Butylated hydroxytoluene modulates DNA methylation in rats

This study was a replication of an earlier Cochran & Ganong study that investigated the perception of nurses and patients regarding the stressors faced by patients in the intensive care unit environment. As the original study was American in origin, one of the aims of the present study was to discover if the results would be replicated in a United Kingdom (UK) intensive care unit. Data collection was by the use of an environmental stress questionnaire that was an adaptation of the original data collection tool modified for use in a UK intensive care unit. The study was undertaken in two intensive care units producing a sample size of 71 patients and 71 nurses. There appears to be a wide variation in the perception of nurses and patients regarding the stress faced by patients in the intensive care unit. Similarities were noted between subject groups as to the nature of the stressors, although nurses tended to rate items over which they believed they had control as being more stressful than did the patients. Patients tended to rate items related to their illness and physical comfort as being most stressful. The results are in keeping with those from the Cochran & Ganong study.     

Are somatic cells inherently deficient in methylation metabolism? A proposed mechanism for DNA methylation loss, senescence and aging

A mechanism of aging is proposed for mammals and other vertebrates. In this mechanism, most somatic cells have inherent deficiencies in methylation metabolism with respect to their capacity to methylate DNA. This leads to incomplete DNA methylation in each cell cycle which, accumulated over many cell cycles, contributes to genetic instability, senescence and cancer. These proposed metabolic deficiencies are present from the time somatic cells are young, yet it is only after many cell divisions that deleterious effects are realized. In nature, most animals have reproduced or have been killed by predators or other environmental hazards before they can be greatly affected by these deficiencies. These deficiencies evolved in animals eating a balance of nutrients from nature. Evidence from the literature is reviewed which establishes that methylation is lost from the DNA of many mammalian somatic cells as they age both in vivo and in vitro, and that DNA methylation levels are influenced by factors, such as diet, that affect methylation metabolism. Partially correcting the proposed deficiencies is considered as a possible molecular mechanism by which caloric restriction extends lifespan. Other possible dietary and transgenic means to correct the proposed deficiencies and extend lifespan are discussed.     

Microsomal function in biliary obstructed rats: effects of S-adenosylmethionine

BACKGROUND/AIMS: S-adenosylmethionine has been reported to have beneficial effects in the treatment of different chronic liver diseases and to protect against different hepatotoxic agents. The aim of this study was to investigate whether S-adenosylmethionine treatment might contribute to improved microsomal function in chronically biliary obstructed rats. METHODS: Secondary biliary cirrhosis was induced by 28 days of bile duct obstruction. Groups of control and cirrhotic animals received S-adenosylmethionine (10 mg/kg per day) through the experimental period. RESULTS: Bile duct obstruction resulted in a marked increase in lipid peroxidation levels and decreases in glutathione concentration, microsomal membrane fluidity, microsomal cytochrome P-450 content, NADPH-cytochrome P-450 reductase activity and the activities of the aniline hydroxylase, aminopyrine demethylase and ethoxycoumarin deethylase. Reductions in glutathione and cytochrome P-450 concentration were not corrected by S-adenosylmethionine, but lipid peroxidation, the decrease in the activities of the various microsomal monooxygenases and the reduction in microsomal membrane fluidity were partially prevented. A significant relationship was found between membrane fluidity and aniline hydroxylase, aminopyrine demethylase or ethoxycoumarin deethylase activities. CONCLUSIONS: S-adenosylmethionine administration partially preserves microsomal function. This effect could be associated to the protection of membrane function by restoring transmethylation reactions.     

