Keyhole Limpet Hemocyanin: Molecular Structure of a Potent Marine Immunoactivator

Objectives: In this short review we present a survey of the available biochemical and electron microscopic data on keyhole limpet hemocyanin (KLH). Results: The b

Morphology and in vitro infectivity of sporozoites of Cryptosporidium parvum.

An important obstacle in studying Cryptosporidium parvum is the lack of a permanent in vitro cultivation system of the parasite. While short-term cultures using various host cell lines have been widely employed, long-term cultures that would facilitate the immortalization of C. parvum isolates have not yet been developed. The description of the complete development of C. parvum in cell-free culture in 2004 has been received with great interest and also with some astonishment. Unfortunately, attempts to reproduce these results with different isolates of C. parvum and also C. hominis have failed. In this report, we provide an alternative interpretation of the nature of a parasite stage that o…

Polymorphism of Amyloid Fibrils and their Complexes with Catalase

Catalase binding to amyloid fibrils has been shown for the Alzheimer’s amyloid-β (Aβ), type 2 diabetes-associated islet amyloid polypeptide (IAPP) and Creutzfeldt-Jakob disease-associated prion protein (PrP). Catalase targets a specific domain with a GAII-like sequence and there are a number of other amyloid fibril-forming proteins that contain related sequences, such as the Parkinson’s associated α-synuclein protein and the Huntington disease protein Huntingtin. Using transmission electron microscopy (TEM) analysis, interactions show specific binding of catalase to some, but not all, fibrillar forms of Aβ, IAPP and PrP fragments, allowing determination of the fibrillar forms that contain a…

Negative Staining and Cryo-negative Staining: Applications in Biology and Medicine

Negative staining is widely applicable to isolated viruses, protein molecules, macromolecular assemblies and fibrils, subcellular membrane fractions, liposomes and artificial membranes, synthetic DNA arrays, and also to polymer solutions and a variety of nanotechnology samples. Techniques are provided for the preparation of the necessary support films (continuous carbon and holey/perforated carbon). The range of suitable negative stains is presented, with some emphasis on the benefit of using ammonium molybdate and of negative stain-trehalose combinations. Protocols are provided for the single droplet negative staining technique (on continuous and holey carbon support films), the floating a…

3D reconstruction of the hemocyanin subunit dimer from the chiton Acanthochiton fascicularis.

Procedures are presented for the purification of the subunit dimer from Acanthochiton fasicularis hemocyanin. Electron microscopy of negatively stained specimens revealed a uniform population of macromolecules possessing the characteristic "boat shape". A 3D reconstruction from this EM data generated a approximately 3 nm resolution model that correlates well with earlier data of the purported subunit dimer, extracted from the 3D reconstruction of the didecamer of Haliotis tuberculata hemocyanin type 1.

Keyhole limpet haemocyanin: negative staining in the presence of trehalose

Abstract Samples of unpurified and purified haemocyanin from the giant keyhole limpet Megathura crenulata have been studied by transmission electron microscopy (TEM) using mixtures of trehalose with the negative stains, uranyl acetate and ammonium molybdate. Trehalose is a known protein preservative during air and freeze drying, UV irradiation and high temperatures, and therefore offers the possibility of protecting proteins during the drying of negatively-stained specimens and their subsequent electron microscopical study. Evidence is presented that trehalose possesses satisfactory stability within the electron beam during conventional room temperature, negative-staining studies. The combi…

Cryo-negative staining

Abstract A procedure is presented for the preparation of thin layers of vitrified biological suspensions in the presence of ammonium molybdate, which we termcryo-negative staining. The direct blotting of sample plus stain solution on holey carbon supports produces thin aqueous films across the holes, which are routinely thiner than the aqueous film produced by conventional negative staining on a continuous carbon layer. Because of this, a higher than usual concentration of negative stain (ca. 16% rather than 2%) is required for cryo-negative staining in order to produce an optimal image contrast. The maintenance of the hydrated state, the absence of adsorption to a carbon film and associate…

Negative Staining of Thinly Spread Biological Samples

Negative staining is widely applicable to isolated viruses, protein molecules, macro-molecular assemblies and fibrils, subcellular membrane fractions, liposomes and artificial membranes, synthetic DNA arrays, and also to polymer solutions. In this chapter, techniques are provided for the preparation of the necessary support films (continuous carbon and holey/perforated carbon). The range of suitable negative stains is presented, with some emphasis on the benefit of using ammonium molybdate and of negative stain-trehalose combinations. Protocols are provided for the single-droplet negative staining technique (on continuous and holey carbon support films), the negative staining-carbon film te…

Alhydrogel® adjuvant, ultrasonic dispersion and protein binding: A TEM and analytical study

