Redistribution of Surface Antigens– a General Property of Animal Cells?

MEMBRANE antigens of animal cells are reported to be localized either in discontinuous discrete areas (H-2^1,2, HL-A³, organ⁴ and immunoglobulin (Ig)^5–9 or continuously (species⁴ and blood group^9,10). These results have been obtained by immunofluorescence (IFL) and electron microscopy performed by double layer principles except in the case of Ig where direct IFL technique has been used. The influence on the localization pattern of marker molecules by variables such as Ig concentration of the reagents used, effects of interactions between Ig molecules in the double layer and temperature dependence of the reaction steps have not been investigated. Redistribution of lymphocyte surface Ig molecules induced by anti-Ig antibody has been described and this was observed to be temperature dependent occurring at 20° C but not at 0° C^11,12. Here I discuss the distribution and localization of different surface antigens (blood group substance A and B, species, H-2 and organ) on several cell types (monkey kidney, human lymphoid cells, HeLa, human thyroid cells, RK13 and L-cells of C3H/K) as recorded by direct and indirect IFL technique. I show that the temperature and Ig concentration used exert an influence on the distribution of these antigens. The cells used in IFL tests were cultured and harvested as described before¹⁰. IFL staining was performed according to Möller1³. The stained cells were examined with a Leitz ‘Orthoplan’ ultraviolet microscope. Direct IFL technique was applied to two test systems. A FITC-conjugated rabbit antiserum to blood group substance A was used on monkey (Macaca fascicularis) kidney cells known to be A-positive¹⁰ and a FITC-conjugated horse anti-human thymocyte globulin were tested on a human lymphoid cell line Robinson (obtained from Dr Moore, Buffalo). In both test systems the staining pattern was completely confluent at all temperatures used: 4° C, 20° C and 37° C. The staining pattern was not related to antibody concentration.     

Surface receptors: Are they involved in transformation of spermatozoa of Ascaris?

Exposure of the spheroidal spermatozoa of Ascaris suum to an extract of the male accessory gland causes their transformation into ameboid cells. We have investigated the mechanism of this transformation, also termed activation, by labeling the proteins of accessory gland extracts with fluorescein isothiocyanate (FITC) or [125I], followed by qualitative localization of the sperm activating substances (SAS) and quantitative measurements of [125I]-SAS binding. Fluorescent patches of FITC-conjugated SAS were localized at the spermatozoan surface and were concentrated primarily at the posterior region. Few fluorescent patches were detectable in the region of the newly formed pseudopodia following transformation. Although spermatozoan transformation occurs within 2-5 min after exposure to SAS, the fluorescent patches became more distinct after a minimum of 8 min and reached maximum density at 15-30 min. Spermatozoa activated with [125I]-SAS became radioactively labeled in direct proportion to the amount of available [125I]-SAS until a saturation level was reached. SDS-polyacrylamide gel electrophoresis combined with autoradiography indicated that the cells bind two SAS components, of small (9,000 MW) and large (56,000 MW) sizes. These same two components were also detectable in a membrane fraction, obtained by differential centrifugation, of the spermatozoa after incubation with [125I]-SAS. binding of the two SAS components was not inhibited by preincubation of the spermatozoa with trypsin or Concanavalin A; however, the 56,000 MW component of SAS was not detectable in autoradiograms of spermatozoa incubated with periodic acid (1.6-10 mM) treated SAS. Such cells also failed to transform into ameboid spermatozoa. These results indicate that the two components of SAS that bind to the spermatozoan surface are possibly responsible for inducing the cell transformations associated with activation.     

Molecular Mechanism of Thioflavin-T Binding to the Surface of β-Rich Peptide Self-Assemblies

A number of small organic molecules have been developed that bind to amyloid fibrils, a subset of which also inhibit fibrillization. Among these, the benzothiol dye Thioflavin-T (ThT) has been used for decades in the diagnosis of protein-misfolding diseases and in kinetic studies of self-assembly (fibrillization). Despite its importance, efforts to characterize the ThT-binding mechanism at the atomic level have been hampered by the inherent insolubility and heterogeneity of peptide self-assemblies. To overcome these challenges, we have developed a minimalist approach to designing a ThT-binding site in a "peptide self-assembly mimic” (PSAM) scaffold. PSAMs are engineered water-soluble proteins that mimic a segment of β-rich peptide self-assembly, and they are amenable to standard biophysical techniques and systematic mutagenesis. The PSAM β-sheet contains rows of repetitive amino acid patterns running perpendicular to the strands (cross-strand ladders) that represent a ubiquitous structural feature of fibril-like surfaces. We successfully designed a ThT-binding site that recapitulates the hallmarks of ThT-fibril interactions by constructing a cross-strand ladder consisting of contiguous tyrosines. The X-ray crystal structures suggest that ThT interacts with the β-sheet by docking onto surfaces formed by a single tyrosine ladder, rather than in the space between adjacent ladders. Systematic mutagenesis further demonstrated that tyrosine surfaces across four or more β-strands formed the minimal binding site for ThT. Our work thus provides structural insights into how this widely used dye recognizes a prominent subset of peptide self-assemblies, and proposes a strategy to elucidate the mechanisms of fibril-ligand interactions.     

