial injury, commonly known as myocardial ischemic-reperfusion injury (IRI) . TherefoLiving organisms have developed antioxidant defense mechanisms against damage due to oxidative stress. These mechanisms in the heart have been extensively studied and the most active endogenous antioxidants involved in this process are superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) ,6. In adThe physiological actions of diet continue to be the focus of interest because of its major role in ischemic heart disease. Dietary antioxidants e.g., vitamin E, beta-carotene, vitamin C have beneficial effects in oxidative stress associated with various cardiovascular diseases, including ischemic heart disease -11. TherElaeis guineensis, is the second major edible oil used worldwide . The doses were selected from the previous studies ,17. DietThe study was approved by Institute Animal Ethics Committee (245 / IAEC / 04) and all animal care and experimental protocols were in compliance with the NIH guidelines for the care and use of the Laboratory Animals . Laboratory bred Wistar rats (150–200 gm) of either sex were maintained under standard laboratory conditions at temperature 25 ± 2°C, relative humidity 50 ± 15% and normal photo period (12 h dark / 12 h light) was used for the study.All chemicals were of analytical grade and chemicals required for sensitive biochemical assays were obtained from Sigma Chemicals . Double distilled water (DDW) was used in all biochemical assays.After one week of acclimatization, rats were randomly divided into three groups, each group containing 16 rats. In control group (C), rats were fed with normal diet for 30 days. In groups (PO5 and PO10), rats were fed with palm olein oil supplemented commercial rat diet for 30 days in two different doses; 5% v / w and 10% v / w of diet. Changes in body weight, food and water intake patterns of rats in all the groups were noted throughout the experimental period. At the end of the 30 days, rats were fasted overnight and half the rats from each group were subjected either to protocol I or to protocol II as described below. Rats were heparinised , and 0.5 h later rats were anesthetized with sodium pentobarbitone  and euthanised.Immediately after euthanization, the hearts were rapidly harvested, washed in ice cold saline, frozen in liquid nitrogen and stored at -80°C until processed for estimations of biochemical parameters. For histopathological studies, heart was stored in 10% buffered formalin (pH 7.2).Group C: Normal diet fed rats (n = 8)Group PO 5: 5% v / w palm olein oil supplemented diet fed rats (n = 8)Group PO 10:10% v / w palm olein oil supplemented diet fed rats (n = 8)3 25; KCl 2.8; KH2PO4 1.2; CaCl2 1.2; MgSO4 0.6, with a pH of 7.4. The buffer solution equilibriated with 95% O2+ 5% CO2 was delivered to the aortic canula at 37°C and 65 mm Hg pressure. Following 10 min. of equilibration period, hearts were subjected to 20 min. of zero flow  and 40 min. of re-flow (reperfusion) [Immediately after euthanization, hearts were rapidly harvested, washed in ice-cold saline, and perfused with the non-recirculating Langendorff's technique , under constant pressure mode with modified Kreb Hensleit's buffer  containirfusion) ,20.Group C IR: Normal diet fed rats subjected to IR injury (n = 8)Group PO5 IR: 5% v/w palm olein oil supplemented diet fed rats subjected to IR injury (n = 8)Group PO10 IR: 10% v/w palm olein oil supplemented diet fed rats subjected to IR injury (n = 8)At the end of each experiment, heart was frozen in liquid nitrogen and stored at -80°C until processed for estimations of biochemical parameters. For histopathological studies, heart was stored in 10% buffered formalin (pH 7.2).Hearts were homogenized in 10% trichloroacetic acid (TCA) at 4°C. 0.2 ml homogenate was pipetted into a test tube, followed by addition of 0.2 ml of 8.1 % sodium dodecyl sulphate (SDS), 1.5 ml of 30% acetic acid (pH-3.5), 1.5 ml of 0.8% thiobarbituric acid (TBA) and volume was made upto 4.0 ml with DDW. Test tubes were boiled at 95°C for 60 min. and then cooled. 