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PubMed2025

An updated compendium and reevaluation of the evidence for nuclear transcription factor occupancy over the mitochondrial genome.

In most eukaryotes, mitochondrial organelles contain their own genome, usually circular, which is the remnant of the genome of the ancestral bacterial endosymbiont that gave rise to modern mitochondria. Mitochondrial genomes are dramatically reduced in their gene content due to the process of endosymbiotic gene transfer to the nucleus; as a result most mitochondrial proteins are encoded in the nucleus and imported into mitochondria. This includes the components of the dedicated mitochondrial transcription and replication systems and regulatory factors, which are entirely distinct from the information processing systems in the nucleus. However, since the 1990s several nuclear transcription factors have been reported to act in mitochondria, and previously we identified 8 human and 3 mouse transcription factors (TFs) with strong localized enrichment over the mitochondrial genome using ChIP-seq (Chromatin Immunoprecipitation) datasets from the second phase of the ENCODE (Encyclopedia of DNA Elements) Project Consortium. Here, we analyze the greatly expanded in the intervening decade ENCODE compendium of TF ChIP-seq datasets (a total of 6,153 ChIP experiments for 942 proteins, of which 763 are sequence-specific TFs) combined with interpretative deep learning models of TF occupancy to create a comprehensive compendium of nuclear TFs that show evidence of association with the mitochondrial genome. We find some evidence for chrM occupancy for 50 nuclear TFs and two other proteins, with bZIP TFs emerging as most likely to be playing a role in mitochondria. However, we also observe that in cases where the same TF has been assayed with multiple antibodies and ChIP protocols, evidence for its chrM occupancy is not always reproducible. In the light of these findings, we discuss the evidential criteria for establishing chrM occupancy and reevaluate the overall compendium of putative mitochondrial-acting nuclear TFs.

Genome, Mitochondrial↗

PubMed2005

Give lipids a START: the StAR-related lipid transfer (START) domain in mammals.

The steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain is a protein module of approximately 210 residues that binds lipids, including sterols. Fifteen mammalian proteins, STARD1-STARD15, possess a START domain and these can be grouped into six subfamilies. Cholesterol, 25-hydroxycholesterol, phosphatidylcholine, phosphatidylethanolamine and ceramides are ligands for STARD1/STARD3/STARD5, STARD5, STARD2/STARD10, STARD10 and STARD11, respectively. The lipids or sterols bound by the remaining 9 START proteins are unknown. Recent studies show that the C-terminal end of the domain plays a fundamental role, forming a lid over a deep lipid-binding pocket that shields the ligand from the external environment. The START domain can be regarded as a lipid-exchange and/or a lipid-sensing domain. Mammalian START proteins have diverse expression patterns and can be found free in the cytoplasm, attached to membranes or in the nucleus. They appear to function in a variety of distinct physiological processes, such as lipid transfer between intracellular compartments, lipid metabolism and modulation of signaling events. Mutation or misexpression of START proteins is linked to pathological processes, including genetic disorders, autoimmune disease and cancer.

Animals↗

PubMed1992

[Experimental study on a skin substitute. Artificial dermis epidermised by human keratinocytes].

In patients with third-degree burns, plastic surgeons must meet emergent requirements for skin coverage. Conventional autografts do not provide enough substance to cover these patients, hence the interest of skin substitutes. The aim of our work is to achieve epidermization of an artificial dermis with a technique of culture of human keratocytes, thus producing in vitro a dermoepidermal skin substitute akin to normal skin, and to implant it in nude mice. Human keratocytes are grown in a definite medium (MCDB 153) on an artificial dermis based on type I and III collagen and glycosaminoglycans, and cross-linked with chitosan. Human keratocytes from thin skin were seeded on the artificial dermis after a last passage in culture fials. Cell density and the number of days in vitro were varied on patches of artificial dermis. After culture, the patches were transferred on nude mice. These were sacrificed on the 7th or 14th day. Cell recolonization of the artificial dermis was then studied, as well as the growth of the epidermis on deep-frozen histologic sections. Primary cultures on an artificial dermis did not show production of a satisfactory epidermis. In some animals grafted with artificial dermis epidermized in a secondary culture, we observed a structure similar to that of normal skin. This work allowed outlining the problems to slove and contemplating the solutions required to develop a total artificial skin.

