Site-directed photochemical disruption of the actin cytoskeleton by actin-binding Rose Bengal-conjugates

The in situ light-induced, non-enzymatic digestion of cytoskeletal actin by a xanthene dye conjugated to heavy meromyosin, anti-actin antibodies and/or anti-myosin antibodies is reported. The dye Rose Bengal was conjugated to either anti-actin antibodies, anti-myosin antibodies or heavy meromyosin. Under our experimental conditions, visible light induced the non-enzymatic breakdown of cytoskeletal actin when mammalian tissue culture cells were probed either with Rose Bengal-conjugated anti-actin and/or anti-myosin antibodies. Similar results were obtained when tissue culture cells were probed with Rose Bengal-conjugated heavy meromyosin before irradiation with visible light. The in situ photochemical reaction depended on the presence of actin-binding Rose Bengal-conjugates.     

Site-directed photoproteolysis of 8-oxoguanine DNA glycosylase 1 (OGG1) by specific porphyrin-protein probe conjugates: a strategy to improve the effectiveness of photodynamic therapy for cancer

The specific light-induced, non-enzymatic photolysis of mOGG1 by porphyrin-conjugated or rose bengal-conjugated streptavidin and porphyrin-conjugated or rose bengal-conjugated first specific or secondary anti-IgG antibodies is reported. The porphyrin chlorin e6 and rose bengal were conjugated to either streptavidin, rabbit anti-mOGG1 primary specific antibody fractions or goat anti-rabbit IgG secondary antibody fractions. Under our experimental conditions, visible light of wavelengths greater than 600 nm induced the non-enzymatic degradation of mOGG1 when this DNA repair enzyme either directly formed a complex with chlorin e6-conjugated anti-mOGG1 primary specific antibodies or indirectly formed complexes with either streptavidin-chlorin e6 conjugates and biotinylated first specific anti-mOGG1 antibodies or first specific anti-mOGG1 antibodies and chlorin e6-conjugated anti-rabbit IgG secondary antibodies. Similar results were obtained when rose bengal was used as photosensitizer instead of chlorin e6. The rate of the photochemical reaction of mOGG1 site-directed by all three chlorin e6 antibody complexes was not affected by the presence of the singlet oxygen scavenger sodium azide. Site-directed photoactivatable probes having the capacity to generate reactive oxygen species (ROS) while destroying the DNA repair system in malignant cells and tumors may represent a powerful strategy to boost selectivity, penetration and efficacy of current photodynamic (PDT) therapy methodologies.     

Selective purification of cardiac myosin by a high-performance salicylate affinity column

The cardiac muscle proteins, myosin and actin, were purified in one step using a salicylate-silica affinity column. The affinity columns were prepared by coupling sodium salicylate via its hydroxyl group to an Altex Ultraffinity-EP column. Crude detergent extracts from guinea pig hearts were passed through the column and the myosin-actin complex was then eluted with excess free salicylate or high salt. The affinity of cardiac myosin for immobilized salicylate was unique as myosin heavy chain from guinea pig leg muscle detergent extracts could not be purified by this procedure. Commercially purified rabbit leg muscle myosin also appeared to have no interaction with the salicylate affinity column, suggesting that the column is specific for cardiac myosin.     

Electrophoretic and computer analysis of skeletal muscle used for cardiac assistance.

