Fragile X expression is decreased by 5-azacytidine and S-adenosylhomocysteine

A lymphoblastoid cell line derived from a patient with fragile X chromosome exhibited fragility only when 5-fluoro-2'-deoxyuridine (FUdR) was added to the culture medium. Addition of methionine with FUdR greatly increased the frequency of fragile X. Addition of 5-azacytidine (an inhibitor of methylation at the level of DNA) or S-adenosylhomocysteine (an inhibitor of the synthesis of the methyl group donor S-adenosylmethionine) reversed the effect of methionine as well as that of FUdR. It is proposed that DNA methylation is in some way involved in the mechanism of fragile X expression.     

Fragile (X) expression: Relationship to the cell cycle

Summary The fragile (X) chromosome demonstrable in individuals with one type of X-linked mental retardation is seldom, if ever, seen in more than 50% of cells of affected individuals. We have devised a model to explain this apparent 50% maximum, one essential feature of which is that the fragile (X) will not be seen in cells in their first division in thymidine-depleted media. The validity of our model was tested on lymphoblastoid cell lines from affected males by treating the cells with fluorodeoxyuridine (FUdR) to induce the marker and/or bromodeoxyuridine (BrdU) to determine the cell cycle. We have evidence that the fragile (X) is present in cells in the first and subsequent these observations our model is not valid and the 50% expression of the fragile site at Xq(28) and other unusual properties of this region of the X chromosome remain unexplained.This work was supported by Grant HD 07879 from the National Institutes of Health     

Parameters affecting the restoration of activity to inactive mutants of thymidylate synthase via subunit exchange: further evidence that thymidylate synthase is a half-of-the-sites activity enzyme

In a previous study we demonstrated that Escherichia coli thymidylate synthase activity could be restored completely by incubating basically inactive mutants of this enzyme at room temperature with R(126)E, another inactive mutant [Maley, F., Pedersen-Lane, J., and Changchien, L.-M. (1995) Biochemistry 34, 1469-1474]. Since only one of the enzyme's two subunits possessed a functional active site and the restoration of activity could be titrated to be equivalent to that of the wild-type enzyme's specific activity, it was proposed that thymidylate synthase was a half-of-the-sites activity enzyme. We now provide additional support for this thesis by presenting an in-depth analysis of some conditions affecting the restoration of enzyme activity. For this purpose, we employed two mutants with marginal thymidylate synthase activity, Y(94)A and R(126)E. The parameters that were examined included pH, concentration of protein, temperature, and urea concentration, all of which influenced the rate of activity restoration. It was found, surprisingly, that by maintaining the amount of each protein constant, while increasing the volume of solution, the rate and total activity restored was greatly enhanced. Increasing the pH from 6.0 to 9.0 markedly increased the rate at which the optimal activity was restored, as did increasing the temperature from 4 to 40 degrees C. A similar effect was obtained when the incubation of the mutants was conducted at 4 degrees C in the presence of 1.5 M urea, a temperature at which activity is restored extremely slowly. Raising the pH to 9.0 resulted in an almost instantaneous restoration of activity at 4 degrees C. The manner in which thymidylate synthase activity is restored from the mutants in the presence of varying concentrations of ethanol, ethylene glycol, and glycerol suggests that changes in subunit interaction and enzyme conformation are in part responsible for the observed differences. Most significantly, at solution levels of 10%, ethanol was found to activate, while ethylene glycol inhibited slightly and glycerol was somewhat more inhibitory. At a concentration of 20%, ethanol inhibited rather strikingly, ethylene glycol was slightly more inhibitory than at 10%, and glycerol was strongly inhibitory. Since the net result of these findings is the suggestion that the restoration of thymidylate synthase activity is due to a separation of the mutant dimers into their respective subunits, followed by their recombination to an active heterodimer, evidence for this phenomenon was sought by separating the recombined dimers using nondenaturating polyacrylamide gel electrophoresis. Sequence analysis of the isolated homo- and heterodimers clearly demonstrated that the active enzyme is a product of subunit exchange, one that is very efficient relative to the wild-type enzyme, which did not exchange subunits unless denatured.     

Brown adipose tissue in humans is activated by elevated plasma catecholamines levels and is inversely related to central obesity

Background

Recent studies have shown that adult human possess active brown adipose tissue (BAT), which might be important in controlling obesity. It is known that ß-adrenoceptor-UCP1 system regulates BAT in rodent, but its influence in adult humans remains to be shown. The present study is to determine whether BAT activity can be independently stimulated by elevated catecholamines levels in adult human, and whether it is associated with their adiposity.

