Effect of vacuum frying and atmospheric frying on the quality and protein oxidation of squid (Loligo chinensis).

Squid are commercial marine species that have high nutritional value. This study aimed to compare the influences of vacuum frying and atmospheric frying on the physicochemical properties and protein oxidation of three main parts (ring, tentacle, and fin) of the squid Loligo chinensis. The results showed that the vacuum-fried (VF) group had lower moisture and total fat contents and looser microstructures than the atmospheric-fried (AF) group. The amino acid contents and molecular weight revealed that the proteins were well preserved during vacuum frying. Carbonyl content in the VF ring, tentacle, and fin samples increased nearly 2.53-, 1.54-, and 2.56-fold, respectively, compared to that in the corresponding fresh group, but these increases were lower than those of the corresponding AF group. In addition, the secondary structures of proteins revealed a slight decrease in the α-helix and ß-turn contents and a significant increase in the ß-sheet content during vacuum frying. Therefore, vacuum frying can be used as an efficient processing method to conserve the high nutritive quality of the product. PRACTICAL APPLICATION: As a developing alternative technology to prepare healthier fried products, vacuum frying has been the focus of recent researches. Vacuum frying produced squid products that had lower TBARS values, carbonyl contents, and Schiff base substances compared to atmospheric frying. And the protein secondary structures of the vacuum-fried group retained better. The study proved that vacuum frying could be an effective method with the advantages of high protein stability and product quality.

Halogenated natural products and anthropogenic persistent organic pollutants in chokka squid (Loligo reynaudii) from three sites along the South Atlantic and Indian Ocean coasts of South Africa.

Chokka squid (Loligo reynaudii) from three sites along the South African coast were analyzed for halogenated natural products (HNPs) and anthropogenic persistent organic pollutants (POPs). HNPs were generally more than one order of magnitude more abundant than POPs. The most prevalent pollutant, i.e. the HNP 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1), was detected in all chokka squid samples with mean concentrations of 105, 98 and 45 ng/g lipid mass, respectively, at the Indian Ocean (site A), between both oceans (site B) and the South Atlantic Ocean (site C). In addition, bromine containing polyhalogenated 1'-methyl-1,2'-bipyrroles (PMBPs), 2,4,6-tribromophenol (2,4,6-TBP, up to 28 ng/g lipid mass), polybrominated methoxy diphenyl ethers, MHC-1, TBMP and other HNPs were also detected. Polychlorinated biphenyls (PCBs) were the predominant class of anthropogenic POPs. PCB 153 was the most abundant PCB congener in chokka squid from the Indian Ocean, and PCB 138 in samples from the South Atlantic Ocean and between both oceans.

Disulfide bridge formation to increase thermostability of DFPase enzyme: A computational study.

Organophosphate compounds bioremediation by use of organophosphorus degradation enzymes such as DFPase is a developing interest in industry and medicine. The most important problem with the bio-catalytic enzymes is their instability on high temperatures. This work carried out to find suitable locations for introducing disulfide bridges in DFPase enzyme. We employed some computational approaches to design the disulfide bridges and evaluate their roles in the enzyme structural thermostability. According to the in silico results, mutant 6 (V24C, C76) increased the enzyme thermostability relative to wild-type.

A comprehensive analysis of the computed tautomer fractions of the imidazole ring of histidines in Loligo vulgaris.

A recently introduced electrostatic-based method to determine the pKa values of ionizable residues and fractions of ionized and tautomeric forms of histidine (His) and acid residues in proteins, at a given fixed pH, is applied here to the analysis of a His-rich protein, namely Loligo vulgaris (pdb id 1E1A), a 314-residue all-ß protein. The average tautomeric fractions for the imidazole ring of each of the six histidines in the sequence were computed using an approach that includes, but is not limited to, molecular dynamic simulations coupled with calculations of the ionization states for all 94 ionizable residues of protein 1E1A in water at pH 6.5 and 300 K. The electrostatic-calculated tautomeric fractions of the imidazole ring of His were compared with predictions obtained from an existent NMR-based methodology. Our results indicate that: (i) the averaged electrostatic-based tautomeric predictions for the imidazole ring of all histidines of Loligo vulgaris are dominated by the Nε2-H rather than the Nδ1-H form, although such preferences from the NMR-based methodology are not so well defined; (ii) the computed average absolute difference between the electrostatic- and the NMR-based tautomeric predictions among all six histidines vary among 0% to 17%; (iii) for the His showing the largest fraction of the neutral form (81%), the absolute difference between the NMR- and electrostatic-based computed tautomeric predictions is only 3%; and (iv) the tautomeric predictions for the imidazole ring of His computed with the NMR-based methodology are stable within a certain, well-defined, range of variations of a tautomer-related parameter.

Preparation and characterization of high purity ß-chitin from squid pens (Loligo chenisis).

