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 (30 ml/yr, 37 ml/yr and 45 ml/yr). 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 (pH 7.4, 37°C) [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 (St. Louis MO, USA). FeTPPS  was from . Rabbit anti-3-NT polyclonal antibodies  were from Upstate (Lake Placid, NY, USA). Anti-rabbit Ig horseradish peroxidase antibodies  were purchased from Stressgen (Victoria BC, Canada). Commercial kits to measure creatinine and urea were from Spinreact (Girona, Spain). All other chemicals were reagent grade and commercially available.Male Wistar rats (Harlan Teklad, Mexico City, Mexico) 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 (GraphPad, San Diego, CA, USA). 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 (E1, E6, E7, and L1), 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 (2, 3, and 4 enzymes) 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 After ttastases , the captastases ]. 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 (RNP1, RRM) 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 (0.45 μm filter, Millipore) 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 (Enzo Biochem, New York, N.Y.) 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 (100 mM potassium acetate-30 mM magnesium acetate-40 mM Tris-acetate, pH 8.1), 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 (Affymetrix, Santa Clara, CA). 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 (SOD, CAT and GPx) 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 [2) and reduce thromboxane A2 (TXA2) levels in tissues [Palm oil, obtained from the fruit of the tropical plant orldwide . Palm olorldwide ,14 and aorldwide  in rats. tissues . HoweverTherefore, the present study was designed to evaluate the effects of dietary palm olein oil on myocardial endogenous antioxidants and on oxidative stress associated with ischemic-reperfusion injury in isolated rat heart model.There was no mortality, changes in body weight as well as food and water intake pattern of rats in any group.There were no significant changes in myocardial TBARS levels in both PO5 (8.5 ± 0.8 nmol / mg protein) and PO10 (8.2 ± 1.2 nmol / mg protein) groups when compared to that of control group (7.8 ± 0.4 nmol / mg protein).There was a significant increase in myocardial CAT activity in both PO5  and PO10  groups as compared to that of control group (34.4 ± 2.1 units / mg protein).There were a significant increase in myocardial SOD activity in PO 5  group when compared to that of the control group (3.5 ± 0.08 units / mg protein). There was no significant change in myocardial SOD activity in PO10 (3.8 ± 0.3 units / mg protein) group.There was a significant (p < 0.01) increase in myocardial GPx activity in PO5 (0.18 ± 0.01 units / mg protein) group when compared to that of the control group (0.13 ± 0.01 units / mg protein). There was no significant change in myocardial GPx activity in PO10 (0.16 ± 0.02 units / mg protein) group.There was a significant (p < 0.05) increase in myocardial TBARS level in the C IR group (9.4 ± 0.3 nmol / mg protein) when compared to that of the control group (7.8 ± 0.4 nmol / mg protein). There was a significant (p < 0.01) decrease in myocardial TBARS levels in both PO5 IR (6.8 ± 0.4 nmol / mg protein) and PO10 IR (5.9 ± 0.6 nmol / mg protein) groups when compared to that of the C IR group.There was a significant (p < 0.05) decrease in myocardial CAT activity (29.1 ± 0.8 units / mg protein) in C IR group when compared to that of the control group (34.4 ± 2.1 units / mg protein). There was a significant rise in CAT activity in PO5 IR  group with no significant change in PO10 IR (36.3 ± 5.6 units / mg protein) group when compared to C IR group.There was a significant decrease in myocardial SOD activity in C IR group  as compared to control group (3.5 ± 0.08 units / mg protein). No significant changes in SOD activities were observed in both PO5 IR (3.1 ± 0.2 units / mg protein) and PO10 IR (3.4 ± 0.3 units / mg protein) groups as compared to C IR group.There was a significant (p < 0.01) decrease in myocardial GPx activity in C IR group (0.11 ± 0.002 units / mg protein) as compared to control group (0.13 ± 0.004 units / mg protein). There was no significant change in myocardial GPx activity in PO5 IR group (0.12 ± 0.01 units / mg protein) with a significant (p < 0.01) increase in myocardial GPx activity in PO10 IR group (0.15 ± 0.01 units / mg protein) as compared to C IR group.Fig. In the present study, a significant increase in myocardial SOD, catalase and GPx activity was observed in the lower dose of palm olein oil fed rats. However, their further augmentation was not observed in the higher dose, i.e., a dose dependent effect was not observed. The finding correlates with the previous studies in which an increase in response was not observed with the increase in the dose of supplemented vitamin E ,27. The Augmentation of endogenous antioxidants (SOD, CAT, GPx) has been recognized as an important pharmacological property, present in natural as well as many synthetic compounds -33. ThisIncrease in myocardial TBARS and