Showing papers in "Cell Biochemistry and Biophysics in 2009"
TL;DR: In this review, primary attention is given to the antioxidant (and prooxidant) activity of polyphenols arising from their interactions with iron both in vitro and in vivo.
Abstract: In this review, primary attention is given to the antioxidant (and prooxidant) activity of polyphenols arising from their interactions with iron both in vitro and in vivo. In addition, an overview of oxidative stress and the Fenton reaction is provided, as well as a discussion of the chemistry of iron binding by catecholate, gallate, and semiquinone ligands along with their stability constants, UV–vis spectra, stoichiometries in solution as a function of pH, rates of iron oxidation by O2 upon polyphenol binding, and the published crystal structures for iron–polyphenol complexes. Radical scavenging mechanisms of polyphenols unrelated to iron binding, their interactions with copper, and the prooxidant activity of iron–polyphenol complexes are briefly discussed.
1,027 citations
TL;DR: The antioxidant activities of sulfur and selenium compounds are discussed, focusing on several antioxidant mechanisms, including ROS scavenging, glutathione peroxidase, and metal-binding antioxidant mechanisms.
Abstract: It is well known that oxidation caused by reactive oxygen species (ROS) is a major cause of cellular damage and death and has been implicated in cancer, neurodegenerative, and cardiovascular diseases. Small-molecule antioxidants containing sulfur and selenium can ameliorate oxidative damage, and cells employ multiple antioxidant mechanisms to prevent this cellular damage. However, current research has focused mainly on clinical, epidemiological, and in vivo studies with little emphasis on the antioxidant mechanisms responsible for observed sulfur and selenium antioxidant activities. In addition, the antioxidant properties of sulfur compounds are commonly compared to selenium antioxidant properties; however, sulfur and selenium antioxidant activities can be quite distinct, with each utilizing different antioxidant mechanisms to prevent oxidative cellular damage. In the present review, we discuss the antioxidant activities of sulfur and selenium compounds, focusing on several antioxidant mechanisms, including ROS scavenging, glutathione peroxidase, and metal-binding antioxidant mechanisms. Findings of several recent clinical, epidemiological, and in vivo studies highlight the need for future studies that specifically focus on the chemical mechanisms of sulfur and selenium antioxidant behavior.
393 citations
TL;DR: A hypothesis of a novel systemic regulatory network, named “NAD World”, for mammalian aging, is developed that provides important insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of metabolic robustness and aging in mammals.
Abstract: For the past several years, it has been demonstrated that the NAD-dependent protein deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis together play a critical role in the regulation of metabolism and possibly aging in mammals. Based on our recent studies on these two critical components, we have developed a hypothesis of a novel systemic regulatory network, named “NAD World”, for mammalian aging. Conceptually, in the NAD World, systemic NAD biosynthesis mediated by intra- and extracellular Nampt functions as a driver that keeps up the pace of metabolism in multiple tissues/organs, and the NAD-dependent deacetylase Sirt1 serves as a universal mediator that executes metabolic effects in a tissue-dependent manner in response to changes in systemic NAD biosynthesis. This new concept of the NAD World provides important insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of metabolic robustness and aging in mammals.
175 citations
TL;DR: This review summarizes the current understanding of the biology of the R7 RGS complexes including their structure/functional organization, protein–protein interactions, and physiological roles.
Abstract: G protein-coupled receptor signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as regulators of G protein signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior, and nociception in mammals. Four proteins in this group, RGS6, RGS7, RGS9, and RGS11, share a common molecular organization of three modules: (i) the catalytic RGS domain, (ii) a GGL domain that recruits Gβ5, an outlying member of the G protein beta subunit family, and (iii) a DEP/DHEX domain that mediates interactions with the membrane anchor proteins R7BP and R9AP. As heterotrimeric complexes, R7 RGS proteins not only associate with and regulate a number of G protein signaling pathway components, but have also been found to form complexes with proteins that are not traditionally associated with G protein signaling. This review summarizes our current understanding of the biology of the R7 RGS complexes including their structure/functional organization, protein–protein interactions, and physiological roles.
134 citations
TL;DR: How checkpoint signaling adjusts cell cycle progression to the emergency situation and the use of specialized pathways promoting replication restart is reviewed, which gives cells more time to deal with the damage.
