Exploring the impact of magnetic fields on biomass production efficiency under aerobic and anaerobic batch fermentation of Saccharomyces cerevisiae.

In this work, the effect of moderate electromagnetic fields (2.5, 10, and 15 mT) was studied using an immersed coil inserted directly into a bioreactor on batch cultivation of yeast under both aerobic and anaerobic conditions. Throughout the cultivation, parameters, including CO2 levels, O2 saturation, nitrogen consumption, glucose uptake, ethanol production, and yeast growth (using OD 600 measurements at 1-h intervals), were analysed. The results showed that 10 and 15 mT magnetic fields not only statistically significantly boosted and sped up biomass production (by 38-70%), but also accelerated overall metabolism, accelerating glucose, oxygen, and nitrogen consumption, by 1-2 h. The carbon balance analysis revealed an acceleration in ethanol and glycerol production, albeit with final concentrations by 22-28% lower, with a more pronounced effect in aerobic cultivation. These findings suggest that magnetic fields shift the metabolic balance toward biomass formation rather than ethanol production, showcasing their potential to modulate yeast metabolism. Considering coil heating, opting for the 10 mT magnetic field is preferable due to its lower heat generation. In these terms, we propose that magnetic field can be used as novel tool to increase biomass yield and accelerate yeast metabolism.

The utility of endometrial thickness measurement in asymptomatic postmenopausal women with endometrial fluid.

The aim of this study was to assess the clinical usefulness of sonographic endometrium thickness measurement in asymptomatic postmenopausal women with endometrial fluid collection. Fifty-two asymptomatic postmenopausal women with endometrial fluid, who underwent endometrial sampling were evaluated. Histopathological findings revealed that 25 (48.1%) women had insufficient tissue, 20 (38.4%) had atrophic endometrium and 7 (13.5%) had endometrial polyps. No case of malignancy was found. There was no statistically significant difference between the various histopathological categories (insufficient tissue, atrophic endometrium and polyp) with regard to the mean single-layer endometrial thickness (1.54 ± 0.87, 2.04 ± 1.76 and 1.79 ± 0.69 mm, respectively, p = 0.436). Out of 44 patients with endometrial thickness of less than 3 mm, 38 (86.4%) had atrophic changes or insufficient tissue and 6 (13.6%) had endometrial polyps. In conclusion, if the endometrial thickness is 3 mm or less, endometrial sampling is not necessary in asymptomatic postmenopausal women with endometrial fluid.

Early postoperative pulmonary complications after heart transplantation.

OBJECTIVE: The aim of this study was to determine the types, incidence, and risk factors for early postoperative pulmonary complications in heart transplant recipients. METHODS: We retrospectively collected data from the records of consecutive heart transplantations from January 2003 to December 2013. A total of 83 patients underwent heart transplantation. The data collected for each case were demographic features, duration of mechanical ventilation, respiratory problems that developed during the intensive care unit (ICU) stay, and early postoperative mortality (<30 d). RESULTS: Of the 72 patients considered, 52 (72.2%) were male. The overall mean age at the time of transplantation was 32.1 ± 16.6 years. Twenty-five patients (34.7%) developed early postoperative respiratory complications. The most frequent problem was pleural effusion (n = 19; 26.4%), followed by atelectasis (n = 6; 8.3%), acute respiratory distress syndrome (n = 5; 6.9%), pulmonary edema (n = 4; 5.6%), and pneumonia (n = 3; 4.2%). Postoperative duration of mechanical ventilation (44.2 ± 59.2 h vs 123.8 ± 190.8 h; P = .005) and the length of postoperative ICU stay (10.1 ± 5.8 h vs 19.8 ± 28.9 h; P = .03) were longer among patients who had respiratory problems. Postoperative length of stay in the hospital (22.3 ± 12.5 d vs 30.3 ± 38.3 d; P = .75) was similar in the 2 groups. The overall mortality rate was 12.5% (n = 9). The patients who had respiratory problems did not show higher mortality than those who did not have respiratory problems (16.0% vs 10.6%; P = .71). CONCLUSIONS: Respiratory complications were relatively common in our cohort of heart transplant recipients. However, these complications were mostly self-limiting and did not result in worse mortality.

Heart rate variability in ß-thalassaemia major with or without cardiac siderosis.