Methodology/Principal Findings

We studied 14 patients with pheochromocytoma and 14 normal subjects who had performed both ¹⁸F-fluorodeoxyglucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT) and plasma total metanephrine (TMN) measurements during 2007–2010. The BAT detection rate and the mean BAT activity were significantly higher in patients with elevated TMN levels (Group A: 6/8 and 6.7±2.1 SUVmean· g/ml) than patients with normal TMN concentrations (Group B: 0/6 and 0.4±0.04 SUVmean· g/ml) and normal subjects (Group C: 0/14 and 0.4±0.03 SUVmean·g/ml). BAT activities were positively correlated with TMN levels (R = 0.83, p<0.0001) and were inversely related to body mass index (R = −0.47, p = 0.010), visceral fat areas (R = −0.39, p = 0.044), visceral/total fat areas (R = −0.52, p = 0.0043) and waist circumferences (R = −0.43, p = 0.019). Robust regression revealed that TMN (R = 0.81, p<0.0001) and waist circumferences (R = −0.009, p = 0.009) were the two independent predictors of BAT activities.

Conclusions/Significance

Brown adipose tissue activity in adult human can be activated by elevated plasma TMN levels, such as in the case of patients with pheochromocytoma, and is negatively associated with central adiposity.     

Polymorphisms of folate pathway enzymes (methylenetetrahydrofolate reductase and thymidylate synthase) and their relationship with thymidylate synthase expression in human astrocytic tumors

Two important polymorphisms of folate cycle enzymes, methylenetetrahydrofolate reductase (MTHFR) C677T and thymidylate synthase (TS) enhancer region (TSER) 28-bp tandem repeat, are related to risk of various types of cancer, including brain tumors, although there are few studies on this subject. A case-control study of these two polymorphisms in astrocytomas of different grades was carried out using polymerase chain reaction-restriction fragment length polymorphism, also determining the immunohistochemical expression of TS. The MTHFR 677 TT genotype was less associated with astrocytic tumors (odds ratio [OR]=0.00; p=0.0238), but the TSER polymorphism did not show any significant association. Combined genotype TT-double repeats/triple repeats (2R/3R) had a protective effect against astrocytomas (OR=0.00; p=0.0388). Expression of TS protein was observed in the majority of cases, with grade IV tumors being the exception. Moreover, the median H-score for the pilocytic astrocytomas was significantly higher when compared with that for diffuse tumors. There was an inverse correlation between the 2R/2R genotype and the highest TS-expressing tumors, and 3R/3R was relatively more frequent among the tumors grouped in the third and fourth quartiles. Our results provide support for the role of MTHFR and TS polymorphism in gliomagenesis, possibly because of the alteration of DNA methylation and repair status. Moreover, high levels of TS expression were detected in these tumors.     

Increased thymidylate synthase (EC 2.1.1.45) activity in normal and neoplastic proliferation

Thymidylate (dTMP) synthase (EC 2.1.1.45) activity was measured in 100,000 x g supernatant fluid with a sensitive, rapid radio assay. The activity in normal rat liver was low (0.098-0.204 nmol/hr/mg protein). dTMP synthase specific activities in rat thymus, spleen, bone marrow, testis, lung, heart, brain, kidney, and small intestine were 6297, 1842, 1500, 788, 215, 76, 61, 39 and 24%, respectively, of that of the liver. The activity in 5-day-old rat liver was 16-fold higher than in adult. dTMP synthase activity increased in rat hepatomas to 7- to 125-fold of that of normal rat liver. There was a significant correlation between the increase in synthase activity and the proliferation rates of the hepatomas. In 8 human colon carcinomas, dTMP synthase activity increased to 2.9- to 8-fold of that of normal human colon mucosa. In leukemic leukocytes from 3 leukemia patients, activity was 8- to 10-fold higher than in normal leukocytes.     

