Escherichia Coli

ERIC Educational Resources Information Center

Goodsell, David S.

2009-01-01

Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

Glycan Engineering for Cell and Developmental Biology.

PubMed

Griffin, Matthew E; Hsieh-Wilson, Linda C

2016-01-21

Cell-surface glycans are a diverse class of macromolecules that participate in many key biological processes, including cell-cell communication, development, and disease progression. Thus, the ability to modulate the structures of glycans on cell surfaces provides a powerful means not only to understand fundamental processes but also to direct activity and elicit desired cellular responses. Here, we describe methods to sculpt glycans on cell surfaces and highlight recent successes in which artificially engineered glycans have been employed to control biological outcomes such as the immune response and stem cell fate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time.

PubMed

Bhat, Supriya V; Sultana, Taranum; Körnig, André; McGrath, Seamus; Shahina, Zinnat; Dahms, Tanya E S

2018-05-29

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Challenges and dreams: physics of weak interactions essential to life.

PubMed

Chien, Peter; Gierasch, Lila M

2014-11-05

Biological systems display stunning capacities to self-organize. Moreover, their subcellular architectures are dynamic and responsive to changing needs and conditions. Key to these properties are manifold weak "quinary" interactions that have evolved to create specific spatial networks of macromolecules. These specific arrangements of molecules enable signals to be propagated over distances much greater than molecular dimensions, create phase separations that define functional regions in cells, and amplify cellular responses to changes in their environments. A major challenge is to develop biochemical tools and physical models to describe the panoply of weak interactions operating in cells. We also need better approaches to measure the biases in the spatial distributions of cellular macromolecules that result from the integrated action of multiple weak interactions. Partnerships between cell biologists, biochemists, and physicists are required to deploy these methods. Together these approaches will help us realize the dream of understanding the biological "glue" that sustains life at a molecular and cellular level. © 2014 Chien and Gierasch. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Noise reduction methods for nucleic acid and macromolecule sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schuller, Ivan K.; Di Ventra, Massimiliano; Balatsky, Alexander

Methods, systems, and devices are disclosed for processing macromolecule sequencing data with substantial noise reduction. In one aspect, a method for reducing noise in a sequential measurement of a macromolecule comprising serial subunits includes cross-correlating multiple measured signals of a physical property of subunits of interest of the macromolecule, the multiple measured signals including the time data associated with the measurement of the signal, to remove or at least reduce signal noise that is not in the same frequency and in phase with the systematic signal contribution of the measured signals.

Cellular stress induces a protective sleep-like state in C. elegans.

PubMed

Hill, Andrew J; Mansfield, Richard; Lopez, Jessie M N G; Raizen, David M; Van Buskirk, Cheryl

2014-10-20

Sleep is recognized to be ancient in origin, with vertebrates and invertebrates experiencing behaviorally quiescent states that are regulated by conserved genetic mechanisms. Despite its conservation throughout phylogeny, the function of sleep remains debated. Hypotheses for the purpose of sleep include nervous-system-specific functions such as modulation of synaptic strength and clearance of metabolites from the brain, as well as more generalized cellular functions such as energy conservation and macromolecule biosynthesis. These models are supported by the identification of synaptic and metabolic processes that are perturbed during prolonged wakefulness. It remains to be seen whether perturbations of cellular homeostasis in turn drive sleep. Here we show that under conditions of cellular stress, including noxious heat, cold, hypertonicity, and tissue damage, the nematode Caenorhabditis elegans engages a behavioral quiescence program. The stress-induced quiescent state displays properties of sleep and is dependent on the ALA neuron, which mediates the conserved soporific effect of epidermal growth factor (EGF) ligand overexpression. We characterize heat-induced quiescence in detail and show that it is indeed dependent on components of EGF signaling, providing physiological relevance to the behavioral effects of EGF family ligands. We find that after noxious heat exposure, quiescence-defective animals show elevated expression of cellular stress reporter genes and are impaired for survival, demonstrating the benefit of stress-induced behavioral quiescence. These data provide evidence that cellular stress can induce a protective sleep-like state in C. elegans and suggest that a deeply conserved function of sleep is to mitigate disruptions of cellular homeostasis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Regulation of the mammalian heat shock factor 1.

PubMed

Dayalan Naidu, Sharadha; Dinkova-Kostova, Albena T

2017-06-01

Living organisms are endowed with the capability to tackle various forms of cellular stress due to the presence of molecular chaperone machinery complexes that are ubiquitous throughout the cell. During conditions of proteotoxic stress, the transcription factor heat shock factor 1 (HSF1) mediates the elevation of heat shock proteins, which are crucial components of the chaperone complex machinery and function to ameliorate protein misfolding and aggregation and restore protein homeostasis. In addition, HSF1 orchestrates a versatile transcriptional programme that includes genes involved in repair and clearance of damaged macromolecules and maintenance of cell structure and metabolism, and provides protection against a broad range of cellular stress mediators, beyond heat shock. Here, we discuss the structure and function of the mammalian HSF1 and its regulation by post-translational modifications (phosphorylation, sumoylation and acetylation), proteasomal degradation, and small-molecule activators and inhibitors. © 2017 Federation of European Biochemical Societies.

Novel approach using DNA-RNA hybrids in RNA nanotechnology | Center for Cancer Research

Cancer.gov

Developing simple approaches to detect interactions, modifications, and cellular locations of macromolecules is essential for understanding biochemical processes. The use of protein fragment complementation assays, also called split-protein systems, is a highly sensitive approach for studying protein interactions in biological systems. In this approach, functional proteins are

Identification of Characteristic Macromolecules of Escherichia coli Genotypes by Atomic Force Microscope Nanoscale Mechanical Mapping

NASA Astrophysics Data System (ADS)

Chang, Alice Chinghsuan; Liu, Bernard Haochih

2018-02-01

The categorization of microbial strains is conventionally based on the molecular method, and seldom are the morphological characteristics in the bacterial strains studied. In this research, we revealed the macromolecular structures of the bacterial surface via AFM mechanical mapping, whose resolution was not only determined by the nanoscale tip size but also the mechanical properties of the specimen. This technique enabled the nanoscale study of membranous structures of microbial strains with simple specimen preparation and flexible working environments, which overcame the multiple restrictions in electron microscopy and label-enable biochemical analytical methods. The characteristic macromolecules located among cellular surface were considered as surface layer proteins and were found to be specific to the Escherichia coli genotypes, from which the averaged molecular sizes were characterized with diameters ranging from 38 to 66 nm, and the molecular shapes were kidney-like or round. In conclusion, the surface macromolecular structures have unique characteristics that link to the E. coli genotype, which suggests that the genomic effects on cellular morphologies can be rapidly identified using AFM mechanical mapping. [Figure not available: see fulltext.

The First Cell Membranes

NASA Technical Reports Server (NTRS)

Deamer, David; Dworkin, Jason P.; Sandford, Scott A.; Bernstein, Max P.; Allamandola, Louis J.

2004-01-01

Organic compounds are synthesized in the interstellar medium and can be delivered to planetary surfaces such as the early Earth, where they mix with endogenous organic mixtures. Some of these compounds are amphiphilic, having polar and non-polar groups on the same molecule. Amphiphilic compounds spontaneously self-assembly into more complex structures such as bimolecular layers, which in turn form closed membranous vesicles. The first forms of cellular life required self-assembled membranes that were likely to be available on the prebiotic Earth. Laboratory simulations show that such vesicles readily encapsulate functional macromolecules, including nucleic acids and polymerases. A goal of future investigations is to fabricate artificial cells as models of the origin of life.

Cannabinoids Modulate Neuronal Activity and Cancer by CB1 and CB2 Receptor-Independent Mechanisms

PubMed Central

Soderstrom, Ken; Soliman, Eman; Van Dross, Rukiyah

2017-01-01

Cannabinoids include the active constituents of Cannabis or are molecules that mimic the structure and/or function of these Cannabis-derived molecules. Cannabinoids produce many of their cellular and organ system effects by interacting with the well-characterized CB1 and CB2 receptors. However, it has become clear that not all effects of cannabinoid drugs are attributable to their interaction with CB1 and CB2 receptors. Evidence now demonstrates that cannabinoid agents produce effects by modulating activity of the entire array of cellular macromolecules targeted by other drug classes, including: other receptor types; ion channels; transporters; enzymes, and protein- and non-protein cellular structures. This review summarizes evidence for these interactions in the CNS and in cancer, and is organized according to the cellular targets involved. The CNS represents a well-studied area and cancer is emerging in terms of understanding mechanisms by which cannabinoids modulate their activity. Considering the CNS and cancer together allow identification of non-cannabinoid receptor targets that are shared and divergent in both systems. This comparative approach allows the identified targets to be compared and contrasted, suggesting potential new areas of investigation. It also provides insight into the diverse sources of efficacy employed by this interesting class of drugs. Obtaining a comprehensive understanding of the diverse mechanisms of cannabinoid action may lead to the design and development of therapeutic agents with greater efficacy and specificity for their cellular targets. PMID:29066974

Accelerated aging in schizophrenia patients: the potential role of oxidative stress.

PubMed

Okusaga, Olaoluwa O

2014-08-01

Several lines of evidence suggest that schizophrenia, a severe mental illness characterized by delusions, hallucinations and thought disorder is associated with accelerated aging. The free radical (oxidative stress) theory of aging assumes that aging occurs as a result of damage to cell constituents and connective tissues by free radicals arising from oxygen-associated reactions. Schizophrenia has been associated with oxidative stress and chronic inflammation, both of which also appear to reciprocally induce each other in a positive feedback manner. The buildup of damaged macromolecules due to increased oxidative stress and failure of protein repair and maintenance systems is an indicator of aging both at the cellular and organismal level. When compared with age-matched healthy controls, schizophrenia patients have higher levels of markers of oxidative cellular damage such as protein carbonyls, products of lipid peroxidation and DNA hydroxylation. Potential confounders such as antipsychotic medication, smoking, socio-economic status and unhealthy lifestyle make it impossible to solely attribute the earlier onset of aging-related changes or oxidative stress to having a diagnosis of schizophrenia. Regardless of whether oxidative stress can be attributed solely to a diagnosis of schizophrenia or whether it is due to other factors associated with schizophrenia, the available evidence is in support of increased oxidative stress-induced cellular damage of macromolecules which may play a role in the phenomenon of accelerated aging presumed to be associated with schizophrenia.

The renal excretion and retention of macromolecules: The chemical structure effect.

PubMed

Rypácek, F; Drobník, J; Chmelar, V; Kálal, J

1982-01-01

Five derivatives of polyaspartamide were used as macromolecular models to study the effect of chemical structure of macromolecules on their renal excretion and retention. The parent polymer was formed solely by N(2-hydroxyethyl)aspartamide units (I) and in its derivatives about 20% of 2-hydroxyethyl groups were randomly replaced by either n-butyl- (II), 2(4-hydroxyphenyl)ethyl- (III, N- dimethylamino propyl- (IV) or the aspartamide unit was modified to free aspartic acid carboxyl (V). The rate of clearance from the serum, the deposition in the kidney tissue in comparison with the deposition in reticuloendothelial system organs-liver and spleen, as well as tissue and cellular localisation of deposits were studied on rabbits and mice taking advantage of fluorescence labelling. The clearance of macromolecular models from the serum compartment by the glomerular filtration is mainly molecular weight controlled, while the retention of macromolecules possessing the same molecular weight by the kidney tubular epithelium is strongly affected chemical modification. About thirty and hundred times higher retentions due to reabsorption in proximal tubule were found with macromolecular models II and III respectively.

Biological effects of electric shock and heat denaturation and oxidation of molecules, membranes, and cellular functions.

PubMed

Tsong, T Y; Su, Z D

1999-10-30

Direct exposure of cells in suspension to intense electric pulses is known to produce damages to cell membranes and supramolecular organizations of cells, and denaturation of macromolecules, much like injuries and tears seen in electric trauma patients. Thus, the system has been used as a laboratory model for investigating the biochemical basis of electric injury. An intense electric pulse can produce two major effects on cells--one caused by the field, or the electric potential, and the other by current, or the electric energy. The field-induced transmembrane potential can produce electro-conformational changes of ion channels and ion pumps and, when the potential exceeds the dielectric strength of the cell membrane (approximately 500 mV for a pulse width of a few ms), electro-conformational damages and electroporations of membrane proteins and lipid bilayers. These events lead to passage of electric current through the membrane-porated cells and to heating of cell membranes and cytoplasmic contents. The subsequent denaturation of cell membranes and cytoplasmic macromolecules brings about many complex biochemical reactions, including oxidation of proteins and lipids. The combined effects may cripple the cells beyond repair. This communication will focus on the thermal effects of electric shock. After a brief review of the current state of knowledge on thermal denaturation of soluble enzymes and muscle proteins, this paper will describe experiments on the thermal denaturation of cellular components and functions, such as nucleosomes, and the electron transport chain and ATP synthetic enzymes of the mitochondrial inner membranes. Data will show that lipid peroxidation and the subsequent loss of the energy-transducing ability of the cells may occur even at moderate temperatures between 40 degrees C and 45 degrees C. However, lipid peroxidation may be prevented with reducing reagents such as mercaptoethanol, dithiothreitol, and ascorbic acid. Reactivation of denatured cellular proteins and functions may also be possible and a strategy for doing so is discussed.

Lactoferricin B inhibits bacterial macromolecular synthesis in Escherichia coli and Bacillus subtilis.

PubMed

Ulvatne, Hilde; Samuelsen, Ørjan; Haukland, Hanne H; Krämer, Manuela; Vorland, Lars H

2004-08-15

Most antimicrobial peptides have an amphipathic, cationic structure, and an effect on the cytoplasmic membrane of susceptible bacteria has been postulated as the main mode of action. Other mechanisms have been reported, including inhibition of cellular functions by binding to DNA, RNA and proteins, and the inhibition of DNA and/or protein synthesis. Lactoferricin B (Lfcin B), a cationic peptide derived from bovine lactoferrin, exerts slow inhibitory and bactericidal activity and does not lyse susceptible bacteria, indicating a possible intracellular target. In the present study incorporation of radioactive precursors into DNA, RNA and proteins was used to demonstrate effects of Lfcin B on macromolecular synthesis in bacteria. In Escherichia coli UC 6782, Lfcin B induces an initial increase in protein and RNA synthesis and a decrease in DNA synthesis. After 10 min, the DNA-synthesis increases while protein and RNA-synthesis decreases significantly. In Bacillus subtilis, however, all synthesis of macromolecules is inhibited for at least 20 min. After 20 min RNA-synthesis increases. The results presented here show that Lfcin B at concentrations not sufficient to kill bacterial cells inhibits incorporation of radioactive precursors into macromolecules in both Gram-positive and Gram-negative bacteria.

Empirical tests of a theory of language, mathematics, and matter.

PubMed

Abler, William L

2008-01-01

In an earlier paper (Abler, 2006), I proposed a theory of language, especially sentences, based on the symmetrical structure of the equation. Here, I use the structure of equations to deduce neural structures (e.g., mirror neurons or intra-cellular macromolecules, or crystals, or resonations) that might generate them. Ultimately, the properties described are a consequence of dimensional properties of matter

Determining the distribution of probes between different subcellular locations through automated unmixing of subcellular patterns.

PubMed

Peng, Tao; Bonamy, Ghislain M C; Glory-Afshar, Estelle; Rines, Daniel R; Chanda, Sumit K; Murphy, Robert F

2010-02-16

Many proteins or other biological macromolecules are localized to more than one subcellular structure. The fraction of a protein in different cellular compartments is often measured by colocalization with organelle-specific fluorescent markers, requiring availability of fluorescent probes for each compartment and acquisition of images for each in conjunction with the macromolecule of interest. Alternatively, tailored algorithms allow finding particular regions in images and quantifying the amount of fluorescence they contain. Unfortunately, this approach requires extensive hand-tuning of algorithms and is often cell type-dependent. Here we describe a machine-learning approach for estimating the amount of fluorescent signal in different subcellular compartments without hand tuning, requiring only the acquisition of separate training images of markers for each compartment. In testing on images of cells stained with mixtures of probes for different organelles, we achieved a 93% correlation between estimated and expected amounts of probes in each compartment. We also demonstrated that the method can be used to quantify drug-dependent protein translocations. The method enables automated and unbiased determination of the distributions of protein across cellular compartments, and will significantly improve imaging-based high-throughput assays and facilitate proteome-scale localization efforts.

Energy parasites trigger oncogene mutation.

PubMed

Pokorný, Jiří; Pokorný, Jan; Jandová, Anna; Kobilková, Jitka; Vrba, Jan; Vrba, Jan

2016-10-01

Cancer initialization can be explained as a result of parasitic virus energy consumption leading to randomized genome chemical bonding. Analysis of experimental data on cell-mediated immunity (CMI) containing about 12,000 cases of healthy humans, cancer patients and patients with precancerous cervical lesions disclosed that the specific cancer and the non-specific lactate dehydrogenase-elevating (LDH) virus antigen elicit similar responses. The specific antigen is effective only in cancer type of its origin but the non-specific antigen in all examined cancers. CMI results of CIN patients display both healthy and cancer state. The ribonucleic acid (RNA) of the LDH virus parasitizing on energy reduces the ratio of coherent/random oscillations. Decreased effect of coherent cellular electromagnetic field on bonding electrons in biological macromolecules leads to elevating probability of random genome reactions. Overlapping of wave functions in biological macromolecules depends on energy of the cellular electromagnetic field which supplies energy to bonding electrons for selective chemical bonds. CMI responses of cancer and LDH virus antigens in all examined healthy, precancerous and cancer cases point to energy mechanism in cancer initiation. Dependence of the rate of biochemical reactions on biological electromagnetic field explains yet unknown mechanism of genome mutation.

Systems and Photosystems: Cellular Limits of Autotrophic Productivity in Cyanobacteria

PubMed Central

Burnap, Robert L.

2014-01-01

Recent advances in the modeling of microbial growth and metabolism have shown that growth rate critically depends upon the optimal allocation of finite proteomic resources among different cellular functions and that modeling growth rates becomes more realistic with the explicit accounting for the costs of macromolecular synthesis, most importantly, protein expression. The “proteomic constraint” is considered together with its application to understanding photosynthetic microbial growth. The central hypothesis is that physical limits of cellular space (and corresponding solvation capacity) in conjunction with cell surface-to-volume ratios represent the underlying constraints on the maximal rate of autotrophic microbial growth. The limitation of cellular space thus constrains the size the total complement of macromolecules, dissolved ions, and metabolites. To a first approximation, the upper limit in the cellular amount of the total proteome is bounded this space limit. This predicts that adaptation to osmotic stress will result in lower maximal growth rates due to decreased cellular concentrations of core metabolic proteins necessary for cell growth owing the accumulation of compatible osmolytes, as surmised previously. The finite capacity of membrane and cytoplasmic space also leads to the hypothesis that the species-specific differences in maximal growth rates likely reflect differences in the allocation of space to niche-specific proteins with the corresponding diminution of space devoted to other functions including proteins of core autotrophic metabolism, which drive cell reproduction. An optimization model for autotrophic microbial growth, the autotrophic replicator model, was developed based upon previous work investigating heterotrophic growth. The present model describes autotrophic growth in terms of the allocation protein resources among core functional groups including the photosynthetic electron transport chain, light-harvesting antennae, and the ribosome groups. PMID:25654078

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis

PubMed Central

Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F.; Lane, Andrew N.; Romick-Rosendale, Lindsey E.; Wells, Susanne I.

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth. PMID:28558019

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis.

PubMed

Matrka, Marie C; Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F; Lane, Andrew N; Romick-Rosendale, Lindsey E; Wells, Susanne I

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth.

Gene expression profiling in the Cynomolgus macaque Macaca fascicularis shows variation within the normal birth range

PubMed Central

2011-01-01

Background Although an adverse early-life environment has been linked to an increased risk of developing the metabolic syndrome, the molecular mechanisms underlying altered disease susceptibility as well as their relevance to humans are largely unknown. Importantly, emerging evidence suggests that these effects operate within the normal range of birth weights and involve mechanisms of developmental palsticity rather than pathology. Method To explore this further, we utilised a non-human primate model Macaca fascicularis (Cynomolgus macaque) which shares with humans the same progressive history of the metabolic syndrome. Using microarray we compared tissues from neonates in the average birth weight (50-75th centile) to those of lower birth weight (5-25th centile) and studied the effect of different growth trajectories within the normal range on gene expression levels in the umbilical cord, neonatal liver and skeletal muscle. Results We identified 1973 genes which were differentially expressed in the three tissue types between average and low birth weight animals (P < 0.05). Gene ontology analysis identified that these genes were involved in metabolic processes including cellular lipid metabolism, cellular biosynthesis, cellular macromolecule synthesis, cellular nitrogen metabolism, cellular carbohydrate metabolism, cellular catabolism, nucleotide and nucleic acid metabolism, regulation of molecular functions, biological adhesion and development. Conclusion These differences in gene expression levels between animals in the upper and lower percentiles of the normal birth weight range may point towards early life metabolic adaptations that in later life result in differences in disease risk. PMID:21999700

Brain delivery research in public-private partnerships: The IMI-JU COMPACT consortium as an example.

PubMed

Meyer, Axel H; Untucht, Christopher; Terstappen, Georg C

2017-07-01

The Blood-Brain Barrier (BBB) represents a major hurdle in the development of treatments for CNS disorders due to the fact that it very effectively keeps drugs, especially biological macromolecules, out of the brain. Concomitantly with the increasing importance of biologics research on the BBB and, more specifically, on brain delivery technologies has intensified in recent years. Public-Private Partnerships (PPPs) represent an innovative opportunity to address such complex challenges as they bring together the best expertise from both industry and academia. Here we present the IMI-JU COMPACT (Collaboration on the Optimisation of Macromolecular Pharmaceutical Access to Cellular Targets) consortium working on nanocarriers for targeted delivery of macromolecules as an example. The scope of the consortium, its goals and the expertise within the consortium are outlined. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders". Copyright © 2016 Elsevier Ltd. All rights reserved.

Cooperative polymerization of α-helices induced by macromolecular architecture

NASA Astrophysics Data System (ADS)

Baumgartner, Ryan; Fu, Hailin; Song, Ziyuan; Lin, Yao; Cheng, Jianjun

2017-07-01

Catalysis observed in enzymatic processes and protein polymerizations often relies on the use of supramolecular interactions and the organization of functional elements in order to gain control over the spatial and temporal elements of fundamental cellular processes. Harnessing these cooperative interactions to catalyse reactions in synthetic systems, however, remains challenging due to the difficulty in creating structurally controlled macromolecules. Here, we report a polypeptide-based macromolecule with spatially organized α-helices that can catalyse its own formation. The system consists of a linear polymeric scaffold containing a high density of initiating groups from which polypeptides are grown, forming a brush polymer. The folding of polypeptide side chains into α-helices dramatically enhances the polymerization rate due to cooperative interactions of macrodipoles between neighbouring α-helices. The parameters that affect the rate are elucidated by a two-stage kinetic model using principles from nucleation-controlled protein polymerizations; the key difference being the irreversible nature of this polymerization.

Structural studies of RNA-protein complexes: A hybrid approach involving hydrodynamics, scattering, and computational methods.

PubMed

Patel, Trushar R; Chojnowski, Grzegorz; Astha; Koul, Amit; McKenna, Sean A; Bujnicki, Janusz M

2017-04-15

The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from experimental hydrodynamic (e.g. analytical ultracentrifugation) and solution scattering measurements (e.g., solution X-ray or neutron scattering), with computational modeling to obtain atomic-level models. While promising, scattering methods rely on aggregation-free, monodispersed preparations and therefore the careful development of a quality control pipeline is fundamental to an unbiased and reliable structural determination. This review article describes hydrodynamic techniques that are highly valuable for homogeneity studies, scattering techniques useful to study the low-resolution shape, and strategies for computational modeling to obtain high-resolution 3D structural models of RNAs, proteins, and RNA-protein complexes. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Measuring In Vivo Protein Dynamics Throughout the Cell Cycle Using Microfluidics.

PubMed

de Leeuw, Roy; Brazda, Peter; Charl Moolman, M; Kerssemakers, J W J; Solano, Belen; Dekker, Nynke H

2017-01-01

Studying the dynamics of intracellular processes and investigating the interaction of individual macromolecules in live cells is one of the main objectives of cell biology. These macromolecules move, assemble, disassemble, and reorganize themselves in distinct manners under specific physiological conditions throughout the cell cycle. Therefore, in vivo experimental methods that enable the study of individual molecules inside cells at controlled culturing conditions have proved to be powerful tools to obtain insights into the molecular roles of these macromolecules and how their individual behavior influence cell physiology. The importance of controlled experimental conditions is enhanced when the investigated phenomenon covers long time periods, or perhaps multiple cell cycles. An example is the detection and quantification of proteins during bacterial DNA replication. Wide-field microscopy combined with microfluidics is a suitable technique for this. During fluorescence experiments, microfluidics offer well-defined cellular orientation and immobilization, flow and medium interchangeability, and high-throughput long-term experimentation of cells. Here we present a protocol for the combined use of wide-field microscopy and microfluidics for the study of proteins of the Escherichia coli DNA replication process. We discuss the preparation and application of a microfluidic device, data acquisition steps, and image analysis procedures to determine the stoichiometry and dynamics of a replisome component throughout the cell cycle of live bacterial cells.

The Biological Macromolecule Crystallization Database and NASA Protein Crystal Growth Archive

PubMed Central

Gilliland, Gary L.; Tung, Michael; Ladner, Jane

1996-01-01

The NIST/NASA/CARB Biological Macromolecule Crystallization Database (BMCD), NIST Standard Reference Database 21, contains crystal data and crystallization conditions for biological macromolecules. The database entries include data abstracted from published crystallographic reports. Each entry consists of information describing the biological macromolecule crystallized and crystal data and the crystallization conditions for each crystal form. The BMCD serves as the NASA Protein Crystal Growth Archive in that it contains protocols and results of crystallization experiments undertaken in microgravity (space). These database entries report the results, whether successful or not, from NASA-sponsored protein crystal growth experiments in microgravity and from microgravity crystallization studies sponsored by other international organizations. The BMCD was designed as a tool to assist x-ray crystallographers in the development of protocols to crystallize biological macromolecules, those that have previously been crystallized, and those that have not been crystallized. PMID:11542472

High throughput screening of ligand binding to macromolecules using high resolution powder diffraction

DOEpatents

Von Dreele, Robert B.; D'Amico, Kevin

2006-10-31

A process is provided for the high throughput screening of binding of ligands to macromolecules using high resolution powder diffraction data including producing a first sample slurry of a selected polycrystalline macromolecule material and a solvent, producing a second sample slurry of a selected polycrystalline macromolecule material, one or more ligands and the solvent, obtaining a high resolution powder diffraction pattern on each of said first sample slurry and the second sample slurry, and, comparing the high resolution powder diffraction pattern of the first sample slurry and the high resolution powder diffraction pattern of the second sample slurry whereby a difference in the high resolution powder diffraction patterns of the first sample slurry and the second sample slurry provides a positive indication for the formation of a complex between the selected polycrystalline macromolecule material and at least one of the one or more ligands.

Intercellular and systemic spread of RNA and RNAi in plants.

PubMed

Nazim Uddin, Mohammad; Kim, Jae-Yean

2013-01-01

Plants possess dynamic networks of intercellular communication that are crucial for plant development and physiology. In plants, intercellular communication involves a combination of ligand-receptor-based apoplasmic signaling, and plasmodesmata and phloem-mediated symplasmic signaling. The intercellular trafficking of macromolecules, including RNAs and proteins, has emerged as a novel mechanism of intercellular communication in plants. Various forms of regulatory RNAs move over distinct cellular boundaries through plasmodesmata and phloem. This plant-specific, non-cell-autonomous RNA trafficking network is also involved in development, nutrient homeostasis, gene silencing, pathogen defense, and many other physiological processes. However, the mechanism underlying macromolecular trafficking in plants remains poorly understood. Current progress made in RNA trafficking research and its biological relevance to plant development will be summarized. Diverse plant regulatory mechanisms of cell-to-cell and systemic long-distance transport of RNAs, including mRNAs, viral RNAs, and small RNAs, will also be discussed. Copyright © 2013 John Wiley & Sons, Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortoleva, Peter J.

Illustrative embodiments of systems and methods for the deductive multiscale simulation of macromolecules are disclosed. In one illustrative embodiment, a deductive multiscale simulation method may include (i) constructing a set of order parameters that model one or more structural characteristics of a macromolecule, (ii) simulating an ensemble of atomistic configurations for the macromolecule using instantaneous values of the set of order parameters, (iii) simulating thermal-average forces and diffusivities for the ensemble of atomistic configurations, and (iv) evolving the set of order parameters via Langevin dynamics using the thermal-average forces and diffusivities.

A rapid method for the preparation of ultrapure, functional lysosomes using functionalized superparamagnetic iron oxide nanoparticles.

PubMed

Walker, Mathew W; Lloyd-Evans, Emyr

2015-01-01

Lysosomes are an emerging and increasingly important cellular organelle. With every passing year, more novel proteins and key cellular functions are associated with lysosomes. Despite this, the methodologies for their purification have largely remained unchanged since the days of their discovery. With little advancement in this area, it is no surprise that analysis of lysosomal function has been somewhat stymied, largely in part by the change in buoyant densities that occur under conditions where lysosomes accumulate macromolecules. Such phenotypes are often associated with the lysosomal storage diseases but are increasingly being observed under conditions where lysosomal proteins or, in some cases, cellular functions associated with lysosomal proteins are being manipulated. These altered lysosomes poise a problem to the classical methods to purify lysosomes that are reliant largely on their correct sedimentation by density gradient centrifugation. Building upon a technique developed by others to purify lysosomes magnetically, we have developed a unique assay using superparamagnetic iron oxide nanoparticles (SPIONs) to purify high yields of ultrapure functional lysosomes from multiple cell types including the lysosomal storage disorders. Here we describe this method in detail, including the rationale behind using SPIONs, the potential pitfalls that can be avoided and the potential functional assays these lysosomes can be used for. Finally we also summarize the other methodologies and the exact reasons why magnetic purification of lysosomes is now the method of choice for lysosomal researchers. Copyright © 2015 Elsevier Inc. All rights reserved.

A Course on Macromolecules.

ERIC Educational Resources Information Center

Horta, Arturo

1985-01-01

Describes a senior-level course that: (1) focuses on the structure and reactions of macromolecules; (2) treats industrial polymers in a unified way; and (3) uses analysis of conformation and conformational statistics as a unifying approach. Also discusses course topics, including polysaccharides, proteins, nucleic acids, and others. (JN)

Environmental pollutants and lifestyle factors induce oxidative stress and poor prenatal development.

PubMed

Al-Gubory, Kaïs H

2014-07-01

Developmental toxicity caused by exposure to a mixture of environmental pollutants has become a major health concern. Human-made chemicals, including xenoestrogens, pesticides and heavy metals, as well as unhealthy lifestyle behaviours, mainly tobacco smoking, alcohol consumption and medical drug abuse, are major factors that adversely influence prenatal development and increase susceptibility of offspring to diseases. There is evidence to suggest that the developmental toxicological mechanisms of chemicals and lifestyle factors involve the generation of reactive oxygen species (ROS) and cellular oxidative damage. Overproduction of ROS induces oxidative stress, a state where increased ROS generation overwhelms antioxidant protection and subsequently leads to oxidative damage of cellular macromolecules. Data on the involvement of oxidative stress in the mechanism of developmental toxicity following exposure to environmental pollutants are reviewed in an attempt to provide an updated basis for future studies on the toxic effect of such pollutants, particularly the notion of increased risk for developmental toxicity due to combined and cumulative exposure to various environmental pollutants. The aims of such studies are to better understand the mechanisms by which environmental pollutants adversely affect conceptus development and to elucidate the impact of cumulative exposures to multiple pollutants on post-natal development and health outcomes. Developmental toxicity caused by exposure to mixture of environmental pollutants has become a major health concern. Human-made chemicals, including xenoestrogens, pesticides and heavy metals, as well as unhealthy lifestyle behaviors, mainly tobacco smoking, alcohol consumption and medical drug abuse, are major factors that adversely influence prenatal development and increase the susceptibility of offspring to development complications and diseases. There is evidence to suggest that the developmental toxicological mechanisms of human-made chemicals and unhealthy lifestyle factors involve the generation of reactive oxygen species (ROS) and cellular oxidative damage. Overproduction of ROS induces oxidative stress, a state where increased generation of ROS overwhelms antioxidant protection and subsequently leads to oxidative damage of cellular macromolecules. Exposure to various environmental pollutants induces synergic and cumulative dose-additive adverse effects on prenatal development, pregnancy outcomes and neonate health. Data from the literature on the involvement of oxidative stress in the mechanism of developmental toxicity following in vivo exposure to environmental pollutants will be reviewed in an attempt to provide an updated basis for future studies on the toxic effect of such pollutants, particularly the notion of increased risk for developmental toxicity due to combined and cumulative exposure to various environmental pollutants. The aims of such studies are to better understand the mechanisms by which environmental pollutants adversely affect conceptus development and to elucidate the impact of cumulative exposures to multiple pollutants on postnatal development and health outcomes. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum*♦

PubMed Central

Yamauchi, Yoshio; Iwamoto, Noriyuki; Rogers, Maximillian A.; Abe-Dohmae, Sumiko; Fujimoto, Toyoshi; Chang, Catherine C. Y.; Ishigami, Masato; Kishimoto, Takuma; Kobayashi, Toshihide; Ueda, Kazumitsu; Furukawa, Koichi; Chang, Ta-Yuan; Yokoyama, Shinji

2015-01-01

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process. PMID:26198636

Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm.

PubMed

Yu, Isseki; Mori, Takaharu; Ando, Tadashi; Harada, Ryuhei; Jung, Jaewoon; Sugita, Yuji; Feig, Michael

2016-11-01

Biological macromolecules function in highly crowded cellular environments. The structure and dynamics of proteins and nucleic acids are well characterized in vitro, but in vivo crowding effects remain unclear. Using molecular dynamics simulations of a comprehensive atomistic model cytoplasm we found that protein-protein interactions may destabilize native protein structures, whereas metabolite interactions may induce more compact states due to electrostatic screening. Protein-protein interactions also resulted in significant variations in reduced macromolecular diffusion under crowded conditions, while metabolites exhibited significant two-dimensional surface diffusion and altered protein-ligand binding that may reduce the effective concentration of metabolites and ligands in vivo. Metabolic enzymes showed weak non-specific association in cellular environments attributed to solvation and entropic effects. These effects are expected to have broad implications for the in vivo functioning of biomolecules. This work is a first step towards physically realistic in silico whole-cell models that connect molecular with cellular biology.

Formation and Biological Targets of Quinones: Cytotoxic versus Cytoprotective Effects

PubMed Central

2016-01-01

Quinones represent a class of toxicological intermediates, which can create a variety of hazardous effects in vivo including, acute cytotoxicity, immunotoxicity, and carcinogenesis. In contrast, quinones can induce cytoprotection through the induction of detoxification enzymes, anti-inflammatory activities, and modification of redox status. The mechanisms by which quinones cause these effects can be quite complex. The various biological targets of quinones depend on their rate and site of formation and their reactivity. Quinones are formed through a variety of mechanisms from simple oxidation of catechols/hydroquinones catalyzed by a variety of oxidative enzymes and metal ions to more complex mechanisms involving initial P450-catalyzed hydroxylation reactions followed by two-electron oxidation. Quinones are Michael acceptors, and modification of cellular processes could occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radical anions leading to the formation of reactive oxygen species (ROS) including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can alter redox balance within cells through the formation of oxidized cellular macromolecules including lipids, proteins, and DNA. This perspective explores the varied biological targets of quinones including GSH, NADPH, protein sulfhydryls [heat shock proteins, P450s, cyclooxygenase-2 (COX-2), glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1, (NQO1), kelch-like ECH-associated protein 1 (Keap1), IκB kinase (IKK), and arylhydrocarbon receptor (AhR)], and DNA. The evidence strongly suggests that the numerous mechanisms of quinone modulations (i.e., alkylation versus oxidative stress) can be correlated with the known pathology/cytoprotection of the parent compound(s) that is best described by an inverse U-shaped dose–response curve. PMID:27617882

PlaMoM: a comprehensive database compiles plant mobile macromolecules

PubMed Central

Guan, Daogang; Yan, Bin; Thieme, Christoph; Hua, Jingmin; Zhu, Hailong; Boheler, Kenneth R.; Zhao, Zhongying; Kragler, Friedrich; Xia, Yiji; Zhang, Shoudong

2017-01-01

In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein–protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/. PMID:27924044

Diffusion in Brain Extracellular Space

PubMed Central

Syková, Eva; Nicholson, Charles

2009-01-01

Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is about 20% and the tortuosity about 1.6 (i.e. free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties are valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain. PMID:18923183

In vitro analysis of metabolic predisposition to drug hypersensitivity reactions.

PubMed Central

Riley, R J; Leeder, J S

1995-01-01

Idiosyncratic hypersensitivity reactions may account for up to 25% of all adverse reactions, and pose a constant problem to physicians because of their unpredictable nature, potentially fatal outcome and resemblance to other disease processes. Current understanding of how drug allergy arises is based largely on the hapten hypothesis: since most drugs are not chemically reactive per se, they must be activated metabolically to reactive species which may become immunogenic through interactions with cellular macromolecules. The role of drug metabolism is thus pivotal to the hapten hypothesis both in activation of the parent compound and detoxification of the reactive species. Although conjugation reactions may occasionally produce potential immunogens (for example, the generation of acylglucuronides from non-steroidal anti-inflammatory drugs such as diclofenac), bioactivation is catalysed most frequently by cytochrome P450 (P450) enzymes. The multifactorial nature of hypersensitivity reactions, particularly the role of often unidentified, reactive drug metabolites in antigen generation, has hampered the routine diagnosis of these disorders by classical immunological methods designed to detect circulating antibodies or sensitized T cells. Similarly, species differences in drug metabolism and immune system regulation have largely precluded the establishment of appropriate animal models with which to examine the immunopathological mechanisms of these toxicities. However, the combined use of in vitro toxicity assays incorporating human tissues and in vivo phenotyping (or, ultimately, in vitro genotyping) methods for drug detoxification pathways may provide the metabolic basis for hypersensitivity reactions to several drugs. This brief review highlights recent efforts to unravel the bases for hypersensitivity reactions to these therapeutic agents (which include anticonvulsants and sulphonamides) using drug metabolism and immunochemical approaches. In particular, examples are provided which illustrate breakthroughs in the identification of the chemical nature of the reactive metabolites which become bound to cellular macromolecules, the enzyme systems responsible for their generation and (possibly) detoxification, and the target proteins implicated in the subsequent immune response. PMID:7813099

Alstonine as a potential fluorescent marker for tiny tumor detection and imaging

NASA Astrophysics Data System (ADS)

Viallet, Pierre M.; Vo-Dinh, Tuan; Salmon, Jean-Marie; Watts, Wendi; Rocchi, Emmanuelle; Isola, Narayana R.; Rebillard, Xavier

1997-06-01

3,4,5,6,16,17-Hexadehydro-16-(methoxycarbolyl)-19(alpha) - methyl-20(alpha) -oxyohimbanium (alstonine) is a fluorescent alcaloid which is known to stain tumor cells more efficiently than normal. The interactions between alstonine and biological macromolecules were first investigated to provide the rationale for preferential labelling. Molecular filtration and spectrosfluorometric techniques with different macromolecules and isopolynucleotides have demonstrated that binding occurs only in the presence of uridyl rings. For the binding affect only the fluorescence intensity of alstonine it can be assumed that it involves only the side chain of the fluorescent compound. The capability for preferential staining was verified in culture using SK-OV-3 cells and rat hepatocarcinoma cells as tumor cells and Mouse fibroblasts or rat liver cells as controls. Techniques of image analysis have demonstrated the efficiency of cellular labelling even in aggregates of rat hepatocarcinoma. These experiments lead the way to the detection of tiny tumors developed on thin visceral walls, using a fiber optic device.

Applying macromolecular crowding to 3D bioprinting: fabrication of 3D hierarchical porous collagen-based hydrogel constructs.

PubMed

Ng, Wei Long; Goh, Min Hao; Yeong, Wai Yee; Naing, May Win

2018-02-27

Native tissues and/or organs possess complex hierarchical porous structures that confer highly-specific cellular functions. Despite advances in fabrication processes, it is still very challenging to emulate the hierarchical porous collagen architecture found in most native tissues. Hence, the ability to recreate such hierarchical porous structures would result in biomimetic tissue-engineered constructs. Here, a single-step drop-on-demand (DOD) bioprinting strategy is proposed to fabricate hierarchical porous collagen-based hydrogels. Printable macromolecule-based bio-inks (polyvinylpyrrolidone, PVP) have been developed and printed in a DOD manner to manipulate the porosity within the multi-layered collagen-based hydrogels by altering the collagen fibrillogenesis process. The experimental results have indicated that hierarchical porous collagen structures could be achieved by controlling the number of macromolecule-based bio-ink droplets printed on each printed collagen layer. This facile single-step bioprinting process could be useful for the structural design of collagen-based hydrogels for various tissue engineering applications.

Hierarchy, determinism, and specificity in theories of development and evolution.

PubMed

Deichmann, Ute

2017-10-16

The concepts of hierarchical organization, genetic determinism and biological specificity (for example of species, biologically relevant macromolecules, or genes) have played a crucial role in biology as a modern experimental science since its beginnings in the nineteenth century. The idea of genetic information (specificity) and genetic determination was at the basis of molecular biology that developed in the 1940s with macromolecules, viruses and prokaryotes as major objects of research often labelled "reductionist". However, the concepts have been marginalized or rejected in some of the research that in the late 1960s began to focus additionally on the molecularization of complex biological structures and functions using systems approaches. This paper challenges the view that 'molecular reductionism' has been successfully replaced by holism and a focus on the collective behaviour of cellular entities. It argues instead that there are more fertile replacements for molecular 'reductionism', in which genomics, embryology, biochemistry, and computer science intertwine and result in research that is as exact and causally predictive as earlier molecular biology.

Multiscale simulation of molecular processes in cellular environments.

PubMed

Chiricotto, Mara; Sterpone, Fabio; Derreumaux, Philippe; Melchionna, Simone

2016-11-13

We describe the recent advances in studying biological systems via multiscale simulations. Our scheme is based on a coarse-grained representation of the macromolecules and a mesoscopic description of the solvent. The dual technique handles particles, the aqueous solvent and their mutual exchange of forces resulting in a stable and accurate methodology allowing biosystems of unprecedented size to be simulated.This article is part of the themed issue 'Multiscale modelling at the physics-chemistry-biology interface'. © 2016 The Author(s).

PlaMoM: a comprehensive database compiles plant mobile macromolecules.

PubMed

Guan, Daogang; Yan, Bin; Thieme, Christoph; Hua, Jingmin; Zhu, Hailong; Boheler, Kenneth R; Zhao, Zhongying; Kragler, Friedrich; Xia, Yiji; Zhang, Shoudong

2017-01-04

In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein-protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Loss of Urinary Macromolecules in Mice Causes Interstitial and Intratubular Renal Calcification Dependent on the Underlying Conditions

NASA Astrophysics Data System (ADS)

Wu, Xue-Ru; Lieske, John C.; Evan, Andrew P.; Sommer, Andre J.; Liaw, Lucy; Mo, Lan

2008-09-01

Urinary protein macromolecules have long been thought to play a role in influencing the various phases of urolithiasis including nucleation, growth, aggregation of mineral crystals and their subsequent adhesion to the renal epithelial cells. However, compelling evidence regarding their precise role was lacking, due partly to the fact that most prior studies were done in vitro and results were highly variable depending on the experimental conditions. The advent of genetic engineering technology has made it possible to study urinary protein macromolecules within an in vivo biological system. Indeed, recent studies have begun to shed light on the net effects of loss of one or more macromolecules on the earliest steps of urolithiasis. This paper focuses on the in vivo consequences of inactivating Tamm-Horsfall protein and/or osteopontin, two major urinary glycoproteins, using the knockout approach. The renal phenotypes of both single and double knockout mice under spontaneous or hyperoxaluric conditions will be described. The functional significance of the urinary macromolecules as critical defense factors against renal calcification will also be discussed.

Molecular mechanisms of toxicity of important food-borne phytotoxins.

PubMed

Rietjens, Ivonne M C M; Martena, Martijn J; Boersma, Marelle G; Spiegelenberg, Wim; Alink, Gerrit M

2005-02-01

At present, there is an increasing interest for plant ingredients and their use in drugs, for teas, or in food supplements. The present review describes the nature and mechanism of action of the phytochemicals presently receiving increased attention in the field of food toxicology. This relates to compounds including aristolochic acids, pyrrolizidine alkaloids, beta-carotene, coumarin, the alkenylbenzenes safrole, methyleugenol and estragole, ephedrine alkaloids and synephrine, kavalactones, anisatin, St. John's wort ingredients, cyanogenic glycosides, solanine and chaconine, thujone, and glycyrrhizinic acid. It can be concluded that several of these phytotoxins cause concern, because of their bioactivation to reactive alkylating intermediates that are able to react with cellular macromolecules causing cellular toxicity, and, upon their reaction with DNA, genotoxicity resulting in tumors. Another group of the phytotoxins presented is active without the requirement for bioactivation and, in most cases, these compounds appear to act as neurotoxins interacting with one of the neurotransmitter systems. Altogether, the examples presented illustrate that natural does not equal safe and that in modern society adverse health effects, upon either acute or chronic exposure to phytochemicals, can occur as a result of use of plant- or herb-based foods, teas, or other extracts.

Enzyme localization, crowding, and buffers collectively modulate diffusion-influenced signal transduction: Insights from continuum diffusion modeling

PubMed Central

Kekenes-Huskey, Peter M.; Eun, Changsun; McCammon, J. A.

2015-01-01

Biochemical reaction networks consisting of coupled enzymes connect substrate signaling events with biological function. Substrates involved in these reactions can be strongly influenced by diffusion “barriers” arising from impenetrable cellular structures and macromolecules, as well as interactions with biomolecules, especially within crowded environments. For diffusion-influenced reactions, the spatial organization of diffusion barriers arising from intracellular structures, non-specific crowders, and specific-binders (buffers) strongly controls the temporal and spatial reaction kinetics. In this study, we use two prototypical biochemical reactions, a Goodwin oscillator, and a reaction with a periodic source/sink term to examine how a diffusion barrier that partitions substrates controls reaction behavior. Namely, we examine how conditions representative of a densely packed cytosol, including reduced accessible volume fraction, non-specific interactions, and buffers, impede diffusion over nanometer length-scales. We find that diffusion barriers can modulate the frequencies and amplitudes of coupled diffusion-influenced reaction networks, as well as give rise to “compartments” of decoupled reactant populations. These effects appear to be intensified in the presence of buffers localized to the diffusion barrier. These findings have strong implications for the role of the cellular environment in tuning the dynamics of signaling pathways. PMID:26342355

Tonal Interface to MacroMolecules (TIMMol): A Textual and Tonal Tool for Molecular Visualization

ERIC Educational Resources Information Center

Cordes, Timothy J.; Carlson, C. Britt; Forest, Katrina T.

2008-01-01

We developed the three-dimensional visualization software, Tonal Interface to MacroMolecules or TIMMol, for studying atomic coordinates of protein structures. Key features include audio tones indicating x, y, z location, identification of the cursor location in one-dimensional and three-dimensional space, textual output that can be easily linked…

Analysis of macromolecules, ligands and macromolecule-ligand complexes

DOEpatents

Von Dreele, Robert B [Los Alamos, NM

2008-12-23

A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development.

PubMed

van Lessen, Max; Shibata-Germanos, Shannon; van Impel, Andreas; Hawkins, Thomas A; Rihel, Jason; Schulte-Merker, Stefan

2017-05-12

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain.

Perfect mixing of immiscible macromolecules at fluid interfaces

NASA Astrophysics Data System (ADS)

Sheiko, Sergei; Matyjaszewski, Krzysztof; Tsukruk, Vladimir; Carrillo, Jan-Michael; Rubinstein, Michael; Dobrynin, Andrey; Zhou, Jing

2014-03-01

Macromolecules typically phase separate unless their shapes and chemical compositions are tailored to explicitly drive mixing. But now our research has shown that physical constraints can drive spontaneous mixing of chemically different species. We have obtained long-range 2D arrays of perfectly mixed macromolecules having a variety of molecular architectures and chemistries, including linear chains, block-copolymer stars, and bottlebrush copolymers with hydrophobic, hydrophilic, and lipophobic chemical compositions. This is achieved by entropy-driven enhancement of steric repulsion between macromolecules anchored on a substrate. By monitoring the kinetics of mixing, we have proved that molecular intercalation is an equilibrium state. The array spacing is controlled by the length of the brush side chains. This entropic templating strategy opens new ways for generating patterns on sub-100 nm length scales with potential application in lithography, directed self-assembly, and biomedical assays. Financial support from the National Science Foundation DMR-0906985, DMR-1004576, DMR-1122483, and DMR-0907515.

Autophagy in protists

PubMed Central

Duszenko, Michael; Ginger, Michael L; Brennand, Ana; Gualdrón-López, Melisa; Colombo, Maria-Isabel; Coombs, Graham H; Coppens, Isabelle; Jayabalasingham, Bamini; Langsley, Gordon; de Castro, Solange Lisboa; Menna-Barreto, Rubem; Mottram, Jeremy C; Navarro, Miguel; Rigden, Daniel J; Romano, Patricia S; Stoka, Veronika; Turk, Boris

2011-01-01

Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles and defense against parasitic invaders. During the past 10–20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target. PMID:20962583

Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm

PubMed Central

Yu, Isseki; Mori, Takaharu; Ando, Tadashi; Harada, Ryuhei; Jung, Jaewoon; Sugita, Yuji; Feig, Michael

2016-01-01

Biological macromolecules function in highly crowded cellular environments. The structure and dynamics of proteins and nucleic acids are well characterized in vitro, but in vivo crowding effects remain unclear. Using molecular dynamics simulations of a comprehensive atomistic model cytoplasm we found that protein-protein interactions may destabilize native protein structures, whereas metabolite interactions may induce more compact states due to electrostatic screening. Protein-protein interactions also resulted in significant variations in reduced macromolecular diffusion under crowded conditions, while metabolites exhibited significant two-dimensional surface diffusion and altered protein-ligand binding that may reduce the effective concentration of metabolites and ligands in vivo. Metabolic enzymes showed weak non-specific association in cellular environments attributed to solvation and entropic effects. These effects are expected to have broad implications for the in vivo functioning of biomolecules. This work is a first step towards physically realistic in silico whole-cell models that connect molecular with cellular biology. DOI: http://dx.doi.org/10.7554/eLife.19274.001 PMID:27801646

Pulmonary vascular clearance of harmful endogenous macromolecules in a porcine model of acute liver failure.

PubMed

Nedredal, Geir I; Elvevold, Kjetil; Chedid, Marcio F; Ytrebø, Lars M; Rose, Christopher F; Sen, Sambit; Smedsrød, Bård; Jalan, Rajiv; Revhaug, Arthur

2016-01-01

Pulmonary complications are common in acute liver failure (ALF). The role of the lungs in the uptake of harmful soluble endogenous macromolecules was evaluated in a porcine model of ALF induced by hepatic devascularization (n = 8) vs. controls (n = 8). In additional experiments, pulmonary uptake was investigated in healthy pigs. Fluorochrome-labeled modified albumin (MA) was applied to investigate the cellular uptake. As compared to controls, the ALF group displayed a 4-fold net increased lung uptake of hyaluronan, and 5-fold net increased uptake of both tissue plasminogen activator and lysosomal enzymes. Anatomical distribution experiments in healthy animals revealed that radiolabeled MA uptake (taken up by the same receptor as hyaluronan) was 53% by the liver, and 24% by the lungs. The lung uptake of LPS was 14% whereas 60% remained in the blood. Both fluorescence and electron microscopy revealed initial uptake of MA by pulmonary endothelial cells (PECs) with later translocation to pulmonary intravascular macrophages (PIMs). Moreover, the presence of PIMs was evident 10 min after injection. Systemic inflammatory markers such as leukopenia and increased serum TNF-α levels were evident after 20 min in the MA and LPS groups. Significant lung uptake of harmful soluble macromolecules compensated for the defect liver scavenger function in the ALF-group. Infusion of MA induced increased TNF-α serum levels and leukopenia, similar to the effect of the known inflammatory mediator LPS. These observations suggest a potential mechanism that may contribute to lung damage secondary to liver disease.

DOMMINO 2.0: integrating structurally resolved protein-, RNA-, and DNA-mediated macromolecular interactions

PubMed Central

Kuang, Xingyan; Dhroso, Andi; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

2016-01-01

Macromolecular interactions are formed between proteins, DNA and RNA molecules. Being a principle building block in macromolecular assemblies and pathways, the interactions underlie most of cellular functions. Malfunctioning of macromolecular interactions is also linked to a number of diseases. Structural knowledge of the macromolecular interaction allows one to understand the interaction’s mechanism, determine its functional implications and characterize the effects of genetic variations, such as single nucleotide polymorphisms, on the interaction. Unfortunately, until now the interactions mediated by different types of macromolecules, e.g. protein–protein interactions or protein–DNA interactions, are collected into individual and unrelated structural databases. This presents a significant obstacle in the analysis of macromolecular interactions. For instance, the homogeneous structural interaction databases prevent scientists from studying structural interactions of different types but occurring in the same macromolecular complex. Here, we introduce DOMMINO 2.0, a structural Database Of Macro-Molecular INteractiOns. Compared to DOMMINO 1.0, a comprehensive database on protein-protein interactions, DOMMINO 2.0 includes the interactions between all three basic types of macromolecules extracted from PDB files. DOMMINO 2.0 is automatically updated on a weekly basis. It currently includes ∼1 040 000 interactions between two polypeptide subunits (e.g. domains, peptides, termini and interdomain linkers), ∼43 000 RNA-mediated interactions, and ∼12 000 DNA-mediated interactions. All protein structures in the database are annotated using SCOP and SUPERFAMILY family annotation. As a result, protein-mediated interactions involving protein domains, interdomain linkers, C- and N- termini, and peptides are identified. Our database provides an intuitive web interface, allowing one to investigate interactions at three different resolution levels: whole subunit network, binary interaction and interaction interface. Database URL: http://dommino.org PMID:26827237

Toxic properties of specific radiation determinant molecules, derived from radiated species

NASA Astrophysics Data System (ADS)

Popov, Dmitri; Maliev, Vecheslav; Kedar, Prasad; Casey, Rachael; Jones, Jeffrey

Introduction: High doses of radiation induce the formation of radiation toxins in the organs of irradiated mammals. After whole body irradiation, cellular macromolecules and cell walls are damaged as a result of long-lived radiation-induced free radicals, reactive oxygen species, and fast, charged particles of radiation. High doses of radiation induce breaks in the chemical bonds of macromolecules and cross-linking reactions via chemically active processes. These processes result in the creation of novel modified macromolecules that possess specific toxic and antigenic properties defined by the type and dose of irradiation by which they are generated. Radiation toxins isolated from the lymph of irradiated animals are classified as hematotoxic, neurotoxic, and enteric non-bacterial (GI) radiation toxins, and they play an important role in the development of hematopoietic, cerebrovascular, and gastrointestinal acute radiation syndromes (ARS). Seven distinct toxins derived from post-irradiated animals have been designated as Specific Radiation Determinants (SRD): SRD-1 (neurotoxic radiation toxin generated by the cerebrovascular form of ARS), SRD-3 (enteric non-bacterial radiation toxins generated by the gastrointestinal form of ARS), and SRD-4 (hematotoxic radiation toxins generated by the hematological, bone marrow form of ARS). SRD-4 is further subdivided into four groups depending on the severity of the ARS induced: SRD-4/1, mild ARS; SRD-4/2, moderate ARS; SRD-4/3, severe ARS; and SRD-4/4, extremely severe ARS. The seventh SRD, SRD-2 is a toxic extract derived from animals suffering from a fourth form of ARS, as described in European literature and produces toxicity primarily in the autonimic nervous system. These radiation toxins have been shown to be responsible for the induction of important pathophysiological, immunological, and biochemical reactions in ARS. Materials and Methods: These studies incorporated the use of statistically significant numbers of a variety of animals. Lymphatic fluid was collected from the thoracic ducts of bovine species exposed to lethal doses of gamma radiation, and the SRDs were separated by size exclusion gel filtration and high-performance liquid chromatography. We compared the toxicity of isolated radiation toxins in a variety of animals. The clinical characteristics of ARS induced by intravenous or intra-muscular injections of radiation toxins were observed. Results: In radiation-na¨ animals (rats, rabbits, and sheep), toxicity was defined ıve by observing the timing and rate of lethality following injections with extracted radiation toxins (SRDs). Preparations of SRD-1 were injected intra-muscularly in doses of 5 or 10 mg/kg body weight. We observed the development of cerebrovascular ARS with 100% lethality at 10-30 minutes after injection. Analysis of the toxicity of different forms of radiation toxins showed that cerebrovascular neurotoxins possess the highest toxicity compared with other forms of radiation toxins. The other SRD's were also injected into radiation-naive animals and observed for subsequent toxicity/lethality, with the other SRDs producing less virulent forms of ARS. However, both the SRD-2- and SRD-3-injected animals also suffered lethality between 2 and 30 days post-injection. Conclusions: We have observed that radiation toxins are transported from the cells and tissues of irradiated organisms to the interstitial blood and lymphatic fluids, and that this migration of radiation toxins occurs hours after irradiation. Upon analysis of the results of our research and literature sources, we postulate that radiation toxins arise from the radiation-induced chemical modification of macromolecules resident in cell membranes and other cellular structures. Furthermore, we postulate that these altered macromolecules are not processed by antigen processing cells, but instead bind to class II MHC molecules and TCR-beta chains. This causes nonspecific activation of T cells, pro-inflammatory agents such as cytokines and isozymes of phospholipase A2 and phospholipase C, and platelet-activating factor. Longer-term effects induced by the altered macromolecules include the activation of cytotoxic T cells, which induces cytolysis in radiation-damaged cells. Activated CD8+ T cells produce tumor necrosis factor-B and additional cytokines. By these mechanisms, we postulate that radiation toxins generate the pathophysiological reactions associated with acute radiation syndromes.

Sensors and regulators of intracellular pH.

PubMed

Casey, Joseph R; Grinstein, Sergio; Orlowski, John

2010-01-01

Protons dictate the charge and structure of macromolecules and are used as energy currency by eukaryotic cells. The unique function of individual organelles therefore depends on the establishment and stringent maintenance of a distinct pH. This, in turn, requires a means to sense the prevailing pH and to respond to deviations from the norm with effective mechanisms to transport, produce or consume proton equivalents. A dynamic, finely tuned balance between proton-extruding and proton-importing processes underlies pH homeostasis not only in the cytosol, but in other cellular compartments as well.

Genetic interactions between Drosophila melanogaster Atg1 and paxillin reveal a role for paxillin in autophagosome formation.

PubMed

Chen, Guang-Chao; Lee, Janice Y; Tang, Hong-Wen; Debnath, Jayanta; Thomas, Sheila M; Settleman, Jeffrey

2008-01-01

Autophagy is a conserved cellular process of macromolecule recycling that involves vesicle-mediated degradation of cytoplasmic components. Autophagy plays essential roles in normal cell homeostasis and development, the response to stresses such as nutrient starvation, and contributes to disease processes including cancer and neurodegeneration. Although many of the autophagy components identified from genetic screens in yeast are well conserved in higher organisms, the mechanisms by which this process is regulated in any species are just beginning to be elucidated. In a genetic screen in Drosophila melanogaster, we have identified a link between the focal adhesion protein paxillin and the Atg1 kinase, which has been previously implicated in autophagy. In mammalian cells, we find that paxillin is redistributed from focal adhesions during nutrient deprivation, and paxillin-deficient cells exhibit defects in autophagosome formation. Together, these findings reveal a novel evolutionarily conserved role for paxillin in autophagy.

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

PubMed

Kim, Sun Yee; Park, Jeen-Woo

2003-03-01

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

Controlling Cellular Endocytosis at the Nanoscale

NASA Astrophysics Data System (ADS)

Battaglia, Giuseppe

2011-03-01

One of the most challenging aspects of drug delivery is the intra-cellular delivery of active agents. Several drugs and especially nucleic acids all need to be delivered within the cell interior to exert their therapeutic action. Small hydrophobic molecules can permeate cell membranes with relative ease, but hydrophilic molecules and especially large macromolecules such as proteins and nucleic acids require a vector to assist their transport across the cell membrane. This must be designed so as to ensure intracellular delivery without compromising cell viability. We have recently achieved this by using pH-sensitive poly(2-(methacryloyloxy)ethyl-phosphorylcholine)- co -poly(2-(diisopropylamino)ethyl methacrylate) (PMPC-PDPA) and poly(ethylene oxide)-co- poly(2-(diisopropylamino)ethyl methacrylate) (PEO-PDPA) diblock copolymers that self-assemble to form vesicles in aqueous solution. These vesicles combine a non-fouling PMPC or PEO block with a pH-sensitive PDPA block and have the ability to encapsulate both hydrophobic molecules within the vesicular membrane and hydrophilic molecules within their aqueous cores. The pH sensitive nature of the PDPA blocks make the diblock copolymers forming stable vesicles at physiological pH but that rapid dissociation of these vesicles occurs between pH 5 and pH 6 to form molecularly dissolved copolymer chains (unimers). We used these vesicles to encapsulate small and large macromolecules and these were successfully delivered intracellularly including nucleic acid, drugs, quantum dots, and antibodies. Dynamic light scattering, zeta potential measurements, and transmission electron microscopy were used to study and optimise the encapsulation processes. Confocal laser scanning microscopy, fluorescence flow cytometry and lysates analysis were used to quantify cellular uptake and to study the kinetics of this process in vitro and in vivo. We show the effective cytosolic delivery of nucleic acids, proteins, hydrophobic molecules, amphiphilic molecules, and hydrophilic molecules without affecting the viability of cells or even triggering inflammatory pathways. Finally we show how size, surface chemistry and surface topology of the vesicles affect their interaction with the cell membrane and hence their cellular uptake. References: C. Lo Presti, M. Massignani, T. Smart, H. Lomas, and G. Battaglia J. Mater. Chem. (2009) 19, 3576-3590 H. Lomas, I. Canton, S. MacNeil, J. Du, S.P. Armes, A.J. Ryan, A.L. Lewis and G. Battaglia Adv. Mater. (2007). 19, 4238-4243 M. Massignani, I. Canton, N. Patikarnmonthon, N. J. Warren, S. P. Armes, A. L. Lewis and G. Battaglia, Nature Prec., 2010, http://hdl.handle.net/10101/npre.2010.4427.1 M. Massignani, C. LoPresti, A. Blanazs, J. Madsen, S. P. Armes, A. L. Lewis and G. Battaglia Small, 2009, 5, 2424-2432. M. Massignani, T. Sun, A. Blanazs, V. Hearnden, I. Canton, P. Desphande, S. Armes, S. MacNeil, A. Lewis and G. Battaglia PLoS One, 2010, 5, e10459.

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development

PubMed Central

van Lessen, Max; Shibata-Germanos, Shannon; van Impel, Andreas; Hawkins, Thomas A; Rihel, Jason; Schulte-Merker, Stefan

2017-01-01

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain. DOI: http://dx.doi.org/10.7554/eLife.25932.001 PMID:28498105

Genetic mouse models of brain ageing and Alzheimer's disease.

PubMed

Bilkei-Gorzo, Andras

2014-05-01

Progression of brain ageing is influenced by a complex interaction of genetic and environmental factors. Analysis of genetically modified animals with uniform genetic backgrounds in a standardised, controlled environment enables the dissection of critical determinants of brain ageing on a molecular level. Human and animal studies suggest that increased load of damaged macromolecules, efficacy of DNA maintenance, mitochondrial activity, and cellular stress defences are critical determinants of brain ageing. Surprisingly, mouse lines with genetic impairment of anti-oxidative capacity generally did not show enhanced cognitive ageing but rather an increased sensitivity to oxidative challenge. Mouse lines with impaired mitochondrial activity had critically short life spans or severe and rapidly progressing neurodegeneration. Strains with impaired clearance in damaged macromolecules or defects in the regulation of cellular stress defences showed alterations in the onset and progression of cognitive decline. Importantly, reduced insulin/insulin-like growth factor signalling generally increased life span but impaired cognitive functions revealing a complex interaction between ageing of the brain and of the body. Brain ageing is accompanied by an increased risk of developing Alzheimer's disease. Transgenic mouse models expressing high levels of mutant human amyloid precursor protein showed a number of symptoms and pathophysiological processes typical for early phase of Alzheimer's disease. Generally, therapeutic strategies effective against Alzheimer's disease in humans were also active in the Tg2576, APP23, APP/PS1 and 5xFAD lines, but a large number of false positive findings were also reported. The 3xtg AD model likely has the highest face and construct validity but further studies are needed. Copyright © 2013 Elsevier Inc. All rights reserved.

Perspective: On the importance of hydrodynamic interactions in the subcellular dynamics of macromolecules

PubMed Central

Skolnick, Jeffrey

2016-01-01

An outstanding challenge in computational biophysics is the simulation of a living cell at molecular detail. Over the past several years, using Stokesian dynamics, progress has been made in simulating coarse grained molecular models of the cytoplasm. Since macromolecules comprise 20%-40% of the volume of a cell, one would expect that steric interactions dominate macromolecular diffusion. However, the reduction in cellular diffusion rates relative to infinite dilution is due, roughly equally, to steric and hydrodynamic interactions, HI, with nonspecific attractive interactions likely playing rather a minor role. HI not only serve to slow down long time diffusion rates but also cause a considerable reduction in the magnitude of the short time diffusion coefficient relative to that at infinite dilution. More importantly, the long range contribution of the Rotne-Prager-Yamakawa diffusion tensor results in temporal and spatial correlations that persist up to microseconds and for intermolecular distances on the order of protein radii. While HI slow down the bimolecular association rate in the early stages of lipid bilayer formation, they accelerate the rate of large scale assembly of lipid aggregates. This is suggestive of an important role for HI in the self-assembly kinetics of large macromolecular complexes such as tubulin. Since HI are important, questions as to whether continuum models of HI are adequate as well as improved simulation methodologies that will make simulations of more complex cellular processes practical need to be addressed. Nevertheless, the stage is set for the molecular simulations of ever more complex subcellular processes. PMID:27634243

Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases

PubMed Central

Wheeler, Richard; Turner, Robert D.; Bailey, Richard G.; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A. S.; Hayhurst, Emma J.; Horsburgh, Malcolm; Hobbs, Jamie K.

2015-01-01

ABSTRACT Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. PMID:26220963

An introduction to the molecular basics of aryl hydrocarbon receptor biology.

PubMed

Abel, Josef; Haarmann-Stemmann, Thomas

2010-11-01

Depending on their chemical structure and properties, environmental chemicals and other xenobiotics that enter the cell can affect cellular function by either nonselective binding to cellular macromolecules or by interference with cellular receptors, which would initiate a more defined cell biological response. One of these intracellular chemosensor molecules is the aryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family that is known to mediate the biochemical and toxic effects of dioxins, polyaromatic hydrocarbons and related compounds. Numerous investigations have revealed that the AhR is not only a master regulator of drug metabolism activated by anthropogenic chemicals, but is also triggered by natural and endogenous ligands and can influence cell biological endpoints such as growth and differentiation. Cutting-edge research has identified new intriguing functions of the AhR, such as during proteasomal degradation of steroid hormone receptors, the cellular UVB stress response and the differentiation of certain T-cell subsets. In this review we provide both a survey of the fundamental basics of AhR biology and an insight into new functional aspects of AhR signaling to further stimulate research on this intriguing transcription factor at the interface between toxicology, cell biology and immunology.

