Uptake Kinetics,Species Differences,and the Determination of Equivalent Combinations of Air Concentration and Exposure Duration for Assessment of Acute Inhalation Toxicity

For acute inhalation toxicity assessment, I develop a conceptual framework for expressing combinations of intensity (air concentration) and duration that produce equivalent toxicity by examining how the shape of the body-burden uptake curve during a bout of inhalation interacts with various pharmacodynamic measures of the critical body burden needed to produce toxicity. If toxicity depends on attaining a critical tissue concentration, three existing empirical approaches—Haber's Law, the ten Berge equation, and pure air-concentration-dependence—are but local approximations to different parts of an overarching mathematical relationship. The compound-specific half-life of elimination determines the range of durations for which each applies: durations of one half-life or shorter follow Haber's Law, exposures of 4 or more half-lives follow pure air-concentration-dependence, and intermediate durations can be approximated by the ten Berge equation. Better animal-to-human extrapolation is achieved if exposure durations are expressed as number of species-specific half-lives. I consider several alternative pharmacodynamic criteria, such as the dependence of toxicity on time spent above a critical tissue concentration, or on the area under the tissue concentration curve, on the tissue concentration of a toxic metabolite, or on the imbalance of damage and repair processes.     

Redox responses in yeast to acetate as the carbon source

Following a shift to medium with acetate as the carbon source, a parental yeast strain exhibited a transient moderate 20% reduction in total cellular [NAD⁺ + NADH] but showed a ∼10-fold increase in the ratio of [NAD⁺]:[NADH] after 36 h. A mutant strain (idhΔ) lacking the tricarboxylic acid cycle enzyme isocitrate dehydrogenase had 50% higher cellular levels of [NAD⁺ + NADH] relative to the parental strain but exhibited similar changes in cofactor concentrations following a shift to acetate medium, despite an inability to grow on that carbon source; essentially all of the cofactor was in the oxidized form within 36 h. The salvage pathway for NAD(H) biosynthesis was found to be particularly important for viability during early transition of the parental strain to stationary phase in acetate medium. However, oxygen consumption was not affected, suggesting that the NAD(H) produced during this time may support other cellular functions. The idhΔ mutant exhibited increased flux through the salvage pathway in acetate medium but was dependent on the de novo pathway for viability. Long-term chronological lifespans of the parental and idhΔ strains were similar, but viability of the mutant strain was dependent on both pathways for NAD(H) biosynthesis.     

On the Nature of the Subjectivity of Living Things

A biosemiotic view of living things is presented that supersedes the mechanistic view of life prevalent in biology today. Living things are active agents with autonomous subjectivity, whose structure is triadic, consisting of the individual organism, its Umwelt and the society. Sociality inheres in every living thing since the very origin of life on the earth. The temporality of living things is guided by the purpose to live, which works as the semantic boundary condition for the processes of embodiment of the subjectivity. Freedom at the molecular and cellular levels allows autonomy and spontaneity to emerge even in single cell organisms, and the presence of the dimension of mind in every living thing is deduced. Living things transcend their individualness, as they live in historically formed higher order structure consisting of the lineage-species and the society. They also transcend materiality, having the dimension of mind.     

Inbreeding levels in Northeast Brazil: Strategies for the prospecting of new genetic disorders

A new autosomal recessive genetic condition, the SPOAN syndrome (an acronym for spastic paraplegia, optic atrophy and neuropathy syndrome), was recently discovered in an isolated region of the State of Rio Grande do Norte in Northeast Brazil, in a population that was identified by the IBGE (Brazilian Institute of Geography and Statistics) as belonging to the Brazilian communities with the highest rates of "deficiencies" (Neri, 2003), a term used to describe diseases, malformations, and handicaps in general. This prompted us to conduct a study of consanguinity levels in five of its municipal districts by directly interviewing their inhabitants. Information on 7,639 couples (corresponding to about 40% of the whole population of the studied districts) was obtained. The research disclosed the existence of very high frequencies of consanguineous marriages, which varied from about 9% to 32%, suggesting the presence of a direct association between genetic diseases such as the SPOAN syndrome, genetic drift and inbreeding levels. This fact calls for the introduction of educational programs for the local populations, as well as for further studies aiming to identify and characterize other genetic conditions. Epidemiological strategies developed to collect inbreeding data, with the collaboration of health systems available in the region, might be very successful in the prospecting of genetic disorders.     

