Methionine Synthetase Research Articles

Applications of Nitrous Oxide for Procedural Sedation in the Pediatric Population

The objective of this review was to provide a general descriptive account of the physical properties, end-organ effects, therapeutic applications, and delivery techniques of nitrous oxide (N2O) as used in the arena of procedural sedation. A computerized bibliographic search regarding the applications of nitrous for provision of sedation and analgesia during procedures with an emphasis on the pediatric population was performed. The end-organ effects of N2O have been well described in the operating room setting. Aside from its effects on the central nervous system of sedation and analgesia, N2O may alter intracerebral dynamics and alter cerebral blood flow and intracranial pressure especially in patients with altered intracranial compliance. Effects on ventilation include a dose-related depression of ventilatory function and control of upper airway patency. These effects are generally limited in the absence of comorbid diseases and potentiated by other sedative and analgesic agents. The more clinically significant respiratory effect of N2O on ventilatory function is a dose-dependent depression of the ventilatory response to hypoxemia. Hemodynamic effects include a mild direct depressant effect on myocardial function, which in the absence of comorbid cardiac disease is generally compensated by stimulation of the sympathetic nervous system. Nitrous oxide may potentially aggravate pulmonary hypertension. Additional physiologic effects on neurologic and hematologic function may result in inactivation of the enzyme, methionine synthetase. Recent concern has also been raised regarding the potential effects of N2O on immune function and its relationship to perioperative surgical site infections. Given differences in the solubility, N2O will diffuse into and significantly expand gas-filled cavities. Chronic exposure of health care works to N2O is also a concern. Although there are limited data in the literature to clearly substantiate concerns regarding the reproductive toxicity of occupational exposure to N2O, appropriate scavenging and use of other techniques are mandatory. Nitrous oxide has been shown to be effective for a variety of minor surgical procedures such as venipuncture, intravenous cannula placement, lumbar puncture, bone marrow aspiration, laceration repair, dental care, and minor dermatologic procedures. It is generally as effective as midazolam, with several studies demonstrating it to be more effective. However, its utility is not as great in severely painful procedures such as fracture reduction. Demonstrated advantages to parenteral sedation include a more rapid onset and a shorter recovery time with the majority of patients preferring it to over other agents or agreeing to its use for subsequent procedures. The literature also suggests increased success rates with simple procedures such as intravenous cannula placement when compared with placebo. In general, life-threatening adverse events have not been reported. Most common adverse effects include dysphoria and vomiting. For more painful procedures, combination with another agent may be used, and in all cases, topical or infiltrative local anesthesia is recommended. In general, N2O is a useful adjunct for procedural sedation. Given the variety of procedures performed in the pediatric patient, ongoing research is required to identify the most appropriate and effective use of this agent. This may be particularly relevant when evaluating its use for procedures associated with significant pain. In these scenarios, the combination of N2O with other agents needs to be evaluated. Given the potential for adverse effects, strict adherence to published guidelines regarding procedural sedation and monitoring is suggested.

Vegetarians, the Good, the Bad, and the Challenges

Becoming a vegetarian has become increasingly popular over the past decade, with many people turning to vegetarianism in an attempt to achieve better health. However this trend is not without it’s controversy. Many doctors and scientists have opposing views on vegetarianism, and this has led to confusion in the public about whether it is indeed the healthier lifestyle choice. The major health advantage for vegetarians include decreased body mass index, waist to hip ratio, blood pressure, plasma total cholesterol (TC), triacylglycerol and LDL-C levels, serum lipoprotein(a) concentration, plasma factor VII activity, ratios of TC/HDL-C, LDL-C/ HDL-C and TAG/HDL-C, and serum ferritin levels. However, being a vegetarian is not without it’s disadvantages and health risks. Studies have shown that vegetarians, especially vegans tend to have lower serum levels of vitamin B12 compared with omnivores. Serum vitamin B12 concentration was significantly negatively correlated with plasma homocysteine. Remethylation of homocysteine to methionine requires vitamin B12 (methylcobalamin form) as a coenzyme for homocysteine methyltransferase (methionine synthetase) and N5-methyltetrahydrofolate as a methyl donor [1]. Vitamin B12 is essential for new cell synthesis, blood formation, maintenance of the nervous system. Vitamin B12, as a coenzyme increases the utilization of folic acid and metabolism of carbohydrate, fat and protein etc. In the vitamins, B12 is the only one containing a mineral (cobalt), it also known as the red vitamin. Seafood, animal meats, eggs and liver are good sources for vitamin B12. Vitamin B12 is not found in plant foods, however, seaweed may contain vitamin B12 analogs which can be counted on as reliable sources of active vitamin B12. Ovo-lacto vegetarians may get vitamin B12 from eggs and dairy products. Vegans could get some very limited vitamin B12 from fermented soybean products, seaweed and edible fungi (like mushrooms) on farms or in the wild which may be contaminated from bacteria in the soil.