Squid pens were extracted by a NaOH solution at 80°C for 10h to remove protein and minerals. The as-prepared ß-chitin had a high molecular weight (8.5±0.1×103kDa), a low protein content (0.63±0.02wt.%), and a negligible amount of minerals. This method avoids the conventional method for the removal of minerals from shrimp and crab shells by HCl. The purity of resulting products was measured by NMR and FTIR. Moreover, the morphology and crystallinity of ß-chitin was characterized by SEM and XRD. The ß-chitin with long chains and high purity is suitable for producing high quantity ß-chitosan for various potential applications.

Evaluation of alternative sources of collagen fractions from Loligo vulgaris squid mantle.

Acid-Solubilized Collagen (ASC) and Pepsin-Solubilized Collagen (PSC) were extracted from the mantle of the common European squid, and were comparatively characterized. ASC and PSC were isolated with an extraction yield of 5.1 and 24.2% (on dry weight basis), respectively. SDS-PAGE showed that the ASC was mostly comprised of α1- and α2-chains; while the PSC presented relevant ß- and γ-components. GPC analysis confirmed that both the ASC and the PSC consisted of fractions characterized by different molecular weight. Thermal denaturation behavior of ASC and PSC were followed by calorimetric and rheological analyses; denaturation temperature was estimated to be 22°C for ASC and 21°C for PSC. Amino acid composition and solubility of collagen were also investigated. Finally, the cytotoxicity of the isolated collagen was evaluated in vitro and no cytotoxic activity caused by the collagen extracts was observed. This study demonstrated that squid mantle has potential as an alternative source of collagen-derived materials.

Beta-chitosan extracted from Loligo Japonica for a potential use to inhibit Newcastle disease.

Beta-chitosan has a parallel structure, which differs from alpha-chitosan's antiparallel structure while producing different properties and difficulties. In this paper, we prepared the beta-chitosan through acid and alkali methods and the resultant material was characterized by elemental analysis, FT-IR, HPLC, XRD, NMR and AFS. To increase the solubility and biological activity of the beta-chitosan, we degraded it through microwave-assisted process. After characterization, we determined that the chitosan had not changed its configuration during the reaction with H2O2 under microwave irradiation. The inhibitory activity of the degraded chitosan for Newcastle disease was revealed by a hemagglutination test and RT-PCR. The yield of the beta-chitosan was approximately 30%, and its molecular weight can be degraded to 1000 to 10,000g/mol. Moreover, the degraded ß-chitosan has higher antiviral activity, reducing the hemagglutination titre to zero, compared with alpha-chitosan. Therefore, beta-chitosan has good development prospects during the development of veterinary drugs for Newcastle disease.

Sulfated polysaccharides from Loligo vulgaris skin: potential biological activities and partial purification.

The characteristics, biological properties, and purification of sulfated polysaccharides extracted from squid (Loligo vulgaris) skin were investigated. Their chemical and physical characteristics were determined using X-ray diffraction and infrared spectroscopic analysis. Sulfated polysaccharides from squid skin (SPSS) contained 85.06% sugar, 2.54% protein, 1.87% ash, 8.07% sulfate, and 1.72% uronic acid. The antioxidant properties of SPSS were investigated based on DPPH radical-scavenging capacity (IC50 = 19.42 mg mL(-1)), hydrogen peroxide-scavenging activity (IC50 = 0.91 mg mL(-1)), and ß-carotene bleaching inhibition (IC50 = 2.79 mg mL(-1)) assays. ACE-inhibitory activity of SPSS was also investigated (IC50 = 0.14 mg mL(-1)). Further antimicrobial activity assays indicated that SPSS exhibited marked inhibitory activity against the bacterial and fungal strains tested. Those polysaccharides did not display hemolytic activity towards bovine erythrocytes. Fractionation by DEAE-cellulose column chromatography showed three major absorbance peaks. Results of this study suggest that sulfated polysaccharides from squid skin are attractive sources of polysaccharides and promising candidates for future application as dietary ingredients.

Polycyclic aromatic hydrocarbons in commercial squids from different geographical origins: levels and risks for human consumption.