OBJECTIVES: Iron cardiomyopathy remains the major cause of death in ß-thalassaemia major. Excessive iron loading could lead to cardiac dysfunction and arrhythmias. Reduced heart rate variability is associated with a higher risk of arrhythmia and sudden death after myocardial infarction and heart failure. Previous data have reported on reduced heart rate variability in patients with marked cardiac iron accumulation. In this study, we compared heart rate variability among ß-thalassaemia major (TM) patients with or without cardiac siderosis. METHODS: Out of 70 ß-thalassaemia major patients with preserved ejection fractions, 38 patients with cardiac T2* magnetic resonance imaging assessment were included in our study. Time domain heart rate variability parameters were analysed from 24-hour recorded electrocardiograms and were compared with the control group. RESULTS: The mean T2* magnetic resonance imaging value was 22.9 ± 13.3 (4.7-47.5). In 21 patients with ß-thalassaemia major, the T2* magnetic resonance imaging values were greater than 20 ms and these patients were considered to be in the early stage of the disease. When we compare these patients with control subjects, the standard deviation of all NN intervals was still significantly lower (133.0 ± 32.2 versus 162.8 ± 32.9, p = 0.001) in ß-thalassaemia major patients despite normal T2* magnetic resonance imaging values. On the contrary, the standard deviation of all NN intervals was not correlated with haemoglobin levels in these patients (p > 0.05). CONCLUSIONS: Heart rate variability parameters were reduced even in ß-thalassaemia major patients without evident cardiac siderosis, as specified by magnetic resonance imaging data. The results of this study show that reduction of heart rate variability may start before cardiac iron loading is demonstrated by T2* magnetic resonance imaging in ß-thalassaemia major.

RIFLE criteria for acute kidney dysfunction following heart transplantation: incidence and risk factors.

BACKGROUND AND PURPOSE: There are few data regarding the occurrence of (RIFLE)-based acute kidney dysfunction (AKD) after heart transplantation (HT) and its risk factors. The aim of this study was to apply RIFLE criteria in patients who developed AKD following HT to compare patients with and without AKD and to determine incidence and risk factors of AKD. PATIENTS AND METHODS: We retrospectively analyzed the records of 65 patients who underwent HT between 2003 and 2012. We investigated 3 levels of renal dysfunction outlined in RIFLE criteria: risk (R), injury (I), and failure (F). Appropriate class was assigned comparing baseline creatinine level to peak levels in the first 7 days after HT. Perioperative variables of heart transplant recipients were collected. RESULTS: The mean age at transplantation was 32.8 ± 16.6 years with 72.7% males. The incidence of AKD was 61%, risk occured in 18%, injury in 16%, and failure in 27% of the patients. Patients who had AKD were significantly older (37.9 ± 15.6 vs 24.6 ± 15.0 years: P = .008), had higher body mass index (24.7 ± 6.7 vs 18.6 ± 4.3; P = .002), and more frequently had history of hypertension (92% vs 8%; P = .011) and smoking (100% vs 0%; P = .008) when compared with those who did not have AKD. When compared with patients who did not develop AKD postoperatively, preoperative higher creatinine levels (1.1 ± 0.3 vs 0.8 ± 0.4; P = .025), intraoperative higher mean arterial pressures (99.2 ± 14.1 vs 89.0 ± 11.4 mm Hg; P = .011), a higher frequency of intraoperative acidosis (81% vs 19%; P = .041), higher lactate levels (5.1 ± 3.8 vs 2.8 ± 1.7 mmol/L; P = .038), and postoperative more frequent use of cyclosporine (91% vs 9%; P = .025) were seen in those who developed AKD. Logistic regression analysis revealed that age (odds ratio [OR], 1.057; 95% confidence interval [CI], 1.010-1.106; P = .018) and use of cyclosporine (OR, 0.099; 95% CI, 0.010-0.935; P = .043) were independent risk factors for AKD. CONCLUSIONS: Our results suggest that based on RIFLE criteria, AKD occur in more than half of HTs postoperatively. Older age and use of cyclosporine are associated with AKD following HT.

Comparison of different estimation techniques for biomass concentration in large scale yeast fermentation.

