Age-Dependent Changes in the Ultrastructure and in the Molecular Composition of Rat Brain Microtubules

An age-dependent increase of a cathepsin D-like protease activity that preferentially degrades high molecular weight microtubule-associated proteins (MAPs) has been previously described. Microtubules (MT) purified from rat brain of different ages in the presence of several protease inhibitors retained undegraded MAPs through cycles of polymerization, and revealed several age-dependent changes in the relative amounts of MAPs and MT-associated kinases. MAP2 immunoreactivity was found significantly lower in MT preparations from aged animals in contrast with a relative increase of tau molecules. In addition, the phosphorylation of MAP2 by its associated cyclic AMP-dependent protein kinase was also altered, consecutively to the partial loss of the enzyme during polymerization cycles and an age-dependent decrease in the ability of the cyclic nucleotide to stimulate MAP2-bound kinase activity. The evidence of an unusually high packing density of sedimented MT from old rat brains further suggested the modification with aging of the physical structure of the arm-like projections of MAPs, in addition to a lower amount in high molecular weight MAPs. These results support the hypothesis of a selective alteration with aging of the mechanical and regulatory properties of brain MT, consecutive to a change in the composition and/or the structure of MAPs.     

Identification of Caveolar Resident Proteins in Ventricular Myocytes Using a Quantitative Proteomic Approach: Dynamic Changes in Caveolar Composition Following Adrenoceptor Activation