depletion of myocardial endogenous antioxidants support the occurrence of oxidative stress in the control hearts following ischemia-reperfusion in the present study. It was also accompanied by tissue injury with marked edema and focal loss of myocardial fibres. Similar changes have been reported earlier to occur following brief period of ischemia followed by reperfusion in rat heart -37. Hear2 /TXA2 ratio [Experimental as well as clinical studies with exogenous antioxidants supplementation have been shown to have protective effect in ischemic heart disease ,39. In tA2 ratio . Palm oiA2 ratio ,43 and iA2 ratio .The observations made in the present study have important nutritional significance for palm olein oil in relation to ischemic heart disease. However, further studies are required to establish the mechanism, underlying the augmentation of tissue antioxidants.The present study, for the first time, demonstrated that long term oral supplementation of palm olein oil caused augmentation of endogenous antioxidants of heart, which were subsequently protected from developing oxidative stress following ischemia-reperfusion.ad libitum.Palm olein oil (Ruchi Gold, India) was obtained from the local market. Commercial rat diet (Ashirwad, India) containing protein: 24%, fat: 5%, fiber: 4%, carbohydrates: 55%, calcium: 0.6%, phosphorus: 0.3% w / w was supplemented with palm olein oil in two different doses [5 % v / w and 10 %v / w of diet]. 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 (NIH Publication #85–23, 1985). 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 (St. Louis, USA). 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 (375 IU / 200 gms, i.p), and 0.5 h later rats were anesthetized with sodium pentobarbitone (60 mg / Kg, i.p) 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 (Hufesco, Hungary), 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 (Nikon, Japan). 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.


While the tat and rev genes encode regulatory proteins absolutely required for virus replication, nef, vif, vpr, vpx and vpu encode for small proteins referred to "auxiliary" (or "accessory"), since their expression is usually dispensable for virus growth in many in vitro systems. However, these auxiliary proteins are essential for viral replication and pathogenesis in vivo. The two vpr- and vpx-related genes are found only in members of the HIV-2/SIVsm/SIVmac group, whereas primate lentiviruses from other lineages (HIV-1, SIVcpz, SIVagm, SIVmnd and SIVsyk) contain a single vpr gene. In this review, we will mainly focus on vpr from HIV-1 and discuss the most recent developments in our understanding of Vpr functions and its role during the virus replication cycle.The genomes of human and simian immunodeficiency viruses (HIV and SIV) encode the gag, pol and env genes and contain at least six supplementary open reading frames termed  Despite its small size, Vpr has been shown to play multiple functions during virus replication, including an effect on the accuracy of the reverse-transcription process, the nuclear import of the viral DNA as a component of the pre-integration complex (PIC), cell cycle progression, regulation of apoptosis, and the transactivation of the HIV-LTR as well as host cell genes  of HIV-1 is a small basic protein 14 kDa) of 96 amino acids, and is well conserved in HIV-1, HIV-2 and SIV [gression . Moreove kDa of 9 disease . Regardicis/trans isomerization are found in the N-terminal domain [Because the full length protein aggregated in aqueous solution, the overall structure of Vpr has been difficult to access , and prel domain . It was l domain . The carl domain ,18-20.Gag region of the gag-encoded Pr55Gag precursor [Gag region of the Pr55Gag precursor is directly involved in the interaction with Vpr [Gag in matrix, capsid, nucleocapsid (NCp7), and p6 mature proteins, Vpr is recruited into the conical core of the virus particle [Gag region of the precursor to the mature NCp7 protein to gain access to the core of the infectious virus particle budding at the cell surface. It seems that Vpr is less avid for the fully processed p6 protein than for the p6Gag region in the context of the p55Gag precursor. Because of this differential avidity, Vpr is recruited into to the core of the particle where it could interact with nucleic acids, NCp7 [Vpr is expressed at a late stage of the virus life cycle, but it is present during the early steps of infection of target cells since it is packaged into virions released from the producing cells. The incorporation of Vpr occurs through a direct interaction with the carboxy-terminal p6recursor -24. Whilrecursor , a leuciwith Vpr ,26. Afteparticle ,28 whereparticle ,30. Inteparticle ,24,31. Sparticle ,28, Vpr ds, NCp7 . Since ids, NCp7 , that main trans in virus-producing cells [trans-complementation also allowed the analysis of mutant of IN without altering assembly, maturation and other subsequent viral events [The incorporation of Vpr has been also used as a unique tool to target cargoes  into viral particles ,35. Thisng cells -38. Thisl events ,39.Furthermore, Vpr fused to the