In this study, previously developed five different state estimation methods are examined and compared for estimation of biomass concentrations at a production scale fed-batch bioprocess. These methods are i. estimation based on kinetic model of overflow metabolism; ii. estimation based on metabolic black-box model; iii. estimation based on observer; iv. estimation based on artificial neural network; v. estimation based on differential evaluation. Biomass concentrations are estimated from available measurements and compared with experimental data obtained from large scale fermentations. The advantages and disadvantages of the presented techniques are discussed with regard to accuracy, reproducibility, number of primary measurements required and adaptation to different working conditions. Among the various techniques, the metabolic black-box method seems to have advantages although the number of measurements required is more than that for the other methods. However, the required extra measurements are based on commonly employed instruments in an industrial environment. This method is used for developing a model based control of fed-batch yeast fermentations.

DNA repair modulates the vulnerability of the developing brain to alkylating agents.

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag(-/-)) or O(6)-methylguanine methyltransferase (Mgmt(-/-)), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt(-/-) neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag(-/-) neurons were for the most part significantly less sensitive than wild type or Mgmt(-/-) neurons to MAM and HN2. Aag(-/-) neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt(-/-) mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag(-/-) or MGMT-overexpressing (Mgmt(Tg+)) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt(Tg+) mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.

Clinical and laboratory aspects of a trichinellosis outbreak in Izmir, Turkey.

Epidemiological, clinical and laboratory data were collected during an outbreak of trichinellosis, which occurred in Izmir, Turkey, between January and March 2004. The source of the infection was raw meatballs made with a mixture of uncooked beef and pork. Of 474 persons who were admitted at the Ataturk Training and Research Hospital during this period with a history of raw meatball consumption, the diagnosis of trichinellosis was confirmed for 154 (32.5%, 87 males and 67 females; mean age 31 years, range 6-67 years). Among persons with a confirmed diagnosis, 79% had myalgia, 77% weakness and malaise, 63% arthralgia, 40% jaw pain, 68% fever, 63% periorbital and/or facial oedema, 49% oedema at the trunk and limb, 42% abdominal pain, 40% nausea and vomiting, 28% diarrhoea, 23% subconjunctival haemorrhage, 25% macular or petechial rash, 4% subungual haemorrhage, 15% cardiac complaints and 0.2% neurological complaints. Nine patients (5.8%) were hospitalised due to severe myalgia (n = 2), high fever (n = 3), neurological manifestations (n = 1), thrombophlebitis (n = 2) and palmar erythema (n = 1). Eosinophilia was present in 88% of the confirmed cases at the admission. Elevated levels of serum creatine phosphokinase, lactic dehydrogenase and aspartate aminotransferase were detected in 72%, 70% and 16% of the confirmed cases, respectively. The seroconversion occurred in most of the infected people between the 4th and 6th weeks after the infection. All of the confirmed cases were treated with mebendazole. People with severe symptoms were treated also with prednisolone (60 mg/day for three days) and those with a moderately severe clinical pattern received a non-steroid anti-inflammatory drug (naproxen sodium, 550 mg/day). All confirmed cases recovered without any clinical sequela.

Role of nucleotide- and base-excision repair in genotoxin-induced neuronal cell death.

Base-excision (BER) and nucleotide-excision (NER) repair play pivotal roles in protecting the genomes of dividing cells from damage by endogenous and exogenous agents (i.e. environmental genotoxins). However, their role in protecting the genome of post-mitotic neuronal cells from genotoxin-induced damage is less clear. The present study examines the role of the BER enzyme 3-alkyladenine DNA glycosylase (AAG) and the NER protein xeroderma pigmentosum group A (XPA) in protecting cerebellar neurons and astrocytes from chloroacetaldehyde (CAA) or the alkylating agent 3-methyllexitropsin (Me-Lex), which produce ethenobases or 3-methyladenine (3-MeA), respectively. Neuronal and astrocyte cell cultures prepared from the cerebellum of wild type (C57BL/6) mice or Aag(-/-) or Xpa(-/-) mice were treated with 0.1-50 microM CAA for 24h to 7 days and examined for cell viability, DNA fragmentation (TUNEL labeling), nuclear changes, and glutathione levels. Aag(-/-) neurons were more sensitive to the acute (>20 microM) and long-term (>5 microM) effects of CAA than comparably treated wild type neurons and this sensitivity correlated with the extent of DNA fragmentation and nuclear changes. Aag(-/-) neurons were also sensitive to Me-Lex at comparable concentrations of CAA. In contrast, Xpa(-/-) neurons were more sensitive than either wild type or Aag(-/-) neurons to CAA (>10 microM), but less sensitive than Aag(-/-) neurons to Me-Lex. Astrocytes from the cerebellum of wild type, Aag(-/-) or Xpa(-/-) mice were essentially insensitive to CAA at the concentrations tested. These studies demonstrate that BER and NER are required to protect neurons from genotoxin-induced cell death.