The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation–contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation.Rat ventricular myocytes were treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MβCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MβCD treatment. Selective activation of α-, β1-, and β2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes.We typically identified 600–850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.Caveolae are specialized invaginated lipid rafts (1), around 50–100 nm in diameter, enriched in cholesterol and sphingolipids, and characterized by the presence of caveolin and cavin proteins. The lipid environment, caveolin content, and morphology of caveolae are central to their diverse functional roles, which include coordination of signal transduction, cholesterol homeostasis, and endocytosis (2). Clustering of elements of particular signal cascades within a caveola promotes efficiency and fidelity of signaling. Although caveolae and noncaveolar rafts coexist, evidence suggests that most proteins are clustered by caveolae in the cardiac cell (3). Caveolin exists as three major isoforms: caveolin 1 and caveolin 2, which are expressed in most cell types, and caveolin 3, which is the muscle-specific isoform. Caveolins 1 and 3 are the predominant forms found in the adult cardiac myocyte (4, 5). Four members of the cavin family of related proteins exist, and all have been detected in the heart (6).One of caveolae''s best-characterized roles is as a signalosome, a compartment that brings together components of signal transduction cascades (including receptors, effectors, and targets (7)). Within caveolae, the 20-residue scaffolding domain of caveolin (CSD)¹ has been proposed to interact with a complementary caveolin-binding motif (CBM) in proteins. This enables oligomeric caveolin to act as a regulatory scaffold for macromolecular signaling complex formation (8). However, the ability of this simple and commonly occurring motif to interact with caveolin (directing proteins to caveolae and regulating their activity) has recently been challenged, because it is often buried within mature proteins (9, 10). Palmitoylation of juxtamembrane cysteine residues has also been proposed to partition proteins to ordered detergent-resistant membranes such as caveolae (11).The organization of proteins in caveolae suggests that they have a key role in regulation of signaling in the heart. We adopt the convention of the field here to assign proteins as caveolar if they are present in buoyant caveolin-containing membrane fractions obtained by sucrose gradient fractionation or in morphologically identifiable caveolae by immunogold electron microscopy. For example, α1- and β2-adrenoceptors (AR) are found exclusively in caveolae-containing membrane fractions of the adult heart (12, 13), whereas β1-AR are in both caveolar and bulk sarcolemmal fractions (14). Cardiac caveolae are also sites of enrichment of G proteins (12, 15), effectors of AR (including adenylyl cyclase V/VI, protein kinase A (RII), GRK2, phospholipase Cβ, PP2A, and eNOS (13–16)), and their downstream targets. Importantly, the distribution of receptors, effectors, and their targets is key to the efficiency and fidelity of their coupling (13, 17, 18). For example, altered β1- and β2-AR responses have been observed following cholesterol depletion (which disrupts caveolae) and severing of normal caveolin 3 interactions with a caveolin 3 CSD peptide (19, 20).A considerable number of cardiac ion transporters are resident in cardiac caveolae: voltage-gated sodium channels (21), L-type calcium channels (16), voltage gated potassium channels (22), ATP-sensitive potassium channels (23), the sodium-calcium exchanger (24) (NCX - although this has been challenged (25)), the sodium potassium ATPase (sodium pump) (26), and the plasma membrane calcium ATPase (PMCA) (27). Physical colocalization of ion transporters in the caveolar compartment may functionally link ion flow by providing a restricted diffusional space (28) and facilitates hormonal regulation of these transporters by placing them physically adjacent to signaling molecules. For example, regulation of a subpopulation of L-type calcium channels by β2-AR requires their colocalization in caveolae (16), and regulation of the cardiac sodium pump by phospholemman (PLM) relies on phosphorylation and dephosphorylation of PLM in caveolae (29, 30). The presence of ion transporters in caveolae is likely to have functional relevance beyond signal transduction because the lipid composition of the bilayer in which an ion transporter resides will influence its activity. Membrane cholesterol modulates many aspects of ion channel function: the sodium pump, for example, is regulated by the cholesterol content of the membranes within which it resides (31).Using traditional biochemical techniques (such as sucrose density gradient fractionation followed by Western blotting), the presence of certain proteins in cardiac caveolae has been shown to be dynamic on an acute timescale (minutes), however, no unbiased assessment of global changes in caveolar composition during signaling has been reported. PKC isoforms translocate into caveolae upon activation (32), and various G-protein coupled receptors translocate in and out of caveolae upon activation: muscarinic M2 (33) receptors into caveolae, and β2-AR out of caveolae (14, 34). Pivotally, this redistribution of proteins has been closely linked with functional responses. For example, the negative inotropic effect of the muscarinic agonist carbachol is ascribed to the ability of M2 receptors to move to caveolae and couple therein to eNOS, which is exclusively found in this compartment (33). Conversely, one suggestion for the limited functional response following β2-AR receptor stimulation is that translocation from the caveolar compartment (where it normally couples to its effector adenylyl cyclase) to clathrin-coated pits terminates its downstream effects by internalization of the receptor (14, 34).Hence, it has been proposed that many signaling proteins and ion transporters are located in caveolae and that the distribution between caveolar and noncaveolar membranes is dynamically regulated. Although quantitative proteomics has been used to highlight the breadth of proteins localized to caveolae in nonexcitable cells (for example (35)), to date no study has used an unbiased proteomic approach to define resident proteins of cardiac caveolae, or changes in the composition of caveolae during signaling events in any cell type.The principal challenge to overcome in using proteomics to characterize a particular membrane compartment is purifying this compartment to homogeneity. The high sensitivity of detection possible in modern proteomics means that even minor contamination of caveolar membranes with another membrane compartment leads to mis-assignment of proteins as caveolar residents. In practice, because contamination of membrane preparations cannot be reduced to zero, an alternative approach has been developed: For proteins to be considered genuinely localized to lipid rafts/caveolae, their presence must be sensitive to cholesterol depletion (36). Hence stable isotope labeling with amino acids in cell culture (SILAC) and cholesterol depletion with methyl-beta-cyclodextrin (MβCD) in noncardiac cultured cells has distinguished genuine caveolar proteins from the mitochondrial, sarco/endoplasmic reticulum, and cytosolic proteins that routinely contaminate caveolae prepared by density gradient centrifugation (35). Using such an approach, contaminating proteins appear in the caveolar fraction whether or not cholesterol is depleted with MβCD, but genuine caveolar residents are specifically depleted. This approach has refuted research that implies that mitochondrial proteins are enriched in caveolae (37, 38), including data from cardiac tissue that suggested that principal components of cardiac caveolae are mitochondrial and structural proteins (39). The current study employed a similar approach—with the modification that because cardiac myocytes cannot be maintained in culture for SILAC (because of marked phenotypic changes), we used iTRAQ-based quantitative proteomics.Here we identify 249 high-confidence caveolar proteins in the cardiac cell and show for the first time that the caveolar proteome is remarkably stable on an acute timescale following adrenoreceptor stimulation, suggesting that signaling complexes are preformed in this microdomain.     

