Hydrogen bonding is a key contributor to the exquisite specificity of the interactions within and between biological macromolecules, and hence accurate modeling of such interactions requires an accurate description of hydrogen bonding energetics. Here we investigate the orientation and distance dependence of hydrogen bonding energetics by combining two quite disparate but complementary approaches: quantum mechanical electronic structure calculations and protein structural analysis. We find a remarkable agreement between the energy landscapes obtained from the electronic structure calculations and the distributions of hydrogen bond geometries observed in protein structures. In contrast, molecular mechanics force fields commonly used for biomolecular simulations do not consistently exhibit close correspondence to either quantum mechanical calculations or experimentally observed hydrogen bonding geometries. These results suggest a route to improved energy functions for biological macromolecules that combines the generality of quantum mechanical electronic structure calculations with the accurate context dependence implicit in protein structural analysis.
Endovascular drug-eluting stents have changed the practice of medicine, and yet it is unclear how they so dramatically reduce restenosis and how to distinguish between the different formulations available. Biological drug potency is not the sole determinant of biological effect. Physicochemical drug properties also play important roles. Historically, two classes of therapeutic compounds emerged: hydrophobic drugs, which are retained within tissue and have dramatic effects, and hydrophilic drugs, which are rapidly cleared and ineffective. Researchers are now questioning whether individual properties of different drugs beyond lipid avidity can further distinguish arterial transport and distribution. In bovine internal carotid segments, tissue-loading profiles for hydrophobic paclitaxel and rapamycin are indistinguishable, reaching load steady state after 2 days. Hydrophilic dextran reaches equilibrium in several hours at levels no higher than surrounding solution concentrations. Both paclitaxel and rapamycin bind to the artery at 30–40 times bulk concentration. Competitive binding assays confirm binding to specific tissue elements. Most importantly, transmural drug distribution profiles are markedly different for the two compounds...
The maintenance of genome integrity and the generation of biological diversity are important biological processes, and both involve homologous recombination. In yeast and animals, homologous recombination requires the function of the RAD51 recombinase. In vertebrates, RAD51 seems to have acquired additional functions in the maintenance of genome integrity, and rad51 mutations cause lethality, but it is not clear how widely these functions are conserved among eukaryotes. We report here a loss-of-function mutant in the Arabidopsis homolog of RAD51, AtRAD51. The atrad51-1 mutant exhibits normal vegetative and flower development and has no detectable abnormality in mitosis. Therefore, AtRAD51 is not necessary under normal conditions for genome integrity. In contrast, atrad51-1 is completely sterile and defective in male and female meioses. During mutant prophase I, chromosomes fail to synapse and become extensively fragmented. Chromosome fragmentation is suppressed by atspo11-1, indicating that AtRAD51 functions downstream of AtSPO11-1. Therefore, AtRAD51 likely plays a crucial role in the repair of DNA double-stranded breaks generated by AtSPO11-1. These results suggest that RAD51 function is essential for chromosome pairing and synapsis at early stages in meiosis in Arabidopsis. Furthermore...
Diverse biochemical rhythms are generated by thousands of cellular oscillators that somehow manage to operate synchronously. In fields ranging from circadian biology to endocrinology, it remains an exciting challenge to understand how collective rhythms emerge in multicellular structures. Using mathematical and computational modeling, we study the effect of coupling through intercell signaling in a population of Escherichia coli cells expressing a synthetic biological clock. Our results predict that a diverse and noisy community of such genetic oscillators interacting through a quorum-sensing mechanism should self-synchronize in a robust way, leading to a substantially improved global rhythmicity in the system. As such, the particular system of coupled genetic oscillators considered here might be a good candidate to provide the first quantitative example of a synchronization transition in a population of biological oscillators.
Promyelocytic leukemia (PML) and Cajal bodies are mobile subnuclear organelles, which are involved in activities like RNA processing, transcriptional regulation, and antiviral defense. A key parameter in understanding their biological functions is their mobility. The diffusion properties of PML and Cajal bodies were compared with a biochemically inactive body formed by aggregates of murine Mx1 by using single-particle tracking methods. The artificial Mx1-yellow fluorescent protein body showed a very similar mobility compared with PML and Cajal bodies. The data are described quantitatively by a mechanism of nuclear body movement consisting of two components: diffusion of the body within a chromatin corral and its translocation resulting from chromatin diffusion. This finding suggests that the body mobility reflects the dynamics and accessibility of the chromatin environment, which might target bodies to specific nuclear subcompartments where they exert their biological function.