Surface Exposed Free Cysteine Suppresses Crystallization of Human γD-Crystallin

Human γD-crystallin (HGD) has remarkable stability against condensation in the human lens, sometimes over a whole lifetime. The native protein has a surface exposed free cysteine that forms dimers (Benedek, 1997; Ramkumar et al., 1864)^1,2 without specific biological function and leads to further protein association and/or aggregation, which creates a paradox for understanding its stability. Previous work has demonstrated that chemical modification of the protein at the free cysteine (C110), increases the temperature at which liquid–liquid phase separation occurs (LLPS), lowers protein solubility and suggests an important role for this amino acid in maintaining its long-term resistance to condensation. Here we demonstrate that mutation of the cysteine does not alter the structure or solubility (liquidus) line for the protein, but dramatically increases the protein crystal nucleation rate following LLPS, suggesting that the free cysteine has a vital role in suppressing crystallization in the human lens.     

Cell Surface Engineering of a β-Galactosidase for Galactooligosaccharide Synthesis

A novel gene encoding transglycosylating β-galactosidase (BGase) was cloned from Penicillium expansum F3. The sequence contained a 3,036-bp open reading frame encoding a 1,011-amino-acid protein. This gene was subsequently expressed on the cell surface of Saccharomyces cerevisiae EBY-100 by galactose induction. The BGase-anchored yeast could directly utilize lactose to produce galactooligosaccharide (GOS), as well as the by-products glucose and a small quantity of galactose. The glucose was consumed by the yeast, and the galactose was used for BGase expression, thus greatly facilitating GOS synthesis. The GOS yield reached 43.64% when the recombinant yeast was cultivated in yeast nitrogen base-Casamino Acids medium containing 100 g/liter initial lactose at 25°C for 5 days. The yeast cells were harvested and recycled for the next batch of GOS synthesis. During sequential operations, both oligosaccharide synthesis and BGase expression were maintained at high levels with GOS yields of over 40%, and approximately 8 U/ml of BGase was detected in each batch.Galactooligosaccharides (GOS) are beneficial for human health as prebiotics that maintain the balance of normal flora in the intestine, enhance lactose tolerance and the digestibility of milk products, reduce serum cholesterol levels, increase Ca²⁺ absorption, synthesize B-complex vitamins, and reduce the risk of cancer (15, 18). Recently, a great deal of attention has been devoted to GOS synthesis, especially via enzymatic transglycosylation, since chemical synthesis of GOS is very tedious (16). GOS can be synthesized by β-galactosidase (BGase) from lactose by glycosyl transfer of one or more galactosyl units onto a galactose moiety of lactose or other structurally related galactosides (10). Both free and immobilized BGases from different microorganisms have been employed for GOS synthesis (12). Based on previous studies, using free enzymes has been associated with limitations, such as low stability and nonreusability of the enzymes. Using immobilized enzymes could overcome these problems, but there are still some drawbacks, including low recovery rates of enzyme activity, the gradual loss of enzyme during the reaction process, finite immobilized carriers, and large mass transfer resistance between some immobilized enzymes and substrates.Recently, an alternative strategy to conventional enzyme immobilization was proposed in which the enzyme is anchored on the cell surfaces of engineered microorganisms, such as Escherichia coli and Saccharomyces cerevisiae (8, 13). S. cerevisiae is a highly advantageous host for cell surface display, as it may allow the accurate folding and glycosylation of recombinant proteins. It is generally regarded as safe in its applications in different fields. Yeast cell surface engineering has been demonstrated using the α-agglutinin receptor of S. cerevisiae to display foreign proteins on the cell surface. There were certain advantages to using cell surface-engineered yeast as an immobilized biocatalyst, e.g., the enzyme was anchored covalently on the cell surface without enzyme loss or additional treatments for immobilization, and the mass transfer resistance between the enzyme and the substrate was sharply reduced in contrast to conventional immobilization methods (17). Several studies have successfully used engineered yeast with immobilized target enzymes as a biocatalyst for a single use, such as the cell surface engineering of a β-glucosidase from Aspergillus oryzae for isoflavone aglycone production and a chitosanase from Paenibacillus fukuinensis for chitooligosaccharide production (7, 19). However, there have been no reports of the use of engineered yeast for consecutive batch production without loss of enzyme activity during the reaction process.The objective of this work was to present a novel approach for GOS synthesis by anchoring BGase from Penicillium expansum F3 on the cell surface of S. cerevisiae as an immobilized enzyme. Figure ​Figure11 shows the main principle of this strategy. The BGase that was cell surface engineered and anchored to yeast (BGase-anchored yeast) could directly utilize lactose for GOS synthesis in batches without loss of enzyme activity. The carbon source (glucose) for cell growth and the inducer (galactose) for enzyme production were the by-products of lactose. The yield of GOS was greatly increased because of the removal of glucose and the continuous expression of BGase. The results showed that this method was especially suitable for GOS synthesis, and it has great promise for industrial oligosaccharide production in the future.Open in a separate windowFIG. 1.Schematic of GOS synthesis by BGase-anchored yeast. The BGase-anchored yeast is represented by modified ovals. The surface BGase converted lactose into GOS, glucose, and a small quantity of galactose. The undesirable glucose was consumed by the yeast for cell growth, and the galactose induced the continuous expression of BGase. Thus, BGase-anchored yeast cells were grown and successively utilized lactose to produce GOS. After a batch reaction, BGase-anchored yeast with higher BGase activity could be harvested and recycled for another batch of GOS synthesis under the same cultivation conditions as the first batch.     