Prevention by thioethers of the hepatotoxicity and covalent binding to macromolecules of N-hydroxy-2-acetylaminofluorene and its sulfate ester in rat liver in vivo and in vitro.

PubMed

van den Goorbergh, J A; de Wit, H; Tijdens, R B; Mulder, G J; Meerman, J H

1987-02-01

In order to find potentially effective compounds that could prevent the covalent binding of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to rat liver macromolecules in vivo, the prevention of the covalent binding to RNA of the sulfate ester of the carcinogen N-OH-AAF by a series of thioethers was investigated in vitro. The most effective thioethers, which inhibited the covalent binding by 70% or more, were studied for their protection against acute hepatotoxicity of N-OH-AAF in the rat in vivo. Three of these thioethers, thiazolidine, methyl 4-(methylthio)benzoate, and 2-(methylthio)benzimidazole significantly decreased the hepatoxicity of N-OH-AAF, by 45, 71 and 83%, respectively. The effects of these thioethers on the covalent binding of N-OH-AAF to cellular macromolecules in vivo were also studied. Methyl 4-(methylthio)benzoate and 2-(methylthio)benzimidazole decreased the adduct formation of N-OH-AAF to DNA by 54 and 44%, respectively, but had no effect on protein adduct formation. Only 2-(methylthio)benzimidazole caused a slight decrease (23%) in the AAF-- protein adduct formation. 2-Acetylaminofluorene (AAF) and methyl 4-(methyl-sulfinyl)benzoate were the main products in the incubation of methyl 4-(methylthio)benzoate with AAF-N-sulfate in vitro. This suggests that the thioether attacks the nitrenium ion which is formed by spontaneous breakdown of AAF-N-sulfate; the formation of a sulfonium--AAF conjugate is postulated which decomposes into AAF and a sulfinyl compound.

X-ray microanalysis of exocrine glands in animal models for cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, R.M.R.; Roomans, G.M.

1985-01-01

Elemental distribution and ultrastructure of the submandibular gland, the parotid gland and the pancreas were investigated in three suggested animal models of the disease cystic fibrosis: the chronically reserpinized rat, the chronically isoproterenol-treated rat, and the chronically pilocarpine-treated rat. To elucidate the cellular mechanism underlying the effects of these treatments, chronic effects of specific alpha - and beta -adrenergic agonists, as well as acute effects of reserpine and various agonists were also investigated. Reserpine, isoproterenol, and pilocarpine cause an increase in the calcium concentration in submandibular gland acinar cells, due to an increased calcium content of the intracellular mucus. Inmore » the parotid gland, reserpine and isoproterenol cause a decrease of the calcium concentration in acinar cells, due to a lower calcium content of the zymogen granules. In the submandibular gland, a decreased cellular Na concentration was noted after chronic treatment with isoproterenol or pilocarpine, and after a single dose of reserpine or isoproterenol. Ultrastructural changes in the exocrine glands investigated included excessive accumulation of intracellular secretory material and formation of abnormal uncondensed secretion granules. A common pattern in the animal models appears to be (1) inhibition of secretion resulting in intracellular accumulation of secretory material, (2) synthesis of secretory macromolecules with altered cation-binding properties.« less

Transcription factor EB: from master coordinator of lysosomal pathways to candidate therapeutic target in degenerative storage diseases.

PubMed

Sardiello, Marco

2016-05-01

The lysosome is the main catabolic hub of the cell. Owing to its role in fundamental processes such as autophagy, plasma membrane repair, mTOR signaling, and maintenance of cellular homeostasis, the lysosome has a profound influence on cellular metabolism and human health. Indeed, inefficient or impaired lysosomal function has been implicated in the pathogenesis of a number of degenerative diseases affecting various organs and tissues, most notably the brain, liver, and muscle. The discovery of the coordinated lysosomal expression and regulation (CLEAR) genetic program and its master controller, transcription factor EB (TFEB), has provided an unprecedented tool to study and manipulate lysosomal function. Most lysosome-based processes-including macromolecule degradation, autophagy, lysosomal exocytosis, and proteostasis-are under the transcriptional control of TFEB. Interestingly, impaired TFEB signaling has been suggested to be a contributing factor in the pathogenesis of several degenerative storage diseases. Preclinical studies based on TFEB exogenous expression to reinstate TFEB activity or promote CLEAR network-based lysosomal enhancement have highlighted TFEB as a candidate therapeutic target for the treatment of various degenerative storage diseases. © 2016 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals, Inc. on behalf of New York Academy of Sciences.

Understanding FRET as a Research Tool for Cellular Studies

PubMed Central

Shrestha, Dilip; Jenei, Attila; Nagy, Péter; Vereb, György; Szöllősi, János

2015-01-01

Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types. PMID:25815593

Mitochondrial oxidative stress in aging and healthspan

PubMed Central

2014-01-01

The free radical theory of aging proposes that reactive oxygen species (ROS)-induced accumulation of damage to cellular macromolecules is a primary driving force of aging and a major determinant of lifespan. Although this theory is one of the most popular explanations for the cause of aging, several experimental rodent models of antioxidant manipulation have failed to affect lifespan. Moreover, antioxidant supplementation clinical trials have been largely disappointing. The mitochondrial theory of aging specifies more particularly that mitochondria are both the primary sources of ROS and the primary targets of ROS damage. In addition to effects on lifespan and aging, mitochondrial ROS have been shown to play a central role in healthspan of many vital organ systems. In this article we review the evidence supporting the role of mitochondrial oxidative stress, mitochondrial damage and dysfunction in aging and healthspan, including cardiac aging, age-dependent cardiovascular diseases, skeletal muscle aging, neurodegenerative diseases, insulin resistance and diabetes as well as age-related cancers. The crosstalk of mitochondrial ROS, redox, and other cellular signaling is briefly presented. Potential therapeutic strategies to improve mitochondrial function in aging and healthspan are reviewed, with a focus on mitochondrial protective drugs, such as the mitochondrial antioxidants MitoQ, SkQ1, and the mitochondrial protective peptide SS-31. PMID:24860647

Using Solid-state NMR to Monitor the Molecular Consequences of Cryptococcus neoformans Melanization with Different Catecholamine Precursors

PubMed Central

Chatterjee, Subhasish; Prados-Rosales, Rafael; Frases, Susana; Itin, Boris; Casadevall, Arturo; Stark, Ruth E.

2012-01-01

Melanins are a class of natural pigments associated with a wide range of biological functions, including microbial virulence, energy transduction, and protection against solar radiation. Because of their insolubility and structural heterogeneity, solid-state nuclear magnetic resonance (NMR) spectroscopy provides an unprecedented means to define the molecular architecture of these enigmatic pigments. The requirement of obligatory catecholamines for melanization of the pathogenic fungus Cryptococcus neoformans also offers unique opportunities for investigating melanin development. In the current study, pigments produced with L-dopa, methyl-L-dopa, epinephrine, and norepinephrine precursors are compared structurally using 13C and 1H magic-angle spinning (MAS) NMR. Striking structural differences were observed for both aromatic and aliphatic molecular constituents of the mature fungal pigment assemblies, thus making it possible to redefine the molecular prerequisites for formation of the aromatic domains of insoluble indole-based biopolymers, to rationalize their distinctive physical characteristics, and to delineate the role of cellular constituents in assembly of the melanized macromolecules with polysaccharides and fatty acyl chain-containing moieties. By achieving an augmented understanding of the mechanisms of C. neoformans melanin biosynthesis and cellular assembly, such studies can guide future drug discovery efforts related to melanin-associated virulence, resistance to tumor therapy, and production of melanin mimetics under cell-free conditions. PMID:22765382

Induced liquid-crystalline ordering in solutions of stiff and flexible amphiphilic macromolecules: Effect of mixture composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glagolev, Mikhail K.; Vasilevskaya, Valentina V., E-mail: vvvas@polly.phys.msu.ru; Khokhlov, Alexei R.

Impact of mixture composition on self-organization in concentrated solutions of stiff helical and flexible macromolecules was studied by means of molecular dynamics simulation. The macromolecules were composed of identical amphiphilic monomer units but a fraction f of macromolecules had stiff helical backbones and the remaining chains were flexible. In poor solvents the compacted flexible macromolecules coexist with bundles or filament clusters from few intertwined stiff helical macromolecules. The increase of relative content f of helical macromolecules leads to increase of the length of helical clusters, to alignment of clusters with each other, and then to liquid-crystalline-like ordering along a singlemore » direction. The formation of filament clusters causes segregation of helical and flexible macromolecules and the alignment of the filaments induces effective liquid-like ordering of flexible macromolecules. A visual analysis and calculation of order parameter relaying the anisotropy of diffraction allow concluding that transition from disordered to liquid-crystalline state proceeds sharply at relatively low content of stiff components.« less

NMR-Fragment Based Virtual Screening: A Brief Overview.

PubMed

Singh, Meenakshi; Tam, Benjamin; Akabayov, Barak

2018-01-25

Fragment-based drug discovery (FBDD) using NMR has become a central approach over the last twenty years for development of small molecule inhibitors against biological macromolecules, to control a variety of cellular processes. Yet, several considerations should be taken into account for obtaining a therapeutically relevant agent. In this review, we aim to list the considerations that make NMR fragment screening a successful process for yielding potent inhibitors. Factors that may govern the competence of NMR in fragment based drug discovery are discussed, as well as later steps that involve optimization of hits obtained by NMR-FBDD.

Recent Applications of Fluorescence Recovery after Photobleaching (FRAP) to Membrane Bio-Macromolecules

PubMed Central

Rayan, Gamal; Guet, Jean-Erik; Taulier, Nicolas; Pincet, Frederic; Urbach, Wladimir

2010-01-01

This review examines some recent applications of fluorescence recovery after photobleaching (FRAP) to biopolymers, while mainly focusing on membrane protein studies. Initially, we discuss the lateral diffusion of membrane proteins, as measured by FRAP. Then, we talk about the use of FRAP to probe interactions between membrane proteins by obtaining fundamental information such as geometry and stoichiometry of the interacting complex. Afterwards, we discuss some applications of FRAP at the cellular level as well as the level of organisms. We conclude by comparing diffusion coefficients obtained by FRAP and several other alternative methods. PMID:22219695

Novel approach using DNA-RNA hybrids in RNA nanotechnology | Center for Cancer Research

Cancer.gov

Developing simple approaches to detect interactions, modifications, and cellular locations of macromolecules is essential for understanding biochemical processes. The use of protein fragment complementation assays, also called split-protein systems, is a highly sensitive approach for studying protein interactions in biological systems. In this approach, functional proteins are split into non-functional fragments, and when attached to possible interacting partners, can reassemble and become functional again. Use of split-protein assays can establish differences between a healthy and a diseased state in the cell as well as determine the outcome of a therapeutic intervention.

Vectorization of Nucleic Acids for Therapeutic Approach: Tutorial Review.

PubMed

Geinguenaud, Frederic; Guenin, Erwann; Lalatonne, Yoann; Motte, Laurence

2016-05-20

Oligonucleotides present a high therapeutic potential for a wide variety of diseases. However, their clinical development is limited by their degradation by nucleases and their poor blood circulation time. Depending on the administration mode and the cellular target, these macromolecules will have to cross the vascular endothelium, to diffuse through the extracellular matrix, to be transported through the cell membrane, and finally to reach the cytoplasm. To overcome these physiological barriers, many strategies have been developed. Here, we review different methods of DNA vectorization, discuss limitations and advantages of the various vectors, and provide new perspectives for future development.

Nutrient acquisition strategies of mammalian cells.

PubMed

Palm, Wilhelm; Thompson, Craig B

2017-06-07

Mammalian cells are surrounded by diverse nutrients, such as glucose, amino acids, various macromolecules and micronutrients, which they can import through transmembrane transporters and endolysosomal pathways. By using different nutrient sources, cells gain metabolic flexibility to survive periods of starvation. Quiescent cells take up sufficient nutrients to sustain homeostasis. However, proliferating cells depend on growth-factor-induced increases in nutrient uptake to support biomass formation. Here, we review cellular nutrient acquisition strategies and their regulation by growth factors and cell-intrinsic nutrient sensors. We also discuss how oncogenes and tumour suppressors promote nutrient uptake and thereby support the survival and growth of cancer cells.

Regulation of singlet oxygen-induced apoptosis by cytosolic NADP+-dependent isocitrate dehydrogenase.

PubMed

Kim, Sun Yee; Lee, Su Min; Tak, Jean Kyoung; Choi, Kyeong Sook; Kwon, Taeg Kyu; Park, Jeen-Woo

2007-08-01

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. Recently, we demonstrated that the control of redox balance and the cellular defense against oxidative damage are the primary functions of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) through supplying NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPc activity in HL-60 cells regulates singlet oxygen-induced apoptosis. When we examined the protective role of IDPc against singlet oxygen-induced apoptosis with HL-60 cells transfected with the cDNA for mouse IDPc in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPc plays an important protective role in apoptosis of HL-60 cells induced by singlet oxygen.

Nickel impact on human health: An intrinsic disorder perspective.

PubMed

Zambelli, Barbara; Uversky, Vladimir N; Ciurli, Stefano

2016-12-01

The interplay of the presence of nickel and protein disorder in processes affecting human health is the focus of the present review. Many systems involving nickel as either a cofactor or as a toxic contaminant are characterized by large disorder. The role of nickel in the biochemistry of bacterial enzymes is discussed here, covering both the beneficial effects of nickel in the human microbiota as well as the role of nickel-depending bacteria in human pathogenesis. In addition, the hazardous health effects caused by nickel exposure to humans, namely nickel-induced carcinogenesis and allergy, are triggered by non-specific interactions of nickel with macromolecules and formation of reactive compounds that mediate cellular damage. Cellular response to nickel is also related to signal transduction cascades. This review thus highlights the most promising systems for future studies aimed at decreasing the adverse effects of nickel on human health. Copyright © 2016 Elsevier B.V. All rights reserved.

Functions of the gene products of Escherichia coli.

PubMed Central

Riley, M

1993-01-01

A list of currently identified gene products of Escherichia coli is given, together with a bibliography that provides pointers to the literature on each gene product. A scheme to categorize cellular functions is used to classify the gene products of E. coli so far identified. A count shows that the numbers of genes concerned with small-molecule metabolism are on the same order as the numbers concerned with macromolecule biosynthesis and degradation. One large category is the category of tRNAs and their synthetases. Another is the category of transport elements. The categories of cell structure and cellular processes other than metabolism are smaller. Other subjects discussed are the occurrence in the E. coli genome of redundant pairs and groups of genes of identical or closely similar function, as well as variation in the degree of density of genetic information in different parts of the genome. PMID:7508076

Extracellular matrix structure.

PubMed

Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

2016-02-01

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

The Molecular Timeline of a Reviving Bacterial Spore

PubMed Central

Sinai, Lior; Rosenberg, Alex; Smith, Yoav; Segev, Einat; Ben-Yehuda, Sigal

2015-01-01

Summary The bacterial spore can rapidly convert from a dormant to a fully active cell. Here we study this remarkable cellular transition in Bacillus subtilis and reveal the identity of the newly synthesized proteins throughout spore revival. Our analysis uncovers a highly ordered developmental program that correlates with the spore morphological changes and reveals the spatial and temporal molecular events fundamental to reconstruct a cell. As opposed to current knowledge, we found that translation takes place during the earliest revival event, termed germination, a process hitherto considered to occur without the need for any macromolecule synthesis. Furthermore, we demonstrate that translation is required for execution of germination and relies on the bona fide translational factors RpmE and Tig. Our study sheds light on the spore revival process and on the vital building blocks underlying cellular awakening, thereby paving the way for designing new antimicrobial agents to eradicate spore-forming pathogens. PMID:25661487

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations: How bacterial secretion systems bias Brownian motion for efficient translocation of macromolecules.

PubMed

Hepp, Christof; Maier, Berenike

2017-10-01

Secretion systems enable bacteria to import and secrete large macromolecules including DNA and proteins. While most components of these systems have been identified, the molecular mechanisms of macromolecular transport remain poorly understood. Recent findings suggest that various bacterial secretion systems make use of the translocation ratchet mechanism for transporting polymers across the cell envelope. Translocation ratchets are powered by chemical potential differences generated by concentration gradients of ions or molecules that are specific to the respective secretion systems. Bacteria employ these potential differences for biasing Brownian motion of the macromolecules within the conduits of the secretion systems. Candidates for this mechanism include DNA import by the type II secretion/type IV pilus system, DNA export by the type IV secretion system, and protein export by the type I secretion system. Here, we propose that these three secretion systems employ different molecular implementations of the translocation ratchet mechanism. © 2017 The Authors. BioEssays Published by WILEY Periodicals, Inc.

Endo-lysosomal and autophagic dysfunction: a driving factor in Alzheimer's disease?

PubMed

Whyte, Lauren S; Lau, Adeline A; Hemsley, Kim M; Hopwood, John J; Sargeant, Timothy J

2017-03-01

Alzheimer's disease (AD) is the most common cause of dementia, and its prevalence will increase significantly in the coming decades. Although important progress has been made, fundamental pathogenic mechanisms as well as most hereditary contributions to the sporadic form of the disease remain unknown. In this review, we examine the now substantial links between AD pathogenesis and lysosomal biology. The lysosome hydrolyses and processes cargo delivered by multiple pathways, including endocytosis and autophagy. The endo-lysosomal and autophagic networks are central to clearance of cellular macromolecules, which is important given there is a deficit in clearance of amyloid-β in AD. Numerous studies show prominent lysosomal dysfunction in AD, including perturbed trafficking of lysosomal enzymes and accumulation of the same substrates that accumulate in lysosomal storage disorders. Examination of the brain in lysosomal storage disorders shows the accumulation of amyloid precursor protein metabolites, which further links lysosomal dysfunction with AD. This and other evidence leads us to hypothesise that genetic variation in lysosomal genes modifies the disease course of sporadic AD. © 2016 International Society for Neurochemistry.

A Study of Aberrant Glycosylation in Simulated Microgravity Using Laser Induced AutoFluorescence and Flow Cytometry

NASA Technical Reports Server (NTRS)

Lawless, B. DeSales

1999-01-01

A number of pathologies and cellular dysfunctions including neoplasms have been correlated with autofluorescence. The complications of aging and diabetes have been associated with the accumulation of non-enzymatic glycosylations of tissue macromolecules. These products are known as the Advanced Glycosylated End Products (AGEs). A physical property associated with AGEs is the emission of 570 mn or 630 nm light energy (autofluorescence) following the absorption of 448 mm energy associated with the argon laser. This investigation sought to assess the induction of argon-laser induced autofluorescence in a variety of in vitro culture systems. Different fluorescence intensities distinguished tumor lines from normal cell populations. Laser-stimulated autofluorescence discriminated primary cultures of lymphocytes grown in the presence of excess glucose as opposed to normal glucose concentrations. The effects of deglycosylating agents upon laser-induced autofluorescence were also assessed. The studies included studies of cell cycle analysis using Propidium Iodide stained DNA of cells grown in simulated microgravity using NASA Bioreactor Vessels in media of normal and elevated glucose concentrations.

The plant cell nucleus: a true arena for the fight between plants and pathogens.

PubMed

Deslandes, Laurent; Rivas, Susana

2011-01-01

Communication between the cytoplasm and the nucleus is a fundamental feature shared by both plant and animal cells. Cellular factors involved in the transport of macromolecules through the nuclear envelope, including nucleoporins, importins and Ran-GTP related components, are conserved among a variety of eukaryotic systems. Interestingly, mutations in these nuclear components compromise resistance signalling, illustrating the importance of nucleocytoplasmic trafficking in plant innate immunity. Indeed, spatial restriction of defence regulators by the nuclear envelope and stimulus-induced nuclear translocation constitute an important level of defence-associated gene regulation in plants. A significant number of effectors from different microbial pathogens are targeted to the plant cell nucleus. In addition, key host factors, including resistance proteins, immunity components, transcription factors and transcriptional regulators shuttle between the cytoplasm and the nucleus, and their level of nuclear accumulation determines the output of the defence response, further confirming the crucial role played by the nucleus during the interaction between plants and pathogens. Here, we discuss recent findings that situate the nucleus at the frontline of the mutual recognition between plants and invading microbes.

What Is Life? What Was Life? What Will Life Be?

NASA Astrophysics Data System (ADS)

Deamer, D.

Our laboratory is exploring self-assembly processes and polymerization reactions of organic compounds in natural geothermal environments and related laboratory simulations. Although the physical environment that fostered primitive cellular life is still largely unconstrained, we can be reasonably confident that liquid water was required, together with a source of organic compounds and energy to drive polymerization reactions. There must also have been a process by which the compounds were sufficiently concentrated to undergo physical and chemical interactions. In earlier work we observed that macromolecules such as nucleic acids and proteins are readily encapsulated in membranous boundaries during wet-dry cycles such as those that would occur at the edges of geothermal springs or tide pools. The resulting structures are referred to as protocells, in that they exhibit certain properties of living cells and are models of the kinds of encapsulated macromolecular systems that would have led toward the first forms of cellular life. However, the assembly of protocells is markedly inhibited by conditions associated with extreme environments: High temperature, high salt concentrations, and low pH ranges. From a biophysical perspective, it follows that the most plausible planetary environment for the origin of cellular life would be an aqueous phase at moderate temperature ranges and low ionic strength, having a pH value near neutrality and divalent cations at submillimolar concentrations. This suggestion is in marked contrast to the view that life most likely began in a geothermal or marine environment, perhaps even the extreme environment of a hydrothermal vent. A more plausible site for the origin of cellular life would be fresh water pools maintained by rain falling on volcanic land masses resembling present-day Hawaii and Iceland. After the first cellular life was able to establish itself in a relatively benign environment, it would rapidly begin to adapt through Darwinian selection to more rigorous environments, including the extreme temperatures, salt concentrations and pH ranges that we now associate with the limits of life on the Earth.

Dietary Modulation of Oxidative Stress in Alzheimer's Disease.

PubMed

Thapa, Arjun; Carroll, Nick J

2017-07-21

Cells generate unpaired electrons, typically via oxygen- or nitrogen-based by-products during normal cellular respiration and under stressed situations. These pro-oxidant molecules are highly unstable and may oxidize surrounding cellular macromolecules. Under normal conditions, the reactive oxygen or nitrogen species can be beneficial to cell survival and function by destroying and degrading pathogens or antigens. However, excessive generation and accumulation of the reactive pro-oxidant species over time can damage proteins, lipids, carbohydrates, and nucleic acids. Over time, this oxidative stress can contribute to a range of aging-related degenerative diseases such as cancer, diabetes, macular degeneration, and Alzheimer's, and Parkinson's diseases. It is well accepted that natural compounds, including vitamins A, C, and E, β-carotene, and minerals found in fruits and vegetables are powerful anti-oxidants that offer health benefits against several different oxidative stress induced degenerative diseases, including Alzheimer's disease (AD). There is increasing interest in developing anti-oxidative therapeutics to prevent AD. There are contradictory and inconsistent reports on the possible benefits of anti-oxidative supplements; however, fruits and vegetables enriched with multiple anti-oxidants (e.g., flavonoids and polyphenols) and minerals may be highly effective in attenuating the harmful effects of oxidative stress. As the physiological activation of either protective or destructive pro-oxidant behavior remains relatively unclear, it is not straightforward to relate the efficacy of dietary anti-oxidants in disease prevention. Here, we review oxidative stress mediated toxicity associated with AD and highlight the modulatory roles of natural dietary anti-oxidants in preventing AD.

Phenomenology based multiscale models as tools to understand cell membrane and organelle morphologies

PubMed Central

Ramakrishnan, N.; Radhakrishnan, Ravi

2016-01-01

An intriguing question in cell biology is “how do cells regulate their shape?” It is commonly believed that the observed cellular morphologies are a result of the complex interaction among the lipid molecules (constituting the cell membrane), and with a number of other macromolecules, such as proteins. It is also believed that the common biophysical processes essential for the functioning of a cell also play an important role in cellular morphogenesis. At the cellular scale—where typical dimensions are in the order of micrometers—the effects arising from the molecular scale can either be modeled as equilibrium or non-equilibrium processes. In this chapter, we discuss the dynamically triangulated Monte Carlo technique to model and simulate membrane morphologies at the cellular scale, which in turn can be used to investigate several questions related to shape regulation in cells. In particular, we focus on two specific problems within the framework of isotropic and anisotropic elasticity theories: namely, (i) the origin of complex, physiologically relevant, membrane shapes due to the interaction of the membrane with curvature remodeling proteins, and (ii) the genesis of steady state cellular shapes due to the action of non-equilibrium forces that are generated by the fission and fusion of transport vesicles and by the binding and unbinding of proteins from the parent membrane. PMID:27087801

Cellular Senescence, Neurological Function, and Redox State.

PubMed

Maciel-Barón, Luis Ángel; Moreno-Blas, Daniel; Morales-Rosales, Sandra Lizbeth; González-Puertos, Viridiana Yazmín; López-Díazguerrero, Norma Edith; Torres, Claudio; Castro-Obregón, Susana; Königsberg, Mina

2018-06-20

Cellular senescence, characterized by permanent cell cycle arrest, has been extensively studied in mitotic cells such as fibroblasts. However, senescent cells have also been observed in the brain. Even though it is recognized that cellular energetic metabolism and redox homeostasis are perturbed in the aged brain and neurodegenerative diseases (NDDs), it is still unknown which alterations in the overall physiology can stimulate cellular senescence induction and their relationship with the former events. Recent Advances: Recent findings have shown that during prolonged inflammatory and pathologic events, the blood-brain barrier could be compromised and immune cells might enter the brain; this fact along with the brain's high oxygen dependence might result in oxidative damage to macromolecules and therefore senescence induction. Thus, cellular senescence in different brain cell types is revised here. Most information related to cellular senescence in the brain has been obtained from research in glial cells since it has been assumed that the senescent phenotype is a feature exclusive to mitotic cells. Nevertheless, neurons with senescence hallmarks have been observed in old mouse brains. Therefore, although this is a controversial topic in the field, here we summarize and integrate the observations from several studies and propose that neurons indeed senesce. It is still unknown which alterations in the overall metabolism can stimulate senescence induction in the aged brain, what are the mechanisms and signaling pathways, and what is their relationship to NDD development. The understanding of these processes will expose new targets to intervene age-associated pathologies.-Antioxid. Redox Signal. 28, 1704-1723.

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

PubMed Central

Gurnev, Philip A.; Nestorovich, Ekaterina M.

2014-01-01

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications. PMID:25153255

Cyto-molecular Tuning of Quantum Dots

NASA Astrophysics Data System (ADS)

Lee, Bong; Suresh, Sindhuja; Ekpenyong, Andrew

Quantum dots (QDs) are semiconductor nanoparticles composed of groups II-VI or III-V elements, with physical dimensions smaller than the exciton Bohr radius, and between 1-10 nm. Their applications and promising myriad applications in photovoltaic cells, biomedical imaging, targeted drug delivery, quantum computing, etc, have led to much research on their interactions with other systems. For biological systems, research has focused on biocompatibility and cytotoxicity of QDs in the context of imaging/therapy. However, there is a paucity of work on how biological systems might be used to tune QDs. Here, we hypothesize that the photo-electronic properties of QDs can be tuned by biological macromolecules following controlled changes in cellular activities. Using CdSe/ZnS core-shell QDs, we perform spectroscopic analysis of optically excited colloidal QDs with and without promyelocytic HL60 cells. Preliminary results show shifts in the emission spectra of the colloidal dispersions with and without cells. We will present results for activated HL60-derived cells where specific macromolecules produced by these cells perturb the electric dipole moments of the excited QDs and the associated electric fields, in ways that constitute what we describe as cyto-molecular tuning. Startup funds from the College of Arts and Sciences, Creighton University (to AEE).

Decoding the mechanical fingerprints of biomolecules.

PubMed

Dudko, Olga K

2016-01-01

The capacity of biological macromolecules to act as exceedingly sophisticated and highly efficient cellular machines - switches, assembly factors, pumps, or motors - is realized through their conformational transitions, that is, their folding into distinct shapes and selective binding to other molecules. Conformational transitions can be induced, monitored, and manipulated by pulling individual macromolecules apart with an applied force. Pulling experiments reveal, for a given biomolecule, the relationship between applied force and molecular extension. Distinct signatures in the force-extension relationship identify a given biomolecule and thus serve as the molecule's 'mechanical fingerprints'. But, how can these fingerprints be decoded to uncover the energy barriers crossed by the molecule in the course of its conformational transition, as well as the associated timescales? This review summarizes a powerful class of approaches to interpreting single-molecule force spectroscopy measurements - namely, analytically tractable approaches. On the fundamental side, analytical theories have the power to reveal the unifying principles underneath the bewildering diversity of biomolecules and their behaviors. On the practical side, analytical expressions that result from these theories are particularly well suited for a direct fit to experimental data, yielding the important parameters that govern biological processes at the molecular level.

Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury

PubMed Central

Azzam, Edouard I.; Jay-Gerin, Jean-Paul; Pain, Debkumar

2013-01-01

Cellular exposure to ionizing radiation leads to oxidizing events that alter atomic structure through direct interactions of radiation with target macromolecules or via products of water radiolysis. Further, the oxidative damage may spread from the targeted to neighboring, non-targeted bystander cells through redox-modulated intercellular communication mechanisms. To cope with the induced stress and the changes in the redox environment, organisms elicit transient responses at the molecular, cellular and tissue levels to counteract toxic effects of radiation. Metabolic pathways are induced during and shortly after the exposure. Depending on radiation dose, dose-rate and quality, these protective mechanisms may or may not be sufficient to cope with the stress. When the harmful effects exceed those of homeostatic biochemical processes, induced biological changes persist and may be propagated to progeny cells. Physiological levels of reactive oxygen and nitrogen species play critical roles in many cellular functions. In irradiated cells, levels of these reactive species may be increased due to perturbations in oxidative metabolism and chronic inflammatory responses, thereby contributing to the long-term effects of exposure to ionizing radiation on genomic stability. Here, in addition to immediate biological effects of water radiolysis on DNA damage, we also discuss the role of mitochondria in the delayed outcomes of ionization radiation. Defects in mitochondrial functions lead to accelerated aging and numerous pathological conditions. Different types of radiation vary in their linear energy transfer (LET) properties, and we discuss their effects on various aspects of mitochondrial physiology. These include short and long-term in vitro and in vivo effects on mitochondrial DNA, mitochondrial protein import and metabolic and antioxidant enzymes. PMID:22182453

Structural and molecular interrogation of intact biological systems

PubMed Central

Chung, Kwanghun; Wallace, Jenelle; Kim, Sung-Yon; Kalyanasundaram, Sandhiya; Andalman, Aaron S.; Davidson, Thomas J.; Mirzabekov, Julie J.; Zalocusky, Kelly A.; Mattis, Joanna; Denisin, Aleksandra K.; Pak, Sally; Bernstein, Hannah; Ramakrishnan, Charu; Grosenick, Logan; Gradinaru, Viviana; Deisseroth, Karl

2014-01-01

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease. PMID:23575631

Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins.

PubMed

Kong, Muwen; Van Houten, Bennett

2017-08-01

Since Robert Brown's first observations of random walks by pollen particles suspended in solution, the concept of diffusion has been subject to countless theoretical and experimental studies in diverse fields from finance and social sciences, to physics and biology. Diffusive transport of macromolecules in cells is intimately linked to essential cellular functions including nutrient uptake, signal transduction, gene expression, as well as DNA replication and repair. Advancement in experimental techniques has allowed precise measurements of these diffusion processes. Mathematical and physical descriptions and computer simulations have been applied to model complicated biological systems in which anomalous diffusion, in addition to simple Brownian motion, was observed. The purpose of this review is to provide an overview of the major physical models of anomalous diffusion and corresponding experimental evidence on the target search problem faced by DNA-binding proteins, with an emphasis on DNA repair proteins and the role of anomalous diffusion in DNA target recognition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Physical effects at the cellular level under altered gravity conditions

NASA Technical Reports Server (NTRS)

Todd, Paul

1992-01-01

Several modifications of differentiated functions of animal cells cultivated in vitro have been reported when cultures have been exposed to increased or decreased inertial acceleration fields by centrifugation, clinorotation, and orbital space flight. Variables modified by clinorotation conditions include inertial acceleration, convection, hydrostatic pressure, sedimentation, and shear stress, which also affect transport processes in the extracellular chemical environment. Autocrine, paracrine and endocrine substances, to which cells are responsive via specific receptors, are usually transported in vitro (and possibly in certain embryos) by convection and in vivo by a circulatory system or ciliary action. Increased inertial acceleration increases convective flow, while microgravity nearly abolishes it. In the latter case the extracellular transport of macromolecules is governed by diffusion. By making certain assumptions it is possible to calculate the Peclet number, the ratio of convective transport to diffusive transport. Some, but not all, responses of cells in vitro to modified inertial environments could be manifestations of modified extracellular convective flow.

Highly Regioregular Polythiophenes for Magneto-Optical Applications

DTIC Science & Technology

2010-07-01

Macromolecules, 2007, 40, 8142-8150 Lieven De Cremer et.al., Macromolecules, 2008, 41, 568-578 Lieven De Cremer et.al., Macromolecules, 2008, 41, 591-598 Marnix...Vangheluwe et.al., Macromolecules, 2008, 41, 1041-1044 David Cornelis et.al., Chem. Mater. 2008, 20, 2133-2143 Palash Gangopadhyay et.al., J. Phys

Rate measurements of the hydrolysis of complex organic macromolecules in cold aqueous solutions: implications for prebiotic chemistry on the early Earth and Titan.

PubMed

Neish, C D; Somogyi, A; Imanaka, H; Lunine, J I; Smith, M A

2008-04-01

Organic macromolecules ("complex tholins") were synthesized from a 0.95 N(2)/0.05 CH(4) atmosphere in a high-voltage AC flow discharge reactor. When placed in liquid water, specific water soluble compounds in the macromolecules demonstrated Arrhenius type first order kinetics between 273 and 313 K and produced oxygenated organic species with activation energies in the range of approximately 60+/-10 kJ mol(-1). These reactions displayed half lives between 0.3 and 17 days at 273 K. Oxygen incorporation into such materials--a necessary step toward the formation of biological molecules--is therefore fast compared to processes that occur on geologic timescales, which include the freezing of impact melt pools and possible cryovolcanic sites on Saturn's organic-rich moon Titan.

Rate Measurements of the Hydrolysis of Complex Organic Macromolecules in Cold Aqueous Solutions: Implications for Prebiotic Chemistry on the Early Earth and Titan

NASA Astrophysics Data System (ADS)

Neish, C. D.; Somogyi, Á.; Imanaka, H .; Lunine, J. I.; Smith, M. A.

2008-04-01

Organic macromolecules (``complex tholins'') were synthesized from a 0.95 N2 / 0.05 CH4 atmosphere in a high-voltage AC flow discharge reactor. When placed in liquid water, specific water soluble compounds in the macromolecules demonstrated Arrhenius type first order kinetics between 273 and 313 K and produced oxygenated organic species with activation energies in the range of ~60 +/- 10 kJ mol-1. These reactions displayed half lives between 0.3 and 17 days at 273 K. Oxygen incorporation into such materials-a necessary step toward the formation of biological molecules-is therefore fast compared to processes that occur on geologic timescales, which include the freezing of impact melt pools and possible cryovolcanic sites on Saturn's organic-rich moon Titan.

Macromolecules Inquiry: Transformation of a Standard Biochemistry Lab

ERIC Educational Resources Information Center

Unsworth, Elizabeth

2014-01-01

Identification of macromolecules in food is a standard introductory high school biology lab. The intent of this article is to describe the conversion of this standard cookbook lab into an inquiry investigation. Instead of verifying the macromolecules found in food, students use their knowledge of the macromolecules in food to determine the…

Cytoskeleton Molecular Motors: Structures and Their Functions in Neuron.

PubMed

Xiao, Qingpin; Hu, Xiaohui; Wei, Zhiyi; Tam, Kin Yip

2016-01-01

Cells make use of molecular motors to transport small molecules, macromolecules and cellular organelles to target region to execute biological functions, which is utmost important for polarized cells, such as neurons. In particular, cytoskeleton motors play fundamental roles in neuron polarization, extension, shape and neurotransmission. Cytoskeleton motors comprise of myosin, kinesin and cytoplasmic dynein. F-actin filaments act as myosin track, while kinesin and cytoplasmic dynein move on microtubules. Cytoskeleton motors work together to build a highly polarized and regulated system in neuronal cells via different molecular mechanisms and functional regulations. This review discusses the structures and working mechanisms of the cytoskeleton motors in neurons.

Mitochondrial dysfunction and tissue injury by alcohol, high fat, nonalcoholic substances and pathological conditions through post-translational protein modifications

PubMed Central

Song, Byoung-Joon; Akbar, Mohammed; Abdelmegeed, Mohamed A.; Byun, Kyunghee; Lee, Bonghee; Yoon, Seung Kew; Hardwick, James P.

2014-01-01

Mitochondria are critically important in providing cellular energy ATP as well as their involvement in anti-oxidant defense, fat oxidation, intermediary metabolism and cell death processes. It is well-established that mitochondrial functions are suppressed when living cells or organisms are exposed to potentially toxic agents including alcohol, high fat diets, smoking and certain drugs or in many pathophysiological states through increased levels of oxidative/nitrative stress. Under elevated nitroxidative stress, cellular macromolecules proteins, DNA, and lipids can undergo different oxidative modifications, leading to disruption of their normal, sometimes critical, physiological functions. Recent reports also indicated that many mitochondrial proteins are modified via various post-translation modifications (PTMs) and primarily inactivated. Because of the recently-emerging information, in this review, we specifically focus on the mechanisms and roles of five major PTMs (namely oxidation, nitration, phosphorylation, acetylation, and adduct formation with lipid-peroxides, reactive metabolites, or advanced glycation end products) in experimental models of alcoholic and nonalcoholic fatty liver disease as well as acute hepatic injury caused by toxic compounds. We also highlight the role of the ethanol-inducible cytochrome P450-2E1 (CYP2E1) in some of these PTM changes. Finally, we discuss translational research opportunities with natural and/or synthetic anti-oxidants, which can prevent or delay the onset of mitochondrial dysfunction, fat accumulation and tissue injury. PMID:25465468

The ribosome as a missing link in the evolution of life.

PubMed

Root-Bernstein, Meredith; Root-Bernstein, Robert

2015-02-21

Many steps in the evolution of cellular life are still mysterious. We suggest that the ribosome may represent one important missing link between compositional (or metabolism-first), RNA-world (or genes-first) and cellular (last universal common ancestor) approaches to the evolution of cells. We present evidence that the entire set of transfer RNAs for all twenty amino acids are encoded in both the 16S and 23S rRNAs of Escherichia coli K12; that nucleotide sequences that could encode key fragments of ribosomal proteins, polymerases, ligases, synthetases, and phosphatases are to be found in each of the six possible reading frames of the 16S and 23S rRNAs; and that every sequence of bases in rRNA has information encoding more than one of these functions in addition to acting as a structural component of the ribosome. Ribosomal RNA, in short, is not just a structural scaffold for proteins, but the vestigial remnant of a primordial genome that may have encoded a self-organizing, self-replicating, auto-catalytic intermediary between macromolecules and cellular life. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Effects of hydration on steric and electric charge-induced interstitial volume exclusion--a model.

PubMed

Øien, Alf H; Justad, Sigrid R; Tenstad, Olav; Wiig, Helge

2013-09-03

The presence of collagen and charged macromolecules like glycosaminoglycans (GAGs) in the interstitial space limits the space available for plasma proteins and other macromolecules. This phenomenon, known as interstitial exclusion, is of importance for interstitial fluid volume regulation. Physical/mathematical models are presented for calculating the exclusion of electrically charged and neutral macromolecules that equilibrate in the interstitium under various degrees of hydration. Here, a central hypothesis is that the swelling of highly electrically charged GAGs with increased hydration shields parts of the neutral collagen of the interstitial matrix from interacting with electrically charged macromolecules, such that exclusion of charged macromolecules exhibits change due to steric and charge effects. GAGs are also thought to allow relatively small neutral, but also charged macromolecules neutralized by a very high ionic strength, diffuse into the interior of GAGs, whereas larger macromolecules may not. Thus, in the model, relatively small electrically charged macromolecules, such as human serum albumin, and larger neutral macromolecules such as IgG, will have quite similar total volume exclusion properties in the interstitium. Our results are in agreement with ex vivo and in vivo experiments, and suggest that the charge of GAGs or macromolecular drugs may be targeted to increase the tissue uptake of macromolecular therapeutic agents. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Proteasome inhibition potentiates antitumor effects of photodynamic therapy in mice through induction of ER stress and unfolded protein response

PubMed Central

Szokalska, Angelika; Makowski, Marcin; Nowis, Dominika; Wilczyński, Grzegorz M.; Kujawa, Marek; Wójcik, Cezary; Młynarczuk-Biały, Izabela; Salwa, Pawel; Bil, Jacek; Janowska, Sylwia; Agostinis, Patrizia; Verfaillie, Tom; Bugajski, Marek; Gietka, Jan; Issat, Tadeusz; Głodkowska, Eliza; Mrówka, Piotr; Stoklosa, Tomasz; Hamblin, Michael R; Mróz, Paweł; Jakóbisiak, Marek; Golab, Jakub

2009-01-01

Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity towards tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including numerous proteins that undergo multiple modifications such as fragmentation, cross-linking and carbonylation that result in protein unfolding and aggregation. Since the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmatic reticulum (ER), aggravated ER stress and potentiated cytotoxicity towards tumor cells. Indeed, we observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response (UPR). Pre-treatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132 and PSI gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60-100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application as bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors. PMID:19435917

New mechanisms of macroion-induced disintegration of charged droplets

NASA Astrophysics Data System (ADS)

Consta, Styliani; Oh, Myong In; Malevanets, Anatoly

2016-10-01

Molecular modeling has revealed that the presence of charged macromolecules (macroions) in liquid droplets dramatically changes the pathways of droplet fission. These mechanisms are not captured by the traditional theories such as ion-evaporation and charge-residue models. We review the general mechanisms by which macroions emerge from droplets and the factors that determine the droplet fission. These mechanisms include counter-intuitive ;star; droplet formations and extrusion of linear macroions from droplets. These findings may play a direct role in determining macromolecule charge states in electrospray mass spectrometry experiments.

Neural control of muscle

NASA Technical Reports Server (NTRS)

Max, S. R.; Markelonis, G. J.

1983-01-01

Cholinergic innervation regulates the physiological and biochemical properties of skeletal muscle. The mechanisms that appear to be involved in this regulation include soluble, neurally-derived polypeptides, transmitter-evoked muscle activity and the neurotransmitter, acetylcholine, itself. Despite extensive research, the interacting neural mechanisms that control such macromolecules as acetylcholinesterase, the acetylcholine receptor and glucose 6-phosphate dehydrogenase remain unclear. It may be that more simplified in vitro model systems coupled with recent dramatic advances in the molecular biology of neurally-regulated proteins will begin to allow researchers to unravel the mechanisms controlling the expression and maintenance of these macromolecules.

[Dermoepidermic junction: a selective, complex and vital barrier].

PubMed

Frede, Silvia C; Dionisio de Cabalier, María E; Zaya, Alejandro; Hliba, Ernesto

2004-01-01

Dermoepidermic junction (DEJ) is a highly complex region, containing a great variety of cellular elements, which despite of having different embriogenesis, interact with each other, generating different substances that keep the function and homeostasis of the greatest organ of the human body. DEJ is regarded as a highly specialized basal lamina, which acts as a highly selective pathway for the migration of cells and macromolecules, inducing cellular differentiation and micro enviromental metabolism modifications. DEJ may be divided into three zones regarding the basal lamina 1--the nearest to epidermic zone, having tonofilaments and hemidesmosomes, which keep anchored basal cells. This region is limited by the lamina densa 2--the intermediate zone, represented exclusively by lamina densa and finally the lamina 3,--the third region--extends from lamina densa to the upper dermis and extracelullar matrix. Despite there is much to learn about DEJ, the knowledge about each molecule and function of every compartment will enable us to know more about the pathogenesis of several dermatologic diseases, with a great prevalence in the clinical practice.

A viral peptide for intracellular delivery

NASA Astrophysics Data System (ADS)

Falanga, Annarita; Tarallo, Rossella; Cantisani, Marco; Della Pepa, Maria Elena; Galdiero, Massimiliano; Galdiero, Stefania

2012-10-01

Biological membranes represent a critical hindrance for administering active molecules which are often unable to reach their designated intracellular target sites. In order to overcome this barrier-like behavior not easily circumvented by many pharmacologically-active molecules, synthetic transporters have been exploited to promote cellular uptake. Linking or complexing therapeutic molecules to peptides that can translocate through the cellular membranes could enhance their internal delivery, and consequently, a higher amount of active compound would reach the site of action. Use of cell penetrating peptides (CPPs) is one of the most promising strategy to efficiently translocate macromolecules through the plasma membrane, and have attracted a lot of attention. New translocating peptides are continuously described and in the present review, we will focus on viral derived peptides, and in particular a peptide (gH625) derived from the herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) that has proved to be a useful delivery vehicle due to its intrinsic properties of inducing membrane perturbation.

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.

PubMed

Treweek, Jennifer B; Chan, Ken Y; Flytzanis, Nicholas C; Yang, Bin; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2015-11-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1-2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

INFLUENCE OF MACROMOLECULES ON CHEMICAL TRANSPORT

EPA Science Inventory

Macromolecules in the pore fluid influence the mobility of hydrophobic compounds through soils. his study evaluated the significance of macromolecules in facilitating chemical transport under laboratory conditions. Partition coefficients between 14C-labeled hexachlorobenzene and ...

AAA+ Machines of Protein Destruction in Mycobacteria.

PubMed

Alhuwaider, Adnan Ali H; Dougan, David A

2017-01-01

The bacterial cytosol is a complex mixture of macromolecules (proteins, DNA, and RNA), which collectively are responsible for an enormous array of cellular tasks. Proteins are central to most, if not all, of these tasks and as such their maintenance (commonly referred to as protein homeostasis or proteostasis) is vital for cell survival during normal and stressful conditions. The two key aspects of protein homeostasis are, (i) the correct folding and assembly of proteins (coupled with their delivery to the correct cellular location) and (ii) the timely removal of unwanted or damaged proteins from the cell, which are performed by molecular chaperones and proteases, respectively. A major class of proteins that contribute to both of these tasks are the AAA+ (ATPases associated with a variety of cellular activities) protein superfamily. Although much is known about the structure of these machines and how they function in the model Gram-negative bacterium Escherichia coli , we are only just beginning to discover the molecular details of these machines and how they function in mycobacteria. Here we review the different AAA+ machines, that contribute to proteostasis in mycobacteria. Primarily we will focus on the recent advances in the structure and function of AAA+ proteases, the substrates they recognize and the cellular pathways they control. Finally, we will discuss the recent developments related to these machines as novel drug targets.

Workshop on High-Field NMR and Biological Applications

NASA Astrophysics Data System (ADS)

Scientists at the Pacific Northwest Laboratory have been working toward the establishment of a new Molecular Science Research Center (MSRC). The primary scientific thrust of this new research center is in the areas of theoretical chemistry, chemical dynamics, surface and interfacial science, and studies on the structure and interactions of biological macromolecules. The MSRC will provide important new capabilities for studies on the structure of biological macromolecules. The MSRC program includes several types of advanced spectroscopic techniques for molecular structure analysis, and a theory and modeling laboratory for molecular mechanics/dynamics calculations and graphics. It is the goal to closely integrate experimental and theoretical studies on macromolecular structure, and to join these research efforts with those of the molecular biological programs to provide new insights into the structure/function relationships of biological macromolecules. One of the areas of structural biology on which initial efforts in the MSRC will be focused is the application of high field, 2-D NMR to the study of biological macromolecules. First, there is interest in obtaining 3-D structural information on large proteins and oligonucleotides. Second, one of the primary objectives is to closely link theoretical approaches to molecular structure analysis with the results obtained in experimental research using NMR and other spectroscopies.

Controlled doping by self-assembled dendrimer-like macromolecules

NASA Astrophysics Data System (ADS)

Wu, Haigang; Guan, Bin; Sun, Yingri; Zhu, Yiping; Dan, Yaping

2017-02-01

Doping via self-assembled macromolecules might offer a solution for developing single atom electronics by precisely placing individual dopants at arbitrary location to meet the requirement for circuit design. Here we synthesize dendrimer-like polyglycerol macromolecules with each carrying one phosphorus atom in the core. The macromolecules are immobilized by the coupling reagent onto silicon surfaces that are pre-modified with a monolayer of undecylenic acid. Nuclear magnetic resonance (NMR) and X-ray photoelectron spectroscopy (XPS) are employed to characterize the synthesized macromolecules and the modified silicon surfaces, respectively. After rapid thermal annealing, the phosphorus atoms carried by the macromolecules diffuse into the silicon substrate, forming dopants at a concentration of 1017 cm-3. Low-temperature Hall effect measurements reveal that the ionization process is rather complicated. Unlike the widely reported simple ionization of phosphorus dopants, nitrogen and carbon are also involved in the electronic activities in the monolayer doped silicon.

Regulated portals of entry into the cell

NASA Astrophysics Data System (ADS)

Conner, Sean D.; Schmid, Sandra L.

2003-03-01

The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. `Endocytosis' encompasses several diverse mechanisms by which cells internalize macromolecules and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of molecular interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiology and thus, must be viewed in a broader context than simple vesicular trafficking.

Magnetic resonance imaging findings of cellular angiofibroma of the tunica vaginalis of the testis: a case report.

PubMed

Ntorkou, Alexandra A; Tsili, Athina C; Giannakis, Dimitrios; Batistatou, Anna; Stavrou, Sotirios; Sofikitis, Nikolaos; Argyropoulou, Maria I

2016-03-31

Cellular angiofibroma represents a rare mesenchymal tumor typically involving the inguinoscrotal area in middle-aged men. Although the origin of this benign tumor is unknown, it is histologically classified as an angiomyxoid tumor. Cellular angiofibroma is characterized by a diversity of pathological and imaging features. An accurate preoperative diagnosis is challenging. Magnetic resonance imaging examination of the scrotum has been reported as a valuable adjunct modality in the investigation of scrotal pathology. The technique by providing both structural and functional information is useful in the differentiation between extratesticular and intratesticular diseases and in the preoperative characterization of the histologic nature of various scrotal lesions. There are few reports in the English literature addressing the magnetic resonance imaging findings of cellular angiofibroma of the scrotum and no reports on functional magnetic resonance imaging data. Here we present the first case of a cellular angiofibroma arising from the tunica vaginalis of the testis and we discuss the value of a multiparametric magnetic resonance protocol, including diffusion-weighted imaging, magnetization transfer imaging and dynamic contrast-enhanced magnetic resonance imaging in the preoperative diagnosis of this rare neoplasm. A 47-year Greek man presented with a painless left scrotal swelling, which had gradually enlarged during the last 6 months. Magnetic resonance imaging of his scrotum displayed a left paratesticular mass, in close proximity to the tunica vaginalis, with heterogeneous high signal intensity on T2-weighted images and no areas of restricted diffusion. The tumor was hypointense on magnetization transfer images, suggestive for the presence of macromolecules. On dynamic contrast-enhanced magnetic resonance imaging the mass showed intense heterogeneous enhancement with a type II curve. Magnetic resonance imaging findings were strongly suggestive of a benign paratesticular tumor, which was confirmed on pathology following lesion excision. Magnetic resonance imaging of the scrotum by combining conventional and functional magnetic resonance data provides useful diagnostic information in the preoperative characterization of scrotal masses. A possible diagnosis of a benign paratesticular tumor based on magnetic resonance imaging features may improve patient care and decrease the number of unnecessary radical surgical explorations.

Contact Kinetics in Fractal Macromolecules.

PubMed

Dolgushev, Maxim; Guérin, Thomas; Blumen, Alexander; Bénichou, Olivier; Voituriez, Raphaël

2015-11-13

We consider the kinetics of first contact between two monomers of the same macromolecule. Relying on a fractal description of the macromolecule, we develop an analytical method to compute the mean first contact time for various molecular sizes. In our theoretical description, the non-Markovian feature of monomer motion, arising from the interactions with the other monomers, is captured by accounting for the nonequilibrium conformations of the macromolecule at the very instant of first contact. This analysis reveals a simple scaling relation for the mean first contact time between two monomers, which involves only their equilibrium distance and the spectral dimension of the macromolecule, independently of its microscopic details. Our theoretical predictions are in excellent agreement with numerical stochastic simulations.

Adsorption of polymethacrylic acid from aqueous solutions on disperse titanium dioxide

NASA Astrophysics Data System (ADS)

Yaremko, Z. M.; Tkachenko, N. G.; Fedushinskaya, L. B.

2011-10-01

The state of macromolecules of polymethacrylic acid adsorbed on the surface of disperse titanium dioxide was assessed using a combination of the differential concentration approach to the determination of adsorption and methods for determining the size of disperse adsorbents by dynamic light scattering and sedimentation analysis in the field of centrifugal forces. Three sections were found on the isotherm of adsorption: in the first, isolated islands of adsorbed macromolecules formed; in the second, layers of macromolecules with a different degree of deformation were observed; in the third, determining the adsorption of macromolecules is complicated by other accompanying processes, and assessing the state of macromolecules in the adsorption layer becomes difficult.

MACROMOLECULES FACILITATE THE TRANSPORT OF TRACE ORGANICS

EPA Science Inventory

Macromolecules in the pore fluid of a soil may influence the mobility of hydrophobic compounds by their partitioning to the macromolecule, which moves with, or even faster than, the water. The mobility is described mathematically by a chemical transport model. The significance of...

Self-assembly in densely grafted macromolecules with amphiphilic monomer units: diagram of states.

PubMed

Lazutin, A A; Vasilevskaya, V V; Khokhlov, A R

2017-11-22

By means of computer modelling, the self-organization of dense planar brushes of macromolecules with amphiphilic monomer units was addressed and their state diagram was constructed. The diagram of states includes the following regions: disordered position of monomer units with respect to each other, strands composed of a few polymer chains and lamellae with different domain spacing. The transformation of lamellae structures with different domain spacing occurred within the intermediate region and could proceed through the formation of so-called parking garage structures. The parking garage structure joins the lamellae with large (on the top of the brushes) and small (close to the grafted surface) domain spacing, which appears like a system of inclined locally parallel layers connected with each other by bridges. The parking garage structures were observed for incompatible A and B groups in selective solvents, which result in aggregation of the side B groups and dense packing of amphiphilic macromolecules in the restricted volume of the planar brushes.

Determining the bacterial cell biology of Planctomycetes.

PubMed

Boedeker, Christian; Schüler, Margarete; Reintjes, Greta; Jeske, Olga; van Teeseling, Muriel C F; Jogler, Mareike; Rast, Patrick; Borchert, Daniela; Devos, Damien P; Kucklick, Martin; Schaffer, Miroslava; Kolter, Roberto; van Niftrik, Laura; Engelmann, Susanne; Amann, Rudolf; Rohde, Manfred; Engelhardt, Harald; Jogler, Christian

2017-04-10

Bacteria of the phylum Planctomycetes have been previously reported to possess several features that are typical of eukaryotes, such as cytosolic compartmentalization and endocytosis-like macromolecule uptake. However, recent evidence points towards a Gram-negative cell plan for Planctomycetes, although in-depth experimental analysis has been hampered by insufficient genetic tools. Here we develop methods for expression of fluorescent proteins and for gene deletion in a model planctomycete, Planctopirus limnophila, to analyse its cell organization in detail. Super-resolution light microscopy of mutants, cryo-electron tomography, bioinformatic predictions and proteomic analyses support an altered Gram-negative cell plan for Planctomycetes, including a defined outer membrane, a periplasmic space that can be greatly enlarged and convoluted, and an energized cytoplasmic membrane. These conclusions are further supported by experiments performed with two other Planctomycetes, Gemmata obscuriglobus and Rhodopirellula baltica. We also provide experimental evidence that is inconsistent with endocytosis-like macromolecule uptake; instead, extracellular macromolecules can be taken up and accumulate in the periplasmic space through unclear mechanisms.

Conversion of psychological stress into cellular stress response: roles of the sigma-1 receptor in the process.

PubMed

Hayashi, Teruo

2015-04-01

Psychiatrists empirically recognize that excessive or chronic psychological stress can result in long-lasting impairments of brain functions that partly involve neuronal cell damage. Recent studies begin to elucidate the molecular pathways activated/inhibited by psychological stress. Activation of the hypothalamic-pituitary-adrenal axis under psychological stress causes inflammatory oxidative stresses in the brain, in part due to elevation of cytokines. Psychological stress or neuropathological conditions (e.g., accumulation of β-amyloids) trigger 'cellular stress responses', which promote upregulation of molecular chaperones to protect macromolecules from degradation. The unfolded protein response, the endoplasmic reticulum (ER)-specific cellular stress response, has been recently implicated in the pathophysiology of neuropsychiatric disorders and the pharmacology of certain clinically used drugs. The sigma-1 receptor is an ER protein whose ligands are shown to exert antidepressant-like and neuroprotective actions. Recent studies found that the sigma-1 receptor is a novel ligand-operated ER chaperone that regulates bioenergetics, free radical generation, oxidative stress, unfolded protein response and cytokine signaling. The sigma-1 receptor also regulates morphogenesis of neuronal cells, such as neurite outgrowth, synaptogenesis, and myelination, which can be perturbed by cellular stress. The sigma-1 receptor may thus contribute to a cellular defense system that protects nervous systems against chronic psychological stress. Findings from sigma receptor research imply that not only cell surface monoamine effectors but also intracellular molecules, especially those at the ER, may provide novel therapeutic targets for future drug developments. © 2014 The Author. Psychiatry and Clinical Neurosciences © 2014 Japanese Society of Psychiatry and Neurology.

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams, Paul  D.; Aertgeerts, Kathleen; Bauer, Cary

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank archive, ~75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery/design, and the goodness-of-fit of ligand models to electron density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide Protein Data Bank/Cambridge Crystallographicmore » Data Centre/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the Protein Data Bank? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.« less

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop

DOE PAGES

Adams, Paul  D.; Aertgeerts, Kathleen; Bauer, Cary; ...

2016-04-05

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank archive, ~75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery/design, and the goodness-of-fit of ligand models to electron density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide Protein Data Bank/Cambridge Crystallographicmore » Data Centre/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the Protein Data Bank? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.« less

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop.

PubMed

Adams, Paul D; Aertgeerts, Kathleen; Bauer, Cary; Bell, Jeffrey A; Berman, Helen M; Bhat, Talapady N; Blaney, Jeff M; Bolton, Evan; Bricogne, Gerard; Brown, David; Burley, Stephen K; Case, David A; Clark, Kirk L; Darden, Tom; Emsley, Paul; Feher, Victoria A; Feng, Zukang; Groom, Colin R; Harris, Seth F; Hendle, Jorg; Holder, Thomas; Joachimiak, Andrzej; Kleywegt, Gerard J; Krojer, Tobias; Marcotrigiano, Joseph; Mark, Alan E; Markley, John L; Miller, Matthew; Minor, Wladek; Montelione, Gaetano T; Murshudov, Garib; Nakagawa, Atsushi; Nakamura, Haruki; Nicholls, Anthony; Nicklaus, Marc; Nolte, Robert T; Padyana, Anil K; Peishoff, Catherine E; Pieniazek, Susan; Read, Randy J; Shao, Chenghua; Sheriff, Steven; Smart, Oliver; Soisson, Stephen; Spurlino, John; Stouch, Terry; Svobodova, Radka; Tempel, Wolfram; Terwilliger, Thomas C; Tronrud, Dale; Velankar, Sameer; Ward, Suzanna C; Warren, Gregory L; Westbrook, John D; Williams, Pamela; Yang, Huanwang; Young, Jasmine

2016-04-05

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank (PDB) archive, ∼75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the PDB? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated. Copyright © 2016 Elsevier Ltd. All rights reserved.

Outcome of the first wwPDB/CCDC/D3R Ligand Validation Workshop

PubMed Central

Adams, Paul D.; Aertgeerts, Kathleen; Bauer, Cary; Bell, Jeffrey A.; Berman, Helen M.; Bhat, Talapady N.; Blaney, Jeff; Bolton, Evan; Bricogne, Gerard; Brown, David; Burley, Stephen K.; Case, David A.; Clark, Kirk L.; Darden, Tom; Emsley, Paul; Feher, Victoria A.; Feng, Zukang; Groom, Colin R.; Harris, Seth F.; Hendle, Jorg; Holder, Thomas; Joachimiak, Andrzej; Kleywegt, Gerard J.; Krojer, Tobias; Marcotrigiano, Joseph; Mark, Alan E.; Markley, John L.; Miller, Matthew; Minor, Wladek; Montelione, Gaetano T.; Murshudov, Garib; Nakagawa, Atsushi; Nakamura, Haruki; Nicholls, Anthony; Nicklaus, Marc; Nolte, Robert T.; Padyana, Anil K.; Peishoff, Catherine E.; Pieniazek, Susan; Read, Randy J.; Shao, Chenghua; Sheriff, Steven; Smart, Oliver; Soisson, Stephen; Spurlino, John; Stouch, Terry; Svobodova, Radka; Tempel, Wolfram; Terwilliger, Thomas C.; Tronrud, Dale; Velankar, Sameer; Ward, Suzanna; Warren, Gregory L.; Westbrook, John D.; Williams, Pamela; Yang, Huanwang; Young, Jasmine

2016-01-01

Summary Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank archive, ~75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery/design, and the goodness-of-fit of ligand models to electron density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide Protein Data Bank/Cambridge Crystallographic Data Centre/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30–31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the Protein Data Bank? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated. PMID:27050687

The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

PubMed

Hansen, Uwe; Hussain, Muzaffar; Villone, Daniela; Herrmann, Mathias; Robenek, Horst; Peters, Georg; Sinha, Bhanu; Bruckner, Peter

2006-05-01

Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

The HADDOCK2.2 Web Server: User-Friendly Integrative Modeling of Biomolecular Complexes.

PubMed

van Zundert, G C P; Rodrigues, J P G L M; Trellet, M; Schmitz, C; Kastritis, P L; Karaca, E; Melquiond, A S J; van Dijk, M; de Vries, S J; Bonvin, A M J J

2016-02-22

The prediction of the quaternary structure of biomolecular macromolecules is of paramount importance for fundamental understanding of cellular processes and drug design. In the era of integrative structural biology, one way of increasing the accuracy of modeling methods used to predict the structure of biomolecular complexes is to include as much experimental or predictive information as possible in the process. This has been at the core of our information-driven docking approach HADDOCK. We present here the updated version 2.2 of the HADDOCK portal, which offers new features such as support for mixed molecule types, additional experimental restraints and improved protocols, all of this in a user-friendly interface. With well over 6000 registered users and 108,000 jobs served, an increasing fraction of which on grid resources, we hope that this timely upgrade will help the community to solve important biological questions and further advance the field. The HADDOCK2.2 Web server is freely accessible to non-profit users at http://haddock.science.uu.nl/services/HADDOCK2.2. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Luminol-Based Chemiluminescent Signals: Clinical and Non-clinical Application and Future Uses

PubMed Central

Khan, Parvez; Idrees, Danish; Moxley, Michael A.; Corbett, John A.; Ahmad, Faizan; von Figura, Guido; Sly, William S.; Waheed, Abdul

2015-01-01

Chemiluminescence (CL) is an important method for quantification and analysis of various macromolecules. A wide range of CL agents such as luminol, hydrogen peroxide, fluorescein, dioxetanes and derivatives of oxalate, and acridinium dyes are used according to their biological specificity and utility. This review describes the application of luminol chemiluminescence (LCL) in forensic, biomedical, and clinical sciences. LCL is a very useful detection method due to its selectivity, simplicity, low cost, and high sensitivity. LCL has a dynamic range of applications, including quantification and detection of macro and micromolecules such as proteins, carbohydrates, DNA, and RNA. Luminol-based methods are used in environmental monitoring as biosensors, in the pharmaceutical industry for cellular localization and as biological tracers, and in reporter gene-based assays and several other immunoassays. Here, we also provide information about different compounds that may enhance or inhibit the LCL along with the effect of pH and concentration on LCL. This review covers most of the significant information related to the applications of luminol in different fields. PMID:24752935

Myosin-1C uses a novel phosphoinositide-dependent pathway for nuclear localization.

PubMed

Nevzorov, Ilja; Sidorenko, Ekaterina; Wang, Weihuan; Zhao, Hongxia; Vartiainen, Maria K

2018-02-01

Accurate control of macromolecule transport between nucleus and cytoplasm underlines several essential biological processes, including gene expression. According to the canonical model, nuclear import of soluble proteins is based on nuclear localization signals and transport factors. We challenge this view by showing that nuclear localization of the actin-dependent motor protein Myosin-1C (Myo1C) resembles the diffusion-retention mechanism utilized by inner nuclear membrane proteins. We show that Myo1C constantly shuttles in and out of the nucleus and that its nuclear localization does not require soluble factors, but is dependent on phosphoinositide binding. Nuclear import of Myo1C is preceded by its interaction with the endoplasmic reticulum, and phosphoinositide binding is specifically required for nuclear import, but not nuclear retention, of Myo1C. Our results therefore demonstrate, for the first time, that membrane association and binding to nuclear partners is sufficient to drive nuclear localization of also soluble proteins, opening new perspectives to evolution of cellular protein sorting mechanisms. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Macromolecular composition of phloem exudate from white lupin (Lupinus albus L.)

PubMed Central

2011-01-01

Background Members of the legume genus Lupinus exude phloem 'spontaneously' from incisions made to the vasculature. This feature was exploited to document macromolecules present in exudate of white lupin (Lupinus albus [L.] cv Kiev mutant), in particular to identify proteins and RNA molecules, including microRNA (miRNA). Results Proteomic analysis tentatively identified 86 proteins from 130 spots collected from 2D gels analysed by partial amino acid sequence determination using MS/MS. Analysis of a cDNA library constructed from exudate identified 609 unique transcripts. Both proteins and transcripts were classified into functional groups. The largest group of proteins comprised those involved in metabolism (24%), followed by protein modification/turnover (9%), redox regulation (8%), cell structural components (6%), stress and defence response (6%) with fewer in other groups. More prominent proteins were cyclophilin, ubiquitin, a glycine-rich RNA-binding protein, a group of proteins that comprise a glutathione/ascorbate-based mechanism to scavenge oxygen radicals, enzymes of glycolysis and other metabolism including methionine and ethylene synthesis. Potential signalling macromolecules such as transcripts encoding proteins mediating calcium level and the Flowering locus T (FT) protein were also identified. From around 330 small RNA clones (18-25 nt) 12 were identified as probable miRNAs by homology with those from other species. miRNA composition of exudate varied with site of collection (e.g. upward versus downward translocation streams) and nutrition (e.g. phosphorus level). Conclusions This is the first inventory of macromolecule composition of phloem exudate from a species in the Fabaceae, providing a basis to identify systemic signalling macromolecules with potential roles in regulating development, growth and stress response of legumes. PMID:21342527

An intrinsic poperty of memory of the Cellular automaton infrastructure of Nature leading to the organization of the physical world as an Internet o things; TOE = IOT

NASA Astrophysics Data System (ADS)

Berkovich, Simon

2015-04-01

The undamental advantage of a Cellular automaton construction foris that it can be viewed as an undetectable absolute frame o reference, in accordance with Lorentz-Poincare's interpretation.. The cellular automaton model for physical poblems comes upon two basic hurdles: (1) How to find the Elemental Rule that, and how to get non-locality from local transformations. Both problems are resolved considering the transfomation rule of mutual distributed synchronization Actually any information proessing device starts with a clocking system. and it turns out that ``All physical phenomena are different aspects of the high-level description of distributed mutual synchronization in a network of digital clocks''. Non-locality comes from two hugely different time-scales of signaling.. The universe is acombinines information and matter processes, These fast spreading diffusion wave solutions create the mechanism of the Holographic Universe. And thirdly Disengaged from synchronization, circular counters can perform memory functions by retaining phases of their oscillations, an idea of Von Neumann'. Thus, the suggested model generates the necessary constructs for the physical world as an Internet of Things. Life emerges due to the specifics of macromolecules that serve as communication means, with the holographic memory...