Skeletal muscle has an inherent plasticity which allows it to undergo fibre type transformation when induced by a specific stimulus. Electrical stimulation has been used here to induce transformation of a predominantly fast type skeletal muscle towards a slow, more fatigue-resistant phenotype, which is more suitable for use in long-term cardiac assistance. Muscle samples from animals electrically stimulated for periods up to 6 months have been analysed by electrophoresis for myosin heavy chain (MHC) and myosin light chain (MLC) fast and slow isoforms. Densitometry and computer analysis have been used to determine the pattern of transformation of the different myosin subunits over this time period. MHC and MLC 2 fast to slow isoform switching preceded that of the alkali light chains (MLC1 and MLC3). After 3 months of stimulation the MHC slow isoform was found to have doubled in concentration relative to the unstimulated control muscle and by 4 months accounted for almost 50% of the total MHC content. The slow isoform accounted for 75% of the MLC2 after 4 months of stimulation. The protein products of mRNA isolated from stimulated muscle samples, translated in vitro and separated by electrophoresis, showed that transformation at the mRNA level preceded that at the protein level. By 2-4 weeks of stimulation MLC2 slow isoform mRNA represented over 60% of the total MLC2 mRNA population. An understanding of the molecular structure of muscle during transformation provides insight into its haemodynamic performance in cardiac assistance.     

Agarose isoelectric focusing for the detection of many isoforms and high molecules in muscle protein analysis

Two-dimensional gel electrophoresis with agarose in the first dimension was established. The technique was reported previously and was now much improved without changing the basic procedure, presenting "a protein map" of chicken skeletal muscle, including almost all components such as myosin heavy chains (200 kDa) and dystrofin (400 kDa). Application of this technique for the analysis of all components was also successful with the liver and lens. Since large molecules can get into the agarose in the first dimension, the study on protein interaction was also reported with troponin components as a model system.     

Highly selective directed assembly of functional actomyosin on Au surfaces

The capability of assembling biomotors onto specific locations of solid substrates is a key for development of biomotor-based nanomechanical systems. We developed a method to direct the assembly of the heavy meromyosin fragment from rabbit skeletal muscle myosin onto specific locations of Au substrates utilizing surface molecular patterns. In this strategy, chemically directed patterns of streptavidin were achieved to direct highly specific assembly of biotinylated heavy meromyosin on the substrates--a strategy applicable for patterning a variety of biotinylated molecules--while BSA was utilized to avoid nonspecific adsorption. In vitro motility assays of filament sliding were used to confirm functionality of assembled actomyosin.     

Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.     

LONG-RANGE TRIPLET-SINGLET ENERGY TRANSFER FROM ENZYME GENERATED TRIPLET ACETONE TO XANTHENE DYES

Enzyme-generated and protected acetone transfers its energy to xanthene dyes by a nontrivial process. The relative populations of the S₁ state are indicative of the operation of a heavy atom effect, being 1:15:100 for fluorescein, eosine and Rose Bengal. On the other hand Stern-Volmer constants and k _ET⁰ values are similar for the three dyes and are remarkably higher than for collisional processes.
It is tentatively suggested that at least for eosine and Rose Bengal, a triplet-triplet cxciton transfer may occur to populate an upper triplet state of the dye followed by heavy-atom enhanced intersystem crossing to the S₁ state.     

Effect of Tomato Peel Extract Grown under Drought Stress Condition in a Sarcopenia Model

Tomatoes and their derivates represent an important source of natural biologically active components. The present study aims to investigate the protective effect of tomato peel extracts, grown in normal (RED-Ctr) or in drought stress (RED-Ds) conditions, on an experimental model of sarcopenia. The phenolic profile and total polyphenols content (TPC) of RED-Ctr and RED-Ds were determined by Ultra High-Performance Liquid Chromatography (UHPLC) analyses coupled to electrospray ionization high-resolution mass spectrometry (ESI-HR-MS). Human skeletal muscle myoblasts (HSMM) were differentiated in myotubes, and sarcopenia was induced by dexamethasone (DEXA) treatment. Differentiation and sarcopenia were evaluated by both real-time PCR and immunofluorescent techniques. Data show that myosin heavy chain 2 (MYH2), troponin T (TNNT1), and miogenin (MYOG) were expressed in differentiated myotubes. 5 μg Gallic Acid Equivalent (GAE/mL) of TPC from RED-Ds extract significantly reduced muscle atrophy induced by DEXA. Moreover, Forkhead BoxO1 (FOXO1) expression, involved in cell atrophy, was significantly decreased by RED-Ds extract. The protective effect of tomato peel extracts depended on their qualitative polyphenolic composition, resulting effectively in the in vitro model of sarcopenia.     