Aluminium-based vaccine adjuvants have been in use since the 1920s. Aluminium hydroxide (alum) that is the chemical basis of Alhydrogel, a widely used adjuvant, is a colloid that binds proteins to the particular surface for efficient presentation to the immune system during the vaccination process. Using conventional TEM and cryo-TEM we have shown that Alhydrogel can be finely dispersed by ultrasonication of the aqueous suspension. Clusters of ultrasonicated aluminium hydroxide micro-fibre crystals have been produced (∼ 10-100 nm), that are significantly smaller than those present the untreated Alhydrogel (∼ 2-12 μm). However, even prolonged ultrasonication did not produce a homogenous susp…

Influence of saline and pH on collagen type I fibrillogenesis in vitro: Fibril polymorphism and colloidal gold labelling

We have produced different collagen type I fibrils by in vitro fibrillogenesis of acetic acid-soluble collagen within the pH range 2.5-9.0, in the presence and absence of 150 mM NaCl. The varying relatively stable molecular assemblies and polymorphic fibrillar end-products produced after 24 h incubation have been assessed and compared by the TEM study of specimens negatively stained with uranyl acetate. In the presence of 150 mM NaCl, the assembly of collagen at low pH (2.5) leads to the formation of initial molecular aggregates that progressively link together at slightly higher pH (5.0) to form sub-fibrils and spindle-shaped D-banded bundles of sub-fibrils. At pH 6.0 these D-banded bundle…

Electron microscopy and biochemical characterization of a 350-kDa annular hemolymph protein from the keyhole limpet Megathura crenulata

The isolation and biochemical characterization of an annular non-hemocyanin hemolymph protein from a marine gastropod, the Californian giant keyhole limpet (Megathura crenulata) is presented. By analytical ultracentrifugation, the protein has a sedimentation coefficient of 12S and molecular mass of approximately 350 kDa. The subunit mass, obtained by SDS/PAGE in the presence of -SH reagent and 8 M urea, is approximately 35 kDa, thereby indicating the presence of 10 subunits in the native molecule. By negative staining, the protein is revealed in one predominant image projection as a pentagonal approximately 8 nm ring-like structure with an approximately 2-nm stain-filled centre and, in anot…

Cholesterol in Alzheimer’s Disease and other Amyloidogenic Disorders

The complex association of cholesterol metabolism and Alzheimer’s disease is presented in depth, including the possible benefits to be gained from cholesterol-lowering statin therapy. Then follows a survey of the role of neuronal membrane cholesterol in Aβ pore formation and Aβ fibrillogenesis, together with the link with membrane raft domains and gangliosides. The contribution of structural studies to Aβ fibrillogenesis, using TEM and AFM, is given some emphasis. The role of apolipoprotein E and its isoforms, in particular ApoE4, in cholesterol and Aβ binding is presented, in relation to genetic risk factors for Alzheimer’s disease. Increasing evidence suggests that cholesterol oxidation p…

The future of transmission electron microscopy (TEM) in biology and medicine.

3-D reconstruction of hemocyanins and other invertebrate hemolymph proteins by cryo-TEM: an overview.

Amphiphile Blockcopolymere mit einem flexiblen Block und einem kettensteifen Segment aus“zylindrischen Bürstenpolymeren”: metallocenkatalysierte Synthese und Bildung von Riesenmicellen

Basic Electron Microscopy

Negative Staining of Thinly Spread Biological Particulates

Topology of the 10 subunits within the decamer of KLH, the hemocyanin of the marine gastropod Megathura crenulata.

Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin isoform 1 (KLH1) decamers and a functional unit-specific monoclonal antibody anti-KLH1-c1. The antibody links hemocyanin molecules at both the collar and the collarless edge of the decamer, indicating a peripheral localization of functional units c. In isoform 2 (KLH2) the positions of functional units c have been identified with the peanut agglutinin (PNA), which has previously been shown to exclusively bind to KLH2-c. Ferritin linked to PNA was used to visualize labeled molecules electron microscopically. The pattern of labeling also indicates a peripheral localization of th…

Formation of two-dimensional crystals of icosahedral RNA viruses.

International audience; The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A…

Quaternary structure of the European spiny lobster (Palinurus elephas) 1x6-mer hemocyanin from cryoEM and amino acid sequence data.

Abstract Arthropod hemocyanins are large respiratory proteins that are composed of up to 48 subunits (8×6-mer) in the 75 kDa range. A 3D reconstruction of the 1×6-mer hemocyanin from the European spiny lobster Palinurus elephas has been performed from 9970 single particles using cryoelectron microscopy. An 8 A resolution of the hemocyanin 3D reconstruction has been obtained from about 600 final class averages. Visualisation of structural elements such as α-helices has been achieved. An amino acid sequence alignment shows the high sequence identity (>80%) of the hemocyanin subunits from the European spiny lobster P. elephas and the American spiny lobster Panulirus interruptus . Comparison of…

Formation, TEM study and 3D reconstruction of the human erythrocyte peroxiredoxin-2 dodecahedral higher-order assembly.

