Pouchitis may increase the risk of dysplasia after restorative proctocolectomy in patients with ulcerative colitis.

AIM: Dysplasia of the pouch mucosa after restorative proctocolectomy is rare. The aim of this study was to establish whether there is a correlation between pouchitis and dysplasia. METHOD: A group of 276 patients treated for ulcerative colitis by restorative proctocolectomy between 1984 and 2009 was analysed. The presence or absence of pouchitis and dysplasia within the pouch was evaluated. RESULTS: Inflammation was diagnosed in 66 (23.9%) patients, low-grade dysplasia in five (1.8%), high-grade dysplasia in three (1.1%), and cancer in one patient (0.4%). The prevalence of low-grade dysplasia was significantly higher in patients with inflammation than in those without (P < 0.04). High-grade dysplasia was significantly more frequent in pouchitis than in non-inflamed pouches (P < 0.01). Logistic regression analysis suggested that the occurrence of mucosal inflammation increased the risk of low grade dysplasia. CONCLUSION: Patients with chronic pouchitis are at risk of dysplasia and require surveillance of the pouch.

Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis.

Genotoxic stress activates complex cellular responses allowing for the repair of DNA damage and proper cell recovery. Although plants are exposed constantly to increasing solar UV irradiation, the signaling cascades activated by genotoxic environments are largely unknown. We have identified an Arabidopsis mutant (mkp1) hypersensitive to genotoxic stress treatments (UV-C and methyl methanesulphonate) due to disruption of a gene that encodes an Arabidopsis homolog of mitogen-activated protein kinase phosphatase (AtMKP1). Growth of the mkp1 mutant under standard conditions is indistinguishable from wild type, indicating a stress-specific function of AtMKP1. MAP kinase phosphatases (MKPs), the potent inactivators of MAP kinases, are considered important regulators of MAP kinase signaling. Although biochemical data from mammalian cell cultures suggests an involvement of MKPs in cellular stress responses, there is no in vivo genetic support for this view in any multicellular organism. The genetic and biochemical data presented here imply a central role for a MAP kinase cascade in genotoxic stress signaling in plants and indicate AtMKP1 to be a crucial regulator of the MAP kinase activity in vivo, determining the outcome of the cellular reaction and the level of genotoxic resistance.

Epigenetic developmental mechanisms in plants: molecules and targets of plant epigenetic regulation.

Genetic approaches to understanding the role of epigenetic regulation of gene expression in plants and its mechanisms have revealed several new components. Rapidly accumulating information from other eukaryotes provides complementary knowledge with important implications for plant research. Comparison of epigenetic events across species is proving critical for defining the mechanisms and functions of epigenetic modification, including those specific to plants.

Gene silencing and DNA methylation processes.

Epigenetic gene silencing results from the inhibition of transcription or from posttranscriptional RNA degradation. DNA methylation is one of the most central and frequently discussed elements of gene silencing in both plants and mammals. Because DNA methylation has not been detected in yeast, Drosophila or Caenorhabditis elegans, the standard genetic workhorses, plants are important models for revealing the role of DNA methylation in the epigenetic regulation of genes in vivo.

Oxidase activity of ceruloplasmin and concentrations of copper and zinc in serum of cancer patients.

A balance between oxidant carcinogens and endogenous antioxidant defence is of particular relevance to the carcinogenesis. Ceruloplasmin (Cp) carries up to 90% of Cu in plasma and performs ferroxidase, antioxidant and amine oxidase activity. Cu and Zn, as trace elements, have been recognized to play an important role as cofactors of SOD. The study presents the relationship of the Cp oxidase activity and concentrations of Cu and Zn in serum of 62 patients with breast (BCA), lung (LCA), gastrointestinal (GICA) and gynecological (GYNCA) cancer. The Cp oxidase activity was determined in serum with o-dianisidine as a substrate. Cu and Zn concentrations in serum were measured by using atomic absorption spectrometry. The results of the study have shown significant increase in the mean serum Cp oxidase activity and total Cu concentrations in all patient groups compared with the control one. The total mean serum Zn concentration was found to be decreased only in LCA group as compared with the control. The effect of the cancer progress on the Cp oxidase activity and concentrations of Cu and Zn was observed within the group of all cancer patients (ALLCA) and within the GICA group. The only significant difference in Cu concentrations among various stages of the disease was observed in GICA between local and distant one. Significant positive correlation coefficients were caLculated for the Cp activity and Cu concentrations in the control group and all patients groups, also according to the cancer progress. Future research is needed to evaLuate the consequences of the elevation of the serum Cp oxidase activity and concentration of Cp, Cu and Zn for the host antioxidant-oxidant balance.

