Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.     

t-Butyl-4-hydroxyanisole, a novel respiratory chain inhibitor. Effects on Trypanosoma cruzi epimastigotes

t-Butyl-4-hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level of cytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.     

In vivo assembly of yeast mitochondrial alpha-ketoglutarate dehydrogenase complex.

The assembly of alpha-ketoglutarate dehydrogenase complex (KGDC) has been studied in wild-type Saccharomyces cerevisiae and in respiratory-deficient strains (pet) with mutations in KGD1 and KGD2, the structural genes for alpha-ketoglutarate dehydrogenase (KE1) and dihydrolipoyl transsuccinylase (KE2) components, respectively. Mutants unable to express KE1 or KE2 form partial complexes similar to those reported in earlier studies on the resolution and reconstitution of bacterial and mammalian KGDC. Thus mutants lacking KE1 assemble a high-molecular-weight subcomplex consisting of a KE2 core particle with bound dihydrolipoyl dehydrogenase (E3). Similarly, mitochondrial extracts of mutants lacking KE2 contain dimeric KE1 and E3. These components, however, are not associated with each other. The partial complexes detected in the mutants are capable of reconstituting normal KGDC when supplied with the missing subunit. Complete restoration of overall alpha-ketoglutarate dehydrogenase activity is achieved by mixing appropriate ratios of mitochondrial extracts from mutants deficient in KE1 and KE2. The reconstitution of enzymatic activity correlates with binding of KE1 to the KE2-E3 particle to form a complex with the same sedimentation properties as wild-type KGDC. Overexpression of KE2 relative to KE1 results in a preponderance of incompletely assembled complexes with substoichiometric contents of KE1. Formation of a complex with a full complement of KE1 therefore depends on a balanced output of KE1 and KE2 from their respective genes. Biochemical screens of a pet mutant collection have led to the identification of a new gene required for the expression of enzymatically active KGDC. Mitochondria of the mutant have all of the catalytic subunits of KGDC. Sedimentation analysis of these components indicates that while the mutant has a stable KE2-E3 subcomplex, the interaction of KE1 with KE2 core is much weaker in the mutant than in the wild type. The gene product responsible for this phenotype, therefore, appears to function at a late stage of assembly of KGDC, most likely by posttranslational modification of one of the subunits.     

Phosphatase activity in Trypanosoma cruzi. Phosphate removal from ATP, phosphorylated proteins and other phosphate compounds

T. cruzi epimastigotes have a lysosomal acid phosphatase (pH 4.0) and acid and alkaline phosphatases (pH 5.5 and 8.0) localized in the cytosolic fraction. The levels of the lysosomal acid phosphatase increase with the age of the cultures, but the cytosolic phosphatases decline after the logarithmic phase of growth. The lysosomal phosphatase preferentially hydrolyses low mol. wt phosphate esters; whereas, the cytosolic alkaline phosphatases primarily act on phosphorylated proteins, and both the cytosolic acid and alkaline phosphatases on uridine nucleotide derivatives. The parasite also contains a microsomal glucose 6-phosphatase, and ATPases (Mg2+ and Ca2+-activated) derived from plasma membranes and mitochondria.     

Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results

Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16–20% of CF newborns, providing linkage and association results from large family and case–control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case–control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy–Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.     