Animals↗

PubMed2000

Inhalation of poorly soluble particles. I. Differences in inflammatory response and clearance during exposure.

Results from animal studies have indicated some uncertainties over the validity of a single general occupational control limit for all types of "particulates (insoluble) not otherwise classified" (PNOC) (ACGIH, 2000). Therefore, to examine the extent to which a given control limit may be valid for nontoxic dusts with different physical characteristics, this study compared the pulmonary effects in rats of inhalation exposure to two poorly soluble dusts of similar density and with relatively low toxicity: titanium dioxide and barium sulfate. The objectives were to compare the dusts in (a) their buildup and clearance in the lungs during inhalation; (b) their transfer to lymph nodes; (c) the changes, with time, in the lavageable cell population; and (d) the pathological change from histology. The exposure aerosol concentrations were selected to achieve similar mass and volume lung burdens for both dusts and to attain "overload" over the common exposure periods of about 4 mo and 7 mo. Despite obtaining similar lung burdens for both dusts, there was significantly more translocation of TiO(2) to the hilar lymph nodes than with BaSO(4). It was also found that clearance of TiO(2) was retarded whereas clearance of BaSO(4) was not. Trends in these data were clarified by the use of a simple model of particle clearance. Retardation of particle clearance and translocation to the lymph nodes are markers of the condition known as "overload" in which the alveolar macrophage-based clearance of particles from the deep lung is impaired. In addition, bronchoalveolar lavage showed that TiO(2) caused significantly more recruitment of inflammatory neutrophils to lungs than BaSO(4). These differences between the dusts were not due to differences in toxicity, solubility, or lung deposition. The explanation that the different responses are due to the different particle size distributions of the two dust types is examined in a companion paper (Tran et al., this issue).

Administration, Inhalation↗

PubMed1985

From the spinning disc to the membrane oxygenator for open-heart surgery.

Gibbon's rotating cylinder could not be enlarged to oxygenate an animal larger than a cat. The spinning disc oxygenator, introduced in 1947, had the capacity to perfuse a dog and the potential to increase oxygenation capacity by addition of more discs. When centers began to do three to four open-heart operations per day, the disposable bubble oxygenator was more practical. Bubble size was optimized to decrease the flow of oxygen relative to the blood flow and reduce trauma to blood. The bubble oxygenator is the type most commonly used today. Use of deep hypothermia with whole blood at an esophageal temperature of 10 degrees C was initially complicated by brain damage due to aggregation of white blood corpuscles and platelets. The introduction of hemodilution permitted safe utilization of hypothermic perfusion. Perfusion of infants should not be carried out at hematocrit below 25 ml/100 m. Early membrane oxygenators used nonporous silicone, or modified silicone membranes. High priming volumes, high pressure drop and marginal gas transfer efficiency characterized these devices. Recent advances in membrane technology have spawned a new generation of membrane oxygenators utilizing microporous polypropylene. In these new oxygenators, with either microporous hollow fibers or sheet membrane, the gas transfer characteristics are far superior to those of types produced in the past. The hollow-fiber devices typically have larger surface areas and higher pressure drop than in the new state-of-the-art flat plate models. An evaluation of one of these new-generation membrane oxygenators gave optimal oxygen and carbon dioxide exchange at a gas flow of 1 l/min of 60% oxygen in air at 30 degrees C and 2 l/min of 80% oxygen in air at normal temperature and rewarming for an adult. Today, after almost 40 years of oxygenator development, these new membrane device can offer better platelet preservation and reduced blood trauma as compared with types developed in the past. The new membrane oxygenators are fast becoming the preferred choice for use in infants and in protracted perfusion.

Age Factors↗

PubMed2004

Overexpression, purification, biochemical characterization, and molecular modeling of recombinant GDP-mannosyltransferase (GumH) from Xylella fastidiosa.