Electrophoretic analysis of electrically trained skeletal muscle.

Sheep latissimus dorsi muscle was electrically trained, thereby inducing fast-to-slow fibre-type transformation. Using a combination of one- and two-dimensional gel electrophoresis techniques with computer analysis, we have analysed altered expression of contractile protein isoforms at protein and mRNA level over a time course of electrical training extending to 5 months. Myosin heavy chain and regulatory myosin light chain analysis showed predominant expression of their slow isoforms (86% and 92%, respectively) after 3 months of training. At the same time point, however, tropomyosin analysis revealed that the slow isoform of the alpha-subunit accounted for 64% of the total alpha expression. Troponin T isoform switching proceeded more slowly over the same time course than tropomyosin and the thick filament proteins studied. Troponin T analysis revealed 5 fast and 2 slow isoforms in the sheep, of which the second slow isoform only became clearly visible after 5 months' training. At this time point the two slow isoforms were more predominant than their fast counterparts. This suggests that a wide heterogeneity of fast and slow isoform combinations are possible in the thin filament of skeletal muscle.     

The water-soluble rose bengal derivatives and their spectroscopic behaviours

The paper presents a series of novel water-soluble derivatives of Rose bengal. Solubility in water of the C-2 Rose bengel esters has been obtained by the attachment of the proper water-soluble moiety to the dye molecule. It is shown that size of the attached molecule has a significiant influence on the spectroscopic properties of the derivatives. The long side chain strongly influences on the position of the absorption λ_max and emission spectre. The side chain creates the microenvironment for the molecule to which is attached. This makes it more difficult the diffusion of H₃OXXX ions into the dye molecule and causes an apparent decrease of pK value for protonation of phenol group of xanthene residue. These types of derivatives make possible to carry out the photooxidation in water even at low pHs.     

From esoteric theory to therapeutic antibodies

Theoretical analyses of amino acid and nucleotide sequences of immunoglobulins have provided a unique approach to the understanding of structure and function of antibodies. Variability plots unambiguously identified that the antibody-combining site is formed by six short complementarity determining regions (CDRs), three each from light and heavy chains. Since three-dimensional (3-D) foldings of framework regions (FRs) are similar among different antibodies, the 3-D configurations of CDRs from a specific antibody can be predicted based on their amino acid sequences. The resulting structure forms a compact surface that fits the antigen molecule tightly. The third CDR of the heavy chain (CDRH3), which is coded by the D-minigene together with the N- and/or P-segments, appears to play a unique role in fine fitting between antibody and antigen molecules. In order to maintain biological activities, the grafting of mouse CDRs onto human FRs should closely match the human FR amino acid sequences with the original mouse antibody. Similarities between human and mouse FR sequences that have been preserved for over 20 million years of evolution can be a useful tool in humanizing mouse antibodies.     

Structural states of dictyostelium myosin

Myosin purified from Dictyostelium amoebae has approximately 10% by weight of RNA associated with it, unless specific steps (DEAE cellulose chromatography or RNase digestion) are taken to remove it. This RNA has significant effects on the structural states formed by the myosin at low ionic strength in the presence of Mg2+. Rapid precipitation of RNA-free myosin by dilution generates bipolar thick filaments (540 nm long, 33 nm thick), often with a bare zone and a 15-nm transverse repeat. Rapid precipitation of myosin with copurified RNA yields linear aggregates of bipolar filaments, showing some lateral association. Slow precipitation of RNA-free myosin by dialysis yields very long filaments or ribbons (greater than 5 micrometer, 30--60 nm wide) in which the myosin may be packed diagonally across the filament, similar to the "side-polar" aggregates formed by other nonmuscle myosins and by smooth muscle myosin (Craig R, Megerman J: J Cell Biol 75:990, 1977; Hinssen H, D'Haese J, Small JV, Sobieszek A: J Ultrastruct Res 64:282, 1978). Slow precipitation of myosin with copurified RNA generates linear filaments with repeat intervals of 290 and 650 nm. Other polyanions were tested for their effects on myosin aggregation. Total RNA and ribosomal RNA from Dictyostelium, when added to RNA-free myosin, also induced the extensive linear aggregation seen with the copurified RNA/myosin complex, although higher concentrations of RNA were required to obtain quantitatively the same effect. DNA and heparin were also effective inducers of linear aggregation, whereas homopolymers of nucleotides and of acidic or basic amino acids were poorly effective.     