We test whether coherent control methods based on ultrashort-pulse phase shaping can be applied when the laser light propagates through biological tissue. Our results demonstrate experimentally that the spectral-phase properties of shaped laser pulses optimized to achieve selective two-photon excitation survive as the laser pulses propagate through tissue. This observation is used to obtain functional images based on selective two-photon excitation of a pH-sensitive chromophore in a sample that is placed behind a slice of biological tissue. Our observation of coherent control through scattering tissue suggests possibilities in multiphoton-based imaging and photodynamic therapy.
Aquaporins mediate rapid selective water transport across biological membranes. Elucidation of their precise physiological roles promises important insight into cellular and organismal osmoregulation. The genome of the yeast Saccharomyces cerevisiae encodes two similar but differentially regulated aquaporins. Here, we show that expression of AQY1 is stimulated during sporulation and that the Aqy1 protein is detectable exclusively in spore membranes. When spores are rapidly frozen, those that lack Aqy1 survive better, providing for a functional test of active spore water channels. Under ambient conditions, lack of Aqy1 reduces spore fitness. Because this reduction is independent from germination conditions, Aqy1 may be important during spore formation rather than subsequent maintenance or germination. Indeed, it seems that Aqy1 is degraded after spores have been formed and during germination. Taken together, Aqy1 is developmentally controlled and may play a role in spore maturation, probably by allowing water outflow. Taken together, we demonstrate a functional role of an aquaporin in gametogenesis, as well as in the formation of durable structures such as spores, a role that may have wider biological and medical implications.
Transitions to new levels of biological complexity often require cooperation among component individuals, but individual selection among those components may favor a selfishness that thwarts the evolution of cooperation. Biological systems with elements of cooperation and conflict are especially challenging to understand because the very direction of evolution is indeterminate and cannot be predicted without knowing which types of selfish mutations and interactions can arise. Here, we investigated the evolution of two bacteriophages (f1 and IKe) experimentally forced to obey a life cycle with elements of cooperation and conflict, whose outcome could have ranged from extinction of the population (due to selection of selfish elements) to extreme cooperation. Our results show the de novo evolution of a conflict mediation system that facilitates cooperation. Specifically, the two phages evolved to copackage their genomes into one protein coat, ensuring cotransmission with each other and virtually eliminating conflict. Thereafter, IKe evolved such extreme genome reduction that it lost the ability to make its own virions independent of f1. Our results parallel a variety of conflict mediation mechanisms existing in nature: evolution of reduced genomes in symbionts...
Molecular biology studies the cause-and-effect relationships among microscopic processes initiated by individual molecules within a cell and observes their macroscopic phenotypic effects on cells and organisms. These studies provide a wealth of information about the underlying networks and pathways responsible for the basic functionality and robustness of biological systems. At the same time, these studies create exciting opportunities for the development of quantitative and predictive models that connect the mechanism to its phenotype then examine various modular structures and the range of their dynamical behavior. The use of such models enables a deeper understanding of the design principles underlying biological organization and makes their reverse engineering and manipulation both possible and tractable The heat shock response presents an interesting mechanism where such an endeavor is possible. Using a model of heat shock, we extract the design motifs in the system and justify their existence in terms of various performance objectives. We also offer a modular decomposition that parallels that of traditional engineering control architectures.
The existence of lipid rafts in biological membranes in vivo is still debated. In contrast, the formation of domains in model systems has been well documented. In giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol, a clear separation of liquid-disordered and sphingomyelin-enriched, liquid-ordered phases could be observed. This phase separation can lead to the fission of the liquid-ordered phase from the vesicle. Here we show that in cholesterol-containing GUVs, the phase separation can involve dynamic redistribution of lipids from one phase into another as a result of a cross-linking perturbation. We found that the molecular structure of a sterol used for the preparation of GUVs determines (i) its ability to induce phase separation and (ii) the curvature (positive or negative) of the formed liquid-ordered phase. As a consequence, the latter can pinch off to the outside or inside of the vesicle. Remarkably, some mixtures of sterols induce liquid-ordered domains exhibiting both positive and negative curvature, which can lead to a new type of budding behavior in GUVs. Our findings could have implications for the role of sterols in various cell-biological processes such as budding of secretory vesicles...
Unraveling cause-and-effect relationships in the nervous system is challenging because some biological processes begin stochastically, take a significant amount of time to unfold, and affect small neuronal subpopulations that can be difficult to isolate and measure. Single-cell approaches are slow, subject to user bias, and sometimes too laborious to achieve sample sizes large enough to detect important effects. Here, we describe an automated imaging and analysis system that enables us to follow the fates of individual cells and intracellular proteins over time. Observations can be quantified in a high-throughput manner with minimal user bias. We have adapted survival analysis methods to determine whether and how factors measured during longitudinal analysis predict a particular biological outcome. The ability to monitor complex processes at single-cell resolution quickly, quantitatively, and over long intervals should have wide applications for biology.
