Microtubule plus-end conformations and dynamics in the periphery of interphase mouse fibroblasts

The plus ends of microtubules (MTs) alternate between phases of growth, pause, and shrinkage, a process called "dynamic instability." Cryo-EM of in vitro-assembled MTs indicates that the dynamic state of the plus end corresponds with a particular MT plus-end conformation. Frayed ("ram's horn like"), blunt, and sheet conformations are associated with shrinking, pausing, and elongating plus ends, respectively. A number of new conformations have recently been found in situ but their dynamic states remained to be confirmed. Here, we investigated the dynamics of MT plus ends in the peripheral area of interphase mouse fibroblasts (3T3s) using electron microscopical and tomographical analysis of cryo-fixed, freeze-substituted, and flat-embedded sections. We identified nine morphologically distinct plus-end conformations. The frequency of these conformations correlates with their proximity to the cell border, indicating that the dynamic status of a plus end is influenced by features present in the periphery. Shifting dynamic instability toward depolymerization with nocodazole enabled us to address the dynamic status of these conformations. We suggest a new transition path from growth to shrinkage via the so-called sheet-frayed and flared ends, and we present a kinetic model that describes the chronology of events taking place in nocodazole-induced MT depolymerization.     

Mechanical model of cytoskeleton structuration during cell adhesion and spreading

The biomechanical behavior of an adherent cell is intimately dependent on its cytoskeleton structure. Several models have been proposed to study this structure taking into account its existing internal forces. However, the structural and geometrical complexities of the cytoskeleton's filamentous networks lead to difficulties for determining a biologically realistic architecture. The objective of this paper is to present a mechanical model, combined with a numerical method, devoted to the form-finding of the cytoskeleton structure (shape and internal forces) when a cell adheres on a substrate. The cell is modeled as a granular medium, using rigid spheres (grains) corresponding to intracellular cross-linking proteins and distant mechanical interactions to reproduce the cytoskeleton filament internal forces. At the initial state (i.e., before adhesion), these interactions are tacit. The adhesion phenomenon is then simulated by considering microtubules growing from the centrosome towards transmembrane integrin-like receptors. The simulated cell shape changes in this process and results in a mechanically equilibrated structure with traction and compression forces, in interaction with the substrate reactions. This leads to a compressive microtubule network and a corresponding tensile actin-filament network. The results provide coherent shape and forces information for developing a mechanical model of the cytoskeleton structure, which can be exploitable in future biomechanical studies of adherent cells.     

Configurational and dynamic properties of different length superhelical DNAs measured by dynamic light scattering

The solution conformation and internal motions of five superhelical DNAs between 2100 and 10200 base-pairs in length have been characterized by dynamic light scattering (DLS). Variations in the diffusion coefficients and rotational relaxation times with molecular weight are both indicative of an anisotropic extended structure of these DNAs; we therefore conclude that under our conditions the interwound superhelical structure prevails. The internal dynamics can be described by a superposition of rotational diffusion and internal relaxation. The latter process is characterized by the internal diffusion of persistence length size segments within the DNA chain and faster bending motions within these segments.     

The system of the epidemic process

An original social-ecological concept of the epidemic process has been constructed on the basis of using social ecology, systemic approach and the basic principles of cybernetics. According to this concept, the epidemic process is regarded as a biosocial, hierarchic, integral system providing for the reproduction of the species of human parasites. At a higher level of organization, the epidemic process is an epidemiological social-ecological system consisting of two interacting subsystems: the biological (epidemiological ecosystem) and the social (social and economic conditions of life of the society) subsystems where the biological subsystem plays the role of the governed object and the social acts as the internal regulator of these interactions. On the basis of this concept a rational structure of the system of epidemiological surveillance over infectious diseases has been proposed according to which each level of the structure of the epidemic process should be subject to adequate monitoring.     

Secondary structure model for the ITS-2 precursor rRNA of strongyloid nematodes of equids: implications for phylogenetic inference

In order to maximise the positional homology in the primary sequence alignment of the second internal transcribed spacer for 30 species of equine strongyloid nematodes, the secondary structures of the precursor ribosomal RNA were predicted using an approach combining an energy minimisation method and comparative sequence analysis. The results indicated that a common secondary structure model of the second internal transcribed spacer of these nematodes was maintained, despite significant interspecific differences (2–56%) in primary sequences. The secondary structure model was then used to refine the primary second internal transcribed spacer sequence alignment. The “manual” and “structure” alignments were both subjected to phylogenetic analysis using three different tree-building methods to compare the effect of using different sequence alignments on phylogenetic inference. The topologies of the phylogenetic trees inferred from the manual second internal transcribed spacer alignment were usually different to those derived from the structure second internal transcribed spacer alignment. The results suggested that the positional homology in the second internal transcribed spacer primary sequence alignment was maximised when the secondary structure model was taken into consideration.     