The production of a higher-order assembly of peroxiredoxin-2 (Prx-2) from human erythrocytes has been achieved during specimen preparation on holey carbon support films, in the presence of ammonium molybdate and polyethylene glycol. TEM study suggested that this assembly is a regular dodecahedron, containing 12 Prx-2 decamers (Mr 2.62 MDa, external diameter approximately 20 nm). This interpretation has been supported by production of a approximately 1.6 nm 3D reconstruction from the negative stain TEM data, with automated docking of the available X-ray data of the Prx-2 decamer. Comparison with other known protein dodecahedral and viral icosahedral structures indicates that this arrangement…

Three-dimensional structure of the anaphase-promoting complex.

The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase, composed of at least 11 subunits, that controls progression through mitosis and G1. Using cryo-electron microscopy and angular reconstitution, we have obtained a three-dimensional model of the human APC at a resolution of 24 A. The APC has a complex asymmetric structure 140 A x 140 A x 135 A in size, in which an outer protein wall surrounds a large inner cavity. We discuss the possibility that this cavity represents a reaction chamber in which ubiquitination reactions take place, analogous to the inner cavities formed by other protein machines such as the 26S proteasome and chaperone complexes. This cag…

Cholesterol Specificity of Some Heptameric β-Barrel Pore-Forming Bacterial Toxins: Structural and Functional Aspects

Apart from the thiol-specific/cholesterol-dependent cytolysin family of toxins (see Chapter 20) there are a number of other unrelated bacterial toxins that also have an affinity for plasma membrane cholesterol. Emphasis is given here on the Vibrio cholerae cytolysin (VCC) and the cytolysins from related Vibrio species. The inhibition of the cytolytic activity of these toxins by prior incubation with extracellular cholesterol or low density lipoprotein emerges as a unifying feature, as does plasma membrane cholesterol depletion. Incubation of VCC with cholesterol produces a heptameric oligomer, which is not equivalent to the pre-pore since it is unable to penetrate the plasma membrane. In st…

Quaternary and subunit structure of Calliphora arylphorin as deduced from electron microscopy, electrophoresis, and sequence similarities with arthropod hemocyanin

Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a …

Cholesterol-Streptolysin O Interaction: An EM Study of Wild-Type and Mutant Streptolysin O

We present transmission electron microscopical data from negatively stained specimens of cholesterol following interaction with the thiol-activated bacterial toxin streptolysin O (SLO) (wild-type and a number of cysteine substitution mutants), with and without chemical modification of the cysteine residues. Two experimental systems were used, one with an aqueous suspension of cholesterol microcrystals and the other with immobilized thin planar cholesterol crystals attached to a carbon film. In both systems the wild-type SLO and two cytolytically active mutants, Cys 530 --Ala (C530A) and Ser 101 --Cys (S101C), readily generated the characteristic SLO arc- and ring-like oligomers on the surfa…

Molybdenum blue: Binding to collagen fibres and microcrystal formation

Collagen fibres have been shown by transmission electron microscopy to progressively bind the polyoxomolybdate ring-complex, termed molybdenum blue. Nucleation of cuboidal molybdenum blue microcrystals occurs on the surface of the collagen fibres, leading eventually to extensive coating of the fibres with microcrystals.

Mass determination, subunit organization and control of oligomerization states of keyhole limpet hemocyanin (KLH).

Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet hemocyanin (KLH; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45-60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains…

Structure, mechanism and regulation of peroxiredoxins.

Peroxiredoxins (Prxs) are a ubiquitous family of antioxidant enzymes that also control cytokine-induced peroxide levels which mediate signal transduction in mammalian cells. Prxs can be regulated by changes to phosphorylation, redox and possibly oligomerization states. Prxs are divided into three classes: typical 2-Cys Prxs; atypical 2-Cys Prxs; and 1-Cys Prxs. All Prxs share the same basic catalytic mechanism, in which an active-site cysteine (the peroxidatic cysteine) is oxidized to a sulfenic acid by the peroxide substrate. The recycling of the sulfenic acid back to a thiol is what distinguishes the three enzyme classes. Using crystal structures, a detailed catalytic cycle has been deriv…

Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study.

The Vibrio cholerae cytolysin (VCC) 63-kDa monomer has been shown to interact in aqueous suspension with cholesterol microcystals to produce a ring/pore-like heptameric oligomer approximately 8 nm in outer diameter. Transmission electron microscopy data were produced from cholesterol samples adsorbed to carbon support films, spread across the holes of holey carbon films, and negatively stained with ammonium molybdate. The VCC oligomers initially attach to the edge of the stacked cholesterol bilayers and with increasing time cover the two planar surfaces. VCC oligomers are also released into solution, with some tendency to cluster, possibly via the hydrophobic membrane-spanning domain. At th…

Native and subunit molecular mass and quarternary structure of the hemoglobin from the primitive branchiopod crustacean Triops cancriformis

