Prevention of neural tube defects by folic acid - awareness among women of childbearing age in Slovakia.

BACKGROUND: Folic acid deficiency plays a central role in the aetiology of many congenital anomalies including neural tube defects. Protective effect of folic acid on embryo may be acquired only if taken periconceptionally. OBJECTIVES: The aim of the study was to investigate the awareness about folic acid among women of childbearing age in Bratislava, Slovakia. METHODS: There were 130 respondents involved in the research (106 pregnant women, 24 female students of medical faculty). Using questionnaire we acquired following data: pregnancy details, interest in diet before and during pregnancy, recommendations regarding nutrition and supplementation pre- and post-conception, knowledge about folic and other acid in 2004 and 2009. RESULTS: More than half of the respondents knew the sources of folic acid. The interest in the nutrition facts of the food dropped from 91 % to 58.5 %. The number of pregnant women advised about correct nutrition and folic acid supplementation before and during pregnancy increased from 16 % to 37 %. Planning the next gravidity with folic acid supplementation became greater than 21 % (38 % in 2009). Nevertheless, only 46 % of these women believed that proper food content with folic acid may prevent congenital anomalies. In a group of students planning to take folic acid periconceptionally the number raised up to 62.5 %. CONCLUSION: The results revealed low knowledge about the effect of folic acid on developing embryo among women of childbearing age. Effective intervention programs are needed with the aim to improve periconceptional intake of folic acid in 2004 and 2009. The results in both periods show low knowledge about this essential vitamin (Tab. 1, Fig. 8, Ref. 31).

New insights into the role of NF1 in cancer.

NF1 proteins are a family of transcription factors that act either as repressors or as activators. Functional studies indicate that NF1 participate in signaling pathways that regulate cell viability, proliferation and differentiation. Participation in regulation of genes important for tumor progression and metastasizing suggests a potential value of NF1 as a prognostic factor for certain types of cancer.

Novel VANGL1 Gene Mutations in 144 Slovakian, Romanian and German Patients with Neural Tube Defects.

Neural tube defects (NTDs) are a group of congenital malformations of the central nervous system occurring at an average rate of 1 per 1,000 human pregnancies worldwide. Numerous genetic and environmental factors are discussed to be relevant in their etiology. In mice, mutants in >200 genes including the planar cell polarity (PCP) pathway are known to cause NTDs, and recently, heterozygous mutations in the human VANGL1 gene have been described in a small subset of patients with NTDs. We performed a VANGL1 mutation analysis in 144 unrelated individuals with NTDs from Slovakia, Romania and Germany and identified 3 heterozygous missense mutations: c.613G>A (p.Gly205Arg) with an open spina bifida (lumbosacral meningomyelocele), c.557G>A (p.Arg186His) with a closed spina bifida (tethered cord and spinal lipoma) and c.518G>A (p.Arg173His) with an unknown NTD. The c.613G>A mutation was also found in a healthy sibling. None of the mutations were described previously. Findings support that heterozygous VANGL1 mutations represent hypomorphs or conditional mutants predisposing to NTDs and occur at a frequency of approximately 2.1% of open and closed spinal NTDs. The mutations (p.Arg173His, p.Arg186His, p.Gly205Arg) modified conserved regions of the VANGL1 protein and shared similarities with previously described mutants, providing further evidence for the presence of mutational hot spots in these patients.

[Epidemiology of neural tube defects]. / Epidemiológia defektov neurálnej rúry.