Mathematical modeling of the circulation in the liver lobule

In this paper, we develop a mathematical model of blood circulation in the liver lobule. We aim to find the pressure and flux distributions within a liver lobule. We also investigate the effects of changes in pressure that occur following a resection of part of the liver, which often leads to high pressure in the portal vein. The liver can be divided into functional units called lobules. Each lobule has a hexagonal cross-section, and we assume that its longitudinal extent is large compared with its width. We consider an infinite lattice of identical lobules and study the two-dimensional flow in the hexagonal cross-sections. We model the sinusoidal space as a porous medium, with blood entering from the portal tracts (located at each of the vertices of the cross-section of the lobule) and exiting via the centrilobular vein (located in the center of the cross-section). We first develop and solve an idealized mathematical model, treating the porous medium as rigid and isotropic and blood as a Newtonian fluid. The pressure drop across the lobule and the flux of blood through the lobule are proportional to one another. In spite of its simplicity, the model gives insight into the real pressure and velocity distribution in the lobule. We then consider three modifications of the model that are designed to make it more realistic. In the first modification, we account for the fact that the sinusoids tend to be preferentially aligned in the direction of the centrilobular vein by considering an anisotropic porous medium. In the second, we account more accurately for the true behavior of the blood by using a shear-thinning model. We show that both these modifications have a small quantitative effect on the behavior but no qualitative effect. The motivation for the final modification is to understand what happens either after a partial resection of the liver or after an implantation of a liver of small size. In these cases, the pressure is observed to rise significantly, which could cause deformation of the tissue. We show that including the effects of tissue compliance in the model means that the total blood flow increases more than linearly as the pressure rises.     

Small artery elasticity is decreased in patients with systemic lupus erythematosus without increased intima media thickness

Introduction  

The objectives of this study were to determine small arterial elasticity (SAE) in systemic lupus erythematosus (SLE) and to investigate its relationship with intima media thickness (IMT), accumulation of advanced glycation end products (AGEs), endothelial activation and inflammation.     

Regulation of the adaptation to zinc deficiency in plants

The molecular mechanisms by which plants sense their micronutrient status, and adapt to their environment in order to ensure a sufficient micronutrient supply, are poorly understood. Zinc is an essential micronutrient for all living organisms. when facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. The molecular regulators controlling this adaptation were recently identified. in this mini-review, we highlight recent progress in understanding the adaptation to zinc deficiency in plants and discuss the future challenges to fully unravel its molecular basis.Key words: adaptation, zinc deficiency, biofortification, molecular regulators, plant nutritionIn an increasingly populated world, agricultural production is an essential element of social development. Agriculture is the primary source of all nutrients required for human life, and nutrient sufficiency is the basis for good health and welfare of the human population.¹ Soils with zinc deficiency are widespread in the world, affecting large areas of cultivated soils in India, Turkey, China, Brazil and Australia,^2,3 making zinc the most common crop micronutrient deficiency.⁴ In addition, risk of inadequate zinc diet and zinc malnutrition are estimated to affect one-third of the global human population, i.e., around two billion people.⁵ Most affected are people living in developing countries, where diets are rich in cereal-based foods. Cereal grains are rich in phytate, which is a potent anti-nutrient, limiting micronutrient bioavailability.⁶ Zinc deficiency in crop production can be easily ameliorated through zinc fertilization, making agronomic biofortification an important strategy,³ however in the poorer regions, the required infrastructure to provide a reliable supply of zinc fertilizers of sufficient quality, is often not available. In those situations, biofortified crops, in which the zinc status of crops is genetically improved by selective breeding or via biotechnology, offer a rural-based intervention that will more likely reach the population.⁷ Different traits can be targeted to developing such improved crops, such as plant zinc deficiency tolerance, zinc use efficiency and the accumulation of zinc in edible parts. However, insufficient knowledge on the molecular mechanisms and the regulation of the zinc homeostasis network in plants is a serious bottleneck when pursuing zinc biofortification.     

A first insight into the genetic diversity of Mycobacterium tuberculosis in Dar es Salaam, Tanzania, assessed by spoligotyping

Background  

Tanzania has a high tuberculosis incidence, and genotyping studies of Mycobacterium tuberculosis in the country are necessary in order to improve our understanding of the epidemic. Spoligotyping is a potentially powerful genotyping method due to fast generation of genotyping results, high reproducibility and low operation costs. The recently constructed SpolDB4 database and the model-based program 'spotclust' can be used to assign isolates to families, subfamilies and variants. The results of a study can thus be analyzed in a global context.