Nucleotides Induced Changes in Skeletal Muscle Myosin by DSC,TMDSC and EPR

Electron paramagnetic resonance (EPR, ST-EPR) and differential scanning calorimetry(DSC) were used in conventional and temperature modulated mode to study internal motions and energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Psoas muscle fibres from rabbit were spin-labelled with an isothiocyanate-based probe molecule at the reactive sulfhydryl site (Cys-707) of the catalytic domain of myosin. In the presence of nucleotides (ATP, ADP, AMP⋅PNP) and ATP or ADP plus orthovanadate, the conventional EPR spectra showed changes in the ordering of the probe molecules in fibres. In MgADP state a new distribution appeared; ATP plus orthovanadate increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like spectrum. In the complex DSC pattern, higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head region The monoton decreasing TMDSC heat capacities show that C _p of biological samples should not be temperature independent. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antibodies to short synthetic peptides for specific recognition of partly denatured protein

On immunization with subclasses of homologous proteins, the presence of immunodominant class-specific structures often prevents the development of subclass specific antibodies. Immunization with synthetic peptides representing specific structures in such a protein can then be used instead. Sequences derived from human IgG2 and Staphylococcus aureus enterotoxin were used as a model to develop general criteria for immunization with synthetic peptides. Peptide sequences were selected on the basis of criteria such as hydrophilic property, surface probability and secondary structure, which together constitute an antigenic index. The carrier proteins keyhole limpet hemocyanin and chicken gammaglobulin were coupled by using either carbodiimide or a maleimide. Peptide-specific responses can be elicited regardless of the sequence. Some coupling conditions influenced the specificity of the antibody responses. The results of immunization with short peptides not representing a natural B-cell determinant of a protein suggest that the frequency of native protein-recognizing antibodies is considerably lower than that of antibodies that recognize the protein in a partly denatured or altered state.     

High-performance ion-exchange chromatography of myosin using a DEAE-5PW column

High-performance ion-exchange chromatography of myosin using a DEAE-5PW packing was used to purify myosin from skeletal, cardiac and smooth muscle. This method produces high-speed resolution (30-min analysis) of myosin from contaminating myofibrillar proteins. The column has a high capacity for binding myosin (up to 1 g) and can be used for small-scale preparation of highly purified myosin. Gel analysis in the presence of sodium dodecyl sulfate showed recovery of myosin with very little contamination of other myofibrillar proteins. Myosin was also recovered from small biopsy samples (0.1 g) by a direct extraction technique with recovery of biological ATPase activity.     

N-acetyl-D-glucosamine kinase interacts with dynein light-chain roadblock type 1 at Golgi outposts in neuronal dendritic branch points