INTRODUCTION: Neural tube defects represent group of congenital diseases with relatively high incidence in population. Authors assess world and Slovak literature and statistical facts about the epidemiology of NTD and compare them with their own results of retrospective study performed in Children's Hospital, Bratislava. MATERIALS AND METHODS: List of patients consists of 250 children (106 boys, 144 girls): X-ray images showing lumbo-sacral part of vertebral column were evaluated retrospectively (X-ray of native abdomen, urological tract or skeleton). Authors assessed presence or non-presence of spina bifida on images, without relation to age, gender or diagnosis of patients. RESULTS: From the total number of 250 radiograms, 72 findings were positive (36 boys and 36 girls), 160 images were negative, 18 were unsuitable for evaluation due to low image quality. The highest diagnostic capture was from urological images - 40% of all positive findings. Incidence of spina bifida in Children's Hospital concluded from X-ray images is quite high - 28.8%. CONCLUSION: According to the data from National Centre of Health Statistics the incidence of open caudal neural tube defects in Slovakia is under 5 per 10 000 live-born children at present time. However the occurrence of occult spina bifida is not known exactly. The high rate of spina bifida presented herein (28%) can be caused by relatively low number of evaluated radiograms. Also the fact that children were not only healthy ones and data were obtained from the western part of Slovakia as well. In conclusion we can say that after accidental finding of caudal neural tube defect consecutive diagnostics should be performed in clinical positive cases - genetics, MRI and consultation with specialists to decide for the optimal follow up of the patient.

[Clinical condition of patients with neural tube defects]. / Klinický stav pacientov s defektami neurálnej rúry.

INTRODUCTION: Neural tube defects are the most common congenital anomalies of central nervous system. Their onset is at the embryonic age of 21 to 28 days. Periconceptional administration of folic acid may reduce the occurrence of neural tube defects up to 70%. Clinical features depend on localization of the defect and malformation of central nervous system, as well as on intensive multidisciplinary care in early stages of development. Open defects (meningomyelocele) present with more severe neurological deficiency early after birth. Closed defect manifestation occurs usually later in life with tethered cord syndrome. PURPOSE: This study evaluates clinical status of the patients with neural tube defects, who are followed in the Children's Faculty Hospital in Bratislava. METHODS: Cross-sectional and also retrospective study was conducted by questionnaire that was given to mothers of children with neural tube defect. Data about perinatal and family history, occurrence of hydrocephalus, scoliosis, joint deformities, dysfunction of urinary system and bowel, as well as social care, were collected. Clinical features were statistically evaluated depending on beginning of the defect or locomotion status. RESULTS: 94 patients with mean age of 12.7 years +/- 6.59 SD were included in the study. Patients with open defects had more severe neurological deficiency with hydrocephalus, more common epilepsy, skeleton deformities, wheelchair-dependency, and dysfunction of urinary tract and bowel. Scoliosis and ability of locomotion significantly correlated with higher lever of defect, while hydrocephalus, continence, urinary infections, clean intermittent catheterization, constipation, scoliosis and joint deformities significantly correlated with the ability of locomotion (p < 0.05). CONCLUSION: For patients with neural tube defects, the level of neurological deficiency is the most important prognostic factor for many other clinical characteristics.

Mutation in the beta subunit of F ATPase allows Kluyveromyces lactis to survive the disruption of the KlPGS1 gene.

The petite-negative yeast Kluyveromyces lactis does not tolerate the loss of phosphatidylglycerol (PG). We demonstrate that the lethality of PG loss is suppressed in strains carrying a mutation in the beta subunit of F(1) ATPase (mgi1-1). Phenotypic characterization shows that the strain lacking the phosphatidylglycerolphosphate synthase gene (KlPGS1) is able to grow only on glucose, but significantly more slowly and to substantially lower densities than the parental mgi1-1 strain. In addition, oxygen consumption in the DeltaKlpgs1 strain is <1% of the parental strain. Western blot analysis of mitochondrial membrane proteins shows that the amounts of some proteins are substantially decreased or even not detectable in this mutant. However, overexpression of the KlPGS1 gene under the inducible GAL1 promoter does not restore the ability of DeltaKlpgs1 cells to grow on galactose, indicating the presence of some other mutations and/or deletions in genes involved in oxidative phosphorylation. We also demonstrate that DeltaKlpgs1 cells do not spontaneously lose mtDNA, but are able to survive its loss after ethidium bromide mutagenesis. Deletion of the cardiolipin synthase gene (KlCLS1) in mgi1-1 has only a minimal effect on mitochondrial physiology, and additional experiments show that this deletion is also viable in wild-type K. lactis.

Induction of micronuclei in rat bone marrow after chronic exposure to lead acetate trihydrate.