Structure and Macromolecular Composition of the Seed Coat of the Musaceae

Mature seed coats of representatives of all three genera ofMusaceae were analysed for macromolecular composition with variousmass spectrometric techniques and compared with scanning electronmicroscopy and light microscopy in combination with histochemicaltechniques. Mass spectrometric techniques are more sensitiveand more specific in identifying macromolecular compounds thanhistochemical methods. The macromolecular ‘fingerprint’of the seed coats of Musaceae showed unique components of aromaticphenols. The seed coat structure of all three genera is homogeneouswithin the Musaceae. It is characteristic at the family leveland most complex within the Zingiberales. Very remarkable arethe separation of the outer cell walls from the exotestal layer,exposing a secondary surface with silica crystals, and the relativelythick mesotesta which protects the seed, e.g. against the bitingforces and passage through the digestive tracts of dispersingagents. Germination takes place with an operculum and is facilitatedby a predetermined rupture layer in the micropylar collar. Themusaceaous seed presents a good example of the solution of conflictingdemands of protection and germination. Musaceae; Musa; Ensete; Musella; seed coat; pyrolysis (gas chromatography) mass spectrometry; histochemistry; anatomy; macromolecules; silica; lignin; cellulose; vegetable polyphenols; operculum; germination     

The Ultrastructure and Development of the Light Line in the Geraniaceae Seed Coat

Abstract: In 32 Geraniaceae species, we investigated the so-called light line in the palisade layer of the seed coat. The light line is an incrustation of electron-dense substances in the secondary cell wall. The existence of different gaps in the line and its complete absence near the chalazal area suggest that this cell wall structure is not responsible for hardseededness in the Geraniaceae seed coat. The multiple light line pattern within idioblasts of the palisade layer is characteristic of this cell type, named multiple light line (mll) cells. The palisade cells and their light line in Geraniaceae differ structurally from those in the Fabaceae.     

Composition and Ultrastructure of Streptomyces venezuelae

Streptomyces venezuelae is a filamentous bacterium with branching vegetative hyphae embedded in the substrate and aerial hyphae bearing spores. The exterior of the spore is inlaid with myriads of tiny rods which can be removed with xylene. The spore wall is approximately 30 nanometers thick. Occasionally, it can be seen that the plasma membrane and the membranous bodies within a spore are connected. The spore's germ plasm is not separated from the cytoplasm by a nuclear envelope. The cell walls of the vegetative hyphae, which are about 15 nanometers thick, are structurally and chemically similar to those of gram-positive bacteria. The numerous internal membranous bodies, some of which arise from the plasma membrane of the vegetative hypha, may be vesicular, whirled, or convoluted. Membranous bodies are usually prominent at the hyphal apices and are associated with septum formation. The germ plasm is an elongate, contorted, centrally placed area of lower electron density than the hyphal cytoplasm. The spores differ from the vegetative hyphae, not only in fine structure, but also in the arginine and leucine contents of their total cellular proteins.     

Ultrastructure and Composition of the Nannochloropsis gaditana Cell Wall

Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (∼75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes.     