Exploring the Spatial and Temporal Organization of a Cell’s Proteome

PubMed Central

Beck, Martin; Topf, Maya; Frazier, Zachary; Tjong, Harianto; Xu, Min; Zhang, Shihua; Alber, Frank

2013-01-01

To increase our current understanding of cellular processes, such as cell signaling and division, knowledge is needed about the spatial and temporal organization of the proteome at different organizational levels. These levels cover a wide range of length and time scales: from the atomic structures of macromolecules for inferring their molecular function, to the quantitative description of their abundance, and distribution in the cell. Emerging new experimental technologies are greatly increasing the availability of such spatial information on the molecular organization in living cells. This review addresses three fields that have significantly contributed to our understanding of the proteome’s spatial and temporal organization: first, methods for the structure determination of individual macromolecular assemblies, specifically the fitting of atomic structures into density maps generated from electron microscopy techniques; second, research that visualizes the spatial distributions of these complexes within the cellular context using cryo electron tomography techniques combined with computational image processing; and third, methods for the spatial modeling of the dynamic organization of the proteome, specifically those methods for simulating reaction and diffusion of proteins and complexes in crowded intracellular fluids. The long-term goal is to integrate the varied data about a proteome’s organization into a spatially explicit, predictive model of cellular processes. PMID:21094684

Surface Characteristics and Adhesion Behavior of Escherichia coli O157:H7: Role of Extracellular Macromolecules

USDA-ARS?s Scientific Manuscript database

Surface macromolecule cleavage experiments were conducted on enterohaemorrhagic Escherichia coli O157:H7 cells to investigate the influence of these macromolecules on cell surface properties. Electrophoretic mobility, hydrophobicity, and titration experiments were carried out on proteinase K treate...

Proposed Mode of Action for Acrolein Respiratory Toxicity Associated with Inhaled Tobacco Smoke.

PubMed

Yeager, R Philip; Kushman, Mary; Chemerynski, Susan; Weil, Roxana; Fu, Xin; White, Marcella; Callahan-Lyon, Priscilla; Rosenfeldt, Hans

2016-06-01

This article presents a mode of action (MOA) analysis that identifies key mechanisms in the respiratory toxicity of inhaled acrolein and proposes key acrolein-related toxic events resulting from the inhalation of tobacco smoke. Smoking causes chronic obstructive pulmonary disorder (COPD) and acrolein has been previously linked to the majority of smoking-induced noncancer respiratory toxicity. In contrast to previous MOA analyses for acrolein, this MOA focuses on the toxicity of acrolein in the lower respiratory system, reflecting the exposure that smokers experience upon tobacco smoke inhalation. The key mechanisms of acrolein toxicity identified in this proposed MOA include (1) acrolein chemical reactivity with proteins and other macromolecules of cells lining the respiratory tract, (2) cellular oxidative stress, including compromise of the important anti-oxidant glutathione, (3) chronic inflammation, (4) necrotic cell death leading to a feedback loop where necrosis-induced inflammation leads to more necrosis and oxidative damage and vice versa, (5) tissue remodeling and destruction, and (6) loss of lung elasticity and enlarged lung airspaces. From these mechanisms, the proposed MOA analysis identifies the key cellular processes in acrolein respiratory toxicity that consistently occur with the development of COPD: inflammation and necrosis in the middle and lower regions of the respiratory tract. Moreover, the acrolein exposures that occur as a result of smoking are well above exposures that induce both inflammation and necrosis in laboratory animals, highlighting the importance of the role of acrolein in smoking-related respiratory disease. Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.

Organometallic macromolecules with piano stool coordination repeating units: chain configuration and stimulated solution behaviour.

PubMed

Cao, Kai; Ward, Jonathan; Amos, Ryan C; Jeong, Moon Gon; Kim, Kyoung Taek; Gauthier, Mario; Foucher, Daniel; Wang, Xiaosong

2014-09-11

Theoretical calculations illustrate that organometallic macromolecules with piano stool coordination repeating units (Fe-acyl complex) adopt linear chain configuration with a P-Fe-C backbone surrounded by aromatic groups. The macromolecules show molecular weight-dependent and temperature stimulated solution behaviour in DMSO.

Quantification of in vivo short echo-time proton magnetic resonance spectra at 14.1 T using two different approaches of modelling the macromolecule spectrum

NASA Astrophysics Data System (ADS)

Cudalbu, C.; Mlynárik, V.; Xin, L.; Gruetter, Rolf

2009-10-01

Reliable quantification of the macromolecule signals in short echo-time 1H MRS spectra is particularly important at high magnetic fields for an accurate quantification of metabolite concentrations (the neurochemical profile) due to effectively increased spectral resolution of the macromolecule components. The purpose of the present study was to assess two approaches of quantification, which take the contribution of macromolecules into account in the quantification step. 1H spectra were acquired on a 14.1 T/26 cm horizontal scanner on five rats using the ultra-short echo-time SPECIAL (spin echo full intensity acquired localization) spectroscopy sequence. Metabolite concentrations were estimated using LCModel, combined with a simulated basis set of metabolites using published spectral parameters and either the spectrum of macromolecules measured in vivo, using an inversion recovery technique, or baseline simulated by the built-in spline function. The fitted spline function resulted in a smooth approximation of the in vivo macromolecules, but in accordance with previous studies using Subtract-QUEST could not reproduce completely all features of the in vivo spectrum of macromolecules at 14.1 T. As a consequence, the measured macromolecular 'baseline' led to a more accurate and reliable quantification at higher field strengths.

New Directions in Biotechnology

NASA Technical Reports Server (NTRS)

2003-01-01

The macromolecule crystallization program within NASA is undergoing considerable pressure, particularly budgetary pressure. While it has shown some successes, they have not lived up to the expectations of others, and technological advances may rapidly overtake the natural advantages offered by crystallization in microgravity. Concomitant with the microgravity effort has been a research program to study the macromolecule crystallization process. It was believed that a better understanding of the process would lead to growth of improved crystals for X-ray diffraction studies. The results of the various research efforts have been impressive in improving our understanding of macromolecule crystallization, but have not led to any improved structures. Macromolecule crystallization for structure determination is "one of", the job being unique for every protein and finished once a structure is obtained. However, the knowledge gained is not lost, but instead lays the foundation for developments in new areas of biotechnology and nanotechnology. In this it is highly analogous to studies into small molecule crystallization, the results of which have led to our present day microelectronics-based society. We are conducting preliminary experiments into areas such as designed macromolecule crystals, macromolecule-inorganic hybrid structures, and macromolecule-based nanotechnology. In addition, our protein crystallization studies are now being directed more towards industrial and new approaches to membrane protein crystallization.

A Robust Biomarker

NASA Technical Reports Server (NTRS)

Westall, F.; Steele, A.; Toporski, J.; Walsh, M. M.; Allen, C. C.; Guidry, S.; McKay, D. S.; Gibson, E. K.; Chafetz, H. S.

2000-01-01

Polymers of bacterial origin, either through cell secretion or the degraded product of cell lysis, form isolated mucoidal strands as well as well-developed biofilms on interfaces. Biofilms are structurally and compositionally complex and are readily distinguishable from abiogenic films. These structures range in size from micrometers to decimeters, the latter occurring as the well-known, mineralised biofilms called stromatolites. Compositionally bacterial polymers are greater than 90 % water, with while the majority of the macromolecules forming the framework of the polymers consisting of polysaccharides (with and some nucteic acids and proteins). These macromolecules contain a vaste amount of functional groups, such as carboxyls, hydroxyls, and phosphoryls which are implicated in cation-binding. It is the elevated metal- binding capacity which provides the bacterial polymer with structural support and also helps to preserves it for up to 3.5 b.y. in the terrestrial rock record. The macromolecules, thus, can become rapidly mineralised and trapped in a mineral matrix. Through early and late diagenesis (bacterial degradation, burial, heat, pressure and time) they break down, losing the functional groups and, gradually, their hydrogen atoms. The degraded product is known as "kerogen". With further diagenesis and metamorphism, all the hydrogen atoms are lost and the carbonaceous matter becomes graphite. until the remnant carbonaceous material become graphitised. This last sentence reads a bit as if ALL these macromolecules break down and end up as graphite., but since we find 441 this is not true for all of the macromolecules. We have traced fossilised polymer and biofilms in rocks from throughout Earth's history, to rocks as old as the oldest being 3.5 b.y.-old. Furthermore, Time of Flight Secondary Ion Mass Spectrometry has been able to identify individual macromolecules of bacterial origin, the identities of which are still being investigated, in all the samples containing fossil biofilm, including the 3.5 b.y..-old carbonaceous cherts from South Africa and Australia. As a result of the unique compositional, structural and "mineralisable" properties of bacterial polymer and biofilms, we conclude that bacterial polymers and biofilms constitute a robust and reliable biomarker for life on Earth and could be a potential biomarker for extraterrestrial life.

JAIL: a structure-based interface library for macromolecules.

PubMed

Günther, Stefan; von Eichborn, Joachim; May, Patrick; Preissner, Robert

2009-01-01

The increasing number of solved macromolecules provides a solid number of 3D interfaces, if all types of molecular contacts are being considered. JAIL annotates three different kinds of macromolecular interfaces, those between interacting protein domains, interfaces of different protein chains and interfaces between proteins and nucleic acids. This results in a total number of about 184,000 database entries. All the interfaces can easily be identified by a detailed search form or by a hierarchical tree that describes the protein domain architectures classified by the SCOP database. Visual inspection of the interfaces is possible via an interactive protein viewer. Furthermore, large scale analyses are supported by an implemented sequential and by a structural clustering. Similar interfaces as well as non-redundant interfaces can be easily picked out. Additionally, the sequential conservation of binding sites was also included in the database and is retrievable via Jmol. A comprehensive download section allows the composition of representative data sets with user defined parameters. The huge data set in combination with various search options allow a comprehensive view on all interfaces between macromolecules included in the Protein Data Bank (PDB). The download of the data sets supports numerous further investigations in macromolecular recognition. JAIL is publicly available at http://bioinformatics.charite.de/jail.

Independent active and thermodynamic processes govern the nucleolus assembly in vivo

PubMed Central

Falahati, Hanieh; Wieschaus, Eric

2017-01-01

Membraneless organelles play a central role in the organization of protoplasm by concentrating macromolecules, which allows efficient cellular processes. Recent studies have shown that, in vitro, certain components in such organelles can assemble through phase separation. Inside the cell, however, such organelles are multicomponent, with numerous intermolecular interactions that can potentially affect the demixing properties of individual components. In addition, the organelles themselves are inherently active, and it is not clear how the active, energy-consuming processes that occur constantly within such organelles affect the phase separation behavior of the constituent macromolecules. Here, we examine the phase separation model for the formation of membraneless organelles in vivo by assessing the two features that collectively distinguish it from active assembly, namely temperature dependence and reversibility. We use a microfluidic device that allows accurate and rapid manipulation of temperature and examine the quantitative dynamics by which six different nucleolar proteins assemble into the nucleoli of Drosophila melanogaster embryos. Our results indicate that, although phase separation is the main mode of recruitment for four of the studied proteins, the assembly of the other two is irreversible and enhanced at higher temperatures, behaviors indicative of active recruitment to the nucleolus. These two subsets of components differ in their requirements for ribosomal DNA; the two actively assembling components fail to assemble in the absence of ribosomal DNA, whereas the thermodynamically driven components assemble but lose temporal and spatial precision. PMID:28115706

Endocellular regulation by free radicals and hydrogen peroxide: key determinants of the inflammatory response.

PubMed

Vitetta, Luis; Linnane, Anthony W

2014-04-01

The formations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) have long been considered as major contributors to the dysregulation of the inflammatory response. Reactive oxygen species and RNS productions often are reported to be associated with the development of chronic diseases and acceleration of the aging process. Mechanistically, this association has linked the phenomena of oxidative stress with the occurrence of random deleterious modifications of macromolecules with progressive development of pro-inflammatory conditions promoting age-associated systemic diseases. On the contrary the so-called random modification of macromolecules is incorrect rather ROS and RNS are molecular regulators (second messengers) and not universal toxins whose overproduction should be annulled by antioxidants. We have previously reviewed the physiological role of superoxide anion (and hydrogen peroxide) and nitric oxide (and peroxynitrite) and concluded that these reactive molecular species behave as pro-oxidant second messengers. Reactive oxygen species and RNS are produced at specific cellular locations and are essential for both the normal physiological function of the metabolome and the regulated inflammatory response. This brings into question the whole concept of the orally administering of antioxidant molecular species to down-regulate or abrogate an overproduction of free radical activity. There are no human clinical trials that demonstrate that small molecules, the so-called antioxidants (e.g., vitamins C, vitamin E and beta-carotene), confer a favorable clinical outcome of long-lasting control of inflammation.

Assessing heterogeneity in oligomeric AAA+ machines.

PubMed

Sysoeva, Tatyana A

2017-03-01

ATPases Associated with various cellular Activities (AAA+ ATPases) are molecular motors that use the energy of ATP binding and hydrolysis to remodel their target macromolecules. The majority of these ATPases form ring-shaped hexamers in which the active sites are located at the interfaces between neighboring subunits. Structural changes initiate in an active site and propagate to distant motor parts that interface and reshape the target macromolecules, thereby performing mechanical work. During the functioning cycle, the AAA+ motor transits through multiple distinct states. Ring architecture and placement of the catalytic sites at the intersubunit interfaces allow for a unique level of coordination among subunits of the motor. This in turn results in conformational differences among subunits and overall asymmetry of the motor ring as it functions. To date, a large amount of structural information has been gathered for different AAA+ motors, but even for the most characterized of them only a few structural states are known and the full mechanistic cycle cannot be yet reconstructed. Therefore, the first part of this work will provide a broad overview of what arrangements of AAA+ subunits have been structurally observed focusing on diversity of ATPase oligomeric ensembles and heterogeneity within the ensembles. The second part of this review will concentrate on methods that assess structural and functional heterogeneity among subunits of AAA+ motors, thus bringing us closer to understanding the mechanism of these fascinating molecular motors.

A Kirkwood-Buff derived force field for alkaline earth halide salts

NASA Astrophysics Data System (ADS)

Naleem, Nawavi; Bentenitis, Nikolaos; Smith, Paul E.

2018-06-01

The activity and function of many macromolecules in cellular environments are coupled with the binding of divalent ions such as calcium or magnesium. In principle, computer simulations can be used to understand the molecular level aspects of how many important macromolecules interact with ions. However, most of the force fields currently available often fail to accurately reproduce the properties of divalent ions in aqueous environments. Here we develop classical non-polarizable force fields for the aqueous alkaline earth metal halides (MX2), where M = Mg2+, Ca2+, Sr2+, Ba2+ and X = Cl-, Br-, I-, which can be used in bimolecular simulations and which are compatible with the Simple Point Charge/Extended (SPC/E) water model. The force field parameters are specifically developed to reproduce the experimental Kirkwood-Buff integrals for aqueous solutions and thereby the experimental activity derivatives, partial molar volumes, and excess coordination numbers. This ensures that a reasonable balance between ion-ion, ion-water, and water-water distributions is obtained. However, this requires a scaling of the cation to water oxygen interaction strength in order to accurately reproduce the integrals. The scaling factors developed for chloride salts are successfully transferable to the bromide and iodide salts. Use of these new models leads to reasonable diffusion constants and dielectric decrements. However, the performance of the models decreases with increasing salt concentration (>4m), and simulations of the pure crystals exhibited unstable behavior.

A Kirkwood-Buff derived force field for alkaline earth halide salts.

PubMed

Naleem, Nawavi; Bentenitis, Nikolaos; Smith, Paul E

2018-06-14

The activity and function of many macromolecules in cellular environments are coupled with the binding of divalent ions such as calcium or magnesium. In principle, computer simulations can be used to understand the molecular level aspects of how many important macromolecules interact with ions. However, most of the force fields currently available often fail to accurately reproduce the properties of divalent ions in aqueous environments. Here we develop classical non-polarizable force fields for the aqueous alkaline earth metal halides (MX 2 ), where M = Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ and X = Cl - , Br - , I - , which can be used in bimolecular simulations and which are compatible with the Simple Point Charge/Extended (SPC/E) water model. The force field parameters are specifically developed to reproduce the experimental Kirkwood-Buff integrals for aqueous solutions and thereby the experimental activity derivatives, partial molar volumes, and excess coordination numbers. This ensures that a reasonable balance between ion-ion, ion-water, and water-water distributions is obtained. However, this requires a scaling of the cation to water oxygen interaction strength in order to accurately reproduce the integrals. The scaling factors developed for chloride salts are successfully transferable to the bromide and iodide salts. Use of these new models leads to reasonable diffusion constants and dielectric decrements. However, the performance of the models decreases with increasing salt concentration (>4m), and simulations of the pure crystals exhibited unstable behavior.

[A review of the current research on prions. The evidence suggests the possibility of transmission of the mad cow disease to humans].

PubMed

Grandien, M; Wahren, B

1998-11-25

Further evidence of the transmissibility of bovine spongiform encephalopathy (BSE) across the species barrier from cow to man has been derived from epidemiological analysis and the characterisation of prion strains. Recent research has shown the persistence of prions after experimental transmission to resistant murine species, and subclinical persistence in cows. The accumulation of pathological prion proteins in tonsils and appendix has been demonstrated prior to clinical confirmation of the presence of the new variant of Creutzfeldt-Jakob disease. Current prion research is focused on the involvement of B lymphocytes as carriers, on the species barrier and cellular receptors, and on macromolecules involved in the conformational change from normal to pathological prion proteins.

Long non-coding RNAs in cancer metabolism.

PubMed

Xiao, Zhen-Dong; Zhuang, Li; Gan, Boyi

2016-10-01

Altered cellular metabolism is an emerging hallmark of cancer. Accumulating recent evidence links long non-coding RNAs (lncRNAs), a still poorly understood class of non-coding RNAs, to cancer metabolism. Here we review the emerging findings on the functions of lncRNAs in cancer metabolism, with particular emphasis on how lncRNAs regulate glucose and glutamine metabolism in cancer cells, discuss how lncRNAs regulate various aspects of cancer metabolism through their cross-talk with other macromolecules, explore the mechanistic conceptual framework of lncRNAs in reprogramming metabolism in cancers, and highlight the challenges in this field. A more in-depth understanding of lncRNAs in cancer metabolism may enable the development of novel and effective therapeutic strategies targeting cancer metabolism. © 2016 WILEY Periodicals, Inc.

The extracellular matrix: A dynamic niche in cancer progression

PubMed Central

Lu, Pengfei; Weaver, Valerie M.

2012-01-01

The local microenvironment, or niche, of a cancer cell plays important roles in cancer development. A major component of the niche is the extracellular matrix (ECM), a complex network of macromolecules with distinctive physical, biochemical, and biomechanical properties. Although tightly controlled during embryonic development and organ homeostasis, the ECM is commonly deregulated and becomes disorganized in diseases such as cancer. Abnormal ECM affects cancer progression by directly promoting cellular transformation and metastasis. Importantly, however, ECM anomalies also deregulate behavior of stromal cells, facilitate tumor-associated angiogenesis and inflammation, and thus lead to generation of a tumorigenic microenvironment. Understanding how ECM composition and topography are maintained and how their deregulation influences cancer progression may help develop new therapeutic interventions by targeting the tumor niche. PMID:22351925

Characterization of Nylon 6 by 15N Solid State NMR

DTIC Science & Technology

1989-05-31

M.; Ritchey, W.; de Boer, E. Macromolecules, 1979, 12, 924. 2. Garroway , A. N.; Ritchey, W. M.; Moniz, W. B.; Macromolecules, 1982, It, 1051. 3...E. Macromolecules, 1982, 15, 1406. 23. Veeman, W. S.; Menger, E. M. Bull. Magn. Reson., 1980,2,77. 24. VanderHart, D. L.; Garroway , A. N. J. Chem

Retention of 14C-labeled multiwall carbon nanotubes by humic acid and polymers: Roles of macromolecule properties

PubMed Central

Zhao, Qing; Petersen, Elijah J.; Cornelis, Geert; Wang, Xilong; Guo, Xiaoying; Tao, Shu; Xing, Baoshan

2016-01-01

Developing methods to measure interactions of carbon nanotubes (CNTs) with soils and sediments and understanding the impact of soil and sediment properties on CNT deposition are essential for assessing CNT environmental risks. In this study, we utilized functionalized carbon-14 labeled nanotubes to systematically investigate retention of multiwall CNTs (MWCNTs) by 3 humic acids, 3 natural biopolymers, and 10 model solid-phase polymers, collectively termed macromolecules. Surface properties, rather than bulk properties of macromolecules, greatly influenced MWCNT retention. As shown via multiple linear regression analysis and path analysis, aromaticity and surface polarity were the two most positive factors for retention, suggesting retention was regulated by π-π stacking and hydrogen bonding interactions. Moreover, MWCNT deposition was irreversible. These observations may explain the high retention of MWCNT in natural soils. Moreover, our findings on the relative contribution of each macromolecule property on CNT retention provide information on macromolecule selection for removal of MWCNTs from wastewater and provide a method for measuring CNT interactions with organic macromolecules. PMID:27458320

The lysosomal membrane protein SCAV-3 maintains lysosome integrity and adult longevity

PubMed Central

Li, Yuan; Chen, Baohui; Zou, Wei; Wang, Xin; Wu, Yanwei; Zhao, Dongfeng; Sun, Yanan; Liu, Yubing

2016-01-01

Lysosomes degrade macromolecules and recycle metabolites as well as being involved in diverse processes that regulate cellular homeostasis. The lysosome is limited by a single phospholipid bilayer that forms a barrier to separate the potent luminal hydrolases from other cellular constituents, thus protecting the latter from unwanted degradation. The mechanisms that maintain lysosomal membrane integrity remain unknown. Here, we identified SCAV-3, the Caenorhabditis elegans homologue of human LIMP-2, as a key regulator of lysosome integrity, motility, and dynamics. Loss of scav-3 caused rupture of lysosome membranes and significantly shortened lifespan. Both of these phenotypes were suppressed by reinforced expression of LMP-1 or LMP-2, the C. elegans LAMPs, indicating that longevity requires maintenance of lysosome integrity. Remarkably, reduction in insulin/insulin-like growth factor 1 (IGF-1) signaling suppressed lysosomal damage and extended the lifespan in scav-3(lf) animals in a DAF-16–dependent manner. Our data reveal that SCAV-3 is essential for preserving lysosomal membrane stability and that modulation of lysosome integrity by the insulin/IGF-1 signaling pathway affects longevity. PMID:27810910

Plasma Protein Oxidation and Its Correlation with Antioxidant Potential During Human Aging

PubMed Central

Pandey, Kanti Bhooshan; Mehdi, Mohd Murtaza; Maurya, Pawan Kumar; Rizvi, Syed Ibrahim

2010-01-01

Previous studies have indicated that the main molecular characteristic of aging is the progressive accumulation of oxidative damages in cellular macromolecules. Proteins are one of the main molecular targets of age-related oxidative stress, which have been observed during aging process in cellular systems. Reactive oxygen species (ROS) can lead to oxidation of amino acid side chains, formation of protein-protein cross-linkages, and oxidation of the peptide backbones. In the present study, we report the age-dependent oxidative alterations in biomarkers of plasma protein oxidation: protein carbonyls (PCO), advanced oxidation protein products (AOPPs) and plasma total thiol groups (T-SH) in the Indian population and also correlate these parameters with total plasma antioxidant potential. We show an age dependent decrease in T-SH levels and increase in PCO and AOPPs level. The alterations in the levels of these parameters correlated significantly with the total antioxidant capacity of the plasma. The levels of oxidized proteins in plasma provide an excellent biomarker of oxidative stress due to the relative long half-life of such oxidized proteins. PMID:20826915

S-Nitrosation of monocarboxylate transporter 1: Inhibition of pyruvate-fueled respiration and proliferation of breast cancer cells

PubMed Central

Diers, Anne R.; Broniowska, Katarzyna A.; Chang, Ching-Fang; Hill, R. Blake; Hogg, Neil

2014-01-01

Summary Energy substrates metabolized through mitochondria (e.g., pyruvate, glutamine) are required for biosynthesis of macromolecules in proliferating cells. Since several mitochondrial proteins are known to be targets of S-nitrosation, we determined whether bioenergetics are modulated by S-nitrosation and defined the subsequent effects on proliferation. The nitrosating agent S-nitroso-L-cysteine (L-CysNO) was used to initiate intracellular S-nitrosation, and treatment decreased mitochondrial function and inhibited proliferation of MCF7 mammary adenocarcinoma cells. Surprisingly, the D isomer of CysNO (D-CysNO) which is not transported into cells also caused mitochondrial dysfunction and limited proliferation. Both L- and D-CysNO also inhibited cellular pyruvate uptake and caused S-nitrosation of thiol groups on monocarboxylate transporter 1, a proton-linked pyruvate transporter. These data demonstrate the importance of mitochondrial metabolism in proliferative responses in breast cancer and highlight a novel role for inhibition of metabolic substrate uptake through S-nitrosation of exofacial protein thiols in cellular responses to nitrosative stress. PMID:24486553

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

PubMed Central

Treweek, Jennifer B; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2016-01-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks. PMID:26492141

Automatic detection and measurement of viral replication compartments by ellipse adjustment

PubMed Central

Garcés, Yasel; Guerrero, Adán; Hidalgo, Paloma; López, Raul Eduardo; Wood, Christopher D.; Gonzalez, Ramón A.; Rendón-Mancha, Juan Manuel

2016-01-01

Viruses employ a variety of strategies to hijack cellular activities through the orchestrated recruitment of macromolecules to specific virus-induced cellular micro-environments. Adenoviruses (Ad) and other DNA viruses induce extensive reorganization of the cell nucleus and formation of nuclear Replication Compartments (RCs), where the viral genome is replicated and expressed. In this work an automatic algorithm designed for detection and segmentation of RCs using ellipses is presented. Unlike algorithms available in the literature, this approach is deterministic, automatic, and can adjust multiple RCs using ellipses. The proposed algorithm is non iterative, computationally efficient and is invariant to affine transformations. The method was validated over both synthetic images and more than 400 real images of Ad-infected cells at various timepoints of the viral replication cycle obtaining relevant information about the biogenesis of adenoviral RCs. As proof of concept the algorithm was then used to quantitatively compare RCs in cells infected with the adenovirus wild type or an adenovirus mutant that is null for expression of a viral protein that is known to affect activities associated with RCs that result in deficient viral progeny production. PMID:27819325

Automatic detection and measurement of viral replication compartments by ellipse adjustment

NASA Astrophysics Data System (ADS)

Garcés, Yasel; Guerrero, Adán; Hidalgo, Paloma; López, Raul Eduardo; Wood, Christopher D.; Gonzalez, Ramón A.; Rendón-Mancha, Juan Manuel

2016-11-01

Viruses employ a variety of strategies to hijack cellular activities through the orchestrated recruitment of macromolecules to specific virus-induced cellular micro-environments. Adenoviruses (Ad) and other DNA viruses induce extensive reorganization of the cell nucleus and formation of nuclear Replication Compartments (RCs), where the viral genome is replicated and expressed. In this work an automatic algorithm designed for detection and segmentation of RCs using ellipses is presented. Unlike algorithms available in the literature, this approach is deterministic, automatic, and can adjust multiple RCs using ellipses. The proposed algorithm is non iterative, computationally efficient and is invariant to affine transformations. The method was validated over both synthetic images and more than 400 real images of Ad-infected cells at various timepoints of the viral replication cycle obtaining relevant information about the biogenesis of adenoviral RCs. As proof of concept the algorithm was then used to quantitatively compare RCs in cells infected with the adenovirus wild type or an adenovirus mutant that is null for expression of a viral protein that is known to affect activities associated with RCs that result in deficient viral progeny production.

Macromolecule diffusion and confinement in prokaryotic cells.

PubMed

Mika, Jacek T; Poolman, Bert

2011-02-01

We review recent observations on the mobility of macromolecules and their spatial organization in live bacterial cells. We outline the major fluorescence microscopy-based methods to determine the mobility and thus the diffusion coefficients (D) of molecules, which is not trivial in small cells. The extremely high macromolecule crowding of prokaryotes is used to rationalize the reported lower diffusion coefficients as compared to eukaryotes, and we speculate on the nature of the barriers for diffusion observed for proteins (and mRNAs) in vivo. Building on in vitro experiments and modeling studies, we evaluate the size dependence of diffusion coefficients for macromolecules in vivo, in case of both water-soluble and integral membrane proteins. We comment on the possibilities of anomalous diffusion and provide examples where the macromolecule mobility may be limiting biological processes. Copyright © 2010 Elsevier Ltd. All rights reserved.

Polyacid macromolecule primers

DOEpatents

Sugama, Toshifumi

1989-01-01

Hydrophylic polyacids, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests.

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

PubMed

Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G; Lai, Michelle; D'Alessio, Joseph A; Theunissen, Jan-Willem

2015-01-01

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis

PubMed Central

Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan

2016-01-01

Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway. PMID:26497474

Neutrophil-generated oxidative stress and protein damage in Staphylococcus aureus

PubMed Central

Beavers, William N.; Skaar, Eric P.

2016-01-01

Staphylococcus aureus is a ubiquitous, versatile and dangerous pathogen. It colonizes over 30% of the human population, and is one of the leading causes of death by an infectious agent. During S. aureus colonization and invasion, leukocytes are recruited to the site of infection. To combat S. aureus, leukocytes generate an arsenal of reactive species including superoxide, hydrogen peroxide, nitric oxide and hypohalous acids that modify and inactivate cellular macromolecules, resulting in growth defects or death. When S. aureus colonization cannot be cleared by the immune system, antibiotic treatment is necessary and can be effective. Yet, this organism quickly gains resistance to each new antibiotic it encounters. Therefore, it is in the interest of human health to acquire a deeper understanding of how S. aureus evades killing by the immune system. Advances in this field will have implications for the design of future S. aureus treatments that complement and assist the host immune response. In that regard, this review focuses on how S. aureus avoids host-generated oxidative stress, and discusses the mechanisms used by S. aureus to survive oxidative damage including antioxidants, direct repair of damaged proteins, sensing oxidant stress and transcriptional changes. This review will elucidate areas for studies to identify and validate future antimicrobial targets. PMID:27354296

Macromolecule mapping of the brain using ultrashort-TE acquisition and reference-based metabolite removal.

PubMed

Lam, Fan; Li, Yudu; Clifford, Bryan; Liang, Zhi-Pei

2018-05-01

To develop a practical method for mapping macromolecule distribution in the brain using ultrashort-TE MRSI data. An FID-based chemical shift imaging acquisition without metabolite-nulling pulses was used to acquire ultrashort-TE MRSI data that capture the macromolecule signals with high signal-to-noise-ratio (SNR) efficiency. To remove the metabolite signals from the ultrashort-TE data, single voxel spectroscopy data were obtained to determine a set of high-quality metabolite reference spectra. These spectra were then incorporated into a generalized series (GS) model to represent general metabolite spatiospectral distributions. A time-segmented algorithm was developed to back-extrapolate the GS model-based metabolite distribution from truncated FIDs and remove it from the MRSI data. Numerical simulations and in vivo experiments have been performed to evaluate the proposed method. Simulation results demonstrate accurate metabolite signal extrapolation by the proposed method given a high-quality reference. For in vivo experiments, the proposed method is able to produce spatiospectral distributions of macromolecules in the brain with high SNR from data acquired in about 10 minutes. We further demonstrate that the high-dimensional macromolecule spatiospectral distribution resides in a low-dimensional subspace. This finding provides a new opportunity to use subspace models for quantification and accelerated macromolecule mapping. Robustness of the proposed method is also demonstrated using multiple data sets from the same and different subjects. The proposed method is able to obtain macromolecule distributions in the brain from ultrashort-TE acquisitions. It can also be used for acquiring training data to determine a low-dimensional subspace to represent the macromolecule signals for subspace-based MRSI. Magn Reson Med 79:2460-2469, 2018. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

Diffusion of macromolecules through sclera.

PubMed

Miao, Heng; Wu, Bi-Dong; Tao, Yong; Li, Xiao-Xin

2013-02-01

To quantify the in vitro permeability coefficient over different topographical locations of porcine sclera to macromolecules with different molecular weight. Fresh equatorial and posterior superotemporal porcine sclera was mounted in a two-chamber diffusion apparatus, and its permeability to fluorescein isothiocyanate (FITC)-conjugated dextrans ranging in molecular weight from 40 kDa to 150 kDa was determined by fluorescence spectrophotometry. The sclera was processed as frozen sections and viewed with a fluorescence microscope. The thickness of the area and the thickness that macromolecules enriched in the surface of sclera were measured. The permeability coefficient (Pc) of porcine sclera to macromolecules was significantly higher (40 kDa, p = 0.028; 70 kDa, p = 0.033; 150 kDa, p = 0.007) in equatorial region than posterior, which could be attributed to the significant difference of thickness (p < 0.001, Kruskal-Wallis) between them. Moreover, linear regression indicated a significant negative relationship (40 kDa, p < 0.001; 70 kDa, p = 0.015; 150 kDa, p < 0.001) between scleral permeability coefficient and thickness. Also, Pc declined significantly with increasing molecular weight (MW, p < 0.001, Kruskal-Wallis). The area that the macromolecules enriched in the scleral surface was thicker for those with larger MW (p < 0.001, Kruskal-Wallis). The maximum MW and size for equatorial and posterior superotemporal scleral tissue were 185.01 KDa and 180.42 KDa, 9.92 nm and 9.67 nm, respectively. The permeability coefficient of porcine sclera has a significant negative relationship with scleral thickness and MW of macromolecules. Larger macromolecules are more likely to accumulate in scleral surface. The difference between topographical locations may have pharmacokinetic implications when considering transscleral diffusion of macromolecules. © 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.

Analysis of diffusion and binding in cells using the RICS approach.

PubMed

Digman, Michelle A; Gratton, Enrico

2009-04-01

The movement of macromolecules in cells is assumed to occur either through active transport or by diffusion. However, the determination of the diffusion coefficients in cells using fluctuation methods or FRAP frequently give diffusion coefficient that are orders of magnitude smaller than the diffusion coefficients measured for the same macromolecule in solution. It is assumed that the cell internal viscosity is partially responsible for this decrease in the apparent diffusion. When the apparent diffusion is too slow to be due to cytoplasm viscosity, it is assumed that weak binding of the macromolecules to immobile or quasi immobile structures is taking place. In this article, we derive equations for fitting of the RICS (Raster-scan Image Correlations Spectroscopy) data in cells to a model that includes transient binding to immobile structures, and we show that under some conditions, the spatio-temporal correlation provided by the RICS approach can distinguish the process of diffusion and weak binding. We apply the method to determine the diffusion in the cytoplasm and binding of Focal Adhesion Kinase-EGFP to adhesions in MEF cells.

Oxygen-Free Biochemistry: The Putative CHN Foundation for Exotic Life in a Hydrocarbon World?

NASA Astrophysics Data System (ADS)

Lv, Kong-Peng; Norman, Lucy; Li, Yi-Liang

2017-11-01

Since Earth's biochemistry is carbon-based and water-borne, the main strategies for searching for life elsewhere are "follow the carbon" and "follow the water." Recently, however, there is a growing focus on the prospect that putative exotic life on other planets could rely on unearthly biochemistries. Here, we hypothesize a novel oxygen-free organic chemistry for supporting potential exotic biosystems, which is named CHN biochemistry. This oxygen-free CHN biochemistry starts from simple oxygen-free species (including hydrocarbons, hydrogen cyanide, and nitriles) and produces a range of functional macromolecules that may function in similar ways to terran macromolecules, such as sugars (cyanosugars), acids (cyanoacids), amino acids (amino cyanoacids), and nucleobases (cyanonucleobases). These CHN macromolecules could further interact with each other to generate higher "cyanoester" and "cyanoprotein" systems. In addition, theoretical calculations indicate that the energy changes of some reactions are consistent with their counterparts in Earth's biochemistry. The CHN biochemistry-based life would be applicable in habitats with a low bioavailability of oxygen, such as the alkane lakes of Titan and non-aquatic liquids on extrasolar bodies.

A 3D Image Filter for Parameter-Free Segmentation of Macromolecular Structures from Electron Tomograms

PubMed Central

Ali, Rubbiya A.; Landsberg, Michael J.; Knauth, Emily; Morgan, Garry P.; Marsh, Brad J.; Hankamer, Ben

2012-01-01

3D image reconstruction of large cellular volumes by electron tomography (ET) at high (≤5 nm) resolution can now routinely resolve organellar and compartmental membrane structures, protein coats, cytoskeletal filaments, and macromolecules. However, current image analysis methods for identifying in situ macromolecular structures within the crowded 3D ultrastructural landscape of a cell remain labor-intensive, time-consuming, and prone to user-bias and/or error. This paper demonstrates the development and application of a parameter-free, 3D implementation of the bilateral edge-detection (BLE) algorithm for the rapid and accurate segmentation of cellular tomograms. The performance of the 3D BLE filter has been tested on a range of synthetic and real biological data sets and validated against current leading filters—the pseudo 3D recursive and Canny filters. The performance of the 3D BLE filter was found to be comparable to or better than that of both the 3D recursive and Canny filters while offering the significant advantage that it requires no parameter input or optimisation. Edge widths as little as 2 pixels are reproducibly detected with signal intensity and grey scale values as low as 0.72% above the mean of the background noise. The 3D BLE thus provides an efficient method for the automated segmentation of complex cellular structures across multiple scales for further downstream processing, such as cellular annotation and sub-tomogram averaging, and provides a valuable tool for the accurate and high-throughput identification and annotation of 3D structural complexity at the subcellular level, as well as for mapping the spatial and temporal rearrangement of macromolecular assemblies in situ within cellular tomograms. PMID:22479430

Steady-State Linear and Non-linear Optical Spectroscopy of Organic Chromophores and Bio-macromolecules

NASA Astrophysics Data System (ADS)

Marazzi, Marco; Gattuso, Hugo; Monari, Antonio; Assfeld, Xavier

2018-04-01

Bio-macromolecules as DNA, lipid membranes and (poly)peptides are essential compounds at the core of biological systems. The development of techniques and methodologies for their characterization is therefore necessary and of utmost interest, even though difficulties can be experienced due to their intrinsic complex nature. Among these methods, spectroscopies, relying on optical properties are especially important to determine their macromolecular structures and behaviors, as well as the possible interactions and reactivity with external dyes – often drugs or pollutants – that can (photo)sensitize the bio-macromolecule leading to eventual chemical modifications, thus damages. In this review, we will focus on the theoretical simulation of electronic spectroscopies of bio-macromolecules, considering their secondary structure and including their interaction with different kind of (photo)sensitizers. Namely, absorption, emission and electronic circular dichroism (CD) spectra are calculated and compared with the available experimental data. Non-linear properties will be also taken into account by two-photon absorption, a highly promising technique (i) to enhance absorption in the red and infra-red windows and (ii) to enhance spatial resolution. Methodologically, the implications of using implicit and explicit solvent, coupled to quantum and thermal samplings of the phase space, will be addressed. Especially, hybrid quantum mechanics/ molecular mechanics (QM/MM) methods are explored for a comparison with solely QM methods, in order to address the necessity to consider an accurate description of environmental effects on spectroscopic properties of biological systems.

Polyacid macromolecule primers

DOEpatents

Sugama, Toshifumi.

1989-12-26

Hydrophilic polyacids are described, such as macromolecules of polyitaconic acid and polyacrylic acid, where such macromolecules have molecular weights >50,000 as primers between a polymeric top coating, such as polyurethane, and an oxidized aluminum or aluminum alloy. A near monolayer of primer is used in polymeric adhesive/oxidized aluminum adhered joint systems in 0.05% primer concentration to give superior results in standard peel tests. 2 figs.

Radiolysis of lignin: Prospective mechanism of high-temperature decomposition

NASA Astrophysics Data System (ADS)

Ponomarev, A. V.

2017-12-01

The range of the radiation-thermal processes resulting in conversion of lignin into monomeric phenols is considered. Statistically the most probable places of macromolecule ionization are aromatic units. Release of phenolic products from a lignin macromolecule is the multistage process beginning via fragmentation of primary cation-radicals. Reactions of electrons and small radicals with macromolecules, also as degradation of cation-radicals, result in formation of phenoxyl radicals. Macroradicals possess lower heat stability in comparison with macromolecules. Thermal decomposition of macroradicals leads to release of monohydric and dihydric phenols. The probability of benzenediols formation increases in the presence of alkanes. As noted, partial transformation of lignin into charcoal is inevitable.

Spectral Photosensitization of Optical Anisotropy in Solid Poly(Vinyl Cinnamate) Films

NASA Astrophysics Data System (ADS)

Kozenkov, V. M.; Spakhov, A. A.; Belyaev, V. V.; Chausov, D. N.; Chigrinov, V. G.

2018-04-01

The possibility and features of formation of sensitized photoinduced optical anisotropy in amorphous films of poly(vinyl cinnamate) and its derivative poly(vinyl-4-metoxicinnamate) under the action of polarized light (including light that is not absorbed by polymer macromolecules themselves) have been investigated. It is found that the effect of induced optical anisotropy is based on the transfer of electron excitation energy from donor (sensitizer) molecules to acceptor molecules and is observed in the course of phototopochemical biomolecular cyclization reaction of cinnamate fragments in polymer macromolecules. The detected photoinduced anisotropy in solid films of poly(vinyl cinnamate) and its derivative poly(vinyl-4-metoxicinnamate) ensures sensitized photo-orientation of low-molecular thermotropic liquid crystals.

Quality control in molecular immunohistochemistry

PubMed Central

2008-01-01

Immunoperoxidase histochemistry is a widespread method of assessing expression of biomolecules in tissue samples. Accurate assessment of the expression levels of genes is critical for the management of disease, particularly as therapy targeted to specific molecules becomes more widespread. Determining the quality of preservation of macromolecules in tissue is important to avoid false negative and false positive results. In this review we discuss (1) issues of sensitivity (false negativity) and specificity (false positivity) of immunohistochemical stains, (2) approaches to better understanding differences in immunostains done by different laboratories (including the recently proposed MISFISHIE specification for tissue localization studies), and (3) approaches to assessing the quality of preservation of macromolecules in tissue, particularly in small biopsy samples. PMID:18648842

[Role of green tea in oxidative stress prevention].

PubMed

Metro, D; Muraca, U; Manasseri, L

2006-01-01

Oxidative stress is a condition caused by an increase of Reactive Oxygen Species (ROS) or by a shortage of the mechanisms of cellular protection and antioxidant defence. ROS have a potential oxidative effect towards various cellular macromolecules: proteins, nucleic acids, proteoglycans, lipids, with consequent damages in several cellular districts and promotion of the ageing process of the organism. However, some substances are able to prevent and/or reduce the damages caused by ROS; therefore, they are defined antioxidant. The present research studied, in a group of subjects, the antioxidant effects of the green tea, that was administered with fruit and vegetables in a strictly controlled diet. 50 subjects were selected and requested to daily consume 2-3 fruit portions (especially pineapple), 3-5 portions of vegetables (especially tomato) and 2-3 glasses of green tea for about 2 months to integrate the controlled basic diet. Some indicators of the oxidative stress were measured in the plasma before and after the integration period. The integration of a basic diet with supplements of fruit, vegetables and green tea turned out to be able in increasing both plasmatic total antioxidant capacity and endogenous antioxidant levels and to reduce the lipid peroxidation of the membranes, suggesting a reduction of the oxidative stress. These data suggest that an adequate supplement of antioxidants can prevent oxidative stress and correlated pathologies.

Crystallization of Macromolecules

PubMed Central

Friedmann, David; Messick, Troy; Marmorstein, Ronen

2014-01-01

X-ray crystallography has evolved into a very powerful tool to determine the three-dimensional structure of macromolecules and macromolecular complexes. The major bottleneck in structure determination by X-ray crystallography is the preparation of suitable crystalline samples. This unit outlines steps for the crystallization of a macromolecule, starting with a purified, homogeneous sample. The first protocols describe preparation of the macromolecular sample (i.e., proteins, nucleic acids, and macromolecular complexes). The preparation and assessment of crystallization trials is then described, along with a protocol for confirming whether the crystals obtained are composed of macromolecule as opposed to a crystallization reagent . Next, the optimization of crystallization conditions is presented. Finally, protocols that facilitate the growth of larger crystals through seeding are described. PMID:22045560

Innovative NMR strategies for complex macromolecules

USDA-ARS?s Scientific Manuscript database

In recent years there has been an increasing research emphasis on complex macromolecular systems. These include polymers with precise control of structures, multicomponent systems with higher degrees of organization, polymers involved in micelles, interfaces, and confined environments, nanochemistr...

Extremophilic polysaccharide nanoparticles for cancer nanotherapy and evaluation of antioxidant properties.

PubMed

Raveendran, Sreejith; Palaninathan, Vivekanandan; Nagaoka, Yutaka; Fukuda, Takahiro; Iwai, Seiki; Higashi, Toshiaki; Mizuki, Toru; Sakamoto, Yasushi; Mohanan, P V; Maekawa, Toru; Kumar, D Sakthi

2015-05-01

Polysaccharides that show finest bioactivities and physicochemical properties are always promising for bionanoscience applications. Mauran is such a macromolecule extracted from halophilic bacterium, Halomonas maura for biotechnology and nanoscience applications. Antioxidant properties of MR/CH nanoparticles were studied using biochemical assays to prove the versatility of these test nanoparticles for biomedical applications. Here, we demonstrate the prospects of extremophilic polysaccharide, mauran based nanoparticles for scavenging reactive oxygen species in both in vitro and ex vivo conditions. 5-fluorouracil loaded MR/CH nanoparticles were tested for anticancer proliferation and compared their therapeutic efficiency using breast adenocarcinoma and glioma cells. Fluorescently labeled nanoparticles were employed to show the cellular uptake of these nanocarriers using confocal microscopic imaging and flow cytometry. Copyright © 2015 Elsevier B.V. All rights reserved.

Opportunities for Live Cell FT-Infrared Imaging: Macromolecule Identification with 2D and 3D Localization

PubMed Central

Mattson, Eric C.; Aboualizadeh, Ebrahim; Barabas, Marie E.; Stucky, Cheryl L.; Hirschmugl, Carol J.

2013-01-01

Infrared (IR) spectromicroscopy, or chemical imaging, is an evolving technique that is poised to make significant contributions in the fields of biology and medicine. Recent developments in sources, detectors, measurement techniques and speciman holders have now made diffraction-limited Fourier transform infrared (FTIR) imaging of cellular chemistry in living cells a reality. The availability of bright, broadband IR sources and large area, pixelated detectors facilitate live cell imaging, which requires rapid measurements using non-destructive probes. In this work, we review advances in the field of FTIR spectromicroscopy that have contributed to live-cell two and three-dimensional IR imaging, and discuss several key examples that highlight the utility of this technique for studying the structure and chemistry of living cells. PMID:24256815

Recent Advances in Transferable Coarse-Grained Modeling of Proteins

PubMed Central

Kar, Parimal; Feig, Michael

2017-01-01

Computer simulations are indispensable tools for studying the structure and dynamics of biological macromolecules. Biochemical processes occur on different scales of length and time. Atomistic simulations cannot cover the relevant spatiotemporal scales at which the cellular processes occur. To address this challenge, coarse-grained (CG) modeling of the biological systems are employed. Over the last few years, many CG models for proteins continue to be developed. However, many of them are not transferable with respect to different systems and different environments. In this review, we discuss those CG protein models that are transferable and that retain chemical specificity. We restrict ourselves to CG models of soluble proteins only. We also briefly review recent progress made in the multi-scale hybrid all-atom/coarse-grained simulations of proteins. PMID:25443957

Three-Dimensional Reconstruction of Nuclear Envelope Architecture Using Dual-Color Metal-Induced Energy Transfer Imaging.

PubMed

Chizhik, Anna M; Ruhlandt, Daja; Pfaff, Janine; Karedla, Narain; Chizhik, Alexey I; Gregor, Ingo; Kehlenbach, Ralph H; Enderlein, Jörg

2017-12-26

The nuclear envelope, comprising the inner and the outer nuclear membrane, separates the nucleus from the cytoplasm and plays a key role in cellular functions. Nuclear pore complexes (NPCs), which are embedded in the nuclear envelope, control transport of macromolecules between the two compartments. Here, using dual-color metal-induced energy transfer (MIET), we determine the axial distance between Lap2β and Nup358 as markers for the inner nuclear membrane and the cytoplasmic side of the NPC, respectively. Using MIET imaging, we reconstruct the 3D profile of the nuclear envelope over the whole basal area, with an axial resolution of a few nanometers. This result demonstrates that optical microscopy can achieve nanometer axial resolution in biological samples and without recourse to complex interferometric approaches.

Molecular Imprinting of Macromolecules for Sensor Applications

PubMed Central

Saylan, Yeşeren; Yilmaz, Fatma; Özgür, Erdoğan; Derazshamshir, Ali; Yavuz, Handan; Denizli, Adil

2017-01-01

Molecular recognition has an important role in numerous living systems. One of the most important molecular recognition methods is molecular imprinting, which allows host compounds to recognize and detect several molecules rapidly, sensitively and selectively. Compared to natural systems, molecular imprinting methods have some important features such as low cost, robustness, high recognition ability and long term durability which allows molecularly imprinted polymers to be used in various biotechnological applications, such as chromatography, drug delivery, nanotechnology, and sensor technology. Sensors are important tools because of their ability to figure out a potentially large number of analytical difficulties in various areas with different macromolecular targets. Proteins, enzymes, nucleic acids, antibodies, viruses and cells are defined as macromolecules that have wide range of functions are very important. Thus, macromolecules detection has gained great attention in concerning the improvement in most of the studies. The applications of macromolecule imprinted sensors will have a spacious exploration according to the low cost, high specificity and stability. In this review, macromolecules for molecularly imprinted sensor applications are structured according to the definition of molecular imprinting methods, developments in macromolecular imprinting methods, macromolecular imprinted sensors, and conclusions and future perspectives. This chapter follows the latter strategies and focuses on the applications of macromolecular imprinted sensors. This allows discussion on how sensor strategy is brought to solve the macromolecules imprinting. PMID:28422082

Iontophoretic transport of charged macromolecules across human sclera.

PubMed

Chopra, Poonam; Hao, Jinsong; Li, S Kevin

2010-03-30

The mechanisms of transscleral iontophoresis have been investigated previously with small molecules in rabbit sclera. The objective of the present study was to examine transscleral iontophoretic transport of charged macromolecules across excised human sclera. Passive and 2mA iontophoretic transport experiments were conducted in side-by-side diffusion cells with human sclera. The effects of iontophoresis upon transscleral transport of model permeants bovine serum albumin (BSA) and polystyrene sulfonic acid (PSS) as well as a model drug bevacizumab (BEV) were determined. Passive and iontophoretic transport experiments of tetraethylammonium (TEA) and salicylic acid (SA) and passive transport experiments of the macromolecules served as the controls. The results of iontophoresis enhanced transport of TEA and SA across human sclera were consistent with those in a previous rabbit sclera study. For the iontophoretic transport of macromolecules BSA and BEV, higher iontophoretic fluxes were observed in anodal iontophoresis as compared to passive and cathodal iontophoresis. This suggests the importance of electroosmosis. For the polyelectrolyte PSS, higher iontophoretic flux was observed in cathodal iontophoresis compared to anodal iontophoresis. Both electroosmosis and electrophoresis affected iontophoretic fluxes of the macromolecules; the relative contributions of electroosmosis and electrophoresis were a function of molecular size and charge of the macromolecules. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Molecular Imprinting of Macromolecules for Sensor Applications.

PubMed

Saylan, Yeşeren; Yilmaz, Fatma; Özgür, Erdoğan; Derazshamshir, Ali; Yavuz, Handan; Denizli, Adil

2017-04-19

Molecular recognition has an important role in numerous living systems. One of the most important molecular recognition methods is molecular imprinting, which allows host compounds to recognize and detect several molecules rapidly, sensitively and selectively. Compared to natural systems, molecular imprinting methods have some important features such as low cost, robustness, high recognition ability and long term durability which allows molecularly imprinted polymers to be used in various biotechnological applications, such as chromatography, drug delivery, nanotechnology, and sensor technology. Sensors are important tools because of their ability to figure out a potentially large number of analytical difficulties in various areas with different macromolecular targets. Proteins, enzymes, nucleic acids, antibodies, viruses and cells are defined as macromolecules that have wide range of functions are very important. Thus, macromolecules detection has gained great attention in concerning the improvement in most of the studies. The applications of macromolecule imprinted sensors will have a spacious exploration according to the low cost, high specificity and stability. In this review, macromolecules for molecularly imprinted sensor applications are structured according to the definition of molecular imprinting methods, developments in macromolecular imprinting methods, macromolecular imprinted sensors, and conclusions and future perspectives. This chapter follows the latter strategies and focuses on the applications of macromolecular imprinted sensors. This allows discussion on how sensor strategy is brought to solve the macromolecules imprinting.

Immersion freezing of birch pollen washing water

NASA Astrophysics Data System (ADS)

Augustin, S.; Wex, H.; Niedermeier, D.; Pummer, B.; Grothe, H.; Hartmann, S.; Tomsche, L.; Clauss, T.; Voigtländer, J.; Ignatius, K.; Stratmann, F.

2013-11-01

Birch pollen grains are known to be ice nucleating active biological particles. The ice nucleating activity has previously been tracked down to biological macromolecules that can be easily extracted from the pollen grains in water. In the present study, we investigated the immersion freezing behavior of these ice nucleating active (INA) macromolecules. Therefore we measured the frozen fractions of particles generated from birch pollen washing water as a function of temperature at the Leipzig Aerosol Cloud Interaction Simulator (LACIS). Two different birch pollen samples were considered, with one originating from Sweden and one from the Czech Republic. For the Czech and Swedish birch pollen samples, freezing was observed to start at -19 and -17 °C, respectively. The fraction of frozen droplets increased for both samples down to -24 °C. Further cooling did not increase the frozen fractions any more. Instead, a plateau formed at frozen fractions below 1. This fact could be used to determine the amount of INA macromolecules in the droplets examined here, which in turn allowed for the determination of nucleation rates for single INA macromolecules. The main differences between the Swedish birch pollen and the Czech birch pollen were obvious in the temperature range between -17 and -24 °C. In this range, a second plateau region could be seen for Swedish birch pollen. As we assume INA macromolecules to be the reason for the ice nucleation, we concluded that birch pollen is able to produce at least two different types of INA macromolecules. We were able to derive parameterizations for the heterogeneous nucleation rates for both INA macromolecule types, using two different methods: a simple exponential fit and the Soccer ball model. With these parameterization methods we were able to describe the ice nucleation behavior of single INA macromolecules from both the Czech and the Swedish birch pollen.

Multistage adsorption of diffusing macromolecules and viruses

NASA Astrophysics Data System (ADS)

Chou, Tom; D'Orsogna, Maria R.

2007-09-01

We derive the equations that describe adsorption of diffusing particles onto a surface followed by additional surface kinetic steps before being transported across the interface. Multistage surface kinetics occurs during membrane protein insertion, cell signaling, and the infection of cells by virus particles. For example, viral entry into healthy cells is possible only after a series of receptor and coreceptor binding events occurs at the cellular surface. We couple the diffusion of particles in the bulk phase with the multistage surface kinetics and derive an effective, integrodifferential boundary condition that contains a memory kernel embodying the delay induced by the surface reactions. This boundary condition takes the form of a singular perturbation problem in the limit where particle-surface interactions are short ranged. Moreover, depending on the surface kinetics, the delay kernel induces a nonmonotonic, transient replenishment of the bulk particle concentration near the interface. The approach generalizes that of Ward and Tordai [J. Chem. Phys. 14, 453 (1946)] and Diamant and Andelman [Colloids Surf. A 183-185, 259 (2001)] to include surface kinetics, giving rise to qualitatively new behaviors. Our analysis also suggests a simple scheme by which stochastic surface reactions may be coupled to deterministic bulk diffusion.

Long Non-Coding RNA Emergence During Renal Cell Carcinoma Tumorigenesis.

PubMed

Liu, Xiaobing; Hao, Yaxing; Yu, Wei; Yang, Xia; Luo, Xing; Zhao, Jiang; Li, Jia; Hu, Xiaoyan; Li, Longkun

2018-05-22

Renal cell carcinoma (RCC) is the most common kidney cancer diagnosed across the globe and has steadily increased in incidence in recent decades. Techniques for diagnosing or treating RCC are limited, and confined mostly to later stages of the disease. Almost all RCC pathological types are resistant to chemotherapeutics and radiation therapy. To this effect, new markers for diagnosis and target therapy are urgently needed. Advanced genome sequencing technologies have revealed long non-coding RNAs (lncRNAs) as a novel marker, transcribed throughout the human genome. The emergence of lncRNAs is an aberrant expression and is involved in the tumorigenesis of RCC. LncRNAs drive cancer phenotypes through their interaction with other cellular macromolecules including DNA, protein, and RNA. Recent research on lncRNA molecular mechanisms has revealed new markers to functionally annotate these cancers' associated transcripts, making them targets for effective diagnosis and therapeutic intervention in the fight against cancer. In this review, we first highlight the common mechanisms that underlie aberrant lncRNA expression in RCC. We go on to discuss the potential translational application of lncRNA research in the diagnosis, prognosis, and treatment of RCC. © 2018 The Author(s). Published by S. Karger AG, Basel.

Transport of Spherical Particles Through Fibrous Media and a Row of Parallel Cylinders: Applications to Glomerular Filtration.

PubMed

Punyaratabandhu, Numpong; Kongoup, Pimkhwan; Dechadilok, Panadda; Katavetin, Pisut; Triampo, Wannapong

2017-12-01

Viewed in renal physiology as a refined filtration device, the glomerulus filters large volumes of blood plasma while keeping proteins within blood circulation. Effects of macromolecule size and macromolecule hydrodynamic interaction with the nanostructure of the cellular layers of the glomerular capillary wall on the glomerular size selectivity are investigated through a mathematical simulation based on an ultrastructural model. The epithelial slit, a planar arrangement of fibers connecting the epithelial podocytes, is represented as a row of parallel cylinders with nonuniform spacing between adjacent fibers. The mean and standard deviation of gap half-width between its fibers are based on values recently reported from electron microscopy. The glomerular basement membrane (GBM) is represented as a fibrous medium containing fibers of two different sizes: the size of type IV collagens and that of glycosaminoglycans (GAGs). The endothelial cell layer is modeled as a layer full of fenestrae that are much larger than solute size and filled with GAGs. The calculated total sieving coefficient agrees well with the sieving coefficients of ficolls obtained from in vivo urinalysis in humans, whereas the computed glomerular hydraulic permeability also falls within the range estimated from human glomerular filtration rate (GFR). Our result indicates that the endothelial cell layer and GBM significantly contribute to solute and fluid restriction of the glomerular barrier, whereas, based on the structure of the epithelial slit obtained from electron microscopy, the contribution of the epithelial slit could be smaller than previously believed.

Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering.

PubMed

Tria, Giancarlo; Mertens, Haydyn D T; Kachala, Michael; Svergun, Dmitri I

2015-03-01

Dynamic ensembles of macromolecules mediate essential processes in biology. Understanding the mechanisms driving the function and molecular interactions of 'unstructured' and flexible molecules requires alternative approaches to those traditionally employed in structural biology. Small-angle X-ray scattering (SAXS) is an established method for structural characterization of biological macromolecules in solution, and is directly applicable to the study of flexible systems such as intrinsically disordered proteins and multi-domain proteins with unstructured regions. The Ensemble Optimization Method (EOM) [Bernadó et al. (2007 ▶). J. Am. Chem. Soc. 129, 5656-5664] was the first approach introducing the concept of ensemble fitting of the SAXS data from flexible systems. In this approach, a large pool of macromolecules covering the available conformational space is generated and a sub-ensemble of conformers coexisting in solution is selected guided by the fit to the experimental SAXS data. This paper presents a series of new developments and advancements to the method, including significantly enhanced functionality and also quantitative metrics for the characterization of the results. Building on the original concept of ensemble optimization, the algorithms for pool generation have been redesigned to allow for the construction of partially or completely symmetric oligomeric models, and the selection procedure was improved to refine the size of the ensemble. Quantitative measures of the flexibility of the system studied, based on the characteristic integral parameters of the selected ensemble, are introduced. These improvements are implemented in the new EOM version 2.0, and the capabilities as well as inherent limitations of the ensemble approach in SAXS, and of EOM 2.0 in particular, are discussed.

Intramolecular Charge Transfer of Conjugated Liquid Crystal Ferrocene Macromolecules - Synthesis and Characterization

DTIC Science & Technology

2016-04-12

AFRL-AFOSR-CL-TR-2016-0012 Intramolecular Charge Transfer of Conjugated Liquid Crystal Ferrocene Macromolecules Ronald Ziolo CIQA Final Report 07/07...3. DATES COVERED (From - To)  15 Aug 2014 to 14 Jan 2016 4. TITLE AND SUBTITLE Intramolecular Charge Transfer of Conjugated Liquid Crystal Ferrocene...characterization of a new series of conjugated macromolecules bearing ferrocene as a highly efficient electron donor material coupled to 2,5-di(alcoxy) benzene

Critical considerations for developing nucleic acid macromolecule based drug products.

PubMed

Muralidhara, Bilikallahalli K; Baid, Rinku; Bishop, Steve M; Huang, Min; Wang, Wei; Nema, Sandeep

2016-03-01

Protein expression therapy using nucleic acid macromolecules (NAMs) as a new paradigm in medicine has recently gained immense therapeutic potential. With the advancement of nonviral delivery it has been possible to target NAMs against cancer, immunodeficiency and infectious diseases. Owing to the complex and fragile structure of NAMs, however, development of a suitable, stable formulation for a reasonable product shelf-life and efficacious delivery is indeed challenging to achieve. This review provides a synopsis of challenges in the formulation and stability of DNA/m-RNA based medicines and probable mitigation strategies including a brief summary of delivery options to the target cells. Nucleic acid based drugs at various stages of ongoing clinical trials are compiled. Copyright © 2016. Published by Elsevier Ltd.

New and unconventional approaches for advancing resolution in biological transmission electron microscopy by improving macromolecular specimen preparation and preservation.

PubMed

Massover, William H

2011-02-01

Resolution in transmission electron microscopy (TEM) now is limited by the properties of specimens, rather than by those of instrumentation. The long-standing difficulties in obtaining truly high-resolution structure from biological macromolecules with TEM demand the development, testing, and application of new ideas and unconventional approaches. This review concisely describes some new concepts and innovative methodologies for TEM that deal with unsolved problems in the preparation and preservation of macromolecular specimens. The selected topics include use of better support films, a more protective multi-component matrix surrounding specimens for cryo-TEM and negative staining, and, several quite different changes in microscopy and micrography that should decrease the effects of electron radiation damage; all these practical approaches are non-traditional, but have promise to advance resolution for specimens of biological macromolecules beyond its present level of 3-10 Å (0.3-1.0 nm). The result of achieving truly high resolution will be a fulfillment of the still unrealized potential of transmission electron microscopy for directly revealing the structure of biological macromolecules down to the atomic level. Published by Elsevier Ltd.

Oxygen-Free Biochemistry: The Putative CHN Foundation for Exotic Life in a Hydrocarbon World?

PubMed

Lv, Kong-Peng; Norman, Lucy; Li, Yi-Liang

2017-11-01

Since Earth's biochemistry is carbon-based and water-borne, the main strategies for searching for life elsewhere are "follow the carbon" and "follow the water." Recently, however, there is a growing focus on the prospect that putative exotic life on other planets could rely on unearthly biochemistries. Here, we hypothesize a novel oxygen-free organic chemistry for supporting potential exotic biosystems, which is named CHN biochemistry. This oxygen-free CHN biochemistry starts from simple oxygen-free species (including hydrocarbons, hydrogen cyanide, and nitriles) and produces a range of functional macromolecules that may function in similar ways to terran macromolecules, such as sugars (cyanosugars), acids (cyanoacids), amino acids (amino cyanoacids), and nucleobases (cyanonucleobases). These CHN macromolecules could further interact with each other to generate higher "cyanoester" and "cyanoprotein" systems. In addition, theoretical calculations indicate that the energy changes of some reactions are consistent with their counterparts in Earth's biochemistry. The CHN biochemistry-based life would be applicable in habitats with a low bioavailability of oxygen, such as the alkane lakes of Titan and non-aquatic liquids on extrasolar bodies. Key Words: Oxygen-free biochemistry-Titan-Hydrocarbons-Hydrogen cyanide-Nitriles. Astrobiology 17, 1173-1181.

Cooperation and selfishness both occur during molecular evolution.

PubMed

Penny, David

2014-11-26

Perhaps the 'selfish' aspect of evolution has been over-emphasised, and organisms considered as basically selfish. However, at the macromolecular level of genes and proteins the cooperative aspect of evolution is more obvious and balances this self-centred aspect. Thousands of proteins must function together in an integrated manner to use and to produce the many molecules necessary for a functioning cell. The macromolecules have no idea whether they are functioning cooperatively or competitively with other genes and gene products (such as proteins). The cell is a giant cooperative system of thousands of genes/proteins that function together, even if it has to simultaneously resist 'parasites'. There are extensive examples of cooperative behavior among genes and proteins in both functioning cells and in the origin of life, so this cooperative nature, along with selfishness, must be considered part of normal evolution. The principles also apply to very large numbers of examples of 'positive interactions' between organisms, including both eukaryotes and akaryotes (prokaryotes). This does not negate in any way the 'selfishness' of genes - but macromolecules have no idea when they are helping, or hindering, other groups of macromolecules. We need to assert more strongly that genes, and gene products, function together as a cooperative unit.

Efficient Intracellular siRNA Delivery by Ethyleneimine-Modified Amphiphilic Macromolecules

PubMed Central

Sparks, Sarah M.; Waite, Carolyn L.; Harmon, Alexander M.; Nusblat, Leora M.; Roth, Charles M.; Uhrich, Kathryn E.

