Sodium Dodecyl Sulfate Extraction Research Articles

Detection of IgG in cerebrospinal fluid for diagnosis of neurocysticercosis: evaluation of saline and SDS extracts from Taenia solium and Taenia crassiceps metacestodes by ELISA and immunoblot assay.

We compared saline (S) and sodium dodecyl sulphate (SDS) extracts from Taenia solium (homologous species - HO) and Taenia crassiceps (heterologous species - HE) metacestodes in order to detect IgG by ELISA and immunoblot assay (IBA) in cerebrospinal fluid (CSF) for the diagnosis of human neurocysticercosis (NC). CSF samples were obtained from 93 patients. Of these, 40 had NC, five had a diagnosis of probable NC, nine had central nervous system schistosomiasis or strongyloidiasis and 39 had other neurological alterations. Samples were analysed by ELISA and the results were compared with IBA in all samples with confirmed and probable NC diagnosis, in all samples with other central nervous system parasitic infection, and in 10 of those with another neurological alterations. ELISA sensitivity was 100%, 85%, 95% and 87.5% for the S-HO, S-HE, SDS-HO and SDS-HE extracts, respectively, and ELISA specificity was 100% for S-HO, S-HE, SDS-HO extracts and 97.9% for SDS-HE antigen. Immunodominant peptides detected by IBA were, by decreasing percentage of recognition: 64-68 and 45 kDa for S-HO; 108-114, 92-95, 64-68, 83 and 88 kDa for S-HE; 64-68, 108-114, 77 and 86 kDa for SDS-HO; and 108-114, 88 and 92-95 kDa for SDS-HE. Overall the homologous antigenic extracts showed higher sensitivity than the heterologous extracts in the diagnosis of NC in CSF samples. The heterologous extracts contained most of the immunodominant peptides presented in the homologous extracts, which are recognized by IgG antibodies in CSF samples.

Reduced Sarcoplasmic Reticulum Ca2+-Uptake and Expression of Phospholamban in Left Ventricular Myocardium of Dogs with Heart Failure

The purpose of this study was to examine the sarcoplasmic reticulum (SR) Ca2+-uptake and the expression of phospholamban (PLB) and Ca2+-ATPase (CAA) in left ventricular (LV) and right ventricular (RV) myocardium of 6 normal (NL) dogs and 6 dogs with chronic heart failure (HF). In addition, gene expression of PLB and CAA was also examined in LV myocardium of NL and HF dogs. HF (LV ejection fraction 23±2%) was produced by multiple sequential intracoronary microembolizations. Oxalate-dependent Ca2+-uptake was measured in isolated membrane vesicles. Using specific dog myocardial monoclonal antibody, the expression of CAA, PLB and calsequestrin (CSQ) were measured in sodium dodecyl sulfate extract prepared from LV and RV tissue. Steady-state mRNA levels were determined by Northern hybridization using specific cDNA clones of PLB, CAA, CSQ, and glyceraldehyde-3-phosphate dehydrogenase (GADPH), a house keeping gene. SR Ca2+-uptake of NL and HF dogs increased with increasing Ca2+concentrations and reached a plateau at 3μm in both LV and RV. Total capacity (134±9v224±10 nmol45Ca/mgprotein/10 min,P<0.05) and maximal velocity (15±2v2 nmol45Ca/mgprotein/min,P<0.05) of the SR to sequester Ca2+was significantly lower in LV myocardium of HF dogs compared to NL, whereas the Hill coefficient and the affinity of the Ca2+-pump for Ca2+were unchanged. LV tissue levels of the PLB and CAA, normalized to noncollagen protein or to CSQ and the PLB and CAA mRNA levels, normalized to CSQ or GADPH mRNA, were also significantly lower in HF dogs compared to NL. In RV myocardial tissue, no significant differences in total capacity of SR to sequester Ca2+, maximal velocity of SR Ca2+-uptake, the affinity and Hill Coefficient of the Ca2+-pump for Ca2+, or tissue levels of PLB and CAA were observed between NL dogs compared to HF dogs. We conclude that SR Ca2+-uptake and SR PLB and CAA protein and gene expression levels are reduced in LV myocardium of dogs with chronic HF. These abnormalities can lead to Ca2+-overload and subsequent global LV dysfunction.

Thermal analysis characterization of aortic tissues for cardiac valve bioprostheses.

