Self-assembling systems based on amphiphilic poly-N-vinylpyrrolidone and their interaction with model proteins

Polymeric particles formed by stearoyl-poly-N-vinylpyrrolidone (PVP-stear) of M_n = 2600 were obtained in aqueous solution, and their shape and size distribution were characterized. The size of the particles was shown to decrease with an increase in the ionic strength of the solution. Interaction of PVP-stear and its aggregates with model proteins (Bowman–Birk soybean proteinase inhibitor (BBI) and its hydrophobized derivatives) was studied. The possibility of inclusion of both native BBI and oleoylic derivative of BBI in the PVP-stear polymeric aggregates was investigated. It was established that polymeric particles with a diameter of 30 nm formed under certain concentration ratios between PVP-stear and poorly soluble dioleoyl BBI are capable of solubilization of dioleoyl BBI as well as prevention of its inactivation at low pH values.     

ATRP synthesis of amphiphilic random,gradient, and block copolymers of 2-(dimethylamino)ethyl methacrylate and n-butyl methacrylate in aqueous media

Amphiphilic random, gradient, and block copolymers of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and n-butyl methacrylate (BMA) were synthesized by atom transfer radical polymerization (ATRP) in water/2-propanol mixtures using a methoxy-poly(ethylene glycol) (MPEG) (M(n) = 2000) macroinitiator. Kinetic studies indicate that the copolymerization is well controlled with molecular weights increasing linearly with conversion. Copolymers with molecular weights up to M(n) = 34000 and low polydispersities (M(w)/M(n) = 1.11-1.47) were prepared. The reactivity ratios were calculated for the copolymerizations catalyzed by CuBr/bpy, (r(DMAEMA) = 1.07, r(BMA) = 1.24). The thermosensitivity and aggregation properties of the random, gradient, and block copolymers significantly depended on the architecture of the copolymers. The lower critical solution temperature of MPEG-b-PDMAEMA(84) was 38 degrees C (5 wt % in water).     

Ionic binding of C3 to the human pathogen Moraxella catarrhalis is a unique mechanism for combating innate immunity

Moraxella catarrhalis ubiquitous surface proteins A1 and A2 (UspA1/A2) interfere with the classical pathway of the complement system by binding C4b-binding protein. In this study we demonstrate that M. catarrhalis UspA1 and A2 noncovalently and in a dose-dependent manner bind both the third component of complement (C3) from EDTA-treated serum and methylamine-treated C3. In contrast, related Moraxella subspecies (n = 13) or other human pathogenic bacteria (n = 13) do not bind C3 or methylamine-treated C3. Experiments with recombinant proteins and M. catarrhalis mutants devoid of UspA1/A2 revealed that UspA1/A2 exert their actions by absorbing and neutralizing C3 from serum and restrain complement activation. UspA2 was responsible for most of the effect, and the Moraxella mutant lacking UspA2 was more sensitive to the lytic effect of human serum compared with the wild type. Interestingly, among the large number of bacteria analyzed, only M. catarrhalis has this unique ability to interfere with the innate immune system of complement by binding C3.     

Electrophoretic and immunologic comparative analysis of Mycoplasma pneumoniae and Mycoplasma genitalium proteins

The Mycoplasma pneumoniae FH strain routinely used in our laboratory for over 25 years as antigen in serological tests, 2 reference M. pneumoniae strains from ATCC (29342 and M129) and 3 isolates of M. pneumoniae obtained in 1995 from pneumonia patients were compared by SDS-PAGE, complement fixation test (CFT) and by Western-immunoblotting against human and rabbit serum samples with high level of mycoplasmal antibodies. On SDS-PAGE all M. pneumoniae strains showed the same number of 23 polypeptides on the gel with identical molecular weights. The same strains on immunoblotting against human and rabbit serum samples showed six bands: 170, 89, 75, 55, 38 and 33 kDa with the strongest antibody staining in 170-(P1 protein) and 89-kDa bands. Because of its known antigenic relationships Mycoplasma genitalium was used for comparison. The pattern of M. genitalium proteins on SDS-PAGE was similar to pattern of M. pneumoniae but distinguishable. On immunoblotting six proteins of M. genitalium (135, 127, 110, 95, 75 and 45 kDa) reacted with human and rabbits immunoglobulins for M. pneumoniae antigens. Furthermore in complement fixation test both antigens, prepared from M. pneumoniae and M. genitalium, reacted as well with human and rabbit immunoglobulins for M. pneumoniae and with rabbit immunoglobulins for M. genitalium. These cross-reactions observed in serological techniques could give false positive results in routine diagnosis of M. pneumoniae infections. In such situations showing on immunoblott of presence in tested serum sample of antibodies to 170- and 89 kDa proteins could confirm M. pneumoniae infection.     

