Human NADH:Ubiquinone Oxidoreductase

NADH:ubiquinone oxidoreductase consists of at least 43 proteins; seven are encoded by the mitochondrial genome, while the remainder are encoded by the nuclear genome. A deficient activity of this enzyme complex is frequently observed in the clinical heterogeneous group of mitochondrial disorders, with Leigh (-like) disease as the main contributor. Enzyme complex activity measurement in skeletal muscle is the mainstay of the diagnostic process. Fibroblast studies are a prerequisite whenever prenatal enzyme diagnosis is considered. Mitochondrial DNA mutations are found in approximately 5–10% of all complex I deficiencies. Recently, all structural nuclear complex I genes have been determined at the cDNA level and several at the gDNA level. A comprehensive mutational analysis study of all complex I nuclear genes in a group of 20 patients exhibiting this deficiency revealed mutations in about 40%. Here, we describe the enzymic methods we use and the recent progress made in genomics and cell biology of human complex I.     

Towards understanding of the complex structure of growing yeast populations

In a growing Saccharomyces cerevisiae population, cell size is finely modulated according to both the chronological and genealogical ages. This generates the complex heterogeneous structure typical of budding yeast populations. In recent years, there has been a growing interest in developing mathematical models capable of faithfully describing population dynamics at the single cell level. A multistaged morphologically structured model has been lately proposed based on the population balance theory. The model was able to describe the dynamics of the generation of a heterogeneous growing yeast population starting from a sub-population of daughter unbudded cells. In this work, which aims at validating the model, the simulated experiment was performed by following the release of a homogeneous population of daughter unbudded cells. A biparametric flow cytometric approach allowed us to analyse the time course joint distribution of DNA and protein contents at the single cell level; this gave insights into the coupling between growth and cell cycle progression that generated the final population structure. The comparison between experimental and simulated size distributions revealed a strong agreement for some unexpected features as well. Therefore, the model can be considered as validated and extendable to more complex situations.     

Atomistic modeling of water diffusion in hydrolytic biomaterials

One of the most promising applications of hydrolytically degrading biomaterials is their use as drug release carriers. These uses, however, require that the degradation and diffusion of drug are reliably predicted, which is complex to achieve through present experimental methods. Atomistic modeling can help in the knowledge-based design of degrading biomaterials with tuned drug delivery properties, giving insights on the small molecules diffusivity at intermediate states of the degradation process. We present here an atomistic-based approach to investigate the diffusion of water (through which hydrolytic degradation occurs) in degrading bulk models of poly(lactic acid) or PLA. We determine the water diffusion coefficient for different swelling states of the polymeric matrix (from almost dry to pure water) and for different degrees of degradation. We show that water diffusivity is highly influenced by the swelling degree, while little or not influenced by the degradation state. This approach, giving water diffusivity for different states of the matrix, can be combined with diffusion-reaction analytical methods in order to predict the degradation path on longer time scales. Furthermore, atomistic approach can be used to investigate diffusion of other relevant small molecules, eventually leading to the a priori knowledge of degradable biomaterials transport properties, helping the design of the drug delivery systems.     

A minimized motile machinery for Mycoplasma genitalium

The cell wall‐less bacterium Mycoplasma genitalium uses specialized adhesins located at the terminal organelle to adhere to host cells and surfaces. The terminal organelle is a polar structure protruding from the cell body that is internally supported by a cytoskeleton and also has an important role in cell motility. We have engineered a M. genitalium null mutant for MG491 protein showing a massive downstream destabilization of proteins involved in the terminal organelle organization. This mutant strain exhibited striking similarities with the previously isolated MG_218 null mutant strain. Upon introduction of an extra copy of MG_318 gene in both strains, the amount of main adhesins P140 and P110 dramatically increased. These strains were characterized by microcinematography, epifluorescence microscopy and cryo‐electron microcopy, revealing the presence of motile cells and filaments in the absence of many proteins considered essential for cell adhesion and motility. These results indicate that adhesin complexes play a major role in the motile machinery of M. genitalium and demonstrate that the rod element of the cytoskeleton core is not the molecular motor propelling mycoplasma cells. These strains containing a minimized motile machinery also provide a valuable cell model to investigate the adhesion and gliding properties of this human pathogen.     

