Creatine biosynthesis and transport in health and disease.

Creatine is physiologically provided equally by diet and by endogenous synthesis from arginine and glycine with successive involvements of arginine glycine amidinotransferase [AGAT] and guanidinoacetate methyl transferase [GAMT]. A specific plasma membrane transporter, creatine transporter [CRTR] (SLC6A8), further enables cells to incorporate creatine and through uptake of its precursor, guanidinoacetate, also directly contributes to creatine biosynthesis. Breakthrough in the role of creatine has arisen from studies on creatine deficiency disorders. Primary creatine disorders are inherited as autosomal recessive (mutations affecting GATM [for glycine-amidinotransferase, mitochondrial]) and GAMT genes) or X-linked (SLC6A8 gene) traits. They have highlighted the role of creatine in brain functions altered in patients (global developmental delay, intellectual disability, behavioral disorders). Creatine modulates GABAergic and glutamatergic cerebral pathways, presynaptic CRTR (SLC6A8) ensuring re-uptake of synaptic creatine. Secondary creatine disorders, addressing other genes, have stressed the extraordinary imbrication of creatine metabolism with many other cellular pathways. This high dependence on multiple pathways supports creatine as a cellular sensor, to cell methylation and energy status. Creatine biosynthesis consumes 40% of methyl groups produced as S-adenosylmethionine, and creatine uptake is controlled by AMP activated protein kinase, a ubiquitous sensor of energy depletion. Today, creatine is considered as a potential sensor of cell methylation and energy status, a neurotransmitter influencing key (GABAergic and glutamatergic) CNS neurotransmission, therapeutic agent with anaplerotic properties (towards creatine kinases [creatine-creatine phosphate cycle] and creatine neurotransmission), energetic and antioxidant compound (benefits in degenerative diseases through protection against energy depletion and oxidant species) with osmolyte behavior (retention of water by muscle). This review encompasses all these aspects by providing an illustrated metabolic account for brain and body creatine in health and disease, an algorithm to diagnose metabolic and gene bases of primary and secondary creatine deficiencies, and a metabolic exploration by (1)H-MRS assessment of cerebral creatine levels and response to therapeutic measures.

Hereditary hemochromatosis in north-eastern Romania.

UNLABELLED: Hereditary hemochromatosis is a genetic disturbance of iron metabolism resulting in iron overload in several organs and their functional failure. Early diagnosis is necessary to start a simple and effective therapy: bleeding. It is the most frequent genetic disease in some populations. Our objectives were 1. An estimate of the expectancy of the disease in the population of our geographic region, and 2. To diagnose the disease by applying the established methods and estimate the efficiency of our diagnosis. METHODS: 1.To estimate the expectancy, we genotyped 200 persons for the most frequent mutations causing the disease: HFE-C282Y and HFE-H63D by PCR-RFLP. 2. To diagnose the disease phenotypically we determined plasma iron level, ferritin level and transferrin saturation index in 549 patients previously diagnosed as chronic hepatitis or cirrhosis and genotyped those with hemochromatosis phenotype. RESULTS: 1. We found allelic frequencies of 1.75% and 13.25% for the HFE-C282Y and H63D mutant alleles respectively. From these frequencies we calculated that a severe case caused by a C282Y/C282Y homozygote can arise in 816 people and a mild case caused by a C282Y/H63D compound heterozygote can arise in 100 people 2. Among 549 patients we found 10 to have the phenotype of hemochromatosis and 3 out of the 10 were found to carry mutations: two in the HFE gene (one homozygous C282Y and one compound heterozygous C282Y/H63D) and one in the hemojuvelin (HJV) gene (a G320V).

In Schizosaccharomyces pombe the 14-3-3 protein Rad24p is involved in negative control of pho1 gene expression.

Expression of Schizosaccharomyces pombe pho1-encoded acid phosphatase is transcriptionally regulated by adenine and phosphate. Four genes, anr1-3 and anr5, encode negative regulators of pho1 expression. Apart from being designated as loci, the anr genes have not been further characterized. In this study we provide evidence that a strain carrying the deletion of rad24, a 14-3-3 protein-encoding gene, exhibits an anr mutant like the phenotype (higher phosphatase activity, higher transcript levels of pho1, lower sensitivity to adenine of pho1 expression) and that rad24 is closely linked, probably allelic, to anr5. By sequencing the two exons of the rad24 gene in a strain carrying the mutant allele anr5-13, we found a T/A-to-C/G transition in the 225th codon of its ORF, causing a leucine-to-serine substitution in a highly conserved region of all proteins of the 14-3-3 family. anr2 and anr3 are not allelic to rad24. The mutant alleles of anr2 and anr3 are recessive to their wild-type alleles and do not belong to the same epistasis group as rad24.