2013-01-01

Summary New materials that can bind and deliver oligonucleotides such as short interfering RNA (siRNA) without toxicity are greatly needed to fulfill the promise of therapeutic gene silencing. Amphiphilic macromolecules (AMs) were functionalized with linear ethyleneimines to create cationic AMs capable of complexing with siRNA. Structurally, the parent AM is formed from a mucic acid backbone whose tetra-hydroxy groups are alkylated with 12-carbon aliphatic chains to form the hydrophobic component of the macromolecule. This alkylated mucic acid is then mono-functionalized with poly(ethylene glycol) (PEG) as a hydrophilic component. The resulting AM contains a free carboxylic acid within the hydrophobic domain. In this work, linear ethyleneimines were conjugated to the free carboxylic acid to produce an AM with one primary amine (1N) or one primary amine and four secondary amines (5N). Further, an AM with amine substitution both to the free carboxylic acid in the hydrophobic domain and also to the adjacent PEG was synthesized to produce a polymer with one primary amine and eight secondary amines (9N), four located on each side of the AM hydrophobic domain. All amine-functionalized AMs formed nanoscale micelles but only the 5N and 9N AMs had cationic zeta potentials, which increased with increasing number of amines. All AMs exhibited less inherent cytotoxicity than linear polyethyleneimine (L-PEI) at concentrations of 10 µM and above. By increasing the length of the cationic ethyleneimine chain and the total number of amines, successful siRNA complexation and cellular siRNA delivery was achieved in a malignant glioma cell line. In addition, siRNA-induced silencing of firefly luciferase was observed using complexes of siRNA with the 9N AM and comparable to L-PEI, yet showed better cell viability at higher concentrations (above 10 µM). This work highlights the promise of cationic AMs as safe and efficient synthetic vectors for siRNA delivery. Specifically, a novel polymer (9N) was identified for efficient siRNA delivery to cancer cells and will be further evaluated. PMID:21793212

Towards the Structure Determination of a Modulated Protein Crystal: The Semicrystalline State of Profilin:Actin

NASA Technical Reports Server (NTRS)

Borgstahl, G.; Lovelace, J.; Snell, E. H.; Bellamy, H.

2003-01-01

One of the remaining challenges to structural biology is the solution of modulated structures. While small molecule crystallographers have championed this type of structure, to date, no modulated macromolecular structures have been determined. Modulation of the molecular structures within the crystal can produce satellite reflections or a superlattice of reflections in reciprocal space. We have developed the data collection methods and strategies that are needed to collect and analyze these data. If the macromolecule's crystal lattice is composed of physiologically relevant packing contacts, structural changes induced under physiological conditions can cause distortion relevant to the function and biophysical processes of the molecule making up the crystal. By careful measurement of the distortion, and the corresponding three-dimensional structure of the distorted molecule, we will visualize the motion and mechanism of the biological macromolecule(s). We have measured the modulated diffraction pattern produced by the semicrystalline state of profilin:actin crystals using highly parallel and highly monochromatic synchrotron radiation coupled with fine phi slicing (0.001-0.010 degrees) for structure determination. These crystals present these crystals present a unique opportunity to address an important question in structural biology. The modulation is believed to be due to the formation of actin helical filaments from the actin beta ribbon upon the pH-induced dissociation of profilin. To date, the filamentous state of actin has resisted crystallization and no detailed structures are available. The semicrystalline state profilin:actin crystals provides a unique opportunity to understand the many conformational states of actin. This knowledge is essential for understanding the dynamics underlying shape changes and motility of eukaryotic cells. Many essential processes, such as cytokinesis, phagocytosis, and cellular migration depend upon the capacity of the actin microfilament system to be restructured in a controlled manner via polymerization, depolymerization, severing, cross-linking, and anchorage. The structure the semicrystalline state of profilin:actin will challenge and validate current models of muscle contraction and cell motility. The methodology and theory under development will be easily extendable to other systems.

Distributions of phytoplankton carbohydrate, protein and lipid in the world oceans from satellite ocean colour.

PubMed

Roy, Shovonlal

2018-06-01

Energy value of phytoplankton regulates the growth of higher trophic species, affecting the tropic balance and sustainability of marine food webs. Therefore, developing our capability to estimate and monitor, on a global scale, the concentrations of macromolecules that determine phytoplankton energy value, would be invaluable. Reported here are the first estimates of carbohydrate, protein, lipid, and overall energy value of phytoplankton in the world oceans, using ocean-colour data from satellites. The estimates are based on a novel bio-optical method that utilises satellite-derived bio-optical fingerprints of living phytoplankton combined with allometric relationships between phytoplankton cells and cellular macromolecular contents. The annually averaged phytoplankton energy value, per cubic metre of sub-surface ocean, varied from less than 0.1 kJ in subtropical gyres, to 0.5-1.0 kJ in parts of the equatorial, northern and southern latitudes, and rising to >10 kJ in certain coastal and optically complex waters. The annually averaged global stocks of carbohydrate, protein and lipid were 0.044, 0.17 and 0.108 gigatonnes, respectively, with monthly stocks highest in September and lowest in June, over 1997-2013. The fractional contributions of phytoplankton size classes e.g., picoplankton, nanoplankton and microplankton to surface concentrations and global stocks of macromolecules varied considerably across marine biomes classified as Longhurst provinces. Among these provinces, the highest annually averaged surface concentrations of carbohydrate, protein, and lipid were in North-East Atlantic Coastal Shelves, whereas, the lowest concentration of carbohydrate or lipid were in North Atlantic Tropical Gyral, and that of protein was in North Pacific Subtropical Gyre West. The regional accuracy of the estimates and their sensitivity to satellite inputs are quantified from the bio-optical model, which show promise for possible operational monitoring of phytoplankton energy value from satellite ocean colour. Adequate in situ measurements of macromolecules and improved retrievals of inherent optical properties from high-resolution satellite images, would be required to validate these estimates at local sites, and to further improve their accuracy in the world oceans.

A mathematical model of single target site location by Brownian movement in subcellular compartments.

PubMed

Kuthan, Hartmut

2003-03-07

The location of distinct sites is mandatory for many cellular processes. In the subcompartments of the cell nucleus, only very small numbers of diffusing macromolecules and specific target sites of some types may be present. In this case, we are faced with the Brownian movement of individual macromolecules and their "random search" for single/few specific target sites, rather than bulk-averaged diffusion and multiple sites. In this article, I consider the location of a distant central target site, e.g. a globular protein, by individual macromolecules executing unbiased (i.e. drift-free) random walks in a spherical compartment. For this walk-and-capture model, the closed-form analytic solution of the first passage time probability density function (p.d.f.) has been obtained as well as the first and second moment. In the limit of a large ratio of the radii of the spherical diffusion space and central target, well-known relations for the variance and the first two moments for the exponential p.d.f. were found to hold with high accuracy. These calculations reinforce earlier numerical results and Monte Carlo simulations. A major implication derivable from the model is that non-directed random movement is an effective means for locating single sites in submicron-sized compartments, even when the diffusion coefficients are comparatively small and the diffusing species are present in one copy only. These theoretical conclusions are underscored numerically for effective diffusion constants ranging from 0.5 to 10.0 microm(2) s(-1), which have been reported for a couple of nuclear proteins in their physiological environment. Spherical compartments of submicron size are, for example, the Cajal bodies (size: 0.1-1.0 microm), which are present in 1-5 copies in the cell nucleus. Within a small Cajal body of radius 0.1 microm a single diffusing protein molecule (with D=0.5 microm(2) s(-1)) would encounter a medium-sized protein of radius 2.5 nm within 1 s with a probability near certainty (p=0.98).

Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes.

PubMed

Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

2016-01-01

Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

PubMed Central

Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

2016-01-01

ABSTRACT Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy. PMID:27046251

Dielectric Relaxation of Water and Water-Plasticized Biomolecules in Relation to Cellular Water Organization, Cytoplasmic Viscosity, and Desiccation Tolerance in Recalcitrant Seed Tissues1

PubMed Central

Sun, Wendell Q.

2000-01-01

To understand the relationship between the organization of cellular water, molecular interactions, and desiccation tolerance, dielectric behaviors of water and water-plasticized biomolecules in red oak (Quercus rubra) seeds were studied during dehydration. The thermally stimulated current study showed three dielectric dispersions: (a) the relaxation of loosely-bound water and small polar groups, (b) the relaxation of tightly-bound water, carbohydrate chains, large polar groups of macromolecules, and (c) the “freezing in” of molecular mobility (glassy state). Seven discrete hydration levels (water contents of 1.40, 0.55, 0.41, 0.31, 0.21, 0.13, and 0.08 g/g dry weight, corresponding to −1.5, −8, −11, −14, −24, −74, and −195 MPa, respectively) were identified according to the changes in thermodynamic and dielectric properties of water and water-plasticized biomolecules during dehydration. The implications of intracellular water organization for desiccation tolerance were discussed. Cytoplasmic viscosity increased exponentially at water content < 0.40 g/g dry weight, which was correlated with the great relaxation slowdown of water-plasticized biomolecules, supporting a role for viscosity in metabolic shutdown during dehydration. PMID:11080297

Synthesis and Characterization of a Chondroitin Sulfate Based Hybrid Bio/Synthetic Biomimetic Aggrecan Macromolecule

NASA Astrophysics Data System (ADS)

Sarkar, Sumona

Lower back pain resulting from intervertebral disc degeneration is one of the leading musculoskeletal disorders confronting our health system. In order to mechanically stabilize the disc early in the degenerative cascade and prevent the need for spinal fusion surgeries, we have proposed the development of a hybrid-bio/synthetic biomimetic proteoglycan macromolecule for injection into the disc in the early stages of degeneration. The goal of this thesis was to incorporate natural chondroitin sulfate (CS) chains into bottle brush polymer synthesis strategies for the fabrication of CS-macromolecules which mimic the proteoglycan structure and function while resisting enzymatic degradation. Both the "grafting-to" and "grafting-through" techniques of bottle brush synthesis were explored. CS was immobilized via a terminal primary amine onto a model polymeric backbone (polyacrylic acid) for investigation of the "grafting-to" strategy and an epoxy-amine step-growth polymerization technique was utilized for the "grafting-through" synthesis of CS-macromolecules with polyethylene glycol backbone segments. Incorporation of a synthetic polymeric backbone at the terminal amine of CS was confirmed via biochemical assays, 1H-NMR and FTIR spectroscopy, and CS-macromolecule size was demonstrated to be higher than that of natural CS via gel permeation chromatography, transmission electron microscopy and viscosity measurements. Further analysis of CS-macromolecule functionality indicated maintenance of natural CS properties such as high fixed charge density, high osmotic potential and low cytotoxicity with nucleus pulposus cells. These studies are the first attempt at the incorporation of natural CS into biomimetic bottle brush structures. CS-macromolecules synthesized via the methods developed in these studies may be utilized in the treatment and prevention of debilitating back pain as well as act as mimetics for other proteoglycans implicated in cartilage, heart valve, and nervous system tissue function.

Preparing Monodisperse Macromolecular Samples for Successful Biological Small-Angle X-ray and Neutron Scattering Experiments

PubMed Central

Jeffries, Cy M.; Graewert, Melissa A.; Blanchet, Clément E.; Langley, David B.; Whitten, Andrew E.; Svergun, Dmitri I

2017-01-01

Small-angle X-ray and neutron scattering (SAXS and SANS) are techniques used to extract structural parameters and determine the overall structures and shapes of biological macromolecules, complexes and assemblies in solution. The scattering intensities measured from a sample contain contributions from all atoms within the illuminated sample volume including the solvent and buffer components as well as the macromolecules of interest. In order to obtain structural information, it is essential to prepare an exactly matched solvent blank so that background scattering contributions can be accurately subtracted from the sample scattering to obtain the net scattering from the macromolecules in the sample. In addition, sample heterogeneity caused by contaminants, aggregates, mismatched solvents, radiation damage or other factors can severely influence and complicate data analysis so it is essential that the samples are pure and monodisperse for the duration of the experiment. This Protocol outlines the basic physics of SAXS and SANS and reveals how the underlying conceptual principles of the techniques ultimately ‘translate’ into practical laboratory guidance for the production of samples of sufficiently high quality for scattering experiments. The procedure describes how to prepare and characterize protein and nucleic acid samples for both SAXS and SANS using gel electrophoresis, size exclusion chromatography and light scattering. Also included are procedures specific to X-rays (in-line size exclusion chromatography SAXS) and neutrons, specifically preparing samples for contrast matching/variation experiments and deuterium labeling of proteins. PMID:27711050

Self-assembly of an amphiphilic macromolecule under spherical confinement: An efficient route to generate hollow nanospheres

NASA Astrophysics Data System (ADS)

Glagoleva, A. A.; Vasilevskaya, V. V.; Yoshikawa, K.; Khokhlov, A. R.

2013-12-01

In general, bio-macromolecules are composed of hydrophilic and hydrophobic moieties and are confined within small cavities, such as cell membranes and intracellular organelles. Here, we studied the self-organization of macromolecules having groups with different affinities to solvents under spherical nano-scale confinement by means of computer modeling. It is shown that depending on the interaction parameters of monomer units composed of side- and main-chain monomer groups along a single linear macromolecule and on cavity size, such amphiphilic polymers undergo the conformational transitions between hollow nanospheres, rod-like and folded cylindrical structures, and a necklace conformation with and without a particular ordering of beads. The diagram of the conformations in the variables the incompatibility parameter of monomer units and the cavity radius is constructed.

Statistics and bioinformatics in nutritional sciences: analysis of complex data in the era of systems biology⋆

PubMed Central

Fu, Wenjiang J.; Stromberg, Arnold J.; Viele, Kert; Carroll, Raymond J.; Wu, Guoyao

2009-01-01

Over the past two decades, there have been revolutionary developments in life science technologies characterized by high throughput, high efficiency, and rapid computation. Nutritionists now have the advanced methodologies for the analysis of DNA, RNA, protein, low-molecular-weight metabolites, as well as access to bioinformatics databases. Statistics, which can be defined as the process of making scientific inferences from data that contain variability, has historically played an integral role in advancing nutritional sciences. Currently, in the era of systems biology, statistics has become an increasingly important tool to quantitatively analyze information about biological macromolecules. This article describes general terms used in statistical analysis of large, complex experimental data. These terms include experimental design, power analysis, sample size calculation, and experimental errors (type I and II errors) for nutritional studies at population, tissue, cellular, and molecular levels. In addition, we highlighted various sources of experimental variations in studies involving microarray gene expression, real-time polymerase chain reaction, proteomics, and other bioinformatics technologies. Moreover, we provided guidelines for nutritionists and other biomedical scientists to plan and conduct studies and to analyze the complex data. Appropriate statistical analyses are expected to make an important contribution to solving major nutrition-associated problems in humans and animals (including obesity, diabetes, cardiovascular disease, cancer, ageing, and intrauterine fetal retardation). PMID:20233650

Cell Chirality Drives Left-Right Asymmetric Morphogenesis.

PubMed

Inaki, Mikiko; Sasamura, Takeshi; Matsuno, Kenji

2018-01-01

Most macromolecules found in cells are chiral, meaning that they cannot be superimposed onto their mirror image. However, cells themselves can also be chiral, a subject that has received little attention until very recently. In our studies on the mechanisms of left-right (LR) asymmetric development in Drosophila , we discovered that cells can have an intrinsic chirality to their structure, and that this "cell chirality" is generally responsible for the LR asymmetric development of certain organs in this species. The actin cytoskeleton plays important roles in the formation of cell chirality. In addition, Myosin31DF ( Myo31DF ), which encodes Drosophila Myosin ID, was identified as a molecular switch for cell chirality. In other invertebrate species, including snails and Caenorhabditis elegans , chirality of the blastomeres, another type of cell chirality, determines the LR asymmetry of structures in the body. Thus, chirality at the cellular level may broadly contribute to LR asymmetric development in various invertebrate species. Recently, cell chirality was also reported for various vertebrate cultured cells, and studies suggested that cell chirality is evolutionarily conserved, including the essential role of the actin cytoskeleton. Although the biological roles of cell chirality in vertebrates remain unknown, it may control LR asymmetric development or other morphogenetic events. The investigation of cell chirality has just begun, and this new field should provide valuable new insights in biology and medicine.

Cell Chirality Drives Left-Right Asymmetric Morphogenesis

PubMed Central

Inaki, Mikiko; Sasamura, Takeshi; Matsuno, Kenji

2018-01-01

Most macromolecules found in cells are chiral, meaning that they cannot be superimposed onto their mirror image. However, cells themselves can also be chiral, a subject that has received little attention until very recently. In our studies on the mechanisms of left-right (LR) asymmetric development in Drosophila, we discovered that cells can have an intrinsic chirality to their structure, and that this “cell chirality” is generally responsible for the LR asymmetric development of certain organs in this species. The actin cytoskeleton plays important roles in the formation of cell chirality. In addition, Myosin31DF (Myo31DF), which encodes Drosophila Myosin ID, was identified as a molecular switch for cell chirality. In other invertebrate species, including snails and Caenorhabditis elegans, chirality of the blastomeres, another type of cell chirality, determines the LR asymmetry of structures in the body. Thus, chirality at the cellular level may broadly contribute to LR asymmetric development in various invertebrate species. Recently, cell chirality was also reported for various vertebrate cultured cells, and studies suggested that cell chirality is evolutionarily conserved, including the essential role of the actin cytoskeleton. Although the biological roles of cell chirality in vertebrates remain unknown, it may control LR asymmetric development or other morphogenetic events. The investigation of cell chirality has just begun, and this new field should provide valuable new insights in biology and medicine. PMID:29666795

ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis.

PubMed

Yamauchi, Yoshio; Yokoyama, Shinji; Chang, Ta-Yuan

2016-01-01

Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Proteomic analysis reveals novel extracellular virulence-associated proteins and functions regulated by the diffusible signal factor (DSF) in Xanthomonas oryzae pv. oryzicola.

PubMed

Qian, Guoliang; Zhou, Yijing; Zhao, Yancun; Song, Zhiwei; Wang, Suyan; Fan, Jiaqin; Hu, Baishi; Venturi, Vittorio; Liu, Fengquan

2013-07-05

Quorum sensing (QS) in Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is mediated by the diffusible signal factor (DSF). DSF-mediating QS has been shown to control virulence and a set of virulence-related functions; however, the expression profiles and functions of extracellular proteins controlled by DSF signal remain largely unclear. In the present study, 33 DSF-regulated extracellular proteins, whose functions include small-protein mediating QS, oxidative adaptation, macromolecule metabolism, cell structure, biosynthesis of small molecules, intermediary metabolism, cellular process, protein catabolism, and hypothetical function, were identified by proteomics in Xoc. Of these, 15 protein encoding genes were in-frame deleted, and 4 of them, including three genes encoding type II secretion system (T2SS)-dependent proteins and one gene encoding an Ax21 (activator of XA21-mediated immunity)-like protein (a novel small-protein type QS signal) were determined to be required for full virulence in Xoc. The contributions of these four genes to important virulence-associated functions, including bacterial colonization, extracellular polysaccharide, cell motility, biofilm formation, and antioxidative ability, are presented. To our knowledge, our analysis is the first complete list of DSF-regulated extracellular proteins and functions in a Xanthomonas species. Our results show that DSF-type QS played critical roles in regulation of T2SS and Ax21-mediating QS, which sheds light on the role of DSF signaling in Xanthomonas.

Functional analysis of lysosomes during mouse preimplantation embryo development.

PubMed

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Ohta, Yuki; Wada, Ayako; Ishida, Yuka; Kito, Seiji; Nishikawa, Tetsu; Minami, Naojiro; Sato, Ken; Kokubo, Toshiaki

2013-01-01

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.

In-situ second harmonic generation by cancer cell targeting ZnO nanocrystals to effect photodynamic action in subcellular space.

PubMed

Gu, Bobo; Pliss, Artem; Kuzmin, Andrey N; Baev, Alexander; Ohulchanskyy, Tymish Y; Damasco, Jossana A; Yong, Ken-Tye; Wen, Shuangchun; Prasad, Paras N

2016-10-01

This paper introduces the concept of in-situ upconversion of deep penetrating near infrared light via second harmonic generation from ZnO nanocrystals delivered into cells to effect photo activated therapies, such as photodynamic therapy, which usually require activation by visible light with limited penetration through biological tissues. We demonstrated this concept by subcellular activation of a photodynamic therapy drug, Chlorin e6, excited within its strong absorption Soret band by the second harmonic (SH) light, generated at 409 nm by ZnO nanocrystals, which were targeted to cancer cells and internalized through the folate-receptor mediated endocytosis. By a combination of theoretical modeling and experimental measurements, we show that SH light, generated in-situ by ZnO nanocrystals significantly contributes to activation of photosensitizer, leading to cell death through both apoptotic and necrotic pathways initiated in the cytoplasm. This targeted photodynamic action was studied using label-free Coherent Anti-Stokes Raman Scattering imaging of the treated cells to monitor changes in the distribution of native cellular proteins and lipids. We found that initiation of photodynamic therapy with upconverted light led to global reduction in the intracellular concentration of macromolecules, likely due to suppression of proteins and lipids synthesis, which could be considered as a real-time indicator of cellular damage from photodynamic treatment. In prospective applications this in-situ photon upconversion could be further extended using ZnO nanocrystals surface functionalized with a specific organelle targeting group, provided a powerful approach to identify and consequently maximize a cellular response to phototherapy, selectively initiated in a specific cellular organelle. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structured crowding and its effects on enzyme catalysis.

PubMed

Ma, Buyong; Nussinov, Ruth

2013-01-01

Macromolecular crowding decreases the diffusion rate, shifts the equilibrium of protein-protein and protein-substrate interactions, and changes protein conformational dynamics. Collectively, these effects contribute to enzyme catalysis. Here we describe how crowding may bias the conformational change and dynamics of enzyme populations and in this way affect catalysis. Crowding effects have been studied using artificial crowding agents and in vivo-like environments. These studies revealed a correlation between protein dynamics and function in the crowded environment. We suggest that crowded environments be classified into uniform crowding and structured crowding. Uniform crowding represents random crowding conditions created by synthetic particles with a narrow size distribution. Structured crowding refers to the highly coordinated cellular environment, where proteins and other macromolecules are clustered and organized. In structured crowded environments the perturbation of protein thermal stability may be lower; however, it may still be able to modulate functions effectively and dynamically. Dynamic, allosteric enzymes could be more sensitive to cellular perturbations if their free energy landscape is flatter around the native state; on the other hand, if their free energy landscape is rougher, with high kinetic barriers separating deep minima, they could be more robust. Above all, cells are structured; and this holds both for the cytosol and for the membrane environment. The crowded environment is organized, which limits the search, and the crowders are not necessarily inert. More likely, they too transmit allosteric effects, and as such play important functional roles. Overall, structured cellular crowding may lead to higher enzyme efficiency and specificity.

Small-angle x-ray scattering study of polymer structure: Carbosilane dendrimers in hexane solution

NASA Astrophysics Data System (ADS)

Shtykova, E. V.; Feigin, L. A.; Volkov, V. V.; Malakhova, Yu. N.; Streltsov, D. R.; Buzin, A. I.; Chvalun, S. N.; Katarzhanova, E. Yu.; Ignatieva, G. M.; Muzafarov, A. M.

2016-09-01

The three-dimensional organization of monodisperse hyper-branched macromolecules of regular structure—carbosilane dendrimers of zero, third, and sixth generations—has been studied by small-angle X-ray scattering (SAXS) in solution. The use of modern methods of SAXS data interpretation, including ab initio modeling, has made it possible to determine the internal architecture of the dendrimers in dependence of the generation number and the number of cyclosiloxane end groups (forming the shell of dendritic macromolecules) and show dendrimers to be spherical. The structural results give grounds to consider carbosilane dendrimers promising objects for forming crystals with subsequent structural analysis and determining their structure with high resolution, as well as for designing new materials to be used in various dendrimer-based technological applications.

Decades of Data: Extracting Trends from Microgravity Crystallization History

NASA Technical Reports Server (NTRS)

Judge, R. A.; Snell, E. H.; Kephart, R.; vanderWoerd, M.

2004-01-01

The reduced acceleration environment of an orbiting spacecraft has been proposed as an ideal environment for biological crystal growth as the first sounding rocket flight in 1981 many crystallization experiments have flown with some showing improvement and others not. To further explore macromolecule crystal improvement in microgravity we have accumulated data from published reports and reports submitted by 63 missions including the Space Shuttle program, unmanned satellites, the Russian Space Station MIR and sounding rocket experiments. While it is not at this point in time a comprehensive record of all flight crystallization experimental results, there is however sufficient information for emerging trends to be identified. In this study the effects of the acceleration environment, the techniques of crystallization, sample molecular weight and the response of individual macromolecules to microgravity crystallization will be investigated.

From the Cover: Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: Fabrication methods and transport studies

NASA Astrophysics Data System (ADS)

McAllister, Devin V.; Wang, Ping M.; Davis, Shawn P.; Park, Jung-Hwan; Canatella, Paul J.; Allen, Mark G.; Prausnitz, Mark R.

2003-11-01

Arrays of micrometer-scale needles could be used to deliver drugs, proteins, and particles across skin in a minimally invasive manner. We therefore developed microfabrication techniques for silicon, metal, and biodegradable polymer microneedle arrays having solid and hollow bores with tapered and beveled tips and feature sizes from 1 to 1,000 μm. When solid microneedles were used, skin permeability was increased in vitro by orders of magnitude for macromolecules and particles up to 50 nm in radius. Intracellular delivery of molecules into viable cells was also achieved with high efficiency. Hollow microneedles permitted flow of microliter quantities into skin in vivo, including microinjection of insulin to reduce blood glucose levels in diabetic rats. transdermal drug delivery | skin | microelectromechanical systems | solid microneedle | hollow needle injection

Macromolecule exchange in Cuscuta-host plant interactions.

PubMed

Kim, Gunjune; Westwood, James H

2015-08-01

Cuscuta species (dodders) are parasitic plants that are able to grow on many different host plants and can be destructive to crops. The connections between Cuscuta and its hosts allow movement of not only water and small nutrients, but also macromolecules including mRNA, proteins and viruses. Recent studies show that RNAs move bidirectionally between hosts and parasites and involve a large number of different genes. Although the function of mobile mRNAs has not been demonstrated in this system, small RNAs are also transmitted and a silencing construct expressed in hosts is able to affect expression of the target gene in the parasite. High throughput sequencing of host-parasite associations has the potential to greatly accelerate understanding of this remarkable interaction. Copyright © 2015 Elsevier Ltd. All rights reserved.

Impacts of Organic Macromolecules, Chlorophyll and Soot on Arctic Sea Ice

NASA Astrophysics Data System (ADS)

Ogunro, O. O.; Wingenter, O. W.; Elliott, S.; Flanner, M.; Dubey, M. K.

2014-12-01

Recent intensification of Arctic amplification can be strongly connected to positive feedback relating black carbon deposition to sea ice surface albedo. In addition to soot deposition on the ice and snow pack, ice algal chlorophyll is likely to compete as an absorber and redistributor of energy. Hence, solar radiation absorption by chlorophyll and some components of organic macromolecules in/under the ice column is currently being examined to determine the level of influence on predicted rate of ice loss. High amounts of organic macromolecules and chlorophyll are produced in global sea ice by the bottom microbial community and also in vertically distributed layers where substantial biological activities take place. Brine channeling in columnar ice can allow for upward flow of nutrients which leads to greater primary production in the presence of moderate light. Modeling of the sea-ice processes in tandem with experiments and field observations promises rapid progress in enhancing Arctic ice predictions. We are designing and conducting global climate model experiments to determine the impact of organic macromolecules and chlorophyll on Arctic sea ice. Influences on brine network permeability and radiation/albedo will be considered in this exercise. Absorption by anthropogenic materials such as soot and black carbon will be compared with that of natural pigments. We will indicate areas of soot and biological absorption dominance in the sense of single scattering, then couple into a full radiation transfer scheme to attribute the various contributions to polar climate change amplification. The work prepares us to study more traditional issues such as chlorophyll warming of the pack periphery and chemical effects of the flow of organics from ice internal communities. The experiments started in the Arctic will broaden to include Antarctic sea ice and shelves. Results from the Arctic simulations will be presented.

Motion of single wandering diblock-macromolecules directed by a PTFE nano-fence: real time SFM observations.

PubMed

Gallyamov, Marat O; Qin, Shuhui; Matyjaszewski, Krzysztof; Khokhlov, Alexei; Möller, Martin

2009-07-21

Using SFM we have observed a peculiar twisting motion of diblock macromolecules pre-collapsed in ethanol vapour during their subsequent spreading in water vapour. The intrinsic asymmetry of the diblock macromolecules has been considered to be the reason for such twisting. Further, friction-deposited PTFE nano-stripes have been employed as nano-trails with the purpose of inducing lateral directed motion of the asymmetric diblock macromolecules under cyclic impact from the changing vapour surroundings. Indeed, some of the macromolecules have demonstrated a certain tendency to orient along the PTFE stripes, and some of the oriented ones have moved occasionally in a directed manner along the trail. However, it has been difficult to reliably record such directed motion at the single molecule level due to some mobility of the PTFE nano-trails themselves in the changing vapour environment. In vapours, the PTFE stripes have demonstrated a distinct tendency towards conjunction. This tendency has manifested itself in efficient expelling of groups of the mobile brush-like molecules from the areas between two PTFE stripes joining in a zip-fastener manner. This different kind of vapour-induced cooperative macromolecular motion has been reliably observed as being directed. The PTFE nano-frame experiences some deformation when constraining the spreading macromolecules. We have estimated the possible force causing such deformation of the PTFE fence. The force has been found to be a few pN as calculated by a partial contribution from every single molecule of the constrained group.

The Biomolecular Crystallization Database Version 4: expanded content and new features.

PubMed

Tung, Michael; Gallagher, D Travis

2009-01-01

The Biological Macromolecular Crystallization Database (BMCD) has been a publicly available resource since 1988, providing a curated archive of information on crystal growth for proteins and other biological macromolecules. The BMCD content has recently been expanded to include 14 372 crystal entries. The resource continues to be freely available at http://xpdb.nist.gov:8060/BMCD4. In addition, the software has been adapted to support the Java-based Lucene query language, enabling detailed searching over specific parameters, and explicit search of parameter ranges is offered for five numeric variables. Extensive tools have been developed for import and handling of data from the RCSB Protein Data Bank. The updated BMCD is called version 4.02 or BMCD4. BMCD4 entries have been expanded to include macromolecule sequence, enabling more elaborate analysis of relations among protein properties, crystal-growth conditions and the geometric and diffraction properties of the crystals. The BMCD version 4.02 contains greatly expanded content and enhanced search capabilities to facilitate scientific analysis and design of crystal-growth strategies.

Low temperature conditioning of garlic (Allium sativum L.) “seed” cloves induces alterations in sprouts proteome

PubMed Central

Dufoo-Hurtado, Miguel D.; Huerta-Ocampo, José Á.; Barrera-Pacheco, Alberto; Barba de la Rosa, Ana P.; Mercado-Silva, Edmundo M.

2015-01-01

Low-temperature conditioning of garlic “seed” cloves substitutes the initial climatic requirements of the crop and accelerates the cycle. We have reported that “seed” bulbs from “Coreano” variety conditioned at 5°C for 5 weeks reduces growth and plant weight as well as the crop yields and increases the synthesis of phenolic compounds and anthocyanins. Therefore, this treatment suggests a cold stress. Plant acclimation to stress is associated with deep changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation. The aim of this work was to study the changes in the protein profiles of garlic “seed” cloves subjected to conditioning at low-temperature using proteomics approach. Two sets of garlic bulbs were used, one set was stored at room temperature (23°C), and the other was conditioned at low temperature (5°C) for 5 weeks. Total soluble proteins were extracted from sprouts of cloves and separated by two-dimensional gel electrophoresis. Protein spots showing statistically significant changes in abundance were analyzed by LC-ESI-MS/MS and identified by database search analysis using the Mascot search engine. The results revealed that low-temperature conditioning of garlic “seed” cloves causes alterations in the accumulation of proteins involved in different physiological processes such as cellular growth, antioxidative/oxidative state, macromolecules transport, protein folding and transcription regulation process. The metabolic pathways affected include protein biosynthesis and quality control system, photosynthesis, photorespiration, energy production, and carbohydrate and nucleotide metabolism. These processes can work cooperatively to establish a new cellular homeostasis that might be related with the physiological and biochemical changes observed in previous studies. PMID:26029231

Spatio-temporal manipulation of small GTPase activity at subcellular level and on timescale of seconds in living cells.

PubMed

DeRose, Robert; Pohlmeyer, Christopher; Umeda, Nobuhiro; Ueno, Tasuku; Nagano, Tetsuo; Kuo, Scot; Inoue, Takanari

2012-03-09

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events(1, 2). Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time(3). In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac(4), Cdc42(4), RhoA(4) and Ras(5), in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells(6). Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level(6).

Low temperature conditioning of garlic (Allium sativum L.) "seed" cloves induces alterations in sprouts proteome.

PubMed

Dufoo-Hurtado, Miguel D; Huerta-Ocampo, José Á; Barrera-Pacheco, Alberto; Barba de la Rosa, Ana P; Mercado-Silva, Edmundo M

2015-01-01

Low-temperature conditioning of garlic "seed" cloves substitutes the initial climatic requirements of the crop and accelerates the cycle. We have reported that "seed" bulbs from "Coreano" variety conditioned at 5°C for 5 weeks reduces growth and plant weight as well as the crop yields and increases the synthesis of phenolic compounds and anthocyanins. Therefore, this treatment suggests a cold stress. Plant acclimation to stress is associated with deep changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation. The aim of this work was to study the changes in the protein profiles of garlic "seed" cloves subjected to conditioning at low-temperature using proteomics approach. Two sets of garlic bulbs were used, one set was stored at room temperature (23°C), and the other was conditioned at low temperature (5°C) for 5 weeks. Total soluble proteins were extracted from sprouts of cloves and separated by two-dimensional gel electrophoresis. Protein spots showing statistically significant changes in abundance were analyzed by LC-ESI-MS/MS and identified by database search analysis using the Mascot search engine. The results revealed that low-temperature conditioning of garlic "seed" cloves causes alterations in the accumulation of proteins involved in different physiological processes such as cellular growth, antioxidative/oxidative state, macromolecules transport, protein folding and transcription regulation process. The metabolic pathways affected include protein biosynthesis and quality control system, photosynthesis, photorespiration, energy production, and carbohydrate and nucleotide metabolism. These processes can work cooperatively to establish a new cellular homeostasis that might be related with the physiological and biochemical changes observed in previous studies.

Poly(ethylene oxide monomethyl ether)- block-poly(propylene succinate) Nanoparticles: Synthesis and Characterization, Enzymatic and Cellular Degradation, Micellar Solubilization of Paclitaxel, and in Vitro and in Vivo Evaluation.

PubMed

Jäger, Alessandro; Jäger, Eliézer; Syrová, Zdeňka; Mazel, Tomas; Kováčik, Lubomír; Raška, Ivan; Höcherl, Anita; Kučka, Jan; Konefal, Rafal; Humajova, Jana; Poučková, Pavla; Štěpánek, Petr; Hrubý, Martin

2018-04-11

Polyester-based nanostructures are widely studied as drug-delivery systems due to their biocompatibility and biodegradability. They are already used in the clinic. In this work, we describe a new and simple biodegradable and biocompatible system as the Food and Drug Administration approved polyesters (poly-ε-caprolactone, polylactic acid, and poly(lactic- co-glycolic acid)) for the delivery of the anticancer drug paclitaxel (PTX) as a model drug. A hydrophobic polyester, poly(propylene succinate) (PPS), was prepared from a nontoxic alcohol (propylene glycol) and monomer from the Krebs's cycle (succinic acid) in two steps via esterification and melt polycondensation. Furthermore, their amphiphilic block copolyester, poly(ethylene oxide monomethyl ether)- block-poly(propylene succinate) (mPEO- b-PPS), was prepared by three steps via esterification followed by melt polycondensation and the addition of mPEO to the PPS macromolecules. Analysis of the in vitro cellular behavior of the prepared nanoparticle carriers (NPs) (enzymatic degradation, uptake, localization, and fluorescence resonance energy-transfer pair degradation studies) was performed by fluorescence studies. PTX was loaded to the NPs of variable sizes (30, 70, and 150 nm), and their in vitro release was evaluated in different cell models and compared with commercial PTX formulations. The mPEO- b-PPS copolymer analysis displays glass transition temperature < body temperature < melting temperature, lower toxicity (including the toxicity of their degradation products), drug solubilization efficacy, stability against spontaneous hydrolysis during transport in bloodstream, and simultaneous enzymatic degradability after uptake into the cells. The detailed cytotoxicity in vitro and in vivo tumor efficacy studies have shown the superior efficacy of the NPs compared with PTX and PTX commercial formulations.

Antimicrobial resistance challenged with metal-based antimicrobial macromolecules.

PubMed

Abd-El-Aziz, Alaa S; Agatemor, Christian; Etkin, Nola

2017-02-01

Antimicrobial resistance threatens the achievements of science and medicine, as it deactivates conventional antimicrobial therapeutics. Scientists respond to the threat by developing new antimicrobial platforms to prevent and treat infections from these resistant strains. Metal-based antimicrobial macromolecules are emerging as an alternative to conventional platforms because they combine multiple mechanisms of action into one platform due to the distinctive properties of metals. For example, metals interact with intracellular proteins and enzymes, and catalyse various intracellular processes. The macromolecular architecture offers a means to enhance antimicrobial activity since several antimicrobial moieties can be conjugated to the scaffold. Further, these macromolecules can be fabricated into antimicrobial materials for contact-killing medical implants, fabrics, and devices. As volatilization or leaching out of the antimicrobial moieties from the macromolecular scaffold is reduced, these medical implants, fabrics, and devices can retain their antimicrobial activity over an extended period. Recent advances demonstrate the potential of metal-based antimicrobial macromolecules as effective platforms that prevent and treat infections from resistant strains. In this review these advances are thoroughly discussed within the context of examples of metal-based antimicrobial macromolecules, their mechanisms of action and biocompatibility. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dermal and transdermal delivery of pharmaceutically relevant macromolecules.

PubMed

Münch, S; Wohlrab, J; Neubert, R H H

2017-10-01

The skin offers an attractive way for dermal and transdermal drug delivery that is why the drug still needs certain qualities to transcend the outermost layer of the skin, the stratum corneum. The requirements are: drugs with a maximum molecular weight of 1kDa, high lipophilicity and a certain polarity. This would restrict the use of a transdermal delivery of macromolecules, which would make the drug more effective in therapeutic administration. Various studies have shown that macromolecules without support do not penetrate the human skin. This effect can be achieved using physical and chemical methods, as well as biological peptides. The most popular physical method is the use of microneedles to create micropores in the skin and release the active agent in different sections. But also, other methods have been tested. Microjets, lasers, electroporation, sonophoresis and iontophoresis are also promising methods to successfully deliver dermal and transdermal macromolecules. Additionally, there are different penetration enhancer groups and biological peptides, which are also considered to be interesting approaches of enabling macromolecules to travel along the skin. All these methods will be described and evaluated in this review article. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantifying Nucleic Acid Ensembles with X-ray Scattering Interferometry.

PubMed

Shi, Xuesong; Bonilla, Steve; Herschlag, Daniel; Harbury, Pehr

2015-01-01

The conformational ensemble of a macromolecule is the complete description of the macromolecule's solution structures and can reveal important aspects of macromolecular folding, recognition, and function. However, most experimental approaches determine an average or predominant structure, or follow transitions between states that each can only be described by an average structure. Ensembles have been extremely difficult to experimentally characterize. We present the unique advantages and capabilities of a new biophysical technique, X-ray scattering interferometry (XSI), for probing and quantifying structural ensembles. XSI measures the interference of scattered waves from two heavy metal probes attached site specifically to a macromolecule. A Fourier transform of the interference pattern gives the fractional abundance of different probe separations directly representing the multiple conformation states populated by the macromolecule. These probe-probe distance distributions can then be used to define the structural ensemble of the macromolecule. XSI provides accurate, calibrated distance in a model-independent fashion with angstrom scale sensitivity in distances. XSI data can be compared in a straightforward manner to atomic coordinates determined experimentally or predicted by molecular dynamics simulations. We describe the conceptual framework for XSI and provide a detailed protocol for carrying out an XSI experiment. © 2015 Elsevier Inc. All rights reserved.

A Method for Decomposition of the Basic Reaction of Biological Macromolecules into Exponential Components

NASA Astrophysics Data System (ADS)

Barabash, Yu. M.; Lyamets, A. K.

2016-12-01

The structural and dynamical properties of biological macromolecules under non-equilibrium conditions determine the kinetics of their basic reaction to external stimuli. This kinetics is multiexponential in nature. This is due to the operation of various subsystems in the structure of macromolecules, as well as the effect of the basic reaction on the structure of macromolecules. The situation can be interpreted as a manifestation of the stationary states of macromolecules, which are represented by monoexponential components of the basic reaction (Monod-Wyman-Changeux model) Monod et al. (J Mol Cell Biol 12:88-118, 1965). The representation of multiexponential kinetics of the basic reaction in the form of a sum of exponential functions (A(t)={sum}_{i=1}^n{a}_i{e}^{-{k}_it}) is a multidimensional optimization problem. To solve this problem, a gradient method of optimization with software determination of the amount of exponents and reasonable calculation time is developed. This method is used to analyze the kinetics of photoinduced electron transport in the reaction centers (RC) of purple bacteria and the fluorescence induction in the granum thylakoid membranes which share a common function of converting light energy.

A new bead-spring model for simulation of semi-flexible macromolecules

NASA Astrophysics Data System (ADS)

Saadat, Amir; Khomami, Bamin

2016-11-01

A bead-spring model for semi-flexible macromolecules is developed to overcome the deficiencies of the current coarse-grained bead-spring models. Specifically, model improvements are achieved through incorporation of a bending potential. The new model is designed to accurately describe the correlation along the backbone of the chain, segmental length, and force-extension behavior of the macromolecule even at the limit of 1 Kuhn step per spring. The relaxation time of different Rouse modes is used to demonstrate the capabilities of the new model in predicting chain dynamics.

ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules

PubMed Central

Ashkenazy, Haim; Abadi, Shiran; Martz, Eric; Chay, Ofer; Mayrose, Itay; Pupko, Tal; Ben-Tal, Nir

2016-01-01

The degree of evolutionary conservation of an amino acid in a protein or a nucleic acid in DNA/RNA reflects a balance between its natural tendency to mutate and the overall need to retain the structural integrity and function of the macromolecule. The ConSurf web server (http://consurf.tau.ac.il), established over 15 years ago, analyses the evolutionary pattern of the amino/nucleic acids of the macromolecule to reveal regions that are important for structure and/or function. Starting from a query sequence or structure, the server automatically collects homologues, infers their multiple sequence alignment and reconstructs a phylogenetic tree that reflects their evolutionary relations. These data are then used, within a probabilistic framework, to estimate the evolutionary rates of each sequence position. Here we introduce several new features into ConSurf, including automatic selection of the best evolutionary model used to infer the rates, the ability to homology-model query proteins, prediction of the secondary structure of query RNA molecules from sequence, the ability to view the biological assembly of a query (in addition to the single chain), mapping of the conservation grades onto 2D RNA models and an advanced view of the phylogenetic tree that enables interactively rerunning ConSurf with the taxa of a sub-tree. PMID:27166375

Mineral-related proteins of sea urchin teeth: Lytechinus variegatus.

PubMed

Veis, Arthur; Barss, Joseph; Dahl, Thomas; Rahima, Mohammed; Stock, Stuart

2002-12-01

Sea urchins have a set of five continuously growing teeth, each of which has a very complex structure. The mineral phase is calcite of varying Mg content, depending on the location within a tooth. The calcium carbonate is present in amorphous, plate-like and rod-like forms. It has been hypothesized that the mineral deposition is a matrix-mediated process, similar to that in vertebrate bone and tooth, wherein certain macromolecules within the organic matrix of the mineralized tissue play an important role in nucleating and controlling the growth habit of the mineral crystals. It has also been hypothesized that the mineral-related macromolecules involved in urchin teeth might bear a direct evolutionary relationship to those of the vertebrate tooth. These hypotheses are explored here by examining the pattern and nature of the mineral distribution, using microCT of intact teeth, and the nature of the mineral-related matrix proteins. The mineral-related proteins were extracted and fractionated by anion exchange chromatography. The relationship of certain fractions to vertebrate matrix proteins was established by immunoblots using antibodies to vertebrate tooth proteins. The antibodies were then used to localize the proteins within the teeth, by immunocytochemistry and histology with specific staining. The microCT data on mineral density has been correlated with the patterns of cellular migration and mineral deposition within the tooth as it grows. It appears that the mineralization within the different tooth compartments might take place under the influence of different matrix proteins. Further studies are in progress to more completely describe the vertebrate-invertebrate immunologically cross-reactive proteins of the urchin teeth. Copyright 2002 Wiley-Liss, Inc.

Perfect mixing of immiscible macromolecules at fluid interfaces

NASA Astrophysics Data System (ADS)

Sheiko, Sergei S.; Zhou, Jing; Arnold, Jamie; Neugebauer, Dorota; Matyjaszewski, Krzysztof; Tsitsilianis, Constantinos; Tsukruk, Vladimir V.; Carrillo, Jan-Michael Y.; Dobrynin, Andrey V.; Rubinstein, Michael

2013-08-01

The difficulty of mixing chemically incompatible substances—in particular macromolecules and colloidal particles—is a canonical problem limiting advances in fields ranging from health care to materials engineering. Although the self-assembly of chemically different moieties has been demonstrated in coordination complexes, supramolecular structures, and colloidal lattices among other systems, the mechanisms of mixing largely rely on specific interfacing of chemically, physically or geometrically complementary objects. Here, by taking advantage of the steric repulsion between brush-like polymers tethered to surface-active species, we obtained long-range arrays of perfectly mixed macromolecules with a variety of polymer architectures and a wide range of chemistries without the need of encoding specific complementarity. The net repulsion arises from the significant increase in the conformational entropy of the brush-like polymers with increasing distance between adjacent macromolecules at fluid interfaces. This entropic-templating assembly strategy enables long-range patterning of thin films on sub-100 nm length scales.

Scalable synthesis of sequence-defined, unimolecular macromolecules by Flow-IEG

PubMed Central

Leibfarth, Frank A.; Johnson, Jeremiah A.; Jamison, Timothy F.

2015-01-01

We report a semiautomated synthesis of sequence and architecturally defined, unimolecular macromolecules through a marriage of multistep flow synthesis and iterative exponential growth (Flow-IEG). The Flow-IEG system performs three reactions and an in-line purification in a total residence time of under 10 min, effectively doubling the molecular weight of an oligomeric species in an uninterrupted reaction sequence. Further iterations using the Flow-IEG system enable an exponential increase in molecular weight. Incorporating a variety of monomer structures and branching units provides control over polymer sequence and architecture. The synthesis of a uniform macromolecule with a molecular weight of 4,023 g/mol is demonstrated. The user-friendly nature, scalability, and modularity of Flow-IEG provide a general strategy for the automated synthesis of sequence-defined, unimolecular macromolecules. Flow-IEG is thus an enabling tool for theory validation, structure–property studies, and advanced applications in biotechnology and materials science. PMID:26269573

Hydrophobic-domain-dependent protein-protein interactions mediate the localization of GPAT enzymes to ER subdomains

USDA-ARS?s Scientific Manuscript database

The endoplasmic reticulum (ER) is a dynamic network that consists of numerous regions or subdomains with discrete morphological features and functional properties, including those involved in protein and oil-body formation, anterograde transport of secretory proteins, the exchange of macromolecules ...

Comparative leaf proteomics of drought-tolerant and -susceptible peanut in response to water stress

USDA-ARS?s Scientific Manuscript database

Water stress (WS) predisposes peanut plants to fungal infection resulting in pre-harvest aflatoxin contamination. Major changes during water stress including oxidative stress, lead to destruction of photosynthetic apparatus and other macromolecules within cells. Two peanut cultivars with diverse dro...

Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm.

PubMed

Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

2016-08-01

There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (D_{tot}) and the displacement distribution functions (P(r,t)) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ, which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ=0.65, while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ. We also investigate the distribution (P(θ,t)) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

Dynamics of highly polydisperse colloidal suspensions as a model system for bacterial cytoplasm

NASA Astrophysics Data System (ADS)

Hwang, Jiye; Kim, Jeongmin; Sung, Bong June

2016-08-01

There are various kinds of macromolecules in bacterial cell cytoplasm. The size polydispersity of the macromolecules is so significant that the crystallization and the phase separation could be suppressed, thus stabilizing the liquid state of bacterial cytoplasm. On the other hand, recent experiments suggested that the macromolecules in bacterial cytoplasm should exhibit glassy dynamics, which should be also affected significantly by the size polydispersity of the macromolecules. In this work, we investigate the anomalous and slow dynamics of highly polydisperse colloidal suspensions, of which size distribution is chosen to mimic Escherichia coli cytoplasm. We find from our Langevin dynamics simulations that the diffusion coefficient (Dtot) and the displacement distribution functions (P (r ,t ) ) averaged over all colloids of different sizes do not show anomalous and glassy dynamic behaviors until the system volume fraction ϕ is increased up to 0.82. This indicates that the intrinsic polydispersity of bacterial cytoplasm should suppress the glass transition and help maintain the liquid state of the cytoplasm. On the other hand, colloids of each kind show totally different dynamic behaviors depending on their size. The dynamics of colloids of different size becomes non-Gaussian at a different range of ϕ , which suggests that a multistep glass transition should occur. The largest colloids undergo the glass transition at ϕ =0.65 , while the glass transition does not occur for smaller colloids in our simulations even at the highest value of ϕ . We also investigate the distribution (P (θ ,t ) ) of the relative angles of displacement for macromolecules and find that macromolecules undergo directionally correlated motions in a sufficiently dense system.

Ligand Binding to Macromolecules: Allosteric and Sequential Models of Cooperativity.

ERIC Educational Resources Information Center

Hess, V. L.; Szabo, Attila

1979-01-01

A simple model is described for the binding of ligands to macromolecules. The model is applied to the cooperative binding by hemoglobin and aspartate transcarbamylase. The sequential and allosteric models of cooperative binding are considered. (BB)

Ocular delivery of macromolecules

PubMed Central

Kim, Yoo-Chun; Chiang, Bryce; Wu, Xianggen; Prausnitz, Mark R.

2014-01-01

Biopharmaceuticals are making increasing impact on medicine, including treatment of indications in the eye. Macromolecular drugs are typically given by physician-administered invasive delivery methods, because non--invasive ocular delivery methods, such as eye drops, and systemic delivery, have low bioavailability and/or poor ocular targeting. There is a need to improve delivery of biopharmaceuticals to enable less-invasive delivery routes, less-frequent dosing through controlled-release drug delivery and improved drug targeting within the eye to increase efficacy and reduce side effects. This review discusses the barriers to drug delivery via various ophthalmic routes of administration in the context of macromolecule delivery and discusses efforts to develop controlled-release systems for delivery of biopharmaceuticals to the eye. The growing number of macromolecular therapies in the eye needs improved drug delivery methods that increase drug efficacy, safety and patient compliance. PMID:24998941

Proteins--The Basis of Life

ERIC Educational Resources Information Center

Wrigley, Colin

2012-01-01

Proteins are a diverse class of biochemical macromolecules, including substances as (apparently) unrelated as silk and sinew, hair and horn, feathers and flagella, enzymes and epidermis, gelatine (jelly) and gluten and gore, spider web, meat and fish muscle. Yet they are unified by being polymers of amino acids. Discovery of the nature of proteins…

Transmembrane domain-dependent protein-protein interactions participate in the localization of GPAT enzymes to ER subdomains

USDA-ARS?s Scientific Manuscript database

The endoplasmic reticulum (ER) is a dynamic network that consists of numerous regions or subdomains with discrete morphological features and functional properties, including those involved in protein and oil-body formation, anterograde transport of secretory proteins, the exchange of macromolecules ...

Integrating Introductory Biology and General Chemistry Laboratories.

ERIC Educational Resources Information Center

Godrick, Elizabeth; Hartman, Standish

2000-01-01

Introduces a science laboratory integrating biology and chemistry courses that includes four modules: (1) the fundamental process of reactions; (2) a semester-long project on the chemical assay of ascorbic acid; (3) human metabolism of Vitamin C; and (4) an open-ended project on the manipulation of macromolecules. (YDS)

A Simple Endpoint Assay for Starch-Degrading Enzymes.

ERIC Educational Resources Information Center

Kroen, William K.

1998-01-01

Since many of the important energy-transferring pathways involve synthesis or degradation of biological macromolecules, observations of the enzymes responsible for starch breakdown provide a useful case study. Provides a short, one-step assay for the enzymes amylase and amyloglucosidase. Topics covered include goals, preparation, assay procedure,…

Species as Stressors: Heterospecific Interactions and the Cellular Stress Response under Global Change.

PubMed

Gunderson, Alex R; King, Emily E; Boyer, Kirsten; Tsukimura, Brian; Stillman, Jonathon H

2017-07-01

Anthropogenic global change is predicted to increase the physiological stress of organisms through changes in abiotic conditions such as temperature, pH, and pollution. However, organisms can also experience physiological stress through interactions with other species, especially parasites, predators, and competitors. The stress of species interactions could be an important driver of species' responses to global change as the composition of biological communities change through factors such as distributional and phenological shifts. Interactions between biotic and abiotic stressors could also induce non-linear physiological stress responses under global change. One of the primary means by which organisms deal with physiological stress is through the cellular stress response (CSR), which is broadly the upregulation of a conserved set of genes that facilitate the removal and repair of damaged macromolecules. Here, we present data on behavioral interactions and CSR gene expression for two competing species of intertidal zone porcelain crab (Petrolisthes cinctipes and Petrolisthes manimaculis). We found that P. cinctipes and P. manimaculis engage in more agonistic behaviors when interacting with heterospecifics than conspecifics; however, we found no evidence that heterospecific interactions induced a CSR in these species. In addition to our new data, we review the literature with respect to CSR induction via species interactions, focusing on predator-prey systems and heterospecific competition. We find extensive evidence for predators to induce cellular stress and aspects of the CSR in prey, even in the absence of direct physical contact between species. Effects of heterospecific competition on the CSR have been studied far less, but we do find evidence that agonistic interactions with heterospecifics can induce components of the CSR. Across all published studies, there is clear evidence that species interactions can lead to cellular stress and induction of the CSR. Nonetheless, our understanding of species-induced cellular stress lags far behind our understanding of abiotic cellular stress. © The Author 2017. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

Development for equipment of the milk macromolecules content detection

NASA Astrophysics Data System (ADS)

Ding, Guochao; Li, Weimin; Shang, Tingyi; Xi, Yang; Gao, Yunli; Zhou, Zhen

Developed an experimental device for rapid and accurate detection of milk macromolecular content. This device developed based on laser scattered through principle, the principle use of the ingredients of the scattered light and transmitted light ratio characterization of macromolecules. Peristaltic pump to achieve automatic input and output of the milk samples, designing weak signal detection amplifier circuit for detecting the ratio with ICL7650. Real-time operating system μC / OS-II is the core design of the software part of the whole system. The experimental data prove that the device can achieve a fast real-time measurement of milk macromolecules.

The spectral properties of DNA and RNA macromolecules at low temperatures: fundamental and applied aspects

NASA Astrophysics Data System (ADS)

Yashchuk, Valeriy M.; Kudrya, Vladislav Yu

2017-03-01

This paper summarizes the results of studies of the spectral properties—optical absorption, fluorescence and phosphorescence—of DNA and RNA macromolecules and synthetic poly-, oligo- and mono-nucleotides, which have been carried out in our laboratory. The system of first excited singlet and triplet energy levels for DNA and RNA is evaluated using low-temperature (4.2 K-77 K) luminescent measurements. The traps of the singlet and triplet electronic excitations in these compounds are identified. An important self-protection mechanism against photo-damage of DNA and RNA by UV photons or penetrative radiation based on the capture of triplet electronic-energy excitations by the most photostable centers—in DNA, the complex formed by neighboring adenosine (A) and thymidine (T) links; in RNA, the adenosine links—is described. It is confirmed that despite similarities in the chemical and partly energy structures DNA is more stable than RNA. The spectral manifestation of the telomeres (the important functional system) in DNA macromolecules is examined. The results obtained on telomere fragments provide the possibility of finding the configuration peculiarities of the triplet excitations traps in DNA macromolecules. The resulting spreading length of the migrating singlet (l s) and triplet (l t) excitations for DNA and RNA macromolecules are evaluated.

Cell-Permeable Parkin Proteins Suppress Parkinson Disease-Associated Phenotypes in Cultured Cells and Animals

PubMed Central

Duong, Tam; Kim, Jaetaek; Ruley, H. Earl; Jo, Daewoong

2014-01-01

Parkinson’s disease (PD) is a neurodegenerative disorder of complex etiology characterized by the selective loss of dopaminergic neurons, particularly in the substantia nigra. Parkin, a tightly regulated E3 ubiquitin ligase, promotes the survival of dopaminergic neurons in both PD and Parkinsonian syndromes induced by acute exposures to neurotoxic agents. The present study assessed the potential of cell-permeable parkin (CP-Parkin) as a neuroprotective agent. Cellular uptake and tissue penetration of recombinant, enzymatically active parkin was markedly enhanced by the addition of a hydrophobic macromolecule transduction domain (MTD). The resulting CP-Parkin proteins (HPM13 and PM10) suppressed dopaminergic neuronal toxicity in cells and mice exposed to 6-hydroxydopamine (6-OHDH) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These included enhanced survival and dopamine expression in cultured CATH.a and SH-SY5Y neuronal cells; and protection against MPTP-induced damage in mice, notably preservation of tyrosine hydroxylase-positive cells with enhanced dopamine expression in the striatum and midbrain, and preservation of gross motor function. These results demonstrate that CP-Parkin proteins can compensate for intrinsic limitations in the parkin response and provide a therapeutic strategy to augment parkin activity in vivo. PMID:25019626

Role of Polyamines in Asthma Pathophysiology

PubMed Central

2018-01-01

Asthma is a complex disease of airways, where the interactions of immune and structural cells result in disease outcomes with airway remodeling and airway hyper-responsiveness. Polyamines, which are small-sized, natural super-cations, interact with negatively charged intracellular macromolecules, and altered levels of polyamines and their interactions have been associated with different pathological conditions including asthma. Elevated levels of polyamines have been reported in the circulation of asthmatic patients as well as in the lungs of a murine model of asthma. In various studies, polyamines were found to potentiate the pathogenic potential of inflammatory cells, such as mast cells and granulocytes (eosinophils and neutrophils), by either inducing the release of their pro-inflammatory mediators or prolonging their life span. Additionally, polyamines were crucial in the differentiation and alternative activation of macrophages, which play an important role in asthma pathology. Importantly, polyamines cause airway smooth muscle contraction and thus airway hyper-responsiveness, which is the key feature in asthma pathophysiology. High levels of polyamines in asthma and their active cellular and macromolecular interactions indicate the importance of the polyamine pathway in asthma pathogenesis; therefore, modulation of polyamine levels could be a suitable approach in acute and severe asthma management. This review summarizes the possible roles of polyamines in different pathophysiological features of asthma. PMID:29316647

The chemistry side of AOP: implications for toxicity ...

EPA Pesticide Factsheets

An adverse outcome pathway (AOP) is a structured representation of the biological events that lead to adverse impacts following a molecular initiating event caused by chemical interaction with a macromolecule. AOPs have been proposed to facilitate toxicity extrapolation across species through understanding of species similarity in the sequence of molecular, cellular, organ and organismal level responses. However, AOPs are non-specific regarding the identity of the chemical initiators, and the range of structures for which an AOP is considered applicable has generally been poorly defined. Applicability domain has been widely understood in the field of QSAR as the response and chemical structure space in which the model makes predictions with a given reliability, and has been traditionally applied to define the similarity of query molecules within the training set. Three dimensional (3D) receptor modeling offers an approach to better define the applicability domain for selected AOPs through determination of the chemical space of the molecular initiating event. Universal 3D-QSAR models were developed for acetylcholinesterase inhibitors and estrogen receptor agonists and antagonists using a combination of fingerprint, molecular docking and structure-based pharmacophore approaches. The models were based on the critical molecular interactions within each receptor ligand binding domain, and included the key amino acid residues responsible for high binding affinity. T

Updating the mitochondrial free radical theory of aging: an integrated view, key aspects, and confounding concepts.

PubMed

Barja, Gustavo

2013-10-20

An updated version of the mitochondrial free radical theory of aging (MFRTA) and longevity is reviewed. Key aspects of the theory are emphasized. Another main focus concerns common misconceptions that can mislead investigators from other specialties, even to wrongly discard the theory. Those different issues include (i) the main reactive oxygen species (ROS)-generating site in the respiratory chain in relation to aging and longevity: complex I; (ii) the close vicinity or even contact between that site and the mitochondrial DNA, in relation to the lack of local efficacy of antioxidants and to sub-cellular compartmentation; (iii) the relationship between mitochondrial ROS production and oxygen consumption; (iv) recent criticisms on the MFRTA; (v) the widespread assumption that ROS are simple "by-products" of the mitochondrial respiratory chain; (vi) the unnecessary postulation of "vicious cycle" hypotheses of mitochondrial ROS generation which are not central to the free radical theory of aging; and (vii) the role of DNA repair concerning endogenous versus exogenous damage. After considering the large body of data already available, two general characteristics responsible for the high maintenance degree of long-lived animals emerge: (i) a low generation rate of endogenous damage: and (ii) the possession of tissue macromolecules that are highly resistant to oxidative modification.

Photodegradable, Photoadaptable Hydrogels via Radical-Mediated Disulfide Fragmentation Reaction.

PubMed

Fairbanks, Benjamin D; Singh, Samir P; Bowman, Christopher N; Anseth, Kristi S

2011-04-26

Various techniques have been adopted to impart a biological responsiveness to synthetic hydrogels for the delivery of therapeutic agents as well as the study and manipulation of biological processes and tissue development. Such techniques and materials include polyelectrolyte gels that swell and deswell with changes in pH, thermosensitive gels that contract at physiological temperatures, and peptide cross-linked hydrogels that degrade upon peptidolysis by cell-secreted enzymes. Herein we report a unique approach to photochemically deform and degrade disulfide cross-linked hydrogels, mitigating the challenges of light attenuation and low quantum yield, permitting the degradation of hydrogels up to 2 mm thick within 120 s at low light intensities (10 mW/cm(2) at 365 nm). Hydrogels were formed by the oxidation of thiol-functionalized 4-armed poly(ethylene glycol) macromolecules. These disulfide cross-linked hydrogels were then swollen in a lithium acylphosphinate photoinitiator solution. Upon exposure to light, photogenerated radicals initiate multiple fragmentation and disulfide exchange reactions, permitting and promoting photodeformation, photowelding, and photodegradation. This novel, but simple, approach to generate photoadaptable hydrogels portends the study of cellular response to mechanically and topographically dynamic substrates as well as novel encapsulations by the welding of solid substrates. The principles and techniques described herein hold implications for more than hydrogel materials but also for photoadaptable polymers more generally.

Investigation of gene expressions in differentiated cell derived bone marrow stem cells during bone morphogenetic protein-4 treatments with Fourier transform infrared spectroscopy

NASA Astrophysics Data System (ADS)

Zafari, Jaber; Jouni, Fatemeh Javani; Ahmadvand, Ali; Abdolmaleki, Parviz; Soodi, Malihe; Zendehdel, Rezvan

2017-02-01

A model was set up to predict the differentiation patterns based on the data extracted from FTIR spectroscopy. For this reason, bone marrow stem cells (BMSCs) were differentiated to primordial germ cells (PGCs). Changes in cellular macromolecules in the time of 0, 24, 48, 72, and 96 h of differentiation, as different steps of the differentiation procedure were investigated by using FTIR spectroscopy. Also, the expression of pluripotency (Oct-4, Nanog and c-Myc) and specific genes (Mvh, Stella and Fragilis) were investigated by real-time PCR. However, the expression of genes in five steps of differentiation was predicted by FTIR spectroscopy. FTIR spectra showed changes in the template of band intensities at different differentiation steps. There are increasing changes in the stepwise differentiation procedure for the ratio area of CH2, which is symmetric to CH2 asymmetric stretching. An ensemble of expert methods, including regression tree (RT), boosting algorithm (BA), and generalized regression neural network (GRNN), was the best method to predict the gene expression by FTIR spectroscopy. In conclusion, the model was able to distinguish the pattern of different steps from cell differentiation by using some useful features extracted from FTIR spectra.

Using whole-exome sequencing to investigate the genetic bases of lysosomal storage diseases of unknown etiology.

PubMed

Wang, Nan; Zhang, Yeting; Gedvilaite, Erika; Loh, Jui Wan; Lin, Timothy; Liu, Xiuping; Liu, Chang-Gong; Kumar, Dibyendu; Donnelly, Robert; Raymond, Kimiyo; Schuchman, Edward H; Sleat, David E; Lobel, Peter; Xing, Jinchuan

2017-11-01

Lysosomes are membrane-bound, acidic eukaryotic cellular organelles that play important roles in the degradation of macromolecules. Mutations that cause the loss of lysosomal protein function can lead to a group of disorders categorized as the lysosomal storage diseases (LSDs). Suspicion of LSD is frequently based on clinical and pathologic findings, but in some cases, the underlying genetic and biochemical defects remain unknown. Here, we performed whole-exome sequencing (WES) on 14 suspected LSD cases to evaluate the feasibility of using WES for identifying causal mutations. By examining 2,157 candidate genes potentially associated with lysosomal function, we identified eight variants in five genes as candidate disease-causing variants in four individuals. These included both known and novel mutations. Variants were corroborated by targeted sequencing and, when possible, functional assays. In addition, we identified nonsense mutations in two individuals in genes that are not known to have lysosomal function. However, mutations in these genes could have resulted in phenotypes that were diagnosed as LSDs. This study demonstrates that WES can be used to identify causal mutations in suspected LSD cases. We also demonstrate cases where a confounding clinical phenotype may potentially reflect more than one lysosomal protein defect. © 2017 Wiley Periodicals, Inc.

Phylogenetic Characterization of Transport Protein Superfamilies: Superiority of SuperfamilyTree Programs over Those Based on Multiple Alignments

PubMed Central

Chen, Jonathan S.; Reddy, Vamsee; Chen, Joshua H.; Shlykov, Maksim A.; Zheng, Wei Hao; Cho, Jaehoon; Yen, Ming Ren; Saier, Milton H.