Aging and DNA methylation in colorectal mucosa and cancer

DNA methylation of promoter-associated CpG islands may function as an alternate mechanism of silencing tumor suppressor genes in multiple neoplasias including colorectal cancer. De novo methylation of genes appears to be an early and frequent event in most neoplasias. For the ER and IGF2 genes, we have previously shown that methylation actually begins in the normal colon mucosa as an age-related event and progresses to hypermethylation in cancer. In this study, we have determined the frequency of age-related methylation in normal colonic mucosa among the genes hypermethylated in colorectal cancer. We studied six genes, including N33, MYOD, p16, HIC-1, THBS1, and CALCA. The N33 gene showed partial methylation in normal colon mucosa, which was age-related (r = 0.7; P = 0.003 using regression analysis). Adenomas and cancers showed further hypermethylation at this locus. Similarly, the MYOD gene showed age-related methylation in normal colon mucosa (r = 0.7; P < 0.00001 using regression analysis) and hypermethylation in cancers. Age-related methylation seems to be gene specific, because p16, THBS1, HIC-1, and CALCA were not affected. Furthermore, this process may also be modulated by tissue-specific factors. Our study suggests that aging is a major contributing factor to hypermethylation in cancer.     

Brain folates and DNA methylation in rats fed a choline deficient diet or treated with low doses of methotrexate

Photorefractive keratectomy (PRK) with the ArF excimer lasers in current use usually approximates the intended corneal curvature by a mean of a delicate step-type pattern that is smooth off afterwards by reepithelialization and tear film. The present study was based on a model eye with axial myopia of -6 D but otherwise the optical and geometric properties of the Gullstrand model eye and was designed to investigate to what extent. (1) corneal step patterns can reduce retinal image contrast and (2) smoothing effects can restore such a loss. METHODS. The corneal surface resulting from PRK in the case of a myopia of -6 D (optical zone diameter 6 mm) is calculated for the parameters of the model eye. The retinal image contrasts of bar patterns are calculated by PSF (point spread function) analysis: varying size of pupil, wavelength, bar width, ablation step height and degree of smoothing. RESULTS. Step height influences retinal image contrast crucially. With step heights above 0.4 micron a massive loss of retinal image contrast must be expected, which can, however, be corrected to a useful extent by surface-smoothing effects. CONCLUSION. This study indicates that PRK with excimer lasers should be performed with low fluence and correspondingly low corneal step heights.     

Enflurane metabolism in rats and man

A-patterns have been treated by various techniques, with variable results. A desinsertion of the superior oblique at its insertion temporal to the superior rectus has proved to be a satisfactory treatment. This is combined with horizontal surgery. The results of this surgery in 137 cases are analysed and the complications outlined.     

Suppression of crossing-over by DNA methylation in Ascobolus

Interpretation of health related quality of life (HRQOL) results in cancer patients is facilitated by knowledge of the levels of HRQOL in the general population. However, direct comparisons can be misleading unless age and gender are considered. We demonstrate the derivation of age- and gender-specific 'expected' values from population reference values by means of simple calculations. This survey included 3000 randomly selected Norwegians above 18 years of age who received the European Organization for Research and Treatment of Cancer Core Quality of Life Questionnaire (EORTC QLQ-C30 (+3) by mail. 1965 responses from 2,892 eligible persons (68%) were received. The population was divided into six disease groups based on self-reported health problems. The observed mean scale scores of the different groups deviated greatly from those obtained in the general population. The score for physical function, for example, was 72 for cancer patients and ranged from 73.3 to 82.5 in other disease groups, as opposed to 89.9 in the general population and 98.9 in those with no health problems. The range for one of the quality of life (QOL) scales was 57.7 to 84.7 compared with 73.7 in the general population. Expected mean scores for the patient groups were computed from the reference values, based on the concept of equivalence of age and gender. The differences between the observed mean scores and the reference values were strongly mediated by this method. The expected scores for physical function then ranged from 83.3 to 93.1 and from 70.3 to 75 for the QOL scale. The impact of age and gender on the reference data from the EORTC QLQ-C30 (+3) obtained in a general population shows that these variables must be considered when interpreting data on HRQOL for cancer patients. The demonstration of how to generate mean values which are adjusted according to the age and gender distribution of a population should increase the usefulness of this questionnaire among clinicians.     

