Molecular Weight Of Ca Research Articles

Photo-induced synthesis of star poly(DL-Lactide)-templated Au and Ag nanoparticles and evaluation of their catalytic performance

In this report, photochemical synthesis of monodispersed gold (Au) and silver (Ag) nanoparticles (NPs) was reported using cholic acid functionalized star poly(DL-lactide) (CA-(PDLLA)) as a capping and reducing agent. CA-(PDLLA) was synthesized by ring-opening polymerization (ROP) of DL-lactide with a feed ratio of 1:120 (cholic acid: DL-lactide). The molecular weight of CA-(PDLLA) was measured by using the gel permeation chromatography (GPC) technique. The thermal properties of the polymer and its nanocomposite materials were studied by thermogravimetric analysis (TGA). The optical properties of Ag and Au NPs were studied by UV–visible absorption spectroscopy. Morphological studies and size of the polymer-nanocomposites were assessed by using field emission scanning electron microscopy (FE-SEM) and high-resolution transmission electron microscopy (HR-TEM) techniques. The percentage composition of Au and Ag present in the polymer-nanocomposites was calculated using energy-dispersive X-ray spectroscopic (EDX) analysis. Finally, the synthesized polymer-metal nanocomposites were used as heterogeneous catalysts for the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) as a model reaction and the apparent rate constant (kapp) values of CA-(PDLLA)-AuNPs and CA-(PDLLA)-AgNPs were found to be 5.76 × 10−4 s−1 and 19.7 × 10−4 s−1, respectively.

Development of a novel agar extraction method using calcium hydroxide and carbon dioxide

A novel agar extraction method was explored as an alternative to the traditional agar extraction method. This method used low concentration of Ca(OH)2 solution to desulfurize and extract Gracilaria agar simultaneously under high temperature homogeneous condition, and used CO2 to neutralize Ca(OH)2. The quality of agar extracted by the Ca(OH)2 (CA) was determined and compared with agar obtained by traditional NaOH extraction methods (NA). Results showed that the Ca(OH)2 extraction allowed increasing the gel strength (up to 1074 g/cm2), while keeping constant agar yield (13.1 %) with respect to the traditional NaOH extraction methods (839 g/cm2, 12.9 %). Interestingly, the presence of sulfate content and 3,6-AG content of CA showed no significant difference with NA, which indicated that the two methods had the same desulfation effect and structural change. On the other hand, the molecular weight of CA (2.67 × 105) was higher than that of NA (2.02 × 105), indicating that the Ca(OH)2 method is a more gentle extraction method with less degradation of agar than traditional methods. In addition, the Ca(OH)2 method is simple, the production cycle is short, the amount of alkali and water is less, and the amount of wastewater is significantly reduced, which showed potential to produce more sustainable agar for food-related applications.

Binding of S100A6 to actin and the actin\u2013tropomyosin complex

S100A6 is a low molecular weight Ca2+-binding protein belonging to the S100 family. Many reports indicate that in the cell S100A6 has an influence on the organization of actin filaments, but so far no direct interaction between S100A6 and actin has been shown. In the present study we investigated binding of S100A6 to actin and the actin–tropomyosin complex. The analyses were performed on G- and F-actin and two tropomyosin isoforms—Tpm1.6 and Tpm1.8. Using purified proteins and a variety of biochemical approaches we have shown that, in a Ca2+-bound form, S100A6 directly interacts with G- and F-actin and with tropomyosin, preferentially with isoform Tpm1.8. S100A6 and tropomyosin bind to the same population of filaments and the presence of tropomyosin on the microfilament facilitates the binding of S100A6. By applying proximity ligation assay we have found that in NIH3T3 fibroblasts S100A6 forms complexes both with actin and with tropomyosin. These results indicate that S100A6, through direct interactions with actin and tropomyosin, might regulate the organization and functional properties of microfilaments.

A new era of secreted phospholipase A2

A new era of secreted phospholipase A₂

Size-tuneable Nanometric MRI Contrast Agents for the Imaging of Molecular Weight Dependent Transport Processes