green fluorescence protein (GFP) has been recently used to tag HIV particles in order to follow intracellular virus behavior during the early steps of infection of target cells ,41.Following virus entry, the viral core is released into the cytoplasm of the target cell and the reverse transcription of the viral RNA takes place in the cytoplasm within a large nucleoprotein complex termed the reverse transcription complex (RTC) containing the two copies of viral RNA and the viral proteins: RT, IN, NCp7, Vpr and a few molecules of the matrix protein -46. It iin vivo mutation rate of HIV-1 by influencing the accuracy of the reverse transcription. The HIV-1 RT is an error-prone RNA dependant DNA polymerase, and quantification of the in vivo rate of forward virus mutation per replication cycle revealed that the mutation rate was as much as fourfold higher in the absence of Vpr expression when measured in actively dividing cells using a genetically engineered system [in vitro and ex vivo in Vpr-expressing cells. While the Trp residue in position 54 located in the exposed loop connecting the second and the third α-helix of HIV-1 Vpr has been shown critical to maintain the interaction with UNG, the Vpr-binding site was mapped within the C-terminal part of UNG2 and occurs through a TrpXXPhe motif. Currently, three distinct cellular partners of Vpr contain a WXXF motif including the TFIIB transcription factor, the adenosine-nucleotide translocator (ANT) and UNG2 [In addition to a potential role in the initiation step of the reverse transcription process , it has d system ,52. Furtd system . This acd system , an enzyand UNG2 ,56.in vitro binding assays suggest that both Vpr and IN associate with UNG to form a trimeric complex (ELR and SB, unpublished results), but further analyses are required to document the nature of the interactions between UNG2, Vpr, IN as well as RT both in virus-producing cells and then in target cells.The association of Vpr with UNG2 in virus-producing cells allows the incorporation of a catalytically active enzyme into HIV-1 particles where UNG2 may directly influence the reverse transcription accuracy , and thiin vitro [HIV-1 and other lentiviruses are unusual among retroviruses in their ability to infect resting or terminally differentiated cells. While Vpr has been shown to facilitate the nuclear import of viral DNA in non-dividing cells, the virion incorporation of UNG2 via Vpr also contributes to the ability of HIV-1 to replicate in primary macrophages. This implies that UNG2 is a cellular factor that plays an important role in the early steps of the HIV-1 replication cycle . This observation is in good agreement with a recent report showing that the misincorporation of uracil into minus strand viral DNA affects the initiation of the plus strand DNA synthesis in vitro . This obin vitro . SimilarIn addition, it is intriguing to note that two viral auxiliary proteins from HIV-1, Vpr and Vif, can both influence the fidelity of viral DNA synthesis. The Vif protein forms a complex with the cellular deaminase APOBEC-3G (CEM15) preventing its encapsidation into virions -63, whilNondividing cells, such as resting T cells and terminally-differentiated macrophages, are important targets for viral replication during the initial stages of infection, since primary infection of these cell populations contributes to the establishment of virus reservoirs, crucial for subsequent virus spread to lymphoid organs and T-helper lymphocytes . InfectiThe exact composition of the PIC is still an area of debate but it contains the viral DNA at least associated with integrase, and many recent studies have confirmed that Vpr is also an integral component of this complex (for reviews, see -77). Of in vitro nuclear import assay, it has been demonstrated that the association with the N-terminal region of hCG1 is required for the docking of Vpr to the NE, whereas the FG-repeat region does not participate in this process [Indeed, Vpr displays evident karyophilic properties and localizes in the nucleus, but a significant fraction is anchored at the NE and can be visualized as a nuclear rim staining in fluorescence microscopy experiments ,79-81. T process . The rol process -86. Alte process . The mol process ,88.in vitro or in transfected cells have shown that Vpr is able to actively promote nuclear import of a reporter protein, such as BSA, β-galastosidase or GFP [Despite the lack of any identifiable canonical nuclear localization signal (NLS), Vpr displays evident karyophilic properties and is rapidly targeted to the host cell nucleus after infection . Even the or GFP ,13,90-94e or GFP ,73. In ae or GFP ,96, but e or GFP ,85. Finae or GFP . Two inde or GFP ,94. These or GFP ,70,80.In conclusion, the nucleophilic property of Vpr and its high affinity for the NPC, associated with its presence in the viral PIC, at least support a role during the docking step of the PIC at the NE, a prerequisite before the translocation of viral DNA into the nucleus. Even though there is no evidence that Vpr directly participates in the translocation process, it is worth noting that purified PICs