Abstract: During every S phase, cells need to duplicate their genomes so that both daughter cells inherit complete copies of genetic information. Given the large size of mammalian genomes and the required precision of DNA replication, genome duplication requires highly fine-tuned corrective and quality control processes. A major threat to the accuracy and efficiency of DNA synthesis is the presence of DNA lesions, caused by both endogenous and exogenous damaging agents. Replicative DNA polymerases, which carry out the bulk of DNA synthesis, evolved to do their job extremely precisely and efficiently. However, they are unable to use damaged DNA as a template and, consequently, are stopped at most DNA lesions. Failure to restart such stalled replication forks can result in major chromosomal aberrations and lead to cell dysfunction or death. Therefore, a well-coordinated response to replication perturbation is essential for cell survival and fitness. Here we review how this response involves activating checkpoint signaling and the use of specialized pathways promoting replication restart. Checkpoint signaling adjusts cell cycle progression to the emergency situation and thus gives cells more time to deal with the damage. Replication restart is mediated by two pathways. Homologous recombination uses homologous DNA sequence to repair or bypass the lesion and is therefore mainly error free. Error-prone translesion synthesis employs specialized, low fidelity polymerases to bypass the damage.
134 citations
TL;DR: The role of vinculin in cell invasion through a 3D extracellular matrix is still fragmentarily investigated in this article, however, the role of Vinculin has been described as an inhibitor of cell migration on planar substrate.
Abstract: Vinculin couples as a focal adhesion protein the extracellular matrix (ECM) through integrins to the actomyosin cytoskeleton. During the last years vinculin has become the focus of cell mechanical measurements and a key protein regulating the transmission of contractile forces. In earlier reports vinculin has been described as an inhibitor of cell migration on planar substrates, because knock-out of vinculin in F9 mouse embryonic carcinoma cells and mouse embryonic fibroblasts showed increased cell motility on 2D substrates. The role of vinculin in cell invasion through a 3D extracellular matrix is still fragmentarily investigated. This review presents vinculin in its role as a regulator of cellular mechanical functions. Contractile force generation is reduced when vinculin is absent, or enhanced when vinculin is present. Moreover, the generation of contractile forces is a prerequisite for cell invasion through a dense 3D ECM, where the pore-size is smaller than the diameter of the cell nucleus (<2 μm). Measurements of cell’s biophysical properties will be presented. In summary, vinculin’s leading role among focal adhesion proteins in regulating the mechanical properties of cells will be discussed.
128 citations
TL;DR: The classical examination of histology slides from a mouse model of breast cancer has been extended in this study to incorporate modern multiphoton excitation and photon-counting techniques and it was found that the properties of the fluorescence from the endogenous fluorophores NADH and FAD were indicative of the pathological state of the tissue.
Abstract: The classical examination of histology slides from a mouse model of breast cancer has been extended in this study to incorporate modern multiphoton excitation and photon-counting techniques. The advantage of such approaches is quantification of potential diagnostic parameters from the fluorescence emission signal, whereby the traditional descriptive staging process is complemented by measurements of fluorescence intensity, lifetime, and spectra. We explored whether the clinical “gold standard” of eosin and hematoxylin stained histology slides would provide optical biomarker signatures of diagnostic value. Alternatively, we examined unstained slides for changes in intensity and/or fluorescence lifetime of relevant endogenous fluorophores. Although eosin provided a strong emission signal and had distinct spectra and lifetime, we found that it was not useful as a fluorescent biological marker, particularly when combined with hematoxylin. Instead, we found that the properties of the fluorescence from the endogenous fluorophores NADH and FAD were indicative of the pathological state of the tissue. Comparing regions of carcinoma in situ to adjacent histologically normal regions, we found that tumor cells produced higher intensity and had a longer fluorescence lifetime. By imaging at 780 nm and 890 nm excitation, we were able to differentiate the fluorescence of FAD from NADH by separating the emission spectra. The shift to a longer lifetime in tumor cells was independent of the free or bound state of FAD and NADH, and of the excitation wavelength. Most forms of cancer have altered metabolism and redox ratios; here we present a method that has potential for early detection of these changes, which are preserved in fixed tissue samples such as classic histopathology slides.
122 citations
TL;DR: The focus will be on updating the recent developments in the field of integrin-growth factor receptor associations and their implications in the vascular processes.
Abstract: A sequence of events in vascular and stromal cells maintained in a highly coordinated manner regulates angiogenesis and tissue remodeling. These processes are mediated by the ability of cells to respond to environmental cues and activate surface integrins. Physiological and pathological processes in vascular biology are dependent on the specificity of important signaling mechanisms that are activated through the association between growth factors, their receptors, integrins, and their specific extracellular matrix ligands. A large body of evidence from in vitro and in vivo models demonstrates the importance of coordination of signals from the extracellular environment that activates specific tyrosine kinase receptors and integrins in order to regulate angiogenic processes in vivo. In addition to complex formation between growth factor receptors and integrins, growth factors and cytokines also directly interact with integrins, depending upon their concentration levels in the environment, and differentially regulate integrin-related processes. Recent studies from a number of laboratories including ours have provided important novel insights into the involvement of many signaling events that improve our existing knowledge on the cross-talk between growth factor receptors and integrins in the regulation of angiogenesis. In this review, our focus will be on updating the recent developments in the field of integrin-growth factor receptor associations and their implications in the vascular processes.
