We have cloned the proteasome and the proteasome activating nucleotidase (PAN) genes from the mesophilic archaeon
Methanosarcina mazei and produced the respective proteins in
Escherichia coli ...cultures. The recombinant complexes were purified to homogeneity and characterized biochemically, structurally, and by mass spectrometry. We found that the degradation of Bodipy-casein by
Methanosarcina proteasomes was activated by
Methanosarcina PAN. Notably, the
Methanosarcina PAN unfolded GFP-SsrA only in the presence of
Methanosarcina proteasomes. Structural analysis by 2D averaging electron microscopy of negatively stained complexes displayed the typical structure for the proteasome, namely four-striped side-views and sevenfold-symmetric top-views, with 15
nm height and 11
nm diameter. The structural analysis of the PAN preparation revealed also four-striped side-views, albeit with a height of 18
nm and sixfold-symmetric top-views with a diameter of 15
nm, which corresponds most likely to a dimer of two hexameric complexes. Mass spectrometric analysis of both the
Methanosarcina and the
Methanocaldococcus PAN proteins indicated hexameric complexes. In summary, we performed a functional and structural characterization of the PAN and proteasome complexes from the archaeon
M. mazei and described unique new structural and functional features.
OBJETIVO: Describir y analizar el proceso de regularización del personal de salud pagado por el Sistema de Protección Social en Salud de México. MATERIAL Y MÉTODOS: Se utilizan datos primarios y ...secundarios provenientes de la evaluación del Sistema de Protección Social en Salud en 2009. RESULTADOS: La regularización mejora las condiciones laborales de los trabajadores pero sus implicaciones para el conjunto del sistema no son necesariamente positivas. CONCLUSIONES: Se requiere considerar la necesidad de que este tipo de inversiones beneficien a todos los actores interesados, principalmente la población asegurada por el sistema.OBJECTIVE: The process of regularization of workers paid by the Social Protection Health System of Mexico is described and analyzed. MATERIALS AND METHODS: Primary and secondary data collected by the external evaluation of the Mexican System for Social Protection in Health in 2009 were used. RESULTS: The regularization clearly improved the labor conditions of workers contracted by the system but a broader systemic implication of regularization does not seem to be necessarily positive. CONCLUSION: It is important to consider the need to guarantee that this type of changes in the contractual conditions of workers benefit all actors, particularly the insured population.
The Archaeon Methanosarcina mazei and related species are of great ecological importance as they are the only organisms fermenting acetate, methylamines and methanol to methane, carbon dioxide and ...ammonia (in case of methylamines). Since acetate is the precursor of 60% of the methane produced on earth these organisms contribute significantly to the production of this greenhouse gas, e.g. in rice paddies. The 4,096,345 base pairs circular chromosome of M. mazei is more than twice as large as the genomes of the methanogenic Archaea currently completely sequenced (Bult et al., 1996; Smith et al., 1997). 3,371 open reading frames (ORFs) were identified. Based on currently available sequence data 376 of these ORFs are Methanosarcina-specific and 1,043 ORFs find their closest homologue in the bacterial domain. 544 of these ORFs reach significant similarity values only in the bacterial domain. They include 56 of the 102 transposases, and proteins involved in gluconeogenesis, proline biosynthesis, transport processes, DNA-repair, environmental sensing, gene regulation, and stress response. Striking examples are the occurrence of the bacterial GroEL/GroES chaperone system and the presence of tetrahydrofolate-dependent enzymes. These findings might indicate that lateral gene transfer has played an important evolutionary role in forging the physiology of this metabolically versatile methanogen.
Intrahepatic cholestasis of pregnancy has a prevalence of 1/1000 to 1/10000. Its etiology is multifactorial, involving genetic an hormonal factors, associated with adverse perinatal and obstetric ...outcomes. Report the case of patient 25 years old, with 32 weeks of gestation, which presents severe pruritus, jaundice, altered liver function tests and lipid profile, with presumptive diagnosis of intrahepatic cholestasis of pregnancy. Making weekly monitoring analytical biochemistry, test of fetal wellbeing, symptomatic management, with abdominal pregnancy termination at 35 weeks, for lack of clinical improvement, increase in metabolic disorders and intrauterine growth restriction, after induction of fetal lung maturity, with good obstetric and perinatal outcome. Definitive diagnosis by liver biopsy.