Allelic losses and DNA methylation at DNA mismatch repair loci in sporadic colorectal cancer

Normal and tumor DNA samples of 35 patients with sporadic colorectal carcinoma were analyzed for microsatellite alterations at 12 markers linked to mismatch repair loci: hMLH1, hMSH2, hMSH3, hMSH6, hPMS1 and hPMS2. Remarkably, no correlation was observed between the replication error phenotype (RER+) and allelic losses at these loci. Hemizygous deletions, seen in 6/35 (17%) informative cases at hMLH1, 4/27 (15%) at hMSH2/hMSH6 and 6/34 (18%) at hMSH3, were rarely found in RER+ tumors. Since mismatch repair protein components act in molecular complexes of defined stoichiometry we propose that hemizygous deletion of the corresponding loci may be involved in colorectal tumorigenesis through defects in cellular functions other than replication error correction. The analysis of the methylation status of the promoter region of hMLH1 revealed that methylation might be an important mechanism of this locus inactivation in RER+ sporadic colorectal cancer.     

Phosphorus metabolism in potassium-deficient rats

Hypophosphatemia as a consequence of potassium deficiency has been reported sporadically. Most cases have been complicated by other factors which might lead to decreased serum phosphorus levels. Therefore, the serum phosphorus in this study was measured in Sprague-Dawley rats with nutritionally induced potassium deficiency. Severe potassium depletion was manifested by hypokalemia (2.4 mEq/liter versus 3.9 mEq/liter in controls) and decreased muscle potassium content. Statistically significant hypophosphatemia did not develop, although decreased muscle phosphorus content was observed. Therefore, hypophosphatemia is not a regular accompaniment of severe potassium deficiency in the rat.     

Protein metabolism in burned rats

Incorporation of [2-14C]glycine was used to estimate serum protein synthesis in four groups of rats. These were the control (group C); 20% body surface burn (group B); 20% burn, seeded with Pseudomonas aeruginosa (group BI); and burned-infected treated topically with mafenide (alpha-amino-p-toluenesulfonamide) acetate (group BIS), a treatment which controls P, aeruginosa burn-wound infection in humans. On the 6th day postburn the relative specific activities of all fractions were increased in the order BI greater than BIS greater than B greater than C, as were the concentrations of the globulins; Serum albumin concentration fell, being lowest in BI. Tissue albumin contents, measured by radioimmunoassay, of eviscerated blood-free bodies of rats were (mg/100 g rat wt): C, 207; B, 294; BI, 256. Analyses of individual tissues showed that the difference was due to increased albumin content in the burn-wound area. The tissue albumin was of normal molecular size and was immunologically reactive. We conclude that the prolonged hypoalbuminemia following burn injury is not a consequence of impaired albumin synthesis, but a result of altered compartmentation.     

Enzymatic DNA methylation as an aging mechanism

By analysis of 70 functional parameters of organism it has been shown that the senescence of human organs starts from the age of 20 +/- 10 years and it has more or less a linear character in the most cases. Judging by the averaged aging rate, human maximal lifespan can be equal to about 145 years. An aging model has been proposed according to it the functional involution is caused by cells destructing and is a result of gradual accumulation of special mutations in genome. The evidences has been obtained that enzymatic DNA methylation is a powerful generator for the 5mC-->T+C transition, constantly occurring with each cell division. It has been found that the age-related loss of the most part or all 5mC residues from DNA coincides with both the Hayflick limit in cell lines and the maximal lifespan of different animal species studied. The rate of DNA hypomethylation is proportional to the cell aging rate in vitro and in vivo. The 5mC-->T transitions contribute more than half of all point mutations, accumulated in genome of vertebrates during evolution. These substitutions occur ten times more often in the methylated sites than in other positions of DNA and they disproportional contribute to the general mutagenesis and to the hereditary human diseases. The conclusion has been made that DNA methylation meet the requirements of the main criterions of an aging biomarker and it can be considered as a mechanism for genetically programmed accumulation of mutations with aging. In immortal cell lines this mechanism starts working in the reverse direction, increasing the 5mC content in DNA.     