蛋白质二级结构预测方法的评价

目前评价蛋白质二级结构预测方法主要考虑预测准确率,并没有充分考虑方法自身参数对方法的影响。本文提出一种新型评价方法,将内在评价与外在评价相结合评价预测方法的优劣。以基于混合并行遗传算法的蛋白质二级结构预测方法为例,通过内在评价,合理选取内在参数——切片长度和组内类别数,有效提高预测准确率,同时,通过外在评价,与其他基于随机算法的蛋白质二级结构预测算法比较和与CASP所提供的结论比较,说明了方法的有效性与正确性,以此验证内在评价和外在评价的客观性、公正性和全面性。     

钩藤钩器官形态发育的解剖学研究

为了解钩藤钩的微观形态发育过程,该文利用石蜡切片的方法,对钩藤营养器官分枝茎钩的微观形态特征进行解剖和比较分析。结果表明:钩与茎之间在解剖学上既有相关性,又各有其自身的特征; 其相关性表现在茎的内部结构大体上决定着其钩的内部结构,充分体现钩是由着生在茎上的侧枝变态发育形成的; 在钩发育中期逐渐形成与茎类似的内部结构; 茎的内部结构组成在数量上比钩更丰富,钩只有初生结构; 钩藤茎与钩的药用成分积累没有本质的区别,均主要分布在韧皮部和木质部及皮层薄壁细胞。由此可知,钩藤钩器官是由茎变态发育形成,但只有初生结构。该研究结果可辅助解决某些仅靠形态学性状难以解决的分类鉴定问题,为判断钩状茎或卷须茎的变态来源提供理论参考依据。     

The structure of organelles of the endocytic pathway in hydrated cryosections of cultured cells

The structure of cellular organelles, in particular those involved in endocytosis, was studied by electron microscopy with hydrated cryosections. In this technique no chemical treatment is used, and the native structure of organelles can be observed in sections viewed at temperatures below -140 degrees C, using a cold stage accessory on the electron microscope. The compartments of the endocytic pathway were prelabeled with gold markers in the living cell, facilitating the identification of different structures in the cryosections. The structure of most identifiable cellular organelles, including those involved in endocytosis, appeared very similar in the hydrated cryosections to that seen after conventional plastic and cryosections of chemically fixed cells. In particular, the internal membranes of the structure we refer to as the prelysosomal compartment (Griffiths et al., Cell 52, 329-341 (1988] could be clearly visualized in these sections indicating that the organization of these membranes is not a consequence of the chemical fixation process.     

Spreading of liposomes at the air/water interface

Two types of film structure are formed when liposomes are spread at the air/water interface. At zero surface pressure, there is a slow transformation of the closed bilayered structure into a lipid monolayer. The internal content of the liposomes is released into the aqueous subphase. In contrast, when multilamellar liposomes are spread against a surface pressure, they retain their internal content at the air/water interface by forming multilayered structures. Among the liposomes which dipped through the interface an important fraction loses its internal content. During the spreading process at zero surface pressure, it seems that the outer layer of the liposome spreads with a better yield as compared with the inner layer. It is possible to use this spreading technique to determine the asymmetrical distribution of lipids across bilayers.     

An X ray diffraction study of frog sciatic nerve myelin in dimethyl sulfoxide.

Reversible structure modification of frog sciatic nerve myelin bathed in Ringer's solution containing dimethyl sulfoxide (DMSO) at a concentration of 33% has been studied by low-angle X-ray diffraction using a linear position-sensitive counter. Fourier images of native myelin layers, derived using low-order reflections measured at various stages of the DMSO treatment, reveal that the bilayer profile of native myelin membrane undergoes a specific asymmetric change prior to the phase transformation: The high-density peak on the extracellular side of the central lipid hydrocarbon layer decreases reversibly as the nerve is permeated by DMSO, while the internal peak and the central layer remain virtually unaltered. The dynamic process by which the contracted phase of myelin is derived from native myelin is speculated on the basis of the observed profile change.     

