Integrity of the methylation cycle is essential for mammalian neural tube closure

BACKGROUND: Closure of the cranial neural tube during embryogenesis is a crucial process in development of the brain. Failure of this event results in the severe neural tube defect (NTD) exencephaly, the developmental forerunner of anencephaly. METHODS: The requirement for methylation cycle function in cranial neural tube closure was tested by treatment of cultured mouse embryos with cycloleucine or ethionine, inhibitors of methionine adenosyl transferase. Embryonic phenotypes were investigated by histological analysis, and immunostaining was performed for markers of proliferation and apoptosis. Methylation cycle intermediates s-adenosylmethionine and s-adenosylhomocysteine were also quantitated by tandem mass spectrometry. RESULTS: Ethionine and cycloleucine treatments significantly reduced the ratio of abundance of s-adenosylmethionine to s-adenosylhomocysteine and are, therefore, predicted to suppress the methylation cycle. Exposure to these inhibitors during the period of cranial neurulation caused a high incidence of exencephaly, in the absence of generalized toxicity, growth retardation, or developmental delay. Reduced neuroepithelial thickness and reduced density of cranial mesenchyme were detected in ethionine-treated but not cycloleucine-treated embryos that developed exencephaly. Reduced mesenchymal density is a potential cause of ethionine-induced exencephaly, although we could not detect a causative alteration in proliferation or apoptosis prior to failure of neural tube closure. CONCLUSIONS: Adequate functioning of the methylation cycle is essential for cranial neural tube closure in the mouse, suggesting that suppression of the methylation cycle could also increase the risk of human NTDs. We hypothesize that inhibition of the methylation cycle causes NTDs due to disruption of crucial reactions involving methylation of DNA, proteins or other biomolecules.     

Exploratory evaluation of several teratogen warning symbols

BACKGROUND: Previous research has noted potential inadequacies in the warning labels and symbols used with some teratogenic medications. A clear teratogen warning symbol represents an important component of risk mitigation for accidental teratogen exposure. METHODS: Several teratogen warning symbols were developed through rapid prototyping and focus groups. A nationally distributed field trial (n = 300) examined the relative effectiveness of 6 candidate symbols, including the symbol in use at the time of the study. Measures included open-ended interpretation, closed-ended preference, and demographic surveys. Each participant was shown a single symbol and asked what it meant, to whom it applied, and what that person should do. Text statements were added to the symbol and participants were asked to reinterpret the warning. Participants were told the intended message of the warning, shown all 6 symbols, and asked to choose the most effective symbol. RESULTS: Four of 6 symbols achieved levels of correct interpretation close to or exceeding the American National Standards Institute (ANSI) benchmark of 85% and none exceeded the ANSI limit of 5% critical confusion. Symbols elicited varying conceptual responses. Respondents considered 1 symbol to be the most effective, by a 4 to 1 margin. Several outcomes varied by age and by ethnicity. CONCLUSIONS: Several symbols emerged as viable alternatives to the current symbol; however, no 1 symbol was clearly found to be the most effective. Instead, the symbol considered "best" depends on the messages that are considered most essential to the warning. Additionally, it appears a symbol without the addition of text can convey most, but possibly not all, of the meaning required of the warning label. Next steps should include further symbol refinement, closer examination of text additions to symbols, and validation of the candidate symbols and warnings through a large-scale field trial.     

Prevalence of periconceptional folic acid use and perceived barriers to the postgestation continuance of supplemental folic acid: survey results from a Teratogen Information Service