The Complex Ultrastructure of the Endolysosomal System

Live-cell imaging reveals the endolysosomal system as a complex and highly dynamic network of interacting compartments. Distinct types of endosomes are discerned by kinetic, molecular, and morphological criteria. Although none of these criteria, or combinations thereof, can capture the full complexity of the endolysosomal system, they are extremely useful for experimental purposes. Some membrane domain specializations and specific morphological characteristics can only be seen by ultrastructural analysis after preparation for electron microscopy (EM). Immuno-EM allows a further discrimination of seemingly identical compartments by their molecular makeup. In this review we provide an overview of the ultrastructural characteristics and membrane organization of endosomal compartments, along with their organizing machineries.The endolysosomal network is required for multiple functions and control of cell homeostasis. It is not only reached by endocytic cargo but also by biosynthetic cargoes. It is an intermediate to degradation, but also essential for recycling, signaling, cell polarity, cilia formation, cytokinesis, and migration (Gould and Lippincott-Schwartz 2009; Taguchi 2013). This multitude of functions can only be ensured by an extremely organized ultrastructure. With the increased understanding of how cellular machinery defines endolysosomal subdomains, the nomenclature of the endolysosomal system has also increased in complexity. We start this review, therefore, with a brief introduction of the terminology of the endolysosomal system.Coated pits and vesicles were described in 1964 (Roth and Porter 1964), and lysosomes were first described by De Duve and Novikoff in the mid-1950s (Novikoff et al. 1956), but the range of organelles in between these beginning and ending stages of endocytosis was only described later (Bhisey and Freed 1971). Electron microscopy (EM) studies by Allen and coworkers on the unicellular ciliate Paramecium caudatum revealed the existence of intracellular compartments that could be loaded with the endocytic marker horseradish peroxidase (HRP) (Allen and Fok 1980). These were named “endosomes.” Parallel studies in mammalian cells, by Pastan, Willingham, and colleagues, also using HRP, described intracellular vacuoles and tubules involved in the transport of transferrin receptor (TfR) (Gonatas et al. 1977; Goud et al. 1981; Willingham and Pastan 1983). These were called “receptosomes” (Willingham and Pastan 1980). Geuze, Slot, and collaborators introduced immunogold labeling, allowing the quantitative localization of multiple proteins within one EM sample (Geuze et al. 1981). When they localized the recycling asialoglycoprotein receptor together with its ligand destined for lysosomal degradation (Geuze et al. 1983), they identified compartments consisting of a vacuole and multiple associated tubules. These were called compartments involved in the uncoupling of receptors and ligands (CURLs) because the vacuoles accumulated the ligand (for degradation) and the tubules the receptor (for recycling). Today the CURL is known as the “early endosome” (EE), which in addition to receptors and ligands is now known to be reached by virtually all components internalized from the cell surface (see Mayor et al. 2014; Cossart and Helenius 2014).In the current literature, different nomenclatures are still used to describe the endolysosomal system, which can sometimes cause some confusion. In this review, based on combined ultrastructural and functional knowledge, we propose the following nomenclature: We refer to the vacuolar domains of EEs as sorting endosomes (SEs) and the tubules emerging from SEs as recycling endosomes (REs). Although in some cells (e.g., melanocytes) (see Delevoye et al. 2009), the RE tubules may stay attached while functioning in recycling, more typically they detach from the SE to form a tubular endosomal network (TEN). The term “endosomal recycling compartment” (ERC) is used to designate the peri-centriolar compartment that can be observed only in some cell types. Late endosomes (LEs), also referred to as multivesicular bodies (MVBs), are rounded compartments filled with intraluminal vesicles (ILVs). Lysosomes are the final compartments of the endocytic pathway, with different morphologies depending on the cell type (schematic representation in Fig. 1). Moreover, in the literature, these terms are differently used because most studies involve light microscopy, which does not provide sufficient resolution to detect all of the distinct domains.Open in a separate windowFigure 1.Schematic and simplified representation of the endolysosomal system showing the different organelles described in this article. Sorting endosomes (SE) are vacuolar compartments often bearing bilayered, flat clathrin coats (brown). Tubules emanate from SE that form the recycling endosomes (RE). The RE may localize to the peri-Golgi area forming the endocytic recycling compartment (ERC) or distribute to the cell periphery. The RE network is complex with multiple sorting sites, thereby the tubular sorting endosome (TSE) or tubular endosomal network (TEN) is also represented. AP1 (red) and AP3 (green) coated buds on RE (ERC/TSE/TEN) are shown. Late endosomes correspond to multivesicular bodies (MVBs) filled with intraluminal vesicles (ILVs). MVBs are fated for fusion with lysosomes. In some cells, a population of MVBs fuse with the plasma membrane, a process during which the ILVs are secreted as exosomes. Gray arrows indicate directions of transport/maturation of compartments. Blue arrows indicate invagination at the endosomal membrane of SE and MVBs required for ILV formation.     