The concentrations of 18 polycyclic aromatic hydrocarbons (PAHs) were determined in five commercially valuable squid species from different geographical origins (Atlantic, Indic and Pacific Oceans). Out of the 18 quantified PAHs (the 16 PAHs considered by US EPA as priority pollutants, dibenzo(a,l)pyrene and benzo(j)fluoranthene) only dibenz(a,h)anthracene was not detected. The total concentrations of PAHs varied by a factor of more than 100-fold, from 0.22 (Loligo gahi) to 60.9 µg/kg ww (Loligo reynaudii). Intra- and inter-specific variability of PAH levels was statistically assessed. Nine carcinogenic (probable/possible) PAHs accounted for 1% (L. reynaudii) to 26% (Loligo opalescens) of the total PAHs content being the main contributors naphthalene (in Loligo duvaucelii, L. reynaudii and Loligo vulgaris species), chrysene (in L. opalescens) and indeno(1,2,3-cd)pyrene (in L. gahi). PAHs source analysis indicated that four of the five zones of capture of the different squid species are significantly affected by both petrogenic and pyrolytic sources. Assessment of the target carcinogenic risks, established by the US EPA, suggested that L. gahi (Atlantic Ocean) and L. opalescens (from Pacific Ocean) may pose additional risks for consumers, if not eaten in moderation, derived from benzo(a)pyrene ingestion.

Thermal mechanisms of millimeter wave stimulation of excitable cells.

Interactions between millimeter waves (MMWs) and biological systems have received increasing attention due to the growing use of MMW radiation in technologies ranging from experimental medical devices to telecommunications and airport security. Studies have shown that MMW exposure alters cellular function, especially in neurons and muscles. However, the biophysical mechanisms underlying such effects are still poorly understood. Due to the high aqueous absorbance of MMW, thermal mechanisms are likely. However, nonthermal mechanisms based on resonance effects have also been postulated. We studied MMW stimulation in a simplified preparation comprising Xenopus laevis oocytes expressing proteins that underlie membrane excitability. Using electrophysiological recordings simultaneously with 60 GHz stimulation, we observed changes in the kinetics and activity levels of voltage-gated potassium and sodium channels and a sodium-potassium pump that are consistent with a thermal mechanism. Furthermore, we showed that MMW stimulation significantly increased the action potential firing rate in oocytes coexpressing voltage-gated sodium and potassium channels, as predicted by thermal terms in the Hodgkin-Huxley model of neurons. Our results suggest that MMW stimulation produces significant thermally mediated effects on excitable cells via basic thermodynamic mechanisms that must be taken into account in the study and use of MMW radiation in biological systems.

Hydrolase stabilization via entanglement in poly(propylene sulfide) nanoparticles: stability towards reactive oxygen species.

In the advancement of green syntheses and sustainable reactions, enzymatic biocatalysis offers extremely high reaction rates and selectivity that goes far beyond the reach of chemical catalysts; however, these enzymes suffer from typical environmental constraints, e.g. operational temperature, pH and tolerance to oxidative environments. A common hydrolase enzyme, diisopropylfluorophosphatase (DFPase, EC 3.1.8.2), has demonstrated a pronounced efficacy for the hydrolysis of a variety of substrates for potential toxin remediation, but suffers from the aforementioned limitations. As a means to enhance DFPase's stability in oxidative environments, enzymatic covalent immobilization within the polymeric matrix of poly(propylene sulfide) (PPS) nanoparticles was performed. By modifying the enzyme's exposed lysine residues via thiolation, DFPase is utilized as a comonomer/crosslinker in a mild emulsion polymerization. The resultant polymeric polysulfide shell acts as a 'sacrificial barrier' by first oxidizing to polysulfoxides and polysulfones, rendering DFPase in an active state. DFPase-PPS nanoparticles thus retain activity upon exposure to as high as 50 parts per million (ppm) of hypochlorous acid (HOCl), while native DFPase is observed as inactive at 500 parts per billion (ppb). This trend is also confirmed by enzyme-generated (chloroperoxidase (CPO), EC 1.11.1.10) reactive oxygen species (ROS) including both HOCl (3 ppm) and ClO(2) (100 ppm).

Properties of film from splendid squid (Loligo formosana) skin gelatin with various extraction temperatures.

Properties of film from splendid squid (Loligo formosana) skin gelatin extracted at different temperatures (50-80°C) were investigated. Tensile strength (TS) and elongation at break (EAB) of films decreased, but water vapour permeability (WVP) increased (P<0.05) as the extraction temperature increased. Increase in transparency value with coincidental decrease in lightness was observed with increasing extraction temperatures. Electrophoretic study revealed that degradation of gelatin became more pronounced with increasing extraction temperatures. As a consequence, their corresponding films had the lower mechanical properties. FTIR spectra of obtained gelatin films revealed the significant loss of molecular order of the triple helix. Thermogravimetric analysis indicated that F80 exhibited the higher heat susceptibility and weight loss. Loosen structure was observed in film prepared from gelatin with increasing extraction temperatures. Thus, the temperature used for gelatin extraction from splendid squid skin directly affected the properties of corresponding films.

The role of protein assembly in dynamically tunable bio-optical tissues.