BACKGROUND: Fewer than 40% of U.S. women are taking folic acid supplements periconceptionally at a time when the risk of neural tube defects (NTDs) can be reduced by supplementation. A better understanding of the vitamin-taking habits of childbearing-age women and effective methods for improving periconceptional supplement use are needed. METHODS: A telephone survey conducted through the California Teratogen Information Service (TIS) between August 2003 and January 2004 assessed the prevalence and characteristics of pregnant callers who did not use folic acid supplements in the periconceptional period, and explored attitudes toward advice to continue vitamin use following pregnancy in order to be protected in a future pregnancy. RESULTS: A total of 327 pregnant women who called the TIS for information agreed to participate in the survey. More than half (53.2%) were not taking folic acid-containing supplements in the periconceptional period. Predictors of lack of use included a higher prepregnancy body mass index, younger maternal age, non-white race/ethnicity, lower education level, and unplanned pregnancy. One-quarter of the women said they would be willing to continue taking vitamins after the pregnancy if advised to do so by a physician. The remainder identified obstacles to following that advice--notably, not planning to become pregnant again and the belief that enough folate is derived from diet alone. CONCLUSIONS: More than half of the callers to the TIS were not compliant with recommendations regarding periconceptional folic acid supplementation. This represents an opportunity for TIS specialists and physicians to intervene in a current pregnancy to encourage maintenance of supplement use in the subsequent interpregnancy interval.     

Failure of homocysteine to induce neural tube defects in a mouse model

BACKGROUND: Folate deficiencies have been associated with many adverse congenital abnormalities. It is not clear, however, whether these defects are due to a folate deficiency or to an increase in homocysteine. Homocysteine has been shown to be teratogenic in the chicken-embryo model and it has been suggested that homocysteine-induced defects are mediated by inhibiting the N-methyl-D-aspartate (NMDA) receptor on neural crest cells. The majority of the teratology studies have been carried out using the chicken embryo model. In an effort to develop a murine model of homocysteine-induced neural tube defects, several inbred mouse strains were treated with homocysteine or the NMDA inhibitor MK801 and the fetuses examined for any induced-NTD. METHODS: Several in-bred mouse strains were administered homocysteine once on gestational day (GD) E8.5 or once daily on GD 6.5-10.5. Additionally, because homocysteine was been reported to mediate its effects through the NMDA receptor, the effect of MK801, an antagonist of this receptor, was also investigated. RESULTS: Regardless of the mouse treatment time, homocysteine failed to induce neural tube defects in our in-bred mouse strains. Homocysteine also failed to increase the number of neural tube defects in the splotch strain, regardless of the genotype. CONCLUSIONS: Irrespective of the mouse strain or treatment, homocysteine failed to induce neural tube defects in our mouse models, which is in contrast to what has been reported in the chicken embryo models.     

Exposure of pregnant mice to chromium picolinate results in skeletal defects in their offspring

BACKGROUND: Chromium(III) picolinate, [Cr(pic)(3)], is a widely marketed dietary supplement. However, Cr(pic)(3) has been associated with oxidative damage to DNA in rats and mutations and DNA fragmentation in cell cultures. In isolated case reports, Cr(pic)(3) supplementation has been said to cause adverse effects, such as anemia, renal failure, liver dysfunction, and neuronal impairment. To date, no studies have been published regarding the safety of chromium picolinate supplementation to a developing fetus, although Cr(pic)(3) has been recommended for pregnant women who are diagnosed with gestational diabetes. METHODS: From gestation days (GD) 6-17, pregnant CD-1 mice were fed diets containing either 200 mg/kg Cr(pic)(3), 200 mg/kg CrCl(3), 174 mg/kg picolinic acid, or the diet only to determine if Cr(pic)(3), CrCl(3), or picolinic acid could cause developmental toxicity. Dams were sacrificed on GD 17, and their litters were examined for adverse effects. RESULTS: The incidence of bifurcated cervical arches was significantly increased in fetuses from the Cr(pic)(3) group as compared to the diet-only group. Fetuses in the picolinic acid-treated group had an incidence double that of the control group; however, this increase was not statistically significant. Fetuses in the CrCl(3) group did not differ from the controls in any variable examined. No maternal toxicity was observed in any of the treatment groups. CONCLUSIONS: High maternal oral exposures to chromium picolinate can cause morphological defects in developing offspring of mice.     