The purpose of the current study was to evaluate size-tuneable polymeric glycol-chitosan (GC)-DTPAGadolinium (Gd) conjugates as MRI contrast agents that can be used as a platform for imaging of molecular weight (MW) dependent transport processes. GC-DTPA-Gd conjugates of precisely controlled MWs were synthesised and evaluated in mice against Gd-DTPA using time series of high-resolution MRI images of trunk, head, and xenograft flank tumours. GC-DTPA modification ratio was one DTPA per 3.9-5.13 of GC monomers. GC-DTAP-Gd provided overall superior contrast compared to Gd-DTPA with the duration of the enhancement depending on MW (≥1hr for 40kD). The kidneys showed early enhancement also, particularly in the renal pelvis, suggesting renal elimination. Imaging of the head with GC-DTPA-Gd allowed detailed anatomical identification of specific blood vessels in particular with the high MW CA agent. Sequential, high-resolution, isotropic imaging of established A431 xenograft flank tumours with DTPA-Gd and GC-DTPA-Gd demonstrated that the initial distribution of the contrast agents was well correlated with blood vessels and supply. In contrast, subsequent tissue transport was primarily by diffusion and limited by CA molecular weight. The data also highlight the role of heterogeneity in CA distribution which was more prominent for the high MW agent. Precise control of glycol chitosan (GC) polymer chemistry facilitates synthesis of a family of Gd-based MRI contrast agents of tuneable MW but otherwise identical physicochemical properties. Such agents allow isotropic high-resolution threedimensional imaging of MW dependent transport processes relevant to the clinical and pre-clinical prediction of drug transport processes.

Reduced expression of psoriasin in human airway cystic fibrosis epithelia

Psoriasin is a low molecular weight Ca2+-binding protein with known antimicrobial activity. Since human airway epithelial cells produce a number of powerful antimicrobial agents as part of their host defence, we investigated whether psoriasin was expressed in human bronchial epithelial cell lines. Expression was investigated in 16HBE14o- cells, derived from a normal individual, and compared to CFBE41o- cells, derived from a cystic fibrosis patient. We also examined psoriasin expression following treatment with factors pertinent to the CF lung-oxidant stress and exposure to pro-inflammatory cytokines. CFBE41o- cells demonstrated much reduced psoriasin levels compared to the 16HBE14o- cells. Increased psoriasin expression was seen following treatment with IL-22 and a cytomix of the pro-inflammatory cytokines IL-1β, TNF-α and IFN-γ; however, the oxidant stressor tert-butyl hydroperoxide had no apparent effect. Over-expression of human psoriasin into both cell lines resulted in increased internalization of Pseudomonas aeruginosa. In conclusion, expression of psoriasin – which has known anti-microbial activity in other systems – appears to be reduced in CFBE410- compared to 16HBE14o- cells, and its expression modified by exposure to pro-inflammatory cytokines.

Long‐Chain Polyacetals From Plant Oils

Plant oil-derived α,ω-diacetals are polycondensated to the novel polyacetals [OCH(2) O(CH(2))(y)](n) (y = 19 and 23) with molecular weight of ca. M(n) = 2 × 10(4) g mol(-1). The long methylene sequences provide substantial melt and crystallization temperatures (T(m) = 88 °C and T(c) = 68 °C for y = 23), and rates of hydrolytic degradation are dramatically lower for the long-chain polyacetals versus a shorter chain analogue (y = 12) studied for comparison.

Purification and characterization of a laccase from the basidiomycete Funalia trogii (Berk.) isolated in Tanzania

A lignolytic basidiomycete fungus, Funalia trogii (Berk.), was isolated from decayed wood in coastal Tanzania and cultivated in submerged culture. Initially screened crude enzyme filtrate showed complete rhemazol brilliant blue - R (RBBR) decolorization 2,2’-azino-bis (3-ethylbenzthiazoline)-6-sulfonate and guaiacol oxidation after 7 days of incubation. The fungal filtrate had maximum laccase activity of 593 U/ml after 15 days of incubation. A laccase was purified by anion exchange and size exclusion chromatography to good purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). The isolated main component had a molecular weight of ca 58 kDa as determined by MS and an isoelectric point (pI) of 3.8. The optimal pH and temperature range for the purified laccase were 4.0 - 5.0 and 50 - 70 oC, respectively, using 2, 6-dimethoxyphenol as a substrate. Key words: Funalia trogii, laccase, anion exchange chromatography, size exclusion chromatography, electrophoresis, isoelectric focusing.

Characterization of Ca 2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca 2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca 2+ or phosphocholine were injected. Binding of Ca 2+ induced a 10-fold higher signal than expected from the molecular weight of Ca 2+. It was interpreted to result from the conformational change that occurs on binding of Ca 2+. Two sites with different characteristics were distinguished: a high-affinity site with K D = 0.03 mM and a low-affinity site with K D = 5.45 mM. The pH dependencies of the two Ca 2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K D = 5 μM at 10 mM Ca 2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca 2+ is an efficient regulator under physiological conditions.