Many branchiopod crustaceans are endowed with extracellular, high-molecular-weight hemoglobins whose exact structural characteristics have remained a matter of conjecture. By using a broad spectrum of techniques, we provide precise and coherent information on the hemoglobin of one of the phylogenetically ‘oldest’ extant branchiopods, the tadpole shrimp Triops cancriformis. The hemoglobin dissociated under reducing conditions into two subunits, designated TcHbA and TcHbB, with masses of 35 775 ± 4 and 36 055 ± 4 Da, respectively, determined by ESI-MS. Nonreducing conditions showed only two disulfide-bridged dimers, a homodimer of TcHbA, designated D1 (71 548 ± 5 Da), and the heterodimer D2 (…

Introduction and Technical Survey: Protein Aggregation and Fibrillogenesis

In this chapter we provided the overall background to the subject of protein aggregation and fibrillogenesis in amyloidogenesis, with introduction and brief discussion of the various topics that are included with the coming chapters. The division of the book into basic science and clinical science sections enables correlation of the topics to be made. The many proteins and peptides that have currently been found to undergo fibrillogenesis are tabulated. A broad technical survey is made, to indicate the vast array of techniques currently available to study aspects of protein oligomerization, aggregation and fibrillogenesis. These are split into three groups and tabulated, as the microscopica…

Polymorphism of amyloid-beta fibrils and its effects on human erythrocyte catalase binding.

The Alzheimer's amyloid-beta (Abeta) peptide exists as a number of naturally occurring forms due to differential proteolytic processing of its precursor molecule. Many of the Abeta peptides of different lengths form fibrils in vitro, which often show polymorphisms in the fibril structure. This study presents a TEM based analysis of fibril formation by eighteen different Abeta peptides ranging in length from 5 to 43 amino acids. Spectrophotometric analysis of Congo red binding to the fibrillar material has been assessed and the binding of human erythrocyte catalase (HEC) to Abeta fibrils has also been investigated by TEM. The results show that a diverse range of Abeta peptides form fibrils a…

Comparison of the decameric structure of peroxiredoxin-II by transmission electron microscopy and X-ray crystallography

Abstract The decameric human erythrocyte protein torin is identical to the thiol-specific antioxidant protein-II (TSA-II), also termed peroxiredoxin-II (Prx-II). Single particle analysis from electron micrographs of Prx-II molecules homogeneously orientated across holes in the presence of a thin film of ammonium molybdate and trehalose has facilitated the production of a ≥20 A 3-D reconstruction by angular reconstitution that emphasises the D5 symmetry of the ring-like decamer. The X-ray structure for Prx-II was fitted into the transmission electron microscopic reconstruction by molecular replacement. The surface-rendered transmission electron microscopy (TEM) reconstruction correlates well…

Experimental Inhibition of Fibrillogenesis and Neurotoxicity by amyloid-beta (Aβ) and Other Disease-Related Peptides/Proteins by Plant Extracts and Herbal Compounds

Amyloid-β (Aβ) fibrillogenesis and associated cyto/neurotoxicity are major pathological events and hallmarks in diseases such as Alzheimer’s disease (AD). The understanding of Aβ molecular pathogenesis is currently a pharmacological target for rational drug design and discovery based on reduction of Aβ generation, inhibition of Aβ fibrillogenesis and aggregation, enhancement of Aβ clearance and amelioration of associated cytotoxicity. Molecular mechanisms for other amyloidoses, such as transthyretin amyloidosis, AL-amyloidosis, as well as α-synuclein and prion protein are also pharmacological targets for current drug therapy, design and discovery. We report on natural herbal compounds and e…

Keyhole Limpet Hemocyanin Type 2 (KLH2): Detection and Immunolocalization of a Labile Functional Unit h

Keyhole limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of …

In Vitro Techniques

Driven in part by the development of genomics, proteomics, and bioinformatics as new disciplines, there has been a tremendous resurgence of interest in physical methods to investigate macromolecular structure and function in the context of living cells. This volume in Methods in Cell Biology is devoted to biophysical techniques in vitro and their applications to cellular biology. The volume covers methods-oriented chapters on fundamental as well as cutting-edge techniques in molecular and cellular biophysics.This book is directed toward the broad audience of cell biologists, biophysicists, pharmacologists, and molecular biologists who employ classical and modern biophysical technologies or …

Isolation and Functional Analysis of Organelles

Double-Shell Vesicles, Strings of Vesicles and Filaments Found in Crosslinked Micellar Solutions of Poly(1,2-butadiene)-block-poly(ethylene oxide) Diblock Copolymers

Micellar structures of amphiphilic poly(1,2-butadiene)-block-poly(ethylene oxide) diblock copolymers have been crosslinked in aqueous solution by γ-irradiation. By transmission electron microscopy (TEM) of neganively stained specimen it is shown that the precominant structures present are sopolymer sesicles (which appear to be double latered. These fived vesules are stable with respect to their share and can be transferred from water into a good solvent for blue filbeks, such as tetrahydrofuran, this demonstrating the effectiveness of the crosslinking. In addition to the resicles a small number of flexible cylindrichl/filimented structures sequentially fused vesicles/strings of vesicles and…

Negative staining and cryo-negative staining of macromolecules and viruses for TEM

In this review we cover the technical background to negative staining of biomolecules and viruses, and then expand upon the different possibilities and limitations. Topics range from conventional air-dry negative staining of samples adsorbed to carbon support films, the variant termed the "negative staining-carbon film" technique and negative staining of samples spread across the holes of holey-carbon support films, to a consideration of dynamic/time-dependent negative staining. For each of these approaches examples of attainable data are given. The cryo-negative staining technique for the specimen preparation of frozen-hydrated/vitrified samples is also presented. A detailed protocol to su…

Controlled cleavage of KLH1 and KLH2 by the V8 protease from Staphylococcus aureus reassociation, electrophoretic and transmission electron microscopy study of peptide fragments.