Left ventricular myocardal activation under ventricular paced beats in chickens Gallus gallus domesticus

The aim of the study was to advance our knowledge regarding the activation process of the ventricular myocardium in birds in which Purkinje fibres penetrate into the ventricular wall to reach the epicardium. A depolarization pattern of the left ventricular free wall was studied in chickens (Gallus gallus) during ventricular paced beats. Duration of the activation process of the left ventricular free wall is significantly increased during ventricular ectopic excitation as compared with sinus rhythm. Its lowest increase occurs during subendocardial pacing of the middle part of the left ventricle, but its greatest increase is observed during subepicardial pacing of the left ventricular base. Multifocality and mosaicity of depolarization of the left ventricular free wall myocardium in chicken are expressed in a considerably less degree during ventricular paced beats in comparison with sinus rhythm. During ventricular paced beats, excitation of the left ventricular free wall is mostly due to the successive spreading of the depolarization wave from pacing sites.     

Segmental bone defects: from cellular and molecular pathways to the development of novel biological treatments

Several conditions in clinical orthopaedic practice can lead to the development of a diaphyseal segmental bone defect, which cannot heal without intervention. Segmental bone defects have been traditionally treated with bone grafting and/or distraction osteogenesis, methods that have many advantages, but also major drawbacks, such as limited availability, risk of disease transmission and prolonged treatment. In order to overcome such limitations, biological treatments have been developed based on specific pathways of bone physiology and healing. Bone tissue engineering is a dynamic field of research, combining osteogenic cells, osteoinductive factors, such as bone morphogenetic proteins, and scaffolds with osteoconductive and osteoinductive attributes, to produce constructs that could be used as bone graft substitutes for the treatment of segmental bone defects. Scaffolds are usually made of ceramic or polymeric biomaterials, or combinations of both in composite materials. The purpose of the present review is to discuss in detail the molecular and cellular basis for the development of bone tissue engineering constructs.     

Differential effects of stretch and compression on membrane currents and [Na+]c in ventricular myocytes

Mechano-electrical feedback was studied in the single ventricular myocytes. A small fraction (approximately 10%) of the cell surface could be stretched or compressed by a glass stylus. Stretch depolarised, shortened the action potential and induced extra systoles. Stretch activated non-selective cation currents (I_ns) showed a linear voltage dependence, a reversal potential of 0 mV, a pure cation selectivity, and were blocked by 8 μM Gd³⁺ or 30 μM streptomycin. Stretch reduced Ca²⁺ and K⁺ (I_K) currents. Local compression of broadwise attached cells activated I_K but not I_ns. Cytochalasin D or colchicin, thought to disrupt the cytoskeleton, suppressed the mechanosensitivity of I_ns and I_K. During stretch, the cytosolic sodium concentration increased with spatial heterogeneities, local hotspots with [Na⁺]_c>24 mM appeared close to surface membrane and t-tubules (pseudoratiometric imaging using Sodium Green fluorescence). Electronprobe microanalysis confirmed this result and indicated that stretch increased total sodium [Na] in cell compartments such as mitochondria, nuclear envelope and nucleus. Our results obtained by local stretch differ from those obtained by end-to-end stretch (literature). We speculate that channels may be activated not only by axial but also by shear stress, and, that stretch can activate channels outside the deformed sarcomeres via second messenger.     

Negative inotropic response to cerium in ventricular papillary muscle is mediated by reactive oxygen species

This study was performed with the objective of assessing the mechanical response of the myocardium to different levels of cerium and delineation of the mechanism underlying the mediation of the functional changes. Rat ventricular papillary muscle was used as the experimental model. Isolated papillary muscles were exposed to different concentrations of CeCl₃ and the force of contraction was measured using a force transducer. Experiments have revealed that the negative inotropic response to CeCl₃ was proportional to its concentration. The inotropic changes were found to be completely reversible at concentrations ≤5μM, and partially reversible at higher concentrations. Neutralization of cerium-induced inotropic changes by the superoxide anion scavenger superoxide dismutase (SOD) at concentrations ≤5 μM indicates that the mechanical changes are mediated by reactive oxygen species. At higher concentrations of Ce³⁺, SOD partially reversed the contractile changes. The beneficial effect of SOD was seen only if the muscles were pretreated with the scavenger prior to the addition of cerium chloride.