2012-01-01

Transport proteins function in the translocation of ions, solutes and macromolecules across cellular and organellar membranes. These integral membrane proteins fall into >600 families as tabulated in the Transporter Classification Database (www.tcdb.org). Recent studies, some of which are reported here, define distant phylogenetic relationships between families with the creation of superfamilies. Several of these are analyzed using a novel set of programs designed to allow reliable prediction of phylogenetic trees when sequence divergence is too great to allow the use of multiple alignments. These new programs, called SuperfamilyTree1 and 2 (SFT1 and 2), allow display of protein and family relationships, respectively, based on thousands of comparative BLAST scores rather than multiple alignments. Superfamilies analyzed include: (1) Aerolysins, (2) RTX Toxins, (3) Defensins, (4) Ion Transporters, (5) Bile/Arsenite/Riboflavin Transporters, (6) Cation: Proton Antiporters, and (7) the Glucose/Fructose/Lactose superfamily within the prokaryotic phosphoenol pyruvate-dependent Phosphotransferase System. In addition to defining the phylogenetic relationships of the proteins and families within these seven superfamilies, evidence is provided showing that the SFT programs outperform programs that are based on multiple alignments whenever sequence divergence of superfamily members is extensive. The SFT programs should be applicable to virtually any superfamily of proteins or nucleic acids. PMID:22286036

An assessment of environmental and toxicological risk to pesticide exposure based on a case-based approach to computing

NASA Astrophysics Data System (ADS)

Coelho, Cristina; Vicente, Henrique; Rosário Martins, M.; Lima, Nelson; Neves, Mariana; Neves, José

2017-01-01

Pesticide environmental fate and toxicity depends on its physical and chemical features, the soil composition, soil adsorption, as well as residues that may be found in different soil slots. Indeed, pesticide degradation in soil may be influenced by either biotic or abiotic factors. In addition, the toxicity of pesticides for living organisms depends on their adsorption, distribution, biotransformation, dissemination of metabolites together with interaction with cellular macromolecules and excretion. Biotransformation may result in the formation of less toxic and/or more toxic metabolites, while other processes determine the balance between toxic and a nontoxic upcoming. Aggregate exposure and risk assessment involve multiple pathways and routes, including the potential for pesticide residues in food and drinking water, in addition to residues from pesticide use in residential and non-occupational environments. Therefore, this work will focus on the development of a decision support system to assess the environmental and toxicological risk to pesticide exposure, built on top of a Logic Programming approach to Knowledge Representation and Reasoning, complemented with a Case Based attitude to computing. The proposed solution is unique in itself, once it caters for the explicit treatment of incomplete, unknown, or even self-contradictory information, either in terms of a qualitative or quantitative setting.

Assembly constraints drive co-evolution among ribosomal constituents.

PubMed

Mallik, Saurav; Akashi, Hiroshi; Kundu, Sudip

2015-06-23

Ribosome biogenesis, a central and essential cellular process, occurs through sequential association and mutual co-folding of protein-RNA constituents in a well-defined assembly pathway. Here, we construct a network of co-evolving nucleotide/amino acid residues within the ribosome and demonstrate that assembly constraints are strong predictors of co-evolutionary patterns. Predictors of co-evolution include a wide spectrum of structural reconstitution events, such as cooperativity phenomenon, protein-induced rRNA reconstitutions, molecular packing of different rRNA domains, protein-rRNA recognition, etc. A correlation between folding rate of small globular proteins and their topological features is known. We have introduced an analogous topological characteristic for co-evolutionary network of ribosome, which allows us to differentiate between rRNA regions subjected to rapid reconstitutions from those hindered by kinetic traps. Furthermore, co-evolutionary patterns provide a biological basis for deleterious mutation sites and further allow prediction of potential antibiotic targeting sites. Understanding assembly pathways of multicomponent macromolecules remains a key challenge in biophysics. Our study provides a 'proof of concept' that directly relates co-evolution to biophysical interactions during multicomponent assembly and suggests predictive power to identify candidates for critical functional interactions as well as for assembly-blocking antibiotic target sites. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Evaluation of cytogenetic and DNA damage in human lymphocytes treated with adrenaline in vitro.

PubMed

Djelić, Ninoslav; Radaković, Milena; Spremo-Potparević, Biljana; Zivković, Lada; Bajić, Vladan; Stevanović, Jevrosima; Stanimirović, Zoran

2015-02-01

Catechol groups can be involved in redox cycling accompanied by generation of reactive oxygen species (ROS) which may lead to oxidative damage of cellular macromolecules including DNA. The objective of this investigation was to evaluate possible genotoxic effects of a natural catecholamine adrenaline in cultured human lymphocytes using cytogenetic (sister chromatid exchange and micronuclei) and the single cell gel electrophoresis (Comet) assay. In cytogenetic tests, six experimental concentrations of adrenaline were used in a range from 0.01-500 μM. There were no indications of genotoxic effects of adrenaline in sister chromatid exchange and micronucleus tests. However, at four highest concentrations of adrenaline (5 μM, 50 μM, 150 μM and 300 μM) we observed a decreased mitotic index and cell-cycle delay. In addition, in the Comet assay we used adrenaline in a range from 0.0005-500 μM, at two treatment times: 15 min or 60 min. In contrast to cytogenetic analysis, there was a dose-dependent increase of DNA damage detected in the Comet assay. These effects were significantly reduced by concomitant treatment with quercetin or catalase. Therefore, the obtained results indicate that adrenaline may exhibit genotoxic effects in cultured human lymphocytes, most likely due to production of reactive oxygen species. Copyright © 2014 Elsevier Ltd. All rights reserved.

Methods and compositions for targeting macromolecules into the nucleus

DOEpatents

Chook, Yuh Min

2013-06-25

The present invention includes compositions, methods and kits for directing an agent across the nuclear membrane of a cell. The present invention includes a Karyopherin beta2 translocation motif in a polypeptide having a slightly positively charged region or a slightly hydrophobic region and one or more R/K/H-X.sub.(2-5)-P-Y motifs. The polypeptide targets the agent into the cell nucleus.

Cell-to-cell communication in plants, animals, and fungi: a comparative review.

PubMed

Bloemendal, Sandra; Kück, Ulrich

2013-01-01

Cell-to-cell communication is a prerequisite for differentiation and development in multicellular organisms. This communication has to be tightly regulated to ensure that cellular components such as organelles, macromolecules, hormones, or viruses leave the cell in a precisely organized way. During evolution, plants, animals, and fungi have developed similar ways of responding to this biological challenge. For example, in higher plants, plasmodesmata connect adjacent cells and allow communication to regulate differentiation and development. In animals, two main general structures that enable short- and long-range intercellular communication are known, namely gap junctions and tunneling nanotubes, respectively. Finally, filamentous fungi have also developed specialized structures called septal pores that allow intercellular communication via cytoplasmic flow. This review summarizes the underlying mechanisms for intercellular communication in these three eukaryotic groups and discusses its consequences for the regulation of differentiation and developmental processes.

Lysosomes as Oxidative Targets for Cancer Therapy.

PubMed

Dielschneider, Rebecca F; Henson, Elizabeth S; Gibson, Spencer B

2017-01-01

Lysosomes are membrane-bound vesicles that contain hydrolases for the degradation and recycling of essential nutrients to maintain homeostasis within cells. Cancer cells have increased lysosomal function to proliferate, metabolize, and adapt to stressful environments. This has made cancer cells susceptible to lysosomal membrane permeabilization (LMP). There are many factors that mediate LMP such as Bcl-2 family member, p53; sphingosine; and oxidative stress which are often altered in cancer. Upon lysosomal disruption, reactive oxygen species (ROS) levels increase leading to lipid peroxidation, mitochondrial dysfunction, autophagy, and reactive iron. Cathepsins are also released causing degradation of macromolecules and cellular structures. This ultimately kills the cancer cell through different types of cell death (apoptosis, autosis, or ferroptosis). In this review, we will explore the contributions lysosomes play in inducing cell death, how this is regulated by ROS in cancer, and how lysosomotropic agents might be utilized to treat cancers.

Proanthocyanidins against Oxidative Stress: From Molecular Mechanisms to Clinical Applications

PubMed Central

Xiong, Xia; Lai, Rui

2018-01-01

Proanthocyanidins (PCs) are naturally occurring polyphenolic compounds abundant in many vegetables, plant skins (rind/bark), seeds, flowers, fruits, and nuts. Numerous in vitro and in vivo studies have demonstrated myriad effects potentially beneficial to human health, such as antioxidation, anti-inflammation, immunomodulation, DNA repair, and antitumor activity. Accumulation of prooxidants such as reactive oxygen species (ROS) exceeding cellular antioxidant capacity results in oxidative stress (OS), which can damage macromolecules (DNA, lipids, and proteins), organelles (membranes and mitochondria), and whole tissues. OS is implicated in the pathogenesis and exacerbation of many cardiovascular, neurodegenerative, dermatological, and metabolic diseases, both through direct molecular damage and secondary activation of stress-associated signaling pathways. PCs are promising natural agents to safely prevent acute damage and control chronic diseases at relatively low cost. In this review, we summarize the molecules and signaling pathways involved in OS and the corresponding therapeutic mechanisms of PCs. PMID:29750172

Quantitative mass imaging of single biological macromolecules.

PubMed

Young, Gavin; Hundt, Nikolas; Cole, Daniel; Fineberg, Adam; Andrecka, Joanna; Tyler, Andrew; Olerinyova, Anna; Ansari, Ayla; Marklund, Erik G; Collier, Miranda P; Chandler, Shane A; Tkachenko, Olga; Allen, Joel; Crispin, Max; Billington, Neil; Takagi, Yasuharu; Sellers, James R; Eichmann, Cédric; Selenko, Philipp; Frey, Lukas; Riek, Roland; Galpin, Martin R; Struwe, Weston B; Benesch, Justin L P; Kukura, Philipp

2018-04-27

The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Histone deacetylase 10 structure and molecular function as a polyamine deacetylase

NASA Astrophysics Data System (ADS)

Hai, Yang; Shinsky, Stephen A.; Porter, Nicholas J.; Christianson, David W.

2017-05-01

Cationic polyamines such as spermidine and spermine are critical in all forms of life, as they regulate the function of biological macromolecules. Intracellular polyamine metabolism is regulated by reversible acetylation and dysregulated polyamine metabolism is associated with neoplastic diseases such as colon cancer, prostate cancer and neuroblastoma. Here we report that histone deacetylase 10 (HDAC10) is a robust polyamine deacetylase, using recombinant enzymes from Homo sapiens (human) and Danio rerio (zebrafish). The 2.85 Å-resolution crystal structure of zebrafish HDAC10 complexed with a transition-state analogue inhibitor reveals that a glutamate gatekeeper and a sterically constricted active site confer specificity for N8-acetylspermidine hydrolysis and disfavour acetyllysine hydrolysis. Both HDAC10 and spermidine are known to promote cellular survival through autophagy. Accordingly, this work sets a foundation for studying the chemical biology of autophagy through the structure-based design of inhibitors that may also serve as new leads for cancer chemotherapy.

A balancing act for autophagin.

PubMed

Till, Andreas; Subramani, Suresh

2010-07-01

Autophagy is a tightly regulated catabolic process whereby cells degrade their constituents to dispose of unwanted cytoplasmic elements and recycle nutrients for cellular remodeling. Studies of autophagy in mammals have elicited substantial interest because it is linked to a range of physiologic and pathologic states. In this issue of the JCI, Mariño et al. uncover a role for autophagy in a balance disorder related to inner ear pathologies. Mice lacking the protease autophagy-related 4B (Atg4b, also known as autophagin-1) exhibited a systemic reduction in autophagy and showed defects in the development of otoconia, organic particles that contain calcium carbonate crystals and proteins and that are essential for balance perception (equilibrioception) in mammals. The intriguing aspect of this work is that an autophagy block impairs the secretion and assembly of otoconial proteins, emphasizing a role for autophagy in functions distinct from macromolecule degradation.

Cell-to-cell communication in plants, animals, and fungi: a comparative review

NASA Astrophysics Data System (ADS)

Bloemendal, Sandra; Kück, Ulrich

2013-01-01

Cell-to-cell communication is a prerequisite for differentiation and development in multicellular organisms. This communication has to be tightly regulated to ensure that cellular components such as organelles, macromolecules, hormones, or viruses leave the cell in a precisely organized way. During evolution, plants, animals, and fungi have developed similar ways of responding to this biological challenge. For example, in higher plants, plasmodesmata connect adjacent cells and allow communication to regulate differentiation and development. In animals, two main general structures that enable short- and long-range intercellular communication are known, namely gap junctions and tunneling nanotubes, respectively. Finally, filamentous fungi have also developed specialized structures called septal pores that allow intercellular communication via cytoplasmic flow. This review summarizes the underlying mechanisms for intercellular communication in these three eukaryotic groups and discusses its consequences for the regulation of differentiation and developmental processes.

Cells producing their own nemesis: understanding methylglyoxal metabolism.

PubMed

Chakraborty, Sangeeta; Karmakar, Kapudeep; Chakravortty, Dipshikha

2014-10-01

Methylglyoxal, which is technically known as 2-oxopropanal or pyruvaldehyde, shows typical reactions of carbonyl compounds as it has both an aldehyde and a ketone functional group. It is an extremely cytotoxic physiological metabolite, which is generated by both enzymatic and nonenzymatic reactions. The deleterious nature of the compound is due to its ability to glycate and crosslink macromolecules like protein and DNA, respectively. However, despite having toxic effects on cellular processes, methylglyoxal retains its efficacy as an anticancer drug. Indeed, methylglyoxal is one of the well-known anticancer therapeutic agents used in the treatment. Several studies on methylglyoxal biology revolve around the manifestations of its inhibitory effects and toxicity in microbial growth and diabetic complications, respectively. Here, we have revisited the chronology of methylglyoxal research with emphasis on metabolism of methylglyoxal and implications of methylglyoxal production or detoxification on bacterial pathogenesis and disease progression. © 2014 International Union of Biochemistry and Molecular Biology.

The stratified syncytium of the vertebrate lens.

PubMed

Shi, Yanrong; Barton, Kelly; De Maria, Alicia; Petrash, J Mark; Shiels, Alan; Bassnett, Steven

2009-05-15

The fusion of cells to generate syncytial tissues is a crucial event in the development of many organisms. In the lens of the vertebrate eye, proteins and other macromolecules diffuse from cell to cell via the large molecule diffusion pathway (LMDP). We used the tamoxifen-induced expression of GFP to investigate the nature and role of the LMDP in living, intact lenses. Our data indicate that the LMPD preferentially connects cells lying within a stratum of the lens cortex and that formation of the LMPD depends on the expression of Lim2, a claudin-like molecule. The conduits for intercellular protein exchange are most likely regions of partial cellular fusion, which are commonly observed in wild-type lenses but rare or absent in Lim2-deficient lenses. The observation that lens tissue constitutes a stratified syncytium has implications for the transparency, refractive function and pathophysiology of the tissue.

Tumor exosomes: cellular postmen of cancer diagnosis and personalized therapy.

PubMed

Sharma, Aman; Khatun, Zamila; Shiras, Anjali

2016-02-01

Nanosized (30-150 nm) extracellular vesicles 'exosomes' are secreted by cells for intercellular communication during normal and pathological conditions. Exosomes carry biomacromolecules from cell-of-origin and, therefore, represent molecular bioprint of the cell. Tumor-derived exosomes or TDEx modulate tumor microenvironment by transfer of macromolecules locally as well as at distant metastatic sites. Due to their biological stability, TDEx are rich source of biomarkers in cancer patients. TDEx focused cancer diagnosis allows liquid biopsy-based tumor typing and may facilitate therapy response monitoring by developing novel exosomes diagnostics. Therefore, efficient and specific capturing of exosomes for subsequent amplification of the biomessages; for example, DNA, RNA, miRNA can reinvent cancer diagnosis. Here, in this review, we discuss advancements in exosomes isolation strategies, presence of exosomes biomarkers and importance of TDEx in gauging tumor heterogeneity for their potential use in cancer diagnosis, therapy.

Zymographic Method for Distinguishing Different Classes of Superoxide Dismutases in Plants.

PubMed

Jamdhade, Ashwini R; Sunkar, Ramanjulu; Hivrale, Vandana K

2017-01-01

In plants, especially in chloroplasts, superoxide radical is generated when an electron is transferred to dimolecular O 2 due to decreased activity of Photosystem I. The superoxide (O 2 - ) radical accumulation is more rampant in plants exposed to abiotic stresses due to oxidation of photosystem components. Excessive superoxide radical accumulation will lead to oxidative damage to the cellular macromolecules. The ubiquitous superoxide dismutases (SODs) represent critical enzymatic antioxidant system present in cells, which can catalyze the disproportion of superoxide (O 2 - ) radical rapidly into hydrogen peroxide (H 2 O 2 ) and molecular oxygen. Depending on the metal cofactor present, the plant SODs are classified into Cu/ZnSOD, MnSOD, and FeSOD. The activity of SODs can be quantified zymographically. Additionally, using this method, different classes of SODs can be distinguished by using H 2 O 2 , KCN, and NaN 3.

The effects of two different ganoderma species (Lingzhi) on gene expression in human monocytic THP-1 cells.

PubMed

Cheng, Chun-Huai; Leung, Albert Y; Chen, Chin-Fu

2010-01-01

Lingzhi (ganoderma) is an important woody mushroom that is known for its medicinal benefits in China since ancient times. The mode of action in humans is still not clear. Using microarray technology, we have compared the ethanol extracts of two different lingzhi (red lingzhi, G. lucidum; and purple lingzhi, G. sinense) for their effects on gene expression profile in human monocytic cells. Our results suggest that at best approximately 25% of target genes are common to the two lingzhi: functionally ranging from cell development, negative regulation of cellular process, and cellular protein metabolic process to signal transduction and transcription. The pathways mediated by purple lingzhi focus on inflammation and immune response, whereas red lingzhi modestly increases levels of expression for genes involved in macromolecule metabolism. Furthermore, our ethanolic extracts of both red and purple lingzhi do not inhibit monocytic cell growth. The extract of red lingzhi does not have significant effect on the genes in the nuclear factor kappa B (NFkappaB) pathway (an important inflammation pathway), whereas the extract of purple lingzhi can increase multiple key genes in the NFkappaB pathway. Altogether, our results suggest that the common mode of action for lingzhi is complex; and different species of Ganoderma can modulate different pathways in human cells.

Disease models for the development of therapies for lysosomal storage diseases.

PubMed

Xu, Miao; Motabar, Omid; Ferrer, Marc; Marugan, Juan J; Zheng, Wei; Ottinger, Elizabeth A

2016-05-01

Lysosomal storage diseases (LSDs) are a group of rare diseases in which the function of the lysosome is disrupted by the accumulation of macromolecules. The complexity underlying the pathogenesis of LSDs and the small, often pediatric, population of patients make the development of therapies for these diseases challenging. Current treatments are only available for a small subset of LSDs and have not been effective at treating neurological symptoms. Disease-relevant cellular and animal models with high clinical predictability are critical for the discovery and development of new treatments for LSDs. In this paper, we review how LSD patient primary cells and induced pluripotent stem cell-derived cellular models are providing novel assay systems in which phenotypes are more similar to those of the human LSD physiology. Furthermore, larger animal disease models are providing additional tools for evaluation of the efficacy of drug candidates. Early predictors of efficacy and better understanding of disease biology can significantly affect the translational process by focusing efforts on those therapies with the higher probability of success, thus decreasing overall time and cost spent in clinical development and increasing the overall positive outcomes in clinical trials. © 2016 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

Modeling of Fluid-Membrane Interaction in Cellular Microinjection Process

NASA Astrophysics Data System (ADS)

Karzar-Jeddi, Mehdi; Diaz, Jhon; Olgac, Nejat; Fan, Tai-Hsi

2009-11-01

Cellular microinjection is a well-accepted method to deliver matters such as sperm, nucleus, or macromolecules into biological cells. To improve the success rate of in vitro fertilization and to establish the ideal operating conditions for a novel computer controlled rotationally oscillating intracytoplasmic sperm injection (ICSI) technology, we investigate the fluid-membrane interactions in the ICSI procedure. The procedure consists of anchoring the oocyte (a developing egg) using a holding pipette, penetrating oocyte's zona pellucida (the outer membrane) and the oolemma (the plasma or inner membrane) using an injection micropipette, and finally to deliver sperm into the oocyte for fertilization. To predict the large deformation of the oocyte membranes up to the piercing of the oolemma and the motion of fluids across both membranes, the dynamic fluid-pipette-membrane interactions are formulated by the coupled Stokes' equations and the continuum membrane model based on Helfrich's energy theory. A boundary integral model is developed to simulate the transient membrane deformation and the local membrane stress induced by the longitudinal motion of the injection pipette. The model captures the essential features of the membranes shown on optical images of ICSI experiments, and is capable of suggesting the optimal deformation level of the oolemma to start the rotational oscillations for piercing into the oolemma.

Effect of posttranslational modifications on enzyme function and assembly.

PubMed

Ryšlavá, Helena; Doubnerová, Veronika; Kavan, Daniel; Vaněk, Ondřej

2013-10-30

The detailed examination of enzyme molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Recently, global analyses of enzymes using methods of contemporary proteomics revealed widespread distribution of PTMs on many key enzymes distributed in all cellular compartments. Critically, patterns of multiple enzymatic and nonenzymatic PTMs within a single enzyme are now functionally evaluated providing a holistic picture of a macromolecule interacting with low molecular mass compounds, some of them being substrates, enzyme regulators, or activated precursors for enzymatic and nonenzymatic PTMs. Multiple PTMs within a single enzyme molecule and their mutual interplays are critical for the regulation of catalytic activity. Full understanding of this regulation will require detailed structural investigation of enzymes, their structural analogs, and their complexes. Further, proteomics is now integrated with molecular genetics, transcriptomics, and other areas leading to systems biology strategies. These allow the functional interrogation of complex enzymatic networks in their natural environment. In the future, one might envisage the use of robust high throughput analytical techniques that will be able to detect multiple PTMs on a global scale of individual proteomes from a number of carefully selected cells and cellular compartments. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Copyright © 2013 Elsevier B.V. All rights reserved.

How do bacteria localize proteins to the cell pole?

PubMed Central

Laloux, Géraldine; Jacobs-Wagner, Christine

2014-01-01

ABSTRACT It is now well appreciated that bacterial cells are highly organized, which is far from the initial concept that they are merely bags of randomly distributed macromolecules and chemicals. Central to their spatial organization is the precise positioning of certain proteins in subcellular domains of the cell. In particular, the cell poles – the ends of rod-shaped cells – constitute important platforms for cellular regulation that underlie processes as essential as cell cycle progression, cellular differentiation, virulence, chemotaxis and growth of appendages. Thus, understanding how the polar localization of specific proteins is achieved and regulated is a crucial question in bacterial cell biology. Often, polarly localized proteins are recruited to the poles through their interaction with other proteins or protein complexes that were already located there, in a so-called diffusion-and-capture mechanism. Bacteria are also starting to reveal their secrets on how the initial pole ‘recognition’ can occur and how this event can be regulated to generate dynamic, reproducible patterns in time (for example, during the cell cycle) and space (for example, at a specific cell pole). Here, we review the major mechanisms that have been described in the literature, with an emphasis on the self-organizing principles. We also present regulation strategies adopted by bacterial cells to obtain complex spatiotemporal patterns of protein localization. PMID:24345373

Shape transitions during clathrin-induced endocytosis

NASA Astrophysics Data System (ADS)

Kumar, Gaurav; Sain, Anirban

2016-12-01

Endocytosis is among the most common transport mechanisms which cells employ to receive macromolecules, the so-called cargo, from its extra cellular environment. Clathrin-mediated endocytosis (CME), in particular, involves the cytoplasmic protein clathrin which induces formation and internalization of clathrin-coated membrane buds that contain extra-cellular cargo. Decades of experimental work have established that the morphology of the clathrin coat evolves with time and induces its curvature on the membrane bud; but energetics of the process remain unclear. Recent experiments by Avinoam et al. [Science 348, 1369 (2015), 10.1126/science.aaa9555] reported that the area of the clathrin coat remains fixed while its curvature increases with time and also the clathrin molecules in the coat turn over rapidly. We show that these observations challenge existing models of coated membrane bud formation. We analyze their data to bring out certain features consistent with the underlying lattice structure of the coat. We hypothesize that membrane curvature inhibits clathrin deposition and propose a kinetic model that explains the area distribution of clathrin coats. We also show that their data on shape evolution of the coated membrane bud can be approximately understood from simple geometric considerations. However, the energetics of the coat formation which controls the kinetics of the process remains a puzzle.

Yeast as a model system to study metabolic impact of selenium compounds

PubMed Central

Herrero, Enrique; Wellinger, Ralf E.

2015-01-01

Inorganic Se forms such as selenate or selenite (the two more abundant forms in nature) can be toxic in Saccharomyces cerevisiae cells, which constitute an adequate model to study such toxicity at the molecular level and the functions participating in protection against Se compounds. Those Se forms enter the yeast cell through other oxyanion transporters. Once inside the cell, inorganic Se forms may be converted into selenide through a reductive pathway that in physiological conditions involves reduced glutathione with its consequent oxidation into diglutathione and alteration of the cellular redox buffering capacity. Selenide can subsequently be converted by molecular oxygen into elemental Se, with production of superoxide anions and other reactive oxygen species. Overall, these events result in DNA damage and dose-dependent reversible or irreversible protein oxidation, although additional oxidation of other cellular macromolecules cannot be discarded. Stress-adaptation pathways are essential for efficient Se detoxification, while activation of DNA damage checkpoint and repair pathways protects against Se-mediated genotoxicity. We propose that yeast may be used to improve our knowledge on the impact of Se on metal homeostasis, the identification of Se-targets at the DNA and protein levels, and to gain more insights into the mechanism of Se-mediated apoptosis. PMID:28357286

COMPUTATIONAL METHODS FOR STUDYING THE INTERACTION BETWEEN POLYCYCLIC AROMATIC HYDROCARBONS AND BIOLOGICAL MACROMOLECULES

EPA Science Inventory

Computational Methods for Studying the Interaction between Polycyclic Aromatic Hydrocarbons and Biological Macromolecules .

The mechanisms for the processes that result in significant biological activity of PAHs depend on the interaction of these molecules or their metabol...

MOLECULAR BIOLOGY OF PHARMACOLOGIC VITREOLYSIS

PubMed Central

Sebag, J

2005-01-01

Purpose Pharmacologic vitreolysis is a promising new therapy to improve vitreoretinal surgery and, ultimately, prevent disease by mitigating the contribution of vitreous to retinopathy. The mechanism of action of the agents being developed for pharmacologic vitreolysis remains unclear. The experiments in this thesis test the hypothesis that pharmacologic vitreolysis agents break down vitreous macromolecules into smaller particles. Methods Microplasmin, hyaluronidase, and collagenase were tested in solutions of hyaluronan (n = 15) and collagen (n = 15), explants of bovine vitreous (n = 15), dissected porcine vitreous (n = 9), and intact porcine eyes (n = 18). There were also 21 controls, totaling 93 specimens. Vitreous macromolecule sizes were determined with dynamic light scattering (DLS), performed at intervals from 10 minutes to 24 hours following injections. Results Studies of DLS reproducibility showed a coefficient of variance of less than 3.3% in all but one specimen. Microplasmin decreased porcine vitreous macromolecule size in a dose-dependent manner (correlation coefficient r = 0.93), with an 85% reduction after a 30-minute exposure to the maximum dose. Hyaluronidase decreased vitreous macromolecule size in hyaluronan solutions by 50% at high (1,000 IU/mL, P < .001) doses and in bovine vitreous by 20%. Collagenase decreased macromolecule size in collagen solutions by 20% at both low (1 mg/mL, P < .001) and high (10 mg/mL, P < .001) doses, but not at all in bovine vitreous. Conclusions Pharmacologic vitreolysis can induce a significant decrease in vitreous macromolecule sizes, depending upon the pharmacologic agents and the experimental model. Broad-spectrum agents were more effective than substrate-specific enzymes. Defining the molecular biology of pharmacologic vitreolysis has implications for surgical developments and may impact upon the design of clinical trials to induce prophylactic posterior vitreous detachment. PMID:17057814

Is it Possible to have the Similar Unit Cell in Crystals of Different form from the same Macromolecule? (A Case Study of Ribosome Crystals)

NASA Technical Reports Server (NTRS)

Karpova, E. A.; Rose, M. Franklin (Technical Monitor)

2000-01-01

Three different types of ribosome crystals were grown by the vapor diffusion technique in hanging drops as described in (1,2). The ribosome is a large asymmetric RNA-protein complex (2.3 million Da), which is protein syntheses machinery of the cell. In this poster we would like to discuss the features of ribosome crystallization. Ribosomes were purified from the thermophilic bacteria Thermus thermophilus by centrifugation (3). Three types of crystals (needle, flat tetragonal and tetragonal-like pyramid) can be grown from the same solution; furthermore, in the same drop using 10-15% 2-methyl-2,4- pentanediol as a precipitant. The crystals appeared in 5-48 hours. The crystals were stable and can co-exist in solution over long period of time. The kinetics of appearance of different crystal forms was different: first the needle crystals were grown, then the tetragonal, and finally the tetragonal pyramids. Later studies of the process of ribosome crystal growth depending on supersaturation showed that low supersaturation results in the appearance of tetragonal plates or tetragonal-like pyramids. An electron microscopy study, together with computer modeling, has shown that crystals of different forms have a high probability of having the same unit cell parameters. According to these experiments the following conclusion can be dranvn: the level of supersaturation of the macromolecule in a crystallizing solution is one of the major factors for forming three-dimensional crystals convenient for X-rays diffraction analysis. From the same macromolecule solution, crystals of different forms can be grown at approximately the same conditions by varying the concentration of macromolecule in the solution. Ion-macromolecule and water-macromolecule interactions, apparently, play the main role in the formation of the unit cell of the crystals.

Hollow Fiber Membrane Modification with Functional Zwitterionic Macromolecules for Improved Thromboresistance in Artificial Lungs

PubMed Central

Ye, Sang-Ho; Arazawa, David T.; Zhu, Yang; Shankarraman, Venkat; Malkin, Alexander D.; Kimmel, Jeremy D.; Gamble, Lara J.; Ishihara, Kazuhiko; Federspiel, William J.; Wagner, William R.

2015-01-01

Respiratory assist devices seek optimized performance in terms of gas transfer efficiency and thromboresistance to minimize device size and reduce complications associated with inadequate blood biocompatibility. The exchange of gas with blood occurs at the surface of the hollow fiber membranes (HFMs) used in these devices. In this study, three zwitterionic macromolecules were attached to HFM surfaces to putatively improve thromboresistance: (1) carboxyl-functionalized zwitterionic phosphorylcholine (PC) and (2) sulfobetaine (SB) macromolecules (mPC or mSB-COOH) prepared by a simple thiol-ene radical polymerization and (3) a low-molecular weight sulfobetaine (SB)-co-methacrylic acid (MA) block copolymer (SBMAb-COOH) prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization. Each macromolecule type was covalently immobilized on an aminated commercial HFM (Celg-A) by a condensation reaction, and HFM surface composition changes were analyzed by X-ray photoelectron spectroscopy. Thrombotic deposition on the HFMs was investigated after contact with ovine blood in vitro. The removal of CO2 by the HFMs was also evaluated using a model respiratory assistance device. The HFMs conjugated with zwitterionic macromolecules (Celg-mPC, Celg-mSB, and Celg-SBMAb) showed expected increases in phosphorus or sulfur surface content. Celg-mPC and Celg-SBMAb experienced rates of platelet deposition significantly lower than those of unmodified (Celg-A, >95% reduction) and heparin-coated (>88% reduction) control HFMs. Smaller reductions were seen with Celg-mSB. The CO2 removal rate for Celg-SBMAb HFMs remained comparable to that of Celg-A. In contrast, the rate of removal of CO2 for heparin-coated HFMs was significantly reduced. The results demonstrate a promising approach to modifying HFMs using zwitterionic macromolecules for artificial lung devices with improved thromboresistance without degradation of gas transfer. PMID:25669307

Electric Birefringence: A Simple Apparatus for Determining Physical Parameters of Macromolecules and Colloids.

ERIC Educational Resources Information Center

Trimm, Harold H.; And Others

1984-01-01

Describes a birefringence apparatus that can be assembled for less than $100 and can be used to measure both the dimensions and dipole moments of many macromolecules. Details are given of the construction and manipulation of the apparatus. (JN)

Solid State Nuclear Magnetic Resonance Studies of the Murchison Organic Macromolecule

NASA Technical Reports Server (NTRS)

Cody, G. D., III; Alexander, C. M. OD.; Tera, F.

2001-01-01

We have used high speed H-1 (DEPTH) and C-13 (VACP MAS-slow spinning) solid state NMR to determine the contributions of protonated vs non-protonated carbon in the Murchison Macromolecule. Additional information is contained in the original extended abstract.

Coding and decoding libraries of sequence-defined functional copolymers synthesized via photoligation

PubMed Central

Zydziak, Nicolas; Konrad, Waldemar; Feist, Florian; Afonin, Sergii; Weidner, Steffen; Barner-Kowollik, Christopher

2016-01-01

Designing artificial macromolecules with absolute sequence order represents a considerable challenge. Here we report an advanced light-induced avenue to monodisperse sequence-defined functional linear macromolecules up to decamers via a unique photochemical approach. The versatility of the synthetic strategy—combining sequential and modular concepts—enables the synthesis of perfect macromolecules varying in chemical constitution and topology. Specific functions are placed at arbitrary positions along the chain via the successive addition of monomer units and blocks, leading to a library of functional homopolymers, alternating copolymers and block copolymers. The in-depth characterization of each sequence-defined chain confirms the precision nature of the macromolecules. Decoding of the functional information contained in the molecular structure is achieved via tandem mass spectrometry without recourse to their synthetic history, showing that the sequence information can be read. We submit that the presented photochemical strategy is a viable and advanced concept for coding individual monomer units along a macromolecular chain. PMID:27901024

Electric field mediated loading of macromolecules in intact yeast cells is critically controlled at the wall level.

PubMed

Ganeva, V; Galutzov, B; Teissié, J

1995-12-13

The mechanism of electric field mediated macromolecule transfer inside an intact yeast cell was investigated by observing, under a microscope, the fluorescence associated to cells after pulsation in a buffer containing two different hydrophilic fluorescent dyes. In the case of a small probe such as propidium iodide, a long lived permeabilized state was induced by the field as classically observed on wall free systems. Penetration of a 70 kDa FITC dextran was obtained only by using drastic conditions and only a very limited number of yeast cells which took up macromolecules remained viable. Most dextrans were trapped in the wall. A dramatic improvement in transfer of dextrans was observed when the cells were treated by dithiothreitol before pulsation. A cytoplasmic protein leakage was detected after the electric treatment suggesting that an irreversible damage took place in the walls of many pulsed cells. Electroloading of macromolecules in intact yeast cells appears to be controlled by a field induced short lived alteration of the envelope organization.

Interphase vs confinement in starch-clay bionanocomposites.

PubMed

Coativy, Gildas; Chevigny, Chloé; Rolland-Sabaté, Agnès; Leroy, Eric; Lourdin, Denis

2015-03-06

Starch-clay bionanocomposites containing 1-10% of natural montmorillonite were elaborated by melt processing in the presence of water. A complex macromolecular dynamics behavior was observed: depending on the clay content, an increase of the glass transition temperature and/or the presence of two overlapped α relaxation peaks were detected. Thanks to a model allowing the prediction of the average interparticle distance, and its comparison with the average size of starch macromolecules, it was possible to associate these phenomena to different populations of macromolecules. In particular, it seems that for high clay content (10%), the slowdown of segmental relaxation due to confinement of the starch macromolecules between the clay tactoïds is the predominant phenomenon. While for lower clay contents (3-5%), a significant modification of chain relaxation seems to occur, due to the formation of an interphase by the starch macromolecules in the vicinity of clay nanoparticles coexisting with the bulk polymer. Copyright © 2014 Elsevier Ltd. All rights reserved.

Coding and decoding libraries of sequence-defined functional copolymers synthesized via photoligation.

PubMed

Zydziak, Nicolas; Konrad, Waldemar; Feist, Florian; Afonin, Sergii; Weidner, Steffen; Barner-Kowollik, Christopher

2016-11-30

Designing artificial macromolecules with absolute sequence order represents a considerable challenge. Here we report an advanced light-induced avenue to monodisperse sequence-defined functional linear macromolecules up to decamers via a unique photochemical approach. The versatility of the synthetic strategy-combining sequential and modular concepts-enables the synthesis of perfect macromolecules varying in chemical constitution and topology. Specific functions are placed at arbitrary positions along the chain via the successive addition of monomer units and blocks, leading to a library of functional homopolymers, alternating copolymers and block copolymers. The in-depth characterization of each sequence-defined chain confirms the precision nature of the macromolecules. Decoding of the functional information contained in the molecular structure is achieved via tandem mass spectrometry without recourse to their synthetic history, showing that the sequence information can be read. We submit that the presented photochemical strategy is a viable and advanced concept for coding individual monomer units along a macromolecular chain.

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.

PubMed

Stockwell, B R; Haggarty, S J; Schreiber, S L

1999-02-01

Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.

Characterization of plasma labile heme in hemolytic conditions

PubMed Central

Gouveia, Zélia; Carlos, Ana R.; Yuan, Xiaojing; Aires-da-Silva, Frederico; Stocker, Roland; Maghzal, Ghassan J.; Leal, Sónia S.; Gomes, Cláudio M.; Todorovic, Smilja; Iranzo, Olga; Ramos, Susana; Santos, Ana C.; Hamza, Iqbal; Gonçalves, João; Soares, Miguel P.

2018-01-01

Extracellular hemoglobin, a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here, we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches, we demonstrate that when generated during hemolytic conditions labile heme is bound to plasma molecules with an affinity higher than 10−7 m and that 2–8% (∼ 2–5 μm) of the total amount of heme detected in plasma can be internalized by bystander cells, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme-binding capacity, that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10−7 m. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme. PMID:28783254

Dynamic Compression Promotes the Matrix Synthesis of Nucleus Pulposus Cells Through Up-Regulating N-CDH Expression in a Perfusion Bioreactor Culture.

PubMed

Xu, Yichun; Yao, Hui; Li, Pei; Xu, Wenbin; Zhang, Junbin; Lv, Lulu; Teng, Haijun; Guo, Zhiliang; Zhao, Huiqing; Hou, Gang

2018-01-01

An adequate matrix production of nucleus pulposus (NP) cells is an important tissue engineering-based strategy to regenerate degenerative discs. Here, we mainly aimed to investigate the effects and mechanism of mechanical compression (i.e., static compression vs. dynamic compression) on the matrix synthesis of three-dimensional (3D) cultured NP cells in vitro. Rat NP cells seeded on small intestinal submucosa (SIS) cryogel scaffolds were cultured in the chambers of a self-developed, mechanically active bioreactor for 10 days. Meanwhile, the NP cells were subjected to compression (static compression or dynamic compression at a 10% scaffold deformation) for 6 hours once per day. Unloaded NP cells were used as controls. The cellular phenotype and matrix biosynthesis of NP cells were investigated by real-time PCR and Western blotting assays. Lentivirus-mediated N-cadherin (N-CDH) knockdown and an inhibitor, LY294002, were used to further investigate the role of N-CDH and the PI3K/Akt pathway in this process. Dynamic compression better maintained the expression of cell-specific markers (keratin-19, FOXF1 and PAX1) and matrix macromolecules (aggrecan and collagen II), as well as N-CDH expression and the activity of the PI3K/Akt pathway, in the 3D-cultured NP cells compared with those expression levels and activity in the cells grown under static compression. Further analysis showed that the N-CDH knockdown significantly down-regulated the expression of NP cell-specific markers and matrix macromolecules and inhibited the activation of the PI3K/Akt pathway under dynamic compression. However, inhibition of the PI3K/Akt pathway had no effects on N-CDH expression but down-regulated the expression of NP cell-specific markers and matrix macromolecules under dynamic compression. Dynamic compression increases the matrix synthesis of 3D-cultured NP cells compared with that of the cells under static compression, and the N-CDH-PI3K/Akt pathway is involved in this regulatory process. This study provides a promising strategy to promote the matrix deposition of tissue-engineered NP tissue in vitro prior to clinical transplantation. © 2018 The Author(s). Published by S. Karger AG, Basel.

Looking inside the box: using Raman microspectroscopy to deconstruct microbial biomass stoichiometry one cell at a time

USGS Publications Warehouse

Hall, Edward K.; Singer, Gabriel A.; Pölzl, Marvin; Hämmerle, Ieda; Schwarz, Christian; Daims, Holger; Maixner, Frank; Battin, Tom J.

2011-01-01

Stoichiometry of microbial biomass is a key determinant of nutrient recycling in a wide variety of ecosystems. However, little is known about the underlying causes of variance in microbial biomass stoichiometry. This is primarily because of technological constraints limiting the analysis of macromolecular composition to large quantities of microbial biomass. Here, we use Raman microspectroscopy (MS), to analyze the macromolecular composition of single cells of two species of bacteria grown on minimal media over a wide range of resource stoichiometry. We show that macromolecular composition, determined from a subset of identified peaks within the Raman spectra, was consistent with macromolecular composition determined using traditional analytical methods. In addition, macromolecular composition determined by Raman MS correlated with total biomass stoichiometry, indicating that analysis with Raman MS included a large proportion of a cell's total macromolecular composition. Growth phase (logarithmic or stationary), resource stoichiometry and species identity each influenced each organism's macromolecular composition and thus biomass stoichiometry. Interestingly, the least variable peaks in the Raman spectra were those responsible for differentiation between species, suggesting a phylogenetically specific cellular architecture. As Raman MS has been previously shown to be applicable to cells sampled directly from complex environments, our results suggest Raman MS is an extremely useful application for evaluating the biomass stoichiometry of environmental microorganisms. This includes the ability to partition microbial biomass into its constituent macromolecules and increase our understanding of how microorganisms in the environment respond to resource heterogeneity.

Looking inside the box: using Raman microspectroscopy to deconstruct microbial biomass stoichiometry one cell at a time

PubMed Central

Hall, Edward K; Singer, Gabriel A; Pölzl, Marvin; Hämmerle, Ieda; Schwarz, Christian; Daims, Holger; Maixner, Frank; Battin, Tom J

2011-01-01

Stoichiometry of microbial biomass is a key determinant of nutrient recycling in a wide variety of ecosystems. However, little is known about the underlying causes of variance in microbial biomass stoichiometry. This is primarily because of technological constraints limiting the analysis of macromolecular composition to large quantities of microbial biomass. Here, we use Raman microspectroscopy (MS), to analyze the macromolecular composition of single cells of two species of bacteria grown on minimal media over a wide range of resource stoichiometry. We show that macromolecular composition, determined from a subset of identified peaks within the Raman spectra, was consistent with macromolecular composition determined using traditional analytical methods. In addition, macromolecular composition determined by Raman MS correlated with total biomass stoichiometry, indicating that analysis with Raman MS included a large proportion of a cell's total macromolecular composition. Growth phase (logarithmic or stationary), resource stoichiometry and species identity each influenced each organism's macromolecular composition and thus biomass stoichiometry. Interestingly, the least variable peaks in the Raman spectra were those responsible for differentiation between species, suggesting a phylogenetically specific cellular architecture. As Raman MS has been previously shown to be applicable to cells sampled directly from complex environments, our results suggest Raman MS is an extremely useful application for evaluating the biomass stoichiometry of environmental microorganisms. This includes the ability to partition microbial biomass into its constituent macromolecules and increase our understanding of how microorganisms in the environment respond to resource heterogeneity. PMID:20703314

Method for selective immobilization of macromolecules on self assembled monolayer surfaces

DOEpatents

Laskin, Julia [Richland, WA; Wang, Peng [Billerica, MA

2011-11-29

Disclosed is a method for selective chemical binding and immobilization of macromolecules on solid supports in conjunction with self-assembled monolayer (SAM) surfaces. Immobilization involves selective binding of peptides and other macromolecules to SAM surfaces using reactive landing (RL) of mass-selected, gas phase ions. SAM surfaces provide a simple and convenient platform for tailoring chemical properties of a variety of substrates. The invention finds applications in biochemistry ranging from characterization of molecular recognition events at the amino acid level and identification of biologically active motifs in proteins, to development of novel biosensors and substrates for stimulated protein and cell adhesion.

Single molecule optical measurements of orientation and rotations of biological macromolecules.

PubMed

Shroder, Deborah Y; Lippert, Lisa G; Goldman, Yale E

2016-11-22

Subdomains of macromolecules often undergo large orientation changes during their catalytic cycles that are essential for their activity. Tracking these rearrangements in real time opens a powerful window into the link between protein structure and functional output. Site-specific labeling of individual molecules with polarized optical probes and measurement of their spatial orientation can give insight into the crucial conformational changes, dynamics, and fluctuations of macromolecules. Here we describe the range of single molecule optical technologies that can extract orientation information from these probes, review the relevant types of probes and labeling techniques, and highlight the advantages and disadvantages of these technologies for addressing specific inquiries.

Associative Interactions in Crowded Solutions of Biopolymers Counteract Depletion Effects.

PubMed

Groen, Joost; Foschepoth, David; te Brinke, Esra; Boersma, Arnold J; Imamura, Hiromi; Rivas, Germán; Heus, Hans A; Huck, Wilhelm T S

2015-10-14

The cytosol of Escherichia coli is an extremely crowded environment, containing high concentrations of biopolymers which occupy 20-30% of the available volume. Such conditions are expected to yield depletion forces, which strongly promote macromolecular complexation. However, crowded macromolecule solutions, like the cytosol, are very prone to nonspecific associative interactions that can potentially counteract depletion. It remains unclear how the cytosol balances these opposing interactions. We used a FRET-based probe to systematically study depletion in vitro in different crowded environments, including a cytosolic mimic, E. coli lysate. We also studied bundle formation of FtsZ protofilaments under identical crowded conditions as a probe for depletion interactions at much larger overlap volumes of the probe molecule. The FRET probe showed a more compact conformation in synthetic crowding agents, suggesting strong depletion interactions. However, depletion was completely negated in cell lysate and other protein crowding agents, where the FRET probe even occupied slightly more volume. In contrast, bundle formation of FtsZ protofilaments proceeded as readily in E. coli lysate and other protein solutions as in synthetic crowding agents. Our experimental results and model suggest that, in crowded biopolymer solutions, associative interactions counterbalance depletion forces for small macromolecules. Furthermore, the net effects of macromolecular crowding will be dependent on both the size of the macromolecule and its associative interactions with the crowded background.

Improved Measurement of B(sub 22) of Macromolecules in a Flow Cell

NASA Technical Reports Server (NTRS)

Wilson, Wilbur; Fanguy, Joseph; Holman, Steven; Guo, Bin

2008-01-01

An improved apparatus has been invented for use in determining the osmotic second virial coefficient of macromolecules in solution. In a typical intended application, the macromolecules would be, more specifically, protein molecules, and the protein solution would be pumped through a flow cell to investigate the physical and chemical conditions that affect crystallization of the protein in question. Some background information is prerequisite to a meaningful description of the novel aspects of this apparatus. A method of determining B22 from simultaneous measurements of the static transmittance (taken as an indication of concentration) and static scattering of light from the same location in a flowing protein solution was published in 2004. The apparatus used to implement the method at that time included a dual-detector flow cell, which had two drawbacks: a) The amount of protein required for analysis of each solution condition was of the order of a milligram - far too large a quantity for a high-throughput analysis system, for which microgram or even nanogram quantities of protein per analysis are desirable. b) The design of flow cell was such that two light sources were used to probe different regions of the flowing solution. Consequently, the apparatus did not afford simultaneous measurements at the same location in the solution and, hence, did not guarantee an accurate determination of B22.

Multifunctional Delivery Systems for Advanced oral Uptake of Peptide/Protein Drugs.

PubMed

Park, Jin Woo; Kim, Sun Jin; Kwag, Dong Sup; Kim, Sol; Park, Jeyoung; Youn, Yu Seok; Bae, You Han; Lee, Eun Seong

2015-01-01

In recent years, advances in biotechnology and protein engineering have enabled the production of large quantities of proteins and peptides as important therapeutic agents. Various researchers have used biocompatible functional polymers to prepare oral dosage forms of proteins and peptides for chronic use and for easier administration to enhance patient compliance. However, there is a need to enhance their safety and effectiveness further. Most macromolecules undergo severe denaturation at low pH and enzymatic degradation in the gastrointestinal tract. The macromolecules' large molecular size and low lipophilicity cause low permeation through the intestinal membrane. The major strategies that have been used to overcome these challenges (in oral drug carrier systems) can be classified as follows: enteric coating or encapsulation with pH-sensitive polymers or mucoadhesive polymers, co-administration of protease inhibitors, incorporation of absorption enhancers, modification of the physicochemical properties of the macromolecules, and site-specific delivery to the colon. This review attempts to summarize the various advanced oral delivery carriers, including nanoparticles, lipid carriers, such as liposomes, nano-aggregates using amphiphilic polymers, complex coacervation of oppositely charged polyelectrolytes, and inorganic porous particles. The particles were formulated and/or surface modified with functional polysaccharides or synthetic polymers to improve oral bioavailability of proteins and peptides. We also discuss formulation strategies to overcome barriers, therapeutic efficacies in vivo, and potential benefits and issues for successful oral dosage forms of the proteins and peptides.

Genome sequence of the algicidal bacterium Kordia algicida OT-1.

PubMed

Lee, Hyun Sook; Kang, Sung Gyun; Kwon, Kae Kyoung; Lee, Jung-Hyun; Kim, Sang-Jin

2011-08-01

Kordia algicida OT-1 is an algicidal bacterium against the bloom-forming microalgae. The genome sequence of K. algicida revealed a number of interesting features, including the degradation of macromolecules, the biosynthesis of carotenoid pigment and secondary metabolites, and the capacity for gliding motility, which might facilitate the understanding of algicidal mechanisms.

Insights into Protein–Ligand Interactions: Mechanisms, Models, and Methods

PubMed Central

Du, Xing; Li, Yi; Xia, Yuan-Ling; Ai, Shi-Meng; Liang, Jing; Sang, Peng; Ji, Xing-Lai; Liu, Shu-Qun

2016-01-01

Molecular recognition, which is the process of biological macromolecules interacting with each other or various small molecules with a high specificity and affinity to form a specific complex, constitutes the basis of all processes in living organisms. Proteins, an important class of biological macromolecules, realize their functions through binding to themselves or other molecules. A detailed understanding of the protein–ligand interactions is therefore central to understanding biology at the molecular level. Moreover, knowledge of the mechanisms responsible for the protein-ligand recognition and binding will also facilitate the discovery, design, and development of drugs. In the present review, first, the physicochemical mechanisms underlying protein–ligand binding, including the binding kinetics, thermodynamic concepts and relationships, and binding driving forces, are introduced and rationalized. Next, three currently existing protein-ligand binding models—the “lock-and-key”, “induced fit”, and “conformational selection”—are described and their underlying thermodynamic mechanisms are discussed. Finally, the methods available for investigating protein–ligand binding affinity, including experimental and theoretical/computational approaches, are introduced, and their advantages, disadvantages, and challenges are discussed. PMID:26821017

Thermodynamics of Macromolecular Association in Heterogeneous Crowding Environments: Theoretical and Simulation Studies with a Simplified Model.

PubMed

Ando, Tadashi; Yu, Isseki; Feig, Michael; Sugita, Yuji

2016-11-23

The cytoplasm of a cell is crowded with many different kinds of macromolecules. The macromolecular crowding affects the thermodynamics and kinetics of biological reactions in a living cell, such as protein folding, association, and diffusion. Theoretical and simulation studies using simplified models focus on the essential features of the crowding effects and provide a basis for analyzing experimental data. In most of the previous studies on the crowding effects, a uniform crowder size is assumed, which is in contrast to the inhomogeneous size distribution of macromolecules in a living cell. Here, we evaluate the free energy changes upon macromolecular association in a cell-like inhomogeneous crowding system via a theory of hard-sphere fluids and free energy calculations using Brownian dynamics trajectories. The inhomogeneous crowding model based on 41 different types of macromolecules represented by spheres with different radii mimics the physiological concentrations of macromolecules in the cytoplasm of Mycoplasma genitalium. The free energy changes of macromolecular association evaluated by the theory and simulations were in good agreement with each other. The crowder size distribution affects both specific and nonspecific molecular associations, suggesting that not only the volume fraction but also the size distribution of macromolecules are important factors for evaluating in vivo crowding effects. This study relates in vitro experiments on macromolecular crowding to in vivo crowding effects by using the theory of hard-sphere fluids with crowder-size heterogeneity.

Decades of Data: Extracting Trends from Microgravity Crystallization History

NASA Technical Reports Server (NTRS)

Judge, Russell A.; Snell, Edward H.; Kephart, Richard; vanderWoerd, Mark; Curreri, Peter A. (Technical Monitor)

2002-01-01

The reduced acceleration environment of an orbiting spacecraft has been posited as an ideal environment for biological crystal growth since buoyancy driven convection and sedimentation are greatly reduced. Since the first sounding rocket flight in 1981 many crystallization experiments have flown with some showing improvement and others not. To further explore macromolecule crystal improvement in microgravity we have accumulated data from published reports and reports submitted by individual investigators to NASA, forming a database called BIOSEArCH (Biological Space Experiment Archive of Crystallization History). To date it contains information from 63 missions including, the Space Shuttle program, unmanned satellites, the Russian Space Station MIR and sounding rocket experiments, containing reports for more than 736 macromolecule experiments. While it is not at this point in time a comprehensive record of all flight crystallization experimental results, there is however sufficient information for emerging trends to be identified. These trends will be highlighted.

[Physical methods and molecular biology].

PubMed

Serdiuk, I N

2009-01-01

The review is devoted to the description of the current state of physical and chemical methods used for studying the structural and functional bases of living processes. Special attention is focused on the physical methods that have opened a new page in the research of the structure of biological macromolecules. They include primarily the methods of detecting and manipulating single molecules using optical and magnetic traps. New physical methods, such as two-dimensional infrared spectroscopy, fluorescence correlation spectroscopy and magnetic resonance microscopy are also analyzed briefly in the review. The path that physics and biology have passed for the latest 55 years shows that there is no single method providing all necessary information on macromolecules and their interactions. Each method provides its space-time view of the system. All physical methods are complementary. It is just complementarity that is the fundamental idea justifying the existence in practice of all physical methods, whose description is the aim of the review.

General Characteristics of the Changes in the Thermal Stability of Proteins and Enzymes After the Chemical Modification of Their Functional Groups

NASA Astrophysics Data System (ADS)

Kutuzova, G. D.; Ugarova, N. N.; Berezin, Ilya V.

1984-11-01

The principal structural and physicochemical factors determining the stability of protein macromolecules in solution and the characteristics of the structure of the proteins from thermophilic microorganisms are examined. The mechanism of the changes in the thermal stability of proteins and enzymes after the chemical modification of their functional side groups and the experimental data concerning the influence of chemical modification on the thermal stability of proteins are analysed. The dependence of the stabilisation effect and of the changes in the structure of protein macromolecules on the degree of modification and on the nature of the modified groups and the groups introduced into proteins in the course of modification (their charge and hydrophobic properties) is demonstrated. The great practical value of the method of chemical modification for the preparation of stabilised forms of biocatalysts is shown in relation to specific examples. The bibliography includes 178 references.

Chemical composition, structural properties, and source apportionment of organic macromolecules in atmospheric PM10 in a coastal city of Southeast China.

PubMed

Chen, Yanting; Du, Wenjiao; Chen, Jinsheng; Hong, Youwei; Zhao, Jinping; Xu, Lingling; Xiao, Hang

2017-02-01

Particulate matter (PM 10 ) associated with the fractions of organic macromolecules, including humic acid (HA), kerogen + black carbon (KB), and black carbon (BC), was determined during summer and winter at urban and suburban sites in a coastal city of southeast China. The organic macromolecules were characterized by elemental analysis (EA), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR), and their sources were identified by using stable carbon/nitrogen isotope (δ 13 C/δ 15 N) and the Hybrid Single Particle Lagrangian Integrated Trajectory (HYSPLIT) Model. The results showed that HA, kerogen (K), and BC accounted for the range of 3.89 to 4.55 % in PM 10 , while they were the dominant fractions of total organic carbon (TOC), ranging from 64.70 to 84.99 %. SEM analysis indicated that BC particles were porous/nonporous and consisted of spherical and non-spherical (i.e., cylindrical and elongate) structures. The FTIR spectra of HA, KB, and BC exhibited similar functional groups, but the difference of various sites and seasons was observed. HA in PM 10 contained a higher fraction of aliphatic structures, such as long-chain fatty and carbohydrates with a carboxylic extremity. The C/N ratio, SEM, and δ 13 C/δ 15 N values provided reliable indicators of the sources of HA, K, and BC in PM 10 . The results suggested that HA and K majorly originated from terrestrial plants, and BC came from the mixture of combustion of terrestrial plants, fossil fuel, and charcoal. The air masses in winter originated from Mongolia (4 %), the northern area of China (48 %), and northern adjacent cities (48 %), suggesting the influence of anthropogenic sources through long-range transport, while the air masses for the summer period came from South China Sea (34 %) and Western Pacific Sea (66 %), representing clean marine air masses with low concentrations of organic macromolecules.

Development of a novel monoclonal antibody against 4-hydroxy-2E,6Z-dodecadienal (4-HDDE)-protein adducts: Immunochemical application in quantitative and qualitative analyses of lipid peroxidation in vitro and ex vivo.

PubMed

Uchida, Koji; Shibata, Takahiro; Toyokuni, Shinya; Daniel, Bareket; Zarkovic, Kamelija; Zarkovic, Neven; Sasson, Shlomo

2018-05-25

Non-enzymatic peroxidation of polyunsaturated fatty acids (PUFA) results in the formation of various α,β-unsaturated aldehydes, of which 4-hydroxyalkenals are abundant. The propensity of n-6 PUFA, such as linoleic acid, γ-linolenic acid and arachidonic acid, to undergo radical-induced peroxidation and generate 4-hydroxy-2E-nonenal (4-HNE) has been widely demonstrated. The ability of the latter to form covalent adducts with macromolecules and modify cellular functions has been linked to numerous pathological processes. Concomitantly, evidence has accumulated on specific signaling properties of low concentrations of 4-HNE that may induce hormetic and protective responses to peroxidation stress in cells. It has long been known that peroxidation of PUFA, and particularly arachidonic acid, also give rise to 4-hydroxy-2E,6Z-dodecadienal (4-HDDE), which is more chemically reactive than 4-HNE. Few studies on 4-HDDE revealed its ability to avidly interact covalently with electronegative moieties in macromolecules and to its ability to selectively activate the transcriptional regulator Peroxisome Proliferator-Activated Receptor (PPAR)-β/δ. The research on 4-HDDE has been impeded due to the lack of available pure 4-HDDE and antibodies that recognize 4-HDDE-modified epitopes in proteins. The purpose of this study was to employ an established procedure to synthesize 4-HDDE and use it to create and characterize a monoclonal antibody against 4-HDDE-modified proteins and establish its application for ELISA and immunohistochemical analysis of cells and tissues and further expand lipid peroxidation research. Copyright © 2018 Elsevier Inc. All rights reserved.

Studies on the metabolism and bioactivation of (S)-nicotine and beta-nicotyrine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shigenaga, M.K.

1989-01-01

(S)-Nicotine has long been suspected of contributing to the chronic toxicities associated with the use of cigarettes and other tobacco products. The possibility that (S)-nicotine could contribute to these chronic toxicities by causing irreversible damage to cellular macromolecules has prompted studies aimed at characterizing the metabolic pathways of (S)-nicotine that form reactive metabolites which bind covalently. In order to study these processes, (S)-5-{sup 3}H-nicotine was synthesized by catalytic tritiolysis of (S)-5-bromonicotine with carrier-free tritium gas, purified by HPLC and characterized by tritium NMR, diode array VV and HPLC chromatographic analysis. The metabolism of (S)-5-{sup 3}H-nicotine by rabbit liver and lungmore » microsomal enzymes produced reactive intermediates which bound covalently to microsomal macromolecules in a time, NADPH and cytochrome P-450 dependent manner. The results of studies employing rabbit lung microsomes and agents which inhibit or alter the expression of the cytochrome P-450 isozyme composition in this tissue indicated that the covalent binding of (S)-nicotine requires (S)-nicotine {Delta}{sup 1{prime},5{prime}}-iminium ion as an obligate intermediate and the catalytic activity of lung cytochrome P-450 isozyme-2. Investigations of the effects of (S)-nicotine and related tobacco alkaloids on the oxidation of the Parkinson's disease inducing agent MPTP by the mitochondrial enzyme MAO-B were prompted by the inverse correlation between cigarette smoking and Parkinson's disease. In the author studies (S)-nicotine A{sup 1{prime},5{prime}}-iminium bisperchlorate inhibited the MAOB catalyzed oxidation of MPTP by a linear-mixed type mechanism. Subsequent studies identified {beta}-nicotyrine as a MAO-B catalyzed oxidation product of (S)-nicotine A{sup 1{prime},5{prime}}-iminium ion.« less

Regulation of germ cell development by intercellular signaling in the mammalian ovarian follicle.

PubMed

Clarke, Hugh J

2018-01-01

Prior to ovulation, the mammalian oocyte undergoes a process of differentiation within the ovarian follicle that confers on it the ability to give rise to an embryo. Differentiation comprises two phases-growth, during which the oocyte increases more than 100-fold in volume as it accumulates macromolecules and organelles that will sustain early embryogenesis; and meiotic maturation, during which the oocyte executes the first meiotic division and prepares for the second division. Entry of an oocyte into the growth phase appears to be triggered when the adjacent granulosa cells produce specific growth factors. As the oocyte grows, it elaborates a thick extracellular coat termed the zona pellucida. Nonetheless, cytoplasmic extensions of the adjacent granulosa cells, termed transzonal projections (TZPs), enable them to maintain contact-dependent communication with the oocyte. Through gap junctions located where the TZP tips meet the oocyte membrane, they provide the oocyte with products that sustain its metabolic activity and signals that regulate its differentiation. Conversely, the oocyte secretes diffusible growth factors that regulate proliferation and differentiation of the granulosa cells. Gap junction-permeable products of the granulosa cells prevent precocious initiation of meiotic maturation, and the gap junctions also enable oocyte maturation to begin in response to hormonal signals received by the granulosa cells. Development of the oocyte or the somatic compartment may also be regulated by extracellular vesicles newly identified in follicular fluid and at TZP tips, which could mediate intercellular transfer of macromolecules. Oocyte differentiation thus depends on continuous signaling interactions with the somatic cells of the follicle. WIREs Dev Biol 2018, 7:e294. doi: 10.1002/wdev.294 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Cellular Differentiation Signaling Pathways > Cell Fate Signaling Early Embryonic Development > Gametogenesis. © 2017 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ben-Dov, Nadav; Korenstein, Rafi, E-mail: korens@post.tau.ac.il

Recently it has been shown that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesicles accompanied by an enhanced uptake of macromolecules. While the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the mechanisms underlying vesicle formation and its scission are still unknown. In light of the critical role of actin in vesicle formation during endocytosis, the present study addresses the involvement of cytoskeletal actin in proton-induced uptake (PIU). The uptake of dextran-FITC is used as a measure for the factual fraction ofmore » inward invaginations that undergo scission from the cell's plasma membrane. Our findings show that the rate of PIU in suspended cells is constant, whereas the rate of PIU in adherent cells is gradually increased in time, saturating at the level possessed by suspended cells. This is consistent with pH induced gradual degradation of stress-fibers in adherent cells. Wortmannin and calyculin-A are able to elevate PIU by 25% in adherent cells but not in suspended cells, while cytochalasin-D, rapamycin and latrunculin-A elevate PIU both in adherent and suspended cells. However, extensive actin depolymerization by high concentrations of latrunculin-A is able to inhibit PIU. We conclude that proton-induced membrane vesiculation is restricted by the actin structural resistance to the plasma membrane bending. Nevertheless, a certain degree of cortical actin restructuring is required for the completion of the scission process. - Highlights: ► Acidification of cells' exterior enhances uptake of macromolecules by the cells. ► Disruption of actin stress fibers leads to enhancement of proton induced uptake. ► Extensive depolymerization of cellular actin attenuates proton-induced uptake.« less

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

PubMed Central

Ramakrishnan, N.; Sunil Kumar, P. B.; Radhakrishnan, Ravi

2014-01-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham - Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies. PMID:25484487

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins.

PubMed

Ramakrishnan, N; Sunil Kumar, P B; Radhakrishnan, Ravi

2014-10-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham - Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies.

Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

NASA Astrophysics Data System (ADS)

Ramakrishnan, N.; Sunil Kumar, P. B.; Radhakrishnan, Ravi

2014-10-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham-Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies.

Single molecule optical measurements of orientation and rotations of biological macromolecules

PubMed Central

Shroder, Deborah Y; Lippert, Lisa G; Goldman, Yale E

2016-01-01

The subdomains of macromolecules often undergo large orientation changes during their catalytic cycles that are essential for their activity. Tracking these rearrangements in real time opens a powerful window into the link between protein structure and functional output. Site-specific labeling of individual molecules with polarized optical probes and measuring their spatial orientation can give insight into the crucial conformational changes, dynamics, and fluctuations of macromolecules. Here we describe the range of single molecule optical technologies that can extract orientation information from these probes, we review the relevant types of probes and labeling techniques, and we highlight the advantages and disadvantages of these technologies for addressing specific inquiries. PMID:28192292

A primer in macromolecular linguistics.

PubMed

Searls, David B

2013-03-01

Polymeric macromolecules, when viewed abstractly as strings of symbols, can be treated in terms of formal language theory, providing a mathematical foundation for characterizing such strings both as collections and in terms of their individual structures. In addition this approach offers a framework for analysis of macromolecules by tools and conventions widely used in computational linguistics. This article introduces the ways that linguistics can be and has been applied to molecular biology, covering the relevant formal language theory at a relatively nontechnical level. Analogies between macromolecules and human natural language are used to provide intuitive insights into the relevance of grammars, parsing, and analysis of language complexity to biology. Copyright © 2012 Wiley Periodicals, Inc.

Microelectrophoretic study of calcium oxalate monohydrate in macromolecular solutions

NASA Technical Reports Server (NTRS)

Curreri, P. A.; Onoda, G. Y., Jr.; Finlayson, B.

1987-01-01

Electrophoretic mobilities were measured for calcium oxalate monohydrate (COM) in solutions containing macromolecules. Two mucopolysaccharides (sodium heparin and chondroitin sulfate) and two proteins (positively charged lysozyme and negatively charged bovine serum albumin) were studied as adsorbates. The effects of pH, calcium oxalate surface charge (varied by calcium or oxalate ion activity), and citrate concentration were investigated. All four macromolecules showed evidence for adsorption. The macromolecule concentrations needed for reversing the surface charge indicated that the mucopolysaccharides have greater affinity for the COM surface than the proteins. Citrate ions at high concentrations appear to compete effectively with the negative protein for surface sites but show no evidence for competing with the positively charged protein.

Fluid Physics and Macromolecular Crystal Growth in Microgravity

NASA Technical Reports Server (NTRS)

Pusey, M.; Snell, E.; Judge, R.; Chayen, N.; Boggon, T.; Helliwell, J.; Rose, M. Franklin (Technical Monitor)

2000-01-01

The molecular structure of biological macromolecules is important in understanding how these molecules work and has direct application to rational drug design for new medicines and for the improvement and development of industrial enzymes. In order to obtain the molecular structure, large, well formed, single macromolecule crystals are required. The growth of macromolecule crystals is a difficult task and is often hampered on the ground by fluid flows that result from the interaction of gravity with the crystal growth process. One such effect is the bulk movement of the crystal through the fluid due to sedimentation. A second is buoyancy driven convection close to the crystal surface. On the ground the crystallization process itself induces both of these flows.

Microphase separation of comb copolymers with two different lengths of side chains

NASA Astrophysics Data System (ADS)

Aliev, M. A.; Kuzminyh, N. Yu.

2009-10-01

The phase behavior of the monodisperse AB comb copolymer melt contained the macromolecules of special architecture is discussed. Each macromolecule is assumed to be composed of two comb blocks which differ in numbers of side chains and numbers of monomer units in these chains. It is shown (by analysis of the structure factor of the melt) that microphase separation at two different length scales in the melt is possible. The large and small length scales correspond to separation between comb blocks and separation between monomer units in repeating fragments of blocks, respectively. The classification diagrams indicated which length scale is favored for a given parameters of chemical structure of macromolecules are constructed.