114 citations
TL;DR: Future challenging opportunities for diagnosis, prevention, and/or therapy of chronic illnesses will require an integrated understanding and identification of developmental phases of inflammation-induced immune dysfunction and age-associated hormonal and physiological readjustments of organ systems.
Abstract: Acute inflammation is a highly regulated defense mechanism of immune system possessing two well-balanced and biologically opposing arms termed apoptosis (‘Yin’) and wound healing (‘Yang’) processes. Unresolved or chronic inflammation (oxidative stress) is perhaps the loss of balance between ‘Yin’ and ‘Yang’ that would induce co-expression of exaggerated or ‘mismatched’ apoptotic and wound healing factors in the microenvironment of tissues (‘immune meltdown’). Unresolved inflammation could initiate the genesis of many age-associated chronic illnesses such as autoimmune and neurodegenerative diseases or tumors/cancers. In this perspective ‘birds’ eye’ view of major interrelated co-morbidity risk factors that participate in biological shifts of growth-arresting (‘tumoricidal’) or growth-promoting (‘tumorigenic’) properties of immune cells and the genesis of chronic inflammatory diseases and cancer will be discussed. Persistent inflammation is perhaps a common denominator in the genesis of nearly all age-associated health problems or cancer. Future challenging opportunities for diagnosis, prevention, and/or therapy of chronic illnesses will require an integrated understanding and identification of developmental phases of inflammation-induced immune dysfunction and age-associated hormonal and physiological readjustments of organ systems. Designing suitable cohort studies to establish the oxido-redox status of adults may prove to be an effective strategy in assessing individual’s health toward developing personal medicine for healthy aging.
102 citations
TL;DR: The molecular basis for signaling via the three BLyS family receptors reveals complex interplay with other B lymphocyte signaling systems, affording the integration of selective and homeostatic processes.
Abstract: The B Lymphocyte Stimulator (BLyS) family of ligands and receptors regulates humoral immunity by controlling B lymphocyte survival and differentiation. Herein, we review the ligands and receptors of this family, their biological functions, and the biochemical processes through which they operate. Pre-immune B lymphocytes rely on BLyS signaling for their survival, whereas antigen experienced B lymphocytes generally interact more avidly with a homologous cytokine, A Proliferation Inducing Ligand (APRIL). The molecular basis for signaling via the three BLyS family receptors reveals complex interplay with other B lymphocyte signaling systems, affording the integration of selective and homeostatic processes. As our understanding of this system advances, molecular targets for manipulating humoral immunity in both health and disease should be revealed.
93 citations
TL;DR: An overview of some commonly used moment-based representations with specific focus on two schemes namely spherical harmonics and their extension, the 3D Zernike descriptors is presented.
Abstract: With structure databases expanding at a rapid rate, the task at hand is to provide reliable clues to their molecular function and to be able to do so on a large scale. This, however, requires suitable encodings of the molecular structure which are amenable to fast screening. To this end, moment-based representations provide a compact and nonredundant description of molecular shape and other associated properties. In this article, we present an overview of some commonly used representations with specific focus on two schemes namely spherical harmonics and their extension, the 3D Zernike descriptors. Key features and differences of the two are reviewed and selected applications are highlighted. We further discuss recent advances covering aspects of shape and property-based comparison at both global and local levels and demonstrate their applicability through some of our studies.
TL;DR: This article will discuss recent findings regarding the function and modulation of SK channels in central neurons.
Abstract: Small conductance (SK) channels are calcium-activated potassium channels that, when cloned in 1996, were thought solely to contribute to the afterhyperpolarisation that follows action potentials, and to control repetitive firing patterns of neurons. However, discoveries over the past few years have identified novel roles for SK channels in controlling dendritic excitability, synaptic transmission and synaptic plasticity. More recently, modulation of SK channel calcium sensitivity by casein kinase 2, and of SK channel trafficking by protein kinase A, have been demonstrated. This article will discuss recent findings regarding the function and modulation of SK channels in central neurons.
TL;DR: Surfactin inhibited proliferation in MCF-7 cells by inducing apoptosis and the elevation of [Ca2+]i may play an important role in the apoptosis, and the mechanism which surfactin caused G2/M arrest seems to be through cell cycle factor regulation.