[The prevalence of type II diabetes mellitus in rural urban population over 35 years of age in Lublin region (Eastern Poland)]. / Badania nad wystepowaniem cukrzycy typu 2 w populacji powyzej 35 roku zycia na wsi i w miescie w regionie lubelskim.

The prevalence of type 2 diabetes mellitus (DM 2) has increased dramatically in the last decade. Data relating to the number of undetected cases of diabetes are underestimated. The aim of the study was to evaluate the prevalence of DM 2, obesity, hypertension, and lipid disturbances in a representative group of urban and rural population in the Lublin region (Eastern Poland). The study was performed in 1998-2001. A two-layer draw was applied: two groups of 3000 people were drawn from the population of Lublin town and from the rural areas each comprising 100,000 inhabitants. In all subjects physical examination was performed and body weight, height, and blood pressure measurements were obtained. Blood samples were taken from the basilic vein to estimate: blood glucose, lipids and insulin concentration. Venous blood glucose concentration was measured using a Glucotrend glucometer. Oral glucose tolerance test (OGTT) after a 75 g-glucose load was performed in subjects without previously diagnosed diabetes mellitus and when the fasting blood glucose was < 8.0 mmol/l (144 mg/l). The LDL-cholesterol level was calculated according to Friedewald formula. DM 2 was identified according to the WHO criteria from 1985. Obesity and hypertension were diagnosed according to the new WHO criteria (Body Mass Index > or = 30 kg/m2, blood pressure > or = 140/90 mm Hg). 3782 subjects: 1809 in the rural area and 1973 in Lublin town were examined. The response rate among rural and urban population was 60.3% and 65.8% respectively. The prevalence of DM 2 was assessed in 17.6% of rural and in 14.1% of urban population. 75% of diabetics in the rural areas and 56% in the town were the newly diagnosed cases. We found impaired glucose tolerance in 30.3% of rural and in 21.6% of urban population, BMI > or = 30 kg/m2 in 30.8% and 30.1%, hypertension in 69.4% (29.2% newly diagnosed) and 68.6% (27.7% newly diagnosed), hypercholesterolaemia (total cholesterol > or = 5.2 mmol/l (200 mg/dl)) in 66.4% and 60%, hyper-LDL-cholesterolaemia (> or = 3.5 mmol/l (135 mg/dl)) in 57.3% and 52.6%, hypo-HDL--cholesterolaemia in 21.7% and 31.4%, hypertriglyceridemia (> or = 2.3 mmol/l (200 mg/dl)) in 15.1% and 22% respectively. This finding indicates the urgent need for introducing a national program for early diagnosis and prevention of DM 2 and concomitant metabolic disturbances.

Gene targeting in Arabidopsis.

Precise modification by gene targeting (GT) provides an important tool for studies of gene function in vivo. Although routine with many organisms, only isolated examples of GT events have been reported for flowering plants. These were at low frequencies precluding reliable estimation of targeting efficiency and evaluation of GT mechanisms. Here we present an unambiguous and straightforward system for detection of GT events in Arabidopsis using an endogenous nuclear gene encoding protoporphyrinogen oxidase (PPO), involved in chlorophyll and heme syntheses. Inhibition of PPO by the herbicide Butafenacil results in rapid plant death. However, the combination of two particular mutations renders PPO highly resistant to Butafenacil. We exploited this feature for selection of GT events by introducing the mutations into the PPO gene by homologous recombination. We have estimated the basal GT frequency to be 2.4 x 10(-3). Approximately one-third of events were true GT (TGT) leading to the anticipated modification of the chromosomal PPO copy. The remaining events could be classified as ectopic GT (EGT) arising by modification of vector DNA by the chromosomal template and its random integration into the Arabidopsis genome. Thus the TGT frequency in our experimental setup is 0.72 x 10(-3). In view of the high efficiency of Arabidopsis transformation, GT experiments of a reasonable size followed by a PCR screen for GT events should also allow for modification of non-selectable targets. Moreover, the system presented here should contribute significantly to future improvement of GT technology in plants.