Cephalopods are nicknamed the "masters of disguise" for their highly evolved camouflage mechanisms, including the hallmark ability to rapidly change the color and reflectance of their skin. Previously, reflectin proteins were identified as the major biomaterial component of iridosomes [1], specialized light-reflecting architectures that contribute intense structural color to squid skin, eyes, and organs [2-5]. Supramolecular assembly of reflectin has been recognized as a key property in the protein's function [6]. Here, we report the first cloning and expression of a specific reflectin protein found in the responsive iridophore cells of the squid Loligo pealeii, which are unique in their ability to switch on/off and change color. We demonstrate that these iridophores can be chemically tuned to reflect the entire visible spectrum. By examining recombinant reflectin, we show that this dynamic optical function is facilitated by the hierarchical assembly of nanoscale protein particles that elicit large volume changes upon condensation. These findings provide insight into the design and synthesis of biomaterials for complex, responsive function in optical applications.

Degradation of the non-palmitoylated invertebrate visual guanine-nucleotide binding protein, iGq alpha(C3,4A), by the ubiquitin-proteasomal pathway is regulated by its activation and translocation to the cytoplasm.

Light-dependent translocation of invertebrate visual guanine-nucleotide binding protein, iGq alpha, from rhabdomeric membranes to the cytoplasm is one of many mechanisms that contribute to light adaptation in the invertebrate eye. We have previously cloned iGq alpha from a Loligo pealei photoreceptor cDNA library and shown that when expressed in HEK 293T cells it is palmitoylated. In this study we compared the activation, cytoplasmic translocation, and turnover of iGq alpha with that of a non-palmitoylated mutant, iGq alpha(C3,4A). In the HEK 293T cells, muscarinic M1 receptors coupled equally well to iGq alpha and iGq alpha(C3,4A) to activate phospholipase C. Activation of iGq alpha(C3,4A), but not iGq alpha, induced translocation of the alpha subunit from the membrane to cytosol with rapid degradation of the soluble protein resulting in a decreased half-life for iGq alpha(C3,4A) of 10 hours compared to 20 hours for iGq alpha. Degradation of iGq alpha(C3,4A) was inhibited by proteasomal inhibitors but not by inhibitors of lysosomal proteases or calpain. The presence of the proteasomal inhibitor led to the accumulation of polyubiquitinated species of either iGq alpha or iGq alpha(C3,4A). Our results suggest that palmitoylation of iGq alpha is required to maintain membrane association of the protein in its active conformation, and whereas membrane-bound and soluble iGq alpha can be polyubiquitinated, membrane association protects the protein from rapid degradation by the proteasomal pathway.

Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris.

The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 A resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.

Structure of the neuronal protein calexcitin suggests a mode of interaction in signalling pathways of learning and memory.

The three-dimensional structure of the neuronal calcium-sensor protein calexcitin from Loligo pealei has been determined by X-ray analysis at a resolution of 1.8A. Calexcitin is up-regulated following Pavlovian conditioning and has been shown to regulate potassium channels and the ryanodine receptor. Thus, calexcitin is implicated in neuronal excitation and plasticity. The overall structure is predominantly helical and compact with a pronounced hydrophobic core between the N and C-terminal domains of the molecule. The structure consists of four EF-hand motifs although only the first three EF hands are involved in binding calcium ions; the C-terminal EF-hand lacks the amino acids required for calcium binding. The overall structure is quite similar to that of the sarcoplasmic calcium-binding protein from Amphioxus although the sequence identity is very low at 31%. The structure shows that the two amino acids of calexcitin phosphorylated by protein kinase C are close to the domain interface in three dimensions and thus phosphorylation is likely to regulate the opening of the domains that is probably required for binding to target proteins. There is evidence that calexcitin is a GTPase and the residues, which have been implicated by mutagenesis in its GTPase activity, are in a short but highly conserved region of 3(10) helix close to the C terminus. This helix resides in a large loop that is partly sandwiched between the N and C-terminal domains suggesting that GTP binding may also require or may cause domain opening. The structure possesses a pronounced electropositive crevice in the vicinity of the 3(10) helix, that might provide an initial docking site for the triphosphate group of GTP. These findings elucidate a number of the reported functions of calexcitin with implications for neuronal signalling.

Crystallization and preliminary X-ray diffraction analysis of calexcitin from Loligo pealei: a neuronal protein implicated in learning and memory.

The neuronal protein calexcitin from the long-finned squid Loligo pealei has been expressed in Escherichia coli and purified to homogeneity. Calexcitin is a 22 kDa calcium-binding protein that becomes up-regulated in invertebrates following Pavlovian conditioning and is likely to be involved in signal transduction events associated with learning and memory. Recombinant squid calexcitin has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P2(1)2(1)2(1). The unit-cell parameters of a = 46.6, b = 69.2, c = 134.8 A suggest that the crystals contain two monomers per asymmetric unit and have a solvent content of 49%. This crystal form diffracts X-rays to at least 1.8 A resolution and yields data of high quality using synchrotron radiation.