Changes of Ultrastructure in Spore Coat of Bacillus thiaminolyticus during Germination and Outgrowth

Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted of at least four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle spore coat layer including two layers of a high and a low electron density, and an inner spore coat layer composing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L -alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological changes, in three steps; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner layer at a prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.     

Caveolar endocytosis and virus entry

The endocytic function of caveolae has been controversial for a long time. However, a real-time-imaging analysis of Simian virus 40 (SV40) 's entry in cells has indicated the existence of caveolar endocytosis during virus entry. The caveolae engulfed SV40 virions begin budding from plasma membrane depending on dynamin. SV40 enclosed in caveolae vesicles move to the caveosome, then to the endoplasmic reticulum. In addition, it was demonstrated that human coronavirus-229E enters the cell through caveolae. This review examines the involvement of caveolae in endocytosis used by the viral entry system.     

Molecular Markers for the agouti Coat Color Locus of the Mouse

The agouti (a) coat color locus of the mouse acts within the microenvironment of the hair follicle to control the relative amount and distribution of yellow and black pigment in the coat hairs. Over 18 different mutations with complex dominance relationships have been described at this locus. The lethal yellow (Ay) mutation is the top dominant of this series and is uniquely associated with an endogenous provirus, Emv-15, in three highly inbred strains. However, we report here that it is unlikely that the provirus itself causes the Ay-associated alteration in coat color, since one strain of mice (YBR-Ay/a) lacks the provirus but still retains a yellow coat color. Using single-copy mouse DNA sequences from the regions flanking Emv-15 we have detected three patterns of restriction fragment length polymorphisms (RFLPs) within this region that can be used as molecular markers for different agouti locus alleles: a wild-type agouti (A) pattern, a pattern which generally cosegregates with the nonagouti (a) mutation, and a pattern which is specific to Emv-15. We have used these RFLPs and a panel of 28 recombinant inbred mouse strains to determine the genetic linkage of these sequences with the agouti locus and have found complete concordance between the two (95% confidence limit of 0.00 to 3.79 centimorgans). We have also physically mapped these sequences by in situ hybridization to band H1 of chromosome 2, thus directly confirming previous assignments of the location of the agouti locus.     

Hormonal Complex and Ultrastructure of Maturing Aesculus hippocastanum Seeds

The content and temporal changes in the endogenous IAA, cytokinins, gibberellin-like compounds (GLC), and ABA were determined during horse chestnut (Aesculus hippocastanum L.) seed development (the stages of embryo axis development, its active growth, and storage compound deposition). The active growth of the embryo was characterized by the highest amounts of free phytohormones. Later, by the end of seed maturation, we observed the accumulation of the bound forms of IAA and ABA and a trend to a decrease in the content of free IAA, zeatin, and GLC (butanol fraction). The electron-microscopic examination of the embryo from the mature seed demonstrated that some structural components of the cytoplasm were similar in the cells of embryo axes and cotyledons. During the entire period of maturation, the embryo cells preserved native vacuoles and protein bodies were not formed. Thus, the structure of cotyledonary and axial cells and the distribution of free and bound phytohormones in the horse-chestnut seeds are similar to those in maturing seeds characterized by exogenous dormancy.     

Molecular Composition and Extinction Coefficient of Native Botulinum Neurotoxin Complex Produced by Clostridium botulinum Hall A Strain

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)^?1cm^?1. These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.     