Acquisition of complement factor H is important for pathogenesis of Streptococcus pyogenes infections: evidence from bacterial in vitro survival and human genetic association

Streptococcus pyogenes (or group A streptococcus [GAS]) is a major human pathogen causing infections, such as tonsillitis, erysipelas, and sepsis. Several GAS strains bind host complement regulator factor H (CFH) via its domain 7 and, thereby, evade complement attack and C3b-mediated opsonophagocytosis. Importance of CFH binding for survival of GAS has been poorly studied because removal of CFH from plasma or blood causes vigorous complement activation, and specific inhibitors of the interaction have not been available. In this study, we found that activation of human complement by different GAS strains (n = 38) correlated negatively with binding of CFH via its domains 5-7. The importance of acquisition of host CFH for survival of GAS in vitro was studied next by blocking the binding with recombinant CFH5-7 lacking the regulatory domains 1-4. Using this fragment in full human blood resulted in death or radically reduced multiplication of all of the studied CFH-binding GAS strains. To study the importance of CFH binding in vivo (i.e., for pathogenesis of streptococcal infections), we used our recent finding that GAS binding to CFH is diminished in vitro by polymorphism 402H, which is also associated with age-related macular degeneration. We showed that allele 402H is suggested to be associated with protection from erysipelas (n = 278) and streptococcal tonsillitis (n = 209) compared with controls (n = 455) (p < 0.05). Taken together, the bacterial in vitro survival data and human genetic association revealed that binding of CFH is important for pathogenesis of GAS infections and suggested that inhibition of CFH binding can be a novel therapeutic approach in GAS infections.     

Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline)

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as β-sheet.     

Oxidative stress in very low birth weight infants as measured by urinary 8-OHdG

Very low birth weight (VLBW) infants can be subjected to oxidative stress in the course of intensive care. We measured 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of oxidative stress, and estimated the degree of oxidative stress in such infants. We also examined if the administered oxygen was related to oxidative stress. Urine samples of 50 Japanese VLBW infants [birth weights: 956.3+/-277.6g, and gestational ages: 28.0+/-2.6 weeks (mean +/- SD)] were collected on various postnatal days and 8-OHdG levels were determined using an ELISA kit. Sixteen term infants served as normal controls. As body weights at sampling increased, the average levels of urinary 8-OHdG decreased. 8-Hydroxydeoxyguanosine levels were: infants under 1000g, 29.5+/-16.4 micromol/mol creatinine (n = 24); 1000-1500g, 23.8+/-14.9 (n = 12); over 1500g, 16.1+/-8.5 (n = 14); and control, 10.9+/-7.2 (n = 16). Significant differences were found between <1000g group and > or = 1500g group (p = 0.0030), <1000g group and control (p < 0.0001), and 1000-1500g group and control (p = 0.0108). Also as postconceptional age at sampling increased, the average levels of 8-OHdG decreased. 8-Hydroxydeoxyguanosine levels were: infants before 252 days (36 weeks) of postconception: 27.4+/-15.5 micromol/mol creatinine (n = 34); after 252 days, 18.2+/-12.5 (n = 16). Differences between <252 days group and control (p < 0.0001), and <252 days group and > or = 252 days groups (p = 0.0253) were statistically significant. Among the three groups based on ambient oxygen concentration (21%, 22-29%, and > or = 30%) there was no significant difference (p = 0.417). The more premature the infants were, the more intense was the oxidative stress, hence, it is the prematurity rather than the administered oxygen which causes oxidative stress in VLBW infants. Drury et al. ["Urinary 8-hydroxydeoxyguanosine in infants and children" Free Radic. Res. 28 (1998) 423-4281 measured urinary 8-OHdG of 28 infants (24-40 weeks gestation) and found no gestation or birthweight related differences. This discrepancy seemed to be because of difference in birth weights and sampling period of the subjects.     