High and low thermal conductivity of amorphous macromolecules

NASA Astrophysics Data System (ADS)

Xie, Xu; Yang, Kexin; Li, Dongyao; Tsai, Tsung-Han; Shin, Jungwoo; Braun, Paul V.; Cahill, David G.

2017-01-01

We measure the thermal conductivity, heat capacity and sound velocity of thin films of five polymers, nine polymer salts, and four caged molecules to advance the fundamental understanding of the lower and upper limits to heat conduction in amorphous macromolecules. The thermal conductivities vary by more than one order of magnitude, from 0.06 W m-1K-1 for [6,6]-phenyl-C71-butyric acid methyl ester to 0.67 W m-1K-1 for poly(vinylphosphonic acid calcium salt). Minimum thermal conductivity calculated from the measured sound velocity and effective atomic density is in good agreement with the thermal conductivity of macromolecules with various molecular structures and intermolecular bonding strength.

Supramolecular Assembly of Comb-like Macromolecules Induced by Chemical Reactions that Modulate the Macromolecular Interactions In Situ.

PubMed

Xia, Hongwei; Fu, Hailin; Zhang, Yanfeng; Shih, Kuo-Chih; Ren, Yuan; Anuganti, Murali; Nieh, Mu-Ping; Cheng, Jianjun; Lin, Yao

2017-08-16

Supramolecular polymerization or assembly of proteins or large macromolecular units by a homogeneous nucleation mechanism can be quite slow and require specific solution conditions. In nature, protein assembly is often regulated by molecules that modulate the electrostatic interactions of the protein subunits for various association strengths. The key to this regulation is the coupling of the assembly process with a reversible or irreversible chemical reaction that occurs within the constituent subunits. However, realizing this complex process by the rational design of synthetic molecules or macromolecules remains a challenge. Herein, we use a synthetic polypeptide-grafted comb macromolecule to demonstrate how the in situ modulation of interactions between the charged macromolecules affects their resulting supramolecular structures. The kinetics of structural formation was studied and can be described by a generalized model of nucleated polymerization containing secondary pathways. Basic thermodynamic analysis indicated the delicate role of the electrostatic interactions between the charged subunits in the reaction-induced assembly process. This approach may be applicable for assembling a variety of ionic soft matters that are amenable to chemical reactions in situ.

Measuring the shapes of macromolecules – and why it matters

PubMed Central

Li, Jie; Mach, Paul; Koehl, Patrice

2013-01-01

The molecular basis of life rests on the activity of biological macromolecules, mostly nucleic acids and proteins. A perhaps surprising finding that crystallized over the last handful of decades is that geometric reasoning plays a major role in our attempt to understand these activities. In this paper, we address this connection between geometry and biology, focusing on methods for measuring and characterizing the shapes of macromolecules. We briefly review existing numerical and analytical approaches that solve these problems. We cover in more details our own work in this field, focusing on the alpha shape theory as it provides a unifying mathematical framework that enable the analytical calculations of the surface area and volume of a macromolecule represented as a union of balls, the detection of pockets and cavities in the molecule, and the quantification of contacts between the atomic balls. We have shown that each of these quantities can be related to physical properties of the molecule under study and ultimately provides insight on its activity. We conclude with a brief description of new challenges for the alpha shape theory in modern structural biology. PMID:24688748

Arachidonic acid with taurine enhances pulmonary absorption of macromolecules without any serious histopathological damages.

PubMed

Miyake, Masateru; Minami, Takanori; Yamazaki, Hiroyuki; Emoto, Chie; Mukai, Tadashi; Toguchi, Hajime

2017-05-01

Therapeutic peptides and protein are being used in several indications; however, their poor permeability still remains to be solved. This study focused on the pulmonary route of macromolecules. First, the effects of arachidonic acid (AA) as an absorption enhancer on drug serum concentration, after intratracheal administration, were investigated in rats. Second, the safety of AA was assessed in rats in an acute toxicity study for 7days. AA enhanced the exposure of both interferon-α (IFN-α) and fluorescein isothiocyanate 4000 (FD-4). In addition, the histopathological analysis indicated that AA caused alveolitis and bronchitis in rats. In combination with Taurine (Tau), these lung injuries were prevented through the histopathological analysis. The combined use of Tau with AA did not show any changes in the pharmacokinetics of FD-4. From these results, we suggest the combined use of AA with Tau as a novel formulation on the pulmonary route of macromolecule drugs. This formulation could improve the bioavailability of macromolecule drugs without any serious local damage to the lungs. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome Sequence of the Algicidal Bacterium Kordia algicida OT-1 ▿

PubMed Central

Lee, Hyun Sook; Kang, Sung Gyun; Kwon, Kae Kyoung; Lee, Jung-Hyun; Kim, Sang-Jin

2011-01-01

Kordia algicida OT-1 is an algicidal bacterium against the bloom-forming microalgae. The genome sequence of K. algicida revealed a number of interesting features, including the degradation of macromolecules, the biosynthesis of carotenoid pigment and secondary metabolites, and the capacity for gliding motility, which might facilitate the understanding of algicidal mechanisms. PMID:21622754

Route of steroid-activated macromolecules through nuclear pores imaged with atomic force microscopy.

PubMed

Oberleithner, H; Schäfer, C; Shahin, V; Albermann, L

2003-02-01

In eukaryotic cells, two concentric membranes, the nuclear envelope (NE), separate the nucleus from the cytoplasm. The NE is punctured by nuclear pore complexes (NPCs; molecular mass 120 MDa) that serve as regulated pathways for macromolecules entering and leaving the nuclear compartment. Transport across NPCs occurs through central channels. Such import and export of macromolecules through individual NPCs can be elicited in the Xenopus laevis oocyte by injecting the mineralocorticoid aldosterone and can be visualized with atomic force microscopy. The electrical NE resistance in intact cell nuclei can be measured in parallel. Resistance increases when macromolecules are engaged with the NPC. This article describe six observations made from these experiments and the conclusions that can be drawn from them. (i) A homogeneous population of macromolecules (approx. 100 kDa) attaches to the cytoplasmic face of the NPC 2 min after aldosterone injection. They are most likely to be aldosterone receptors. After a few minutes, they have disappeared. (ii) Large plugs (approx. molecular mass 1 MDa) appear in the central channels 20 min after hormone injection. They are most likely to be ribonucleoproteins exiting the nucleus. (iii) Electrical resistance measurements in isolated nuclei reveal transient electrical NE resistance peaks: an early (2 min) peak and a late (20 min) peak. Electrical peaks reflect macromolecule interaction with the NPC. (iv) Spironolactone blocks both the early and late peaks. This indicates that classic aldosterone receptors are involved in the pregenomic (early) and post-genomic (late) responses. (v) Actinomycin D and, independently, RNase A block the late electrical peak, confirming that plugs are genomic in nature. (vi) Intracellular calcium chelation blocks both early and late electrical peaks. Thus, the release of calcium from internal stores, which is known to be the first intracellular signal in response to aldosterone, is a prerequisite for the late genomic response.

EPS in Environmental Microbial Biofilms as Examined by Advanced Imaging Techniques

NASA Astrophysics Data System (ADS)

Neu, T. R.; Lawrence, J. R.

2006-12-01

Biofilm communities are highly structured associations of cellular and polymeric components which are involved in biogenic and geogenic environmental processes. Furthermore, biofilms are also important in medical (infection), industrial (biofouling) and technological (biofilm engineering) processes. The interfacial microbial communities in a specific habitat are highly dynamic and change according to the environmental parameters affecting not only the cellular but also the polymeric constituents of the system. Through their EPS biofilms interact with dissolved, colloidal and particulate compounds from the bulk water phase. For a long time the focus in biofilm research was on the cellular constituents in biofilms and the polymer matrix in biofilms has been rather neglected. The polymer matrix is produced not only by different bacteria and archaea but also by eukaryotic micro-organisms such as algae and fungi. The mostly unidentified mixture of EPS compounds is responsible for many biofilm properties and is involved in biofilm functionality. The chemistry of the EPS matrix represents a mixture of polymers including polysaccharides, proteins, nucleic acids, neutral polymers, charged polymers, amphiphilic polymers and refractory microbial polymers. The analysis of the EPS may be done destructively by means of extraction and subsequent chemical analysis or in situ by means of specific probes in combination with advanced imaging. In the last 15 years laser scanning microscopy (LSM) has been established as an indispensable technique for studying microbial communities. LSM with 1-photon and 2-photon excitation in combination with fluorescence techniques allows 3-dimensional investigation of fully hydrated, living biofilm systems. This approach is able to reveal data on biofilm structural features as well as biofilm processes and interactions. The fluorescent probes available allow the quantitative assessment of cellular as well as polymer distribution. For this purpose lectin-binding- analysis has been suggested as a suitable approach to image glycoconjugates within the polymer matrix of biofilm communities. More recently synchrotron radiation is increasingly recognized as a powerful tool for studying biological samples. Hard X-ray excitation can be used to map elemental composition whereas IR imaging allows examination of biological macromolecules. A further technique called soft X-ray scanning transmission microscopy (STXM) has the advantage of both techniques and may be employed to detect elements as well as biomolecules. Using the appropriate spectra, near edge X-ray absorption fine structure (NEXAFS) microscopy allows quantitative chemical mapping at 50 nm resolution. In this presentation the applicability of LSM and STXM will be demonstrated using several examples of different environmental biofilm systems. The techniques in combination provide a new view of complex microbial communities and their interaction with the environment. These advanced imaging techniques offer the possibility to study the spatial structure of cellular and polymeric compounds in biofilms as well as biofilm microhabitats, biofilm functionality and biofilm processes.

Sedimentation Coefficient, Frictional Coefficient, and Molecular Weight: A Preparative Ultracentrifuge Experiment for the Advanced Undergraduate Laboratory.

ERIC Educational Resources Information Center

Halsall, H. B.; Wermeling, J. R.

1982-01-01

Describes an experiment using a high-speed preparative centrifuge and calculator to demonstrate effects of the frictional coefficient of a macromolecule on its rate of transport in a force field and to estimate molecular weight of the macromolecule using an empirical relationship. Background information, procedures, and discussion of results are…

Force spectroscopy of biomolecular folding and binding: theory meets experiment

NASA Astrophysics Data System (ADS)

Dudko, Olga

2015-03-01

Conformational transitions in biological macromolecules usually serve as the mechanism that brings biomolecules into their working shape and enables their biological function. Single-molecule force spectroscopy probes conformational transitions by applying force to individual macromolecules and recording their response, or ``mechanical fingerprints,'' in the form of force-extension curves. However, how can we decode these fingerprints so that they reveal the kinetic barriers and the associated timescales of a biological process? I will present an analytical theory of the mechanical fingerprints of macromolecules. The theory is suitable for decoding such fingerprints to extract the barriers and timescales. The application of the theory will be illustrated through recent studies on protein-DNA interactions and the receptor-ligand complexes involved in blood clot formation.

Chains are more flexible under tension

PubMed Central

Carrillo, Jan-Michael Y.; Rubinstein, Michael

2010-01-01

The mechanical response of networks, gels, and brush layers is a manifestation of the elastic properties of the individual macromolecules. Furthermore, the elastic response of macromolecules to an applied force is the foundation of the single-molecule force spectroscopy techniques. The two main classes of models describing chain elasticity include the worm-like and freely-jointed chain models. The selection between these two classes of models is based on the assumptions about chain flexibility. In many experimental situations the choice is not clear and a model describing the crossover between these two limiting classes is therefore in high demand. We are proposing a unified chain deformation model which describes the force-deformation curve in terms of the chain bending constant K and bond length b. This model demonstrates that the worm-like and freely-jointed chain models correspond to two different regimes of polymer deformation and the crossover between these two regimes depends on the chain bending rigidity and the magnitude of the applied force. Polymer chains with bending constant K>1 behave as a worm-like chain under tension in the interval of the applied forces f ≤ KkBT/b and as a freely-jointed chain for f ≥ KkBT/b (kB is the Boltzmann constant and T is the absolute temperature). The proposed crossover expression for chain deformation is in excellent agreement with the results of the molecular dynamics simulations of chain deformation and single-molecule deformation experiments of biological and synthetic macromolecules. PMID:21415940

Exosomes in Cancer Development, Metastasis and Drug Resistance: A Comprehensive Review

PubMed Central

Azmi, Asfar S.; Bao, Bin; Sarkar, Fazlul H.

2013-01-01

Trafficking of biological material across membranes is an evolutionary conserved mechanism and is part of any normal cell homeostasis. Such transport is comprised of active, passive, export through microparticles and vesicular transport (exosomes) that collectively maintain proper compartmentalization of important micro and macromolecules. In pathological states, such as cancer, aberrant activity of export machinery results in expulsion of a number of key proteins and microRNAs resulting in their misexpression. Exosome mediated expulsion of intracellular drugs could be another barrier in the proper action of most of the commonly used therapeutics, targeted agents and their intracellular metabolites. Over the last decade, a number of studies have revealed that exosomes cross-talk and/or influence major tumor related pathways such as hypoxia driven EMT, cancer stemness, angiogenesis and metastasis involving many cell types within the tumor microenvironment. Emerging evidence suggest that exosome secreted proteins can also propel fibroblast growth, resulting in Desmoplastic reaction (DR); a major barrier in effective cancer drug delivery. This comprehensive review highlights the advancements in the understanding of the biology of exosomes secretions and the consequence on cancer drug resistance. We propose that the successful combination of cancer treatments to tackle exosome mediated drug resistance requires an interdisciplinary understanding of these cellular exclusion mechanisms, and how secreted biomolecules are involved in cellular cross-talk within the tumor microenvironment. PMID:23709120

Identification of small molecule inhibitors of cytokinesis and single cell wound repair

PubMed Central

Clark, Andrew G.; Sider, Jenny R.; Verbrugghe, Koen; Fenteany, Gabriel; von Dassow, George; Bement, William M.

2013-01-01

Screening of small molecule libraries offers the potential to identify compounds that inhibit specific biological processes and, ultimately, to identify macromolecules that are important players in such processes. To date, however, most screens of small molecule libraries have focused on identification of compounds that inhibit known proteins or particular steps in a given process, and have emphasized automated primary screens. Here we have used “low tech” in vivo primary screens to identify small molecules that inhibit both cytokinesis and single cell wound repair, two complex cellular processes that possess many common features. The “diversity set”, an ordered array of 1990 compounds available from the National Cancer Institute, was screened in parallel to identify compounds that inhibit cytokinesis in D. excentricus (sand dollar) embryos and single cell wound repair in X. laevis (frog) oocytes. Two small molecules were thus identified: Sph1 and Sph2. Sph1 reduces Rho activation in wound repair and suppresses formation of the spindle midzone during cytokinesis. Sph2 also reduces Rho activation in wound repair and may inhibit cytokinesis by blocking membrane fusion. The results identify two small molecules of interest for analysis of wound repair and cytokinesis, reveal that these processes are more similar than often realized and reveal the potential power of low tech screens of small molecule libraries for analysis of complex cellular processes. PMID:23125193

Simultaneous assay for plasmin and DNase using radiolabeled human fibroblasts on microcarriers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boswell, G.S.; Dimitrijevich, S.D.; Gracy, R.W.

1989-10-01

A critical step in tissue and wound repair is the removal of eschar--accumulation of denatured cellular and extracellular macromolecules. Enzymatic debridement using a combination of plasmin (fibrinolysin) and DNase has been successfully utilized on a variety of types of wounds. Monitoring the activity of these enzymes by measuring the rate of fibrinolysis, or by viscometric changes due to DNA hydrolysis, is exceedingly cumbersome, time consuming, and, at best, only semiquantitative. Although spectrophotometric assays using synthetic substrates offer several advantages, they do not allow extrapolation of the data to the more complex natural substrates encountered in vivo. We have, therefore, developedmore » an in vitro radioisotopic assay for the simultaneous and quantitative measurement of the hydrolytic activity of both plasmin and DNase. Double labeled ((3H)thymidine, (14C)leucine) human dermal fibroblasts grown on microcarrier beads are utilized as sources of nucleic acid and protein substrates. The assay meets all the criteria of analytical validity, is sensitive and rapid, and is amenable to adaptation for analysis of other hydrolytic enzymes. The method offers a direct evaluation of the enzymatic debridement of wounds using actual human cellular substrates. Moreover, the microcarriers provide a greatly increased surface area for cell attachment and growth, are amenable to rapid separation from the cells by simple mechanical methods, and are ideally suited to analytical manipulations.« less

Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi

PubMed Central

Henderson, Colin J.; Wolf, C. Roland; Kitteringham, Neil; Powell, Helen; Otto, Diana; Park, B. Kevin

2000-01-01

Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This toxic reaction is associated with metabolic activation by the P450 system to form a quinoneimine metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione, a reaction catalyzed in part by the glutathione S-transferases (GST), such as GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we examined acetaminophen metabolism and liver damage in mice nulled for GstP (GstP1/P2(−/−)). Contrary to our expectations, instead of being more sensitive, GstP null mice were highly resistant to the hepatotoxic effects of this compound. No significant differences between wild-type (GstP1/P2(+/+)) mice and GstP1/P2(−/−) nulls in either the rate or route of metabolism, particularly to glutathione conjugates, or in the levels of covalent binding of acetaminophen-reactive metabolites to cellular protein were observed. However, although a similar rapid depletion of hepatic reduced glutathione (GSH) was found in both GstP1/P2(+/+) and GstP1/P2(−/−) mice, GSH levels only recovered in the GstP1/P2(−/−) mice. These data demonstrate that GstP does not contribute in vivo to the formation of glutathione conjugates of acetaminophen but plays a novel and unexpected role in the toxicity of this compound. This study identifies new ways in which GST can modulate cellular sensitivity to toxic effects and suggests that the level of GST Pi may be an important and contributing factor in the sensitivity of patients with acetaminophen-induced hepatotoxicity. PMID:11058152

Biomaterial design for specific cellular interactions: Role of surface functionalization and geometric features

NASA Astrophysics Data System (ADS)

Kolhar, Poornima

The areas of drug delivery and tissue engineering have experienced extraordinary growth in recent years with the application of engineering principles and their potential to support and improve the field of medicine. The tremendous progress in nanotechnology and biotechnology has lead to this explosion of research and development in biomedical applications. Biomaterials can now be engineered at a nanoscale and their specific interactions with the biological tissues can be modulated. Various design parameters are being established and researched for design of drug-delivery carriers and scaffolds to be implanted into humans. Nanoparticles made from versatile biomaterial can deliver both small-molecule drugs and various classes of bio-macromolecules, such as proteins and oligonucleotides. Similarly in the field of tissue engineering, current approaches emphasize nanoscale control of cell behavior by mimicking the natural extracellular matrix (ECM) unlike, traditional scaffolds. Drug delivery and tissue engineering are closely connected fields and both of these applications require materials with exceptional physical, chemical, biological, and biomechanical properties to provide superior therapy. In the current study the surface functionalization and the geometric features of the biomaterials has been explored. In particular, a synthetic surface for culture of human embryonic stem cells has been developed, demonstrating the importance of surface functionalization in maintaining the pluripotency of hESCs. In the second study, the geometric features of the drug delivery carriers are investigated and the polymeric nanoneedles mediated cellular permeabilization and direct cytoplasmic delivery is reported. In the third study, the combined effect of surface functionalization and geometric modification of carriers for vascular targeting is enunciated. These studies illustrate how the biomaterials can be designed to achieve various cellular behaviors and control the interactions with cells in vivo .

Multiscale diffusion of a molecular probe in a crowded environment: a concept

NASA Astrophysics Data System (ADS)

Currie, Megan; Thao, Chang; Timerman, Randi; Welty, Robb; Berry, Brenden; Sheets, Erin D.; Heikal, Ahmed A.

2015-08-01

Living cells are crowded with macromolecules and organelles. Yet, it is not fully understood how macromolecular crowding affects the myriad of biochemical reactions, transport and the structural stability of biomolecules that are essential to cellular function and survival. These molecular processes, with or without electrostatic interactions, in living cells are therefore expected to be distinct from those carried out in test tube in dilute solutions where excluded volumes are absent. Thus there is an urgent need to understand the macromolecular crowding effects on cellular and molecular biophysics towards quantitative cell biology. In this report, we investigated how biomimetic crowding affects both the rotational and translation diffusion of a small probe (rhodamine green, RhG). For biomimetic crowding agents, we used Ficoll-70 (synthetic polymer), bovine serum albumin and ovalbumin (proteins) at various concentrations in a buffer at room temperature. As a control, we carried out similar measurements on glycerolenriched buffer as an environment with homogeneous viscosity as a function of glycerol concentration. The corresponding bulk viscosity was measured independently to test the validity of the Stokes-Einstein model of a diffusing species undergoing a random walk. For rotational diffusion (ps-ns time scale), we used time-resolved anisotropy measurements to examine potential binding of RhG as a function of the crowding agents (surface structure and size). For translational diffusion (μs-s time scale), we used fluorescence correlation spectroscopy for single-molecule fluctuation analysis. Our results allow us to examine the diffusion model of a molecular probe in crowded environments as a function of concentration, length scale, homogeneous versus heterogeneous viscosity, size and surface structures. These biomimetic crowding studies, using non-invasive fluorescence spectroscopy methods, represent an important step towards understanding cellular biophysics and quantitative cell biology.

Co-evolution of proteins and solutions: protein adaptation versus cytoprotective micromolecules and their roles in marine organisms.

PubMed

Yancey, Paul H; Siebenaller, Joseph F

2015-06-01

Organisms experience a wide range of environmental factors such as temperature, salinity and hydrostatic pressure, which pose challenges to biochemical processes. Studies on adaptations to such factors have largely focused on macromolecules, especially intrinsic adaptations in protein structure and function. However, micromolecular cosolutes can act as cytoprotectants in the cellular milieu to affect biochemical function and they are now recognized as important extrinsic adaptations. These solutes, both inorganic and organic, have been best characterized as osmolytes, which accumulate to reduce osmotic water loss. Singly, and in combination, many cosolutes have properties beyond simple osmotic effects, e.g. altering the stability and function of proteins in the face of numerous stressors. A key example is the marine osmolyte trimethylamine oxide (TMAO), which appears to enhance water structure and is excluded from peptide backbones, favoring protein folding and stability and counteracting destabilizers like urea and temperature. Co-evolution of intrinsic and extrinsic adaptations is illustrated with high hydrostatic pressure in deep-living organisms. Cytosolic and membrane proteins and G-protein-coupled signal transduction in fishes under pressure show inhibited function and stability, while revealing a number of intrinsic adaptations in deep species. Yet, intrinsic adaptations are often incomplete, and those fishes accumulate TMAO linearly with depth, suggesting a role for TMAO as an extrinsic 'piezolyte' or pressure cosolute. Indeed, TMAO is able to counteract the inhibitory effects of pressure on the stability and function of many proteins. Other cosolutes are cytoprotective in other ways, such as via antioxidation. Such observations highlight the importance of considering the cellular milieu in biochemical and cellular adaptation. © 2015. Published by The Company of Biologists Ltd.

Improved antimicrobial therapy with cationic tetra- and octa-substituted phthalocyanines

NASA Astrophysics Data System (ADS)

Angelov, I.; Mantareva, V.; Kussovski, V.; Woehrle, D.; Borisova, E.; Avramov, L.

2008-12-01

Photodynamic therapy (PDT) today is an innovative and not yet widespread light-drug initiated treatment that is based on the photoactive compound irradiated with proper light to produce oxygen species that are toxic to the pathogenic biological objects- bacteria, viruses, tumor cells. The obstacles that limited the efficacy of PDT concern to the selectivity and multi-drug resistance prolong time for cellular release and side effects of skin photosensitivity for commercial porphyrin originated photosensitizers (PS). Now there are very intensive investigations for introducing in practice a new, with a least side effects PSs for PDT. The usefulness of the more extended macromolecules structured with proper substituents refers not only to the improved optical properties like far-red and with intensive absorption and emission capacity, but mainly to the ability for selective delivery and adhesion to the target cells, such as bacteria or other pathogens. The present study focuses on the charge effect of photodynamic agent on the uptake capacity toward gram-negative bacteria cells and their further photoinactivation. The multi-drug resistant microorganism Aeromanas hydrophilla, which is causing diseases to fishes and humans, is treated. The new octa-cationic phthalocyanines are designed to compare PDT efficacy to the efficacy of tetra-substituted derivatives with the same functional peripheral substituents. The higher cellular accumulation to the bacteria cells as a result of the high number of positive charges of photosensitizer, leading to the better adhesion to the cellular membranes and improved photoinactivation of bacteria causing superficial and intraorgan infections. These results set a base of a rationale design of covalently octa-substituted phthalocyanines with positive charge for a successful treatment of microorganisms.

A prospective analysis of co-processed non-ionic surfactants in enhancing permeability of a model hydrophilic drug.

PubMed

Alvi, Mohammed M; Chatterjee, Parnali

2014-04-01

Paracellular route is a natural pathway for the transport of many hydrophilic drugs and macromolecules. The purpose of this study was to prospectively evaluate the ability of novel co-processed non-ionic surfactants to enhance the paracellular permeability of a model hydrophilic drug metformin using Caco-2 (human colonic adenocarcinoma) cell model. A three-tier screen was undertaken to evaluate the co-processed blends based on cytotoxicity, cellular integrity, and permeability coefficient. The relative contribution of the paracellular and the transcellular route in overall transport of metformin by co-processed blends was determined. Immunocytochemistry was conducted to determine the distribution of tight-junction protein claudin-1 after incubation with the co-processed blends. It was found that novel blends of Labrasol and Transcutol-P enhanced metformin permeability by approximately twofold with transient reduction in the transepithelia electrical resistance (TEER) and minimal cytotoxicity compared with the control, with the paracellular pathway as the major route of metformin transport. Maximum permeability of metformin (∼10-fold) was mediated by Tween-20 blends along with >75% reduction in the TEER which was irreversible over 24-h period. A shift in metformin transport from the paracellular to the transcellular route was observed with some Tween-20 blends. Immunocytochemical analysis revealed rearrangement of the cellular borders and fragmentation on treatment with Tween-20 blends. In conclusion, cytotoxicity, cellular integrity, and permeability of the hydrophilic drugs can be greatly influenced by the polyoxyethylene residues and medium chain fatty acids in the non-ionic surfactants at clinically relevant concentrations and therefore should be thoroughly investigated prior to their inclusion in formulations.

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

PubMed Central

Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

2006-01-01

Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

Thin-film optical shutter. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matlow, S.L.

1981-02-01

A specific embodiment of macroconjugated macromolecules, the poly (p-phenylene)'s, has been chosen as the one most likely to meet all of the requirements of the Thin Film Optical Shutter project (TFOS). The reason for this choice is included. In order to be able to make meaningful calculations of the thermodynamic and optical properties of the poly (p-phenylene)'s a new quantum mechanical method was developed - Equilibrium Bond Length (EBL) Theory. Some results of EBL Theory are included.

Fast, accurate semiempirical molecular orbital calculations for macromolecules

NASA Astrophysics Data System (ADS)

Dixon, Steven L.; Merz, Kenneth M., Jr.

1997-07-01

A detailed review of the semiempirical divide-and-conquer (D&C) method is given, including a new approach to subsetting, which involves dual buffer regions. Comparisons are drawn between this method and other semiempirical macromolecular schemes. D&C calculations are carried out using a basic 32 Mbyte memory workstation on a variety of peptide systems, including proteins containing up to 1960 atoms. Aspects of storage and SCF convergence are addressed, and parallelization of the D&C algorithm is discussed.

Recent in vivo advances in cell-penetrating peptide-assisted drug delivery.

PubMed

Kurrikoff, Kaido; Gestin, Maxime; Langel, Ülo

2016-01-01

Delivery of macromolecular drugs is an important field in medical research. However, macromolecules are usually unable to cross the cell membrane without the assistance of a delivery system. Cell penetrating peptides (CPPs) are unique tools to gain access to the cell interior and deliver a bioactive cargo into the cytosol or nucleus. In addition to macromolecular delivery, CPPs have been used to deliver smaller bioactive molecules. Therefore CPPs have become an intensive field of research for medical treatment. In this review, we highlight studies that include CPP in vivo disease models. We review different strategies and approaches that have been used, with specific attention on recent publications. The approaches that have been used include CPP-cargo covalent conjugation strategies and nanoparticle strategies. Various additional strategies have been used to achieve disease targeting, including active targeting, passive targeting, and combined active/passive strategies. As a result, delivery of various types of molecule has been achieved, including small drug molecules, proteins and nucleic acid-based macromolecules (e.g. siRNA, antisense nucleotides and plasmid DNA). Despite recent advances in the field, confusions surrounding CPP internalization mechanisms and intracellular trafficking are hindering the development of new and more efficient vectors. Nevertheless, the recent increase in the number of publications containing in vivo CPP utilization looks promising that the number of clinical trials would also increase in the near future.

Stress-tolerance of baker's-yeast (Saccharomyces cerevisiae) cells: stress-protective molecules and genes involved in stress tolerance.

PubMed

Shima, Jun; Takagi, Hiroshi

2009-05-29

During the fermentation of dough and the production of baker's yeast (Saccharomyces cerevisiae), cells are exposed to numerous environmental stresses (baking-associated stresses) such as freeze-thaw, high sugar concentrations, air-drying and oxidative stresses. Cellular macromolecules, including proteins, nucleic acids and membranes, are seriously damaged under stress conditions, leading to the inhibition of cell growth, cell viability and fermentation. To avoid lethal damage, yeast cells need to acquire a variety of stress-tolerant mechanisms, for example the induction of stress proteins, the accumulation of stress protectants, changes in membrane composition and repression of translation, and by regulating the corresponding gene expression via stress-triggered signal-transduction pathways. Trehalose and proline are considered to be critical stress protectants, as is glycerol. It is known that these molecules are effective for providing protection against various types of environmental stresses. Modifications of the metabolic pathways of trehalose and proline by self-cloning methods have significantly increased tolerance to baking-associated stresses. To clarify which genes are required for stress tolerance, both a comprehensive phenomics analysis and a functional genomics analysis were carried out under stress conditions that simulated those occurring during the commercial baking process. These analyses indicated that many genes are involved in stress tolerance in yeast. In particular, it was suggested that vacuolar H+-ATPase plays important roles in yeast cells under stress conditions.

Localization and expression of MreB in Vibrio parahaemolyticus under different stresses.

PubMed

Chiu, Shen-Wen; Chen, Shau-Yan; Wong, Hin-chung

2008-11-01

MreB, the homolog of eukaryotic actin, may play a vital role when prokaryotes cope with stress by altering their spatial organization, including their morphology, subcellular architecture, and localization of macromolecules. This study investigates the behavior of MreB in Vibrio parahaemolyticus under various stresses. The behavior of MreB was probed using a yellow fluorescent protein-MreB conjugate in merodiploid strain SC9. Under normal growth conditions, MreB formed helical filaments in exponential-phase cells. The shape of starved or stationary-phase cells changed from rods to small spheroids. The cells differentiated into the viable but nonculturable (VBNC) state with small spherical cells via a "swelling-waning" process. In all cases, drastic remodeling of the MreB cytoskeleton was observed. MreB helices typically were loosened and fragmented into short filaments, arcs, and spots in bacteria under these stresses. The disintegrated MreB exhibited a strong tendency to attach to the cytoplasmic membrane. The expression of mreB generally declined in bacteria in the stationary phase and under starvation but was upregulated during the initial periods of cold shock and VBNC state differentiation and decreased afterwards. Our findings demonstrated the behavior of MreB in the morphological changes of V. parahaemolyticus under intrinsic or extrinsic stresses and may have important implications for studying the cellular stress response and aging.

Updating the Mitochondrial Free Radical Theory of Aging: An Integrated View, Key Aspects, and Confounding Concepts

PubMed Central

2013-01-01

Abstract An updated version of the mitochondrial free radical theory of aging (MFRTA) and longevity is reviewed. Key aspects of the theory are emphasized. Another main focus concerns common misconceptions that can mislead investigators from other specialties, even to wrongly discard the theory. Those different issues include (i) the main reactive oxygen species (ROS)-generating site in the respiratory chain in relation to aging and longevity: complex I; (ii) the close vicinity or even contact between that site and the mitochondrial DNA, in relation to the lack of local efficacy of antioxidants and to sub-cellular compartmentation; (iii) the relationship between mitochondrial ROS production and oxygen consumption; (iv) recent criticisms on the MFRTA; (v) the widespread assumption that ROS are simple “by-products” of the mitochondrial respiratory chain; (vi) the unnecessary postulation of “vicious cycle” hypotheses of mitochondrial ROS generation which are not central to the free radical theory of aging; and (vii) the role of DNA repair concerning endogenous versus exogenous damage. After considering the large body of data already available, two general characteristics responsible for the high maintenance degree of long-lived animals emerge: (i) a low generation rate of endogenous damage: and (ii) the possession of tissue macromolecules that are highly resistant to oxidative modification. Antioxid. Redox Signal. 19, 1420–1445. PMID:23642158

Effects of soft interactions and bound mobility on diffusion in crowded environments: a model of sticky and slippery obstacles

NASA Astrophysics Data System (ADS)

Stefferson, Michael W.; Norris, Samantha L.; Vernerey, Franck J.; Betterton, Meredith D.; E Hough, Loren

2017-08-01

Crowded environments modify the diffusion of macromolecules, generally slowing their movement and inducing transient anomalous subdiffusion. The presence of obstacles also modifies the kinetics and equilibrium behavior of tracers. While previous theoretical studies of particle diffusion have typically assumed either impenetrable obstacles or binding interactions that immobilize the particle, in many cellular contexts bound particles remain mobile. Examples include membrane proteins or lipids with some entry and diffusion within lipid domains and proteins that can enter into membraneless organelles or compartments such as the nucleolus. Using a lattice model, we studied the diffusive movement of tracer particles which bind to soft obstacles, allowing tracers and obstacles to occupy the same lattice site. For sticky obstacles, bound tracer particles are immobile, while for slippery obstacles, bound tracers can hop without penalty to adjacent obstacles. In both models, binding significantly alters tracer motion. The type and degree of motion while bound is a key determinant of the tracer mobility: slippery obstacles can allow nearly unhindered diffusion, even at high obstacle filling fraction. To mimic compartmentalization in a cell, we examined how obstacle size and a range of bound diffusion coefficients affect tracer dynamics. The behavior of the model is similar in two and three spatial dimensions. Our work has implications for protein movement and interactions within cells.

Oxidative stress in obstructive sleep apnea and intermittent hypoxia--revisited--the bad ugly and good: implications to the heart and brain.

PubMed

Lavie, Lena

2015-04-01

Obstructive sleep apnea (OSA), characterized by intermittent hypoxia (IH), is linked with increased reactive oxygen species/reactive nitrogen species (ROS/RNS) and oxidative stress, which adversely affect the associated cardio-/cerebro-vascular disease in OSA. Yet, animal and a small number of human studies support activation of cardio-/cerebro-protective mechanisms as well. ROS/RNS are intricate and multifaceted molecules with multiple functions. At low-moderate concentrations ROS/RNS are considered "good", by regulating vital cellular functions. At higher levels, they are considered "bad" by promoting oxidative stress and damaging vital macromolecules through ischemia and reperfusion (I/R) injury. Subsequently, ROS/RNS can get "ugly" by eliciting sterile inflammation and a multitude of deadly pathologies. What makes ROS/RNS good, bad, or ugly? A dynamic interplay between a large number of factors determines the outcomes. These include the types of ROS/RNS produced, their quantity, duration, frequency, intracellular localization, micro-environmental antioxidants, as well as the genetic make-up and life style related variables. This review presents the currently available data on redox biology in physiological/pathophysiological conditions and in OSA/IH, in order to better understand the apparently contradictory findings on damage vs. repair. These findings are discussed within the context of the prevailing views on I/R associated ROS/RNS, and their potential implications to OSA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of gold nanoparticle mediated photoporation: vapor nanobubbles outperform direct heating for delivering macromolecules in live cells.

PubMed

Xiong, Ranhua; Raemdonck, Koen; Peynshaert, Karen; Lentacker, Ine; De Cock, Ine; Demeester, Jo; De Smedt, Stefaan C; Skirtach, Andre G; Braeckmans, Kevin

2014-06-24

There is a great interest in delivering macromolecular agents into living cells for therapeutic purposes, such as siRNA for gene silencing. Although substantial effort has gone into designing nonviral nanocarriers for delivering macromolecules into cells, translocation of the therapeutic molecules from the endosomes after endocytosis into the cytoplasm remains a major bottleneck. Laser-induced photoporation, especially in combination with gold nanoparticles, is an alternative physical method that is receiving increasing attention for delivering macromolecules in cells. By allowing gold nanoparticles to bind to the cell membrane, nanosized membrane pores can be created upon pulsed laser illumination. Depending on the laser energy, pores are created through either direct heating of the AuNPs or by vapor nanobubbles (VNBs) that can emerge around the AuNPs. Macromolecules in the surrounding cell medium can then diffuse through the pores directly into the cytoplasm. Here we present a systematic evaluation of both photoporation mechanisms in terms of cytotoxicity, cell loading, and siRNA transfection efficiency. We find that the delivery of macromolecules under conditions of VNBs is much more efficient than direct photothermal disturbance of the plasma membrane without any noticeable cytotoxic effect. Interestingly, by tuning the laser energy, the pore size could be changed, allowing control of the amount and size of molecules that are delivered in the cytoplasm. As only a single nanosecond laser pulse is required, we conclude that VNBs are an interesting photoporation mechanism that may prove very useful for efficient high-throughput macromolecular delivery in live cells.

Macromolecular Competition Titration Method: Accessing Thermodynamics of the Unmodified Macromolecule–Ligand Interactions Through Spectroscopic Titrations of Fluorescent Analogs

PubMed Central

Bujalowski, Wlodzimierz; Jezewska, Maria J.

2011-01-01

Analysis of thermodynamically rigorous binding isotherms provides fundamental information about the energetics of the ligand–macromolecule interactions and often an invaluable insight about the structure of the formed complexes. The Macromolecular Competition Titration (MCT) method enables one to quantitatively obtain interaction parameters of protein–nucleic acid interactions, which may not be available by other methods, particularly for the unmodified long polymer lattices and specific nucleic acid substrates, if the binding is not accompanied by adequate spectroscopic signal changes. The method can be applied using different fluorescent nucleic acids or fluorophores, although the etheno-derivatives of nucleic acid are especially suitable as they are relatively easy to prepare, have significant blue fluorescence, their excitation band lies far from the protein absorption spectrum, and the modification eliminates the possibility of base pairing with other nucleic acids. The MCT method is not limited to the specific size of the reference nucleic acid. Particularly, a simple analysis of the competition titration experiments is described in which the fluorescent, short fragment of nucleic acid, spanning the exact site-size of the protein–nucleic acid complex, and binding with only a 1:1 stoichiometry to the protein, is used as a reference macromolecule. Although the MCT method is predominantly discussed as applied to studying protein–nucleic acid interactions, it can generally be applied to any ligand–macromolecule system by monitoring the association reaction using the spectroscopic signal originating from the reference macromolecule in the presence of the competing macromolecule, whose interaction parameters with the ligand are to be determined. PMID:21195223

[Dynamics of biomacromolecules in coherent electromagnetic radiation field].

PubMed

Leshcheniuk, N S; Apanasevich, E E; Tereshenkov, V I

2014-01-01

It is shown that induced oscillations and periodic displacements of the equilibrium positions occur in biomacromolecules in the absence of electromagnetic radiation absorption, due to modulation of interaction potential between atoms and groups of atoms forming the non-valence bonds in macromolecules by the external electromagnetic field. Such "hyperoscillation" state causes inevitably the changes in biochemical properties of macromolecules and conformational transformation times.

Solid-State 15N NMR of 15N-Labeled Nylon 6 and Nylon 11

DTIC Science & Technology

1990-05-22

S. Veeman, E. M. Menger, W. Ritchey, and E. de Boer, Macromolecules, 1979, 12, 924. 2. A. N. Garroway , W. M. Ritchey and W. B. Moniz, Macromolecules...S. Veeman and E. M. Menger, Bull. Magn. Reson., 1980, 2, 77. 26. D. L. VanderHart and A. N. Garroway , J. Chem. Phys., 1979, 71, 2773. 27. M. D

Formation and characterization of calcium orthophosphates in the presence of two different acidic macromolecules

NASA Astrophysics Data System (ADS)

Pelin, Irina M.; Maier, Vasilica; Suflet, Dana M.; Popescu, Irina; Darie-Nita, Raluca N.; Aflori, Magdalena; Butnaru, Maria

2017-10-01

The synthetic nanocrystalline calcium orthophosphates have a notable bioactivity due to the chemical similarity with biological apatite from calcified tissues. In mineralized tissues, the highly ordered structures come from organized assemblies of biomacromolecules and inorganic nanoparticles. One of the purposes of this work was to study the effect of two types of acidic macromolecules: atelocollagen and phosphorylated curdlan onto calcium orthophosphates formation after 30 days of maturation at 2 ± 2 °C. The resulted samples after a long aging time, either calcium orthophosphates or composites, were first investigated by FT-IR spectroscopy and X-ray diffractometry and the results indicated that precipitated hydroxyapatite with low crystallinity was obtained when the synthesis was performed in the presence of phosphorylated curdlan. The macromolecules influenced the morphology of the particles as shown by scanning and transmission electron microscopy. The presence of macromolecules as demonstrated by thermal investigation also influenced the rheological properties of the samples. The second purpose of the work was to evaluate the cytotoxicity of the samples using the MTT assay, and the results revealed very good cells viability. The preliminary results are encouraging regarding the use of these materials for further tests in order to develop injectable bone substitutes.

Singlet Molecular Oxygen Generation by Light-Activated DHN-Melanin of the Fungal Pathogen Mycosphaerella fijiensis in Black Sigatoka Disease of Bananas

PubMed Central

Beltrán-García, Miguel J.; Prado, Fernanda M.; Oliveira, Marilene S.; Ortiz-Mendoza, David; Scalfo, Alexsandra C.; Pessoa, Adalberto; Medeiros, Marisa H. G.; White, James F.; Di Mascio, Paolo

2014-01-01

In pathogenic fungi, melanin contributes to virulence, allowing tissue invasion and inactivation of the plant defence system, but has never been implicated as a factor for host cell death, or as a light-activated phytotoxin. Our research shows that melanin synthesized by the fungal banana pathogen Mycosphaerella fijiensis acts as a virulence factor through the photogeneration of singlet molecular oxygen O2 (1Δg). Using analytical tools, including elemental analysis, ultraviolet/infrared absorption spectrophometry and MALDI-TOF mass spectrometry analysis, we characterized both pigment content in mycelia and secreted to the culture media as 1,8-dihydroxynaphthalene (DHN)-melanin type compound. This is sole melanin-type in M. fijiensis. Isolated melanins irradiated with a Nd:YAG laser at 532 nm produced monomol light emission at 1270 nm, confirming generation of O2 (1Δg), a highly reactive oxygen specie (ROS) that causes cellular death by reacting with all cellular macromolecules. Intermediary polyketides accumulated in culture media by using tricyclazole and pyroquilon (two inhibitors of DHN-melanin synthesis) were identified by ESI-HPLC-MS/MS. Additionally, irradiation at 532 nm of that mixture of compounds and whole melanized mycelium also generated O2 (1Δg). A pigmented-strain generated more O2 (1Δg) than a strain with low melanin content. Banana leaves of cultivar Cavendish, naturally infected with different stages of black Sigatoka disease, were collected from field. Direct staining of the naturally infected leaf tissues showed the presence of melanin that was positively correlated to the disease stage. We also found hydrogen peroxide (H2O2) but we cannot distinguish the source. Our results suggest that O2 (1Δg) photogenerated by DHN-melanin may be involved in the destructive effects of Mycosphaerella fijiensis on banana leaf tissues. Further studies are needed to fully evaluate contributions of melanin-mediated ROS to microbial pathogenesis. PMID:24646830

From the Cover: Disease-Induced Disparities in Formation of the Nanoparticle-Biocorona and the Toxicological Consequences

PubMed Central

Shannahan, Jonathan H.; Fritz, Kristofer S.; Raghavendra, Achyut J.; Podila, Ramakrishna; Persaud, Indushekar; Brown, Jared M.

2016-01-01

Nanoparticle (NP) association with macromolecules in a physiological environment forms a biocorona (BC), which alters NP distribution, activity, and toxicity. While BC formation is dependent on NP physicochemical properties, little information exists on the influence of the physiological environment. Obese individuals and those with cardiovascular disease exist with altered serum chemistry, which is expected to influence BC formation and NP toxicity. We hypothesize that a BC formed on NPs following incubation in hyperlipidemic serum will result in altered NP–BC protein content, cellular association, and toxicity compared to normal serum conditions. We utilized Fe3O4 NPs, which are being developed as MRI contrast and tumor targeting agents to test our hypothesis. We used rat aortic endothelial cells (RAECs) within a dynamic flow in vitro exposure system to more accurately depict the in vivo environment. A BC was formed on 20nm PVP-suspended Fe3O4 NPs following incubation in water, 10% normal or hyperlipidemic rat serum. Addition of BCs resulted in increased hydrodynamic size and decreased surface charge. More cholesterol associated with Fe3O4 NPs after incubation in hyperlipidemic as compared with normal serum. Using quantitative proteomics, we identified unique differences in BC protein components between the 2 serum types. Under flow conditions, formation of a BC from both serum types reduced RAECs association of Fe3O4 NPs. Addition of BCs was found to exacerbate RAECs inflammatory gene responses to Fe3O4 NPs (Fe3O4-hyperlipidemic > Fe3O4-normal > Fe3O4) including increased expression of IL-6, TNF-α, Cxcl-2, VCAM-1, and ICAM-1. Overall, these findings demonstrate that disease-induced variations in physiological environments have a significant impact NP-BC formation, cellular association, and cell response. PMID:27255384

Coagulation Factor XIIIA (F13A1): Novel Perspectives in Treatment and Pharmacogenetics.

PubMed

Gemmati, Donato; Vigliano, Marco; Burini, Francesco; Mari, Rosella; El Mohsein, Hodeib Hossam Abd; Parmeggiani, Francesco; Serino, Maria L

2016-01-01

Factor XIII (FXIII) is a key molecule in the field of blood coagulation and in the last decades it has weakened attention within the field of angiogenesis and tissue repair. FXIII positively influences wound healing in several tissues by exerting multiple plasma and cellular functions. In the field of haemostasis, FXIII cross-links the neo formed fibrin fibers and supports platelet adhesion to the damaged sub-endothelium warranting a solid architecture. In addition, the pro-angiogenic functions of FXIII are directed by the interaction of vascular endothelial growth factor receptor 2 (VEGFR2) and the integrin αVβ3, on the cell membrane, favouring an important step in the formation of granulation tissue at the wound site for optimal tissue healing. Conversely, the same mechanisms could lead to undesired increased neovascularisation, for example in inflammatory bowel disease or in the retinal degenerative pathologies. The classical symptoms of FXIII deficiency span from intracranial haemorrhage to delay bleeding or the staying of chronic wounds in the skin including impaired mucosal healing. In this view, FXIII bridges primary haemostasis, coagulation and definite tissue healing. Another important recently discovered function ascribed to FXIII is its ability to limit bacterial spreading from the lesion by incorporating specific macromolecules addressed to cellular infiltration, favouring in turn cell migration and survival, as observed also in fibrin-heart cultures for stem cell recruitment. In the field of the novel prognostic biomarkers, the monitoring of the residual circulating FXIII level during acute myocardial infarction has been considered predictive of the post-myocardial infarction healing. Accordingly, adequate FXIII levels can drive and predict the prognosis of complex diseases and the outcome of the associated therapies or interventions. In addition, peculiar pharmacogenetics aspects of the FXIII gene are of extraordinary interest. The present review accounts for the recognized role of FXIII in the healing process and gives some examples on how to use it as prognostic biological/ molecular marker or as potential tailored therapeutic molecule in complex diseases.

Impact Mediated Loading Cytoplasmic Loading of Macromolecules into Adherent Cells

NASA Technical Reports Server (NTRS)

Clarke, Mark S. F.; Feeback, Daniel L.; Vanderburg, Charles R.

2003-01-01

The advent of modern molecular biology, including the development of gene array technologies, has resulted in an explosion of information concerning the specific genes activated during normal cellular development, as well as those associated with a variety of pathological conditions. These techniques have served as a highly efficient, broacI.-based screening approach for those specific genes involved. in regulating normal cellular physiology and identifying candidate genes directly associated with the etiology of specific disease states. However, this approach provides information at the transcriptional' level only and does not necessarily indicate . that the gene in question is in fact translated ito a protein, or whether or not post-translational modification of the protein occurs. The critical importance of post-translational modification (i.e. phosphorylation, glycosylation, sialyation, etc.) to protein function has been recognized with regard to a number of proteins involved in a variety of important disease states. For example, altered glycosylation of beta-amyloid precursor protein results in an increase in the amount of beta-amyloid peptide generated and hence secreted as insoluble extracellular amyloid deposits (Georgopoulou, McLaughlin et al. 2001; Walter, Fluhrer et al. 2001), a pathological hal1nark of Alzheimer's disease. Abnormal phosphorylaion of synapsin I has been linked to alterations in synaptic vesicle trafficking leading to defective neurotransmission in Huntington's disease (Lievens, Woodman et al. 2002). Altered phosphorylation of the TAU protein involved in microtubule function has been linked to a number of neurodegenative diseases such as Alzheimer's disease (Billingsley and Kincaid 1997; Sanchez, Alvarez-Tllada et a1. 2001). Aberrant siaIyation of cell/I surface antigens has been detected in a number of different tumor cell types and has been linked to the acquisition of a neoplastic phenotype (Sell 1990), while improper' sia1yation of sodium channels in cardiac tissue has been linked to heart failure (Ufret-Vincenty, Baro et al. 2001; Fozzard and Kyle 2002).

Renew or die: The molecular mechanisms of peptidoglycan recycling and antibiotic resistance in Gram-negative pathogens.

PubMed

Domínguez-Gil, Teresa; Molina, Rafael; Alcorlo, Martín; Hermoso, Juan A

2016-09-01

Antimicrobial resistance is one of the most serious health threats. Cell-wall remodeling processes are tightly regulated to warrant bacterial survival and in some cases are directly linked to antibiotic resistance. Remodeling produces cell-wall fragments that are recycled but can also act as messengers for bacterial communication, as effector molecules in immune response and as signaling molecules triggering antibiotic resistance. This review is intended to provide state-of-the-art information about the molecular mechanisms governing this process and gather structural information of the different macromolecular machineries involved in peptidoglycan recycling in Gram-negative bacteria. The growing body of literature on the 3D structures of the corresponding macromolecules reveals an extraordinary complexity. Considering the increasing incidence and widespread emergence of Gram-negative multidrug-resistant pathogens in clinics, structural information on the main actors of the recycling process paves the way for designing novel antibiotics disrupting cellular communication in the recycling-resistance pathway. Copyright © 2016. Published by Elsevier Ltd.

Giant viruses: The difficult breaking of multiple epistemological barriers.

PubMed

Claverie, Jean-Michel; Abergel, Chantal

2016-10-01

The discovery of the first "giant virus", Mimivirus, in 2003 could solely have been that of an exceptional freak, a blind alley of evolution as occasionally encountered in biology, albeit without conceptual significance. On the contrary, once broken this epistemological barrier, additional unrelated families of giant viruses such as the Pandoraviruses, the Pithoviruses and most recently Mollivirus, were quickly unraveled, suggesting that an entire chapter of microbiology had been ignored since Pasteur and Ivanovski. In this article, we examine to what extent the giant viruses challenge previous definitions of viruses, the diversity of forms they could take, and how they might have evolved from extinct ancestral cellular lineages. Inspired by the epistemology of Gaston Bachelard, we will also suggest the reasons for which giant viruses laid hidden in plain sight for more than a century. Finally, we propose a new definition for "viruses" that paradoxically emphasize the fact that they do not encode a single universally shared macromolecule or biochemical function. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Digital Quantification of Proteins and mRNA in Single Mammalian Cells.

PubMed

Albayrak, Cem; Jordi, Christian A; Zechner, Christoph; Lin, Jing; Bichsel, Colette A; Khammash, Mustafa; Tay, Savaş

2016-03-17

Absolute quantification of macromolecules in single cells is critical for understanding and modeling biological systems that feature cellular heterogeneity. Here we show extremely sensitive and absolute quantification of both proteins and mRNA in single mammalian cells by a very practical workflow that combines proximity ligation assay (PLA) and digital PCR. This digital PLA method has femtomolar sensitivity, which enables the quantification of very small protein concentration changes over its entire 3-log dynamic range, a quality necessary for accounting for single-cell heterogeneity. We counted both endogenous (CD147) and exogenously expressed (GFP-p65) proteins from hundreds of single cells and determined the correlation between CD147 mRNA and the protein it encodes. Using our data, a stochastic two-state model of the central dogma was constructed and verified using joint mRNA/protein distributions, allowing us to estimate transcription burst sizes and extrinsic noise strength and calculate the transcription and translation rate constants in single mammalian cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Investigating molecular crowding within nuclear pores using polarization-PALM

PubMed Central

Fu, Guo; Tu, Li-Chun; Zilman, Anton

2017-01-01

The key component of the nuclear pore complex (NPC) controlling permeability, selectivity, and the speed of nucleocytoplasmic transport is an assembly of natively unfolded polypeptides, which contain phenylalanine-glycine (FG) binding sites for nuclear transport receptors. The architecture and dynamics of the FG-network have been refractory to characterization due to the paucity of experimental methods able to probe the mobility and density of the FG-polypeptides and embedded macromolecules within intact NPCs. Combining fluorescence polarization, super-resolution microscopy, and mathematical analyses, we examined the rotational mobility of fluorescent probes at various locations within the FG-network under different conditions. We demonstrate that polarization PALM (p-PALM) provides a rich source of information about low rotational mobilities that are inaccessible with bulk fluorescence anisotropy approaches, and anticipate that p-PALM is well-suited to explore numerous crowded cellular environments. In total, our findings indicate that the NPC’s internal organization consists of multiple dynamic environments with different local properties. PMID:28949296

Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface

PubMed Central

Siegrist, M. Sloan; Swarts, Benjamin M.; Fox, Douglas M.; Lim, Shion An; Bertozzi, Carolyn R.

2015-01-01

The cell surface is the essential interface between a bacterium and its surroundings. Composed primarily of molecules that are not directly genetically encoded, this highly dynamic structure accommodates the basic cellular processes of growth and division as well as the transport of molecules between the cytoplasm and the extracellular milieu. In this review, we describe aspects of bacterial growth, division and secretion that have recently been uncovered by metabolic labeling of the cell envelope. Metabolite derivatives can be used to label a variety of macromolecules, from proteins to non-genetically-encoded glycans and lipids. The embedded metabolite enables precise tracking in time and space, and the versatility of newer chemoselective detection methods offers the ability to execute multiple experiments concurrently. In addition to reviewing the discoveries enabled by metabolic labeling of the bacterial cell envelope, we also discuss the potential of these techniques for translational applications. Finally, we offer some guidelines for implementing this emerging technology. PMID:25725012

The molecular mechanism of nuclear transport revealed by atomic-scale measurements

PubMed Central

Hough, Loren E; Dutta, Kaushik; Sparks, Samuel; Temel, Deniz B; Kamal, Alia; Tetenbaum-Novatt, Jaclyn; Rout, Michael P; Cowburn, David

2015-01-01

Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG)-rich repeats line the pore and interact with TFs. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with TFs. We used nuclear magnetic resonance spectroscopy to address these issues. We show that FG repeats are highly dynamic IDPs, stabilized by the cellular environment. Fast transport of TFs is supported because the rapid motion of FG motifs allows them to exchange on and off TFs extremely quickly through transient interactions. Because TFs uniquely carry multiple pockets for FG repeats, only they can form the many frequent interactions needed for specific passage between FG repeats to cross the NPC. DOI: http://dx.doi.org/10.7554/eLife.10027.001 PMID:26371551

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

PubMed Central

Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J.; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I.

2015-01-01

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro. PMID:25736020

Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.

PubMed

Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I

2015-03-04

Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thornton-Manning, J.R.; Hotchkiss, J.A.; Ding, Xinxin

The nasal mucosa, the first tissue of contact for inhaled xenobiotics, possesses substantial enobiotic-metabolizing capacti. Enzymes of the nasal cavity may metabolize xenobiotics to innocuous, more water-soluble compounds that are eliminated from the body, or they may bioactivate them to toxic metabolites. These toxic metabolites may find to cellular macromolecules in the nasal cavity or be transported to other parts of the body where they may react. Nasal carcinogenesis in rodents often results from bioactivation of xenobiotics. The increased incidences of nasal tumors associated with certain occupations suggest that xenobiotic bioactivation may be important in human nasal cancer etiology, asmore » well. The increasing popularity of the nose as a route of drug administration makes information concerning nasal drug metabolism and disposition vital to accomplish therapeutic goals. For these reasons, the study of xenobiotic-met abolizing capacity of the nasal cavity is an important area of health-related research. In the present study, we have confirmed the presence of CYP2A6 mRNA in human respiratory mucosa.« less

Reverse depletion effects and the determination of ligand density on some spherical bioparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Chunxiang; Liu, Yanhui, E-mail: ionazati@itp.ac.cn; Fan, Yangtao

In cell environments crowded with macromolecules, the depletion effects act and assist in the assembly of a wide range of cellular structures, from the cytoskeleton to the chromatin loop, which are well accepted. But a recent quantum dot experiment indicated that the dimensions of the receptor–ligand complex have strong effects on the size-dependent exclusion of proteins in cell environments. In this article, a continuum elastic model is constructed to resolve the competition between the dimension of the receptor–ligand complex and depletion effects in the endocytosis of a spherical virus-like bioparticle. Our results show that the depletion effects do not alwaysmore » assist endocytosis of a spherical virus-like bioparticle; while the dimension of the ligand–receptor complex is larger than the size of a small bioparticle in cell environments, the depletion effects do not work and reverse effects appear. The ligand density covered on the virus can be identified quantitatively.« less

Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves.

PubMed

Nicolas, William J; Grison, Magali S; Trépout, Sylvain; Gaston, Amélia; Fouché, Mathieu; Cordelières, Fabrice P; Oparka, Karl; Tilsner, Jens; Brocard, Lysiane; Bayer, Emmanuelle M

2017-06-12

Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.

Quantifying quality in DNA self-assembly

PubMed Central

Wagenbauer, Klaus F.; Wachauf, Christian H.; Dietz, Hendrik

2014-01-01

Molecular self-assembly with DNA is an attractive route for building nanoscale devices. The development of sophisticated and precise objects with this technique requires detailed experimental feedback on the structure and composition of assembled objects. Here we report a sensitive assay for the quality of assembly. The method relies on measuring the content of unpaired DNA bases in self-assembled DNA objects using a fluorescent de-Bruijn probe for three-base ‘codons’, which enables a comparison with the designed content of unpaired DNA. We use the assay to measure the quality of assembly of several multilayer DNA origami objects and illustrate the use of the assay for the rational refinement of assembly protocols. Our data suggests that large and complex objects like multilayer DNA origami can be made with high strand integration quality up to 99%. Beyond DNA nanotechnology, we speculate that the ability to discriminate unpaired from paired nucleic acids in the same macromolecule may also be useful for analysing cellular nucleic acids. PMID:24751596

A national facility for biological cryo-electron microscopy

PubMed Central

Saibil, Helen R.; Grünewald, Kay; Stuart, David I.

2015-01-01

Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback. PMID:25615867

Toxicokinetics of ethers used as fuel oxygenates.

PubMed

Dekant, W; Bernauer, U; Rosner, E; Amberg, A

2001-10-15

The toxicokinetics and biotransformation of methyl-tert.butyl ether (MTBE), ethyl-tert.butyl ether (ETBE) and tert.amyl-methyl ether (TAME) in rats and humans are summarized. These ethers are used as gasoline additives in large amounts, and thus, a considerable potential for human exposure exists. After inhalation exposure MTBE, ETBE and TAME are rapidly taken up by both rats and humans; after termination of exposure, clearance by exhalation and biotransformation to urinary metabolites is rapid in rats. In humans, clearance by exhalation is slower in comparison to rats. Biotransformation of MTBE and ETBE is both qualitatively and quantitatively similar in humans and rats after inhalation exposure under identical conditions. The extent of biotransformation of TAME is also quantitatively similar in rats and humans; the metabolic pathways, however, are different. The results suggest that reactive and potentially toxic metabolites are not formed during biotransformation of these ethers and that toxic effects of these compounds initiated by covalent binding to cellular macromolecules are unlikely.

ULK1, mammalian target of rapamycin, and mitochondria: linking nutrient availability and autophagy.

PubMed

Kundu, Mondira

2011-05-15

A fundamental function of autophagy conserved from yeast to mammals is mobilization of macromolecules during times of limited nutrient availability, permitting organisms to survive under starvation conditions. In yeast, autophagy is initiated following nitrogen or carbon deprivation, and autophagy mutants die rapidly under these conditions. Similarly, in mammals, autophagy is upregulated in most organs following initiation of starvation, and is critical for survival in the perinatal period following abrupt termination of the placental nutrient supply. The nutrient-sensing kinase, mammalian target of rapamycin, coordinates cellular proliferation and growth with nutrient availability, at least in part by regulating protein synthesis and autophagy-mediated degradation. This review focusses on the regulation of autophagy by Tor, a mammalian target of rapamycin, and Ulk1, a mammalian homolog of Atg1, in response to changes in nutrient availability. Given the importance of mitochondria in maintaining bioenergetic homestasis, and potentially as a source of membrane for autophagosomes during starvation, possible roles for mitochondria in this process are also discussed.

Fetal oxidative stress mechanisms of neurodevelopmental deficits and exacerbation by ethanol and methamphetamine.

PubMed

Wells, Peter G; Bhatia, Shama; Drake, Danielle M; Miller-Pinsler, Lutfiya

2016-06-01

In utero exposure of mouse progeny to alcohol (ethanol, EtOH) and methamphetamine (METH) causes substantial postnatal neurodevelopmental deficits. One emerging pathogenic mechanism underlying these deficits involves fetal brain production of reactive oxygen species (ROS) that alter signal transduction, and/or oxidatively damage cellular macromolecules like lipids, proteins, and DNA, the latter leading to altered gene expression, likely via non-mutagenic mechanisms. Even physiological levels of fetal ROS production can be pathogenic in biochemically predisposed progeny, and ROS formation can be enhanced by drugs like EtOH and METH, via activation/induction of ROS-producing NADPH oxidases (NOX), drug bioactivation to free radical intermediates by prostaglandin H synthases (PHS), and other mechanisms. Antioxidative enzymes, like catalase in the fetal brain, while low, provide critical protection. Oxidatively damaged DNA is normally rapidly repaired, and fetal deficiencies in several DNA repair proteins, including oxoguanine glycosylase 1 (OGG1) and breast cancer protein 1 (BRCA1), enhance the risk of drug-initiated postnatal neurodevelopmental deficits, and in some cases deficits in untreated progeny, the latter of which may be relevant to conditions like autism spectrum disorders (ASD). Risk is further regulated by fetal nuclear factor erythroid 2-related factor 2 (Nrf2), a ROS-sensing protein that upregulates an array of proteins, including antioxidative enzymes and DNA repair proteins. Imbalances between conceptal pathways for ROS formation, versus those for ROS detoxification and DNA repair, are important determinants of risk. Birth Defects Research (Part C) 108:108-130, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Simultaneous optimization of biomolecular energy function on features from small molecules and macromolecules

PubMed Central

Park, Hahnbeom; Bradley, Philip; Greisen, Per; Liu, Yuan; Mulligan, Vikram Khipple; Kim, David E.; Baker, David; DiMaio, Frank

2017-01-01

Most biomolecular modeling energy functions for structure prediction, sequence design, and molecular docking, have been parameterized using existing macromolecular structural data; this contrasts molecular mechanics force fields which are largely optimized using small-molecule data. In this study, we describe an integrated method that enables optimization of a biomolecular modeling energy function simultaneously against small-molecule thermodynamic data and high-resolution macromolecular structural data. We use this approach to develop a next-generation Rosetta energy function that utilizes a new anisotropic implicit solvation model, and an improved electrostatics and Lennard-Jones model, illustrating how energy functions can be considerably improved in their ability to describe large-scale energy landscapes by incorporating both small-molecule and macromolecule data. The energy function improves performance in a wide range of protein structure prediction challenges, including monomeric structure prediction, protein-protein and protein-ligand docking, protein sequence design, and prediction of the free energy changes by mutation, while reasonably recapitulating small-molecule thermodynamic properties. PMID:27766851

Electronic method for autofluorography of macromolecules on two-D matrices

DOEpatents

Davidson, Jackson B.; Case, Arthur L.

1983-01-01

A method for detecting, localizing, and quantifying macromolecules contained in a two-dimensional matrix is provided which employs a television-based position sensitive detection system. A molecule-containing matrix may be produced by conventional means to produce spots of light at the molecule locations which are detected by the television system. The matrix, such as a gel matrix, is exposed to an electronic camera system including an image-intensifier and secondary electron conduction camera capable of light integrating times of many minutes. A light image stored in the form of a charge image on the camera tube target is scanned by conventional television techniques, digitized, and stored in a digital memory. Intensity of any point on the image may be determined from the number at the memory address of the point. The entire image may be displayed on a television monitor for inspection and photographing or individual spots may be analyzed through selected readout of the memory locations. Compared to conventional film exposure methods, the exposure time may be reduced 100-1000 times.

Recent advances in jointed quantum mechanics and molecular mechanics calculations of biological macromolecules: schemes and applications coupled to ab initio calculations.

PubMed

Hagiwara, Yohsuke; Tateno, Masaru

2010-10-20

We review the recent research on the functional mechanisms of biological macromolecules using theoretical methodologies coupled to ab initio quantum mechanical (QM) treatments of reaction centers in proteins and nucleic acids. Since in most cases such biological molecules are large, the computational costs of performing ab initio calculations for the entire structures are prohibitive. Instead, simulations that are jointed with molecular mechanics (MM) calculations are crucial to evaluate the long-range electrostatic interactions, which significantly affect the electronic structures of biological macromolecules. Thus, we focus our attention on the methodologies/schemes and applications of jointed QM/MM calculations, and discuss the critical issues to be elucidated in biological macromolecular systems. © 2010 IOP Publishing Ltd

Method for detecting and diagnosing disease caused by pathological protein aggregation

DOEpatents

Stevens, Fred J.; Myatt, Elizabeth A.; Solomon, Alan

2000-01-01

A method is provided for detecting pathological macromolecules in a patient, comprising obtaining body fluid from the patient, pretreating the body fluid, subjecting the pretreated body fluid to size-exclusion chromatography to create an excluded fluid, and analyzing the excluded fluid to detect macromolecules having a predetermined molecular weight. The method also allows for comparing elution spectra with reference spectra of suspect pathologic proteins.

A Versatile Technique for the In Vivo Imaging of Human Tumor Xenografts Using Near-Infrared Fluorochrome-Conjugated Macromolecule Probes

PubMed Central

Suemizu, Hiroshi; Kawai, Kenji; Higuchi, Yuichiro; Hashimoto, Haruo; Ogura, Tomoyuki; Itoh, Toshio; Sasaki, Erika; Nakamura, Masato

2013-01-01

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging. PMID:24358218

Ice nucleation rates of single protein complexes and single macromolecules

NASA Astrophysics Data System (ADS)

Stratmann, F.; Wex, H.; Niedermeier, D.; Hartmann, S.; Augustin, S.; Clauss, T.; Voigtlaender, J.; Pummer, B.; Grothe, H.