N-acetylglucosamine kinase (GlcNAc kinase or NAGK) is a ubiquitously expressed enzyme in mammalian cells. Recent studies have shown that NAGK has an essential structural, non-enzymatic role in the upregulation of dendritogenesis. In this study, we conducted yeast two-hybrid screening to search for NAGK-binding proteins and found a specific interaction between NAGK and dynein light-chain roadblock type 1 (DYNLRB1). Immunocytochemistry (ICC) on hippocampal neurons using antibodies against NAGK and DYNLRB1 or dynein heavy chain showed some colocalization, which was increased by treating the live cells with a crosslinker. A proximity ligation assay (PLA) of NAGK-dynein followed by tubulin ICC showed the localization of PLA signals on microtubule fibers at dendritic branch points. NAGK-dynein PLA combined with Golgi ICC showed the colocalization of PLA signals with somal Golgi facing the apical dendrite and with Golgi outposts in dendritic branch points and distensions. NAGK-Golgi PLA followed by tubulin or DYNLRB1 ICC showed that PLA signals colocalize with DYNLRB1 at dendritic branch points and at somal Golgi, indicating a tripartite interaction between NAGK, dynein and Golgi. Finally, the ectopic introduction of a small peptide derived from the C-terminal amino acids 74–96 of DYNLRB1 resulted in the stunting of hippocampal neuron dendrites in culture. Our data indicate that the NAGK-dynein-Golgi tripartite interaction at dendritic branch points functions to regulate dendritic growth and/or branching.     

Intramolecular formal [4+2] cycloaddition reactions of secondary and tertiary aryldiacetylene alcohols

Thermal and thionyl chloride induced cycloaromatizations of secondary and tertiary aryldiacetylene alcohols were studied. The secondary alcohol did not respond to thionyl chloride, but in all the other cases presumptive biradical intermediates evolved either by intramolecular radical coupling or, when derived from a xanthene scaffold, by intramolecular radical acylation to a diketone that finally afforded a p-methylene-quinone.     

Chromatographic analysis of tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle.

Tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle all exist as dimeric, ca. 100% alpha-helical coiled-coil species in benign media. Two major tropomyosin isoforms from each muscle source have been identified and can be conveniently designated alpha (fast) and beta (slow) based on electrophoretic mobility under denaturing conditions. The ratio of alpha to beta chains is ca. 3-4:1 for rabbit skeletal and ca. 1:1 for chicken gizzard and earthworm tropomyosins. Each chain from the former two muscle sources has been sequenced, thus providing a molecular basis for interpreting the in vivo population of homo- and hetero-dimers. The characteristics of each purified tropomyosin in weak-anion exchange, strong-cation exchange and reversed-phase high-performance liquid chromatography are described. Binding to and/or elution from the reversed-phase matrix results in dissociation into highly helical monomeric chains. This mode of chromatography separates the alpha and beta chains of earthworm and chicken gizzard tropomyosins, but not those of the rabbit protein. Both anion- and cation-exchange chromatography use mild (benign) elution conditions under which the native, in vivo dimer population should be preserved. Only the rabbit protein exhibited peak separation on the anion-exchange resin, with peak assignment corresponding to the known molecular organization of homo- and hetero-dimers. In strong cation-exchange analysis, all three tropomyosins exhibit a chromatographic transition near pH 6.5, possibly the result of histidine(s) titration. Collectively, the chromatographic data confirm the present understanding of the in vivo mixture of dimers for tropomyosin from rabbit skeletal and chicken gizzard. It is concluded that native earthworm tropomyosin exists predominantly as an alpha beta hetero-dimer.     

DSC and EPR study on AMP.PNP,BeFx and AlF4 containing myosin nucleotide complexes

Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated skeletal muscle fibres to study the effect of the binding of nucleotides and nucleotide analogues to myosin. The thermal unfolding of muscle fibres in rigor showed three discrete domain regions with thermal stability of 52.2, 58.8 and 67.8°C. AMP.PNP and ATP plus AlF₃ or BeF₂ affected markedly the transitions, which implies the strong interaction between AMP.PNP or nucleotide analogues and catalytic domain of myosin, and a partial dissociation of heads from actin. ADP.BeFx and states model the transition states of the ATP hydrolysis cycle which precede the powerstroke of the muscle fibres. Spectrum deconvolution on isothiocyanate-labelled fibres in AMP.PNP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The myosin heads which showed high degree of order were in the strongly binding ADP-state. The spectra in - and ADP.BeFx state reflected random orientation of labels with increased rotational mobility in comparison with rigor. The results suggest that myosin in muscle fibres in ADP.BeFx state exists in two forms. This revised version was published online in July 2006 with corrections to the Cover Date.