also dock at the NE before nuclear translocation using a pathway also distinct from the NLS and M9 nuclear import pathways . One canGag precursor during the late budding step of the virus life cycle [In addition to its nonconventional NLS for targeting into the nucleus, Vpr is a dynamic mobile protein able to shuttle between the nucleus and cytoplasmic compartments ,99,100. fe cycle ,100.S. pombe fission yeast as well as S. cerevisiae overexpressing HIV-1 Vpr are also blocked in the G2 phase of the cell cycle [vpr gene from SIV similarly provoked a G2 arrest [A further important biological activity of SIV and HIV Vpr proteins is related to their ability to induce an arrest in the G2 phase of the cell cycle of infected proliferating human and simian T cells ,101-105.ll cycle -109, sup2 arrest . The bio2 arrest . In agreThe determinants of the G2 arrest activity are mainly located in the C-terminal unstructured basic region of HIV-1 Vpr and phosphorylation of the protein is required ,113. RegS. pombe suggest the involvement of PP2A and Wee1 in the Vpr-induced cell cycle arrest [The protein phosphatase 2A (PP2A) has been shown to be directly associated with Vpr via its B55α subunit . PP2A ise arrest . Intrigue arrest . As mente arrest . It seeme arrest ,131).+ T cells in AIDS patients, which results in a weakened immune system, impairing its ability to fight infections. The major mechanism for CD4+ T cell depletion is programmed cell death, or apoptosis, that can be induced by HIV through multiple pathways of both infected cells and non-infected "bystander" cells (for review, see [in vitro systems. In addition, transgenic mice expressing Vpr under the control of the CD4 promoter show both CD4 and CD8 T cell depletion associated with thymic atrophy [HIV infection causes a depletion of CD4iew, see ). Even t atrophy . However atrophy ,135.Initially proposed as a consequence of the prolonged cell cycle arrest -140, othc and apoptosis inducing factors, in the cytosol. Cytochrome c then associates with Apaf-1 in a complex with caspase-9 to create the apoptosome, allowing activation of effector caspases, such as caspase-3, and subsequently the final execution of the apoptotic process  . This diiew, see ). While iew, see ,147,149,iew, see . These rin vitro and ex vivo, its real contribution with other viral determinants, such as gp120 envelope, Tat, Nef and the viral protease, in the physiopathology of AIDS needs to be further documented during the course of HIV infection [Although the causal role of Vpr in the induction of apoptosis is evident both nfection . Howevernfection . Conversnfection , indicatIn addition, soluble Vpr protein is found in the sera as well as in the cerebrospinal fluid of HIV-infected patients, and was proposed to play a role related to its pro-apoptotic activity in AIDS-associated dementia ,155. Thecis-acting elements, including NF-κB, Sp1, C/EBP and the GRE enhancer sequences found in the LTR promotor [Waf1 and survivin [The first reported function of Vpr was a modest transcriptional activity on the viral LTR promotor as well as on heterologous cellular promotors ,162. Whipromotor -170. Alssurvivin -173. Finsurvivin , the glusurvivin ,175, thesurvivin ,176, andsurvivin . This lasurvivin .cis-acting elements alone but it associates with Sp1 in the context of the G/C box array [26 and LQQLL68 motifs contained within the first and third α-helical domains of HIV-1 Vpr [Vpr displays high affinity for nucleic acids but no specific DNA sequence targeted by Vpr has been yet identified ,29. Inteox array , as wellox array , indicatox array , suggestox array ,175. TheIV-1 Vpr ,180.vpr expression revealed that Vpr induces up and down regulation of various cell genes [Vpr may also function as an adaptor molecule for an efficient recruitment of transcriptional co-activators (GRE, p300/CBP...) to the HIV-1 LTR promotor and thus enhances viral replication. Additionally, it may be involved in the activation of host cell genes inducing cellular pathways in relation with the AIDS pathogenesis. Indeed, cDNA microarray analysis using isogenic HIV-1 either with or without ll genes .in vivo is likely crucial. While major efforts have been made during the last years to define the molecular mechanisms and cellular targets of Vpr, additional work is needed for the complete understanding of its wide range of activities. An important issue now is to define the precise contribution of each activity to the viral replication and pathogenesis during the natural course of HIV infection. The involvement of Vpr in key processes of the early steps the viral life cycle  represents a good target for developing novel therapeutic strategies for AIDS therapy. In addition, this viral factor represents a valuable tool to elucidate many fundamental cellular processes.By interfering with many distinct cellular pathways all along the virus life cycle, it is now evident that Vpr's contribution to the overall pathogenesis of HIV-1 infection HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; CypA, cyclophilin A; nup, nucleoporin; PIC, pre-integration complex; RTC, reverse transcription complex.