Lead increasingly contributes to pollution of the environment and may play a role in the development of adverse effects in the human and animal body. Data concerning its mutagenic, clastogenic, and carcinogenic properties have been conflicting. In this study, we evaluated the frequency of micronuclei in bone marrow erythrocytes of rats treated with lead acetate trihydrate. Outbred Wistar rats were exposed to a daily dose of 100 mg/L drinking water for 125 days. The mean value of the total number of micronuclei observed in polychromatic erythrocytes of female rats was significantly higher than that found in the control group (13.375 +/- 2.722 against 9.625 +/- 3.204 micronuclei/1000 cells; P = 0.024 in ANOVA). In exposed female animals, no significant reduction of the ratio of polychromatic to normochromatic erythrocytes was observed (0.990 +/- 0.228 against 1.208 +/- 0.195; P = 0.060 in ANOVA). The effects of lead acetate trihydrate in male rats are both cytotoxic and genotoxic because of a decrease in ratio of polychromatic to normochromatic erythrocytes (0.715 +/- 0.431 against 1.343 +/- 0.306; P = 0.023, ANOVA followed by Tukey test) and an increase in frequency of micronucleated polychromatic erythrocytes (24.167 +/- 7.859 against 4.0 +/- 4.528 micronuclei/1000 cells; P < or = 0.001, ANOVA followed by Tukey test), respectively.

Anti-apoptotic proteins-targets for chemosensitization of tumor cells and cancer treatment.

Apoptosis or programmed cell death is an essential process not only for the normal development and function of multi-cellular organisms but it is also an important phenomenon in tumor cells killing. Numerous studies have indicated that non surgical cancer therapies eliminate tumor cells by activating apoptosis. The central regulators of apoptosis are proteins of the Bcl-2 family. In a wide variety of human tumors, the increased expression of anti-apoptotic proteins (Bcl-2 and Bcl-(XL)) was found. Moreover, it was revealed that high levels of these proteins block the action of many chemotherapeutic drugs. Due to this fact the inhibition of anti-apoptotic function of Bcl-2 proteins could be a strategy for either to restore the normal apoptotic process in tumor cells or to make them susceptible for chemo- and radio-therapeutic treatment. Three alternative therapeutic strategies for the repression of cytoprotective activity of anti-apoptotic proteins in tumor cells are reviewed in this article.

Lactate utilization in mitochondria prevents Bax cytotoxicity in yeast Kluyveromyces lactis.

In a search for the physiological conditions able to suppress the disruption of electron transport through the inner mitochondrial membrane induced by Bax, we found that respiratory substrate - lactate completely abolished Bax toxicity in yeast Kluyveromyces lactis. The effect of lactate was dependent on the presence of cytochrome c, as no effect was observed in the cytochrome c null strain. The investigation of lactate effect on markers of Bax toxicity showed that: (i) oxidation of lactate did not affect the decrease in oxygen consumption, but (ii) lactate was able to diminish the generation of reactive oxygen species and simultaneously to suppress Bax-induced cell death. We show that suppression of Bax lethality in K. lactis can be, in addition to anti-apoptotic proteins, achieved also by the utilization of lactate in the mitochondria.

The delivery of ADP/ATP carrier protein to mitochondria probed by fusions with green fluorescent protein and beta-galactosidase.

The import of proteins into mitochondria is an essential process, largely investigated in vitro with isolated mitochondria and radioactively labeled precursors. In this study, we used intact cells and fusions with genes encoding two reporter proteins, green fluorescent protein (GFP) and beta-galactosidase (lacZ), to probe the import of the ADP/ATP carrier (AAC). Typical mitochondrial fluorescence was observed with AAC-GFP fusions containing at least one complete transmembrane loop. This confirms the results of in vitro analysis demonstrating that an internal targeting signal was present in each one of the three transmembrane loops of the carrier. The fusions of AAC fragments to beta-galactosidase demonstrated that the targeting signal was capable of delivering the reporter molecule to the mitochondrial surface, but not to internalize it to a protease-inaccessible location. The delivery to a protease-inaccessible location required the presence of more distal sequences present within the third (C-terminal) transmembrane loop of the carrier molecule. The results of our study provide an alternative for investigation in a natural context of mitochondrial protein import in cells when the isolation of intact, functional mitochondria is not achievable.