The reassociation behaviour of protease V8-cleaved peptides from KLH1 and KLH2, the two hemocyanin isoforms from the giant keyhole limpet Megathura crenulata, has been studied by transmission electron microscopy of negatively stained specimens and SDS/PAGE. Reassociation of the complete mixture of protease cleavage products and of combinations of peptide fragments purified by HPLC was performed in the presence of 100 mm CaCl2 and 100 mm MgCl2 at pH 7.4, over a period of 1 to 4 weeks. The V8 protease splits KLH1 into peptide fragments containing the functional units abc, def, defg, defgh, g and h. This mixture of peptide fragments reassociated to form helical tubular polymers, with a diamete…

Transmission electron microscopy in molecular structural biology: A historical survey.

In this personal, historic account of macromolecular transmission electron microscopy (TEM), published data from the 1940s through to recent times is surveyed, within the context of the remarkable progress that has been achieved during this time period. The evolution of present day molecular structural biology is described in relation to the associated biological disciplines. The contribution of numerous electron microscope pioneers to the development of the subject is discussed. The principal techniques for TEM specimen preparation, thin sectioning, metal shadowing, negative staining and plunge-freezing (vitrification) of thin aqueous samples are described, with a selection of published im…

Keyhole limpet hemocyanin (KLH), II: Characteristic reassociation properties of purified KLH1 and KLH2.

Subunits of the two types of keyhole limpet hemocyanin (KLH1 and KLH2), purified by gel filtration chromatography and preparative polyacrylamide gel electrophoresis from Immucothel, have been used for macromolecular reassociation studies. In-vitro reassociation has been achieved with a standardized system using a Tris-saline stabilizing buffer at pH 7.4 containing 100 mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation has been monitored and the varying oligomeric forms of KLH1 and KLH2 produced have been studied by transmission electron microscopy, using specimens negatively stained with 5% ammonium molybdate containing 1% trehalose. Specimens ha…

Placental endogenous retrovirus (ERV): structural, functional, and evolutionary significance

Summary That endogenous retrovirus (ERV) is present within the placenta of humans and other mammals has been known for the past 25 years, but the significance of this observation is still not fully understood. Much molecular biological data have emerged in recent years to support the earlier electron microscopic data on the presence of placental ERV. The evidence for ERV in animal and human placental tissue is presented, then integrated with data on the the presence of ERV in a range of other tissues, in particular teratocarcinoma cells. Placental invasiveness and maternal immunosuppression are then discussed in relation to metalloproteinase secretion, the immunosuppressive potential of ret…

Nanoparticles Built of Cross-Linked Heterotelechelic, Amphiphilic Poly(dimethylsiloxane)-b-poly(ethylene oxide) Diblock Copolymers

Novel short chain α,ω-heterotelechelic amphiphilic poly(dimethylsiloxane)-b-poly(ethylene oxide) diblock copolymers (PDMS−PEO) with total molecular weights below 10 000 g/mol are synthesized, characterized, and used as basic constituent parts for functionalized nanoparticles. The self-assembly of the amphiphilic diblock copolymer in water as a solvent selective for the PEO block leads to the formation of spherical and cylindrical micellar structures with diameters between 10 and 25 nm. The core of the micelles is built of the hydrophobic PDMS chains, whereas the corona is set up by the hydrophilic PEO blocks. By using α,ω-heterotelechelic diblock copolymers, it is possible to fix the core o…

Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile apicomplexan organelle.

From an EM study of thin sections, the rod-like microneme organelles within conventionally glutaraldehyde fixed Cryptosporidium parvum sporozoites have been shown to undergo a shape change to a more spherical structure when the sporozoites age in vitro for a period of approximately 12 to 24 h. This correlates with the shape change of intact sporozoites, from motile hence viable thin banana-shaped cells to swollen pear-shaped cells, shown by differential interference contrast light microscopy of unstained unfixed and glutaraldehyde-fixed samples, as well as by thin section EM of fixed sporozoites. From negatively stained EM specimens of unfixed and fixed sporozoites the cellular shape change…

Fibril formation and toxicity of the non-amyloidogenic rat amylin peptide.