We have compared the substitution pattern of the glucocerebrosidase gene (GBA) and the glucocerebrosidase pseudogene (psGBA), two highly homologous regions under different selective pressures and ...within the same genomic background. Mutations in GBA may lead to Gaucher disease, an inborn metabolic disorder. Disease-causing mutations and neutral variation in the gene have been compared to neutral variation in the pseudogene. This comparison offers a unique opportunity to better understand the action of purifying selection, since the differences between mutational patterns can be attributed to different selective pressures. A similar frequency of CpG dinucleotides was observed in GBA and in psGBA, and CpG pairs were mutated with the same high frequency in both regions. However, nucleotides not in CpG pairs were more likely to contribute to disease-causing mutation than to accepted polymorphisms. This pattern, which resulted in a lower transition to transversion ratio in the gene, may be due to CpG avoidance on critical regions within exons.
We have obtained haplotypes from the autosomal glucocerebrosidase pseudogene (psGBA) for 100 human chromosomes from worldwide populations, as well as for four chimpanzee and four gorilla chromosomes. ...In humans, in a 5420-nucleotide stretch analyzed, variation comprises 17 substitutions, a 3-bp deletion, and a length polymorphism at a polyadenine tract. The substitution rate on the pseudogene (1.23 +/- 0.22 x 10(-9) per nucleotide and year) is within the range of previous estimates considering phylogenetic estimations. Recombination within the pseudogene was recognized, although the low variability of this locus prevented an accurate measure of recombination rates. At least 13% of the psGBA sequence could be attributed to gene conversion from the contiguous GBA gene, whereas the reciprocal event has been shown to lead to Gaucher disease. Human psGBA sequences showed a recent coalescence time (approximately 200,000 yr ago), and the most ancestral haplotype was found only in Africans; both observations are compatible with the replacement hypothesis of human origins. In a deeper timeframe, phylogenetic analysis showed that the duplication event that created psGBA could be dated at approximately 27 million years ago, in agreement with previous estimates.
Friedreich's ataxia (FRDA) is the most frequent hereditary ataxia in the Caucasian population (Harding 1983), with an estimated incidence of 1 in 50,000 and a calculated carrier frequency of 1 in ...120. It is a progressive neurodegenerative disease characterized by a variety of different symptoms, the most important being ataxia of gait and limbs, dysarthria, and areflexia (Harding 1981). Onset of the disease is typically in late childhood, almost always before age 25. FRDA represents the first autosomal recessive inherited trinucleotide disease (Campuzano et al. 1996), the primary cause of which is an unstable GAA repeat expansion within an Alu sequence in the first intron of the X25 gene. This results in a marked reduction in the steady-state level of mature X25 mRNA and, consequently, of the encoded product, frataxin (Bidichandani, Ashizawa, and Patel 1997; Campuzano et al. 1997). The GAA motif is polymorphic with bimodal size distribution in the normal population (83% of normal alleles have 6-12 repeats, 17% have 12-36 repeats), but it is amplified as many as 1,000 times in FRDA patients (Campuzano et al. 1996; Cossee et al. 1997; Montermini et al. 1997a). More than 95% of patients are known to be homozygous for the expanded allele, with a few cases of point mutations in the X25 gene (always in heterozygous individuals) (Campuzano et al. 1996; Duerr et al. 1996; Filla et al. 1996; Monros et al. 1997). Subsequent analysis has demonstrated that clinical variability in FRDA, including age at onset as well as severity and extent of disease involvement, is related to the size of the expanded alleles. Milder forms of the disease are associated with shorter expansions (Duerr et al. 1996; Filla et al. 1996; Monros et al. 1997; Montermini et al. 1997b). In order to understand the evolutionary context of the X25 GAA trinucleotide repeat, we analyzed the Alu element inserted in intron 1 of this gene (Alu-X25) in several primate species.
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