Nano-coating of β-galactosidase onto the Surface of Lactose by Using an Ultrasound-assisted Technique

We nano-coated powdered lactose particles with the enzyme β-galactosidase using an ultrasound-assisted technique. Atomization of the enzyme solution did not change its activity. The amount of surface-attached β-galactosidase was measured through its enzymatic reaction product D-galactose using a standardized method. A near-linear increase was obtained in the thickness of the enzyme coat as the treatment proceeded. Interestingly, lactose, which is a substrate for β-galactosidase, did not undergo enzymatic degradation during processing and remained unchanged for at least 1 month. Stability of protein-coated lactose was due to the absence of water within the powder, as it was dry after the treatment procedure. In conclusion, we were able to attach the polypeptide to the core particles and determine precisely the coating efficiency of the surface-treated powder using a simple approach.     

Chronic Morphine Reduces Surface Expression of δ-Opioid Receptors in Subregions of Rostral Striatum

The delta opioid receptor (DOPr), whilst not the primary target of clinically used opioids, is involved in development of opioid tolerance and addiction. There is growing evidence that DOPr trafficking is involved in drug addiction, e.g., a range of studies have shown increased plasma membrane DOPr insertion during chronic treatment with opioids. The present study used a transgenic mouse model in which the C-terminal of the DOPr is tagged with enhanced-green fluorescence protein to examine the effects of chronic morphine treatment on surface membrane expression in striatal cholinergic interneurons that are implicated in motivated learning following both chronic morphine and morphine sensitization treatment schedules in male mice. A sex difference was noted throughout the anterior striatum, which was most prominent in the nucleus accumbens core region. Incontrast with previous studies in other neurons, chronic exposure to a high dose of morphine for 6 days had no effect, or slightly decreased (anterior dorsolateral striatum) surface DOPr expression. A morphine sensitization schedule produced similar results with a significant decrease in surface DOPr expression in nucleus accumbens shell. These results suggest that chronic morphine and morphine sensitisation treatment may have effects on instrumental reward-seeking behaviours and learning processes related to drug addiction, via effects on striatal DOPr function.     

Surface coating — No improvement of shear stress resistance of anchorage dependent mammalian cells

Summary The growth of adherent Baby Hamster Kidney cells (BHK 21) on surfaces coated with different substances (FCS, Con. A, PHA, cholesterol, collagen) was investigated. A positive influence on cell viability could only be observed when using a fetal calf serum (FCS) coat. To evaluate the effects of coating on shear stress sensitivity, experiments in a home made flow chamber were carried out. For comparison further investigations were conducted for several serum concentrations in the medium. The results demonstrate that the latter had a more beneficial effect on shear stress resistance than coating.     

Computational Design of the β-Sheet Surface of a Red Fluorescent Protein Allows Control of Protein Oligomerization

Computational design has been used with mixed success for the design of protein surfaces, with directed evolution heretofore providing better practical solutions than explicit design. Directed evolution, however, requires a tractable high-throughput screen because the random nature of mutation does not enrich for desired traits. Here we demonstrate the successful design of the β-sheet surface of a red fluorescent protein (RFP), enabling control over its oligomerization. To isolate the problem of surface design, we created a hybrid RFP from DsRed and mCherry with a stabilized protein core that allows for monomerization without loss of fluorescence. We designed an explicit library for which 93 of 96 (97%) of the protein variants are soluble, stably fluorescent, and monomeric. RFPs are heavily used in biology, but are natively tetrameric, and creating RFP monomers has proven extremely difficult. We show that surface design and core engineering are separate problems in RFP development and that the next generation of RFP markers will depend on improved methods for core design.     