Involuntary movements in infantile cobalamin deficiency appearing after treatment.

Involuntary movements may be a symptom in most infants who present with neurologic syndrome of infantile cobalamin (vitamin B12) deficiency. In this report, two infants with cobalamin deficiency are presented. These patients also developed a striking movement disorder that appeared a few days after treatment with intramuscular cobalamin. The movement disorder was characterized by severe involuntary movements, which were a combination of tremor and myoclonus particularly involving tongue, face, pharynx, and legs. The neurologic symptoms improved within a few days after the administration of clonazepam. In each patient the mother was also cobalamin deficient and the infant was solely breast-fed. The cause of involuntary movements that can appear rarely after treatment in infantile cobalamin deficiency is not known. Besides initial neurologic presenting symptoms of cobalamin deficiency, the occurrence of involuntary movements after treatment should also receive attention. This movement disorder may disappear spontaneously, or an additional treatment may be an alternative approach if the symptoms are severe.

Spontaneously immortalized cell lines obtained from adult Atm null mice retain sensitivity to ionizing radiation and exhibit a mutational pattern suggestive of oxidative stress.

The study of Ataxia-telangiectasia (A-T) has benefited significantly from mouse models with knockout mutations for the Atm (A-T mutation) locus. While these models have proven useful for in vivo studies, cell cultures from Atm null embryos have been reported to grow poorly and then senesce. In this study, we initiated primary cultures from adult ears and kidneys of Atm homozygous mice and found that these cultures immortalized readily without loss of sensitivity to ionizing radiation and other Atm related cell cycle defects. A mutational analysis for loss of expression of an autosomal locus showed that ionizing radiation had a mutagenic effect. Interestingly, some spontaneous mutants exhibited a mutational pattern that is characteristic of oxidative mutagenesis. This result is consistent with chronic oxidative stress in Atm null cells. In total, the results demonstrate that permanent cell lines can be established from the tissues of adult mice homozygous for Atm and that these cell lines will exhibit expected and novel consequences of this deficiency.

Recovery of ammonia as struvite from anaerobic digester effluents.

The effects of environmental conditions on ammonia removal as struvite (Magnesium ammonium phosphate, MAP) were studied in a laboratory scale batch reactor. MAP precipitation was carried out by adding phosphoric acid and magnesium source either as MgCl, or MgO. The effect of temperature, pH, MgN:P ratios were studied. Temperature did not significantly affect ammonia removal between 25-40 degrees C and over 90% removal was obtained. The effect of pH, however,was significant and highest removal was reached at pH 8.5-9.0. The various stoichiometric ratios of ammonium to Mg and P have been tested and slight excess of Mg and P found to be beneficial for higher recovery of ammonia as struvite. However further increase in Mg and P ratios did not result in further ammonia removal which is also costly for the practical application of the process. When MgO was used as M source,the ammonia recovery was 60-70% whereas the useMgCl2 has increased this figure up to 95%. In addition a two step purification process was developed to recover MAP crystals from impurities of the anaerobic digester. Firstly, precipitates were dissolved in acid and impurities were removed by centrifugation. The clarified supernatant was re-precipitated by adjusting its pH with caustic. It was shown that in the two steps process white MAP crystals could be obtained with over 85% recovery to be used for another applications. The economical analysis of the process has shown that ammonia in the digester effluents can be recovered at the cost of $7.5-8.0 kg(-1) NH4+-N. The rate of reaction is very fast and is completed almost in minutes. This simplifies the process design resulting in a smaller reaction vessel.

Somatic cell mutations: can they provide a link between aging and cancer?