Carbanilation of cereal beta-glucans for molecular weight determination and conformational studies

Cereal beta-glucans can form aggregates in aqueous solution. The presence of aggregates in cereal beta-glucan solutions led to inaccurate determination of molecular weights and it was believed that intermolecular hydrogen bonding caused the aggregation. To eliminate aggregates, a carbanilation method for molecular weight determination of cereal beta-glucans was developed. Wheat beta-glucan samples were selected for investigation. The carbanilation method can prevent intermolecular hydrogen bonding by blocking hydroxyl groups with phenyl carbamate groups. The carbanilates of cereal beta-glucans were prepared by the reaction of cereal beta-glucans with phenylisocyanate catalyzed by DMSO and pyridine. To avoid degradation during the carbanilation reaction, relatively mild conditions were used, which led to incomplete substitution (DS: approximately 2). However, after the carbanilation reaction, the carbanilates dissolved completely in 1,4-dioxane solution without any detectable aggregates, which allowed accurate molecular weight determination. The degree of substitution (DS) of carbanilates was determined by both a nitrogen content method and an FT-IR method. The FT-IR method proved to be the more effective for DS estimation. Using this method, the converted molecular weights of cereal beta-glucans were in good agreement with the results measured in 0.5M NaOH solution, which previously was shown to be a good solvent for cereal beta-glucans. After the carbanilation reaction, conformational changes of carbanilates were studied by static and dynamic light scattering techniques. The fractal dimension (d(f)=2.27) and the structure sensitive parameters (rho >2) suggested a porous globular structure for partially carbanilated beta-glucans.     

Binding of complement inhibitor C4b-binding protein contributes to serum resistance of Porphyromonas gingivalis

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the alpha-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.     

A new low molecular-weight protein effector of human complement from the venom of the Central Asian cobra Naja naja oxiana

Six protein effectors of human complement were isolated from the whole venom of the Central Asian cobra Naja naja oxiana. Three of them have acidic properties and molecular weights of 61 000, 5000 and 3000, and the rest are basic proteins with molecular weights of 54 000, 9000 and 7000. Two low molecular weight basic proteins CFB-II and CFB-III are isolated in as high amounts as 115 and 85 mg per g of dry venom. All the effectors inhibit the classical pathway of complement activation and, with the exception of CFB-II and CFB-III, the alternative pathway. The latter, on the contrary, enhances the alternative pathway of activation. N-Terminal sequence determination for CFB-III demonstrated its identity to the earlier characterized cytotoxin II. The action of CFB-III on the classical pathway of complement activation consists in the component C4 inactivation. A mechanism for the CFB-III activation of the alternative pathway is proposed implying the CFB-III induced transformation of the C3 component into a C3b-like one producing a soluble C3 convertase.     

Calcium-Dependent Calmodulin Binding to Chromaffin Granule Membranes: Presence of a 65-Kilodalton Calmodulin-Binding Protein

The presence of calmodulin-binding sites on chromaffin granule membranes has been investigated. Saturable, high-affinity 125I-calmodulin-binding sites (KD = 9.8 nM; Bmax = 25 pmol/mg protein) were observed in the presence of 10(-4) M free calcium. A second, nonsaturable, calmodulin-binding activity could also be detected at 10(-7) M free calcium. No binding occurred at lower calcium levels. When chromaffin granule membranes were delipidated by solvent extraction, calmodulin binding was observed at 10(-4) M free calcium. However no binding was detected at lower calcium concentrations. Thus it appears that a calcium concentration of 10(-7) M promotes the binding of calmodulin to some solvent-soluble components of the chromaffin granule membrane. Calmodulin-binding proteins associated with the granule membrane identified by photoaffinity cross-linking. A calmodulin-binding protein complex, of molecular weight 82K, was formed in the presence of 10(-4) M free calcium. This cross-linked product was specific because it was not detected either in the absence of calcium, in the presence of nonlabeled calmodulin, or in the absence of cross-linker activation. When solvent-treated membranes were used, a second, specific, calmodulin-binding protein complex (70K) was formed. Since the apparent molecular weight of calmodulin in our electrophoresis system was 17K, these experiments suggested the presence of two calmodulin-binding proteins, of molecular weights 65K and 53K, in the chromaffin granule membrane. This result was confirmed by the use of calmodulin-affinity chromatography. When detergent-solubilized membranes were applied on the column in the presence of calcium, two polypeptides of apparent molecular weights of 65K and 53K were specifically eluted by EGTA buffers. Since detergent treatments or solvent extractions are necessary to detect the 53K calmodulin-binding protein, it is concluded that only the 65K calmodulin-binding polypeptide may play a role in the interaction between calmodulin and secretory granules in chromaffin cells.     