Abstract: Surfactin, purified from Bacillus subtilis natto TK-1, inhibited proliferation of human breast cancer MCF-7 cells in a dose- and time-dependent manner, with IC(50) at 24, 48, and 72 h of 82.6, 27.3, and 14.8 microM, respectively. Surfactin-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change by acridine orange/ethidium bromide staining and Transferase-mediated dUTP Nick End-labeling assay. [Ca(2+)]i measurement revealed that surfactin induced a sustained increase in concentration of intracellular [Ca(2+)]i. Flow cytometric analysis also demonstrated that surfactin caused time-dependent apoptosis of MCF-7 cells through cell arrest at G(2)/M phase. Western blot revealed that surfactin induced accumulation of the tumor suppressor p53 and cyclin kinase inhibitor p21(waf1/cip1), and inhibited the activity of the G(2)-specific kinase, cyclin B1/p34(cdc2). Based on our findings, surfactin inhibited proliferation in MCF-7 cells by inducing apoptosis and the elevation of [Ca(2+)]i may play an important role in the apoptosis. The mechanism which surfactin caused G(2)/M arrest seems to be through cell cycle factor regulation.
TL;DR: Findings suggest that dicer and miRNAs especially miR-188 are involved in Hcy-induced cardiac remodeling.
Abstract: Elevated level of homocysteine (Hcy) called hyperhomocysteinemia (HHcy) is one of the major risk factors for chronic heart failure. Although the role of Hcy in cardiac remodeling is documented, the regulatory mechanism involved therein is still nebulous. MicroRNAs (miRNAs) and dicer have been implicated in regulation of cardiovascular diseases. Dicer is the only known enzyme involved in miRNA maturation. We investigated the involvement of dicer and miRNA in Hcy-induced cardiac remodeling. HL-1 cardiomyocytes were cultured in different doses of Hcy. Total RNA was isolated and RT-PCR and real-time PCR was performed for dicer, MMP-2,-9, TIMP-1,-3, and NOX-4. MiRNA microarray was used for analyzing the differential expression of miRNAs. Individual miRNA assay was also done. Western blotting was used to assess the MMP-9 expression in HHcy cardiomyocytes. The RT-PCR results suggest that dicer expression is enhanced in HHcy cardiomyocytes suggesting its involvement in cardiac remodeling caused due to high dose of Hcy. On the other hand, high dose of Hcy increased NOX-4 expression, a marker for oxidative stress. Additionally, HHcy cardiomyocytes showed elevated levels of MMP-2,-9 and TIMP-1,-3, and reduced expression of TIMP-4, suggesting cardiac remodeling due to oxidative stress. The miRNA microarray assay revealed differential expression of 11 miRNAs and among them miR-188 show dramatic downregulation. These findings suggest that dicer and miRNAs especially miR-188 are involved in Hcy-induced cardiac remodeling.
TL;DR: The biological roles of S100A11 and its possible mechanism in the processes of inflammation, regulation of enzyme activity, and cell growth regulation are described, corresponding to a variety of its target proteins.
Abstract: S100A11, as a member of S100 protein family, while featuring the common identities as the other EF-hand Ca(2+)-binding family members, has its own individual characteristics. S100A11 is widely expressed in multiple tissues, and is located in cytoplasm, nucleus, and even cell periphery. S100A11 exists as a non-covalent homodimer with an antiparallel conformation. Ca(2+) binding to S100A11 would trigger conformational changes which would expose the hydrophobic cleft of S100A11 and facilitate its interaction with target proteins. Since S100A11 appears to lack enzymatic activity, in this article, corresponding to a variety of its target proteins, we systematically describe the biological roles of S100A11 and its possible mechanism in the processes of inflammation, regulation of enzyme activity, and cell growth regulation. As a dual cell growth mediator, S100A11 acts as either a tumor suppressor or promoter in many different types of tumors and would play respective roles in influencing the proliferation of the cancer cells. We intend to illustrate the biological function of the S100 protein, and shed light on the further research, which will provide us with a better understanding of it.
TL;DR: The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T- type calcium channels with structurally diverse antagonists.
Abstract: Low-voltage-activated (T-type) calcium channels play a role in diverse physiological responses including neuronal burst firing, hormone secretion, and cell growth. To better understand the biological role and therapeutic potential of the target, a number of structurally diverse antagonists have been identified. Multiple drug interaction sites have been identified for L-type calcium channels, suggesting a similar possibility exists for the structurally related T-type channels. Here, we radiolabel a novel amide T-type calcium channel antagonist (TTA-A1) and show that several known antagonists, including mibefradil, flunarizine, and pimozide, displace binding in a concentration-dependent manner. Further, we identify a novel quinazolinone T-type antagonist (TTA-Q4) that enhanced amide radioligand binding, increased affinity in a saturable manner and slowed dissociation. Functional evaluation showed these compounds to be state-dependent antagonists which show a positive allosteric interaction. Consistent with slowing dissociation, the duration of efficacy was prolonged when compounds were co-administered to WAG/Rij rats, a genetic model of absence epilepsy. The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T-type calcium channels with structurally diverse antagonists.