2012-12-01

With our flow-tube LACIS (Leipzig Aerosol cloud Interaction Simulator), we measured immersion freezing of droplets containing biological ice nucleating (IN) agents. From our measurements, we were able to deduce ice nucleation rates for single IN protein complexes (for Snomax) and for IN macromolecules (in the case of Birch pollen). For the measurements, aerosol particles were produced from solutions/suspensions of either Snomax (deadened and partly fractionalized pseudomonas syringae bacteria) or of Birch pollen washing water (BW in the following). All particles were dried and size selected before entering LACIS. In LACIS, particles were activated to droplets, and we measured the fraction of all droplets that froze (F(ice)) as function of temperature. For Snomax, a strong increase in F(ice) was observed around -7 to -10°C, for BW around -19 to -25°C, respectively. After this initial steep increase, F(ice) stayed constant for both examined substances down to -35°C. We found that the values of F(ice) in the plateau region depended on the dry particle size. The initial solution used to generate the particles contained parts of bacteria with ice active protein complexes on them in the case of Snomax, or IN macromolecules in the case of BW (Pummer et al., 2011). We show that the distribution of the IN proteins or IN molecules in the aerosol particles follows the Poisson distribution. With this knowledge, derivation of the ice nucleation rates for single IN protein complexes or for single IN macromolecules is possible. Combining the Poisson distribution with a stochastic model and using the derived nucleation rates, we can reproduce not only our measurements for both examined substances, but also past measurements done for Snomax and even pseudomonas syringae bacteria. As an additional peculiarity, we seem to observe two different macromolecules being ice active for Birch trees growing in Central Europe or Northern Europe, with the latter initiating freezing at slightly warmer temperatures. Pummer, B. G. et al. (2012), Suspendable macromolecules are responsible for ice nucleation activity of birch and conifer pollen, Aerosol Chem. Phys., 12, 2541-2550.

LORES: Low resolution shape program for the calculation of small angle scattering profiles for biological macromolecules in solution

NASA Astrophysics Data System (ADS)

Zhou, J.; Deyhim, A.; Krueger, S.; Gregurick, S. K.

2005-08-01

A program for determining the low resolution shape of biological macromolecules, based on the optimization of a small angle neutron scattering profile to experimental data, is presented. This program, termed LORES, relies on a Monte Carlo optimization procedure and will allow for multiple scattering length densities of complex structures. It is therefore more versatile than utilizing a form factor approach to produce low resolution structural models. LORES is easy to compile and use, and allows for structural modeling of biological samples in real time. To illustrate the effectiveness and versatility of the program, we present four specific biological examples, Apoferritin (shell model), Ribonuclease S (ellipsoidal model), a 10-mer dsDNA (duplex helix) and a construct of a 10-mer DNA/PNA duplex helix (heterogeneous structure). These examples are taken from protein and nucleic acid SANS studies, of both large and small scale structures. We find, in general, that our program will accurately reproduce the geometric shape of a given macromolecule, when compared with the known crystallographic structures. We also present results to illustrate the lower limit of the experimental resolution which the LORES program is capable of modeling. Program summaryTitle of program:LORES Catalogue identifier: ADVC Program summary URL:http://cpc.cs.qub.ac.uk/summaries/ADVC Program obtainable from: CPC Program Library, Queen's University of Belfast, N. Ireland Computer:SGI Origin200, SGI Octane, SGI Linux, Intel Pentium PC Operating systems:UNIX64 6.5 and LINUX 2.4.7 Programming language used:C Memory required to execute with typical data:8 MB No. of lines in distributed program, including test data, etc.:2270 No. of bytes in distributed program, including test data, etc.:13 302 Distribution format:tar.gz External subprograms used:The entire code must be linked with the MATH library

The Science of Pizza: The Molecular Origins of Cheese, Bread, and Digestion Using Interactive Activities for the General Public

ERIC Educational Resources Information Center

Rowat, Amy C.; Rosenberg, Daniel; Hollar, Kathryn A.; Stone, Howard A.

2010-01-01

We describe a presentation on the science of pizza, which is designed for the general public including children ages 6 and older. The presentation focuses on the science of making and digesting cheese and bread. We highlight 4 major scientific themes: (1) how macromolecules such as carbohydrates and proteins are composed of atoms and small…

Induced polymersome formation from a diblock PS-b-PAA polymer via encapsulation of positively charged proteins and peptides.

PubMed

Hvasanov, David; Wiedenmann, Jörg; Braet, Filip; Thordarson, Pall

2011-06-14

In contrast to simple salts or negatively charged macromolecules, positively charged proteins and peptides including cytochrome c (yeast) and poly-L-lysine are efficiently encapsulated while inducing the formation of polymersomes from polystyrene(140)-b-poly(acrylic acid)(48) (PS(140)-b-PAA(48)). This journal is © The Royal Society of Chemistry 2011

Metabolic features of the cell danger response.

PubMed

Naviaux, Robert K

2014-05-01

The cell danger response (CDR) is the evolutionarily conserved metabolic response that protects cells and hosts from harm. It is triggered by encounters with chemical, physical, or biological threats that exceed the cellular capacity for homeostasis. The resulting metabolic mismatch between available resources and functional capacity produces a cascade of changes in cellular electron flow, oxygen consumption, redox, membrane fluidity, lipid dynamics, bioenergetics, carbon and sulfur resource allocation, protein folding and aggregation, vitamin availability, metal homeostasis, indole, pterin, 1-carbon and polyamine metabolism, and polymer formation. The first wave of danger signals consists of the release of metabolic intermediates like ATP and ADP, Krebs cycle intermediates, oxygen, and reactive oxygen species (ROS), and is sustained by purinergic signaling. After the danger has been eliminated or neutralized, a choreographed sequence of anti-inflammatory and regenerative pathways is activated to reverse the CDR and to heal. When the CDR persists abnormally, whole body metabolism and the gut microbiome are disturbed, the collective performance of multiple organ systems is impaired, behavior is changed, and chronic disease results. Metabolic memory of past stress encounters is stored in the form of altered mitochondrial and cellular macromolecule content, resulting in an increase in functional reserve capacity through a process known as mitocellular hormesis. The systemic form of the CDR, and its magnified form, the purinergic life-threat response (PLTR), are under direct control by ancient pathways in the brain that are ultimately coordinated by centers in the brainstem. Chemosensory integration of whole body metabolism occurs in the brainstem and is a prerequisite for normal brain, motor, vestibular, sensory, social, and speech development. An understanding of the CDR permits us to reframe old concepts of pathogenesis for a broad array of chronic, developmental, autoimmune, and degenerative disorders. These disorders include autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD), asthma, atopy, gluten and many other food and chemical sensitivity syndromes, emphysema, Tourette's syndrome, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), epilepsy, suicidal ideation, organ transplant biology, diabetes, kidney, liver, and heart disease, cancer, Alzheimer and Parkinson disease, and autoimmune disorders like lupus, rheumatoid arthritis, multiple sclerosis, and primary sclerosing cholangitis. © 2013. Published by Elsevier B.V. All rights reserved.

Biomolecular Analysis Capability for Cellular and Omics Research on the International Space Station

NASA Technical Reports Server (NTRS)

Guinart-Ramirez, Y.; Cooley, V. M.; Love, J. E.

2016-01-01

International Space Station (ISS) assembly complete ushered a new era focused on utilization of this state-of-the-art orbiting laboratory to advance science and technology research in a wide array of disciplines, with benefits to Earth and space exploration. ISS enabling capability for research in cellular and molecular biology includes equipment for in situ, on-orbit analysis of biomolecules. Applications of this growing capability range from biomedicine and biotechnology to the emerging field of Omics. For example, Biomolecule Sequencer is a space-based miniature DNA sequencer that provides nucleotide sequence data for entire samples, which may be used for purposes such as microorganism identification and astrobiology. It complements the use of WetLab-2 SmartCycler"TradeMark", which extracts RNA and provides real-time quantitative gene expression data analysis from biospecimens sampled or cultured onboard the ISS, for downlink to ground investigators, with applications ranging from clinical tissue evaluation to multigenerational assessment of organismal alterations. And the Genes in Space-1 investigation, aimed at examining epigenetic changes, employs polymerase chain reaction to detect immune system alterations. In addition, an increasing assortment of tools to visualize the subcellular distribution of tagged macromolecules is becoming available onboard the ISS. For instance, the NASA LMM (Light Microscopy Module) is a flexible light microscopy imaging facility that enables imaging of physical and biological microscopic phenomena in microgravity. Another light microscopy system modified for use in space to image life sciences payloads is initially used by the Heart Cells investigation ("Effects of Microgravity on Stem Cell-Derived Cardiomyocytes for Human Cardiovascular Disease Modeling and Drug Discovery"). Also, the JAXA Microscope system can perform remotely controllable light, phase-contrast, and fluorescent observations. And upcoming confocal microscopy capability will allow for optical sectioning of biological tissues to determine microanatomical localization of biomarkers. Furthermore, NASA's geneLAB effort addresses integration of genomic, epigenomic, transcriptomic, proteomic and metabolomic datasets, by applying an innovative open source science platform for multi-investigator high throughput utilization of the ISS. In sum, the expanding ISS capability for analysis of biomolecules is enabling innovative research in a broad spectrum of areas such as cellular and molecular biology, biotechnology, tissue engineering, biomedicine, and Omics, providing manifold benefits for humanity.

The effects of osmotic stress on the structure and function of the cell nucleus.

PubMed

Finan, John D; Guilak, Farshid

2010-02-15

Osmotic stress is a potent regulator of the normal function of cells that are exposed to osmotically active environments under physiologic or pathologic conditions. The ability of cells to alter gene expression and metabolic activity in response to changes in the osmotic environment provides an additional regulatory mechanism for a diverse array of tissues and organs in the human body. In addition to the activation of various osmotically- or volume-activated ion channels, osmotic stress may also act on the genome via a direct biophysical pathway. Changes in extracellular osmolality alter cell volume, and therefore, the concentration of intracellular macromolecules. In turn, intracellular macromolecule concentration is a key physical parameter affecting the spatial organization and pressurization of the nucleus. Hyper-osmotic stress shrinks the nucleus and causes it to assume a convoluted shape, whereas hypo-osmotic stress swells the nucleus to a size that is limited by stretch of the nuclear lamina and induces a smooth, round shape of the nucleus. These behaviors are consistent with a model of the nucleus as a charged core/shell structure pressurized by uneven partition of macromolecules between the nucleoplasm and the cytoplasm. These osmotically-induced alterations in the internal structure and arrangement of chromatin, as well as potential changes in the nuclear membrane and pores are hypothesized to influence gene transcription and/or nucleocytoplasmic transport. A further understanding of the biophysical and biochemical mechanisms involved in these processes would have important ramifications for a range of fields including differentiation, migration, mechanotransduction, DNA repair, and tumorigenesis. (c) 2009 Wiley-Liss, Inc.

Flexible Charged Macromolecules on Mixed Fluid Lipid Membranes: Theory and Monte Carlo Simulations

PubMed Central

Tzlil, Shelly; Ben-Shaul, Avinoam

2005-01-01

Fluid membranes containing charged lipids enhance binding of oppositely charged proteins by mobilizing these lipids into the interaction zone, overcoming the concomitant entropic losses due to lipid segregation and lower conformational freedom upon macromolecule adsorption. We study this energetic-entropic interplay using Monte Carlo simulations and theory. Our model system consists of a flexible cationic polyelectrolyte, interacting, via Debye-Hückel and short-ranged repulsive potentials, with membranes containing neutral lipids, 1% tetravalent, and 10% (or 1%) monovalent anionic lipids. Adsorption onto a fluid membrane is invariably stronger than to an equally charged frozen or uniform membrane. Although monovalent lipids may suffice for binding rigid macromolecules, polyvalent counter-lipids (e.g., phosphatidylinositol 4,5 bisphosphate), whose entropy loss upon localization is negligible, are crucial for binding flexible macromolecules, which lose conformational entropy upon adsorption. Extending Rosenbluth's Monte Carlo scheme we directly simulate polymer adsorption on fluid membranes. Yet, we argue that similar information could be derived from a biased superposition of quenched membrane simulations. Using a simple cell model we account for surface concentration effects, and show that the average adsorption probabilities on annealed and quenched membranes coincide at vanishing surface concentrations. We discuss the relevance of our model to the electrostatic-switch mechanism of, e.g., the myristoylated alanine-rich C kinase substrate protein. PMID:16126828

Lost in Transit: Long-Distance Trafficking and Phloem Unloading of Protein Signals in Arabidopsis Homografts[OPEN

PubMed Central

Gustin, Marie-Paule; Molnar, Attila; Oparka, Karl J.

2016-01-01

In addition to moving sugars and nutrients, the phloem transports many macromolecules. While grafting and aphid stylectomy experiments have identified many macromolecules that move in the phloem, the functional significance of phloem transport of these remains unclear. To gain insight into protein trafficking, we micrografted Arabidopsis thaliana scions expressing GFP-tagged chloroplast transit peptides under the 35S promoter onto nontransgenic rootstocks. We found that plastids in the root tip became fluorescent 10 d after grafting. We obtained identical results with the companion cell-specific promoter SUC2 and with signals that target proteins to peroxisomes, actin, and the nucleus. We were unable to detect the respective mRNAs in the rootstock, indicating extensive movement of proteins in the phloem. Outward movement from the root protophloem was restricted to the pericycle-endodermis boundary, identifying plasmodesmata at this interface as control points in the exchange of macromolecules between stele and cortex. Intriguingly, signals directing proteins to the endoplasmic reticulum and Golgi apparatus from membrane-bound ribosomes were not translocated to the root. It appears that many organelle-targeting sequences are insufficient to prevent the loss of their proteins into the translocation stream. Thus, nonspecific loss of proteins from companion cells to sieve elements may explain the plethora of macromolecules identified in phloem sap. PMID:27600534

Using a water-confined carbon nanotube to probe the electricity of sequential charged segments of macromolecules

NASA Astrophysics Data System (ADS)

Wang, Yu; Zhao, Yan-Jiao; Huang, Ji-Ping

2012-07-01

The detection of macromolecular conformation is particularly important in many physical and biological applications. Here we theoretically explore a method for achieving this detection by probing the electricity of sequential charged segments of macromolecules. Our analysis is based on molecular dynamics simulations, and we investigate a single file of water molecules confined in a half-capped single-walled carbon nanotube (SWCNT) with an external electric charge of +e or -e (e is the elementary charge). The charge is located in the vicinity of the cap of the SWCNT and along the centerline of the SWCNT. We reveal the picosecond timescale for the re-orientation (namely, from one unidirectional direction to the other) of the water molecules in response to a switch in the charge signal, -e → +e or +e → -e. Our results are well understood by taking into account the electrical interactions between the water molecules and between the water molecules and the external charge. Because such signals of re-orientation can be magnified and transported according to Tu et al. [2009 Proc. Natl. Acad. Sci. USA 106 18120], it becomes possible to record fingerprints of electric signals arising from sequential charged segments of a macromolecule, which are expected to be useful for recognizing the conformations of some particular macromolecules.

Explicit Solvent Simulations of Friction between Brush Layers of Charged and Neutral Bottle-Brush Macromolecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carrillo, Jan-Michael; Brown, W Michael; Dobrynin, Andrey

2012-01-01

We study friction between charged and neutral brush layers of bottle-brush macromolecules using molecular dynamics simulations. In our simulations the solvent molecules were treated explicitly. The deformation of the bottle-brush macromolecules under the shear were studied as a function of the substrate separation and shear stress. For charged bottle-brush layers we study effect of the added salt on the brush lubricating properties to elucidate factors responsible for energy dissipation in charged and neutral brush systems. Our simulations have shown that for both charged and neutral brush systems the main deformation mode of the bottle-brush macromolecule is associated with the backbonemore » deformation. This deformation mode manifests itself in the backbone deformation ratio, , and shear viscosity, , to be universal functions of the Weissenberg number W. The value of the friction coefficient, , and viscosity, , are larger for the charged bottle-brush coatings in comparison with those for neutral brushes at the same separation distance, D, between substrates. The additional energy dissipation generated by brush sliding in charged bottle-brush systems is due to electrostatic coupling between bottle-brush and counterion motion. This coupling weakens as salt concentration, cs, increases resulting in values of the viscosity, , and friction coefficient, , approaching corresponding values obtained for neutral brush systems.« less

Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation

NASA Astrophysics Data System (ADS)

Li, Fengfu; Carlsson, David; Lohmann, Chris; Suuronen, Erik; Vascotto, Sandy; Kobuch, Karin; Sheardown, Heather; Munger, Rejean; Nakamura, Masatsugu; Griffith, May

2003-12-01

Our objective was to determine whether key properties of extracellular matrix (ECM) macromolecules can be replicated within tissue-engineered biosynthetic matrices to influence cellular properties and behavior. To achieve this, hydrated collagen and N-isopropylacrylamide copolymer-based ECMs were fabricated and tested on a corneal model. The structural and immunological simplicity of the cornea and importance of its extensive innervation for optimal functioning makes it an ideal test model. In addition, corneal failure is a clinically significant problem. Matrices were therefore designed to have the optical clarity and the proper dimensions, curvature, and biomechanical properties for use as corneal tissue replacements in transplantation. In vitro studies demonstrated that grafting of the laminin adhesion pentapeptide motif, YIGSR, to the hydrogels promoted epithelial stratification and neurite in-growth. Implants into pigs' corneas demonstrated successful in vivo regeneration of host corneal epithelium, stroma, and nerves. In particular, functional nerves were observed to rapidly regenerate in implants. By comparison, nerve regeneration in allograft controls was too slow to be observed during the experimental period, consistent with the behavior of human cornea transplants. Other corneal substitutes have been produced and tested, but here we report an implantable matrix that performs as a physiologically functional tissue substitute and not simply as a prosthetic device. These biosynthetic ECM replacements should have applicability to many areas of tissue engineering and regenerative medicine, especially where nerve function is required. regenerative medicine | tissue engineering | cornea | implantation | innervation

In situ phosphorylation of proteins in MCTs microdissected from rat kidney: Effect of AVP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Homma, S.; Gapstur, S.M.; Yusufi, N.K.

1988-04-01

Adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, the authors developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, semipermeabilization, increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment alsomore » did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with {gamma}-({sup 32}P)ATP resulted in corporation of {sup 32}P{sub i} into two major protein bands detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in {sup 32}P{sub i} incorporation into multiple protein bands. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.« less

Mechanism of Facilitated Diffusion during a DNA Search in Crowded Environments.

PubMed

Krepel, Dana; Gomez, David; Klumpp, Stefan; Levy, Yaakov

2016-11-03

The key feature explaining the rapid recognition of a DNA target site by its protein lies in the combination of one- and three-dimensional (1D and 3D) diffusion, which allows efficient scanning of the many alternative sites. This facilitated diffusion mechanism is expected to be affected by cellular conditions, particularly crowding, given that up to 40% of the total cellular volume may by occupied by macromolecules. Using coarse-grained molecular dynamics and Monte Carlo simulations, we show that the crowding particles can enhance facilitated diffusion and accelerate search kinetics. This effect originates from a trade-off between 3D and 1D diffusion. The 3D diffusion coefficient is lower under crowded conditions, but it has little influence because the excluded volume effect of molecular crowding restricts its use. Largely prevented from using 3D diffusion, the searching protein dramatically increases its use of the hopping search mode, which results in a higher linear diffusion coefficient. The coefficient of linear diffusion also increases under crowded conditions as a result of increased collisions between the crowding particles and the searching protein. Overall, less 3D diffusion coupled with an increase in the use of the hopping and speed of 1D diffusion results in faster search kinetics under crowded conditions. Our study shows that the search kinetics and mechanism are modulated not only by the crowding occupancy but also by the properties of the crowding particles and the salt concentration.

Oxidative stress and pathology in muscular dystrophies: focus on protein thiol oxidation and dysferlinopathies.

PubMed

Terrill, Jessica R; Radley-Crabb, Hannah G; Iwasaki, Tomohito; Lemckert, Frances A; Arthur, Peter G; Grounds, Miranda D

2013-09-01

The muscular dystrophies comprise more than 30 clinical disorders that are characterized by progressive skeletal muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for pathogenesis generally remains unknown. It is considered that disturbed levels of reactive oxygen species (ROS) contribute to the pathology of many muscular dystrophies. Reactive oxygen species and oxidative stress may cause cellular damage by directly and irreversibly damaging macromolecules such as proteins, membrane lipids and DNA; another major cellular consequence of reactive oxygen species is the reversible modification of protein thiol side chains that may affect many aspects of molecular function. Irreversible oxidative damage of protein and lipids has been widely studied in Duchenne muscular dystrophy, and we have recently identified increased protein thiol oxidation in dystrophic muscles of the mdx mouse model for Duchenne muscular dystrophy. This review evaluates the role of elevated oxidative stress in Duchenne muscular dystrophy and other forms of muscular dystrophies, and presents new data that show significantly increased protein thiol oxidation and high levels of lipofuscin (a measure of cumulative oxidative damage) in dysferlin-deficient muscles of A/J mice at various ages. The significance of this elevated oxidative stress and high levels of reversible thiol oxidation, but minimal myofibre necrosis, is discussed in the context of the disease mechanism for dysferlinopathies, and compared with the situation for dystrophin-deficient mdx mice. © 2013 The Authors Journal compilation © 2013 FEBS.

Marine-Derived Metabolites of S-Adenosylmethionine as Templates for New Anti-Infectives

PubMed Central

Sufrin, Janice R.; Finckbeiner, Steven; Oliver, Colin M.

2009-01-01

S-Adenosylmethionine (AdoMet) is a key biochemical co-factor whose proximate metabolites include methylated macromolecules (e.g., nucleic acids, proteins, phospholipids), methylated small molecules (e.g., sterols, biogenic amines), polyamines (e.g., spermidine, spermine), ethylene, and N-acyl-homoserine lactones. Marine organisms produce numerous AdoMet metabolites whose novel structures can be regarded as lead compounds for anti-infective drug design. PMID:19841722

Bioinspired synthesis and self-assembly of hybrid organic–inorganic nanomaterials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Honghu

Nature is replete with complex organic–inorganic hierarchical materials of diverse yet specific functions. These materials are intricately designed under physiological conditions through biomineralization and biological self-assembly processes. Tremendous efforts have been devoted to investigating mechanisms of such biomineralization and biological self-assembly processes as well as gaining inspiration to develop biomimetic methods for synthesis and self-assembly of functional nanomaterials. In this work, we focus on the bioinspired synthesis and self-assembly of functional inorganic nanomaterials templated by specialized macromolecules including proteins, DNA and polymers. The in vitro biomineralization process of the magnetite biomineralizing protein Mms6 has been investigated using small-angle X-ray scattering.more » Templated by Mms6, complex magnetic nanomaterials can be synthesized on surfaces and in the bulk. DNA and synthetic polymers have been exploited to construct macroscopic two- and three-dimensional (2D and 3D) superlattices of gold nanocrystals. Employing X-ray scattering and spectroscopy techniques, the self-assembled structures and the self-assembly mechanisms have been studied, and theoretical models have been developed. Our results show that specialized macromolecules including proteins, DNA and polymers act as effective templates for synthesis and self-assembly of nanomaterials. These bottom-up approaches provide promising routes to fabricate hybrid organic–inorganic nanomaterials with rationally designed hierarchical structures, targeting specific functions.« less

The highly efficient T7 RNA polymerase: A wonder macromolecule in biological realm.

PubMed

Borkotoky, Subhomoi; Murali, Ayaluru

2018-05-27

The study of bacteriophage has always been of keen interest for biologists to understand the fundamentals of biology. Bacteriophage T7 was first isolated in 1945 and its first comprehensive genetic map of was published in 1969. Since then, it gained immense attention of researchers and became a prime model system for experimental biologists. The major gene product of T7 phage, T7 RNA polymerase (T7RNAP), continues to attract researchers since a long time due to its high and specific processivity with a single subunit structure and its capability of transcribing a complete gene without additional proteins. Since the first review article in 1993 there has been around nine reviews on this polymerase till year 2009, most of which focussed on particular aspects of T7RNAP such as structure and function. However, this review encapsulates a broad view on T7RNAP, one of the simplest macromolecule catalyzing RNA synthesis including recent updates on its applications, structure, activators and inhibitors. Thus this brief review bridges the huge gap on the recent updates on this polymerase and will help the biologists in their endeavours that include the use of T7RNAP. Copyright © 2017. Published by Elsevier B.V.

Insights into the key roles of epigenetics in matrix macromolecules-associated wound healing.

PubMed

Piperigkou, Zoi; Götte, Martin; Theocharis, Achilleas D; Karamanos, Nikos K

2017-10-24

Extracellular matrix (ECM) is a dynamic network of macromolecules, playing a regulatory role in cell functions, tissue regeneration and remodeling. Wound healing is a tissue repair process necessary for the maintenance of the functionality of tissues and organs. This highly orchestrated process is divided into four temporally overlapping phases, including hemostasis, inflammation, proliferation and tissue remodeling. The dynamic interplay between ECM and resident cells exerts its critical role in many aspects of wound healing, including cell proliferation, migration, differentiation, survival, matrix degradation and biosynthesis. Several epigenetic regulatory factors, such as the endogenous non-coding microRNAs (miRNAs), are the drivers of the wound healing response. microRNAs have pivotal roles in regulating ECM composition during wound healing and dermal regeneration. Their expression is associated with the distinct phases of wound healing and they serve as target biomarkers and targets for systematic regulation of wound repair. In this article we critically present the importance of epigenetics with particular emphasis on miRNAs regulating ECM components (i.e. glycoproteins, proteoglycans and matrix proteases) that are key players in wound healing. The clinical relevance of miRNA targeting as well as the delivery strategies designed for clinical applications are also presented and discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

High-resolution NMR spectroscopy of encapsulated proteins dissolved in low-viscosity fluids

PubMed Central

Nucci, Nathaniel V.; Valentine, Kathleen G.; Wand, A. Joshua

2014-01-01

High-resolution multi-dimensional solution NMR is unique as a biophysical and biochemical tool in its ability to examine both the structure and dynamics of macromolecules at atomic resolution. Conventional solution NMR approaches, however, are largely limited to examinations of relatively small (< 25 kDa) molecules, mostly due to the spectroscopic consequences of slow rotational diffusion. Encapsulation of macromolecules within the protective nanoscale aqueous interior of reverse micelles dissolved in low viscosity fluids has been developed as a means through which the ‘slow tumbling problem’ can be overcome. This approach has been successfully applied to diverse proteins and nucleic acids ranging up to 100 kDa, considerably widening the range of biological macromolecules to which conventional solution NMR methodologies may be applied. Recent advances in methodology have significantly broadened the utility of this approach in structural biology and molecular biophysics. PMID:24656086

Isothermal titration calorimetry for measuring macromolecule-ligand affinity.

PubMed

Duff, Michael R; Grubbs, Jordan; Howell, Elizabeth E

2011-09-07

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

PubMed Central

Duff,, Michael R.; Grubbs, Jordan; Howell, Elizabeth E.

2011-01-01

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given. PMID:21931288

Analysis of the conductivity of plasmodesmata by microinjection.

PubMed

Kragler, Friedrich

2015-01-01

Pressure microinjection can be used to introduce fluorescent dyes and labeled macromolecules into single cells. The method allows measuring transport activity of macromolecules such as proteins and RNA molecules within and between cells. Routinely, plant mesophyll cells are injected with fluorescent dextran molecules of specific sizes to measure an increase of the size exclusion limit of plasmodesmata in the presence of a co-injected or expressed protein. The mobility of a macromolecule can also be addressed directly by injecting a recombinant protein that itself is labeled with fluorescent dye and following its transport to neighboring cells. This chapter describes a pressure microinjection protocol successfully applied to Nicotiana leaves. This protocol requires basic skills and experience in handling a microscope equipped with an imaging system, a micromanipulator, and a microinjection system attached to an upright microscope. Using this equipment, a trained person can inject approximately 10-20 mesophyll cells per hour.

Sparsity of the normal matrix in the refinement of macromolecules at atomic and subatomic resolution.

PubMed

Jelsch, C

2001-09-01

The normal matrix in the least-squares refinement of macromolecules is very sparse when the resolution reaches atomic and subatomic levels. The elements of the normal matrix, related to coordinates, thermal motion and charge-density parameters, have a global tendency to decrease rapidly with the interatomic distance between the atoms concerned. For instance, in the case of the protein crambin at 0.54 A resolution, the elements are reduced by two orders of magnitude for distances above 1.5 A. The neglect a priori of most of the normal-matrix elements according to a distance criterion represents an approximation in the refinement of macromolecules, which is particularly valid at very high resolution. The analytical expressions of the normal-matrix elements, which have been derived for the coordinates and the thermal parameters, show that the degree of matrix sparsity increases with the diffraction resolution and the size of the asymmetric unit.

An Overview of Biological Macromolecule Crystallization

PubMed Central

Krauss, Irene Russo; Merlino, Antonello; Vergara, Alessandro; Sica, Filomena

2013-01-01

The elucidation of the three dimensional structure of biological macromolecules has provided an important contribution to our current understanding of many basic mechanisms involved in life processes. This enormous impact largely results from the ability of X-ray crystallography to provide accurate structural details at atomic resolution that are a prerequisite for a deeper insight on the way in which bio-macromolecules interact with each other to build up supramolecular nano-machines capable of performing specialized biological functions. With the advent of high-energy synchrotron sources and the development of sophisticated software to solve X-ray and neutron crystal structures of large molecules, the crystallization step has become even more the bottleneck of a successful structure determination. This review introduces the general aspects of protein crystallization, summarizes conventional and innovative crystallization methods and focuses on the new strategies utilized to improve the success rate of experiments and increase crystal diffraction quality. PMID:23727935

Interspecies scaling and prediction of human clearance: comparison of small- and macro-molecule drugs

PubMed Central

Huh, Yeamin; Smith, David E.; Feng, Meihau Rose

2014-01-01

Human clearance prediction for small- and macro-molecule drugs was evaluated and compared using various scaling methods and statistical analysis.Human clearance is generally well predicted using single or multiple species simple allometry for macro- and small-molecule drugs excreted renally.The prediction error is higher for hepatically eliminated small-molecules using single or multiple species simple allometry scaling, and it appears that the prediction error is mainly associated with drugs with low hepatic extraction ratio (Eh). The error in human clearance prediction for hepatically eliminated small-molecules was reduced using scaling methods with a correction of maximum life span (MLP) or brain weight (BRW).Human clearance of both small- and macro-molecule drugs is well predicted using the monkey liver blood flow method. Predictions using liver blood flow from other species did not work as well, especially for the small-molecule drugs. PMID:21892879

Regulation of calcite crystal morphology by intracrystalline acidic proteins and glycoproteins.

PubMed

Albeck, S; Addadi, I; Weiner, S

1996-01-01

Many biologically formed calcite crystals contain intracrystalline macromolecules. The ways in which they interact with growing calcite crystals were evaluated by monitoring changes in the morphology of calcite crystals grown in vitro in their presence. Macromolecules were extracted from within isolated prisms from the prismatic layer of the shell of the mollusk Atrina rigida and from spines of the sea urchin Paracentrotus lividus. Two modes of interaction were identified; the interaction of highly acidic proteins with calcite planes perpendicular to the c crystallographic axis and the interaction of glycoproteins with planes roughly parallel to the c axis. By different preparative procedures we demonstrated that the polysaccharide moieties of the sea urchin spine glycoproteins are directly involved in the latter mode of interactions. We suggest that organisms utilize the abilities of these macromolecules to interact in different ways with calcite crystals, and in so doing fine-tune aspects of the control of crystal growth in vivo.

Macromolecule Crystal Quality Improvement in Microgravity: The Role of Impurities

NASA Technical Reports Server (NTRS)

Judge, Russell A.; Snell, Edward H.; Pusey, Marc L.; Sportiello, Michael G.; Todd, Paul; Bellamy, Henry; Borgstahl, Gloria E.; Pokros, Matt; Cassanto, John M.

2000-01-01

While macromolecule impurities may affect crystal size and morphology the over-riding question is; "How do macromolecule impurities effect crystal X-ray quality and diffraction resolution?" In the case of chicken egg white lysozyme, crystals can be grown in the presence of a number of impurities without affecting diffraction resolution. One impurity however, the lysozyme dimer, does negatively impact the X-ray crystal properties. Crystal quality improvement as a result of better partitioning of this impurity during crystallization in microgravity has been reported'. In our recent experimental work dimer partitioning was found to be not significantly different between the two environments. Mosaicity analysis of pure crystals showed a reduced mosaicity and increased signal to noise for the microgravity grown crystals. Dimer incorporation however, did greatly reduce the resolution limit in both ground and microgravity grown crystals. These results indicate that impurity effects in microgravity are complex and may rely on the conditions or techniques employed.

Sequence-controlled methacrylic multiblock copolymers via sulfur-free RAFT emulsion polymerization

NASA Astrophysics Data System (ADS)

Engelis, Nikolaos G.; Anastasaki, Athina; Nurumbetov, Gabit; Truong, Nghia P.; Nikolaou, Vasiliki; Shegiwal, Ataulla; Whittaker, Michael R.; Davis, Thomas P.; Haddleton, David M.

2017-02-01

Translating the precise monomer sequence control achieved in nature over macromolecular structure (for example, DNA) to whole synthetic systems has been limited due to the lack of efficient synthetic methodologies. So far, chemists have only been able to synthesize monomer sequence-controlled macromolecules by means of complex, time-consuming and iterative chemical strategies such as solid-state Merrifield-type approaches or molecularly dissolved solution-phase systems. Here, we report a rapid and quantitative synthesis of sequence-controlled multiblock polymers in discrete stable nanoscale compartments via an emulsion polymerization approach in which a vinyl-terminated macromolecule is used as an efficient chain-transfer agent. This approach is environmentally friendly, fully translatable to industry and thus represents a significant advance in the development of complex macromolecule synthesis, where a high level of molecular precision or monomer sequence control confers potential for molecular targeting, recognition and biocatalysis, as well as molecular information storage.

Delta L: An Apparatus for Measuring Macromolecule Crystal Growth Rates in Microgravity

NASA Technical Reports Server (NTRS)

Judge, Russell A.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

Strongly diffracting high quality macromolecule crystals of suitable volume are keenly sought for X-ray diffraction analysis so that high-resolution molecular structure data can be obtained. Such data is of tremendous value to medical research, agriculture and commercial biotechnology. In previous studies by many investigators microgravity has been reported in some instances to improve biological macromolecule X-ray crystal quality while little or no improvement was observed in other cases. A better understanding of processes effecting crystal quality improvement in microgravity will therefore be of great benefit in optimizing crystallization success in microgravity. In ground based research with the protein lysozyme we have previously shown that a population of crystals grown under the same solution conditions, exhibit a variation in X-ray diffraction properties (Judge et al., 1999). We have also observed that under the same solution conditions, individual crystals will grow at slightly different growth rates. This phenomenon is called growth rate dispersion. For small molecule materials growth rate dispersion has been directly related to crystal quality (Cunningham et al., 1991; Ristic et al., 1991). We therefore postulate that microgravity may act to improve crystal quality by reducing growth rate dispersion. If this is the case then as different, Materials exhibit different degrees of growth rate dispersion on the ground then growth rate dispersion could be used to screen which materials may benefit the most from microgravity crystallization. In order to assess this theory the Delta L hardware is being developed so that macromolecule crystal growth rates can be measured in microgravity. Crystal growth rate is defined as the change or delta in crystal size (defined as a characteristic length, L) over time; hence the name of the hardware. Delta L will consist of an optics, a fluids, and a data acquisition sub-assemblies. The optics assembly will consist of a video microscope camera mounted on three axis computer controlled translation stages. The fluids assembly consists of macromolecule and precipitant reservoirs, a temperature controlled growth cell and waste container, The data acquisition is achieved by using a frame-gabber, with images being stored on a hard drive. In operation, macromolecule and precipitant solution will be injected into the temperature controlled growth cell. As macromolecule crystals grow, the video microscope camera controlled by the translation stages, will be used to locate and record images of individual crystals, returning to the same crystals at specific time intervals. The images will be stored on the hard drive and used to calculate the crystal growth rate. To prevent vibrations interfering in the crystal growth rate measurements (Snell et al., 1997) Delta L will be used in connection with the Glovebox Integrated Microgravity Isolation Technology (g-LIMIT) inside the Microgravity Science Glovebox (MSG), onboard the International Space Station (ISS).

Nuclear Overhauser Enhancement Imaging of Glioblastoma at 7 Tesla: Region Specific Correlation with Apparent Diffusion Coefficient and Histology

PubMed Central

Windschuh, Johannes; Meissner, Jan-Eric; Zaiss, Moritz; Eidel, Oliver; Kickingereder, Philipp; Nowosielski, Martha; Wiestler, Benedikt; Sahm, Felix; Floca, Ralf Omar; Neumann, Jan-Oliver; Wick, Wolfgang; Heiland, Sabine; Bendszus, Martin; Schlemmer, Heinz-Peter; Ladd, Mark Edward; Bachert, Peter; Radbruch, Alexander

2015-01-01

Objective To explore the correlation between Nuclear Overhauser Enhancement (NOE)-mediated signals and tumor cellularity in glioblastoma utilizing the apparent diffusion coefficient (ADC) and cell density from histologic specimens. NOE is one type of chemical exchange saturation transfer (CEST) that originates from mobile macromolecules such as proteins and might be associated with tumor cellularity via altered protein synthesis in proliferating cells. Patients and Methods For 15 patients with newly diagnosed glioblastoma, NOE-mediated CEST-contrast was acquired at 7 Tesla (asymmetric magnetization transfer ratio (MTRasym) at 3.3ppm, B1 = 0.7 μT). Contrast enhanced T1 (CE-T1), T2 and diffusion-weighted MRI (DWI) were acquired at 3 Tesla and coregistered. The T2 edema and the CE-T1 tumor were segmented. ADC and MTRasym values within both regions of interest were correlated voxelwise yielding the correlation coefficient rSpearman (rSp). In three patients who underwent stereotactic biopsy, cell density of 12 specimens per patient was correlated with corresponding MTRasym and ADC values of the biopsy site. Results Eight of 15 patients showed a weak or moderate positive correlation of MTRasym and ADC within the T2 edema (0.16≤rSp≤0.53, p<0.05). Seven correlations were statistically insignificant (p>0.05, n = 4) or yielded rSp≈0 (p<0.05, n = 3). No trend towards a correlation between MTRasym and ADC was found in CE-T1 tumor (-0.310.05, n = 6). The biopsy-analysis within CE-T1 tumor revealed a strong positive correlation between tumor cellularity and MTRasym values in two of the three patients (rSp patient3 = 0.69 and rSp patient15 = 0.87, p<0.05), while the correlation of ADC and cellularity was heterogeneous (rSp patient3 = 0.545 (p = 0.067), rSp patient4 = -0.021 (p = 0.948), rSp patient15 = -0.755 (p = 0.005)). Discussion NOE-imaging is a new contrast promising insight into pathophysiologic processes in glioblastoma regarding cell density and protein content, setting itself apart from DWI. Future studies might be based on the assumption that NOE-mediated CEST visualizes cellularity more accurately than ADC, especially in the CE-T1 tumor region. PMID:25789657

Nuclear Overhauser Enhancement imaging of glioblastoma at 7 Tesla: region specific correlation with apparent diffusion coefficient and histology.

PubMed

Paech, Daniel; Burth, Sina; Windschuh, Johannes; Meissner, Jan-Eric; Zaiss, Moritz; Eidel, Oliver; Kickingereder, Philipp; Nowosielski, Martha; Wiestler, Benedikt; Sahm, Felix; Floca, Ralf Omar; Neumann, Jan-Oliver; Wick, Wolfgang; Heiland, Sabine; Bendszus, Martin; Schlemmer, Heinz-Peter; Ladd, Mark Edward; Bachert, Peter; Radbruch, Alexander

2015-01-01

To explore the correlation between Nuclear Overhauser Enhancement (NOE)-mediated signals and tumor cellularity in glioblastoma utilizing the apparent diffusion coefficient (ADC) and cell density from histologic specimens. NOE is one type of chemical exchange saturation transfer (CEST) that originates from mobile macromolecules such as proteins and might be associated with tumor cellularity via altered protein synthesis in proliferating cells. For 15 patients with newly diagnosed glioblastoma, NOE-mediated CEST-contrast was acquired at 7 Tesla (asymmetric magnetization transfer ratio (MTRasym) at 3.3ppm, B1 = 0.7 μT). Contrast enhanced T1 (CE-T1), T2 and diffusion-weighted MRI (DWI) were acquired at 3 Tesla and coregistered. The T2 edema and the CE-T1 tumor were segmented. ADC and MTRasym values within both regions of interest were correlated voxelwise yielding the correlation coefficient rSpearman (rSp). In three patients who underwent stereotactic biopsy, cell density of 12 specimens per patient was correlated with corresponding MTRasym and ADC values of the biopsy site. Eight of 15 patients showed a weak or moderate positive correlation of MTRasym and ADC within the T2 edema (0.16≤rSp≤0.53, p<0.05). Seven correlations were statistically insignificant (p>0.05, n = 4) or yielded rSp≈0 (p<0.05, n = 3). No trend towards a correlation between MTRasym and ADC was found in CE-T1 tumor (-0.310.05, n = 6). The biopsy-analysis within CE-T1 tumor revealed a strong positive correlation between tumor cellularity and MTRasym values in two of the three patients (rSppatient3 = 0.69 and rSppatient15 = 0.87, p<0.05), while the correlation of ADC and cellularity was heterogeneous (rSppatient3 = 0.545 (p = 0.067), rSppatient4 = -0.021 (p = 0.948), rSppatient15 = -0.755 (p = 0.005)). NOE-imaging is a new contrast promising insight into pathophysiologic processes in glioblastoma regarding cell density and protein content, setting itself apart from DWI. Future studies might be based on the assumption that NOE-mediated CEST visualizes cellularity more accurately than ADC, especially in the CE-T1 tumor region.

The role of endocytic pathways in cellular uptake of plasma non-transferrin iron

PubMed Central

Sohn, Yang-Sung; Ghoti, Hussam; Breuer, William; Rachmilewitz, Eliezer; Attar, Samah; Weiss, Guenter; Cabantchik, Z. Ioav

2012-01-01

Background In transfusional siderosis, the iron binding capacity of plasma transferrin is often surpassed, with concomitant generation of non-transferrin-bound iron. Although implicated in tissue siderosis, non-transferrin-bound iron modes of cell ingress remain undefined, largely because of its variable composition and association with macromolecules. Using fluorescent tracing of labile iron in endosomal vesicles and cytosol, we examined the hypothesis that non-transferrin-bound iron fractions detected in iron overloaded patients enter cells via bulk endocytosis. Design and Methods Fluorescence microscopy and flow cytometry served as analytical tools for tracing non-transferrin-bound iron entry into endosomes with the redox-reactive macromolecular probe Oxyburst-Green and into the cytosol with cell-laden calcein green and calcein blue. Non-transferrin-bound iron-containing media were from sera of polytransfused thalassemia major patients and model iron substances detected in thalassemia major sera; cell models were cultured macrophages, and cardiac myoblasts and myocytes. Results Exposure of cells to ferric citrate together with albumin, or to non-transferrin-bound iron-containing sera from thalassemia major patients caused an increase in labile iron content of endosomes and cytosol in macrophages and cardiac cells. This increase was more striking in macrophages, but in both cell types was largely reduced by co-exposure to non-transferrin-bound iron-containing media with non-penetrating iron chelators or apo-transferrin, or by treatment with inhibitors of endocytosis. Endosomal iron accumulation traced with calcein-green was proportional to input non-transferrin-bound iron levels (r2=0.61) and also preventable by pre-chelation. Conclusions Our studies indicate that macromolecule-associated non-transferrin-bound iron can initially gain access into various cells via endocytic pathways, followed by iron translocation to the cytosol. Endocytic uptake of plasma non-transferrin-bound iron is a possible mechanism that can contribute to iron loading of cell types engaged in bulk/adsorptive endocytosis, highlighting the importance of its prevention by iron chelation. PMID:22180428

Proteomic and Biochemical Changes during Senescence of Phalaenopsis 'Red Dragon' Petals.

PubMed

Chen, Cong; Zeng, Lanting; Ye, Qingsheng

2018-04-28

Phalaenopsis flowers are some of the most popular ornamental flowers in the world. For most ornamental plants, petal longevity determines postharvest quality and garden performance. Therefore, it is important to have insight into the senescence mechanism of Phalaenopsis . In the present study, a proteomic approach combined with ultrastructural observation and activity analysis of antioxidant enzymes was used to profile the molecular and biochemical changes during pollination-induced petal senescence in Phalaenopsis “Red Dragon”. Petals appeared to be visibly wilting at 24 h after pollination, accompanied by the mass degradation of macromolecules and organelles during senescence. In addition, 48 protein spots with significant differences in abundance were found by two-dimensional electrophoresis (2-DE) and subjected to matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS). There were 42 protein spots successfully identified and homologous to known functional protein species involved in key biological processes, including antioxidant pathways, stress response, protein metabolism, cell wall component metabolism, energy metabolism, cell structure, and signal transduction. The activity of all reactive oxygen species (ROS)-scavenging enzymes was increased, keeping the content of ROS at a low level at the early stage of senescence. These results suggest that two processes, a counteraction against increased levels of ROS and the degradation of cellular constituents for maintaining nutrient recycling, are activated during pollination-induced petal senescence in Phalaenopsis . The information provides a basis for understanding the mechanism regulating petal senescence and prolonging the florescence of Phalaenopsis .

KPNA2 is a nuclear export protein that contributes to aberrant localisation of key proteins and poor prognosis of breast cancer.

PubMed

Alshareeda, A T; Negm, O H; Green, A R; Nolan, C C; Tighe, P; Albarakati, N; Sultana, R; Madhusudan, S; Ellis, I O; Rakha, E A

2015-06-09

It is recognised that modulations of the nuclear import of macromolecules have a role in changing cellular phenotypes and carcinogenesis. We and others have noticed that aberrant subcellular localisation of DNA damage response (DDR) proteins in breast cancer (BC) is associated with loss-of-function phenotype. This study aims to investigate the biological and clinical significance of the nucleocytoplasmic transport protein karyopherin α-2 (KPNA2), and its role in controlling DDR proteins subcellular localisation in BC. A large (n=1494) and well-characterised series of early-stage invasive BC with a long-term follow-up was assessed for KPNA2 protein by using immunohistochemistry. KPNA2 expression was associated with the subcellular localisation of key DDR proteins that showed cytoplasmic expression including BRCA1, RAD51, SMC6L1, γH2AX, BARD1, UBC9, PIAS1 and CHK1. High level of KPNA2 was associated not only with cytoplasmic localisation of these proteins but also with their low/negative nuclear expression. Positive KPNA2 expression was associated with negative oestrogen receptor and triple-negative phenotype. Survival analysis showed that KPNA2 was associated with poor outcome (P<0.0001), but this effect was not independent of other prognostic variables. This study provides further evidence for the complexity of DDR mechanism in BC, and that KNPA2 has a role in the aberrant subcellular localisation of DDR proteins with subsequent impaired function.

Proteomic and Biochemical Changes during Senescence of Phalaenopsis ‘Red Dragon’ Petals

PubMed Central

Chen, Cong; Zeng, Lanting; Ye, Qingsheng

2018-01-01

Phalaenopsis flowers are some of the most popular ornamental flowers in the world. For most ornamental plants, petal longevity determines postharvest quality and garden performance. Therefore, it is important to have insight into the senescence mechanism of Phalaenopsis. In the present study, a proteomic approach combined with ultrastructural observation and activity analysis of antioxidant enzymes was used to profile the molecular and biochemical changes during pollination-induced petal senescence in Phalaenopsis “Red Dragon”. Petals appeared to be visibly wilting at 24 h after pollination, accompanied by the mass degradation of macromolecules and organelles during senescence. In addition, 48 protein spots with significant differences in abundance were found by two-dimensional electrophoresis (2-DE) and subjected to matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS). There were 42 protein spots successfully identified and homologous to known functional protein species involved in key biological processes, including antioxidant pathways, stress response, protein metabolism, cell wall component metabolism, energy metabolism, cell structure, and signal transduction. The activity of all reactive oxygen species (ROS)-scavenging enzymes was increased, keeping the content of ROS at a low level at the early stage of senescence. These results suggest that two processes, a counteraction against increased levels of ROS and the degradation of cellular constituents for maintaining nutrient recycling, are activated during pollination-induced petal senescence in Phalaenopsis. The information provides a basis for understanding the mechanism regulating petal senescence and prolonging the florescence of Phalaenopsis. PMID:29710804

iMODS: internal coordinates normal mode analysis server.

PubMed

López-Blanco, José Ramón; Aliaga, José I; Quintana-Ortí, Enrique S; Chacón, Pablo

2014-07-01

Normal mode analysis (NMA) in internal (dihedral) coordinates naturally reproduces the collective functional motions of biological macromolecules. iMODS facilitates the exploration of such modes and generates feasible transition pathways between two homologous structures, even with large macromolecules. The distinctive internal coordinate formulation improves the efficiency of NMA and extends its applicability while implicitly maintaining stereochemistry. Vibrational analysis, motion animations and morphing trajectories can be easily carried out at different resolution scales almost interactively. The server is versatile; non-specialists can rapidly characterize potential conformational changes, whereas advanced users can customize the model resolution with multiple coarse-grained atomic representations and elastic network potentials. iMODS supports advanced visualization capabilities for illustrating collective motions, including an improved affine-model-based arrow representation of domain dynamics. The generated all-heavy-atoms conformations can be used to introduce flexibility for more advanced modeling or sampling strategies. The server is free and open to all users with no login requirement at http://imods.chaconlab.org. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Protein Crystallization.

PubMed

McPherson, Alexander

2017-01-01

Protein crystallization was discovered by chance nearly 200 years ago and was developed in the late nineteenth century as a powerful purification tool, and a demonstration of chemical purity. The crystallization of proteins, nucleic acids, and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule, but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by manipulation of various parameters that include temperature, ionic strength, and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors, or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch, and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years due to the advent of practical, easy-to-use screening kits, and the application of laboratory robotics.

Solubility Limits in Lennard-Jones Mixtures: Effects of Disparate Molecule Geometries.

PubMed

Dyer, Kippi M; Perkyns, John S; Pettitt, B Montgomery

2015-07-23

In order to better understand general effects of the size and energy disparities between macromolecules and solvent molecules in solution, especially for macromolecular constructs self-assembled from smaller molecules, we use the first- and second-order exact bridge diagram extensions of the HNC integral equation theory to investigate single-component, binary, ternary, and quaternary mixtures of Lennard-Jones fluids. For pure fluids, we find that the HNCH3 bridge function integral equation (i.e., exact to third order in density) is necessary to quantitatively predict the pure gas and pure liquid sides of the coexistence region of the phase diagram of the Lennard-Jones fluid. For the mixtures, we find that the HNCH2 bridge function integral equation is sufficient to qualitatively predict solubility in the binary, ternary, and quaternary mixtures, up to the nominal solubility limit. The results, as limiting cases, should be useful to several problems, including accurate phase diagram predictions for complex mixtures, design of self-assembling nanostructures via solvent controls, and the solvent contributions to the conformational behavior of macromolecules in complex fluids.

Drive the Car(go)s-New Modalities to Control Cargo Trafficking in Live Cells.

PubMed

Mondal, Payel; Khamo, John S; Krishnamurthy, Vishnu V; Cai, Qi; Zhang, Kai

2017-01-01

Synaptic transmission is a fundamental molecular process underlying learning and memory. Successful synaptic transmission involves coupled interaction between electrical signals (action potentials) and chemical signals (neurotransmitters). Defective synaptic transmission has been reported in a variety of neurological disorders such as Autism and Alzheimer's disease. A large variety of macromolecules and organelles are enriched near functional synapses. Although a portion of macromolecules can be produced locally at the synapse, a large number of synaptic components especially the membrane-bound receptors and peptide neurotransmitters require active transport machinery to reach their sites of action. This spatial relocation is mediated by energy-consuming, motor protein-driven cargo trafficking. Properly regulated cargo trafficking is of fundamental importance to neuronal functions, including synaptic transmission. In this review, we discuss the molecular machinery of cargo trafficking with emphasis on new experimental strategies that enable direct modulation of cargo trafficking in live cells. These strategies promise to provide insights into a quantitative understanding of cargo trafficking, which could lead to new intervention strategies for the treatment of neurological diseases.

Understanding the nano- and macromechanical behaviour, the failure and fatigue mechanisms of advanced and natural polymer fibres by Raman/IR microspectrometry

NASA Astrophysics Data System (ADS)

Colomban, Philippe

2013-03-01

The coupled mechanical and Raman/infrared (IR) analysis of the (nano)structure and texture of synthetic and natural polymer fibres (polyamides (PA66), polyethylene terephthalate (PET), polypropylene (PP), poly(paraphenylene benzobisoxazole) (PBO), keratin/hair, Bombyx mori, Gonometa rufobrunea/postica Antheraea/Tussah silkworms and Nephila Madagascarensis spider silks) is applied so as to differentiate between crystalline and amorphous macromolecules. Bonding is very similar in the two cases but a broader distribution of conformations is observed for the amorphous macromolecules. These conclusions are then used to discuss the modifications induced by the application of a tensile or compressive stress, including the effects of fatigue. Detailed attention is paid to water and the inter-chain coupling for which the importance of hydrogen bonding is reconsidered. The significant role of the ‘amorphous’ bonds/domains in the process of fracture/fatigue is shown. Invited talk at the 6th International Workshop on Advanced Materials Science and Nanotechnology IWAMSN2012, 30 October-2 November 2012, Ha Long, Vietnam.

Electronic method for autofluorography of macromolecules on two-D matrices. [Patent application

DOEpatents

Davidson, J.B.; Case, A.L.

1981-12-30

A method for detecting, localizing, and quantifying macromolecules contained in a two-dimensional matrix is provided which employs a television-based position sensitive detection system. A molecule-containing matrix may be produced by conventional means to produce spots of light at the molecule locations which are detected by the television system. The matrix, such as a gel matrix, is exposed to an electronic camera system including an image-intensifier and secondary electron conduction camera capable of light integrating times of many minutes. A light image stored in the form of a charge image on the camera tube target is scanned by conventional television techniques, digitized, and stored in a digital memory. Intensity of any point on the image may be determined from the number at the memory address of the point. The entire image may be displayed on a television monitor for inspection and photographing or individual spots may be analyzed through selected readout of the memory locations. Compared to conventional film exposure methods, the exposure time may be reduced 100 to 1000 times.

Strongly Phase-Segregating Block Copolymers with Sub-20 nm Features

DTIC Science & Technology

2013-07-19

PERSON 19b. TELEPHONE NUMBER Francis Doyle Kristian Kempe, Kato L. Killops, Justin E. Poelma, Hyunjung Jung, Joona Bang, Richard Hoogenboom , Helen...Hyunjung Jung,# Joona Bang,# Richard Hoogenboom ,▽ Helen Tran,○ Craig J. Hawker,*,∥,¶ Ulrich S. Schubert,*,†,‡,◆ and Luis M. Campos*,○ †Laboratory of Organic...Macromolecules 2011, 44, 5825. (30) Wiesbrock, F.; Hoogenboom , R.; Leenen, M. A. M.; Meier, M. A. R.; Schubert, U. S. Macromolecules 2005, 38, 5025

Thermally Cross-Linked Anion Exchange Membranes from Solvent Processable Isoprene Containing Ionomers

DTIC Science & Technology

2015-01-15

isoprene determined by 1H NMR of each copolymer. Hydration Macromolecules Article DOI: 10.1021/ma502362a Macromolecules XXXX, XXX, XXX−XXX B number (λ) is...C. This is attributed to the decomposition of the TMA groups. Slight weight loss at lower temperatures is presumably due to the loss of trapped water...that at sufficiently high hydration levels the diffusion coefficient of ions approach their dilute solution diffusivity limits.30 Since conductivity is

Membrane-active macromolecules kill antibiotic-tolerant bacteria and potentiate antibiotics towards Gram-negative bacteria

PubMed Central

Uppu, Divakara S. S. M.; Konai, Mohini M.; Sarkar, Paramita; Samaddar, Sandip; Fensterseifer, Isabel C. M.; Farias-Junior, Celio; Krishnamoorthy, Paramanandam; Shome, Bibek R.; Franco, Octávio L.

2017-01-01

Chronic bacterial biofilms place a massive burden on healthcare due to the presence of antibiotic-tolerant dormant bacteria. Some of the conventional antibiotics such as erythromycin, vancomycin, linezolid, rifampicin etc. are inherently ineffective against Gram-negative bacteria, particularly in their biofilms. Here, we report membrane-active macromolecules that kill slow dividing stationary-phase and antibiotic tolerant cells of Gram-negative bacteria. More importantly, these molecules potentiate antibiotics (erythromycin and rifampicin) to biofilms of Gram-negative bacteria. These molecules eliminate planktonic bacteria that are liberated after dispersion of biofilms (dispersed cells). The membrane-active mechanism of these molecules forms the key for potentiating the established antibiotics. Further, we demonstrate that the combination of macromolecules and antibiotics significantly reduces bacterial burden in mouse burn and surgical wound infection models caused by Acinetobacter baumannii and Carbapenemase producing Klebsiella pneumoniae (KPC) clinical isolate respectively. Colistin, a well-known antibiotic targeting the lipopolysaccharide (LPS) of Gram-negative bacteria fails to kill antibiotic tolerant cells and dispersed cells (from biofilms) and bacteria develop resistance to it. On the contrary, these macromolecules prevent or delay the development of bacterial resistance to known antibiotics. Our findings emphasize the potential of targeting the bacterial membrane in antibiotic potentiation for disruption of biofilms and suggest a promising strategy towards developing therapies for topical treatment of Gram-negative infections. PMID:28837596

A rapid and practical technique for real-time monitoring of biomolecular interactions using mechanical responses of macromolecules

NASA Astrophysics Data System (ADS)

Tarhan, Mehmet C.; Lafitte, Nicolas; Tauran, Yannick; Jalabert, Laurent; Kumemura, Momoko; Perret, Grégoire; Kim, Beomjoon; Coleman, Anthony W.; Fujita, Hiroyuki; Collard, Dominique

2016-06-01

Monitoring biological reactions using the mechanical response of macromolecules is an alternative approach to immunoassays for providing real-time information about the underlying molecular mechanisms. Although force spectroscopy techniques, e.g. AFM and optical tweezers, perform precise molecular measurements at the single molecule level, sophisticated operation prevent their intensive use for systematic biosensing. Exploiting the biomechanical assay concept, we used micro-electro mechanical systems (MEMS) to develop a rapid platform for monitoring bio/chemical interactions of bio macromolecules, e.g. DNA, using their mechanical properties. The MEMS device provided real-time monitoring of reaction dynamics without any surface or molecular modifications. A microfluidic device with a side opening was fabricated for the optimal performance of the MEMS device to operate at the air-liquid interface for performing bioassays in liquid while actuating/sensing in air. The minimal immersion of the MEMS device in the channel provided long-term measurement stability (>10 h). Importantly, the method allowed monitoring effects of multiple solutions on the same macromolecule bundle (demonstrated with DNA bundles) without compromising the reproducibility. We monitored two different types of effects on the mechanical responses of DNA bundles (stiffness and viscous losses) exposed to pH changes (2.1 to 4.8) and different Ag+ concentrations (1 μM to 0.1 M).

A natural and readily available crowding agent: NMR studies of proteins in hen egg white.

PubMed

Martorell, Gabriel; Adrover, Miquel; Kelly, Geoff; Temussi, Piero Andrea; Pastore, Annalisa

2011-05-01

In vitro studies of biological macromolecules are usually performed in dilute, buffered solutions containing one or just a few different biological macromolecules. Under these conditions, the interactions among molecules are diffusion limited. On the contrary, in living systems, macromolecules of a given type are surrounded by many others, at very high total concentrations. In the last few years, there has been an increasing effort to study biological macromolecules directly in natural crowded environments, as in intact bacterial cells or by mimicking natural crowding by adding proteins, polysaccharides, or even synthetic polymers. Here, we propose the use of hen egg white (HEW) as a simple natural medium, with all features of the media of crowded cells, that could be used by any researcher without difficulty and inexpensively. We present a study of the stability and dynamics behavior of model proteins in HEW, chosen as a prototypical, readily accessible natural medium that can mimic cytosol. We show that two typical globular proteins, dissolved in HEW, give NMR spectra very similar to those obtained in dilute buffers, although dynamic parameters are clearly affected by the crowded medium. The thermal stability of one of these proteins, measured in a range comprising both heat and cold denaturation, is also similar to that in buffer. Our data open new possibilities to the study of proteins in natural crowded media. Copyright © 2010 Wiley-Liss, Inc.

Vasoactive intestinal peptide stimulates tracheal submucosal gland secretion in ferret

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peatfield, A.C.; Barnes, P.J.; Bratcher, C.

1983-07-01

We studied the effect of vasoactive intestinal peptide (VIP) on the output of 35S-labeled macromolecules from ferret tracheal explants either placed in beakers or suspended in modified Ussing chambers. In Ussing chamber experiments, the radiolabel precursor, sodium (35S)sulfate, and all drugs were placed on the submucosal side of the tissue. Washings were collected at 30-min intervals from the luminal side and were dialyzed to remove unbound 35S, leaving radiolabeled macromolecules. Vasoactive intestinal peptide at 3 X 10(-7) M stimulated bound 35S output by a mean of + 252.6% (n . 14). The VIP response was dose-dependent with a near maximalmore » response and a half maximal response at approximately 10(-6) M and 10(-8), M, respectively. The VIP effect was not inhibited by a mixture of tetrodotoxin, atropine, I-propranolol, and phentolamine. Vasoactive intestinal peptide had no effect on the electrical properties of the of the tissues. We conclude that VIP stimulates output of sulfated-macromolecules from ferret tracheal submucosal glands without stimulating ion transport. Our studies also suggest that VIP acts on submucosal glands via specific VIP receptors. Vasoactive intestinal peptide has been shown to increase intracellular levels of cyclic AMP, and we suggest that this may be the mechanism for its effect on the output of macromolecules. This mechanism may be important in the neural regulation of submucosal gland secretion.« less

Ultrasound Microbubble Treatment Enhances Clathrin-Mediated Endocytosis and Fluid-Phase Uptake through Distinct Mechanisms.

PubMed

Fekri, Farnaz; Delos Santos, Ralph Christian; Karshafian, Raffi; Antonescu, Costin N

2016-01-01

Drug delivery to tumors is limited by several factors, including drug permeability of the target cell plasma membrane. Ultrasound in combination with microbubbles (USMB) is a promising strategy to overcome these limitations. USMB treatment elicits enhanced cellular uptake of materials such as drugs, in part as a result of sheer stress and formation of transient membrane pores. Pores formed upon USMB treatment are rapidly resealed, suggesting that other processes such as enhanced endocytosis may contribute to the enhanced material uptake by cells upon USMB treatment. How USMB regulates endocytic processes remains incompletely understood. Cells constitutively utilize several distinct mechanisms of endocytosis, including clathrin-mediated endocytosis (CME) for the internalization of receptor-bound macromolecules such as Transferrin Receptor (TfR), and distinct mechanism(s) that mediate the majority of fluid-phase endocytosis. Tracking the abundance of TfR on the cell surface and the internalization of its ligand transferrin revealed that USMB acutely enhances the rate of CME. Total internal reflection fluorescence microscopy experiments revealed that USMB treatment altered the assembly of clathrin-coated pits, the basic structural units of CME. In addition, the rate of fluid-phase endocytosis was enhanced, but with delayed onset upon USMB treatment relative to the enhancement of CME, suggesting that the two processes are distinctly regulated by USMB. Indeed, vacuolin-1 or desipramine treatment prevented the enhancement of CME but not of fluid phase endocytosis upon USMB, suggesting that lysosome exocytosis and acid sphingomyelinase, respectively, are required for the regulation of CME but not fluid phase endocytosis upon USMB treatment. These results indicate that USMB enhances both CME and fluid phase endocytosis through distinct signaling mechanisms, and suggest that strategies for potentiating the enhancement of endocytosis upon USMB treatment may improve targeted drug delivery.

Is light-emitting diode phototherapy (LED-LLLT) really effective?

PubMed Central

Kim, Won-Serk; Calderhead, R Glen

2011-01-01

Background: Low level light therapy (LLLT) has attracted attention in many clinical fields with a new generation of light-emitting diodes (LEDs) which can irradiate large targets. To pain control, the first main application of LLLT, have been added LED-LLLT in the accelerated healing of wounds, both traumatic and iatrogenic, inflammatory acne and the patient-driven application of skin rejuvenation. Rationale and Applications: The rationale behind LED-LLLT is underpinned by the reported efficacy of LED-LLLT at a cellular and subcellular level, particularly for the 633 nm and 830 nm wavelengths, and evidence for this is presented. Improved blood flow and neovascularization are associated with 830 nm. A large variety of cytokines, chemokines and macromolecules can be induced by LED phototherapy. Among the clinical applications, non-healing wounds can be healed through restoring the collagenesis/collagenase imbalance in such examples, and ‘normal’ wounds heal faster and better. Pain, including postoperative pain, postoperative edema and many types of inflammation can be significantly reduced. Experimental and clinical evidence: Some personal examples of evidence are offered by the first author, including controlled animal models demonstrating the systemic effect of 830 nm LED-LLLT on wound healing and on induced inflammation. Human patients are presented to illustrate the efficacy of LED phototherapy on treatment-resistant inflammatory disorders. Conclusions: Provided an LED phototherapy system has the correct wavelength for the target cells, delivers an appropriate power density and an adequate energy density, then it will be at least partly, if not significantly, effective. The use of LED-LLLT as an adjunct to conventional surgical or nonsurgical indications is an even more exciting prospect. LED-LLLT is here to stay. PMID:24155530

A molecule-centered method for accelerating the calculation of hydrodynamic interactions in Brownian dynamics simulations containing many flexible biomolecules

PubMed Central

Elcock, Adrian H.

2013-01-01

Inclusion of hydrodynamic interactions (HIs) is essential in simulations of biological macromolecules that treat the solvent implicitly if the macromolecules are to exhibit correct translational and rotational diffusion. The present work describes the development and testing of a simple approach aimed at allowing more rapid computation of HIs in coarse-grained Brownian dynamics simulations of systems that contain large numbers of flexible macromolecules. The method combines a complete treatment of intramolecular HIs with an approximate treatment of the intermolecular HIs which assumes that the molecules are effectively spherical; all of the HIs are calculated at the Rotne-Prager-Yamakawa level of theory. When combined with Fixman’s Chebyshev polynomial method for calculating correlated random displacements, the proposed method provides an approach that is simple to program but sufficiently fast that it makes it computationally viable to include HIs in large-scale simulations. Test calculations performed on very coarse-grained models of the pyruvate dehydrogenase (PDH) E2 complex and on oligomers of ParM (ranging in size from 1 to 20 monomers) indicate that the method reproduces the translational diffusion behavior seen in more complete HI simulations surprisingly well; the method performs less well at capturing rotational diffusion but its discrepancies diminish with increasing size of the simulated assembly. Simulations of residue-level models of two tetrameric protein models demonstrate that the method also works well when more structurally detailed models are used in the simulations. Finally, test simulations of systems containing up to 1024 coarse-grained PDH molecules indicate that the proposed method rapidly becomes more efficient than the conventional BD approach in which correlated random displacements are obtained via a Cholesky decomposition of the complete diffusion tensor. PMID:23914146

Electrostatic, elastic and hydration-dependent interactions in dermis influencing volume exclusion and macromolecular transport.