Full-length native rat amylin 1-37 has previously been widely shown to be unable to form fibrils and to lack the toxicity of the human amylin form leading to its use as a non-amyloidogenic control peptide. A recent study has suggested that rat amylin 1-37 forms amyloidogenic β-sheet structures in the presence of the human amylin form and suggested that this property could promote toxicity. Using TEM analysis we show here fibril formation by synthetic rat amylin 1-37 and 8-37 peptides when the lyophilized HPLC purified peptides are initially dissolved in 20 mM Tris-HCl. Dissolution of synthetic rat amylin 1-37 and 8-37 peptides in H(2)O or phosphate buffered saline failed to produce fibrils.…

Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel® followed by separation of KLH1 and KLH2

Abstract Studies of keyhole limpet hemocyanin (KLH) normally require purification of functional complexes directly from living animals. An alternative procedure is described wherein a commercial preparation of KLH which is fully dissociated into its subunits (Immucothel®, biosyn Arzneimittel GmbH) is reassociated in the presence of a high concentration of calcium and magnesium. The reassociation products, when observed by electron microscopy, consist of didecamers, multidecamers and flexible tubules of varying length. The two forms of KLH described previously and designated KLH1 and KLH2, are present in the reassocated mixture as homo-oligomers/polymers and can be separated by selective dis…

Micelle Formation from Amphiphilic“Cylindrical Brush”—Coil Block Copolymers Prepared by Metallocene Catalysis

Cholesterol binding to amyloid-β fibrils: A TEM study

There is increasing interest in the role of brain cholesterol in Alzheimer's disease and the contribution of cholesterol to the formation of amyloid plaques. This paper presents a TEM study showing the binding of soluble approximately 10 nm diameter cholesterol-PEG 600 micelles to amyloid-beta(1-42) (Abeta(1-42)) fibrils formed either in the presence of this cholesterol derivative or to preformed fibrils generated under four different fibrillogenesis conditions. Specimens negatively stained with uranyl acetate revealed that during 24 h fibrillogenesis at 37 degrees C the cholesterol-PEG micelles bound periodically to Abeta(1-42) protofibrils and apparently also formed a thin smooth unbroken…

Membrane Protein Complexes: Structure and Function

This edited book contains a compilation of 14 advanced academic chapters dealing with the structure and function of membrane protein complexes. This rapidly advancing important field of study closely parallels those on soluble protein complexes, and viral protein and nucleoprotein complexes.Diverse topics are included in this book, ranging from membrane–bound enzymes to ion channels, proton pumps and photosystems. Data from X-ray crystallography, cryo-electron microscopy and other biophysical and biochemical techniques are presented throughout the book. There is extensive use of colour figures of protein structures. Throughout the book structure and function are closely correlated.The two e…

Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 Å resolution by electron cryomicroscopy and angular reconstitution † 1 †This article is dedicated to the memory of Anneke van Heel. 1Edited by M.F. Moody

Abstract A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to ∼45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins o…

Keyhole limpet haemocyanin (KLH): Purification of intact KLH1 through selective dissociation of KLH2

Abstract Keyhole limpet haemocyanin (KLH) from almost all newly captive animals contains a mixture of KLH1 and KLH2. We show that the dissociation of KLH2 can be produced during EM specimen preparation by the negative staining-carbon film (NS-CF) procedure and in solution by ammonium molybdate-PEG solutions at slightly acidic pHs. The KLH2 multidecamers split apart in the pH range 7.5-6.5 and in the pH range 6.5-6.0 the individual decamers break open and start to dissociate. At pH 5.9 the dissociation of KLH2 yields predominantly a mixture of single subunits and what appear to be subunit dimers. Over the pH range 7.0-5.7 the KLH1 didecamer remains stable. Separation of intact KLH1, in the f…

Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and htH.

Keyhole limpet hemocyanin (KLH) is a well-established immune stimulant and hapten carrier, and Haliotis tuberculata hemocyanin (HtH) is a related product. Biologically, KLH and HtH are blue copper proteins which serve as oxygen carriers in the blood of the keyhole limpet Megathura crenulata and the abalone H. tuberculata, respectively, two marine gastropods. Both hemocyanins occur as two distinct isoforms, termed KLH1 KLH2, HtH1, and HtH2. Each of these molecules is based on a very large polypeptide chain, the subunit (molecular mass ca 400 kDa), which is folded into a series of eight globular functional units (molecular mass ca 50 kDa each). Twenty copies of this subunit form a cylindrical…

Visualizing In Vitro Type I Collagen Fibrillogenesis by Transmission Electron Microscopy

Techniques and protocols for the in vitro formation of collagen type I fibrils and the extensive biochemical variation of the fibrillogenesis conditions are presented. In all cases, the incubation and fibrillogenesis product can be readily monitored by transmission electron microscopic study of negatively stained specimens. Representative TEM data is presented and discussed within the context of the products of the fibrillogenesis protocols, from which the extensive biochemical and structural possibilities of this integrated approach can be appreciated.

The Contribution of Microscopy to the Study of Alzheimer’s Disease, Amyloid Plaques and Aβ Fibrillogenesis

A broad survey is presented in this chapter, dealing with the impact that microscopy has made to the study of Alzheimer’s disease, amyloid plaques and amyloid-β fibrillogenesis. This includes classical light microscopy and the modern immunolabelling and confocal microscopies, together with the contribution of transmission electron microscopy and atomic force microscopy. Whilst usefully standing alone, the individual microscopies often contribute most effectively when they are integrated with cellular, biophysical and molecular approaches.