A short DNA methyltransferase isoform restores methylation in vivo

Two murine DNA methyltransferase isoforms (MTases) have been observed, a longer form in somatic and embryonic stem (ES) cells and a shorter form in oocytes and preimplantation embryos. While the longer MTase is associated with maintenance methyltransferase activity in replicating cells, little is known about the shorter form. We present genetic and biochemical evidence that both isoforms are expressed from the same Dnmt1 gene by using different translation initiation sites in exons 1 and 4. We further demonstrate that the shorter isoform can functionally rescue Dnmt1 null ES cells that have a hypomethylated genome. These rescued ES cells differentiate in vivo into a variety of cell types, unlike the Dnmt1 null ES cells that die upon induction of differentiation. These results show that the shorter isoform can substitute for the longer maintenance MTase in ES and differentiated cells. Our data further indicate that the shorter MTase isoform found in oocytes is fully functional in vivo and may play an active role in the regulation of DNA methylation and the establishment of imprinting patterns.     

DNA methylation and the promotion of flowering by vernalization

We have tested the hypothesis that the promotion of flowering by prolonged exposure to low temperatures (vernalization) is mediated by DNA demethylation [Burn, J. E., Bagnall, D. J., Metzger, J. M., Dennis, E. S. & Peacock, W. J. (1993) Proc. Natl. Acad. Sci. USA 90, 287-291]. Arabidopsis plants that have reduced levels of DNA methylation because of the presence of a methyltransferase (METI) antisense gene flowered earlier than untransformed control plants, without the need for a cold treatment. Decreased DNA methylation mutants (ddm1) also flowered earlier than the wild-type progenitor under conditions where they respond to vernalization. We conclude that demethylation of DNA is sufficient to cause early flowering, and we have found that the promotion of flowering is directly proportional to the decrease in methylation in METI antisense lines. The early-flowering phenotype was inherited in sexual progeny, even when the antisense transgene had been lost by segregation. Methyltransferase antisense plants with low DNA methylation levels responded to a low-temperature treatment by flowering even earlier than their untreated siblings indicating that the promotion of flowering by cold and by demethylation was additive when neither treatment saturated the early-flowering response. As in untransformed control plants, the cold-induced early-flowering signal was reset in progeny of METI antisense plants. These observations suggest that the demethylation brought about by a METI antisense can account for some properties of vernalization, but not for the need for revernalization in each generation.     

DNA methylation and developmental genes in lymphomagenesis--more questions than answers?

There is now considerable evidence suggesting that alterations in the DNA methylating machinery play an important role in tumorigenesis and tumour progression. For example, focal hypermethylation and generalised genomic demethylation are features of many different types of neoplasms. It is thought that tumorigenesis and tumour progression may be caused by hypermethylation induced mutational events and silencing of genes which control cellular proliferation and/or demethylation induced reactivation of genes which may only be required during embryological development. Consequently, we have begun to investigate the role of DNA methylation and developmental genes in malignant lymphoproliferative diseases. Previously, in all cases of non-Hodgkins lymphoma and leukemia studied, we have shown that the myogenic developmental gene Myf-3 is abnormally hypermethylated. In this review we discuss the possible significance of these findings since in vitro studies suggest that Myf-3 may play an important role in control of the cell cycle and therefore lymphomagenesis. In vitro and in vivo evidence suggests that PAX genes may also have oncogenic potential. The PAX family of developmental genes are involved in cellular differentiation, proliferation and cell migration. Expression of PAX3 in particular is associated with cellular mobility. Our previous studies have indicated that alternate regional expression of PAX genes may be controlled by DNA methylation. Therefore, we have proposed that abnormal methylation profiles of PAX3 may be associated with neoplastic transformation and/or metastatic potential. Results thus far reveal that the paired box of PAX3 is abnormally hypermethylated and the homeobox abnormally hypomethylated in lymphomas and leukemias. These new findings are consistent with our postulate and support the idea that inappropriate methylation induced activation or inactivation of developmental genes such as Myf-3 and PAX3 play an important role in lymphomagenesis and disease progression and that inspection of the methylation status of other developmental genes is warranted.     