Elevated levels of intrachromosomal homologous recombination in Arabidopsis overexpressing the MIM gene.

The Arabidopsis MIM gene encodes a protein belonging to the SMC family (structure maintenance of chromosomes) which is required for intrachromosomal homologous recombination (ICR). Both ICR and MIM gene expression are enhanced by DNA-damaging treatments, suggesting that MIM is a factor limiting DNA repair by homologous recombination (HR) under genotoxic stress. We tested this hypothesis by measuring the levels of recombination in the mim mutant under genotoxic stress, using methyl methanesulfonate. Although the mutant clearly showed diminished basal and induced levels of ICR, enhancement of ICR by DNA-damaging treatments was similar to that observed in the wild type. This suggests that the MIM gene product is required for DNA repair by HR, but is not critical for HR induction. To determine whether enhanced availability of MIM would increase basal HR levels in Arabidopsis, we examined ICR frequencies in transgenic Arabidopsis strains overexpressing the MIM gene after ectopic insertion of additional MIM copies. Two independent lines showed a twofold increase in ICR frequency relative to the wild type. Thus MIM is required for efficient ICR in plants, and its manipulation can be used to change homologous recombination frequencies. Since MIM is one of the components responsible for chromatin dynamics, our results suggest that the chromatin environment determines the frequency of homologous recombination.

Lipid peroxidation and activities of antioxidant enzymes in the diabetic kidney: effect of treatment with angiotensin convertase inhibitors.

Effects of the angiotensin convertase inhibitors captopril (CAP) and enalapril (ENA) on the malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD) and catalase in the kidneys of rats with streptozotocin-induced diabetes were studied. Induction of diabetes resulted in an increase of MDA concentration and progressive decreases of SOD and catalase activities after 6 and 12 weeks. CAP and ENA administration did not affect body weight changes or blood glucose and HbA1c contents in diabetic rats but decreased albuminuria and kidney weight increase, attenuated lipid peroxidation, and prevented the decreases in SOD and catalase activities. These results confirm the oxidative stress in streptozotocin-induced experimental diabetes and point to the beneficial antioxidant effects of angiotensin convertase inhibitors.

Transcriptional gene silencing mutants.

Genetic approaches to identify molecular components of transcriptional gene silencing (TGS) in plants have yielded several Arabidopsis thaliana mutants and identified the first genes involved. All mutations found affect the maintenance of silencing and reactivate silent genes in trans. The mutations fall into two categories: ddm1 and hog release silencing in association with decreased levels of DNA methylation, while sil and mom reactivate genes without changing the methylation state. While plants homozygous over several generations for hog, sil or mom exhibit no morphological changes, ddm1-type mutants accumulate developmental abnormalities. The mutants indicate that TGS in plants is controlled by several genetic components and possibly by multiple independent pathways. The DDM1 gene was assigned to the SWI2/SNF2 gene family of chromatin-remodelling proteins, the MOM gene is a novel protein and the other loci have not yet been characterized.

Endogenous targets of transcriptional gene silencing in Arabidopsis.

Transcriptional gene silencing (TGS) frequently inactivates foreign genes integrated into plant genomes but very likely also suppresses an unknown subset of chromosomal information. Accordingly, RNA analysis of mutants impaired in silencing should uncover endogenous targets of this epigenetic regulation. We compared transcripts from wild-type Arabidopsis carrying a silent transgene with RNA from an isogenic transgene-expressing TGS mutant. Two cDNA clones were identified representing endogenous RNA expressed only in the mutant. The synthesis of these RNAs was found to be released in several mutants affected in TGS, implying that TGS in general and not a particular mutation controls the transcriptional activity of their templates. Detailed analysis revealed that the two clones are part of longer transcripts termed TSI (for transcriptionally silent information). Two major classes of related TSI transcripts were found in a mutant cDNA library. They are synthesized from repeats present in heterochromatic pericentromeric regions of Arabidopsis chromosomes. These repeats share sequence homology with the 3' terminal part of the putative retrotransposon Athila. However, the transcriptional activation does not include the transposon itself and does not promote its movement. There is no evidence for a general release of silencing from retroelements. Thus, foreign genes in plants encounter the epigenetic control normally directed, at least in part, toward a subset of pericentromeric repeats.