Immunonegative Staining: Epitope Localization on Macromolecules

Relevant literature relating to immunonegative staining is reviewed and integrated with current research of the author and others. The immunonegative staining procedure has been utilized for the study of epitope localization on immune complexes formed from keyhole limpet hemocyanin type 2 (KLH2) di- and multidecamers, and the 20S and 26S proteasome from Xenopus laevis. The IgG linkage pattern of molecules in small immune complexes is considered to provide the most reliable indication of epitope location. For both KLH2 and the 20S proteasome, using domain-specific monoclonal antibodies and a 32-kDa (p32) subunit-specific polyclonal antibody, respectively, it is shown that epitopes (KLH2, sub…

Comparative 11A structure of two molluscan hemocyanins from 3D cryo-electron microscopy

Abstract Hemocyanins are giant extracellular proteins that transport oxygen in the hemolymph of many molluscs. Molluscan hemocyanins are cylindrical decamers or didecamers of a 350–400 kDa subunit that contains seven or eight different covalently linked globular functional units (FUs), arranged in a linear manner. Each FU carries a single copper active site and reversibly binds one dioxygen molecule. As a consequence, the decamer can carry up to 70 or 80 O 2 molecules. Although complete sequence information is now available from several molluscan hemocyanins, many details of the quaternary structure are still unclear, including the topology of the 10 subunits within the decamer. Here we sho…

Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites.

Abstract Sporozoites of the apicomplexan parasite Cryptosporidium parvum were subjected to cell disruption and subcellular fractionation using a sucrose density step gradient. With this procedure, highly enriched preparations of the parasite membrane, the micronemes, dense granules and amylopectin granules were produced. No separate fraction containing rhoptries was obtained, however this organelle was found in defined fractions of the gradient, still associated with the apical tip of the sporozoites. Using negative staining, the internal structure of the micronemes was revealed by transmission electron microscopy. Micronemes and dense granules showed characteristic protein compositions by …

The structure of Yersinia pestis Caf1 polymer in free and adjuvant bound states

Caf1 of the plague bacterium, Yersinia pestis is a polymeric virulence factor and vaccine component, formed from monomers by a donor strand exchange (DSE) mechanism. Here, EM images of Caf1 reveal flexible polymers up to 1.5 microm long (4MDa). The bead-like structures along the polymer are 5.8 + or - 1 nm long and correspond to single Caf1 proteins. Short polymers often form circles, presumably by DSE. We also provide the first images of proteins bound to alhydrogel adjuvant. Caf1, hemocyanin and anthrax PA are all resolved clearly and Caf1 exhibits adjuvant bound stretches with long intervening loops draped from the edges.

Preparation of Thin-Film Frozen-Hydrated/ Vitrified Biological Specimens for Cryoelectron Microscopy

Oligomerization and hemolytic properties of the C-terminal domain of pyolysin, a cholesterol-dependent cytolysin

Pyolysin (PLO) belongs to the homologous family of the cholesterol- dependent cytolysins (CDCs), which bind to cell membranes containing cholesterol to form oligomeric pores of large size. The CDC monomer structure consists of 4 domains. Among these, the C-terminal domain 4 has been implicated in membrane binding of the monomer, while the subsequent processes of oligomerization and membrane insertion have primarily been assigned to other domains of the molecule. Recombinantly expressed or proteolytic fragments that span domain 4 of the CDCs streptolysin O and perfringolysin O bind to membranes but fail to oligomerize, and they inhibit the activity of the respective wild-type toxins. We repo…

Immunoelectron Microscopy of Hemocyanin from the Keyhole Limpet (Megathura crenulata): A Parallel Subunit Model

Abstract Immunoelectron microscopy has been performed using negatively stained immune complexes of keyhole limpet hemocyanin (KLH) subunit 2 di- and multidecamers with domain-specific monoclonal antibodies. One antibody (KLH2 a macr 1) links the hemocyanin molecules in a side-to-side pattern, whereas the other antibody (KLH2 fg macr 1) links the molecules end-to-end. From existing knowledge of the domain sequence of KLH subunit 2, these data provide support for a parallel arrangement of subunits within each decamer. Ten N-terminal a macr: domains are then present at the noncollar region of each decamer with 10 C-terminal g macr domains at the collar region. The immunonegative staining data …

Molecular organization of selected prokaryotic S-layer proteins.

Regular crystalline surface layers (S-layers) are widespread among prokaryotes and probably represent the earliest cell wall structures. S-layer genes have been found in approximately 400 different species of the prokaryotic domains bacteria and archaea. S-layers usually consist of a single (glyco-)protein species with molecular masses ranging from about 40 to 200 kDa that form lattices of oblique, tetragonal, or hexagonal architecture. The primary sequen ces of hyperthermophilic archaeal species exhibit some characteristic signatures. Further adaptations to their specific environments occur by various post-translational modifications, such as linkage of glycans, lipids, phosphate, and sulf…

Engineering a 2D protein-DNA crystal.