Effects of Space Flight on the Expression of Liver Proteins in the Mouse

Raw data derived from mass spectroscopic (MS) analyses of formalin-fixed paraffin-embedded (FFPE) tissue sections of the essential metabolic organ, liver, allocated by the provider (Amgen) from mice subjected to 13 days of microgravity on NASA Flight STS-118 were analyzed by two different search engines, using shotgun proteomics. With the eight statistically significant readouts in hand, Ingenuity Pathway Analysis (IPA) was employed to visualize probable biologic pathway relationships among proteins that might be associated with alterations in liver biochemistry due to space flight. Most noteworthy was the finding of up-regulation of the first urea cycle enzyme carbamoylphosphate synthetase, consistent with increased amino acid catabolism resulting from gravitational changes, and/ or other stress associated with missions in space. Down-regulation of fructose-bisphosphate aldolase B, regucalcin, ribonuclease UK114, alpha enolase, glycine N-methyltransferase and S-adenosyl methionine synthetase isoform type-1 was observed. 60 kDa heat shock protein was elevated.

Biosynthetic and Iron Metabolism Is Regulated by Thiol Proteome Changes Dependent on Glutaredoxin-2 and Mitochondrial Peroxiredoxin-1 in Saccharomyces cerevisiae

Redoxins are involved in maintenance of thiol redox homeostasis, but their exact sites of action are only partly known. We have applied a combined redox proteomics and transcriptomics experimental strategy to discover specific functions of two interacting redoxins: dually localized glutaredoxin 2 (Grx2p) and mitochondrial peroxiredoxin 1 (Prx1p). We have identified 139 proteins showing differential postranslational thiol redox modifications when the cells do not express Grx2p, Prx1p, or both and have mapped the precise cysteines involved in each case. Some of these modifications constitute functional switches that affect metabolic and signaling pathways as the primary effect, leading to gene transcription remodeling as the secondary adaptive effect as demonstrated by a parallel high throughput gene expression analysis. The results suggest that in the absence of Grx2p, the metabolic flow toward nucleotide and aromatic amino acid biosynthesis is slowed down by redox modification of the key enzymes Rpe1p (D-ribulose-5-phosphate 3-epimerase), Tkl1p (transketolase) and Aro4p (3-deoxy-D-arabino-heptulosonate-7-phosphate synthase). The glycolytic mainstream is then diverted toward carbohydrate storage by induction of trehalose and glycogen biosynthesis genes. Porphyrin biosynthesis may also be compromised by inactivation of the redox-sensitive cytosolic enzymes Hem12p (uroporphyrinogen decarboxylase) and Sam1p (S-adenosyl methionine synthetase) and a battery of respiratory genes sensitive to low heme levels are induced. Genes of the Aft1p-dependent iron regulon were induced specifically in the absence of Prx1p despite optimal mitochondrial Fe-S biogenesis, suggesting dysfunction of the mitochondria to the cytosol signaling pathway. Strikingly, requirement of Grx2p for these events places dithiolic Grx2 in the framework of iron metabolism.