Two multistep extractions were achieved on porcine aortic tissues to obtain acellular matrices used for cardiac bioprostheses. The evaluation of structural modifications and the possible damage of extracellular matrix fibrous proteins were investigated by means of thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Protein-water interactions and degradation temperatures were determined by TGA. DSC was used to characterize protein thermal transitions (glass transition and denaturation), which provided information on the dynamic structure of the aortic tissue components. Sodium dodecyl sulfate (SDS) extraction had a destructuring effect, while Triton and cholate treatments did not affect the structural integrity of either elastin and collagen. A DSC comparison showed that SDS destabilizes the collagen triple helical domain and swells the elastin network.

Glycerol is a suberin monomer. New experimental evidence for an old hypothesis

The monomer composition of the esterified part of suberin can be determined using gas chromatography-mass spectroscopy technology and is accordingly believed to be well known. However, evidence was presented recently indicating that the suberin of green cotton (Gossypium hirsutum cv Green Lint) fibers contains substantial amounts of esterified glycerol. This observation is confirmed in the present report by a sodium dodecyl sulfate extraction of membrane lipids and by a developmental study, demonstrating the correlated accumulation of glycerol and established suberin monomers. Corresponding amounts of glycerol also occur in the suberin of the periderm of cotton stems and potato (Solanum tuberosum) tubers. A periderm preparation of wound-healing potato tuber storage parenchyma was further purified by different treatments. As the purification proceeded, the concentration of glycerol increased at about the same rate as that of alpha,omega-alkanedioic acids, the most diagnostic suberin monomers. Therefore, it is proposed that glycerol is a monomer of suberins in general and can cross-link aliphatic and aromatic suberin domains, corresponding to the electron-translucent and electron-opaque suberin lamellae, respectively. This proposal is consistent with the reported dimensions of the electron-translucent suberin lamellae.

Type XVII Collagen (BP180) and LAD-1 are Present as Separate Trimeric Complexes

This study characterized the high molecular mass BP180 complex that is observed when unheated sodium dodecyl sulfate extracts of human skin or keratinocytes are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. In heated extracts BP180 is present as a monomer with a molecular weight of 180 kDa, in unheated extracts BP180 runs at a molecular weight position over 500 kDa. By preincubating the unheated extracts at temperatures between 31 degrees C and 40 degrees C, the high molecular weight complex could be "melted" down to monomeric BP180. Under the conditions employed the T1/2 of the dissociation process was between 35 degrees C and 36 degrees C. The temperature resistance of the high molecular weight complex was used to analyze its molecular composition by performing two-dimensional electrophoresis with a "low-temperature" first dimension step and a "high-temperature" second dimension step. Silver staining and immunoblotting of the two-dimensional gels revealed the high molecular weight complex to be composed of solely BP180, indicating that the complex is the nondissociated homotrimeric form of BP180. The 120 kDa linear IgA dermatosis antigen (LAD-1) is an collagenous anchoring filament protein with homology to the extracellular collagenous part of BP180. Two-dimensional immunoblotting showed that LAD-1, as BP180, is also present as a high molecular mass complex and does not form mixed complexes with BP180.

A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica.

A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library. The cDNA library was constructed in the expression vector lambda gt 11, with the RNA isolated from actively growing mycelial cells. The deduced amino acid sequence shows that the encoded protein contains an N-terminal hydrophobic signal peptide. We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein. Northern hybridization identified YWP1 transcript only when Y. lipolytica was growing in the mycelial form. The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is solubilized following partial degradation of beta-glucan by Zymolyase digestion. The protein is localized in the outer surface on the tip of the growing mycelial cells and is found partially cryptic in sub-apical locations, suggesting that it participates directly in the mycelial wall architecture.

The Mechanism of Detergent Solubilization of Liposomes and Protein-Containing Membranes

The present study explores intermediate stages in detergent solubilization of liposomes and Ca 2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized (∼C 12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca 2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca 2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C 12E 8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca 2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and β- d-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.