1.0 ml of DDW and 5.0 ml of n-butanol: pyridine (15:1 v / v) mixture was added to the test tubes and centrifuge at the 4,000 × g for 10 min. The absorbance of developed colour in organic layer was measured at 532 nm.Commercially available 1, 1, 3, 3-tetraethoxypropane  was used as a standard for MDA. Data is expressed as nmol / mg protein.Hearts were homogenized at 4°C (1:10) in 50 mmol/l potassium phosphate buffer (pH- 7.4) and centrifuged at 3,000 × g for 10 min. Supernatant (50 μl) was added to a 3.0 ml cubette that contained 1.95 ml of 50 mM phosphate buffer (pH-7.0). Then1.0 ml of 30 mM hydrogen peroxide was added and changes in absorbance were measured for 30 sec. at 240 nm at an interval of 15 sec. Catalase activity is expressed as units / mg protein as compared to the standard.Hearts were homogenized in 0.25 M tris sucrose buffer and centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was fractionated by 50% ammonium sulphate and dialysed overnight. Aliquots of the supernatant (100 μl) was added to sodium pyrophosphate buffer (pH-8.3) followed by addition of 0.1 ml of 186 μM phenazine methosulphate, 0.3 ml of 300 mM nitroblue tetrazolium and 0.2 ml of 780 μM NADH. Reaction mixture was incubated for 90 sec. at 30°C and stopped the reaction by adding 1.0 ml of glacial acetic acid. 4.0 ml of n-butanol was then added and centrifuged at 3,000 × g for 10 min. The absorbance of organic layer was measured at 560 nm. SOD activity is expressed as units / mg protein as compared to the standard.Hearts were homogenized at 4°C in 0.25 M phosphate buffer saline (pH-7.0). Homogenate was centrifuged at 15,000 × g for 60 min. at 4°C and supernatant were assayed for the GPx activity. GPx activity was in a 1.0 ml cubette containing 400 μl of 0.25 M potassium phosphate buffer (pH-7.0), 200 μl of sample, 100 μl of 10 mM GSH, 100 μl of 2.5 mM NADPH and 100 μl of glutathione reductase (6 U / ml). Hydrogen peroxide (100 μl of 12 mM) was then added and change in absorbance was measured at an interval of 1 min for 5 min at 366 nm. GPx activity is expressed as units / mg protein as compared to the standard.Protein concentration was measured by Bradford method .Heart tissue was fixed in 10% buffered formalin, routinely processed and embedded in paraffin. Paraffin sections (3 μm) were cut on glass slides and stained with hematoxylin and eosin (H&E), periodic acid Schiff (PAS) reagent and examined under a light microscope . Histopathological study was carried out by one of the authors (AKD), blinded to the groups.All values are expressed as mean ± SE. One way ANOVA followed by Bonferroni test was applied to test for significance of biochemical data of the different groups. Significance is set at p < 0.05.2: prostacyclin; TXA2: thromboxane A2 ; SE: standard error; ANOVA: analysis of variance; MDA: malondialdehyde; TCA: tricarboxylic acid; SDS: sodium dodecyl sulphate; DDW: double distilled water; NADH: nicotinamide adenine dinucleotide reduced; TBA: thiobarbituric acid; NADPH: nicotinamide adenine dinucleotide phosphate reduced; GSH: reduced glutathione; PAS; periodic acid Schiff reagent; H&E: hematoxylin and eosin.IRI: ischemic-reperfusion injury; PO: palm olein oil; PO5: rats fed with 5% v/w palm olein oil supplemented diet; PO10: rats fed with 10% v/w palm olein oil supplemented diet; C: rats fed with normal diet; C IR: rats fed with normal diet subjected to ischemic-reperfusion injury; PO5 IR: rats fed with 5% v/w palm olein oil supplemented diet subjected to ischemic-reperfusion injury; PO10 IR: rats fed with 10% v/w palm olein oil supplemented diet subjected to ischemic-reperfusion injury; TBARS: thiobarbituric acid reactive substances; SOD: superoxide dismutase; CAT: catalase; GPX: glutathione peroxidase; IHD: ischemic heart disease; ROS: reactive oxygen species; PGIDN carried out the animal experimentation, biochemical estimation and statistical analysis of results. SS and MT participated in the design of the study and statistical analysis. AKD carried out the light microscopic study. SKM conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.