Conformational Changes Leading to T7 DNA Delivery upon Interaction with the Bacterial Receptor

The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity. Bacteriophage T7 belongs to the Podoviridae family and has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Here we characterize the kinetics and structure of bacteriophage T7 DNA delivery process. We show that T7 recognizes lipopolysaccharides (LPS) from Escherichia coli rough strains through the fibers. Rough LPS acts as the main phage receptor and drives DNA ejection in vitro. The structural characterization of the phage tail after ejection using cryo-electron microscopy (cryo-EM) and single particle reconstruction methods revealed the major conformational changes needed for DNA delivery at low resolution. Interaction with the receptor causes fiber tilting and opening of the internal tail channel by untwisting the nozzle domain, allowing release of DNA and probably of the internal head proteins.     

Finding non-crystallographic symmetry in density maps of macromolecular structures

The internal symmetry of a macromolecule is both an important aspect of its function and a useful feature in obtaining a structure by X-ray crystallography and other techniques. A method is presented for finding internal symmetry and other non-crystallographic symmetry in a structure based on patterns of density in a density map for that structure. Regions in map that are similar are identified by cutting out a sphere of density from a region that has high local variation and using an FFT-based correlation search to find other regions that match. The relationships among correlated regions are then refined to maximize their correlations and are found to accurately represent non-crystallographic symmetry in the map.     

The fine structure of calcified Mandl's corpuscles in teleost fish scales

Calcified Mandl's corpuscles present in the internal layer (or fibrillary plate) of the teleost fish scale were studied by transmission and scanning electron microscopy for a better understanding of this special type of mineralization process. The corpuscles show a great variability in their structure, form and surface features depending on the arrangement of the collagen fibrils in the internal layer of the different fish species studied, on the localization of the corpuscles in the scale and on the technical treatment to which the scale is subjected.     

Hydrophobic interaction and a model for the elasticity of elastin

The thermodynamics of the elastic process in the rubberlike protein elastin have been investigated by microcalorimetery. The results indicate that the reversible heat liberated upon the extension of water-swollen elastin at room temperature is much largerthan the stored elastic energy, indicating a large than the stored elastic energy, indicating a large, negative internal energy change for stretching. The ratio of the measured internal energy change to the stored energy varies inversely wiht extension, and at 22° C it is ?91 for 2% extension and ?3 for 70% extension. The interanl energy change also varies dramatically with temperature over the range of 2–65° C it is zero. The temperature dependence for internal energy change is virtually identical to the temperature dependence for internal energy changes associated with the breaking of hydrophobic interactions, and it is suggested that the measured internal energy change can be attributed entirely to hte absorption of water onto nonpolar groups in the elastin network. Calculatons based on this assumption indicate that the free-energy change associated with this solvent–polymer process is large and positive. It is concluded that the absorption of water onto hydrophobic groups contributes to the elasticity of elastin, particularly at extensions of less than about 70%. The implications of this elastic mechanism are discussed in terms of the random-network model for elastin structure.     

Penetration of membrane-containing double-stranded-DNA bacteriophage PM2 into Pseudoalteromonas hosts

The icosahedral bacteriophage PM2 has a circular double-stranded DNA (dsDNA) genome and an internal lipid membrane. It is the only representative of the Corticoviridae family. How the circular supercoiled genome residing inside the viral membrane is translocated into the gram-negative marine Pseudoalteromonas host has been an intriguing question. Here we demonstrate that after binding of the virus to an abundant cell surface receptor, the protein coat is most probably dissociated. During the infection process, the host cell outer membrane becomes transiently permeable to lipophilic gramicidin D molecules proposing fusion with the viral membrane. One of the components of the internal viral lipid core particle is the integral membrane protein P7, with muralytic activity that apparently aids the process of peptidoglycan penetration. Entry of the virion also causes a limited depolarization of the cytoplasmic membrane. These phenomena differ considerably from those observed in the entry process of bacteriophage PRD1, a dsDNA virus, which uses its internal membrane to make a cell envelope-penetrating tubular structure.     

Evidence for cell generation controlled proliferation in the small intestinal crypt

In this paper we discuss the hypothesis that cell proliferation is controlled by the number of generations after leaving an 'eternal' stem cell. The theory is based on a simulation of the kinetic behaviour of cells in the intestinal crypts. There is evidence of three, four and five generations of cells which are allowed to enter mitosis in the lower and upper part of the normal intestinal tract, and in some disease states, respectively. We suggest an internal proliferation control: some kind of knowledge that cells carry from generation to generation. It is an open question what sets and changes the generation counter: internal genetic information or external influences such as growth factors or chalones. The geometric shape of the epithelial tissue in the intestinal tract can be understood as the steady state of a highly dynamic process. Age and death are determined from the beginning; cell-cell interaction or communication is not necessary and can be neglected. Our theory will be illustrated using the intestinal crypts as they are easily accessible, of a simple structure and completely described in the literature.     