TL;DR: Investigating the effect of intermittent high glucose on the expression of IL-18, MCP-1, and PAI-1 and adiponectin in 3T3-L1 adipocytes found this effect seems to be related to over-production of ROS.
Abstract: Elevated circulating concentrations of interleukin-18 (IL-18), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) and decrease of adiponectin are associated with obesity-related diseases. The mechanism that mediates the aberrant production of the adipokines remains poorly understood. The aim of this study was to investigate the effect of intermittent high glucose on the expression of IL-18, MCP-1, and PAI-1 and adiponectin in 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated for 24 h in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l and a daily alternating 5 or 20 mmol/l glucose, with or without the addition of 1.0 mmol/l N-acetylcysteine (NAC). The expression and secretion of IL-18, MCP-1, PAI-1, and adiponectin were determined by real-time RT-PCR and ELISA, respectively. The production of reactive oxygen species (ROS) and 8-hydroxydeoxyguanosine (8-OHdG) were measured. Stable high glucose significantly increased expression and secretion of IL-18, MCP-1, and PAI-1, and reduced adiponectin expression and secretion compared to normal glucose conditions. These effects were significantly greater under intermittent high glucose conditions compared to stable high glucose. The level of ROS and 8-OHdG were significantly elevated under both intermittent and stable high glucose conditions, the effect being greater under intermittent high glucose. The intermittent glucose was more effective in triggering the generation of ROS than stable high glucose. The adding of the NAC, a specific pharmacological inhibitor of ROS, normalized the expression of these adipokines and the levels of ROS and 8-OHdG under both stable and intermittent glucose conditions. Intermittent high glucose induces a greater aberrant production of key adipokines than stable high glucose, and this effect seems to be related to over-production of ROS.
TL;DR: A mechanical parts list that include track, energy conversion machinery, and moving parts for translocating motors is proposed that may have evolved as nature’s design strategy for these molecular engines.
Abstract: Translocating motors generate force and move along a biofilament track to achieve diverse functions including gene transcription, translation, intracellular cargo transport, protein degradation, and muscle contraction. Advances in single molecule manipulation experiments, structural biology, and computational analysis are making it possible to consider common mechanical design principles of these diverse families of motors. Here, we propose a mechanical parts list that include track, energy conversion machinery, and moving parts. Energy is supplied not just by burning of a fuel molecule, but there are other sources or sinks of free energy, by binding and release of a fuel or products, or similarly between the motor and the track. Dynamic conformational changes of the motor domain can be regarded as controlling the flow of free energy to and from the surrounding heat reservoir. Multiple motor domains are organized in distinct ways to achieve motility under imposed physical constraints. Transcending amino acid sequence and structure, physically and functionally similar mechanical parts may have evolved as nature’s design strategy for these molecular engines.
TL;DR: It is shown that neither cell doubling time nor the cell cycle of RPMI 7932 cells was affected by the frequency of the GHz radiation and duration of the exposure, in the conditions above reported.
Abstract: The potential antiproliferative effects of low power millimeter waves (MMWs) at 42.20 and 53.57 GHz on RPMI 7932 human skin melanoma cells were evaluated in vitro in order to ascertain if these two frequencies, comprised in the range of frequency used in millimeter wave therapy, would have a similar effect when applied in vivo to malignant melanoma tumours. Cells were exposed for 1 h exposure/day and to repeated exposure up to a total of four treatments. Plane wave incident power densities <1 mW/cm(2) were used in the MMWs-exposure experiments so that the radiations did not cause significant thermal effects. Numerical simulations of Petri dish reflectivity were made using the equations for the reflection coefficient of a multilayered system. Such analysis showed that the power densities transmitted into the aqueous samples were < or = 0.3 mW/cm(2). Two very important and general biological endpoints were evaluated in order to study the response of melanoma cells to these radiations, i.e. cell proliferation and cell cycle. Herein, we show that neither cell doubling time nor the cell cycle of RPMI 7932 cells was affected by the frequency of the GHz radiation and duration of the exposure, in the conditions above reported.
TL;DR: The results showed that the biophysical characteristics of immature and mature DCs were severely impaired by HCC compared with those under normal conditions, including the increased osmotic fragilities, decreased cell membrane fluidities, increased membrane viscoelastic properties, dysfunction and increased expression of cytoskeleton protein F-actin.