Uncovering the Human Methyltransferasome

We present a comprehensive analysis of the human methyltransferasome. Primary sequences, predicted secondary structures, and solved crystal structures of known methyltransferases were analyzed by hidden Markov models, Fisher-based statistical matrices, and fold recognition prediction-based threading algorithms to create a model, or profile, of each methyltransferase superfamily. These profiles were used to scan the human proteome database and detect novel methyltransferases. 208 proteins in the human genome are now identified as known or putative methyltransferases, including 38 proteins that were not annotated previously. To date, 30% of these proteins have been linked to disease states. Possible substrates of methylation for all of the SET domain and SPOUT methyltransferases as well as 100 of the 131 seven-β-strand methyltransferases were surmised from sequence similarity clusters based on alignments of the substrate-specific domains.

M14.1 Human placental digitalis-like factor in preeclampsia and its potential regulation

Folate, a vitamin of the B group involved in one-carbon group metabolism, plays an important role in DNA synthesis and methylation. Several polymorphisms in the genes involved in folate uptake and biotransformations have been shown to be associated to the risk of cancer and to anticancer drug response. We studied common polymorphisms in MTHFR (N5,10-methylene-tetrahydrofolate reductase), MTHFD1 (N5,10-methylene-tetrahydrofolate dehydrogenase), MTR (methionine synthetase) and SLC19A1 (reduced folate carrier) in the panel of 60 human tumour cell lines established by the NCI for anticancer drug screening and we tentatively associated these polymorphisms with gene expression and drug cytotoxicity as extracted from the public database of the Developmental Therapeutic Programme. We observed a consistent and highly significant association between the presence of the variant C allele of the A>C1298 polymorphism of MTHFR and the sensitivity to many anticancer drugs belonging to the classes of antifolates, antimetabolites, alkylating agents and, to a lesser extent, topoisomerase inhibitors. In contrast, the T variant allele of the C>T677 variation of MTHFR was rather associated to lower sensitivity of the cell lines towards anticancer drugs (alkylating agents, antifolates and antimetabolites) but with much lower effects than the A>C1298 variation. The polymorphisms of the other genes studied were not associated with differences in anticancer drug sensitivity, but the expression of the SLC19A1 gene was significantly correlated with the sensitivity to several drugs (antifolates, thiopurines, nitrosoureas, and DACH-platinum drugs). We concluded that the NCI-60 panel may constitute a good starting point for implementing clinical studies aimed at discovering and validating predictive genetic markers of drug efficacy and/or toxicity.

Validation of Housekeeping Genes for Gene Expression Studies in Symbiodinium Exposed to Thermal and Light Stress

Unicellular photosynthetic algae (dinoflagellate) from the genus Symbiodinium live in mutualistic symbiosis with reef-building corals. Cultured Symbiodinium sp. (clade C) were exposed to a range of environmental stresses that included elevated temperatures (29°C and 32°C) under high (100μmol quanta m(-2)s(-1) Photosynthetic Active Radiation) and low (10μmol quanta m(-2)s(-1)) irradiances. Using real-time RT-PCR the stability of expression for the nine selected putative housekeeping genes (HKGs) was tested. The most stable expression pattern was identified for cyclophilin and S-adenosyl methionine synthetase (SAM) followed by S4 ribosomal protein (Rp-S4), Calmodulin (Cal), and Cytochrome oxidase subunit 1 (Cox), respectively. Thermal stress alone resulted in the highest expression stability for Rp-S4 and SAM, with a minimum of two reference genes required for data normalization. For Symbiodinium exposed to both, light and thermal stresses, at least five reference genes were recommended by geNorm analysis. In parallel, the expression of Hsp90 for Symbiodinium in culture and in symbiosis within coral host (Acropora millepora) was evaluated using the most stable HKGs. Our results revealed a drop in Hsp90 expression after an 18 h-period and a 24 h-period of exposure to elevated temperatures indicating the similar Hsp90 expression profile in symbiotic and non-symbiotic environments. This study provides the first list of the HKGs and will provide a useful reference in future gene expression studies in symbiotic dinoflagellates.

Cloning and expression of S-Adenosyl Methionine (SAMe) Synthetase gene in recombinant E. coli strain for large scale production of SAMe

Adenosyl Methionine (SAMe) Synthetase is an enzyme which catalyses the synthesis of S-Adenosyl Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies.