Hypocalcemia in Infants and Children

Hypocalcemia in Infants and Children

Purification of bovine pancreatic phospholipase A 2 by an affinity ultrafiltration technique

Phospholipase A 2 (PLA 2; EC 3.1.1.4) is a lipolytic enzyme that hydrolyze phosphoglycerides at the acyl ester bond at the sn-2-position into the corresponding lysophospholipid and fatty acid. Mammalian PLA 2 enzymes are subdivided into three groups: low molecular weight Ca 2+-dependent enzymes (sPLA 2), high molecular weight Ca 2+-dependent enzymes (cPLA 2) and the Ca 2+-independent isoforms (iPLA 2). The object of this study is to discover the synthesis of an affinity complex which could be used in an ultrafiltration system for the biospecific affinity isolation of phospholipase A 2 from mixtures containing other proteins. For this purpose, first, the development of a macromolecular water soluble resin was attempted, based on chitosan bearing phosphatidylethanolamine, a phospholipase A 2 substrate. Then, together with ultrafiltration techniques, the macroligand was employed to purify phospholipase A 2 from bovine pancreas. The enzyme was purified 79-fold and had a high activity yield 76.3%. The enzymatic properties obtained and showed nearly similarity in terms of optimum temperature and pH, molecular weight, Ca 2+-dependence with bovine and other mammalian pancreatic PLA 2's.

Calexcitin‐like immunoreactivity in the pond snail Lymnaea stagnalis

AbstractCalexcitin (CE) is a low molecular weight Ca2+‐ and guanosine triphosphate‐ binding protein, which is phosphorylated during associative learning in both vertebrates and invertebrates. The purpose of this study was to determine the presence of CE in the central nervous system (CNS) of the pond snail Lymnaea stagnalis, which can acquire classical and operant conditioning. Immunoblotting of CE showed that the anti‐CE antibody prepared from squid can detect Lymnaea CE. In the cerebral ganglia, CE‐like immunoreactivity was exhibited in two pairs of cell clusters that receive taste signals from the superior or median lip nerves. In both pedal ganglia, CE‐like immunoreactivity was detected in 1‐4 cell of the PeA clusters, which are involved in the withdrawal response. Our results therefore showed that CE is involved in the feeding and withdrawal neural networks, suggesting that CE may function in associative learning of feeding and withdrawal behavior in L. stagnalis.

Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana.

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.

Expression of a novel high molecular-weight myosin light chain kinase in endothelium.

Myosin light chain phosphorylation results in cellular contraction and is a critical component of agonist-mediated endothelial cell (EC) junctional gap formation and permeability. We have shown that this reaction is catalyzed by a novel high molecular-weight Ca2+/calmodulin-dependent nonmuscle myosin light chain kinase (MLCK) isoform recently cloned in human endothelium (Am. J. Respir. Cell Mol. Biol., 1997;16:489-494). To characterize EC MLCK expression further in cultured and adult tissues, we employed immunoblotting techniques and reverse transcriptase-polymerase chain reaction to demonstrate that freshly isolated and cultured human macro- and microvascular EC express only the EC MLCK isoform (214 kD), which is distinct from smooth-muscle MLCK isoforms (130 to 150 kD). Immunocytochemical studies demonstrated the presence of the high molecular-weight MLCK isoform in adult human cardiac endothelium using anti-MLCK antibodies, which preferentially recognize the high molecular-weight EC MLCK isoform. Monitoring of MLCK expression in different cell types with antibodies generated against a unique human EC MLCK N-terminal sequence revealed a high level of expression of the 214-kD enzyme in endothelium, minimal level of expression in smooth muscle, and no expression in skeletal muscle. These data suggest that the novel 214-kD kinase, the only MLCK isoform found in endothelium, may be preferentially expressed in this nonmuscle tissue.

Equilibrium concentration profiles of physically end tethered polystyrene molecules at the air-polymer interface

Deuteriopolystyrene of molecular weight ca 32 000 g mol −1 has been functionalized at one end with a fluorocarbon. On mixing this polymer with an unfunctionalized hydrogenous polystyrene a surface excess layer of the deuteriopolymer forms on annealing at 423 K. The surface excess as a function of deuteriopolymer content has been obtained using nuclear reaction analysis and neutron reflectometry, and the shape of the profile from neutron reflectometry alone. From previous secondary ion mass spectral data and by comparison with predictions of surface enrichment theory, it is concluded that the deuteriopolystyrene is tethered by the fluorine label to the air surface. The parameters of this polymer ‘brush’ obtained experimentally are the surface volume fraction of labelled polymer, the brush height, the surface excess and the layer thickness. The surface excess and surface volume fraction of the deuteriopolymer and the adsorption isotherm (surface excess at a function of equilibrium bulk volume fraction) have been compared to the predictions of a self-consistent field theory. Best agreement is obtained with a sticking energy of 1.6 k B T; however, there appear to be some disparities when compared to the limiting ‘dry’ brush predictions. There is evidence from neutron reflectometry of brush formation before annealing of the polymer films.

Cloning and characterization of novel rat and mouse low molecular weight Ca(2+)-dependent phospholipase A2s containing 16 cysteines.