Abstract: Dendritic cells (DCs) are potent antigen-presenting cells and induce antigen-specific immune responses in the organism. The dysfunction of DCs has been implicated in tumor-bearing host. In order to elucidate the effects of tumor microenvironment on the functions of DCs from interdisciplinary aspects, we characterized the biophysical properties of DCs co-cultured with hepatocellular carcinoma cells (HCC). The results showed that the biophysical characteristics of immature and mature DCs were severely impaired by HCC compared with those under normal conditions, including the increased osmotic fragilities, decreased cell membrane fluidities, increased membrane viscoelastic properties, dysfunction and increased expression of cytoskeleton protein F-actin, as well as the deteriorated transendothelium migration. The impaired biophysical properties of DCs may be one of many aspects of the immune escape mechanisms of tumors. These results are clinically and instructionally significant with regard to how to enhance efficiency of the anti-tumor therapy based on DCs.
TL;DR: An experimental approach in which a focused continuous near-infrared laser beam was used to activate single rat hippocampal neurons by transiently elevating the local temperature, finding that small temperature changes were sufficient to produce significant changes in neuronal excitability.
Abstract: Optical control of neuronal activity has a number of advantages over electrical methods and can be conveniently applied to intact individual neurons in vivo. In this study, we demonstrated an experimental approach in which a focused continuous near-infrared (CNI) laser beam was used to activate single rat hippocampal neurons by transiently elevating the local temperature. Reversible changes in the amplitude and kinetics of neuronal voltage-gated Na and K channel currents were recorded following irradiation with a single-mode 980 nm CNI-laser. Using single-channel recordings under controlled temperatures as a means of calibration, it was estimated that temperature at the neuron rose by 14°C in 500 ms. Computer simulation confirmed that small temperature changes of about 5°C were sufficient to produce significant changes in neuronal excitability. The method should be broadly applicable to studies of neuronal activity under physiological conditions, in particular studies of temperature-sensing neurons expressing thermoTRP channels.
TL;DR: The role of steered molecular dynamics simulations in providing details at the atomic level on a group of protein domains, which play a fundamental role in signal exchange by responding properly to mechanical strain, is focused on.
Abstract: Efficient communication between the cell and its external environment is of the utmost importance to the function of multicellular organisms. While signaling events can be generally characterized as information exchange by means of controlled energy conversion, research efforts have hitherto mainly been concerned with mechanisms involving chemical and electrical energy transfer. Here, we review recent computational efforts addressing the function of mechanical force in signal transduction. Specifically, we focus on the role of steered molecular dynamics (SMD) simulations in providing details at the atomic level on a group of protein domains, which play a fundamental role in signal exchange by responding properly to mechanical strain. We start by giving a brief introduction to the SMD technique and general properties of mechanically stable protein folds, followed by specific examples illustrating three general regimes of signal transfer utilizing mechanical energy: purely mechanical, mechanical to chemical, and chemical to mechanical. Whenever possible the physiological importance of the example at hand is stressed to highlight the diversity of the processes in which mechanical signaling plays a key role. We also provide an overview of future challenges and perspectives for this rapidly developing field.
TL;DR: Conclusively, after validation in clinical samples, overexpression of genes like BRCA1, p53, p21, GST, MDR1 and TOPOIIα could be used as a prognostic biomarker for detection of acquired resistance in breast cancer and as therapeutic targets for the improvement of breast cancer treatment strategies.
Abstract: This study was designed to investigate the molecular changes that may develop during exposure of breast cancer cells to anticancer agents and that may lead to acquired resistance. We used two breast cancer cell lines, a parental (MCF7/WT) and a doxorubicin-resistant (MCF7/DOX) one. Cell survival, cell cycle distribution and RT-PCR expression level of genes involved in DNA damage response, MDR1, GST and TOPOIIα were measured. MCF7/DOX cells were five-fold more resistant to doxorubicin (DOX) than the MCF7/WT cells. DOX treatment causes arrest of MCF7/DOX cells in G1 and G2 phases of cell cycle whereas MCF7/WT cells were arrested in S-phase. The molecular changes in both cell lines due to DOX treatment could be classified into: (1) the basal level of p53, p21, BRCA1, GST and TOPOIIα mRNA was higher in MCF7/DOX than MCF7/WT. During DOX treatment, the expression level of these genes decreased in both cell lines but the rate of down-regulation was faster in MCF7/WT than MCF7/DOX cells. (2) The expression level of MDR1 was the same in both cell lines but 48 and 72 h of drug treatment, MDR1 disappeared in MCF7/WT but still expressed in MCF7/DOX. (3) There was no change in the expression level of BAX, FAS and BRCA2 in both cell lines. Conclusively, after validation in clinical samples, overexpression of genes like BRCA1, p53, p21, GST, MDR1 and TOPOIIα could be used as a prognostic biomarker for detection of acquired resistance in breast cancer and as therapeutic targets for the improvement of breast cancer treatment strategies.