Inhibition of nitric oxide synthesis during induced cholestasis ameliorates hepatocellular injury by facilitating S-nitrosothiol homeostasis

Cholestatic liver injury following extra- or intrahepatic bile duct obstruction causes nonparenchymal cell proliferation and matrix deposition leading to end-stage liver disease and cirrhosis. In cholestatic conditions, nitric oxide (NO) is mainly produced by a hepatocyte-inducible NO synthase (iNOS) as a result of enhanced inflow of endotoxins to the liver and also by accumulation of bile salts in hepatocytes and subsequent hepatocellular injury. This study was aimed to investigate the role of NO and S-nitrosothiol (SNO) homeostasis in the development of hepatocellular injury during cholestasis induced by bile duct ligation (BDL) in rats. Male Wistar rats (200–250 g) were divided into four groups (n=10 each), including sham-operated (SO), bile duct-ligated (BDL), tauroursodeoxycholic acid (TUDCA, 50 mg/kg) and S-methylisothiourea (SMT, 25 mg/kg) treated. After 7 days, BDL rats showed elevated serum levels of γ-glutamiltranspeptidase, aspartate aminotransferase, alanine aminotransferase, LDH, and bilirubin, bile duct proliferation and fibrosis, compared with the SO group. TUDCA treatment did not significantly alter these parameters, but the iNOS inhibitor SMT ameliorated hepatocellular injury, as shown by lower levels of circulating hepatic enzymes and bilirubin, and a decreased grade of bile duct proliferation and fibrosis. Both TUDCA and SMT treatments reversed Mrp2 canalicular pump expression to control levels. However, only SMT treatment significantly lowered the increased levels of plasma NO and S-nitrosation (S-nitrosylation) of liver proteins in BDL rats. Moreover, BDL resulted in a reduction of the S-nitrosoglutathione reductase (GSNOR/Adh5) enzymatic activity and a downregulation of the GSNOR/Adh5 mRNA expression that was reverted by SMT, but not TUDCA, treatment. A total of 25 liver proteins, including S-adenosyl methionine synthetase, betaine-homocysteine S-methyltransferase, Hsp90 and protein disulfide isomerase, were found to be S-nitrosated in BDL rats. In conclusion, the inhibition of NO production during induced cholestasis ameliorates hepatocellular injury. This effect is in part mediated by the improvement of cell proficiency in maintaining SNO homeostasis.

Quantitative Proteomic Analysis of CMS-Related Changes in Honglian CMS Rice Anther

Honglian (HL) cytoplasmic male sterility (CMS) is one of the rice CMS types and has been widely used in hybrid rice production in China. The CMS line (Yuetai A, YTA) has a Yuetai B (maintainer line, YTB) nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of Yuetai B. The fertility of hybrid (HL-6) was restored by restorer gene in nuclear genome of restorer line (9311). We used isotope-code affinity tag (ICAT) technology to perform the protein profiling of uninucleate stage rice anther and identify the CMS-HL related proteins. Two separate ICAT analyses were performed in this study: (1) anthers from YTA versus anthers from YTB, and (2) anthers from YTA versus anthers from HL-6. Based on the two analyses, a total of 97 unique proteins were identified and quantified in uninucleate stage rice anther under the error rate of less than 10%, of which eight proteins showed abundance changes of at least twofold between YTA and YTB. Triosephosphate isomerase, fructokinase II, DNA-binding protein GBP16 and ribosomal protein L3B were over-expressed in YTB, while oligopeptide transporter, floral organ regulator 1, kinase and S-adenosyl-L: -methionine synthetase were over-expressed in YTA. Reduction of the proteins associated with energy production and lesser ATP equivalents detected in CMS anther indicated that the low level of energy production played an important role in inducing CMS-HL.