The antiapoptotic protein Bcl-x(L) prevents the cytotoxic effect of Bax, but not Bax-induced formation of reactive oxygen species, in Kluyveromyces lactis.

The murine proapoptotic protein Bax was expressed in Kluyveromyces lactis to investigate its effect on cell survival and production of reactive oxygen species (ROS). Bax expression decreased the number of cells capable of growing and forming colonies, and it increased the number of cells producing ROS, as detected by both dihydrorhodamine 123 fluorescence and the intracellular content of SH groups. Mutation in the beta-subunit of F(1)-ATPase, or mitochondrial deficiency resulting from deletion of mtDNA (rho(0) mutant), increased the sensitivity to Bax, indicating that Bax cytotoxicity does not require mitochondrial respiratory-chain functions. The antiapoptotic protein Bcl-x(L), when co-expressed with Bax, localized to the mitochondria and prevented Bax cytotoxicity. However, this co-expression did not prevent the production of ROS. These data suggest that in K. lactis cells expressing Bax, ROS are not the sine qua non of cell death and that the antiapoptotic function of Bcl-x(L) is not limited to its antioxidant property.

A carbon-source-responsive element is required for regulation of the hypoxic ADP/ATP carrier (AAC3) isoform in Saccharomyces cerevisiae.

The mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is encoded by three genes that are differentially expressed under different physiological conditions. We investigated the transcriptional control of AAC3, an oxygen-repressed isoform. By deletion analysis, DNA electrophoretic mobility-shift assays, DNase I footprinting and site-directed mutagenesis, we have identified a promoter region (upstream repressing sequence 1, URS(1)) involved in a carbon-source-dependent repression of AAC3. It is different from the previously characterized oxygen-dependent ROX1 (regulation by oxygen 1) repressor-binding region (URS(2)). The complex character of URS(1) includes the presence of two different cis-acting sequences: (i) a RAP1 (repressor activator protein 1)-binding site that is capable of binding the RAP1 protein in vitro and (ii) two putative ethanol-repression sequences, the modification of which derepresses the AAC3 gene. These findings demonstrate that the hypoxic AAC3 gene is regulated by two upstream repressor sites; one controlled by oxygen and haem, the other by the carbon source. Both sites function to completely switch off the expression of the AAC3 isoform when ATP is made by oxidative phosphorylation, and they modulate AAC3 expression when import of glycolytic ATP into mitochondria is required.

The cytotoxic action of Bax on yeast cells does not require mitochondrial ADP/ATP carrier but may be related to its import to the mitochondria.

The effect of the expression of murine Bax protein on growth and vitality was examined in Saccharomyces cerevisiae and compared with the effect of Bax in mutant cells lacking functional mitochondria. The cytotoxic effect of Bax on yeast does not require functional oxidative phosphorylation, respiration, or mitochondrial proteins (ADP/ATP carriers) implicated in the formation of the permeability transition pore in mammalian mitochondria. In the wild type S. cerevisiae the expression of Bax does not result in a severe effect on mitochondrial membrane potential and respiration. On the basis of Bax induced differences in the fluorescence of green fluorescent protein fused to mitochondrial proteins, it is proposed that Bax may interfere with one essential cellular process in yeast: the mitochondrial protein import pathway that is specific for the proteins of the mitochondrial carrier family.

Fatty acids induced uncoupling of Saccharomyces cerevisiae mitochondria requires an intact ADP/ATP carrier.