A New Strategy To Determine the Actual Protein Content of Poly(lactide-co-glycolide) Microspheres

Bovine serum albumin, lysozyme, and trypsin inhibitor were first encapsulated into poly-d,l-lactide-co-glycolide (PLGA) microspheres and then a new strategy was used to quantitate the actual levels of proteins in the microspheres. The proper combination of water-miscible dimethyl sulfoxide and 0.05N-NaOH containing 0.5% sodium dodecyl sulfate (SDS) made it possible to solubilize both PLGA microspheres and proteins in a single phase. A total protein assay conveniently provided accurate information on the amount of protein encapsulated into the microspheres. In contrast to conventional techniques making use of acetonitrile, dichloromethane, and SDS extraction methods, this new method did not necessitate polymer precipitation, filtration, and protein extraction into other phases. These features were a great advantage in recovering proteins without any loss due to experimental processes. As a consequence, the new method reported in this study provided accurate data for the actual level of protein in PLGA microspheres, regardless of the pattern of protein distribution inside microspheres or the characteristics of microspheres. The experiment relying on the use of a radiolabeled protein also validated the reliability of this new method.

SR Ca(2+)-ATPase activity and expression in ventricular myocardium of dogs with heart failure.

The purpose of this study was to examine the activity and expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase in left ventricular (LV) myocardium of dogs with chronic heart failure (HF). LV and right ventricular (RV) tissue specimens were obtained from six normal (NL) control dogs and six dogs with chronic HF (LV ejection fraction, 23 +/- 2%) produced by multiple sequential intracoronary microembolizations. Thapsigargin-sensitive Ca(2+)-ATPase activity was measured in isolated SR membrane fractions prepared from LV and RV myocardium. Ca(2+)-ATPase expression, using a specific dog myocardium monoclonal antibody, was measured in sodium dodecyl sulfate (SDS) extract prepared from LV and RV myocardium. Ca(2+)-ATPase activity in both ventricles of NL or HF dogs increased with increasing Ca2+ concentration and reached a plateau at 3 microM Ca2+. The maximal velocity (Vmax, mumol Pi released.min-1.mg-1) of Ca(2+)-ATPase activity was significantly lower in LV of HF dogs compared with NL (0.15 +/- 0.01 vs. 0.23 +/- 0.01, P < 0.05), whereas the affinity of the Ca2+ pump for Ca2+ was unchanged. LV tissue levels of Ca(2+)-ATPase (densitometric units/5 micrograms noncollagen protein) were also significantly lower in LV myocardium of HF dogs compared with NL (3.52 +/- 0.43 vs. 5.53 +/- 0.47, P < 0.05). No significant differences in Ca(2+)-ATPase activity or expression were observed in RV myocardium of HF dogs compared with NL. We conclude that SR Ca(2+)-ATPase activity and protein levels are reduced in LV myocardium of dogs with chronic HF. This abnormality of the SR Ca2+ pump of the failed LV can result in impaired Ca2+ uptake and ultimately to Ca2+ overload and global LV dysfunction.

Activation of MAP kinases in airway smooth muscle.

To test the hypothesis that mitogen-activated protein (MAP) kinases are activated by contractile agonists in intact nonproliferating airway smooth muscle, kinase activities were compared in resting and stimulated canine tracheal smooth muscle. Kinase activities in sodium dodecyl sulfate extracts were assayed by a gel renaturation method. Myelin basic protein kinase activities corresponding to ERK1 and ERK2 immunoreactive proteins were activated twofold above the basal level within 5 min by 1 microM carbachol. MAP kinase activity assayed in crude homogenates using a synthetic peptide substrate (APRTPGGRR) also increased twofold above basal in muscles stimulated with 1 microM carbachol. Two protein kinases separated by Mono-Q chromatography were identified on Western blots as ERK1 and ERK2 MAP kinases. Carbachol stimulation increased caldesmon phosphorylation in intact muscle, and purified caldesmon was a substrate for activated murine ERK2 MAP kinase. Activated ERK2 MAP kinase added to Triton X-100-permeabilized fibers potentiated Ca2+-induced contraction. The results show that ERK MAP kinases are activated after stimulation of muscarinic receptors in airway smooth muscle, which is consistent with coupling of MAP kinases to phosphorylation of caldesmon in vivo.

A Novel Cell Wall Protein Specific to the Mycelial Form of Yarrowia lipolytica

A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library. The cDNA library was constructed in the expression vector λgt11, with the RNA isolated from actively growing mycelial cells. The deduced amino acid sequence shows that the encoded protein contains an N-terminal hydrophobic signal peptide. We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein. Northern hybridization identified YWP1 transcript only when Y. lipolytica was growing in the mycelial form. The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is solubilized following partial degradation of β-glucan by Zymolyase digestion. The protein is localized in the outer surface on the tip of the growing mycelial cells and is found partially cryptic in sub-apical locations, suggesting that it participates directly in the mycelial wall architecture.