Involvement of gene polymorphisms of the folate pathway enzymes in gene expression and anticancer drug sensitivity using the NCI-60 panel as a model

Folate, a vitamin of the B group involved in one-carbon group metabolism, plays an important role in DNA synthesis and methylation. Several polymorphisms in the genes involved in folate uptake and biotransformations have been shown to be associated to the risk of cancer and to anticancer drug response. We studied common polymorphisms in MTHFR (N 5,10-methylene-tetrahydrofolate reductase), MTHFD1 (N 5,10-methylene-tetrahydrofolate dehydrogenase), MTR (methionine synthetase) and SLC19A1 (reduced folate carrier) in the panel of 60 human tumour cell lines established by the NCI for anticancer drug screening and we tentatively associated these polymorphisms with gene expression and drug cytotoxicity as extracted from the public database of the Developmental Therapeutic Programme. We observed a consistent and highly significant association between the presence of the variant C allele of the A>C1298 polymorphism of MTHFR and the sensitivity to many anticancer drugs belonging to the classes of antifolates, antimetabolites, alkylating agents and, to a lesser extent, topoisomerase inhibitors. In contrast, the T variant allele of the C>T677 variation of MTHFR was rather associated to lower sensitivity of the cell lines towards anticancer drugs (alkylating agents, antifolates and antimetabolites) but with much lower effects than the A>C1298 variation. The polymorphisms of the other genes studied were not associated with differences in anticancer drug sensitivity, but the expression of the SLC19A1 gene was significantly correlated with the sensitivity to several drugs (antifolates, thiopurines, nitrosoureas, and DACH-platinum drugs). We concluded that the NCI-60 panel may constitute a good starting point for implementing clinical studies aimed at discovering and validating predictive genetic markers of drug efficacy and/or toxicity.

Transcobalamin II deficiency at birth

Transcobalamin II deficiency (# MIM 275350) is a rare, recessively inherited disorder of cobalamin transport that leads to intracellular cobalamin depletion with secondary impairment of methionine synthetase and methyl-malonyl CoA mutase activities. Affected individuals may suffer from long-term neurological sequelae if therapy with intramuscular hydroxocobalamin is not initiated promptly. We report two sisters with complete absence of transcobalamin due to homozygosity for a novel mutation (c.insC110) in the TCN2 gene that leads to a premature stop codon and non-functional protein. The older sister, now 4.5 years old, presented at 6 weeks of age with pancytopenia, protein losing enteropathy and a rapidly declining clinical course. Prompt therapy with 1 mg hydroxocobalamin/day led to full recovery within days. Her now 1.5 year old sister was diagnosed shortly after birth and was started on hydroxocobalamin prior to onset of clinical symptoms. Interestingly, urinary methylmalonic acid excretion was increased significantly during the first days of life suggesting that functional cobalamin deficiency is present also during fetal life, although not giving rise to clinical symptoms until well after birth.

Maternal polymorphisms for methyltetrahydrofolate reductase and methionine synthetase reductase and risk of children with Down syndrome

The purpose of this research was to study factors that are involved in centromeric hypomethylation in the pathogenesis of Down syndrome (DS). This was a case-control study to evaluate the association between methyltetrahydrofolate reductase (MTHFR) C677T and methionine synthetase-reductase (MTRR) A66G polymorphisms and the risk of DS; we compared mothers in Italy who had children with DS and matched control subjects. Seventy-four cases of DS caused by an error in maternal meiosis were compared with 184 matched control subjects. The frequencies of the MTHFR C677T polymorphism were similar between the 2 groups. As regards the MTRR A66G polymorphism, the presence of the mutated G allele either in the heterozygous or homozygous form was significantly more common among mothers of children with DS than among control subjects (odds ratio, 2.21; 95% CI, 1.11-4.40). In a population with a high prevalence of the mutated T allele, maternal MTRR A66G, but not MTHFR, polymorphisms are associated with Down syndrome.