A novel rat 4.4-kilobase (kb) cDNA encoding a low molecular weight Ca(2+)-dependent phospholipase A2 (PLA2) and its murine homologue were cloned. The rat and mouse cDNA predict a mature protein of 130 amino acids (M(r) = 14, 763) preceded by a 28-amino acid prepro-peptide. The deduced amino acid sequences encode a protein containing 16 cysteines which distinguishes them from both mammalian group I and II PLA2s and the recently described group of mammalian PLA2s containing 12 cysteines. A rat RNA blot hybridized with the rat cDNA exhibited an abundant 2.3-kb and a less abundant 5-kb transcript in testis. When the rat cDNA was expressed using an Epstein-Barr virus-based vector in human 293s cells, PLA2 activity accumulated in the culture medium. Conditioned medium optimally hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli at neutral to alkaline pH with 1-7 mM Ca2+. In assays with individual substrates, L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol was hydrolyzed more efficiently than L-alpha-1-palmitoyl-2-oleoyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, or L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine.

Cloning and recombinant expression of a novel human low molecular weight Ca(2+)-dependent phospholipase A2.

Extensive biochemical studies of phospholipase A2s (PLA2s) over the last two decades indicate that there are likely to be several distinct PLA2 genes in mammals. Here we report the cloning of a 1-kilobase pair cDNA encoding a novel human low molecular weight PLA2. The cDNA appears to encode a 118-amino acid mature peptide (M(r) = 13,592) preceded by a 20-residue prepeptide. The deduced amino acid sequence encodes a protein that lacks one of the seven disulfide bridges found in similar PLA2s and, therefore, represents a class of enzymes distinct from the mammalian group I and group II enzymes. An RNA blot hybridized with the cDNA exhibited a putative 1.2-kilobase pair transcript in heart and, less abundantly, in lung, as well as multiple putative transcripts in placenta. When the cDNA was expressed using an Epstein-Barr virus-based vector in human 293s cells, PLA2 activity accumulated in the culture medium. Conditioned medium optimally hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli at neutral to alkaline pH with 10 mM or greater Ca2+. In assays done with individual substrates, L-alpha-1-palmitoyl-2-oleoyl phosphatidylcholine was more efficiently hydrolyzed than L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine, or L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol.

Ryanodine-affinity chromatography purifies 106 kD Ca 2+ release channels from skeletal and cardiac sarcoplasmic reticulum

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca 2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the ‘physiological’ Ca 2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca 2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca 2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca 2+ release channels which are a minor component (0.1–0.3%) of SR proteins.

Purification and characterization of a novel 12,000-Da calcium binding protein from smooth muscle

A new low molecular weight calcium binding protein, designated 12-kDa CaBP, has been isolated from chicken gizzard using a phenyl-Sepharose affinity column followed by ion-exchange and gel filtration chromatographies. The isolated protein was homogeneous and has a molecular weight of 12,000 based on sodium dodecyl sulfate-gel electrophoresis. The amino acid composition of this protein is similar to but distinct from other known low molecular weight Ca 2+ binding proteins. Ca 2+ binding assays using Arsenazo III (Sigma) indicated that the protein binds 1 mol of Ca 2+/mol of protein. The 12-kDa CaBP underwent a conformational change upon binding Ca 2+, as revealed by uv difference spectroscopy and circular dichroism studies in the aromatic and far-ultraviolet range. Addition of Ca 2+ to the 12-kDa CaBP labeled with 2- p-toluidinylnaphthalene-6-sulfonate (TNS) resulted in a sevenfold increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 463 to 445 nm. Hence, the probe in the presence of Ca 2+ moves to a more nonpolar microenvironment. Like calmodulin and other related Ca 2+ binding proteins, this protein also exposes a hydrophobic site upon binding calcium. Fluorescence titration with Ca 2+ using TNS-labeled protein revealed the presence of a single high affinity calcium binding site ( k d ≈ 1 × 10 −6 M).

IgE‐Binding Proteins in Almonds (Prunus amygdalus); Identification by Immunoblotting with Sera from Almond‐Allergic Adults

ABSTRACTThe allergenicity of three raw almond varieties (Nonpareil, Mission and Carmel) and three types of processed almonds (blanched, blanched‐roasted, and almond butter) were examined and compared by immunoblotting using sera eight almond‐allergic individuals. Serum from the 8 individuals exhibited unique IgE‐binding patterns with most of the binding activity occurring with proteins of molecular weights between 30,000 and 70,000 daltons. Two major allergens were identified with molecular weights of ca 70,000 and 45,000–50,000 daltons, with each of these proteins binding to sera from 4 of the 8 allergic individuals. The immunoreactivity of the larger one was reduced by heat processing, while the smaller one was not affected.