Evolution and diversity of the Golgi

The Golgi is an ancient and fundamental eukaryotic organelle. Evolutionary cell biological studies have begun establishing the repertoire, processes, and level of complexity of membrane-trafficking machinery present in early eukaryotic cells. This article serves as a review of the literature on the topic of Golgi evolution and diversity and reports a novel comparative genomic survey addressing Golgi machinery in the widest taxonomic diversity of eukaryotes sampled to date. Finally, the article is meant to serve as a primer on the rationale and design of evolutionary cell biological studies, hopefully encouraging readers to consider this approach as an addition to their cell biological toolbox. It is clear that the major machinery involved in vesicle trafficking to and from the Golgi was already in place by the time of the divergence of the major eukaryotic lineages, nearly 2 billion years ago. Much of this complexity was likely generated by an evolutionary process involving gene duplication and coevolution of specificity encoding membrane-trafficking proteins. There have also been clear cases of loss of Golgi machinery in some lineages as well as innovation of novel machinery. The Golgi is a wonderfully complex and diverse organelle and its continued exploration promises insight into the evolutionary history of the eukaryotic cell.     

Exploring the Effect of Hesperetin–HSPC Complex—A Novel Drug Delivery System on the In Vitro Release, Therapeutic Efficacy and Pharmacokinetics

Hesperetin is known to exhibit a variety of pharmacological activities in mammalian cell systems. Although it shows appreciable bioavailability when administered orally, its faster elimination from body creates the need of frequent administration to maintain effective plasma concentration. To overcome this limitation, a phospholipid complex of hesperetin was prepared and evaluated for antioxidant activity and pharmacokinetic profile. The hesperetin content of the complex was determined by a spectrophotometer and the surface characteristics of the complex were studied by means of microscope. The antioxidant activity was evaluated in carbon-tetrachloride-intoxicated rats at a dose level of 100 mg/kg body weight, p.o. The complex was studied for in vitro drug release characteristics and effect of complexation on serum concentration of hesperetin in rats was also studied along with main pharmacokinetic parameters. The results showed that the complex has a sustained release property and enhanced antioxidant activity (P < 0.05 and <0.01) as compared to free hesperetin at the same dose level. Pharmacokinetic study depicted that the complex has higher relative bioavailability and acted for a longer period of time. The study therefore suggests that phospholipid complex of hesperetin produced better antioxidant activity than free drug at the same dose level and the effect persisted for a longer period of time, which may be helpful in solving the problems of faster elimination of the molecule.     

Continuum heterogeneous biofilm model--a simple and accurate method for effectiveness factor determination

We present a novel analytical approach to describe biofilm processes considering continuum variation of both biofilm density and substrate effective diffusivity. A simple perturbation and matching technique was used to quantify biofilm activity using the steady-state diffusion-reaction equation with continuum variable substrate effective diffusivity and biofilm density, along the coordinate normal to the biofilm surface. The procedure allows prediction of an effectiveness factor, η, defined as the ratio between the observed rate of substrate utilization (reaction rate with diffusion resistance) and the rate of substrate utilization without diffusion limitation. Main assumptions are that (i) the biofilm is a continuum, (ii) substrate is transferred by diffusion only and is consumed only by microorganisms at a rate according to Monod kinetics, (iii) biofilm density and substrate effective diffusivity change in the x direction, (iv) the substrate concentration above the biofilm surface is known, and (v) the substratum is impermeable. With this approach one can evaluate, in a fast and efficient way, the effect of different parameters that characterize a heterogeneous biofilm and the kinetics of the rate of substrate consumption on the behavior of the biological system. Based on a comparison of η profiles the activity of a homogeneous biofilm could be as much as 47.8% higher than that of a heterogeneous biofilm, under the given conditions. A comparison of η values estimated for first order kinetics and η values obtained by numerical techniques showed a maximum deviation of 1.75% in a narrow range of modified Thiele modulus values. When external mass transfer resistance, is also considered, a global effectiveness factor, η(0) , can be calculated. The main advantage of the approach lies in the analytical expression for the calculation of the intrinsic effectiveness factor η and its implementation in a computer program. For the test cases studied convergence was achieved quickly after four or five iterations. Therefore, the simulation and scale-up of heterogeneous biofilm reactors can be easily carried out.     