Dicer is an RNase III-family nuclease that initiates RNA interference (RNAi) and related phenomena by generation of the small RNAs that determine the specificity of these gene silencing pathways. We have previously shown that Dicer is essential for mammalian development, with Dicer-deficient mice dying at embryonic day 7.5 with a lack of detectable multipotent stem cells. To permit a more detailed investigation of the biological roles of Dicer, we have generated embryonic stem cell lines in which their single Dicer gene can be conditionally inactivated. As expected, Dicer loss compromises maturation of microRNAs and leads to a defect in gene silencing triggered by long dsRNAs. However, the absence of Dicer does not affect the ability of small interfering RNAs to repress gene expression. Of interest, Dicer loss does compromise the proliferation of ES cells, possibly rationalizing the phenotype previously observed in Dicer-null animals. Dicer loss also affects the abundance of transcripts from mammalian centromeres but does so without a pronounced affect on histone modification status at pericentric repeats or methylation of centromeric DNA. These studies provide a conditional model of RNAi deficiency in mammals that will permit the dissection of the biological roles of the RNAi machinery in cultured mammalian cells.
van Dijk, Melissa R.; Douradinha, Bruno; Franke-Fayard, Blandine; Heussler, Volker; van Dooren, Maaike W.; van Schaijk, Ben; van Gemert, Geert-Jan; Sauerwein, Robert W.; Mota, Maria M.; Waters, Andrew P.; Janse, Chris J.
Fonte: National Academy of SciencesPublicador: National Academy of Sciences
Immunization with Plasmodium sporozoites that have been attenuated by γ-irradiation or specific genetic modification can induce protective immunity against subsequent malaria infection. The mechanism of protection is only known for radiation-attenuated sporozoites, involving cell-mediated and humoral immune responses invoked by infected hepatocytes cells that contain long-lived, partially developed parasites. Here we analyzed sporozoites of Plasmodium berghei that are deficient in P36p (p36p-), a member of the P48/45 family of surface proteins. P36p plays no role in the ability of sporozoites to infect and traverse hepatocytes, but p36p- sporozoites abort during development within the hepatocyte. Immunization with p36p- sporozoites results in a protective immunity against subsequent challenge with infectious wild-type sporozoites, another example of a specifically genetically attenuated sporozoite (GAS) conferring protective immunity. Comparison of biological characteristics of p36p- sporozoites with radiation-attenuated sporozoites demonstrates that liver cells infected with p36p- sporozoites disappear rapidly as a result of apoptosis of host cells that may potentiate the immune response. Such knowledge of the biological characteristics of GAS and their evoked immune responses are essential for further investigation of the utility of an optimized GAS-based malaria vaccine.
The radiation-induced bystander effect is defined as “the induction of biological effects in cells that are not directly traversed by a charged particle but are in close proximity to cells that are.” Although these bystander effects have been demonstrated with a variety of biological endpoints in both human and rodent cell lines (as well as in 3D tissue samples), the mechanism of the phenomenon is not known. Although gap junction communication and the presence of soluble mediator(s) are both known to play important roles in the bystander response, the precise signaling molecules have yet to be identified. By using the Columbia University charged particle beam in conjunction with a strip dish design, we show here that the cyclooxygenase-2 (COX-2, also known as prostaglandin endoperoxide synthase-2) signaling cascade plays an essential role in the bystander process. Treatment of bystander cells with NS-398, which suppresses COX-2 activity, significantly reduced the bystander effect. Because the critical event of the COX-2 signaling is the activation of the mitogen-activated protein kinase pathways, our finding that inhibition of the extracellular signal-related kinase phosphorylation suppressed bystander response further confirmed the important role of mitogen-activated protein kinase signaling cascade in the bystander process. These results provide evidence that the COX-2-related pathway...
Birds are of great interest for a variety of research purposes, and effective methods for manipulating the avian genome would greatly accelerate progress in fields that rely on birds as model systems for biological research, such as developmental biology and behavioral neurobiology. Here, we describe a simple and effective method for producing transgenic birds. We used lentiviral vectors to produce transgenic quails that express GFP driven by the human synapsin gene I promoter. Expression of GFP was specific to neurons and consistent across multiple generations. Expression was sufficient to allow visualization of individual axons and dendrites of neurons in vivo by intrinsic GFP fluorescence. Tissue-specific transgene expression at high levels provides a powerful tool for biological research and opens new avenues for genetic manipulation in birds.