Viral complement regulators: the expert mimicking swindlers

The complement system is a principal bastion of innate immunity designed to combat a myriad of existing as well as newly emerging pathogens. Since viruses are obligatory intracellular parasites, they are continuously exposed to host complement assault and, therefore, have imbibed various strategies to subvert it. One of them is molecular mimicry of the host complement regulators. Large DNA viruses such as pox and herpesviruses encode proteins that are structurally and functionally similar to human regulators of complement activation (RCA), a family of proteins that regulate complement. In this review, we have presented the structural and functional aspects of virally encoded RCA homologs (vRCA), in particular two highly studied vRCAs, vaccinia virus complement control protein (VCP) and Kaposi's sarcoma-associated herpesvirus complement regulator (kaposica). Importance of these evasion molecules in viral pathogenesis and their role beyond complement regulation are also discussed.     

Proteins induced by various types of double-stranded RNA and interferon type I in human fibroblasts. Correlation with antiviral activity of the substances]

Protein induction by new antiviral preparations of dsRNAs (larifan, ridostin, rifastin and poly(A).poly(U)) and recombinant beta-interferon in human fibroblasts (M19) was studied. The common gene products: 88, 80, 68, 58, 56, 52, 50 and 26 kD were detected in the spectra of the induced cytoplasmic polypeptides. At the same time the sets of the induced proteins had individual distinctions in various preparations. Induction of the 56-kD protein was more essential in the action of dsRNAs than that of interferon. The antiviral activity of dsRNAs and interferon preparations correlated with a relative increase in the synthesis of proteins with molecular weights of 88, 80 and 58 kD. The study results are in agreement with the fact that the dsRNAs have interferon-independent pathways of antiviral action with participation of 56- and 58-kD protein genes.     

Staphylococcus epidermidis polysaccharide intercellular adhesin activates complement

Staphylococcus epidermidis is a frequent cause of nosocomial infections. The central virulence factor of S. epidermidis is biofilm formation. Polysaccharide intercellular adhesin (PIA) constitutes the major biofilm matrix-component. PIA and biofilm have been implicated in S. epidermidis evasion of host immune defence. We examined the effects of S. epidermidis PIA on the inflammatory response with focus on complement activation. We used a human whole-blood ex vivo model of infection and compared the effects of a PIA-positive S. epidermidis strain (SE1457) and its PIA-negative isogenic mutant (M10). The independent effect of purified PIA on complement activation was investigated. In glucose-rich media, the mutant formed a proteinacious DNA-rich biofilm, whereas SE1457 formed a thick PIA-biofilm. In biofilm growth, SE1457 induced a stronger activation of the complement system compared with M10. We verified that purified PIA was independently responsible for a strong activation of the complement system. In contrast, M10 induced higher granulocyte activation by expression of CD11b and higher secretion of cytokines. We conclude that PIA has potent pro-inflammatory properties by activating the complement system. However, in a complex balance of the immune response, the decreased activation of granulocytes and cytokines by a PIA biofilm may limit host eradication of S. epidermidis.     

Preparation and characterization of two isozymes of choline acetyltransferase from squid head ganglia. II. Self-association, molecular weight determinations, and studies with inactivating antisera.

The two isozymes of choline acetyltransferase (Acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) from head ganglia of Loligo pealei have been examined by polyacrylamide gel electrophoresis, gel chromatography, and equilibrium sedimentation in the ultracentrifuge. Inactivating antisera, prepared to both native and dithiothreitol-treated isozymes 1 and 2 of squid choline acetyltransferase, were used to demonstrate the immunologic identity of isozymes 1 and 2. Each isozyme appeared to contain two non-identical catalytically active subunits, with molecular weights of approx. 37 000 and 56 000. A staining method was developed to visualize choline acetyltransferase activity in acrylamide gels. The method is based on the formation of a precipitate of manganese ferrocyanide at sites where free coenzyme A is released. By this method, and by analysis of gel slices, it was found that each of the isozymes can form aggregates of several different sizes. The formation of immune precipitates with the aggregates showed the identity of the multiple bands of enzyme protein resolved on disc gel electrophoresis. Isozyme 1 was most active as a small aggregate, whereas isozyme 2 was most active as a large aggregate. Both chromatography on Sephadex G-200 and isoelectric focusing yielded a number of active species with molecular weights ranging from 35 000 to 300 000. In addition, we demonstrated the dissociation of enzyme protein in the presence of 1.0 - 10(-2) M dithiothreitol, the formation of multiple precipitin bands by aged enzyme, and the identity of the different isoelectric fractions of each of the isozymes.     