The GumH enzyme from Xylella fastidiosa catalyzes the transfer reaction of a mannose from GDP-mannose to the carrier lipid cellobiose-pyrophosphate-polyprenol (Glc(2)-PP-Lip), an intermediary in the reaction for the synthesis of the exopolysaccharide (EPS) fastidian gum. The gumH gene was subcloned in the pMal-c2x vector, allowing the expression of the GumH-MBP fusion protein. Various attempts were made to obtain protein with the necessary degree of purity for crystallographic studies but the yield was very low. The gumH gene was then subcloned in the pET28a vector allowing the expression of the GumH enzyme in fusion with a histidine-rich peptide. The protein was purified and characterized. The three-dimensional structure of the X. fastidiosa GumH enzyme was modeled by threading studies. The model consists of N- and C-terminal domains similar in size and topology and separated by a deep cleft, which includes the EX(7)E motif that can be involved in the catalysis of GumH.

Amino Acid Motifs↗

PubMed2006

Vicariance and dispersal effects on phylogeographic structure and speciation in a widespread estuarine invertebrate.

Vicariance and dispersal can strongly influence population genetic structure and allopatric speciation, but their importance in the origin of marine biodiversity is unresolved. In transitional estuarine environments, habitat discreteness and dispersal barriers could enhance divergence and provide insight to evolutionary mechanisms underlying marine and freshwater biodiversity. We examined this by assessing phylogeographic structure in the widespread amphipod Gammarus tigrinus across 13 estuaries spanning its northwest Atlantic range from Quebec to Florida. Mitochondrial cytochrome c oxidase I and nuclear internal transcribed spacer 1 phylogenies supported deep genetic structure consistent with Pliocene separation and cryptic northern and southern species. This break occurred across the Virginian-Carolinian coastal biogeographic zone, where an oceanographic discontinuity may restrict gene flow. Ten estuarine populations of the northern species occurred in four distinct clades, supportive of Pleistocene separation. Glaciation effects on genetic structure of estuarine populations are largely unknown, but analysis of molecular variance (AMOVA) supported a phylogeographic break among clades in formerly glaciated versus nonglaciated areas across Cape Cod, Massachusetts. This finding was concordant with patterns in other coastal species, though there was no significant relationship between latitude and genetic diversity. This supports Pleistocene vicariance events and divergence of clades in different northern glacial refugia. AMOVA results and private haplotypes in most populations support an allopatric distribution across estuaries. Clade mixture zones are consistent with historical colonization and human-mediated transfer. An isolation-by-distance model of divergence was detected after we excluded a suspected invasive haplotype in the St. Lawrence estuary. The occurrence of cryptic species and divergent population structure support limited dispersal, dispersed habitat distribution, and historical factors as important determinants of estuarine speciation and diversification.

Amphipoda↗

PubMed2006

Engineering luminescent quantum dots for in vivo molecular and cellular imaging.

Semiconductor quantum dots are luminescent nanoparticles that are under intensive development for use as a new class of optical imaging contrast agents. Their novel properties such as optical tunability, improved photostability, and multicolor light emission have opened new opportunities for imaging living cells and in vivo animal models at unprecedented sensitivity and spatial resolution. Combined with biomolecular engineering strategies for tailoring the particle surfaces at the molecular level, bio-conjugated quantum dot probes are well suited for imaging single-molecule dynamics in living cells, for monitoring protein-protein interactions within specific intracellular locations, and for detecting diseased sites and organs in deep tissue. In this article, we describe the engineering principles for preparing high-quality quantum dots and for conjugating the dots to biomolecular ligands. We also discuss recent advances in using quantum dots for in vivo molecular and cellular imaging.

Animals↗

PubMed2005

Fate of rising CO2 droplets in seawater.