大蒜花叶病毒外壳蛋白基因cDNA的克隆和序列分析

我们从自然发病的大蒜中分离得到了大蒜花叶病毒。以其基因组ＲＮＡ为模板合成了３＇末端部分ｃＤＮＡ。从中选出一批插入片段在２．０ｋｂ以上的重组克隆,经Ｎｏｒｔｈｅｒｎ点杂交分析证实了所选克隆与基因组ＲＮＡ同源。通过对若干个克隆的插入片段两端部分序列的测定,选出一个克隆ｐＧＭ４９５,其插入片段的长度约为２．４ｋｂ,３′末端存有一个Ｐｏｌｙ（Ａ）结构,它应包含了编码该病毒外壳蛋白全部序列。序列测定的结果表明,这个ｃＤＮＡ片段全长为２３７９ｂｐ,其中含有与酶切图谱分析结果相符的ＥｅｏＲＩ、ＰｓｔＩ及ＢａｍＨＩ酶切位点。第一个终止密码子ＴＡＡ与３′ｇ末端相距２６４ｂｐ,我们根据碱基序列推定的氨基酸序列与其它已发表的Ｐｏｔｙｖｉｒｕｓ的外壳蛋白氨基酸序列以及外壳蛋白翻译后加工的蛋白酶专一切点相比较后推测,编码该病毒外壳蛋白序列可能起始于３′末端上游的１１７０ｂｐ处,共编码３０２个氨基酸,其分子量为３６ｋＤ,略大于ＳＤＳ－ＰＡＧＥ所测定的３３ｋＤ,非编码区域长２６４ｂｐ,富含ＡＴ,并有多个终止密码子的存在。趾３′末端３２～２７ｂｐ处有一个ＡＡＴＡＡ序列。     

Caveolar Fatty Acids and Acylation of Caveolin-1

Purpose

Caveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.

Methods

Caveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.

Results

In contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5×10⁷ cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.

Conclusion

Caveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.     

Purification, Ultrastructure, and Composition of Axial Filaments from Leptospira

The ultrastructure of three strains of water Leptospira was studied by negative staining, thin sectioning, and freeze-etching. The cells possessed a triple-layered sheath which covered two independent axial filaments, one inserted subterminally in each end of the cell. The protoplasmic cylinder was surrounded by a triple-layered cell wall and possessed ribosomes, lamellar structures, and a typical procaryotic nuclear region. The axial filament was comprised of several component structures. An axial fibril, with a diameter of 20 to 25 nm, consisted of a solid inner core (13 to 16 nm in diameter) surrounded by a coat. A terminal knob (40 to 70 nm in length) was connected to a series of disc insertion structures at the terminal end of the axial fibril. The axial fibril was surrounded by a helical outer coat (35 to 60 nm in diameter) which was composed of a continuously coiled fiber, 3 to 4 nm in diameter, embedded in an electron-dense material. A procedure for the purification of the axial fibrils was presented and their ultrastructural, physical, and chemical properties were determined. Similarities in ultrastructural, physical, and chemical properties were noted between the axial fibrils and bacterial flagella. A schematic model of the leptospiral axial filament is presented, and a mechanism is proposed for its function as a locomotor organelle.     

Microtubule-Dependent Modulation of Adhesion Complex Composition

The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding α₅β₁ integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, α₅-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, α₅-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183.     

Molecular Modeling Study of the Norfloxacin-DNA Complex

Abstract

Molecular modeling and molecular dynamics were performed to investigate the interaction of norfloxacin with the DNA oligonucleotide 5′-d(ATACGTAT)₂. Eight quinolone-DNA binding structures were built by molecular modeling on the basis of experimental results. A 100ps molecular dynamics calculation was carried out on two groove binding models and six partially intercalating models. The resulting average structures were compared with each other and to free DNA structure as a reference. The favorable binding mode of norfloxacin to a DNA substrate was pursued by structural assess including steric hindrance, presence of hydrogen-bonding, non-bonding energies of the complex and presence of abnormal structural distortion. Although two of the intercalative models showed the highest binding energy and the lowest non-bonding interaction energy, they presented structural features which contrast with experimental results. On the other hand, one groove binding model demonstrated the most acceptable structure when the experimental observation was accounted. In this model, hydrogen bonding of the carbonyl and carboxyl group of the norfloxacin rings with the DNA bases was present, and norfloxacin binds to the amine group of the guanine base which protrudes toward the minor groove of B-DNA.