Mapping proteins from various structural regions of human kidney by two-dimensional electrophoresis]

The protein composition of various structural divisions of human kidney was studied using two-dimensional electrophoresis. Two-dimensional electrophoregrams of the cortical substance of human kidney revealed 165 polypeptide fractions within the pH range of 4.5-7.5, having molecular masses of 10 to 330 kDa. Electrophoresis of glomerular proteins gave 155 fractions with M(r) = 15-300 kDa, whereas fractionation of glomerular basement membrane proteins gave 40 fractions with M(r) = 30-330 kDa within the same range of pH. The M(r) values for all fractions and the relative electrophoretic mobility in the forward direction were determined. A comparative analysis of the electrophoregrams was conducted. The data obtained were used to construct two-dimensional maps of the cortical substance and glomerular proteins of human kidney.     

Cell surface expression of the C3b/C4b receptor (CR1) protects Chinese hamster ovary cells from lysis by human complement.

The C3b/C4b receptor, also known as complement receptor type 1 (CR1, CD35), is a single chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. A series of recombinant proteins derived from CR1 has been prepared and assessed for the capacity to inhibit complement lysis of the host Chinese hamster ovary (CHO) cells. The full-length recombinant CR1 inhibited human complement-mediated CHO cell lysis, and the efficiency of inhibition was directly proportional to the number of receptors/cell. The SCR 15-18 of CR1, but not SCR 15-16, inhibited complement lysis of the host CHO cell, bound monomeric C3b (Kd,app = 6.5 x 10(-7) M), and dimeric C3b (Kd = 1.8 x 10(-8) M), and served as a cofactor in the proteolysis of C3b by factor I, confirming and extending the observations of Fearon and colleagues (Kalli, K. R., Hsu, P., Bartow, T. J., Ahearn, J. M., Matsumoto, A. K., Klickstein, L. B., and Fearon, D. T. (1991) J. Exp. Med. 174, 1451-1460). The SCR 1-4 of CR1, but not SCR 1-2, also inhibited complement lysis of the host CHO cell, indicating that more than two SCR are necessary and that four SCR are sufficient for optimal C4b binding to CR1. Thus, the structural requirements for C4b binding are analogous to those for C3b binding, namely, four SCR of CR1 form the binding sites for each of these proteins. CR1 has long been recognized to regulate extrinsic complement activation, that is, to bind to and promote the degradation of fluid phase C3b and of C3b attached to immune complex. These results demonstrate that CR1 is also an intrinsic regulator of complement activation in that, under appropriate conditions, CR1 inhibits complement-mediated lysis of the cell on which it is expressed.     

Characteristics of the aggregative state of the substrate in the reaction of 5-lipoxygenase oxidation of linoleic acid