A novel cell wall protein specific to the mycelial form of Yarrowia lipolytica

A cDNA clone specifying a cell wall protein was isolated from a Yarrowia lipolytica cDNA library. The cDNA library was constructed in the expression vector λgt11, with the RNA isolated from actively growing mycelial cells. The deduced amino acid sequence shows that the encoded protein contains an N-terminal hydrophobic signal peptide. We have designated this protein YWP1 for Yarrowia lipolytica cell Wall Protein. Northern hybridization identified YWP1 transcript only when Y. lipolytica was growing in the mycelial form. The encoded protein seems to be covalently bound to the glucan cell wall since it is not released from the cell walls by sodium dodecyl sulphate extraction, but it is solubilized following partial degradation of β-glucan by Zymolyase digestion. The protein is localized in the outer surface on the tip of the growing mycelial cells and is found partially cryptic in sub-apical locations, suggesting that it participates directly in the mycelial wall architecture.

Cholera toxin induces synthesis of phospholipase A2-activating protein

The mechanism of cholera toxin (CT)-stimulated arachidonate metabolism was evaluated. CT caused rapid in vitro synthesis of prostaglandin E2 (PGE2) in murine smooth muscle-like cells (BC3H1), reaching maximal levels within 3 to 4 min. In comparison, cyclic AMP (cAMP) levels were unchanged, and addition of dibutyryl cAMP did not affect PGE2 synthesis. CT-induced PGE2 synthesis was prevented by actinomycin D or cycloheximide, indicating a need for de novo protein synthesis. Northern blot analysis of total RNA from BC3H1 cells revealed that exposure to CT resulted in an increase in abundance of mRNA encoding phospholipase A2 (PLA2)-activating protein (PLAP). PLAP is a regulatory protein that increases the enzymatic activity of cellular PLA(2), which in turn causes increased hydrolysis of arachidonate from membrane phospholipids. Furthermore, CT evoked the accumulation of PLAP mRNA in J774 (murine monocyte/macrophage) and Caco-2 (human intestinal epithelial) cells in vitro, but the responses were more delayed than that of BC3H1 cells. A protein band of approximately 35 kDa, which corresponded to the size of PLAP, was observed in sodium dodecyl sulfate extracts of Caco-2 cells by Western blot (immunoblot) analysis using affinity-purified antibodies to PLAP synthetic peptides. Synthesis of PLAP protein was increased after 2 h of exposure to CT. Exposure of mouse intestinal loops to either CT or live Salmonella typhimurium for 3 h increased mucosal PLAP mRNA levels. The role of PLAP in CT-induced PGE2 synthesis provides an attractive explanation for the reported suppression of CT-induced intestinal secretion by inhibitors of protein synthesis.

A Diagnostic Test to Confirm Interspecific Allium Hybrids Using Random Amplified Polymorphic DNA from Crude Leaf DNA Extracts.

A crude extraction procedure for DNA was modified and used for small leaf samples (2-4 mg) in Allium. Six random decamers (OPD 5, 7, 14, 15, 18 and 19) generated characteristic random amplified polymorphic DNA (RAPD) profiles from leaf DNA of Allium tuberosum Rottl. ex Spreng. 'Tender pole' obtained using a crude micro extraction process These RAPD profiles were roughly similar to those of DNA purified using a sodium dodecyl sulfate (SDS) extraction procedure.Crude extracts of leaf DNA were prepared from three accessions of A. tuberosum 'Tender pole', 'five 'Yoshitake hybrids' (A. chinense G. Don × A. thunbergii G. Don) and seven putative trispecific hybrids between these two groups. DNA amplification with four random decamers (OPD 14, 15, 18 and 19) by polymerase chain reaction produced a total of 186 bands : 49 bands were found only in the RAPD profiles of the putative hybrid and the corresponding male parent (M bands), whereas 58 bands were found in both female parent and trispecific hybrid plants (F bands). One of the seven putative hybrids had no M band, indicating that it had no relationship to its putative male parent and that it may have been the product of an unwanted pollination.