TL;DR: This review summarizes applicable systems biology and mathematical modeling techniques including ordinary differential equations-based models, principal component analysis, and Bayesian networks that have been or could be applied to better understand the link between biomaterial properties and intracellular signaling.
Abstract: Bioactive materials present important micro-environmental cues that induce specific intracellular signaling responses which ultimately determine cell behavior. For example, vascular endothelial cells on a normal vessel wall resist inflammation and thrombosis, but the same cells seeded on an artificial vascular graft or stent do not. What makes these cells behave so differently when they are adhered to different materials? Intracellular signaling from integrins and other cell-surface receptors is an important part of the answer, but these signaling responses constitute a highly-branched, interconnected network of molecules. In order to perform rational design of biomaterials, one must understand how altering the properties of the material (micro-environment) causes changes in cell behavior, and this in turn requires understanding the complex signaling response. Systems biology and mathematical modeling aid analysis of the connectivity of this network. This review summarizes applicable systems biology and mathematical modeling techniques including ordinary differential equations-based models, principal component analysis, and Bayesian networks. Next covered is biomaterials research which studies the intracellular signaling responses generated by variation of biomaterial properties. Finally, the review details ways in which modeling has been or could be applied to better understand the link between biomaterial properties and intracellular signaling.
TL;DR: It is concluded that coronary endothelial membranes can be conveniently labeled with colloidal silica, however, due to the ionic nature of interaction of colloid silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity.
Abstract: The endothelial cell (EC) membrane is an important interface, which plays a crucial role in signal transduction. Our aim was to selectively purify luminal EC membrane proteins from the coronary vasculature of the isolated perfused mouse heart and analyze its composition with mass spectrometry (MS). To specifically label coronary ECs in the intact heart, the colloidal silica method was applied, which is based on the binding of positively charged colloidal silica to the surface of EC membranes. Transmission electron microscopy revealed the specific labeling of ECs of macro and microvessels. Two different methods of tissue homogenization (Teflon pestle and ultra blade) together with density centrifugation were used for membrane protein enrichment. Enrichment and purity was controlled by Western blot analysis using the EC-specific protein caveolin 1 and various intracellular marker proteins. The ultra blade method resulted in a tenfold enrichment of caveolin 1, while there was negligible contamination as judged by Western blot. However, protein yield was low and required pooling of ten hearts for MS. When enriched endothelial membrane proteins were digested with trypsin and analyzed by LC-MS, a total of 56 proteins could be identified, of which only 12 were membrane proteins. We conclude that coronary endothelial membranes can be conveniently labeled with colloidal silica. However, due to the ionic nature of interaction of colloidal silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity.
TL;DR: In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence, and inhibition of T-AG duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.
Abstract: The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.
TL;DR: Examining many of the biophysical properties of β-thalassemia major red blood cells (RBCs) found them to be effective, low cost, and fast techniques, therefore, it is suggested that these techniques could be applied for β-Thalassemiamajor screening purposes.
Abstract: Thalassemia is the world's most common hereditary disease; therefore, more interest has been devoted for the development of the screening procedure of this disease In beta-thalassemia major, the subject of the current study, impaired biosynthesis of beta-globin leads to accumulation of unpaired alpha-globin chain The objective of the present study, was to examine many of the biophysical properties of beta-thalassemia major red blood cells (RBCs) and to study the possibility of use of any of them as a preliminary screening tool for beta-thalassemia The percentage of normal hemolysis, osmotic fragility test, turbidity test, rheological properties, and dielectric properties, were studied in 20 regularly blood transfused thalassemia major patients who were under chelation therapy and their status were compared with those of 10 healthy subjects There was an increase in the percentage of hemolysis for beta-thalassemia by 1146% compared to the normal RBCs The fragility curve for beta-thalassemia RBCs showed a shift toward lower NaCl concentration compared to the normal curve The average osmotic fragility (H(50): the NaCl concentration producing 50% homolysis) for beta-thalassemia was found to be 321 +/- 067 g/l, whereas for normal RBCs it was 55 +/- 031 g/l The turbidity curve of the beta-thalassemic RBCs showed a shift toward higher detergent concentration of the normal curve, with higher value for the average membrane solubilization (S(50)) The viscosity value of whole blood beta-thalassemia was found to be 3916 +/- 056 cp whereas for normal blood was 2516 +/- 036 cp The relative permittivity, dielectric loss, and AC conductivity of RBCs decreased significantly compared to normal samples This could be attributed to the loss of the insulating properties of the membrane and loss of its surface charge of thalassemic RBCs As can be noticed, several factors showed clear difference between thalassemic and normal blood samples Some of these parameters could be measured immediately after sample withdrawal and require short time to perform the measurements This offers the advantages of being effective, low cost, and fast techniques, therefore, we suggest that these techniques could be applied for beta-thalassemia major screening purposes
TL;DR: The traditional codon table is reviewed and a novel superposition of the BLOSUM62 matrix and an allowed point mutation matrix is presented, which depicts an important aspect of the true genetic code—its ability to tolerate mutations and mistranslations.