Disruption of the plant gene MOM releases transcriptional silencing of methylated genes.

Epigenetic modifications change transcription patterns in multicellular organisms to achieve tissue-specific gene expression and inactivate alien DNA such as transposons or transgenes. In plants and animals, DNA methylation is involved in heritability and flexibility of epigenetic states, although its function is far from clear. We have isolated an Arabidopsis gene, MOM, whose product is required for the maintenance of transcriptional gene silencing. Mutation of this gene or depletion of its transcript by expression of antisense RNA reactivates transcription from several previously silent, heavily methylated loci. Despite this, the dense methylation at these reactivated loci is maintained even after nine generations, indicating that transcriptional activity and methylation pattern are inherited independently. The predicted MOM gene product is a nuclear protein of 2,001 amino acids containing a region similar to part of the ATPase region of the SWI2/SNF2 family, members of which are involved in chromatin remodelling. MOM is the first known molecular component that is essential for transcriptional gene silencing and does not affect methylation pattern. Thus, it may act downstream of methylation in epigenetic regulation, or be part of a new pathway that does not require methylation marks.

Prospects for the precise engineering of plant genomes by homologous recombination.

The targeting of chromosomal genes via homologous recombination (HR) is an essential tool of reverse genetics as applied for the functional assay of genes within complex genomes. However, in higher plants, foreign DNA integrates almost exclusively at random, non-homologous sites. A variety of environmental parameters known to influence levels of HR do not increase targeting frequencies when combined in gene-targeting experiments. The identification of cellular factors that may control the level of chromosomal HR in plant somatic cells is required. Plant genes encoding proteins similar to those involved in HR in other organisms can be found in the expanding sequence databases. Evidence for evolutionary conservation should help to decipher mechanisms of plant HR and possibly detect limiting factors. At present, however, only one genetic locus influencing levels of chromosomal recombination in plants has been well defined. Here we summarise current knowledge of HR and the status of gene targeting (GT) in plants, focusing on genetic approaches to molecular factors regulating HR levels.

An SMC-like protein is required for efficient homologous recombination in Arabidopsis.

In plants, the observed low frequency of gene targeting and intrachromosomal recombination contrasts markedly with the efficient extrachromosomal recombination of DNA. Thus, chromatin accessibility can have a major influence on the recombination frequency of chromosomal DNA in vivo. An Arabidopsis mutant hypersensitive to a range of DNA-damaging treatments (UV-C, X-rays, methyl methanesulfonate and mitomycin C) is also defective in somatic intrachromosomal homologous recombination. The wild-type gene encodes a protein closely related to the structural maintenance of chromosomes (SMC) family involved in structural changes in chromosomes. Although loss of SMC function is lethal in other eukaryotes, growth of the Arabidopsis mutant is normal in the absence of genotoxic treatments. This suggests a surprisingly specialized function for this protein in plants, and provides the first in vivo evidence for the involvement of an SMC protein in recombinational DNA repair. It is possible that SMC-like proteins in plants alleviate suppressive chromatin structure limiting homologous recombination in somatic cells.

Involvement of Arabidopsis thaliana ribosomal protein S27 in mRNA degradation triggered by genotoxic stress.

A recessive Arabidopsis mutant with elevated sensitivity to DNA damaging treatments was identified in one out of 800 families generated by T-DNA insertion mutagenesis. The T-DNA generated a chromosomal deletion of 1287 bp in the promoter of one of three S27 ribosomal protein genes (ARS27A) preventing its expression. Seedlings of ars27A developed normally under standard growth conditions, suggesting wild-type proficiency of translation. However, growth was strongly inhibited in media supplemented with methyl methane sulfate (MMS) at a concentration not affecting the wild type. This inhibition was accompanied by the formation of tumor-like structures instead of auxiliary roots. Wild-type seedlings treated with increasing concentrations of MMS up to a lethal dose never displayed such a trait, neither was this phenotype observed in ars27A plants in the absence of MMS or under other stress conditions. Thus, the hypersensitivity and tumorous growth are mutant-specific responses to the genotoxic MMS treatment. Another important feature of the mutant is its inability to perform rapid degradation of transcripts after UV treatment, as seen in wild-type plants. Therefore, we propose that the ARS27A protein is dispensable for protein synthesis under standard conditions but is required for the elimination of possibly damaged mRNA after UV irradiation.