Effects of genetic variants on lipid parameters and dyslipidemia in a Chinese population

A number of recent genome-wide association (GWA) studies have identified several novel genetic determinants of plasma lipid and lipoprotein concentrations in European populations. However, it is still unclear whether these loci identified in Caucasian GWA studies also exert the same effect on lipid and lipoprotein concentrations in a Chinese population. We genotyped 10 single-nucleotide polymorphisms (SNPs) in nine loci in a Chinese Han population sample (n = 4,192) and assessed the associations of these SNPs with metabolic traits, using linear regression adjusted for age, gender, diabetes status, and body mass index. Three variants (rs12654264, P ～ 1.7 × 10(-6); rs3764261, P ～ 7.1 × 10(-7); and rs4420638, P ～ 1.1 × 10(-3)) showed strong evidence for association with total cholesterol; four variants (rs780094, P ～ 1.8 × 10(-11); rs17145738, P ～ 5.0 × 10(-7); rs326, P ～ 2.3 × 10(-6); and rs439401, P ～ 2.2 × 10(-5)) showed strong evidence for association with triglycerides, four variants (rs17145738, P ～ 1.9 × 10(-4); rs326, P ～ 9.7 × 10(-4); rs1800588, P ～ 1.5 × 10(-7); and rs3764261, P ～ 4.3 × 10(-14)) showed strong evidence for association with HDL-cholesterol (HDL-C), two variants (rs12654264, P ～ 2.3 × 10(-5); and rs4420638, P ～ 3.6 × 10(-4)) showed strong evidence for association with LDL-C, and four variants (rs326, P ～ 2.8 × 10(-3); rs1800588, P ～ 6.1 × 10(-4); rs3764261, P ～ 2.0 × 10(-3); and rs4420638, P ～ 9.4 × 10(-5)) showed strong evidence for association with total cholesterol-HDL-C-related ratio. These SNPs generated strong combined effects on lipid traits and dyslipidemia. Our findings indicate that the variants that associated with metabolic traits in Europeans may also play a role in a Chinese Han population.     

Ubiquitin is a novel substrate for human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (k_cat = 2 s^− 1) followed by a slow cleavage between residues 72 and 73 (k_cat = 0.07 s^−  1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG <  0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.     

Structural basis for the allosteric regulation and substrate recognition of human cytosolic 5'-nucleotidase II

Cytosolic 5′-nucleotidase II (cN-II) catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5′-monophosphates and participates in the regulation of purine nucleotide pools within the cell. It interferes with the phosphorylation-dependent activation of nucleoside analogues used in the treatment of cancer and viral diseases. It is allosterically activated by a number of phosphate-containing cellular metabolites such as ATP, diadenosine polyphosphates, and 2,3-bisphosphoglycerate, which couple its activity with the metabolic state of the cell. We present seven high-resolution structures of human cN-II, including a ligand-free form and complexes with various substrates and effectors. These structures reveal the structural basis for the allosteric activation of cN-II, uncovering a mechanism where an effector-induced disorder-to-order transition generates rearrangements within the catalytic site and the subsequent coordination of the catalytically essential magnesium. Central to the activation is the large transition of the catalytically essential Asp356. This study also provides the structural basis for the substrate specificity of cN-II, where Arg202, Asp206, and Phe157 seem to be important residues for purine/pyrimidine selectivity. These structures provide a comprehensive structural basis for the design of cN-II inhibitors. They also contribute to the understanding of how the nucleotide salvage pathway is regulated at a molecular level.     

Crystal structure of the passenger domain of the Escherichia coli autotransporter EspP

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β-domain” responsible for translocation of the passenger across the bacterial outer membrane. Here, we present the 2.5-Å crystal structure of the passenger domain of the extracellular serine protease EspP, produced by the pathogen Escherichia coli O157:H7 and a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs). Like the previously structurally characterized SPATE passenger domains, the EspP passenger domain contains an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. We describe the structure of the EspP passenger domain in the context of previous results and provide an alternative hypothesis for the function of the β-helix within SPATEs.