The sequestration of fossil fuel CO2 in the deep ocean has been discussed by a number of workers, and direct ocean experiments have been carried out to investigate the fate of rising CO2 droplets in seawater. However, no applicable theoretical models have been developed to calculate the dissolution rate of rising CO2 droplets with or without hydrate shells. Such models are important for the evaluation of the fate of CO2 injected into oceans. Here, I adapt a convective dissolution model to investigate the dynamics and kinetics of a single rising CO2 droplet (or noninteracting CO2 droplets) in seawater. The model has no free parameters; all of the required parameters are independently available from literature. The input parameters include: the initial depth, the initial size of the droplet, the temperature as a function of depth, density of CO2 liquid, the solubility of CO2 liquid or hydrate, the diffusivity of CO2, and viscosity of seawater. The effect of convection in enhancing mass transfer is treated using relations among dimensionless numbers. The calculated dissolution rate for CO2 droplets with a hydrate shell agrees with data in the literature. The theory can be used to explore the fate of CO2 injected into oceans under various temperature and pressure conditions.

Carbon Dioxide↗

PubMed1997

X-ray crystallography reveals a large conformational change during guanyl transfer by mRNA capping enzymes.

We have solved the crystal structure of an mRNA capping enzyme at 2.5 A resolution. The enzyme comprises two domains with a deep, but narrow, cleft between them. The two molecules in the crystallographic asymmetric unit adopt very different conformations; both contain a bound GTP, but one protein molecule is in an open conformation while the other is in a closed conformation. Only in the closed conformation is the enzyme able to bind manganese ions and undergo catalysis within the crystals to yield the covalent guanylated enzyme intermediate. These structures provide direct evidence for a mechanism that involves a significant conformational change in the enzyme during catalysis.

Binding Sites↗

PubMed2003

Crystal structure of Mycobacterium tuberculosis MenB, a key enzyme in vitamin K2 biosynthesis.

Bacterial enzymes of the menaquinone (Vitamin K2) pathway are potential drug targets because they lack human homologs. MenB, 1,4-dihydroxy-2-naphthoyl-CoA synthase, the fourth enzyme in the biosynthetic pathway leading from chorismate to menaquinone, catalyzes the conversion of O-succinylbenzoyl-CoA (OSB-CoA) to 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA). Based on our interest in developing novel tuberculosis chemotherapeutics, we have solved the structures of MenB from Mycobacterium tuberculosis and its complex with acetoacetyl-coenzyme A at 1.8 and 2.3 A resolution, respectively. Like other members of the crotonase superfamily, MenB folds as an (alpha3)2 hexamer, but its fold is distinct in that the C terminus crosses the trimer-trimer interface, forming a flexible part of the active site within the opposing trimer. The highly conserved active site of MenB contains a deep pocket lined by Asp-192, Tyr-287, and hydrophobic residues. Mutagenesis shows that Asp-192 and Tyr-287 are essential for enzymatic catalysis. We postulate a catalytic mechanism in which MenB enables proton transfer within the substrate to yield an oxyanion as the initial step in catalysis. Knowledge of the active site geometry and characterization of the catalytic mechanism of MenB will aid in identifying new inhibitors for this potential drug target.

Acyl Coenzyme A↗

PubMedDate not supplied

Low-selenium diet, bone, and articular cartilage in rats.

The pathophysiology of secondary osteoarthritis remains largely obscure. Our attention has been drawn to Kashin-Beck disease (KBD), which has been attributed to Se deficiency. To obtain information regarding the prevention, prediction of progression, and treatment of this condition, we performed histological and biochemical studies on bone and articular cartilage specimens obtained from rats fed a low-Se diet. A low-Se diet was prepared and fed to Wistar rats for 3-11 mo, after which the rats were killed under general anesthesia, and their articular cartilages were studied microscopically and electron microscopically. The bone mineral density (BMD) of the femur was determined by the microdensitometry method and ash weight. In addition, serum Se, Ca, P, Alk Phos, T3, T4, and urinary Se were measured. In the low-Se group, impaired weight gain was observed from the 5th mo, and head alopecia was found in 60% of the animals. Microscopically, no clear changes in the articular chondrocytes were apparent, whereas with the electron microscope, chondrocytes in the deep layer showed degeneration of nuclei and endoplasmic reticular ballooning. From the 5th mo, a decrease in BMD (ash weight) was noted. Serum Se concentrations, alkaline phosphatase activity, and urine Se concentrations were decreased in the Se-deficient rats, whereas serum Ca, P, T3, and T4 values did not differ from those of a control group. Also, a decrease in sulfotransferase activity, which is involved in transfer in the process of synthesis of glycosaminoglycan, which is a proteoglycan carbohydrate chain, was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed1990

A new approach to pharmacokinetic parameters: estimation of cefuroxime during haemodialysis.