Choline deficiency and methotrexate treatment induces marked but reversible changes in hepatic folate concentrations, serum homocysteine and DNA methylation rates in rats

OBJECTIVE: The study compared the effects of feeding rats a choline deficient (CD) diet or injecting low doses of methotrexate (MTX) on hepatic folate concentration and distribution, homocysteine (Hcy) concentration and DNA methylation. METHODS: Thirty rats were divided into three groups and were fed either a choline sufficient (CS) or deficient diet (CD), or injected with low doses of MTX (0.1 mg/kg/day) for 2 weeks. Half the animals of each group were sacrificed and the remaining CD and MTX animals were fed repletion diets without methotrexate administration for two additional weeks. RESULTS: CD or MTX resulted in a significantly lower folate concentrations (25-50%) compared to the control group. Folate distribution in the treated animals was associated with elongation of the glutamate chains: higher proportion of hexa (from 14%, control, to 35%, choline, p < 0.05), hepta (from 5% to 16%, p < 0.05), and appearance of octaglutamyl folates. MTX administration resulted in a similar pattern of hepatic folate distribution. Two weeks following the MTX administration and the restoration of an adequate choline diet for 2 weeks restored the hepatic folate levels to the control animals. CONCLUSIONS: Results are discussed based on the possibility that CD and MTX treatment appear to impair the capacity of tissues to incorporate folate in only 2 weeks and affect other biomarkers of one-carbon metabolism such as Hcy concentration and DNA methylation. This adverse picture was partially reversed in a relative short time by simply feeding an adequate CS diet and discontinuing MTX injections.     

The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylation of B12

BACKGROUND: In both mammalian and microbial species, B12-dependent methionine synthase catalyzes methyl transfer from methyltetrahydrofolate (CH3-H4folate) to homocysteine. The B12 (cobalamin) cofactor plays an essential role in this reaction, accepting the methyl group from CH3-H4folate to form methylcob(III)alamin and in turn donating the methyl group to homocysteine to generate methionine and cob(I)alamin. Occasionally the highly reactive cob(I)alamin intermediate is oxidized to the catalytically inactive cob(II)alamin form. Reactivation to sustain enzyme activity is achieved by a reductive methylation, requiring S-adenosylmethionine (AdoMet) as the methyl donor and, in Esherichia coli, flavodoxin as an electron donor. The intact system is controlled and organized so that AdoMet, rather than methyltetrahydrofolate, is the methyl donor in the reactivation reaction. AdoMet is not wasted as a methyl donor in the catalytic cycle in which methionine is synthesized from homocysteine. The structures of the AdoMet binding site and the cobalamin-binding domains (previously determined) provide a starting point for understanding the methyl transfer reactions of methionine synthase. RESULTS: We report the crystal structure of the 38 kDa C-terminal fragment of E.coli methionine synthase that comprises the AdoMet-binding site and is essential for reactivation. The structure, which includes residues 901-1227 of methionine synthase, is a C-shaped single domain whose central feature is a bent antiparallel betasheet. Database searches indicate that the observed polypeptide has no close relatives. AdoMet binds near the center of the inner surface of the domain and is held in place by both side chain and backbone interactions. CONCLUSIONS: The conformation of bound AdoMet, and the interactions that determine its binding, differ from those found in other AdoMet-dependent enzymes. The sequence Arg-x-x-x-Gly-Tyr is critical for the binding of AdoMet to methionine synthase. The position of bound AdoMet suggests that large areas of the C-terminal and cobalamin-binding fragments must come in contact in order to transfer the methyl group of AdoMet to cobalamin. The catalytic and activation cycles may be turned off and on by alternating physical separation and approach of the reactants.