Method for protein tagging in Schizosaccharomyces pombe.

Tagging is a useful method for the investigation of proteins. It allows the localization of the proteins in the cell, their purification in order to investigate their function and the determination of their expression. The aim of the present study was to tag the Rad32 protein of fission yeast (which is the homologue of Mre11 protein from humans) at its N-terminus. Rad32p as well as Mre11p are involved in the repair of DNA double strand breaks and in the DNA damage checkpoint. We carried out this tagging using the Cre-loxp recombination system. In a first step, a 2 kb DNA fragment was integrated upstream of the initiating codon of rad32 gene. This fragment encoded the TAP-tag (tandem affinity purification), a loxp site, a selectable marker (sup3-5), an exogenous promoter (nmt1) and a second loxp site, in this sequence. Following transformation of this DNA fragment into S. pombe cells, rad32 was under the control of the artificial promotor, which allows a controlled expression of the gene by thiamine. In a second step, the cells were transformed with a plasmid coding for Cre recombinase, which catalyses the excision of the DNA sequence between the two loxp sites, removing the marker and the artificial promotor. Thus the tag became attached to the rad32 gene upstream of the ATG, placing the gene under the control of its native promotor. The strain thus obtained will be subsequently used for evidencing the tagged protein by Western blotting and then for its purification in order to investigate its function.

[Mutagenic aspects of the lipoprotein lipase gene]. / Aspecte mutagenice la nivelul genei lipoprotein lipazei.

Lipoprotein lipase (LPL) is an enzyme involved in the metabolism of triglyceride-rich lipoproteins (chylomicrons and VLDL), participating also in the remodeling of HDL particles. It is encoded by a gene located on chromosome 8 (8p22), containing 10 exons. Abnormalities in this gene lead to the development of LPL enzyme deficiency. About 100 mutations have been described in the LPL gene, the most frequent being Asp9Asn, Gly188Glu and Asn291Ser. Mutations in the homozygous form are associated with type I hyperlipoproteinemia (familial chylomicronemia). Mutations in the heterozygous state have a significant incidence in population (3-7%) and lead to a decrease in the LPL activity with up to 50%, which causes the modification of the plasma lipid profile, meaning an increase in triglycerides and a decrease in HDL cholesterol. Both modifications represent cardiovascular risk factors, so that the carriers of LPL gene mutations have an increased predisposition to develop coronary heart disease. Moreover, the association of heterozygous mutations in the LPL gene in individuals with genetic-type hyperlipoproteinemias may aggravate the perturbations of the lipid profile and, consequently, they can increase the cardiovascular risk in these patients. The accumulated data may be considered an argument for the importance of investigating LPL gene mutations in the population, in order to detect precociously the individuals with an increased atherogenic predisposition and to decide on the appropriate therapy.

[Method for the molecular diagnosis of lipoprotein lipase genetic deficiency]. / Metoda de diagnostic molecular al deficitului genetic al lipoprotein lipazei.

Modifications of plasma lipid profile is one of the major causes of a high cardiovascular risk. They can be the consequences of mutations in the gene encoding lipoprotein lipase (LPL), an enzyme that has an important role in the metabolism of plasma lipoproteins. The aim of the present study was to put into practice a method for detecting the Gly188Glu mutation in the LPL gene. The search was performed on a group of 107 patients with cardiovascular diseases and/or dyslipidemias. DNA investigation consisted, in a first stage, in the enzymatic digestion of exon 5 of the LPL gene, previously amplified by the PCR reaction, with the AvaII restriction endonuclease. Three of the subjects were further investigated by the sequencing of exon 5, in order to search for the presence of other mutations. We didn't detect the Gly188Glu mutation in none of the cases, and no other mutation in exon 5 was found in the three patients tested by DNA sequencing. We conclude that the amplification-restriction method can be used for the detection of known mutations in the LPL gene, allowing an early identification of the subjects with a high cardiovascular risk and the onset of the appropriate therapy. In order to detect mutations which don't affect the recognition sequence of a restriction enzyme and eventually new mutations, the sequencing of that gene is recommended.

Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it. It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation. The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development. A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions. This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program. The events of the program that were affected by inositol starvation were investigated. Commitment to mating and production of pheromone M were shown not to be inositol-dependent. A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation. In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent. The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.