Preliminary proteomics analysis of the total proteins of flower bud induction of apple trees

Apple is one of the most important fruit trees in the world. Nevertheless, mainly due to its long juvenile period, its breeding work constantly falls far behind other crops. So the aim of this study is to reveal the mechanism of apple flower bud differentiation, shorten the juvenile period and accelerate its breeding process. Proteomics technology (including two-dimensional gel electrophoresis (2-DE), biomass spectrometry and bioinformatics) was applied to work on the specific protein of flower bud and leaf bud after the brachyblasts of ‘Fuji’ stopped growth for 3–9 weeks. The results showed that the morphodifferentiation of flower bud did not begin until the seventh week after the brachyblast stopped growth. Furthermore, compared with the leaf bud, flower bud had significant changes in the expression of 283 protein spots in quality and quantity on 2-DE maps. Among the 283 protein spots, four protein spots (16.4, 30.2, 40.3 and 65.1 kD) were characteristic of the flower bud in the archae-stage (initial inflorescence appeared) at the beginning of flower-bud differentiation, three (39.3, 60.2 and 66.3 kD) in the post-stage (Lateral-flower appears) and one (77.1 kD) in the sepal stage on 2-DE maps. Analysis by peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry also identified and forecasted functionally by blasting different databases. In the four specific proteins, it was found that spots No. 256 (16.4 kD) and 298 (30.2 kD) were unknown proteins, spot Nos. 327 (40.3 kD) was identified as the synthesis enzyme protein and spot No. 367 (40.3 kD) was identified as a RNA-binding protein involved in transcription. When flower bud started to differentiate morphologically, we detected four specific proteins which were 16.4, 30.2, 40.3 and 65.1 kD. Three specific proteins 39.3, 60.2 and 66.3 kD were observed at side flower-appearing stage. When calyx began to emerge, there was one specific protein: 77.1 kD. The proteins 16.4 kD and 30.2 kD were two unknown specific proteins. The 40.3 kD protein was related to methionine synthetase while 65.1 kD protein, a RNA-binding protein, was related to transcription.

Purification and characterization of S-adenosyl methionine synthetase from Pichia ciferrii

Purification and characterization of S-adenosyl methionine synthetase from Pichia ciferrii

Effect of nitrous oxide on plasma homocysteine and folate in patients undergoing major surgery

BackgroundNitrous oxide (N2O) inhibits methionine synthetase resulting in elevated plasma homocysteine (Hcy) concentration after surgery. In epidemiological studies, hyperhomocysteinaemia is associated with increased risk of cardiovascular disease and dementia. MethodsBlood samples were obtained to measure plasma folate and Hcy concentrations from two centres participating in a multicentre randomized trial investigating the effects of N2O on the outcome after major surgery. The effect of N2O and duration of anaesthesia on plasma Hcy, and the relationship between hyperhomocysteinaemia and outcomes were assessed. ResultsWe enrolled 394 patients. The N2O Group had an increase in plasma Hcy concentration after surgery when compared with the N2O-free Group: 11.1 (3.8) vs 8.5 (4.0) μmol litre−1, P<0.0005. Postoperative hyperhomocysteinaemia was associated with an increased risk of major complications: risk ratio (RR) 2.8 (95% CI: 1.4–5.4), P=0.002 and cardiovascular events, RR 5.1 (95% CI: 3.1–8.5), P<0.0005. There was a significant association between duration of anaesthesia and the relative change in plasma Hcy concentration, particularly in the N2O Group: r=0.42, P<0.001. ConclusionsN2O increases plasma Hcy concentration; this effect is greater with a longer duration of anaesthesia. Hyperhomocysteinaemia is a risk factor for major postoperative complications. N2O-induced increases in plasma Hcy concentration may be a cause of postoperative cardiovascular morbidity.

Direct Genotyping of Single Nucleotide Polymorphisms in Methyl Metabolism Genes Using Probe-Free High-Resolution Melting Analysis