Endocytotic activity of bladder superficial urothelial cells is inversely related to their differentiation stage

The composition of the apical plasma membrane of bladder superficial urothelial cells is dramatically modified during cell differentiation, which is accompanied by the change in the dynamics of endocytosis. We studied the expression of urothelial differentiation-related proteins uroplakins and consequently the apical plasma membrane molecular composition in relation to the membrane-bound and fluid-phase endocytosis in bladder superficial urothelial cells. By using primary urothelial cultures in the environment without mechanical stimuli, we studied the constitutive endocytosis. Four new findings emerge from our study. First, in highly differentiated superficial urothelial cells with strong uroplakin expression, the endocytosis of fluid-phase endocytotic markers was 43% lower and the endocytosis of membrane-bound markers was 86% lower compared to partially differentiated cells with weak uroplakin expression. Second, superficial urothelial cells have 5–15-times lower endocytotic activity than MDCK cells. Third, in superficial urothelial cells the membrane-bound markers are delivered to lysosomes, while fluid-phase markers are seen only in early endocytotic compartments, suggesting their kiss-and-run recycling. Finally, we provide the first evidence that in highly differentiated cells the uroplakin-positive membrane regions are excluded from internalization, suggesting that uroplakins hinder endocytosis from the apical plasma membrane in superficial urothelial cells and thus maintain optimal permeability barrier function.     

Tyrosyl kinase activity is inversely related to prostatic acid phosphatase activity in two human prostate carcinoma cell lines.

Alterations in prostatic acid phosphatase (PAcP), a phosphotyrosyl phosphatase, corresponded to changes in overall tyrosyl kinase activity. PAcP added to extracts of prostate carcinoma cells with a low endogenous level of PAcP activity and elevated tyrosyl kinase activity decreased the tyrosyl kinase activity. On the other hand, when PAcP activity was decreased by the addition of androgens to cells, there was a corresponding increase in tyrosyl kinase activity.     

Age-associated reduction of prostacyclin and thromboxane synthesis is inversely related to plasma cholesterol levels: modulation by dietary cholesterol supplementation

Abnormalities of vasoactive eicosanoid synthesis with age are reported. We observed an age-associated reduction of vascular prostacyclin production and thrombin-stimulated thromboxane A2 production in blood. Amounts of production of these eicosanoids were inversely related to plasma cholesterol levels. However, there were no such relationships in rats supplemented with cholesterol. Dietary cholesterol supplementation induced a reduction of thromboxane A2/prostacyclin ratio regardless of age. These results suggest that age-associated changes of blood cholesterol levels are closely linked with vasoactive eicosanoid synthesis and that excessive consumption of cholesterol may induce a compensatory reaction by reducing the thromboxane A2/prostacyclin ratio.     

Carotid plaque age is a feature of plaque stability inversely related to levels of plasma insulin

Background

The stability of atherosclerotic plaques determines the risk for rupture, which may lead to thrombus formation and potentially severe clinical complications such as myocardial infarction and stroke. Although the rate of plaque formation may be important for plaque stability, this process is not well understood. We took advantage of the atmospheric ¹⁴C-declination curve (a result of the atomic bomb tests in the 1950s and 1960s) to determine the average biological age of carotid plaques.

Methodology/Principal Finding

The cores of carotid plaques were dissected from 29 well-characterized, symptomatic patients with carotid stenosis and analyzed for ¹⁴C content by accelerator mass spectrometry. The average plaque age (i.e. formation time) was 9.6±3.3 years. All but two plaques had formed within 5–15 years before surgery. Plaque age was not associated with the chronological ages of the patients but was inversely related to plasma insulin levels (p = 0.0014). Most plaques were echo-lucent rather than echo-rich (2.24±0.97, range 1–5). However, plaques in the lowest tercile of plaque age (most recently formed) were characterized by further instability with a higher content of lipids and macrophages (67.8±12.4 vs. 50.4±6.2, p = 0.00005; 57.6±26.1 vs. 39.8±25.7, p<0.0005, respectively), less collagen (45.3±6.1 vs. 51.1±9.8, p<0.05), and fewer smooth muscle cells (130±31 vs. 141±21, p<0.05) than plaques in the highest tercile. Microarray analysis of plaques in the lowest tercile also showed increased activity of genes involved in immune responses and oxidative phosphorylation.

Conclusions/Significance

Our results show, for the first time, that plaque age, as judge by relative incorporation of ¹⁴C, can improve our understanding of carotid plaque stability and therefore risk for clinical complications. Our results also suggest that levels of plasma insulin might be involved in determining carotid plaque age.     

Thymidylate synthase gene expression is stimulated by some (but not all) introns.