5-lipoxygenase (EC 1.13.11.12) oxidizes polyunsaturated fatty acids by molecular oxygen. The enzyme acts in close contact with the cell membranes, which main components are ionic and non-ionic lipids. In order to investigate the kinetic parameters of 5-lipoxygenase reaction in vitro, extremely hydrophobic fatty acid substrate (linoleic acid) should be solubilized in the reaction mixture. We used Lubrol PX ("Sigma" Chem. Co), as a non-ionic detergent consisted of oligoethylene glycol and fatty alcohol. Linoleic acid and Lubrol PX formed mixed micelles thus solubilizing the fatty acid substrate in a buffer with appropriate pH. We have studied the sizes and shapes of mixed micelles Lubrol PX/linoleic acid (aggregates type 1) and Lubrol PX/linoleic acid/SDS (aggregates type 2; SDS was an effective activator of potato tuber 5-lipoxygenase) by means of gel-filtration and laser light scattering techniques. The parameters under investigation were molecular weights, Stocks radii and shapes of the mixed micelles. The average molecular weights and Stocks radii of the mixed micelles type 1 determined by mean of gel-filtration on Sephadex G-200 were 95,142 +/- 5184 Da and 3.45 +/- 0.11 nm, respectively. The same parameters for the mixed micelles type 2 were 73,694 +/- 893 Da and 3.02 +/- 0.02 nm, respectively. The strong similarity in physicochemical parameters for both types of mixed micelles indicated that SDS did not influence the size and shape of mixed micelles of Lubrol PX and linoleic acid. The activatory action of SDS on potato tuber lipoxygenase may be a result of electrostatic effect or direct participation of SDS in enzymatic catalysis. The laser light scattering technique allowed to determine two main fraction of particles in type 1 system with hydrodynamic diameters 2.6 and 5.7 nm and relative contribution to light scattering 13 and 87%, respectively. The particles with d = 5.7 nm were interpreted as the mixed micelles. The particles with d = 2.6 nm were interpreted as isolated molecules of Lubrol PX, linoleic acid and (or) their premicellar aggregates. The data obtained are to be used in creation of reliable physical and mathematical models of 5-lipoxygenase.     

Complement System Activation by Amyloid Aggregates of Aβ(1-40) and Aβ(1-42) Peptides: Facts and Hypotheses

Biophysics - Abstract—Here, we consider the problem of the activation of the complement system by amyloid aggregates, in particular, amyloid fibrils of the Aβ(1-40) and Aβ(1-42)...     

Two calcium regulation systems in squid (Ommastrephes sloani pacificus) muscle. Preparation of calcium-sensitive myosin and troponin-tropomyosin.

The Ca-regulatory system in squid mantle muscle was studied. The findings were as follows. (a) Squid mantle myosin B (squid myosin B) was Ca-sensitive, and its Ca-sensitivity was unaffected by addition of a large amount of rabbit skeletal myosin (skeletal myosin) or rabbit skeletal F-actin (skeletal F-actin). (b) Squid myosin was prepared from the mantle muscle. It showed a heavy chain component and two light chain components in the SDS-gel electrophoretic pattern: the molecular weights of the latter two were 17,000 and 15,000. Actomyosin reconstituted from squid myosin and skeletal (or squid) actin showed Ca-sensitivity in superprecipitation and Mg-ATPase assays. EDTA- treatment had no effect on the Ca-sensitivity of squid myosin. (c) Squid mantle actin (squid actin) was prepared by the method of Spudich and Watt. Hybrid actomyosin reconstituted by using the pure squid actin preparation with skeletal myosin showed no Ca-sensitivity in Mg-ATPase assay, whereas that reconstituted using crude squid actin showed marked Ca-sensitivity. The crude squid actin contained four protein components which were capable of associating with F-actin in 0.1 M KCl, 1 mM MgCl2 and 20 mM Tris-maleate (pH7.5). (d) Native tropomyosin was prepared from squid mantle muscle, and it conferred Ca-sensitivity on skeletal actomyosin as well as on a hybrid actomyosin reconstituted from squid actin and skeletal myosin. (e) Squid native tropomyosin was separated into troponin and tropomyosin fractions by placing it in 0.4 M LiCl at pH 4.7. The troponin fraction was further purified by DEAE-cellulose chromatography. Squid troponin thus obtained was different in mobility from rabbit skeletal or carp dorsal troponin; three bands of squid troponin corresponded to molecular weights of 52,000, 28,000, and 24,000 daltons. It could confer Ca-sensitivity in the presence of tropomyosin on skeletal actomyosin as well as on a hybrid reconstituted from squid actin and skeletal myosin. (f) Squid myosin B, and two hybrid actomyosins were compared as regards Ca and Sr requirements for their Mg-ATPase activities. The myosin-linked regulatory system rather than the thin-filament-linked regulatory system was predominant in squid myosin B. Squid myosin B required higher Ca2+ and Sr2+ concentrations for Mg-ATPase activity; half-maximal activation of Mg-ATPase was obtained at 0.8 micron Ca2+ and 28 micron Sr2+ with skeletal myosin B, and at 2.5 micron Ca2+ and 140 micron Sr2+ with squid myosin B.