A linear three-compartment model is proposed as a means of estimating disposition and extracorporeal elimination pharmacokinetic parameters during haemodialysis. It was created by connecting a compartment, corresponding to the amount of drug present in the dialysis cell, to the central compartment of the standard two-compartment model from which elimination would normally take place. The transfer rate constants between the central compartment and the 'dialysis cell' and vice versa were given in terms of the plasma flow, central compartment volume, and volume of plasma contained in the dialysis cell, and thus cannot be considered to be independent parameters. The product of the rate constant for elimination from the dialysis cell, k30, and the plasma volume within the cell was used to quantify the extracorporeal elimination and termed intrinsic dialyser clearance, CLint,D. The model was used to interpret the plasma level curves obtained after administering 750 mg cefuroxime by IV bolus to a group of patients undergoing haemodialysis. The distribution parameters estimated by non-linear regression were similar to those given in the literature; however, in five of the twelve cases, no estimate of cefuroxime elimination from the central compartment (k10) could be derived. On the other hand, the estimates of the elimination rate constant (k30) from the deep peripheral compartment were greater than the values given in the literature, ranging from 50.6 h-1 to 151.0 h-1 and CLint,D ranging from 3.1 h-1 to 8.90 l h-1. The error in measuring the flow of plasma which reaches the dialyser, its influence upon the estimation of the model parameters and the relationship between the parameters themselves and those customarily used in the study of drug haemodialysis are all discussed.

Cefuroxime↗

PubMed2005

Response of CR-39 to medium energy electron irradiation.

The effect of 5-15 keV electron irradiation on the etch rate of CR-39 nuclear track detectors has been investigated for surface doses from 0.09 to 13.85 J/cm2. Even before etching, electrons produce a surface depression proportional to the surface dose raised to the power 0.78 +/- 0.01. Etch rate enhancement was observed in the surface region (about 4 micrometers deep) and was well approximated by a Gaussian function of depth. The depth and thickness of the etch rate enhancement increase with increasing electron energy, but are independent of dose. The maximum etch rate is proportional to the peak volumetric dose raised to the power of 1.45 +/- 0.06, independent of electron energy. No signs of a saturated response were found in the etch rate. This enhanced etching rate may significantly affect nuclear track identification in situations with an electron background.

Dose-Response Relationship, Radiation↗

PubMed2001

Optimized shielding for space radiation protection.

Future deep space mission and International Space Station exposures will be dominated by the high-charge and -energy (HZE) ions of the Galactic Cosmic Rays (GCR). A few mammalian systems have been extensively tested over a broad range of ion types and energies. For example, C3H10T1/2 cells, V79 cells, and Harderian gland tumors have been described by various track-structure dependent response models. The attenuation of GCR induced biological effects depends strongly on the biological endpoint, response model used, and material composition. Optimization of space shielding is then driven by the nature of the response model and the transmission characteristics of the given material.

Aluminum↗

PubMed2006

Solution structure of a post-transition state analog of the phosphotransfer reaction between the A and B cytoplasmic domains of the mannitol transporter IIMannitol of the Escherichia coli phosphotransferase system.