Abstract: The standard codon table is a primary tool for basic understanding of molecular biology. In the minds of many, the table’s orderly arrangement of bases and amino acids is synonymous with the true genetic code, i.e., the biological coding principle itself. However, developments in the field reveal a much more complex and interesting picture. In this article, we review the traditional codon table and its limitations in light of the true complexity of the genetic code. We suggest the codon table be brought up to date and, as a step, we present a novel superposition of the BLOSUM62 matrix and an allowed point mutation matrix. This superposition depicts an important aspect of the true genetic code—its ability to tolerate mutations and mistranslations.
TL;DR: Testing the ability of a technology to control protein position in budding yeast using a chemical inducer of dimerization to direct the nucleocytoplasmic transport of 16 representative kinases and transcription factors found that 12 targets are susceptible to re-positioning, suggesting that this method might be applicable to a range of targets.
Abstract: In eukaryotes, reversible shuttling between the nucleus and cytoplasm is an important regulatory mechanism, particularly for many kinases and transcription factors. Inspired by the natural system, we recently developed a technology to control protein position in budding yeast using a chemical inducer of dimerization (CID). In this method, a nuclear export or localization signal is reversibly appended to a protein of interest by the CID, which effectively places its subcellular location under direct control of the chemical stimulus. Here, we explicitly tested the ability of this system to direct the nucleocytoplasmic transport of a panel of 16 representative kinases and transcription factors. From this set, we found that 12 targets (75%) are susceptible to re-positioning, suggesting that this method might be applicable to a range of targets. Interestingly, the four proteins that resisted mislocalization (Fun20p, Hcm1p, Pho4p, and Ste12p) are known to engage in a large number of protein-protein contacts. We suspect that, for these highly connected targets, the strength of the chemical signal may be insufficient to drive mislocalization and that proteins with relatively few partners might be most amenable to this approach. Collectively, these studies provide a necessary framework for the design of large-scale applications.
TL;DR: It is suggested that the elevated content of SM observed in plasma membranes of 3D fibroblasts could be responsible for an increased rigidity and possibly reduced permeability of cells cultured in 3D environment.
Abstract: The differences in the surface active properties of native lipids extracted from plasma membranes of cells cultured as a monolayer and in three-dimensional (3D) matrix were investigated. This experimental model was chosen because most of the current knowledge on cellular physiological processes is based on studies performed with conventional monolayer two-dimensional (2D) cell cultures, where cells are forced to adjust to unnaturally rigid surfaces that differ significantly from the natural matrix surrounding cells in living organisms. Differences between monolayer and 3D cells were observed in the lipid composition of plasma membranes and especially in the level of the two major microdomain-forming lipids—sphingomyelin (SM) and cholesterol, which were significantly elevated in 3D cells. The obtained results showed that culturing of cells in in vivo-like environment affected the surface active properties of plasma membrane lipids at interfaces which might influence certain membrane-associated interface processes. The detected differences in the lipid levels in 2D and 3D cell extracts affected significantly the behavior of the model lipid monolayers at the air–water interface (Langmuir monolayers) which resulted in different values of the monolayer equilibrium (γeq) and dynamic (γmax, γmin) surface tension and surface potential. Compensation of the SM content in extracts of 2D cell cultures up to a level close to the one measured in 3D cells approximated the monolayer properties to the values observed for 3D cells. These results implied that the interactions between the cells and the surrounding medium affected the level of plasma membrane SM and other lipids, which had a strong impact on the surface properties of lipid monolayers, such as γeq, γmax, and γmin, the compression/decompression curve shape, the hysteresis area during cycling of the monolayers, etc. We suggest that the elevated content of SM observed in plasma membranes of 3D fibroblasts could be responsible for an increased rigidity and possibly reduced permeability of cells cultured in 3D environment. The current results provide useful information that should be taken into account in the interpretation of the membrane physico-chemical properties of cells cultured under different conditions.