Observation on Flower Bud Differentiation of Crape Myrtle in Red Soil Environment

Flower bud differentiation is a key component of plant blooming biology and understanding how it works is vital for flowering regulation and plant genetic breeding, increasing the number and quality of flowering. Red soil is the most widely covered soil type in the world, and it is also the most suitable soil type for crape myrtle planting. The flower buds of crape myrtle (Lagerstroemia indica) planted in red soil were employed as experimental materials in this study, and the distinct periods of differentiation were identified using stereomicroscopy and paraffin sectioning. We optimized the steps of dehydration, transparency, embedding, sectioning and staining when employing paraffin sections. When seen under a microscope, this optimization can make the cell structure of paraffin sections obvious, the tissue structure complete, and the staining clear and natural. The flower bud differentiation process is divided into 7 periods based on anatomical observations of the external morphology and internal structure during flower bud differentiation: undifferentiated period, start of differentiation period, inflorescence differentiation period, calyx differentiation period, petal differentiation period, stamen differentiation period, and pistil differentiation period. The differentiation time is concentrated from the end of May to mid-June. Crape myrtle flower bud differentiation is a complicated process, and the specific regulatory mechanism and affecting elements need to be investigated further.     

Viral IRES RNA structures and ribosome interactions

In eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide 'cap' on the mRNA and also proteins that recruit and position the ribosome. Many pathogenic viruses use an alternative, cap-independent mechanism that substitutes RNA structure for the cap and many proteins. The RNAs driving this process are called internal ribosome-entry sites (IRESs) and some are able to bind the ribosome directly using a specific 3D RNA structure. Recent structures of IRES RNAs and IRES-ribosome complexes are revealing the structural basis of viral IRES' 'hijacking' of the protein-making machinery. It now seems that there are fundamental differences in the 3D structures used by different IRESs, although there are some common features in how they interact with ribosomes.     

Imaging of the mouse lung with scanning laser optical tomography (SLOT)

The current study focuses on the use of scanning laser optical tomography (SLOT) in imaging of the mouse lung ex vivo. SLOT is a highly efficient fluorescence microscopy technique allowing rapid scanning of samples of a size of several millimeters, thus enabling volumetric visualization by using intrinsic contrast mechanisms of previously fixed lung lobes. Here, we demonstrate the imaging of airways, blood vessels, and parenchyma from whole, optically cleared mouse lung lobes with a resolution down to the level of single alveoli using absorption and autofluorescence scan modes. The internal structure of the lung can then be analyzed nondestructively and quantitatively in three-dimensional datasets in any preferred planar orientation. Moreover, the procedure preserves the microscopic structure of the lung and allows for subsequent correlative histologic studies. In summary, the current study has shown that SLOT is a valuable technique to study the internal structure of the mouse lung.     

Morphology and affinities of Eridostracina: Palaeozoic ostracods with moult retention

Ostracods are by far the most abundant living group of arthropods in the fossil record. Traditionally, eridostracines were classified as members of the Class Ostracoda. They have also been considered to represent extinct marine spinicaudatan (conchostracan) branchiopods. The ostracod affinity of the Eridostracina is evident in a number of features such as the muscle scars pattern, the hinge structure, the presence of an adductorial sulcus reflected as a ridge on the internal surface and the separation at the dorsal margin of successive valves. The eridostracines might be a polyphyletic group, containing aberrant representatives of ostracods, with ancestors probably among the conchoprimitid, leperditellid and beyrichioidean ostracod species. The Eridostracina represent an extinct group of small marine crustaceans with a multilayer structure of the calcified carapace, formed through the retention of unshed moults during the growth process. Details of the morphology of the eridostracine Cryptophyllus socialis from the Upper Devonian of Russia are reconstructed using the process of exfoliation of successive exuviae. ‘Double-sided’ hingement structures were found in the accumulated exuviae. It is suggested that the main function of these structures was the strengthening of the connection between the accumulated valves. The hingement of Cryptophyllus represents a vestigial structure, which has lost its original function as a pivot, a role documented in the ancestors of that genus. Tubular structures were found attached to the internal side of the calcite layer. It is suggested that they also represent vestigial pore canals, having lost their original function as sensory receptors. External surfaces of the attached exuviae bear imprints of the tubular structures of the overlying exuviae. These imprints originated probably due to the strong pressure of the new cuticle against the old one, during the very short moulting time. During this process, the freshly formed cuticle was at its final size, but still soft and non-calcified. A number of three-dimensionally preserved cell-like structures were recovered inside the interlayer chambers.