The solution structure of the post-transition state complex between the isolated cytoplasmic A (IIAMtl) and phosphorylated B (phospho-IIBMtl) domains of the mannitol transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-554 of IIAMtl was mutated to glutamine to block phosphoryl transfer activity, and the active site Cys-384 of IIBMtl (residues of IIBMtl are denoted in italic type) was substituted by serine to permit the formation of a stable phosphorylated form of IIBMtl. The two complementary interaction surfaces are predominantly hydrophobic, and two methionines on IIBMtl, Met-388 and Met-393, serve as anchors by interacting with two deep pockets on the surface of IIAMtl. With the exception of a salt bridge between the conserved Arg-538 of IIAMtl and the phosphoryl group of phospho-IIBMtl, electrostatic interactions between the two proteins are limited to the outer edges of the interface, are few in number, and appear to be weak. This accounts for the low affinity of the complex (Kd approximately 3.7 mm), which is optimally tuned to the intact biological system in which the A and B domains are expressed as a single polypeptide connected by a flexible 21-residue linker. The phosphoryl transition state can readily be modeled with no change in protein-protein orientation and minimal perturbations in both the backbone immediately adjacent to His-554 and Cys-384 and the side chains in close proximity to the phosphoryl group. Comparison with the previously solved structure of the IIAMtl-HPr complex reveals how IIAMtl uses the same interaction surface to recognize two structurally unrelated proteins and explains the much higher affinity of IIAMtl for HPr than IIBMtl.

Amino Acid Sequence↗

PubMed1997

A clinical and microbiological study of deep carious lesions during stepwise excavation using long treatment intervals.

Concern about the survival of microorganisms in deep carious lesions may often lead to unnecessary exposure of the pulp during final excavation. There are reasons, therefore, to initiate systematic studies on the alternative procedure known as stepwise excavation. Clinical evaluation of stepwise excavation was performed on 31 deep carious lesions considered to result in pulp perforation by traditional excavation. This study examines the clinical and microbiological alterations during the final excavation performed during long intervals (6-12 months) after the initial treatment that included peripheral dentine excavation and removal of the central cariogenic biomass and the superficial necrotic dentine. The dentine colour and consistency were assessed by means of standardized scales before application of a Ca(OH)2 compound and a temporary sealing for 6-12 months. Reassessments were performed before the after final excavation. Microbiological dentine samples were obtained in 19 randomly selected lesions by a sterile bur, transferred to and diluted in reduced transport fluid, and plated on tryptic soy agar. After anaerobic incubation at 37 degrees C for 7 days, total colony-forming units per millilitre were counted from (1) peripheral excavated and hard dentine (control), (2) central demineralized dentine before and final excavation, and (3) central dentine after the final excavation. Six samples of central demineralized dentine were without any cultivable flora increasing to 9 samples after the final excavation. The clinical dentine changes occurring during stepwise excavation were characterized by enhanced hardness of the dentine which was associated with a marked reduction in bacterial growth after the final excavation. Despite the presence of bacteria in the excavated dentine none of the carious lesions resulted in pulp perforation, suggesting that the initial removal of the cariogenic biomass appears to be essential for control of caries progression. Stepwise excavation is not only an appropriate treatment of deep carious lesions but is also considered a suitable model for microbiological studies to determine the bacteria persisting in clinically excavated lesions.

Anaerobiosis↗

PubMed1995

A role for quaternary structure in the substrate specificity of leucine dehydrogenase.

BACKGROUND: Glutamate, phenylalanine and leucine dehydrogenases catalyze the NAD(P)(+)-linked oxidative deamination of L-amino acids to the corresponding 2-oxoacids, and sequence homology between these enzymes clearly indicates the existence of an enzyme superfamily related by divergent evolution. We have undertaken structural studies on a number of members of this family in order to investigate the molecular basis of their differential amino acid specificity. RESULTS: We have solved the X-ray structure of the leucine dehydrogenase from Bacillus sphaericus to a resolution of 2.2 A. Each subunit of this octameric enzyme contains 364 amino acids and folds into two domains, separated by a deep cleft. The nicotinamide ring of the NAD+ cofactor binds deep in this cleft, which is thought to close during the hydride transfer step of the catalytic cycle. CONCLUSIONS: Comparison of the structure of leucine dehydrogenase with a hexameric glutamate dehydrogenase has shown that these two enzymes share a related fold and possess a similar catalytic chemistry. A mechanism for the basis of the differential amino acid specificity between these enzymes involves point mutations in the amino acid side-chain specificity pocket and subtle changes in the shape of this pocket caused by the differences in quaternary structure.

Amino Acid Oxidoreductases↗