Neuronal remodeling is a fundamental process by which the brain responds to environmental influences, e.g., during stress. In the hippocampus, chronic stress causes retraction of dendrites in CA3 pyramidal neurons. We have recently identified the glycoprotein M6a as a stress-responsive gene in the hippocampal formation. This gene is down-regulated in the hippocampus of both socially and physically stressed animals, and this effect can be reversed by antidepressant treatment. In the present work, we analyzed the biological function of the M6a protein. Immunohistochemistry showed that the M6a protein is abundant in all hippocampal subregions, and subcellular analysis in primary hippocampal neurons revealed its presence in membrane protrusions (filopodia/spines). Transfection experiments revealed that M6a overexpression induces neurite formation and increases filopodia density in hippocampal neurons. M6a knockdown with small interference RNA methodology showed that M6a low-expressing neurons display decreased filopodia number and a lower density of synaptophysin clusters. Taken together, our findings indicate that M6a plays an important role in neurite/filopodium outgrowth and synapse formation. Therefore, reduced M6a expression might be responsible for the morphological alterations found in the hippocampus of chronically stressed animals. Potential mechanisms that might explain the biological effects of M6a are discussed.
Mapping biological pathways across microbial genomes is a highly important technique in functional studies of biological systems. Existing methods mainly rely on sequence-based orthologous gene mapping, which often leads to suboptimal mapping results because sequence-similarity information alone does not contain sufficient information for accurate identification of orthology relationship. Here we present an algorithm for pathway mapping across microbial genomes. The algorithm takes into account both sequence similarity and genomic structure information such as operons and regulons. One basic premise of our approach is that a microbial pathway could generally be decomposed into a few operons or regulons. We formulated the pathway-mapping problem to map genes across genomes to maximize their sequence similarity under the constraint that the mapped genes be grouped into a few operons, preferably coregulated in the target genome. We have developed an integer-programming algorithm for solving this constrained optimization problem and implemented the algorithm as a computer software program, p-map. We have tested p-map on a number of known homologous pathways. We conclude that using genomic structure information as constraints could greatly improve the pathway-mapping accuracy over methods that use sequence-similarity information alone.
Biological molecular assemblies are excellent models for the development of nanoengineered systems with desirable biomedical properties. Here we report an approach for fabrication of spontaneous, biologically active molecular networks consisting of bacteriophage (phage) directly assembled with gold (Au) nanoparticles (termed Au–phage). We show that when the phage are engineered so that each phage particle displays a peptide, such networks preserve the cell surface receptor binding and internalization attributes of the displayed peptide. The spontaneous organization of these targeted networks can be manipulated further by incorporation of imidazole (Au–phage–imid), which induces changes in fractal structure and near-infrared optical properties. The networks can be used as labels for enhanced fluorescence and dark-field microscopy, surface-enhanced Raman scattering detection, and near-infrared photon-to-heat conversion. Together, the physical and biological features within these targeted networks offer convenient multifunctional integration within a single entity with potential for nanotechnology-based biomedical applications.
We analyzed a very large set of molecular interactions that had been derived automatically from biological texts. We found that published statements, regardless of their verity, tend to interfere with interpretation of the subsequent experiments and, therefore, can act as scientific “microparadigms,” similar to dominant scientific theories [Kuhn, T. S. (1996) The Structure of Scientific Revolutions (Univ. Chicago Press, Chicago)]. Using statistical tools, we measured the strength of the influence of a single published statement on subsequent interpretations. We call these measured values the momentums of the published statements and treat separately the majority and minority of conflicting statements about the same molecular event. Our results indicate that, when building biological models based on published experimental data, we may have to treat the data as highly dependent-ordered sequences of statements (i.e., chains of collective reasoning) rather than unordered and independent experimental observations. Furthermore, our computations indicate that our data set can be interpreted in two very different ways (two “alternative universes”): one is an “optimists’ universe” with a very low incidence of false results (<5%)...
Variation among populations in gene expression should be related to the accumulation of random-neutral changes and evolution by natural selection. The following evolutionary analysis has general applicability to biological and medical science because it accounts for genetic relatedness and identifies patterns of expression variation that are affected by natural selection. To identify genes evolving by natural selection, we allocate the maximum among-population variation to genetic distance and then examine the remaining variation relative to a hypothesized important ecological parameter (temperature). These analyses measure the expression of metabolic genes in common-gardened populations of the fish Fundulus heteroclitus whose habitat is distributed along a steep thermal gradient. Although much of the variation in gene expression fits a null model of neutral drift, the variation in expression for 22% of the genes that regress with habitat temperature was far greater than could be accounted for by genetic distance alone. The most parsimonious explanation for among-population variation for these genes is evolution by natural selection. In addition, many metabolic genes have patterns of variation incongruent with neutral evolution: They have too much or too little variation. These patterns of biological variation in expression may reflect important physiological or ecological functions.