Cytosine methylation at CG and CNG sites is not a prerequisite for the initiation of transcriptional gene silencing in plants, but it is required for its maintenance.

Transgenes integrated into plant chromosomes, and/or endogenous plant genes, may be subjected to epigenetic silencing at the transcriptional or post-transcriptional level. Transcriptional inactivation is correlated with hypermethylation of CG/CNG sites at the silent loci. It is not known whether local hypermethylation is part of the inactivation process, or just an outcome of the silent state. To address this issue, we generated transgenic tobacco lines containing a selectable marker gene controlled by a derivative of the 35S promoter of the cauliflower mosaic virus (CaMV) devoid of CG and CNG methylation acceptor sites. Silencing was triggered by crossing to the silencer locus of tobacco line 271. This line contains inactive and methylated copies of the 35S promoter and is able to silence homologous promoter copies at ectopic chromosomal positions. The mutated promoter lacking CG/CNG methylation acceptor sites was as susceptible to Trans-silencing as the unmodified 35S promoter control. Thus, methylation at CG and CNG sites is not a prerequisite for the initiation of epigenetic gene inactivation. Interestingly, while methylation of the remaining cytosines is usually only slightly affected by silencing, it was significantly increased in the absence of CG/CNG sequences. Since this sequence preference is the same as that of known methyltransferases, this may imply that silencing is accompanied or directly followed by recruitment of methyltransferase, which, in the absence of cytosines in the optimal sequence context, modifies other C residues in the affected area. However, silencing without CG/CNG methylation was immediately relieved in the absence of the silencer. Thus, CG/CNG methylation is probably essential for the maintenance of previously established epigenetic states.

The genetics of epigenetics.

The formation of silent epialleles is accompanied by local hypermethylation of the DNA template, and genetically altered, methylation-deficient Arabidopsis mutants generate an increased number of epimutations. But what came first - methylation or epigenetic change?

Plant genes: the genetics of epigenetics.

The formation of silent epialleles is accompanied by local hypermethylation of the DNA template, and genetically altered, methylation-deficient Arabidopsis mutants generate an increased number of epimutations. But what came first--methylation or epigenetic change?

Release of epigenetic gene silencing by trans-acting mutations in Arabidopsis.

Gene silencing in plants inactivates trans-genes introduced into plants and/or endogenous homologous genes. This stable but potentially reversible loss of gene activity resembles epigenetic changes that occur in normal development. The stability of silencing implies the involvement of trans-acting components, although none of them have been identified so far. Here we report the finding of second-site mutations interfering with maintenance of the silent state. We mutagenized Arabidopsis thaliana plants carrying a silent transgene encoding hygromycin phosphotransferase (hpt) and therefore show a heritable hygromycin-sensitive phenotype. The M2 generation was screened for hygromycin resistance. Eight putative mutants (som1 to 8) were found that expressed the transgene and transmitted the expressed state to their progeny. All mutations were shown to reactivate a silent transgenic test locus in trans. The level of DNA methylation at the hpt locus and at centromeric repeats was found to be reduced in the som mutants. Complementation crosses indicated complex epigenetic interactions among the som mutant alleles and with the previously described ddm1 allele, which elicits DNA hypomethylation [Vongs, A., Kakutani, T, Martiensen, R.A. & Richards, E.J. (1993) Science 260, 1926-1928]. Som mutants can be classified into three groups: (i) allelic or interacting with ddm1 and with each other (som 1,4, and 5), (ii) nonallelic with ddm1 and som mutants of group A (som2), and (iii) mutants with slow resilencing after out-crosses, which hinders their classification (som 3, 6, 7 and 8).