High-resolution melting (HRM) shows great promise for high-throughput, rapid genotyping of individual polymorphic loci. We have developed HRM assays for genotyping single nucleotide polymorphisms (SNP) in several key genes that are involved in methyl metabolism and may directly or indirectly affect the methylation status of the DNA. The SNPs are in the 5,10-methylenetetrahydrofolate reductase (MTHFR; C677T and A1298C), methionine synthetase (MTR; 5-methyltetrahydrofolate-homocysteine methyltransferase; A2756G), and DNA methyltransferase 3b (DNMT3b; C46359T and C31721T) loci. The choice of short amplicons led to greater melting temperature (Tm) differences between the two homozygous genotypes, which allowed accurate genotyping without the use of probes or spiking with control DNA. In the case of MTHFR, there is a second rarer SNP (rs4846051) close to the A1298C SNP that may result in inaccurate genotyping. We masked this second SNP by placing the primer over it and choosing a base at the polymorphic position that was equally mismatched to both alleles. The HRM assays were done on HRM capable real-time PCR machines rather than stand-alone HRM machines. Monitoring the amplification allows ready identification of samples that may give rise to aberrant melting curves because of PCR abnormalities. We show that samples amplifying markedly late can give rise to shifted melting curves without alteration of shapes and potentially lead to misclassification of genotypes. In conclusion, rapid and high-throughput SNP analysis can be done with probe-free HRM if sufficient attention is paid to amplicon design and quality control to omit aberrantly amplifying samples.

Vitamin Β12 and Folic Acid Effects on Polyamine Metabolism in Rat Liver

Abstract Polyamines, spennine, spermidine and putrescinc, small aliphatic nitrous bases, are normal constituents of microbial, plant and animal cells, where they fulfill an array of physiological roles. Metabolism of polyamines is associated with growth and differentiation of mammalian cells, spermine related to RNA and spermidine to DNA metabolism. Methyl-cobalamin, the coenzyme of methionine synthetase, catalyses the recycling of homocysteine to methionine using 5-methyltctrahydrofolate. By acceleration of methionine biosynthesis these vitamins may influence spermidine and spermine synthesis. Liver tissue is rich in polyamines and it is the place of vitamin B12 and folic acid deposition. Polyamine oxidase (PAO, EC 1.5.3) and diamino oxidase (DAO, EC 1.4.3.6) participate in the process of degradation and interconversion of spermine, spermidine and putrescinc. The idea of our work was to examine the effccts of cobalamin and folic acid on the polyamine metabolism. We have examined the amount of spermine, spermidine and putrescine in liver tissue. At the same time we have examined the activities of PAO and DAO, the catabolic enzymes of polyamine metabolism. Our result suggest that the supplementation of experimental animals with vitamin B12 alone or together with folic acid augmentais spermidine and spermine levels in rat liver, at the same time the amount of putrescine does not change. The application of vitamin cobalamin to experimental animals alone increases PAO; the supplementation of experimental animals with vitamin Β12 together with folic acid causes opposite effect - the decrease of PAO activity. DAO activity significantly decreases under the influence of cobalamin and also with cobalamin and folic acid in combination. Our experimental results indicate the importance of cobalamin and folic acid in polyamine metabolism.

Chaperomics: In Vivo GroEL Function Defined

A recent proteome analysis of protein folding inside cells of Escherichia coli predicts that only 84 of the approximately 2400 cytosolic proteins expressed in minimal media depend absolutely on the GroEL/GroES chaperone system to avoid aggregation. These proteins are enriched in alpha/beta domains and 13 are essential for growth.

Chronic ethanol consumption increases homocysteine accumulation in hepatocytes

Results of previous studies have shown that chronic ethanol administration impairs methionine synthetase activity and decreases S-adenosylmethionine levels in the liver, indicating interference with homocysteine remethylation. The purpose of the present study was to investigate the effects of chronic ethanol feeding on the accumulation of homocysteine (Hcy), a potentially toxic agent. The research was divided into two experiments. In Experiment A, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 2 weeks of feeding, and the release of Hcy into the medium was determined. Hepatocytes obtained from ethanol-fed rats released twice as much Hcy into the medium as did those obtained from controls. When hepatocytes were challenged by a methionine load, a marked increase in Hcy generation was observed, and the increase was further enhanced in hepatocytes obtained from ethanol-fed rats. In Experiment B, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 4 weeks of feeding (the feeding time required for significant formation of alcoholic fatty liver in rats). In this experiment, similar results were obtained with Hcy generation as in Experiment A. In Experiment B, supplementation of the incubation medium with betaine prevented the increase in generation of Hcy by methionine-treated control cells as well as the generation of Hcy by cells of ethanol-treated rats. These results indicate that betaine may have the potential as a therapeutic agent against toxic Hcy formation.