Cancers increase during aging in mammals, and an accumulating body of evidence suggests that mutational events too do likewise. Mutational events are intimately involved in the malignant process. One current view is that mutator phenotypes are required in malignant cells for a sufficient number of critical target genes to be affected. These mutator phenotypes are believed to result from underlying deficiencies in genes necessary to maintain genomic stability. This review will provide a framework for a discussion of cancer and aging by detailing with a pair of wise approach studies that address the relations between aging, cancer, and mutations. Results from these studies will be used to suggest that a mutator phenotype develops in the cells of older individuals in the absence of an underlying genetic deficiency. Instead, it is proposed that a mixture of chromosomal aberrations, DNA damage, and chronic exposure to genotoxic forces, including oxidative stress, provide the basis for this age-accelerated mutator phenotype.

Oral cyclic megadose methylprednisolone therapy for chronic immune thrombocytopenic purpura in childhood.

The objectives of this study were to investigate the effectiveness of oral megadose methylprednisolone (OMMP) therapy in children with chronic immune thrombocytopenic purpura (ITP). Twenty-two patients were given oral methylprednisolone daily for 7 d (30 mg/kg for 3 d and then 20 mg/kg for 4 d). OMMP therapy was repeated once per month if the platelet count was less than 20,000/mm3 at the 30th day of therapy, for up to six courses. The number of platelets of all patients increased gradually during the OMMP therapy, with a peak number at the 7th day, then decreased until the 14th day, and remained relatively stable until 12 months. During the study no patient had a platelet count less than 20,000/mm3 at the 3rd day and 50,000/mm3 at the 7th day. Although the number of platelets was gradually decreased between the 7th and 14th days, it remained above 100,000/mm3 for at least 12 months in the nine patients, and above 20,000/mm3 in the four patients. None of these 13 patients required hospitalization or therapy during the follow-up period. All of the patients tolerated the medication well. None of them reported side-effects that were severe enough to discontinue therapy. We conclude that OMMP therapy is a safe, easy and effective therapy in children with refractory chronic ITP, and it may provide long-term remission in about two thirds of the patients.

Biallelic methylation and silencing of mouse Aprt in normal kidney cells.

Heritable gene silencing is an important mechanism of tumor suppressor gene inactivation in a variety of human cancers. In the present study, we show that methylation-associated silencing of the autosomal adenine phosphoribosyltransferase (Aprt) locus occurs in primary mouse kidney cells. Aprt-deficient cells were isolated from mice that were heterozygous for Aprt, i.e., they contained one wild-type Aprt allele and one targeted allele bearing an insertion of the bacterial neo gene. Although silencing of the wild-type allele alone was sufficient for the cells to become completely Aprt-deficient, biallelic methylation of the promoter region was found to occur. Moreover, despite the absence of selective pressure against the targeted allele, phenotypic silencing of the inserted neo gene accompanied silencing of the wild-type Aprt allele. A potential role for allelic homology in these events is discussed.

The effect of propolis extract in experimental chemical corneal injury.

PURPOSE: To investigate the effect of propolis, a natural resin produced by honeybees, in the treatment of alkali-injured rabbit eyes. METHOD: A corneal alkali burn was induced by applying filter paper immersed in 1 N NaOH on the central axis of the right corneas of 24 rabbits for 30 s. The animals were divided into three treatment groups: group 1 (control) was given 0.3% tobramycin and phosphate-buffered solution; group 2 was treated with antibiotic coverage and topical 1% ethanolic extract of propolis; group 3 received antibiotic coverage and topical 1% dexamethasone. The dexamethasone-treated group was maintained as the positive control. Drugs were instilled 4 times a day for 7 days. The corneal inflammation was evaluated by calculating the average inflammatory index obtained from clinical observation of the ciliary hyperemia, central and peripheral corneal edema at 24 h, 48 h, on day 5 and day 7, before sacrificing the animals. Then, the corneas underwent routine histological examination. RESULTS: The effects of dexamethasone and propolis on healing of injured corneas were similar (p>0.05) and significantly better than controls at 24 h (p<0.01, p< 0.05, respectively), and on day 7 (p<0.05) with respect to the inflammatory index. On histological observation, inflammatory cell infiltration was lower as compared to control in both the dexamethasone and propolis groups (p<0.001) and similar with each other (p>0.05). CONCLUSIONS: Our study shows that propolis has an anti-inflammatory effect comparable to dexamethasone in chemical corneal injury.

Tandem B1 elements located in a mouse methylation center provide a target for de novo DNA methylation.