PubMed

Øien, Alf H; Wiig, Helge

2016-07-07

Interstitial exclusion refers to the limitation of space available for plasma proteins and other macromolecules based on collagen and negatively charged glycosaminoglycans (GAGs) in the interstitial space. It is of particular importance to interstitial fluid and plasma volume regulation. Here we present a novel mechanical and mathematical model of the dynamic interactions of structural elements within the interstitium of the dermis at the microscopic level that may explain volume exclusion of charged and neutral macroparticles. At this level, the interstitium is considered to consist of elements called extracellular matrix (ECM) cells, again containing two main interacting structural components on a fluid background including anions and cations setting up osmotic forces: one smaller GAG component, having an intrinsic expansive electric force, and one bigger collagen component, having an intrinsic elastic force. Because of size differences, the GAG component interacts with a fraction of the collagen component only at normal hydration. This fraction, however, increases with rising hydration as a consequence of the modeled form of the interaction force between the GAGs and collagen. Collagen is locally displaced at variable degrees as hydration changes. Two models of GAGs are considered, having largely different geometries which demands different, but related, forms of GAG-collagen interaction forces. The effects of variable fixed charges on GAGs and of GAG density in tissue are evaluated taking into account observed volume exclusion properties of charged macromolecules as a function of tissue hydration. The presented models may improve our biophysical understanding of acting forces influencing tissue fluid dynamics. Such knowledge is significant when evaluating the transport of electrically charged and neutral macromolecules into and through the interstitium, and therefore to drug uptake and the therapeutic effects of macromolecular agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Diffusion within the cytoplasm: a mesoscale model of interacting macromolecules.

PubMed

Trovato, Fabio; Tozzini, Valentina

2014-12-02

Recent experiments carried out in the dense cytoplasm of living cells have highlighted the importance of proteome composition and nonspecific intermolecular interactions in regulating macromolecule diffusion and organization. Despite this, the dependence of diffusion-interaction on physicochemical properties such as the degree of poly-dispersity and the balance between steric repulsion and nonspecific attraction among macromolecules was not systematically addressed. In this work, we study the problem of diffusion-interaction in the bacterial cytoplasm, combining theory and experimental data to build a minimal coarse-grained representation of the cytoplasm, which also includes, for the first time to our knowledge, the nucleoid. With stochastic molecular-dynamics simulations of a virtual cytoplasm we are able to track the single biomolecule motion, sizing from 3 to 80 nm, on submillisecond-long trajectories. We demonstrate that the size dependence of diffusion coefficients, anomalous exponents, and the effective viscosity experienced by biomolecules in the cytoplasm is fine-tuned by the intermolecular interactions. Accounting only for excluded volume in these potentials gives a weaker size-dependence than that expected from experimental data. On the contrary, adding nonspecific attraction in the range of 1-10 thermal energy units produces a stronger variation of the transport properties at growing biopolymer sizes. Normal and anomalous diffusive regimes emerge straightforwardly from the combination of high macromolecular concentration, poly-dispersity, stochasticity, and weak nonspecific interactions. As a result, small biopolymers experience a viscous cytoplasm, while the motion of big ones is jammed because the entanglements produced by the network of interactions and the entropic effects caused by poly-dispersity are stronger. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Single DNA molecules on freestanding and supported cationic lipid bilayers: diverse conformational dynamics controlled by the local bilayer properties

NASA Astrophysics Data System (ADS)

Herold, Christoph; Schwille, Petra; Petrov, Eugene P.

2016-02-01

We present experimental results on the interaction of DNA macromolecules with cationic lipid membranes with different properties, including freestanding membranes in the fluid and gel state, and supported lipid membranes in the fluid state and under conditions of fluid-gel phase coexistence. We observe diverse conformational dynamics of membrane-bound DNA molecules controlled by the local properties of the lipid bilayer. In case of fluid-state freestanding lipid membranes, the behaviour of DNA on the membrane is controlled by the membrane charge density: whereas DNA bound to weakly charged membranes predominantly behaves as a 2D random coil, an increase in the membrane charge density leads to membrane-driven irreversible DNA collapse and formation of subresolution-sized DNA globules. On the other hand, electrostatic binding of DNA macromolecules to gel-state freestanding membranes leads to completely arrested diffusion and conformational dynamics of membrane-adsorbed DNA. A drastically different picture is observed in case of DNA interaction with supported cationic lipid bilayers: When the supported bilayer is in the fluid state, membrane-bound DNA molecules undergo 2D translational Brownian motion and conformational fluctuations, irrespectively of the charge density of the supported bilayer. At the same time, when the supported cationic membrane shows fluid-gel phase coexistence, membrane-bound DNA molecules are strongly attracted to micrometre-sized gel-phase domains enriched with the cationic lipid, which results in 2D compaction of the membrane-bound macromolecules. This DNA compaction, however, is fully reversible, and disappears as soon as the membrane is heated above the fluid-gel coexistence. We also discuss possible biological implications of our experimental findings.

Coated substrate apparatus and method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, Zhenan; Diao, Ying; Mannsfeld, Stefan Christian Bernhardt

A coated substrate is formed with aligned objects such as small molecules, macromolecules and nanoscale particulates, such as inorganic, organic or inorganic/organic hybrid materials. In accordance with one or more embodiments, an apparatus or method involves an applicator having at least one surface patterned with protruded or indented features, and a coated substrate including a solution-based layer of objects having features and morphology attributes arranged as a function of the protruded or indented features.

REGULATION OF COAL POLYMER DEGRADATION BY FUNGI

DOE Office of Scientific and Technical Information (OSTI.GOV)

John A. Bumpus

1998-11-30

A variety of lignin degrading fungi mediate solubilization and subsequent biodegradation of coal macromolecules (a.k.a. coal polymer) from highly oxidized low rank coals such as leonardites. It appears that oxalate or possibly other metal chelators (i.e., certain Krebs Cycle intermediates) mediate solubilization of low rank coals while extracellular oxidases have a role in subsequent oxidation of solubilized coal macromolecule. These processes are under nutritional control. For example, in the case of P. chrysosporium, solubilization of leonardite occurred when the fungi were cultured on most but not all nutrient agars tested and subsequent biodegradation occurred only in nutrient nitrogen limited cultures.more » Lignin peroxidases mediate oxidation of coal macromolecule in a reaction that is dependent on the presence of veratryl alcohol and hydrogen peroxide. Kinetic evidence suggests that veratryl alcohol is oxidized to the veratryl alcohol cation radical which then mediates oxidation of the coal macromolecule. Results by others suggest that Mn peroxidases mediate formation of reactive Mn{sup 3+} complexes which also mediate oxidation of coal macromolecule. A biomimetic approach was used to study solubilization of a North Dakota leonardite. It was found that a concentration {approximately}75 mM sodium oxalate was optimal for solubilization of this low rank coal. This is important because this is well above the concentration of oxalate produced by fungi in liquid culture. Higher local concentrations probably occur in solid agar cultures and thus may account for the observation that greater solubilization occurs in agar media relative to liquid media. The characteristics of biomimetically solubilized leonardite were similar to those of biologically solubilized leonardite. Perhaps our most interesting observation was that in addition to oxalate, other common Lewis bases (phosphate/hydrogen phosphate/dihydrogen phosphate and bicarbonate/carbonate ions) are able to mediate substantial solubilization of leonardite at physiological pH values. Lastly, we present evidence that some fungi appear to possess coal solubilization ability in which the initial events of solubilization is not mediated by oxalate ion.« less

Digital X-ray camera for quality evaluation three-dimensional topographic reconstruction of single crystals of biological macromolecules

NASA Technical Reports Server (NTRS)

Borgstahl, Gloria (Inventor); Lovelace, Jeff (Inventor); Snell, Edward Holmes (Inventor); Bellamy, Henry (Inventor)

2008-01-01

The present invention provides a digital topography imaging system for determining the crystalline structure of a biological macromolecule, wherein the system employs a charge coupled device (CCD) camera with antiblooming circuitry to directly convert x-ray signals to electrical signals without the use of phosphor and measures reflection profiles from the x-ray emitting source after x-rays are passed through a sample. Methods for using said system are also provided.

Basal-body-associated macromolecules: a continuing debate.

PubMed

Pierre Mignot, J; Brugerolle, G; Didier, P; Bornens, M

1993-07-01

Controversy over the possibility that centrioles/basal bodies contain nucleic acids has overshadowed results demonstrating other macromolecules in the lumen of these organelles. Glycogen particles, which are known to be present within the lumen of the centriole/basal body of sperm cells, have now been found in basal bodies of protists belonging to three different groups. Here, we extend the debate on a role for RNA in basal body/centriole function and speculate on the origin and the function of centriolar glycogen.

Importance of the Debye Screening Length on Nanowire Field Effect Transistor Sensors

PubMed Central

Stern, Eric; Wagner, Robin; Sigworth, Fred J.; Breaker, Ronald; Fahmy, Tarek M.; Reed, Mark A.

2009-01-01

Nanowire field effect transistors (NW-FETs) can serve as ultrasensitive detectors for label-free reagents. The NW-FET sensing mechanism assumes a controlled modification in the local channel electric field created by the binding of charged molecules to the nanowire surface. Careful control of the solution Debye length is critical for unambiguous selective detection of macromolecules. Here we show the appropriate conditions under which the selective binding of macromolecules is accurately sensed with NW-FET sensors. PMID:17914853

Importance of the Debye screening length on nanowire field effect transistor sensors.

PubMed

Stern, Eric; Wagner, Robin; Sigworth, Fred J; Breaker, Ronald; Fahmy, Tarek M; Reed, Mark A

2007-11-01

Nanowire field effect transistors (NW-FETs) can serve as ultrasensitive detectors for label-free reagents. The NW-FET sensing mechanism assumes a controlled modification in the local channel electric field created by the binding of charged molecules to the nanowire surface. Careful control of the solution Debye length is critical for unambiguous selective detection of macromolecules. Here we show the appropriate conditions under which the selective binding of macromolecules is accurately sensed with NW-FET sensors.

Growing Hyperbranched Polymers Using Natural Sunlight

PubMed Central

Yan, Jun-Jie; Sun, Jiao-Tong; You, Ye-Zi; Wu, De-Cheng; Hong, Chun-Yan

2013-01-01

In nature, a sapling can grow into a big tree under irradiation of sunlight. In chemistry, a similar concept that a small molecule only exposing to sunlight grows into a hyperbranched macromolecule has not been realized by now. The achievement of the concept will be fascinating and valuable for polymer synthesis wherein sunlight is inexpensive, abundant, renewable, and nonpolluting. Herein, we report a new strategy in which small monomers can directly grow into big hyperbranched macromolecule under irradiation of sunlight without any catalyst. PMID:24100948

Nucleotide-induced asymmetry within ATPase activator ring drives σ54-RNAP interaction and ATP hydrolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang

2013-12-10

It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase andmore » the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.« less

In vitro preparation and assessment of radical reducing peptide from Octopus aegina using digestive proteases.

PubMed

Sudhakar, Sekar; Nazeer, Rasool Abdul

2017-07-01

Antioxidant peptides protect biological macromolecules against radical damages. The use of these peptides was evaluated using free radicals scavenging assays [2,2-diphenyl-1 picrylhydrazyl (DPPH) and hydroxyl] with the help of UV-visible and electron spin resonance (ESR) spectroscopy methods. The Octopus aegina mantle protein were tested upon hydrolysis using gastrointestinal enzymes up to 12 h, where pepsin hydrolysate exhibited superior properties (DPPH: 44.39±0.67% and hydroxyl: 38.84±1.07%) compared with trypsin and α-chymotrypsin. Consequently, the antioxidant activity of the purified hydrolysate increased on a successive purification, and the peptide sequence was determined to be 368.9 Da with Gly-Glu-Tyr amino acids. Tripeptide exerted free radical scavenging efficiency in DNA damage, lipid peroxidation and cellular destruction (MCF7 cells) under stress condition. The results obtained with octopus antioxidant peptide suggested its role as an adjunct in food and pharmaceutical industries. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Huntingtons Disease: The Value of Transcranial Meganetic Stimulation

PubMed

Medina, F J; Túnez, I

2010-01-01

Huntington's disease (HD) is a genetic neurodegenerative process whose etiology is based on a localized disturbance in the short arm of chromosome 4 that encodes the huntingtin protein (Htt). The elongation of triple CAG for glutamine characterizes this change. Mutated Htt (mHtt) causes the appearance of intracellular aggregates inducing alterations in mitochondrial metabolism in the form of reactive oxygen species (ROS) and ATP depletion. The oxidative imbalance caused by mHtt leads the neurons to a state of oxidative stress resulting in damage to macromolecules and cellular death. Since the discovery of certain mechanisms underlying the pathogenesis of HD, several therapeutic procedures have been shown to delay or slow the evolution of the condition and have demonstrated the biochemical and molecular mechanism involved. The studies have reported that transcranial magnetic stimulation (TMS) may improve motor and other symptoms associated with neurodegenerative and neuropsychiatric processes such as major depression, schizophrenia, epilepsy, neuropathic pain, amyotrophic lateral sclerosis, progressive muscle atrophy, multiple sclerosis, stroke, Alzheimer's disease, Parkinson's disease or HD. This study focuses on the effect of TMS on oxidative stress and neurogenesis in studies and its possible usefulness in HD.

Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity

PubMed Central

Im, Michelle; Dagnino, Lina

2018-01-01

The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity. PMID:29568383

Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity.

PubMed

Im, Michelle; Dagnino, Lina

2018-03-02

The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity.

Thermodynamics and solvent linkage of macromolecule-ligand interactions

PubMed Central

Duff, Michael R.; Howell, Elizabeth E.

2014-01-01

Binding involves two steps, desolvation and association. While water is ubiquitous and occurs at high concentration, it is typically ignored. In vitro experiments typically use infinite dilution conditions, while in vivo, the concentration of water is decreased due to the presence of high concentrations of molecules in the cellular milieu. This review discusses isothermal titration calorimetry approaches that address the role of water in binding. For example, use of D2O allows the contribution of solvent reorganization to the enthalpy component to be assessed. Further, the addition of osmolytes will decrease the water activity of a solution and allow effects on Ka to be determined. In most cases, binding becomes tighter in the presence of osmolytes as the desolvation penalty associated with binding is minimized. In other cases, the osmolytes prefer to interact with the ligand or protein, and if their removal is more difficult than shedding water, then binding can be weakened. These complicating layers can be discerned by different slopes in ln(Ka) vs osmolality plots and by differential scanning calorimetry in the presence of the osmolyte. PMID:25462561

Investigating biomolecular recognition at the cell surface using atomic force microscopy.

PubMed

Wang, Congzhou; Yadavalli, Vamsi K

2014-05-01

Probing the interaction forces that drive biomolecular recognition on cell surfaces is essential for understanding diverse biological processes. Force spectroscopy has been a widely used dynamic analytical technique, allowing measurement of such interactions at the molecular and cellular level. The capabilities of working under near physiological environments, combined with excellent force and lateral resolution make atomic force microscopy (AFM)-based force spectroscopy a powerful approach to measure biomolecular interaction forces not only on non-biological substrates, but also on soft, dynamic cell surfaces. Over the last few years, AFM-based force spectroscopy has provided biophysical insight into how biomolecules on cell surfaces interact with each other and induce relevant biological processes. In this review, we focus on describing the technique of force spectroscopy using the AFM, specifically in the context of probing cell surfaces. We summarize recent progress in understanding the recognition and interactions between macromolecules that may be found at cell surfaces from a force spectroscopy perspective. We further discuss the challenges and future prospects of the application of this versatile technique. Copyright © 2014 Elsevier Ltd. All rights reserved.

In-depth proteomic analysis of shell matrix proteins of Pinctada fucata

PubMed Central

Liu, Chuang; Li, Shiguo; Kong, Jingjing; Liu, Yangjia; Wang, Tianpeng; Xie, Liping; Zhang, Rongqing

2015-01-01

The shells of pearl oysters, Pinctada fucata, are composed of calcite and aragonite and possess remarkable mechanical properties. These shells are formed under the regulation of macromolecules, especially shell matrix proteins (SMPs). Identification of diverse SMPs will lay a foundation for understanding biomineralization process. Here, we identified 72 unique SMPs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteins extracted from the shells of P. fucata combined with a draft genome. Of 72 SMPs, 17 SMPs are related to both the prismatic and nacreous layers. Moreover, according to the diverse domains found in the SMPs, we hypothesize that in addition to controlling CaCO3 crystallization and crystal organization, these proteins may potentially regulate the extracellular microenvironment and communicate between cells and the extracellular matrix (ECM). Immunohistological localization techniques identify the SMPs in the mantle, shells and synthetic calcite. Together, these proteomic data increase the repertoires of the shell matrix proteins in P. fucata and suggest that shell formation in P. fucata may involve tight regulation of cellular activities and the extracellular microenvironment. PMID:26608573

Media additives to promote spheroid circularity and compactness in hanging drop platform.

PubMed

Leung, Brendan M; Lesher-Perez, Sasha Cai; Matsuoka, Toshiki; Moraes, Christopher; Takayama, Shuichi

2015-02-01

Three-dimensional spheroid cultures have become increasingly popular as drug screening platforms, especially with the advent of different high throughput spheroid forming technologies. However, comparing drug efficacy across different cell types in spheroid culture can be difficult due to variations in spheroid morphologies and transport characteristics. Improving the reproducibility of compact, circular spheroids contributes to standardizing and increasing the fidelity of the desired gradient profiles in these drug screening three-dimensional tissue cultures. In this study we discuss the role that circularity and compaction has on spheroids, and demonstrate the impact methylcellulose (MethoCel) and collagen additives in the culture media can contribute to more compact and circular spheroid morphology. We demonstrate that improved spheroid formation is not a simple function of increased viscosity of the different macromolecule additives, suggesting that other macromolecular characteristics contribute to improved spheroid formation. Of the various macromolecular additives tested for hanging drop culture, MethoCel provided the most desirable spheroid formation. Additionally, the higher viscosity of MethoCel-containing media improved the ease of imaging of cellular spheroids within hanging drop cultures by reducing motion-induced image blur.

Effects of coarse-graining on fluctuations in gene expression

NASA Astrophysics Data System (ADS)

Pedraza, Juan; Paulsson, Johan

2008-03-01

Many cellular components are present in such low numbers per cell that random births and deaths of individual molecules can cause significant `noise' in concentrations. But biochemical events do not necessarily occur in steps of individual molecules. Some processes are greatly randomized when synthesis or degradation occurs in large bursts of many molecules in a short time interval. Conversely, each birth or death of a macromolecule could involve several small steps, creating a memory between individual events. Here we present generalized theory for stochastic gene expression, formulating the variance in protein abundance in terms of the randomness of the individual events, and discuss the effective coarse-graining of the molecular hardware. We show that common molecular mechanisms produce gestation and senescence periods that can reduce noise without changing average abundances, lifetimes, or any concentration-dependent control loops. We also show that single-cell experimental methods that are now commonplace in cell biology do not discriminate between qualitatively different stochastic principles, but that this in turn makes them better suited for identifying which components introduce fluctuations.

[The correlations between aging of the human body, oxidative stress and reduced efficiency of repair systems].

PubMed

Michalak, Aleksandra; Krzeszowiak, Jakub; Markiewicz-Górka, Iwona

2014-12-15

The article presents an current knowledge overview about the importance of oxidative stress and reduced efficiency of repair processes during the aging process of the human body. Oxidative damage to cellular macromolecules (proteins, lipids, nucleic acids), are formed under the influence of reactive oxygen species (ROS). They are the part of important mechanism which is responsible for the process of aging and the development of many diseases. The most important effects result from DNA damage, due to the mutations formation, which can lead to the development of tumors. However, a well-functioning repair systems (i.a. homologous recombination) remove the damage and prevent harmful changes in the cells. Lipid peroxidation products also cause oxidative modification of nucleic acids (and proteins). Proteins and fats also have repair systems, but much simpler than those responsible for the repair of nucleic acids. Unfortunately, with increasing age, they are more weakened, which contributes to increase numbers of cell damage, and consequently development of diseases specific to old age: cancer, neurodegenerative diseases or atherosclerosis.

Binding abilities of polyaminocyclodextrins: polarimetric investigations and biological assays

PubMed Central

Russo, Marco; La Corte, Daniele; Pisciotta, Annalisa; Riela, Serena; Alduina, Rosa

2017-01-01

Three polyaminocyclodextrin materials, obtained by direct reaction between heptakis(6-deoxy-6-iodo)-β-cyclodextrin and the proper linear polyamines, were investigated for their binding properties, in order to assess their potential applications in biological systems, such as vectors for simultaneous drug and gene cellular uptake or alternatively for the protection of macromolecules. In particular, we exploited polarimetry to test their interaction with some model p-nitroaniline derivatives, chosen as probe guests. The data obtained indicate that binding inside the host cavity is mainly affected by interplay between Coulomb interactions and conformational restraints. Moreover, simultaneous interaction of the cationic polyamine pendant bush at the primary rim was positively assessed. Insights on quantitative aspects of the interaction between our materials and polyanions were investigated by studying the binding with sodium alginate. Finally, the complexation abilities of the same materials towards polynucleotides were assessed by studying their interaction with the model plasmid pUC19. Our results positively highlight the ability of our materials to exploit both the cavity and the polycationic branches, thus functioning as bimodal ligands. PMID:29564010

Concentrated Solutions of Single-Chain Nanoparticles: A Simple Model for Intrinsically Disordered Proteins under Crowding Conditions.

PubMed

Moreno, Angel J; Lo Verso, Federica; Arbe, Arantxa; Pomposo, José A; Colmenero, Juan

2016-03-03

By means of large-scale computer simulations and small-angle neutron scattering (SANS), we investigate solutions of single-chain nanoparticles (SCNPs), covering the whole concentration range from infinite dilution to melt density. The analysis of the conformational properties of the SCNPs reveals that these synthetic nano-objects share basic ingredients with intrinsically disordered proteins (IDPs), as topological polydispersity, generally sparse conformations, and locally compact domains. We investigate the role of the architecture of the SCNPs in their collapse behavior under macromolecular crowding. Unlike in the case of linear macromolecules, which experience the usual transition from self-avoiding to Gaussian random-walk conformations, crowding leads to collapsed conformations of SCNPs resembling those of crumpled globules. This behavior is already found at volume fractions (about 30%) that are characteristic of crowding in cellular environments. The simulation results are confirmed by the SANS experiments. Our results for SCNPs--a model system free of specific interactions--propose a general scenario for the effect of steric crowding on IDPs: collapse from sparse conformations at high dilution to crumpled globular conformations in cell environments.

Databases and archiving for cryoEM

PubMed Central

Patwardhan, Ardan; Lawson, Catherine L.

2017-01-01

Cryo-EM in structural biology is currently served by three public archives – EMDB for 3DEM reconstructions, PDB for models built from 3DEM reconstructions and EMPIAR for the raw 2D image data used to obtain the 3DEM reconstructions. These archives play a vital role for both the structural community and the wider biological community in making the data accessible so that results may be reused, reassessed and integrated with other structural and bioinformatics resources. The important role of the archives is underpinned by the fact that many journals mandate the deposition of data to PDB and EMDB on publication. The field is currently undergoing transformative changes where on the one hand high-resolution structures are becoming a routine occurrence while on the other hand electron tomography is enabling the study of macromolecules in the cellular context. Concomitantly the archives are evolving to best serve their stakeholder communities. In this chapter we describe the current state of the archives, resources available for depositing, accessing, searching, visualising and validating data, on-going community-wide initiatives and opportunities and challenges for the future. PMID:27572735

Quantifying the assembly of multicomponent molecular machines by single-molecule total internal reflection fluorescence microscopy

PubMed Central

Boehm, Elizabeth M.; Subramanyam, Shyamal; Ghoneim, Mohamed; Washington, M. Todd; Spies, Maria

2016-01-01

Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order (i.e., ternary, quaternary, etc.) complexes using multi-color setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches has become today. PMID:27793278

Quantitative spatiotemporal analysis of antibody fragment diffusion and endocytic consumption in tumor spheroids.

PubMed

Thurber, Greg M; Wittrup, K Dane

2008-05-01

Antibody-based cancer treatment depends upon distribution of the targeting macromolecule throughout tumor tissue, and spatial heterogeneity could significantly limit efficacy in many cases. Antibody distribution in tumor tissue is a function of drug dosage, antigen concentration, binding affinity, antigen internalization, drug extravasation from blood vessels, diffusion in the tumor extracellular matrix, and systemic clearance rates. We have isolated the effects of a subset of these variables by live-cell microscopic imaging of single-chain antibody fragments against carcinoembryonic antigen in LS174T tumor spheroids. The measured rates of scFv penetration and retention were compared with theoretical predictions based on simple scaling criteria. The theory predicts that antibody dose must be large enough to drive a sufficient diffusive flux of antibody to overcome cellular internalization, and exposure time must be long enough to allow penetration to the spheroid center. The experimental results in spheroids are quantitatively consistent with these predictions. Therefore, simple scaling criteria can be applied to accurately predict antibody and antibody fragment penetration distance in tumor tissue.

Quantitative Spatiotemporal Analysis of Antibody Fragment Diffusion and Endocytic Consumption in Tumor Spheroids

PubMed Central

Thurber, Greg M.; Wittrup, K. Dane

2010-01-01

Antibody-based cancer treatment depends upon distribution of the targeting macromolecule throughout tumor tissue, and spatial heterogeneity could significantly limit efficacy in many cases. Antibody distribution in tumor tissue is a function of drug dosage, antigen concentration, binding affinity, antigen internalization, drug extravasation from blood vessels, diffusion in the tumor extracellular matrix, and systemic clearance rates. We have isolated the effects of a subset of these variables by live-cell microscopic imaging of single-chain antibody fragments against carcinoembryonic antigen in LS174T tumor spheroids. The measured rates of scFv penetration and retention were compared with theoretical predictions based on simple scaling criteria. The theory predicts that antibody dose must be large enough to drive a sufficient diffusive flux of antibody to overcome cellular internalization, and exposure time must be long enough to allow penetration to the spheroid center. The experimental results in spheroids are quantitatively consistent with these predictions. Therefore, simple scaling criteria can be applied to accurately predict antibody and antibody fragment penetration distance in tumor tissue. PMID:18451160

ULK1, Mammalian Target of Rapamycin, and Mitochondria: Linking Nutrient Availability and Autophagy

PubMed Central

2011-01-01

Abstract A fundamental function of autophagy conserved from yeast to mammals is mobilization of macromolecules during times of limited nutrient availability, permitting organisms to survive under starvation conditions. In yeast, autophagy is initiated following nitrogen or carbon deprivation, and autophagy mutants die rapidly under these conditions. Similarly, in mammals, autophagy is upregulated in most organs following initiation of starvation, and is critical for survival in the perinatal period following abrupt termination of the placental nutrient supply. The nutrient-sensing kinase, mammalian target of rapamycin, coordinates cellular proliferation and growth with nutrient availability, at least in part by regulating protein synthesis and autophagy-mediated degradation. This review focusses on the regulation of autophagy by Tor, a mammalian target of rapamycin, and Ulk1, a mammalian homolog of Atg1, in response to changes in nutrient availability. Given the importance of mitochondria in maintaining bioenergetic homestasis, and potentially as a source of membrane for autophagosomes during starvation, possible roles for mitochondria in this process are also discussed. Antioxid. Redox Signal. 14, 1953–1958. PMID:21235397

Kinetic attractor phase diagrams of active nematic suspensions: the dilute regime.

PubMed

Forest, M Gregory; Wang, Qi; Zhou, Ruhai

2015-08-28

Large-scale simulations by the authors of the kinetic-hydrodynamic equations for active polar nematics revealed a variety of spatio-temporal attractors, including steady and unsteady, banded (1d) and cellular (2d) spatial patterns. These particle scale activation-induced attractors arise at dilute nanorod volume fractions where the passive equilibrium phase is isotropic, whereas all previous model simulations have focused on the semi-dilute, nematic equilibrium regime and mostly on low-moment orientation tensor and polarity vector models. Here we extend our previous results to complete attractor phase diagrams for active nematics, with and without an explicit polar potential, to map out novel spatial and dynamic transitions, and to identify some new attractors, over the parameter space of dilute nanorod volume fraction and nanorod activation strength. The particle-scale activation parameter corresponds experimentally to a tunable force dipole strength (so-called pushers with propulsion from the rod tail) generated by active rod macromolecules, e.g., catalysis with the solvent phase, ATP-induced propulsion, or light-activated propulsion. The simulations allow 2d spatial variations in all flow and orientational variables and full spherical orientational degrees of freedom; the attractors correspond to numerical integration of a coupled system of 125 nonlinear PDEs in 2d plus time. The phase diagrams with and without the polar interaction potential are remarkably similar, implying that polar interactions among the rodlike particles are not essential to long-range spatial and temporal correlations in flow, polarity, and nematic order. As a general rule, above a threshold, low volume fractions induce 1d banded patterns, whereas higher yet still dilute volume fractions yield 2d patterns. Again as a general rule, varying activation strength at fixed volume fraction induces novel dynamic transitions. First, stationary patterns saturate the instability of the isotropic state, consisting of discrete 1d banded or 2d cellular patterns depending on nanorod volume fraction. Increasing activation strength further induces a sequence of attractor bifurcations, including oscillations superimposed on the 1d and 2d stationary patterns, a uniform translational motion of 1d and 2d oscillating patterns, and periodic switching between 1d and 2d patterns. These results imply that active macromolecular suspensions are capable of long-range spatial and dynamic organization at isotropic equilibrium concentrations, provided particle-scale activation is sufficiently strong.

The effect of macromolecular crowding on the structure of the protein complex superoxide dismutase

NASA Astrophysics Data System (ADS)

Rajapaksha Mudalige, Ajith Rathnaweera

Biological environments contain between 7 - 40% macromolecules by volume. This reduces the available volume for macromolecules and elevates the osmotic pressure relative to pure water. Consequently, biological macromolecules in their native environments tend to adopt more compact and dehydrated conformations than those in vitro. This effect is referred to as macromolecular crowding and constitutes an important physical difference between native biological environments and the simple solutions in which biomolecules are usually studied. We used small angle scattering (SAS) to measure the effects of macromolecular crowding on the size of a protein complex, superoxide dismutase (SOD). Crowding was induced using 400 MW polyethylene glycol (PEG), triethylene glycol (TEG), methyl-alpha-glucoside (alpha-MG) and trimethylamine N-oxide (TMAO). Parallel small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS) allowed us to unambiguously attribute apparent changes in radius of gyration to changes in the structure of SOD. For a 40% PEG solution, we find that the volume of SOD was reduced by 9%. SAS coupled with osmotic pressure measurements allowed us to estimate a compressibility modulus for SOD. We believe this to be the first time the osmotic compressibility of a protein complex was measured. Molecular Dynamics (MD) simulations are widely used to obtain insights on biomolecular processes. However, it is not clear whether MD is capable of predicting subtle effects of macromolecular crowding. We used our experimentally observed compressibility of SOD to evaluate the ability of MD to predict macromolecular crowding. Effects of macromolecular crowding due to PEG on SOD were modeled using an all atom MD simulation with the CHARMM forcefield and the crystallographically resolved structures of SOD and PEG. Two parallel MD simulations were performed for SOD in water and SOD in 40% PEG for over 150~ns. Over the period of the simulation the SOD structure in 40% PEG did not change compared to the SOD structure in water. It therefore appears that under the conditions of our simulations MD could not describe the experimentally observed effects of macromolecular crowding. In a separate project, we measured the rate of diffusive transport in excised porcine corneal stroma using FCS for fluorescent labeled dextran molecules with hydrodynamic radii ranging from 1.3 to 34 nm. Dextran molecules diffuse more slowly in cornea as compared to buffer solution. The reduction in diffusion coefficient is modest however (67% smaller), and is uniform over the range of sizes that we measured. Diffusion coefficients measured parallel vs. perpendicular to the collagen lamellae were indistinguishable. This indicates that diffusion in the corneal stroma is not highly anisotropic. Delivery of therapeutic agents to the eye requires efficient transport through cellular and extracellular barriers. Our measurements bring important insights into how macromolecular and nanoparticle therapeutics might permeate through the eyes.

Cellular internalization mechanism and intracellular trafficking of filamentous M13 phages displaying a cell-penetrating transbody and TAT peptide.

PubMed

Kim, Aeyung; Shin, Tae-Hwan; Shin, Seung-Min; Pham, Chuong D; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

2012-01-01

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01%) than that of TAT-M13 (0.001 ≈ 0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

PubMed Central

Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

2012-01-01

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631

Smooth deuterated cellulose films for the visualisation of adsorbed bio-macromolecules

PubMed Central

Su, Jielong; Raghuwanshi, Vikram S.; Raverty, Warwick; Garvey, Christopher J.; Holden, Peter J.; Gillon, Marie; Holt, Stephen A.; Tabor, Rico; Batchelor, Warren; Garnier, Gil

2016-01-01

Novel thin and smooth deuterated cellulose films were synthesised to visualize adsorbed bio-macromolecules using contrast variation neutron reflectivity (NR) measurements. Incorporation of varying degrees of deuteration into cellulose was achieved by growing Gluconacetobacter xylinus in deuterated glycerol as carbon source dissolved in growth media containing D2O. The derivative of deuterated cellulose was prepared by trimethylsilylation(TMS) in ionic liquid(1-butyl-3-methylimidazolium chloride). The TMS derivative was dissolved in toluene for thin film preparation by spin-coating. The resulting film was regenerated into deuterated cellulose by exposure to acidic vapour. A common enzyme, horseradish peroxidase (HRP), was adsorbed from solution onto the deuterated cellulose films and visualized by NR. The scattering length density contrast of the deuterated cellulose enabled accurate visualization and quantification of the adsorbed HRP, which would have been impossible to achieve with non-deuterated cellulose. The procedure described enables preparing deuterated cellulose films that allows differentiation of cellulose and non-deuterated bio-macromolecules using NR. PMID:27796332

Adsorption properties of the nanozirconia/anionic polyacrylamide system-Effects of surfactant presence, solution pH and polymer carboxyl groups content

NASA Astrophysics Data System (ADS)

Wiśniewska, Małgorzata; Chibowski, Stanisław; Urban, Teresa

2016-05-01

The adsorption mechanism of anionic polyacrylamide (PAM) on the nanozirconia surface was examined. The effects of solution pH, carboxyl groups content in macromolecules and anionic surfactant (sodium dodecyl sulfate-SDS) addition were determined. The more probable structure of polymer adsorption layer was characterized based on the data obtained from spectrophotometry, viscosimetry and potentiometric titration methods. The adsorbed amount of polymer, size of macromolecules in the solution and surface charge density of ZrO2 particles in the absence and presence of PAM were assessed, respectively. Analysis of these results indicated that the increase of solution pH and content of carboxyl groups in the polymeric chains lead to more expanded conformations of adsorbing macromolecules. As a result, the adsorption of anionic polyacrylamide decreased. The SDS presence caused the significant increase of PAM adsorbed amount at pH 3, whereas at pH 6 and 9 the surfactant addition resulted in reduction of polymer adsorption level.

Polar Nature of Biomimetic Fluorapatite/Gelatin Composites: A Comparison of Bipolar Objects and the Polar State of Natural Tissue.

PubMed

Burgener, Matthias; Putzeys, Tristan; Gashti, Mazeyar Parvinzadeh; Busch, Susanne; Aboulfadl, Hanane; Wübbenhorst, Michael; Kniep, Rüdiger; Hulliger, Jürg

2015-09-14

The correspondence of the state of alignment of macromolecules in biomimetic materials and natural tissues is demonstrated by investigating a mechanism of electrical polarity formation: An in vitro grown biomimetic FAp/gelatin composite is investigated for its polar properties by second harmonic (SHGM) and scanning pyroelectric microscopy (SPEM). Hexagonal prismatic seed crystals formed in gelatin gels represent a monodomain polar state, due to aligned mineralized gelatin molecules. Later growth stages, showing dumbbell morphologies, develop into a bipolar state because of surface recognition by gelatin functionality: A reversal of the polar alignment of macromolecules, thus, takes place close to that basal plane of the seed. In natural hard tissues (teeth and bone investigated by SPEM) and the biomimetic FAp/gelatin composite, we find a surprising analogy in view of growth-induced states of polarity: The development of polarity in vivo and in vitro can be explained by a Markov-type mechanism of molecular recognition during the attachment of macromolecules.

The electrokinetic behavior of calcium oxalate monohydrate in macromolecular solutions

NASA Technical Reports Server (NTRS)

Curreri, P. A.; Onoda, G. Y., Jr.; Finlayson, B.

1988-01-01

Electrophoretic mobilities were measured for calcium oxalate monohydrate (COM) in solutions containing macromolecules. Two mucopolysaccharides (sodium heparin and chrondroitin sulfate) and two proteins (positively charged lysozyme and negatively charged bovine serum albumin) were studied as adsorbates. The effects of pH, calcium oxalate surface charge (varied by calcium or oxalate ion activity), and citrate concentration were investigated. All four macromolecules showed evidence for chemical adsorption. The macromolecule concentrations needed for reversing the surface charge indicated that the mucopopolysacchrides have greater affinity for the COM surface than the proteins. The amount of proteins that can chemically adsorb appears to be limited to approximately one monomolecular layer. When the surface charge is high, an insufficient number of proteins can chemically adsorb to neutralize or reverse the surface charge. The remaining surface charge is balanced by proteins held near the surface by longer range electrostatic forces only. Citrate ions at high concentrations appear to compete effectively with the negative protein for surface sites but show no evidence for competing with the positively charged protein.

Principles and Overview of Sampling Methods for Modeling Macromolecular Structure and Dynamics

PubMed Central

Moffatt, Ryan; Ma, Buyong; Nussinov, Ruth

2016-01-01

Investigation of macromolecular structure and dynamics is fundamental to understanding how macromolecules carry out their functions in the cell. Significant advances have been made toward this end in silico, with a growing number of computational methods proposed yearly to study and simulate various aspects of macromolecular structure and dynamics. This review aims to provide an overview of recent advances, focusing primarily on methods proposed for exploring the structure space of macromolecules in isolation and in assemblies for the purpose of characterizing equilibrium structure and dynamics. In addition to surveying recent applications that showcase current capabilities of computational methods, this review highlights state-of-the-art algorithmic techniques proposed to overcome challenges posed in silico by the disparate spatial and time scales accessed by dynamic macromolecules. This review is not meant to be exhaustive, as such an endeavor is impossible, but rather aims to balance breadth and depth of strategies for modeling macromolecular structure and dynamics for a broad audience of novices and experts. PMID:27124275

Small-Molecule-Based Self-Assembled Ligands for G-Quadruplex DNA Surface Recognition.

PubMed

Rivera-Sánchez, María Del C; García-Arriaga, Marilyn; Hobley, Gerard; Morales-de-Echegaray, Ana V; Rivera, José M

2017-10-31

Most drugs are small molecules because of their attractive pharmacokinetics, manageable development and manufacturing, and effective binding into the concave crevices of bio-macromolecules. Despite these features, they often fall short when it comes to effectively recognizing the surfaces of bio-macromolecules. One way to overcome the challenge of biomolecular surface recognition is to develop small molecules that become self-assembled ligands (SALs) prior to binding. Herein, we report SALs made from 8-aryl-2'-deoxyguanosine derivatives forming precise hydrophilic supramolecular G-quadruplexes (SGQs) with excellent size, shape, and charge complementarity to G-quadruplex DNA (QDNA). We show that only those compounds forming SGQs act as SALs, which in turn differentially stabilize QDNAs from selected oncogene promoters and the human telomeric regions. Fluorescence resonance energy-transfer melting assays are consistent with spectroscopic, calorimetric, and light scattering studies, showing the formation of a "sandwichlike" complex QDNA·SGQ·QDNA. These results open the door for the advent of SALs that recognize QDNAs and potentially the surfaces of other bio-macromolecules such as proteins.

AquaSAXS: a web server for computation and fitting of SAXS profiles with non-uniformally hydrated atomic models.

PubMed

Poitevin, Frédéric; Orland, Henri; Doniach, Sebastian; Koehl, Patrice; Delarue, Marc

2011-07-01

Small Angle X-ray Scattering (SAXS) techniques are becoming more and more useful for structural biologists and biochemists, thanks to better access to dedicated synchrotron beamlines, better detectors and the relative easiness of sample preparation. The ability to compute the theoretical SAXS profile of a given structural model, and to compare this profile with the measured scattering intensity, yields crucial structural informations about the macromolecule under study and/or its complexes in solution. An important contribution to the profile, besides the macromolecule itself and its solvent-excluded volume, is the excess density due to the hydration layer. AquaSAXS takes advantage of recently developed methods, such as AquaSol, that give the equilibrium solvent density map around macromolecules, to compute an accurate SAXS/WAXS profile of a given structure and to compare it to the experimental one. Here, we describe the interface architecture and capabilities of the AquaSAXS web server (http://lorentz.dynstr.pasteur.fr/aquasaxs.php).

Thermochemolysis: A New Sample Preparation Approach for the Detection of Organic Components of Complex Macromolecules in Mars Rocks via Gas Chromatography Mass Spectrometry in SAM on MSL

NASA Technical Reports Server (NTRS)

Eugenbrode, J.; Glavin, D.; Dworkin, J.; Conrad, P.; Mahaffy, P.

2011-01-01

Organic chemicals, when present in extraterrestrial samples, afford precious insight into past and modern conditions elsewhere in the Solar System . No single technology identifies all molecular components because naturally occurring molecules have different chemistries (e.g., polar vs. non-polar, low to high molecular weight) and interface with the ambient sample chemistry in a variety of modes (i.e., organics may be bonded, absorbed or trapped by minerals, liquids, gases, or other organics). More than 90% of organic matter in most natural samples on Earth and in meteorites is composed of complex macromolecules (e.g. biopolymers, complex biomolecules, humic substances, kerogen) because the processes that tend to break down organic molecules also tend towards complexation of the more recalcitrant components. Thus, methodologies that tap the molecular information contained within macromolecules may be critical to detecting extraterrestrial organic matter and assessing the sources and processes influencing its nature.

Principles and Overview of Sampling Methods for Modeling Macromolecular Structure and Dynamics.

PubMed

Maximova, Tatiana; Moffatt, Ryan; Ma, Buyong; Nussinov, Ruth; Shehu, Amarda

2016-04-01

Investigation of macromolecular structure and dynamics is fundamental to understanding how macromolecules carry out their functions in the cell. Significant advances have been made toward this end in silico, with a growing number of computational methods proposed yearly to study and simulate various aspects of macromolecular structure and dynamics. This review aims to provide an overview of recent advances, focusing primarily on methods proposed for exploring the structure space of macromolecules in isolation and in assemblies for the purpose of characterizing equilibrium structure and dynamics. In addition to surveying recent applications that showcase current capabilities of computational methods, this review highlights state-of-the-art algorithmic techniques proposed to overcome challenges posed in silico by the disparate spatial and time scales accessed by dynamic macromolecules. This review is not meant to be exhaustive, as such an endeavor is impossible, but rather aims to balance breadth and depth of strategies for modeling macromolecular structure and dynamics for a broad audience of novices and experts.

Validating metal binding sites in macromolecule structures using the CheckMyMetal web server

PubMed Central

Zheng, Heping; Chordia, Mahendra D.; Cooper, David R.; Chruszcz, Maksymilian; Müller, Peter; Sheldrick, George M.

2015-01-01

Metals play vital roles in both the mechanism and architecture of biological macromolecules. Yet structures of metal-containing macromolecules where metals are misidentified and/or suboptimally modeled are abundant in the Protein Data Bank (PDB). This shows the need for a diagnostic tool to identify and correct such modeling problems with metal binding environments. The "CheckMyMetal" (CMM) web server (http://csgid.org/csgid/metal_sites/) is a sophisticated, user-friendly web-based method to evaluate metal binding sites in macromolecular structures in respect to 7350 metal binding sites observed in a benchmark dataset of 2304 high resolution crystal structures. The protocol outlines how the CMM server can be used to detect geometric and other irregularities in the structures of metal binding sites and alert researchers to potential errors in metal assignment. The protocol also gives practical guidelines for correcting problematic sites by modifying the metal binding environment and/or redefining metal identity in the PDB file. Several examples where this has led to meaningful results are described in the anticipated results section. CMM was designed for a broad audience—biomedical researchers studying metal-containing proteins and nucleic acids—but is equally well suited for structural biologists to validate new structures during modeling or refinement. The CMM server takes the coordinates of a metal-containing macromolecule structure in the PDB format as input and responds within a few seconds for a typical protein structure modeled with a few hundred amino acids. PMID:24356774

Diffusion profile of macromolecules within and between human skin layers for (trans)dermal drug delivery.

PubMed

Römgens, Anne M; Bader, Dan L; Bouwstra, Joke A; Baaijens, Frank P T; Oomens, Cees W J

2015-10-01

Delivering a drug into and through the skin is of interest as the skin can act as an alternative drug administration route for oral delivery. The development of new delivery methods, such as microneedles, makes it possible to not only deliver small molecules into the skin, which are able to pass the outer layer of the skin in therapeutic amounts, but also macromolecules. To provide insight into the administration of these molecules into the skin, the aim of this study was to assess the transport of macromolecules within and between its various layers. The diffusion coefficients in the epidermis and several locations in the papillary and reticular dermis were determined for fluorescein dextran of 40 and 500 kDa using a combination of fluorescent recovery after photobleaching experiments and finite element analysis. The diffusion coefficient was significantly higher for 40 kDa than 500 kDa dextran, with median values of 23 and 9 µm(2)/s in the dermis, respectively. The values only marginally varied within and between papillary and reticular dermis. For the 40 kDa dextran, the diffusion coefficient in the epidermis was twice as low as in the dermis layers. The adopted method may be used for other macromolecules, which are of interest for dermal and transdermal drug delivery. The knowledge about diffusion in the skin is useful to optimize (trans)dermal drug delivery systems to target specific layers or cells in the human skin. Copyright © 2015 Elsevier Ltd. All rights reserved.

Auditory Pattern Memory: Mechanisms of Tonal Sequence Discrimination by Human Observers.

DTIC Science & Technology

1987-09-30

different task, and Macmillan, Kaplan, and Creelman (1977) in a study of categorica l percept ion. Tanrr ’s model included a short-term decaying...components, J. Acoust. Soc. of Am., 76, 1037-1044. 34 Macmillan, N. A., Kaplan H. L., and Creelman , C. D. The psychophysics of categorical perception... Psychological Review, 1977, 84, 452-471. Sankoff, D., and Kruskal, J. B. (1983). Time Warps, String Edits, and Macromolecules: The Theory and Practice of

Improved data visualization techniques for analyzing macromolecule structural changes.

PubMed

Kim, Jae Hyun; Iyer, Vidyashankara; Joshi, Sangeeta B; Volkin, David B; Middaugh, C Russell

2012-10-01

The empirical phase diagram (EPD) is a colored representation of overall structural integrity and conformational stability of macromolecules in response to various environmental perturbations. Numerous proteins and macromolecular complexes have been analyzed by EPDs to summarize results from large data sets from multiple biophysical techniques. The current EPD method suffers from a number of deficiencies including lack of a meaningful relationship between color and actual molecular features, difficulties in identifying contributions from individual techniques, and a limited ability to be interpreted by color-blind individuals. In this work, three improved data visualization approaches are proposed as techniques complementary to the EPD. The secondary, tertiary, and quaternary structural changes of multiple proteins as a function of environmental stress were first measured using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering, respectively. Data sets were then visualized as (1) RGB colors using three-index EPDs, (2) equiangular polygons using radar charts, and (3) human facial features using Chernoff face diagrams. Data as a function of temperature and pH for bovine serum albumin, aldolase, and chymotrypsin as well as candidate protein vaccine antigens including a serine threonine kinase protein (SP1732) and surface antigen A (SP1650) from S. pneumoniae and hemagglutinin from an H1N1 influenza virus are used to illustrate the advantages and disadvantages of each type of data visualization technique. Copyright © 2012 The Protein Society.

Improved data visualization techniques for analyzing macromolecule structural changes

PubMed Central

Kim, Jae Hyun; Iyer, Vidyashankara; Joshi, Sangeeta B; Volkin, David B; Middaugh, C Russell

2012-01-01

The empirical phase diagram (EPD) is a colored representation of overall structural integrity and conformational stability of macromolecules in response to various environmental perturbations. Numerous proteins and macromolecular complexes have been analyzed by EPDs to summarize results from large data sets from multiple biophysical techniques. The current EPD method suffers from a number of deficiencies including lack of a meaningful relationship between color and actual molecular features, difficulties in identifying contributions from individual techniques, and a limited ability to be interpreted by color-blind individuals. In this work, three improved data visualization approaches are proposed as techniques complementary to the EPD. The secondary, tertiary, and quaternary structural changes of multiple proteins as a function of environmental stress were first measured using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering, respectively. Data sets were then visualized as (1) RGB colors using three-index EPDs, (2) equiangular polygons using radar charts, and (3) human facial features using Chernoff face diagrams. Data as a function of temperature and pH for bovine serum albumin, aldolase, and chymotrypsin as well as candidate protein vaccine antigens including a serine threonine kinase protein (SP1732) and surface antigen A (SP1650) from S. pneumoniae and hemagglutinin from an H1N1 influenza virus are used to illustrate the advantages and disadvantages of each type of data visualization technique. PMID:22898970

Recognition- and Reactivity-Based Fluorescent Probes for Studying Transition Metal Signaling in Living Systems

PubMed Central

2015-01-01

Conspectus Metals are essential for life, playing critical roles in all aspects of the central dogma of biology (e.g., the transcription and translation of nucleic acids and synthesis of proteins). Redox-inactive alkali, alkaline earth, and transition metals such as sodium, potassium, calcium, and zinc are widely recognized as dynamic signals, whereas redox-active transition metals such as copper and iron are traditionally thought of as sequestered by protein ligands, including as static enzyme cofactors, in part because of their potential to trigger oxidative stress and damage via Fenton chemistry. Metals in biology can be broadly categorized into two pools: static and labile. In the former, proteins and other macromolecules tightly bind metals; in the latter, metals are bound relatively weakly to cellular ligands, including proteins and low molecular weight ligands. Fluorescent probes can be useful tools for studying the roles of transition metals in their labile forms. Probes for imaging transition metal dynamics in living systems must meet several stringent criteria. In addition to exhibiting desirable photophysical properties and biocompatibility, they must be selective and show a fluorescence turn-on response to the metal of interest. To meet this challenge, we have pursued two general strategies for metal detection, termed “recognition” and “reactivity”. Our design of transition metal probes makes use of a recognition-based approach for copper and nickel and a reactivity-based approach for cobalt and iron. This Account summarizes progress in our laboratory on both the development and application of fluorescent probes to identify and study the signaling roles of transition metals in biology. In conjunction with complementary methods for direct metal detection and genetic and/or pharmacological manipulations, fluorescent probes for transition metals have helped reveal a number of principles underlying transition metal dynamics. In this Account, we give three recent examples from our laboratory and collaborations in which applications of chemical probes reveal that labile copper contributes to various physiologies. The first example shows that copper is an endogenous regulator of neuronal activity, the second illustrates cellular prioritization of mitochondrial copper homeostasis, and the third identifies the “cuprosome” as a new copper storage compartment in Chlamydomonas reinhardtii green algae. Indeed, recognition- and reactivity-based fluorescent probes have helped to uncover new biological roles for labile transition metals, and the further development of fluorescent probes, including ones with varied Kd values and new reaction triggers and recognition receptors, will continue to reveal exciting and new biological roles for labile transition metals. PMID:26215055

Recognition- and reactivity-based fluorescent probes for studying transition metal signaling in living systems.

PubMed

Aron, Allegra T; Ramos-Torres, Karla M; Cotruvo, Joseph A; Chang, Christopher J

2015-08-18

Metals are essential for life, playing critical roles in all aspects of the central dogma of biology (e.g., the transcription and translation of nucleic acids and synthesis of proteins). Redox-inactive alkali, alkaline earth, and transition metals such as sodium, potassium, calcium, and zinc are widely recognized as dynamic signals, whereas redox-active transition metals such as copper and iron are traditionally thought of as sequestered by protein ligands, including as static enzyme cofactors, in part because of their potential to trigger oxidative stress and damage via Fenton chemistry. Metals in biology can be broadly categorized into two pools: static and labile. In the former, proteins and other macromolecules tightly bind metals; in the latter, metals are bound relatively weakly to cellular ligands, including proteins and low molecular weight ligands. Fluorescent probes can be useful tools for studying the roles of transition metals in their labile forms. Probes for imaging transition metal dynamics in living systems must meet several stringent criteria. In addition to exhibiting desirable photophysical properties and biocompatibility, they must be selective and show a fluorescence turn-on response to the metal of interest. To meet this challenge, we have pursued two general strategies for metal detection, termed "recognition" and "reactivity". Our design of transition metal probes makes use of a recognition-based approach for copper and nickel and a reactivity-based approach for cobalt and iron. This Account summarizes progress in our laboratory on both the development and application of fluorescent probes to identify and study the signaling roles of transition metals in biology. In conjunction with complementary methods for direct metal detection and genetic and/or pharmacological manipulations, fluorescent probes for transition metals have helped reveal a number of principles underlying transition metal dynamics. In this Account, we give three recent examples from our laboratory and collaborations in which applications of chemical probes reveal that labile copper contributes to various physiologies. The first example shows that copper is an endogenous regulator of neuronal activity, the second illustrates cellular prioritization of mitochondrial copper homeostasis, and the third identifies the "cuprosome" as a new copper storage compartment in Chlamydomonas reinhardtii green algae. Indeed, recognition- and reactivity-based fluorescent probes have helped to uncover new biological roles for labile transition metals, and the further development of fluorescent probes, including ones with varied Kd values and new reaction triggers and recognition receptors, will continue to reveal exciting and new biological roles for labile transition metals.

Endocytosis of glycosylphosphatidylinositol-anchored proteins

PubMed Central

2009-01-01

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) represent an interesting amalgamation of the three basic kinds of cellular macromolecules viz. proteins, carbohydrates and lipids. An unusually hybrid moiety, the GPI-anchor is expressed in a diverse range of organisms from parasites to mammalian cells and serves to anchor a large number of functionally diverse proteins and has been the center of attention in scientific debate for some time now. Membrane organization of GPI-APs into laterally-organized cholesterol-sphingolipid ordered membrane domains or "rafts" and endocytosis of GPI-APs has been intensely debated. Inclusion into or exclusion from these membrane domains seems to be the critical factor in determining the endocytic mechanisms and intracellular destinations of GPI-APs. The intracellular signaling as well as endocytic trafficking of GPI-APs is critically dependent upon the cell surface organization of GPI-APs, and the associations with these lipid rafts play a vital role during these processes. The mechanism of endocytosis for GPI-APs may differ from other cellular endocytic pathways, such as those mediated by clathrin-coated pits (caveolae), and is necessary for unique biological functions. Numerous intracellular factors are involved in and regulate the endocytosis of GPI-APs, and these may be variably dependent on cell-type. The central focus of this article is to describe the significance of the endocytosis of GPI-APs on a multitude of biological processes, ranging from nutrient-uptake to more complex immune responses. Ultimately, a thorough elucidation of GPI-AP mediated signaling pathways and their regulatory elements will enhance our understanding of essential biological processes and benefit as components of disease intervention strategies. PMID:19832981

Cytochrome P450s and molecular epidemiology

NASA Astrophysics Data System (ADS)

Gonzalez, Frank J.; Gelboin, Harry V.

1993-03-01

Cytochrome P450 (P450) represent a superfamily of heme-containing monooxygenases that are found throughout the animal and plant kingdoms and in many microorganisms. A number of these enzymes are involved in biosynthetic pathways of steroid synthesis but in mammals the vast majority of P450s function to metabolize foreign chemicals or xenobiotics. In the classical phase I reactions on the latter, a membrane-bound P450 will hydroxylate a compound, usually hydrophobic in nature, and the hydroxyl group will serve as a substrate for the various transferases or phase II enzymes that attach hydrophilic substituents such as glutathione, sulfate or glucuronic acid. Some chemicals, however, are metabolically-activated by P450s to electrophiles capable of reacting with cellular macromolecules. The cellular concentrations of the chemical and P450, reactivity of the active metabolite with nucleic acid and the repairability of the resultant adducts, in addition to the nature of the cell type, likely determines whether a chemical will be toxic and kill the cell or will transform the cell. Immunocorrelative and cDNA-directed expression have been used to define the substrate specificities of numerous human P450s. Levels of expression of different human P450 forms have been measured by both in vivo and in vitro methodologies leading to the realization that a large degree of interindividual differences occur in P450 expression. Reliable procedures for measuring P450 expression in healthy and diseased subjects will lead to prospective and case- cohort studies to determine whether interindividual differences in levels of P450 are associated with susceptibility or resistance to environmentally-based disease.

Protein diffusion along DNA: on the effect of roadblocks and crowders

NASA Astrophysics Data System (ADS)

Krepel, Dana; Levy, Yaakov

2016-12-01

Rapid recognition by a protein of its DNA target site is achieved through a combination of one- and three-dimensional (1D and 3D) diffusion, which allows efficient scanning of the many alternative sites. This facilitated diffusion mechanism is expected to be affected by cellular conditions, particularly crowding, given that up to 40% of the total cellular volume may by occupied by macromolecules. Both experimental and theoretical studies showed that crowding particles can enhance facilitated diffusion and accelerate search kinetics. This effect may originate from crowding forcing a trade-off between 3D and 1D diffusion. In this study, using coarse-grained molecular dynamic simulations, we investigate how the molecular properties of the crowders may modulate the effect exerted by crowding on a searcher protein. We show that crowders with an affinity to the DNA are less effective search facilitators than particles whose contribution is solely entropic. Crowders that have affinity to DNA may occupy DNA sites and thereby function as obstacles or roadblocks that slow down the searcher protein, and they may also produce a smaller excluded volume effect and so reduce usage of the hopping searching mode in favor of less-effective 3D diffusion in the bulk. We discuss how strong repulsive interactions between the crowding particles themselves may affect the overall dynamics of the crowders and their excluded volume effect. Our study shows that search kinetics and its mechanism are modulated not only by salt concentration and crowding occupancy, but also by the properties of the crowding particles.

Elucidating the role of osmotic, ionic and major salt responsive transcript components towards salinity tolerance in contrasting chickpea (Cicer arietinum L.) genotypes.

PubMed

Singh, Jogendra; Singh, Vijayata; Sharma, P C

2018-05-01

The growth of chickpea ( Cicer arietinum L.) is extremely hampered by salt stress. Understanding of physio-biochemical and molecular attributes along with morphological traits contributing to the salinity tolerance is important for developing salt tolerant chickpea varieties. To explore these facts, two genotypes CSG8962 and HC5 with contrasting salt tolerance were evaluated in the salinity stress (Control and 120 mM NaCl) conditions. CSG8962 maintained lower Na/K ratio in root and shoot, trammeled Na translocation to the shoots from roots compared to HC5 which ascribed to better exclusion of salt from its roots and compartmentation in the shoot. In chickpea, salt stress specifically induced genes/sequences involved at several levels in the salt stress signaling pathway. Higher induction of trehalose 6 phosphate synthase and protein kinase genes pertaining to the osmotic and signaling modules, respectively, were evident in CSG8962 compared to HC5. Further transcripts of late embryogenesis abundant, non-specific lipid transfer protein, HI and 219 genes/sequences were also highly induced in CSG8962 compared to HC5 which emphasizes the better protection of cellular membranous network and membrane-bound macromolecules under salt stress. This further suppressed the stress enhanced electrolyte leakage, loss of turgidity, promoted the higher compatible solute accumulation and maintained better cellular ion homoeostasis in CSG8962 compared to HC5. Our study further adds to the importance of these genes in salt tolerance by comparing their behavior in contrasting chickpea genotypes.

Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress

PubMed Central

Nunes, Paula; Roth, Isabelle; Meda, Paolo; Féraille, Eric; Brown, Dennis; Hasler, Udo

2015-01-01

Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking. PMID:26045497

Deep learning-based subdivision approach for large scale macromolecules structure recovery from electron cryo tomograms

PubMed Central

Xu, Min; Chai, Xiaoqi; Muthakana, Hariank; Liang, Xiaodan; Yang, Ge; Zeev-Ben-Mordehai, Tzviya; Xing, Eric P.

2017-01-01

Abstract Motivation: Cellular Electron CryoTomography (CECT) enables 3D visualization of cellular organization at near-native state and in sub-molecular resolution, making it a powerful tool for analyzing structures of macromolecular complexes and their spatial organizations inside single cells. However, high degree of structural complexity together with practical imaging limitations makes the systematic de novo discovery of structures within cells challenging. It would likely require averaging and classifying millions of subtomograms potentially containing hundreds of highly heterogeneous structural classes. Although it is no longer difficult to acquire CECT data containing such amount of subtomograms due to advances in data acquisition automation, existing computational approaches have very limited scalability or discrimination ability, making them incapable of processing such amount of data. Results: To complement existing approaches, in this article we propose a new approach for subdividing subtomograms into smaller but relatively homogeneous subsets. The structures in these subsets can then be separately recovered using existing computation intensive methods. Our approach is based on supervised structural feature extraction using deep learning, in combination with unsupervised clustering and reference-free classification. Our experiments show that, compared with existing unsupervised rotation invariant feature and pose-normalization based approaches, our new approach achieves significant improvements in both discrimination ability and scalability. More importantly, our new approach is able to discover new structural classes and recover structures that do not exist in training data. Availability and Implementation: Source code freely available at http://www.cs.cmu.edu/∼mxu1/software. Contact: mxu1@cs.cmu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:28881965

Overview of the cellular and molecular basis of kidney fibrosis

PubMed Central

Eddy, Allison A

2014-01-01

The common pathogenetic pathway of progressive injury in patients with chronic kidney disease (CKD) is epitomized as normal kidney parenchymal destruction due to scarring (fibrosis). Understanding the fundamental pathways that lead to renal fibrosis is essential in order to develop better therapeutic options for human CKD. Although complex, four cellular responses are pivotal. (1) An interstitial inflammatory response that has multiple consequences—some harmful and others healing. (2) The appearance of a unique interstitial cell population of myofibroblasts, primarily derived from kidney stromal cells (fibroblasts and pericytes), that are the primary source of the various extracellular matrix proteins that form interstitial scars. (3) Tubular epithelial cells that have variable and time-dependent roles as early responders to injury and later as victims of fibrosis due to the loss of their regenerative abilities. (4) Loss of interstitial capillary integrity that compromises oxygen delivery and leads to a vicious cascade of hypoxia–oxidant stress that accentuates injury and fibrosis. In the absence of adequate angiogenic responses, a healthy interstitial capillary network is not maintained. The fibrotic ‘scar' that typifies CKD is an interesting consortium of multifunctional macromolecules that not only change in composition and structure over time, but can be degraded via extracellular and intracellular proteases. Although transforming growth factor beta appears to be the primary driver of kidney fibrosis, a vast array of additional molecules may have modulating roles. The importance of genetic and epigenetic factors is increasingly appreciated. An intriguing but incompletely understood cardiorenal syndrome underlies the high morbidity and mortality rates that develop in association with progressive kidney fibrosis. PMID:25401038

Peptide-directed self-assembly of hydrogels

PubMed Central

Kopeček, Jindřich; Yang, Jiyuan

2009-01-01

This review focuses on the self-assembly of macromolecules mediated by the biorecognition of peptide/protein domains. Structures forming α-helices and β-sheets have been used to mediate self-assembly into hydrogels of peptides, reactive copolymers and peptide motifs, block copolymers, and graft copolymers. Structural factors governing the self-assembly of these molecules into precisely defined three-dimensional structures (hydrogels) are reviewed. The incorporation of peptide motifs into hybrid systems, composed of synthetic and natural macromolecules, enhances design opportunities for new biomaterials when compared to individual components. PMID:18952513

Extracellular vesicle communication pathways as regulatory targets of oncogenic transformation.

PubMed

Choi, Dongsic; Lee, Tae Hoon; Spinelli, Cristiana; Chennakrishnaiah, Shilpa; D'Asti, Esterina; Rak, Janusz

2017-07-01

Pathogenesis of human cancers bridges intracellular oncogenic driver events and their impact on intercellular communication. Among multiple mediators of this 'pathological connectivity' the role of extracellular vesicles (EVs) and their subsets (exosomes, ectosomes, oncosomes) is of particular interest for several reasons. The release of EVs from cancer cells represents a unique mechanism of regulated expulsion of bioactive molecules, a process that also mediates cell-to-cell transfer of lipids, proteins, and nucleic acids. Biological effects of these processes have been implicated in several aspects of cancer-related pathology, including tumour growth, invasion, angiogenesis, metastasis, immunity and thrombosis. Notably, the emerging evidence suggests that oncogenic mutations may impact several aspects of EV-mediated cell-cell communication including: (i) EV release rate and protein content; (ii) molecular composition of cancer EVs; (iii) the inclusion of oncogenic and mutant macromolecules in the EV cargo; (iv) EV-mediated release of genomic DNA; (v) deregulation of mechanisms responsible for EV biogenesis (vesiculome) and (vi) mechanisms of EV uptake by cancer cells. Intriguingly, EV-mediated intercellular transfer of mutant and oncogenic molecules between subpopulations of cancer cells, their indolent counterparts and stroma may exert profound biological effects that often resemble (but are not tantamount to) oncogenic transformation, including changes in cell growth, clonogenicity and angiogenic phenotype, or cause cell stress and death. However, several biological barriers likely curtail a permanent horizontal transformation of normal cells through EV-mediated mechanisms. The ongoing analysis and targeting of EV-mediated intercellular communication pathways can be viewed as a new therapeutic paradigm in cancer, while the analysis of oncogenic cargo contained in EVs released from cancer cells into biofluids is being developed for clinical use as a biomarker and companion diagnostics. Indeed, studies are underway to further explore the multiple links between molecular causality in cancer and various aspects of cellular vesiculation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Validation of ligands in macromolecular structures determined by X-ray crystallography

PubMed Central

Horský, Vladimír; Svobodová Vařeková, Radka; Bendová, Veronika

2018-01-01

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) play a crucial role in structure-guided drug discovery and design, and also provide atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. The quality with which small-molecule ligands have been modelled in Protein Data Bank (PDB) entries has been, and continues to be, a matter of concern for many investigators. Correctly interpreting whether electron density found in a binding site is compatible with the soaked or co-crystallized ligand or represents water or buffer molecules is often far from trivial. The Worldwide PDB validation report (VR) provides a mechanism to highlight any major issues concerning the quality of the data and the model at the time of deposition and annotation, so the depositors can fix issues, resulting in improved data quality. The ligand-validation methods used in the generation of the current VRs are described in detail, including an examination of the metrics to assess both geometry and electron-density fit. It is found that the LLDF score currently used to identify ligand electron-density fit outliers can give misleading results and that better ligand-validation metrics are required. PMID:29533230

Introduction to protein crystallization

PubMed Central

McPherson, Alexander; Gavira, Jose A.

2014-01-01

Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid–liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies. PMID:24419610