(Figure Presented) Weaving with DNA: A DNA-binding protein was used to control the structure of a self-assembled 2D crystal. In the absence of protein, four oligonucleotides hybridize to form a Kagome lattice of interwoven double helices with p3 symmetry (see image). Addition of protein RuvA during assembly changes the symmetry and connectivity to give a DNA-protein crystal with an approximately square unit cell. © 2005 Wiley-VCH Verlag GmbH and Co. KGaA.

Papillomavirus assembly requires trimerization of the major capsid protein by disulfides between two highly conserved cysteines.

ABSTRACT We have used viruslike particles (VLPs) of human papillomaviruses to study the structure and assembly of the viral capsid. We demonstrate that mutation of either of two highly conserved cysteines of the major capsid protein L1 to serine completely prevents the assembly of VLPs but not of capsomers, whereas mutation of all other cysteines leaves VLP assembly unaffected. These two cysteines form intercapsomeric disulfides yielding an L1 trimer. Trimerization comprises about half of the L1 molecules in VLPs but all L1 molecules in complete virions. We suggest that trimerization of L1 is indispensable for the stabilization of intercapsomeric contacts in papillomavirus capsids.

Negative staining across holes: application to fibril and tubular structures.

The negative staining technique, when used with holey carbon support films, presents superior imaging conditions than is the case when samples are adsorbed to continuous carbon films. A demonstration of this negative staining approach is presented, using ammonium molybdate in combination with trehalose, applied to several fibrillar and tubular samples. Fibrils formed from the amyloid-beta peptide and the protease inhibitor pepstain A spread very well unsupported across holes and the different polymorphic fibril forms can be readily assessed. However, tubular forms of amyloid-beta have a tendency to be flattened, due to surface tension forces prior to and during specimen drying. Sub-fibril a…

Keyhole Limpet Hemocyanin (KLH): Slow In Vitro Reassociation of KLH1 and KLH2 from Immucothel®

Abstract Following our in vitro reassociation of keyhole limpet hemocyanin subunits in the presence of high concentrations (100 mM each) of calcium and magnesium chloride (Harris et al., 1997a, Micron 28, 31–41; 1997b, Micron 28, 43–56), we have now extended our investigations by using a buffer system containing a lower concentration of the two divalent cations (10 mM each). Reassociation of mixed KLH subunits present in the commercially available product Immucothel® was performed using a standardized buffer solution containing 50 mM Tris–HCl, 150 mM NaCl, 10 mM CaCl2 and 10 mM MgCl2 (pH 7.4) over a minimum period of one week, at 4°C. This solution was selected as being close to our KLH sta…

Book review

Routine preparation of air-dried negatively stained and unstained specimens on holey carbon support films: a review of applications.

Several representative examples are given of the successful application of negative staining across the holes of holey carbon support films using 5% (w/v) ammonium molybdate solution containing trehalose. The inclusion of 0.1% (w/v) trehalose is considered to be most satisfactory, although good data have also been obtained in the presence of 0.01 and 1.0% (w/v) trehalose. The examples given fall into the following groups: protein molecules in the absence of polyethylene glycol (PEG), protein molecules in the presence of PEG (Mr 1000), lipoproteins, lipids and membranes, filaments and tubules, viruses in the absence of PEG, viruses in the presence of PEG, aqueous polymer solutions, and final…

A discoidal lipoprotein from the coelomic fluid of the polychaete Nereis virens.

A discoidal lipoprotein was isolated from the coelomic fluid of the polychaete, Nereis virens, by density gradient centrifugation. The lipoprotein was present in both sexes and moved as a uniform band in an agarose gel. The average diameter of the lipoprotein particles determined by electron microscopy was 42 nm with a thickness of 10 nm. SDS electrophoresis showed two apoprotein subunits with molecular masses of 247 and 85 kDa, respectively. In lectin blots, both apoproteins were reactive with Concanavalin A indicating the presence of N-glycans. The small subunit was also reactive with peanut lectin, indicating additional O-glycosylation. The total lipid content was 48% and consisted mainl…

The collagen type I segment long spacing (SLS) and fibrillar forms: Formation by ATP and sulphonated diazo dyes.

The collagen type I segment long spacing (SLS) crystallite is a well-ordered rod-like molecular aggregate, ∼300nm in length, which is produced in vitro under mildly acidic conditions (pH 2.5-3.5) in the presence of 1mM ATP. The formation of the SLS crystallite amplifies the inherent linear structural features of individual collagen heterotrimers, due to the punctate linear distribution and summation of the bulkier amino acid side chains along the length of individual collagen heterotrimers. This can be correlated structurally with the 67nm D-banded collagen fibril that is found in vivo, and formed in vitro. Although first described many years ago, the range of conditions required for ATP-in…