A cis-acting methylation center that signals de novo DNA methylation is located upstream of the mouse Aprt gene. In the current study, two approaches were taken to determine if tandem B1 repetitive elements found at the 3' end of the methylation center contribute to the methylation signal. First, bisulfite genomic sequencing demonstrated that CpG sites within the B1 elements were methylated at relative levels of 43% in embryonal stem cells deficient for the maintenance DNA methyltransferase when compared with wild type embryonal stem cells. Second, the ability of the B1 elements to signal de novo methylation upon stable transfection into mouse embryonal carcinoma cells was examined. This approach demonstrated that the B1 elements were methylated de novo to a high level in the embryonal carcinoma cells and that the B1 elements acted synergistically. The results from these experiments provide strong evidence that the tandem B1 repetitive elements provide a significant fraction of the methylation center signal. By extension, they also support the hypothesis that one role for DNA methylation in mammals is to protect the genome from expression and transposition of parasitic elements.

Fully expanded FMR1 CGG repeats exhibit a length- and differentiation-dependent instability in cell hybrids that is independent of DNA methylation.

The fragile X syndrome is characterized at the molecular level by expansion and methylation of a CGG trinucleotide repeat located within the FMR1 locus. The tissues of most full mutation carriers are mosaic for repeat size, but these mutational patterns tend to be well conserved when comparing multiple tissues within an individual. Moreover, full mutation alleles are stable in cultured fibroblasts. These observations have been used to suggest that fragile X CGG repeat instability normally is limited to a period during early embryogenesis. DNA methylation of the repeat region is also believed to occur during early development, and some experimental evidence indicates that this modification may stabilize the repeats. To study the behavior of full mutation alleles in mitotic cells, we generated human-mouse somatic cell hybrids that carry both methylated and unmethylated full mutation FMR1 alleles. We observed considerable repeat instability and analyzed repeat dynamics in the hybrids as a function of DNA methylation, repeat length and cellular differentiation. Our results indicate that although DNA methylation does correlate with stability in primary human fibroblasts, it does not do so in the cell hybrids. Instead, repeat stability in the hybrids is dependent on repeat length, except in an undifferentiated cellular background where large alleles are maintained with a high degree of stability. This stability is lost when the cells undergo differentiation. These results indicate that the determinants of CGG repeat stability are more complex than generally believed, and suggest an unexpected role for cellular differentiation in this process.

The establishment and maintenance of DNA methylation patterns in mouse somatic cells.

Somatic cell DNA methylation patterns in mammals are established during embryonic development and are then maintained somewhat faithfully for the remainder of the individual's lifetime. Pattern formation can be divided into a series of linked steps that include demethylation, de novo methylation, methylation spreading, methylation blocking, and maintenance methylation. In this review, these steps will be combined to present a model for the formation and maintenance of a methylation pattern in the 5' region of the mouse Aprt gene. This model suggests that an apparently 'stable' methylation pattern results from a dynamic equilibrium between forces that promote and inhibit methylation spreading.

Hypomethylation of an expanded FMR1 allele is not associated with a global DNA methylation defect.

The vast majority of fragile-X full mutations are heavily methylated throughout the expanded CGG repeat and the surrounding CpG island. Hypermethylation initiates and/or stabilizes transcriptional inactivation of the FMR1 gene, which causes the fragile X-syndrome phenotype characterized, primarily, by mental retardation. The relation between repeat expansion and hypermethylation is not well understood nor is it absolute, as demonstrated by the identification of nonretarded males who carry hypomethylated full mutations. To better characterize the methylation pattern in a patient who carries a hypomethylated full mutation of approximately 60-700 repeats, we have evaluated methylation with the McrBC endonuclease, which allows analysis of numerous sites in the FMR1 CpG island, including those located within the CGG repeat. We report that the expanded-repeat region is completely free of methylation in this full-mutation male. Significantly, this lack of methylation appears to be specific to the expanded FMR1 CGG-repeat region, because various linked and unlinked repetitive-element loci are methylated normally. This finding demonstrates that the lack of methylation in the expanded CGG-repeat region is not associated with a global defect in methylation of highly repeated DNA sequences. We also report that de novo methylation of the expanded CGG-repeat region does not occur when it is moved via microcell-mediated chromosome transfer into a de novo methylation-competent mouse embryonal carcinoma cell line.