Linear bulk diffusion into heterogeneous tissue

In this paper an expression is derived which describes the transient overall uptake of an inert solute by a section of tissue excised with parallel faces and placed upon an impermeable base. The approach diverges from the conventional analyses for perfused tissue (Morales and Smith,Bull. Math. Biophysics,6, 125–141, 1944;7, 47–99, 1945) because the extravascular zone is regarded as a heterogeneous diffusion medium. Account for this is taken by regarding tissue as effectively composed of two phases—a continuous (extracellular) phase similar to water, and a dispersed phase comprising cells of irregular profile. In both phases the relevant mode of uptake is taken as bulk diffusion rather than surface permeation, thus emphasizing the influence of the internal geometry of the tissue upon its overall exchange response.     

Streptomyces species producing the streptovaricin complex

Morphological, cultural and physiological-biochemical properties ofStreptomyces sp. strain 1000 and its antibiotic production were investigated. Antibiotics 1011 (identical with the streptovaricin complex) and 1012 (with antibacterial action) were isolated from the cultural broth of this strain. The overproducing natural variant 1011 was isolated from the population of a strain producing antibiotic 1011 at a concentration of 1000 mg/L (activity of the parent strain represents 41 mg/L only). Comparative taxonomical characteristic ofStreptomyces sp. strain 1000 with strains fromS. spectabilis showed that the strain 1000 differed in some properties and antibiotic production being considered as a new variant ofS. spectabilis. The strain shows an expressed antibiotic activity against G+ as well as G− bacterial and yeasts.     

Diffusion approximation of the stochastic process of microtubule assembly

Microtubules are protein polymers that guide intracellular motility. Stochastic switching of a microtubule between states of elongation, shortening, and pause is described in detail by the dynamic instability (DI) model. Recently we have described the dynamics of microtubules phenomenologically as generalized diffusion of their ends. Genesis of the diffusion dynamics and accuracy of diffusion model are studied in this work. It is shown that wandering of the end of a microtubule undergoing DI asymptotically approaches the Wiener diffusion process. Accuracy of the diffusion approximation is evaluated by comparing its predictions with results of simulation of DI. Stationary distributions of microtubule length and lifetime that are predicted by both models differ qualitatively between two cell types considered. However, predictions of the diffusion model are in each case practically identical to predictions of the DI model being also consistent with experimental data. The peculiar stochastic process of microtubule assembly thus converges at cell scale to a kind of widespread-in-nature diffusion process. This result is considered an example of qualitative change in dynamical properties in transition from the molecular to cellular level of biological organization. Additionally, it suggests employment of diffusion process theory in studying functions of microtubules in the cell.     

Functional membrane diffusion of G-protein coupled receptors

G-protein-coupled receptor function involves interactions between the receptor, G-proteins and effectors in the cell plasma membrane. The main biochemical processes have been individually identified but the mechanisms governing the successive protein–protein interactions of this complex multi-molecular machinery have yet to be established. We discuss advances in understanding the functional dynamics of the receptor resulting from diffusion measurements, and in the context of the plasma membrane organization. Aurélie Baker and Aude Saulière contributed equally to this work. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Effects of pH on electron transport system (ETS) activity and oxygen consumption in Gammarus fossarum, Asellus aquaticus and Niphargus sphagnicolus