We previously described the construction of an intronless mouse thymidylate synthase (TS) minigene that has the normal 5' and 3' flanking regions of the gene linked to full length TS cDNA. Transfection of the minigene into ts- hamster V79 cells led to low level expression of normal mouse TS mRNA and protein. In the present study we analyzed the effect of introns on the expression of the TS minigene in transient transfection assays. Inclusion of introns 5 and 6 at their normal locations in the coding region led to an 8-9-fold stimulation of the level of TS and TS mRNA. Almost all of introns 5 and 6 could be deleted without diminishing the stimulatory effect. Inclusion of intron 3 also stimulated the expression of the minigene, although to a lesser extent than introns 5 and 6. However, inclusion of intron 4 had no stimulatory effect. Analysis of minigenes that contained various combinations of introns revealed that the stimulatory effects of the introns were not additive.     

The distance between Escherichia coli genes is related to gene expression levels.

It is shown that the distance between Escherichia coli genes oriented in the same direction on the chromosome is positively correlated to the expression level of the downstream gene. It is suggested that this could be a strategy to avoid interference between ribosomes translating two different genes.     

Fragile (X) X-linked mental retardation. II. Frequency and replication pattern of fragile (X)(q28) in heterozygotes

The frequency of cytologic expression and the replication pattern of the fragile (X) [fra(X)] were investigated in 28 fra(X) heterozygotes, of which 25 agreed to psychological assessment. One-third of the heterozygotes in this study are mentally retarded. The intellectually impaired carriers had a higher frequency of fra(X) and a higher proportion of early-replicating fra(X) than the normally intelligent carriers. The early-replicating fra(X) accounted for 39% of the variability in IQ and the late-replicating fra(X) for 12%. Age had a minimal inverse effect on fra(X) expression and replication pattern. Thus, it appears that mental retardation in females heterozygous for the fra(X) may largely be a function of the proportion of cells with an early-replicating, active X chromosome possessing the fragile site.     

The metabolic function of hepatocytes differentiated from human mesenchymal stem cells is inversely related to cellular glutathione levels

Differentiation of mesenchymal stem cells (MSCs) to hepatocytes‐like cells is associated with alteration in the level of reactive oxygen species (ROS) and antioxidant defense system. Here, we report the role of glutathione in the functions of hepatocytes derived from MSCs. The stem cells undergoing differentiation were treated with glutathione modifiers [buthionine sulfoxide (BSO) or N‐acetyl cysteine (NAC)], and hepatocytes were collected on day 14 of differentiation and analysed for their biological and metabolic functions. Differentiation process has been performed in presence of glutathione modifiers viz. BSO and NAC. Depending on the level of cellular glutathione, the proliferation rate of MSCs was affected. Glutathione depletion by BSO resulted in increased levels of albumin and ROS in hepatocytes. Whereas, albumin and ROS were inhibited in cells treated with glutathione precursor (NAC). The metabolic function of hepatocytes was elevated in BSO‐treated cells as judged by increased urea, transferrin, albumin, alanine transaminase and aspartate transaminase secretions in the media. However, the metabolic activity of the hepatocytes was inhibited when glutathione was increased by NAC. We conclude that the efficiency of metabolic function of hepatocytes is inversely related to the levels of cellular glutathione. These data may suggest a novel role of glutathione in regulation of metabolic function of hepatocytes. Copyright © 2013 John Wiley & Sons, Ltd.     

The induction of X-chromosomal aneuploidy by 5-fluorodeoxyuridine (FUdR) fed to Drosophila melanogaster females.

After feeding FUdR (5-fluorodeoxyuridine) to female Drosophila melanogaster, highly significant increases in the frequencies of both XO and XXY exceptions were observed in their offspring. The XXY exceptions and part of the XO exceptions result from maternal nondisjunction of the X-chromosomes. Part of the XO exceptions can be assumed to be produced by X-chromosome breakage followed by bridge formation. The analysis of the brood pattern observed suggests that interphase cells (premeiotic oocytes, oogonia) are especially sensitive in the induction of both XO and XXY exceptions by FUdR. In addition, and contrary to the results obtained with other objects, FUdR seems to induce chromosomal damage (presumably chromatid and/or isochromatid breaks) not only in interphase but also in prophase cells. The mechanisms of the induction of X-chromosomal aneuploids by FUdR are discussed.