Fatty acids stimulate the oxidation rate of mitochondria isolated from the wild-type Saccharomyces cerevisiae, but do not affect significantly the respiration of mitochondria isolated from mutants, in which the ADP/ATP carrier (AAC) was either modified (R96H) or deleted (delta aac2). Similarly as in mammalian mitochondria, the transmembrane electrical potential difference (delta psi) in the wild-type yeast mitochondria was dissipated by low concentrations of free fatty acids, and this was partially inhibited by bongkrecate. In contrast to the wild-type mitochondria, the addition of increasing concentrations of fatty acids to the op1 (R96H) mutant mitochondria abolished only a small portion of delta psi, as compared to the change induced by classical uncouplers. The different effects of fatty acids on both, the respiration and the delta psi of mitochondria isolated from the wild-type and the aac mutants, respectively, demonstrates that the intact AAC is essential for the fatty acids induced H+ permeability of mitochondrial membrane.

Transcription of the AAC1 gene encoding an isoform of mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is regulated by oxygen in a heme-independent manner.

The mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is encoded by three independent genes. AAC1, AAC2, and AAC3. In this work, we analysed the 5' upstream region of AAC1 by sequencing and by mapping the transcription initiation site of the gene. By monitoring the level of AAC1 mRNA and the beta-galactosidase activity of AAC1-lacZ fusion constructs, we showed that expression of AAC1 is subjected to regulation by oxygen. In contrast to the other two AAC genes, the effect of oxygen on AAC1 is not mediated by heme and heme-dependent transcription factors. The AAC1 expression was reduced eightfold in anaerobically grown cells compared to expression in cells grown aerobically, but it was not affected by the nature of carbon source used for growth. The data presented show that AAC1 expression, while constitutive under all aerobic conditions tested, is repressed during anaerobiosis in a heme-independent manner.

Expression of the AAC2 gene encoding the major mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is controlled at the transcriptional level by oxygen, heme and HAP2 factor.

Expression of the Saccharomyces cerevisiae AAC2 gene encoding the major mitochondrial ADP/ATP carrier was examined. The intracellular level of the carrier protein, as well as the level of the AAC2-gene-specific mRNA, is influenced by the presence or absence of oxygen or of heme, and it is subject to carbon-source control. In addition, the expression of AAC2 gene requires the products of the HAP2 and HAP3 genes, but not that of the HAP1 gene. The 5'-flanking region of the gene was isolated, sequenced and fused to the lacZ reporter gene in order to study the effect of carbon sources and of specific deletion mutations on expression of the gene in yeast transformants. The expression of the reporter gene reveals that the AAC2 gene possesses a strong inducible promoter. The promoter analysis, combined with expression studies in the wild-type as well as in various mutant strains, identified an upstream activation site (UAS) contained within a sequence between -393 bp and -268 bp, and several major initiation sites of AAC2 mRNA between -105bp and -95 bp. Deletion analysis also shows that the TATA boxes located 45 bp and 104 bp upstream of the 5'-ends of AAC2 mRNA are not essential for the transcription. The UAS of the AAC2 gene is required for activation by HAP2 and heme and for release from glucose repressin. A restriction fragment containing the UAS conferred oxygen and carbon source regulation when placed upstream of another yeast gene encoding ADP/ATP carrier (AAC3), deleted of its regulatory sequences. The UAS of the AAC2 gene contains at least two distinct motifs for DNA-binding transcriptional activators, including one which is identical with the core HAP2/3/4 binding motif, and a second one with the ABF1 consensus binding sequence. Our results indicate that these sequences mediate the effects of the respective transactivator on the oxygen- and carbon-source-dependent transcription of the AAC2 gene.

Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor.

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor. The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation, is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme.

Yeast ADP/ATP carrier (AAC) proteins exhibit similar enzymatic properties but their deletion produces different phenotypes.

Saccharomyces cerevisiae strains expressing a single type of ADP/ATP carrier (AAC) protein were prepared from a mutant in which all AAC genes were disrupted, by transformation with plasmids containing a chosen AAC gene. As demonstrated by measurements of [14C]ADP specific binding and transport, all three translocator proteins, AAC1, AAC2 and AAC3 when present in the mitochondrial membrane, exhibited similar translocation properties. The disruption of some AAC genes, however, resulted in phenotypes indicating that the function of these proteins in whole cells can be quite different. Specifically, we found that the disruption of AAC1 gene, but not AAC2 and AAC3, resulted in a change in colony phenotype.