SUPERFICIAL – Surface mapping of proteins via structure-based peptide library design

Background  

The determination of protein surfaces and the detection of binding sites are essential to our understanding of protein-protein interactions. Such binding sites can be characterised as linear and non-linear, the non-linear sites being prevailant. Conventional mapping techniques with arrays of synthetic peptides have limitations with regard to the location of discontinuous or non-linear binding sites of proteins.     

Functional Demonstration of Connexin—Protein Binding Using Surface Plasmon Resonance

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.     

Surface–sediment diatom assemblages of the urban ponds of St. Petersburg,Russia

The diatom assemblages of the surface sediments have been studied in 53 urban ponds and lakes of St. Petersburg for the first time. In total, 350 species and infraspecific taxa have been registered; the species of Achnanthidium, Cocconeis, Cyclostephanos, Cyclotella, Gomphonema, Lemnicola, Navicula, Nitzschia, and Stephanodiscus genera were the most common. The wide distribution of small centric planktonic taxa, Cyclostephanos dubius, C. invisitatus, Cyclotella pseudostelligera, Stephanodiscus hantzschii, and S. minutulus, is preconditioned by the depletion of dissolved silica, which is a result of the high phosphorus load. The high abundance of macrophytes in the shallowest sites favors the dominance of epiphytic Fragilaria, Staurosira, and Staurosirella taxa. Bottom-living diatoms in the shallow eutrophic and hypereutrophic urban ponds are light-limited; this is a result of either macrophytes or phytoplankton development. Alkaliphilous and alkalibiontic species prevail among the pH-indicative species, while eutraphentic and hypereutraphentic diatoms, as well as those thriving in a wide range of trophic conditions, dominate over other trophic groups. The β- and α-mesosaprobic species are the most common saprobity-indicative taxa.     

Radiating Amyloid Fibril Formation on the Surface of Lipid Membranes through Unit-Assembly of Oligomeric Species of α-Synuclein

Background

Lewy body in the substantia nigra is a cardinal pathological feature of Parkinson''s disease. Despite enormous efforts, the cause-and-effect relationship between Lewy body formation and the disorder is yet to be explicitly unveiled.

Methodology/Principal Findings

Here, we showed that radiating amyloid fibrils (RAFs) were instantly developed on the surface of synthetic lipid membranes from the β-sheet free oligomeric species of α-synuclein through a unit-assembly process. The burgeoning RAFs were successfully matured by feeding them with additional oligomers, which led to concomitant dramatic shrinkage and disintegration of the membranes by pulling off lipid molecules to the extending fibrils. Mitochondria and lysosomes were demonstrated to be disrupted by the oligomeric α-synuclein via membrane-dependent fibril formation.

Conclusion

The physical structure formation of amyloid fibrils, therefore, could be considered as detrimental to the cells by affecting membrane integrity of the intracellular organelles, which might be a molecular cause for the neuronal degeneration observed in Parkinson''s disease.     

Modeling of a Graphene Nanoribbon–based Microfluidic Surface Plasmon Resonance Biosensor

Plasmonics - A surface plasmon resonance (SPR) biosensor based on a graphene nanoribbon array in a microfluidic flow cell operating in a flow-over format is studied. The optical response of the...     

Surface carbohydrates of Rhizobium I. β-1, 2-Glucans

Because of increased interest in surface carbohydrates of Rhizobium in relation to host specificity, phenol — water extractions were carried out of whole cells of Rhizobium strains of the species R. leguminosarum, R. phaseoli, R. trifolii and R. meliloti.Fractionation of the crude extracts with cetavlon afforded polysaccharide mixtures, which were essentially free of RNA and acidic exopolysaccharide (EPS). They could be separated into a high molecular weight heteropolysaccharide fraction of lipopolysaccharide (LPS) nature and a low molecular weight glucan fraction. Glucan turned out to be the principal polysaccharide component of the cells (up to 10% of the dry cell weight), when cultivated in carbohydrate-rich media, and to be present as firmly attached capsular material.Glucan (mol wt 3000) structure was elucidated by methylation and periodate oxidation techniques. Methylation yielded 3, 4, 6-tri-O-methyl-d-glucose, characterized by GLC-MS, as the only product of hydrolysis of the fully methylated glucan. The glucan consumed 1 mole of periodate per mole anhydroglucose unit and gave sophorose on partial hydrolysis. From these data a linear -1,2-linked glucan structure was deduced. The occurrence of -1,2-glucan and the implications for the specific binding properties of Rhizobium cells are discussed.