1. Electron transport system (ETS) activity and oxygen consumption were measured in three crustacean species (Gammarus fossarum, Asellus aquaticus and Niphargus sphagnicolus) that differ in their pH tolerance. Measurements were made under four different pH regimes: strongly acid (pH 4.5–5.0), weakly acid (5.5–6.0), ‘neutral’ (7.0–7.8) and alkaline (8.5–9.0). 2. The significantly lower ETS activity/respiration (ETS/R) ratios observed in strongly acid water than in neutral and alkaline water indicate an effect of acidity on metabolism of the organisms. The main reason for the lower ratios is not increased oxygen consumption (except for G. fossarum in strongly acid water) but decreased ETS activity. Metabolic potential was lower in strongly and weakly acid water than in neutral water. Therefore, efficient exploitation of metabolic potential (i.e. of relatively large production of ATP with the existing enzyme machinery) probably enables N. sphagnicolus and A. aquaticus to survive in an acid environment. 3. Increased oxygen consumption of G. fossarum in strongly acid water indicates an acid stress that leads to the collapse of metabolism and, consequent death of the animals. 4. Although N. sphagnicolus is found exclusively in permanently acid water, no negative effect of alkaline water on metabolism was observed. This species can, therefore, be best considered as an acid‐resistant species, not an acidobiont.     

Methane in Water: An Ab Initio Study

Abstract

In preceding publications we discussed some properties of pure water in condensed phases using an ab initio approach. Here this study is used as a basis of comparison for analysing the behaviour of water as a solvent in the presence of an apolar molecule. Our analysis is focused on the process of organization of the hydrogen bonding network around the solute. For this purpose we perform some ab initio calculations for a system of 32 water molecules and one methane molecule at 300 K; in particular, the average molecular dipole moment of water is determined and the result is compared with that of pure water. Next the attention is switched to the methane molecule; related properties such as excluded volume and sphericity of its shape are illustrated and discussed. A comparison with results obtained using classical approaches suggests that some classical models of water can be considered to be still valid when they are used to analyse the water-methane system.     

Cyclin‐dependent kinase activity maintains the shoot apical meristem cells in an undifferentiated state

As the shoot apex produces most of the cells that comprise the aerial part of the plant, perfect orchestration between cell division rates and fate specification is essential for normal organ formation and plant development. However, the inter‐dependence of cell‐cycle machinery and meristem‐organizing genes is still poorly understood. To investigate this mechanism, we specifically inhibited the cell‐cycle machinery in the shoot apex by expression of a dominant negative allele of the A‐type cyclin‐dependent kinase (CDK) CDKA;1 in meristematic cells. A decrease in the cell division rate within the SHOOT MERISTEMLESS domain of the shoot apex dramatically affected plant growth and development. Within the meristem, a subset of cells was driven into the differentiation pathway, as indicated by premature cell expansion and onset of endo‐reduplication. Although the meristem structure and expression patterns of the meristem identity genes were maintained in most plants, the reduced CDK activity caused splitting of the meristem in some plants. This phenotype correlated with the level of expression of the dominant negative CDKA;1 allele. Therefore, we propose a threshold model in which the effect of the cell‐cycle machinery on meristem organization is determined by the level of CDK activity.     

Diffusion MRI: a new strategy for assessment of cancer therapeutic efficacy

The use of anatomical imaging in clinical oncology practice traditionally relies on comparison of patient scans acquired before and following completion of therapeutic intervention. Therapeutic success is typically determined from inspection of gross anatomical images to assess changes in tumor size. Imaging could provide significant additional insight into therapeutic impact if a specific parameter or combination of parameters could be identified which reflect tissue changes at the cellular or physiologic level. This would provide an early indicator or treatment response/outcome in an individual patient before completion of therapy. Moreover, response of a tumor to therapeutic intervention may be heterogeneous. The use of imaging could assist in delineating therapeutic-induced spatial heterogeneity within a tumor mass by providing information related to specific regions that are resistant or responsive to treatment. Largely untapped potential resides in exploratory methods such as diffusion MRI, which is a nonvolumetric intravoxel measure of tumor response based upon water molecular mobility. Alterations in water mobility reflect changes in tissue structure at the cellular level. While the clinical utility of diffusion MRI for oncologic practice is still under active investigation, this overview on the use of diffusion MRI for the evaluation of brain tumors will serve to introduce how this approach may be applied in the future for the management of patients with solid tumors.