Zymogram and Preliminary Characterization of Lactobacillus helveticus Autolysins

The autolysins of Lactobacillus helveticus ISLC5 were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymogram). By using lyophilized Micrococcus luteus cells or heated whole cells of L. helveticus ISLC5 (0.2% [wt/vol]) as a substrate, several lytic activities were detected in the whole-cell SDS extract of strain ISLC5 (i) one activity at 42.4 kDa, which was named autolysin A, and (ii) six other activities having very similar molecular weights (29.1, 29.6, 30, 30.8, 31.7, and 32.8 kDa), which were named autolysins B (B1 through B6, respectively). As regards the temporal distribution of the enzymes, autolysins A and B were detected in the cells harvested from the beginning of the exponential growth phase. Autolysin A appeared to be associated only with viable cells, whereas the autolysins B remained associated with the cell envelope several days after the complete loss of culture viability. When SDS-treated walls of L. helveticus ISLC5 were used as a substrate, a supplementary lytic activity appeared at 37.5 kDa; it was considered a peptidoglycan hydrolase, since it was not able to induce lysis of whole-cell substrate. The autolysins of 30 other strains of L. helveticus from various geographical origins were also analyzed by zymogram; all the activity profiles obtained were similar to that of strain ISLC5 in terms of the number of lytic bands and their apparent molecular weights. Only the relative intensities of the lytic bands corresponding to autolysins A and B were variable depending on the strains. This observation suggested that autolysins are highly conserved enzymes. A concentrated crude lysate of the virulent bacteriophage 832-B1 infecting L. helveticus was also analyzed by zymogram; one lytic activity with an apparent molecular weight of 31.7 kDa, very close to the weights of the autolysins B, was observed. Finally, the autolysins of L. helveticus ISLC5 were successfully extracted from whole cells by using a 1 M lithium chloride solution; they were partially purified by precipitation, selective resolubilization, and gel filtration chromatography, which led to a 20-fold increase in specific activity.

Distribution of IS901 in strains of Mycobacterium avium complex from swine by using IS901-detecting primers that discriminate between M. avium and Mycobacterium intracellulare

The presence of the mycobacterial insertion sequence IS901 was studied by PCR with reference strains of Mycobacterium avium complex; 122 veterinary strains of mycobacteria, mainly M. avium complex, isolated from swine; and 15 clinical strains. Four kinds of DNA extraction methods for PCR were compared. Use of the commercial extraction matrix allowed for the faster and easier preparation of PCR-amplifiable DNA than use of NaOH heating extraction or sodium dodecyl sulfate extraction of pretreated mycobacteria. It also provided more effective protection than boiling extraction against the destruction of DNA. Four reference strains of serovars 1 to 3 possessed IS901. Nine reference strains of serovars 1, 4 to 6, 8 to 11, and 21 possessed only IS901 insertion sites. A novel PCR product was found in the other reference strains of serovars 7, 12 to 17, 19, and 20 and two clinical strains of serovar 15. It is suggested that the primers that amplified the insertion portion of IS901 divided the M. avium complex into M. avium, Mycobacterium intracellulare, and other mycobacteria. None of the 110 strains of M. avium complex isolated from swine possessed IS901. It is suggested that the absence of IS901 might be characteristic of swine-derived strains of M. avium complex.

Rapid Dephosphorylation of τ in Heat‐Shocked Fetal Rat Cerebral Explants: Prevention and Hyperphosphorylation by Inhibitors of Protein Phosphatases PP1 and PP2A

We heat shocked 21- and 35-day-old fetal rat cerebral explants at 45 degrees C for 18 min and performed immunocytochemistry and immunoblot analysis of sodium dodecyl sulfate extracts using the monoclonal anti-tau antibodies Tau-1, Tau-5, Tau-46, and PHF-1 and the peroxidase-antiperoxidase technique or 125I-labeled protein A. Tau-1 and PHF-1 recognize nonphosphorylated and phosphorylated epitopes, respectively, and both Tau-5 and Tau-46 recognize phosphate-independent epitopes. tau immunoreactivity was confined to neurons and increased in heat-shocked perikarya but not axons. At 0 h after heat shocking, there was dephosphorylation of tau exemplified by (1) faster migration of tau isoforms with resultant loss or attenuation of the 60- and 52-kDa tau isoforms recognized by all four anti-tau antibodies and concomitant accentuation of the fastest moving 50-kDa tau isoform recognized by Tau-1, Tau-5, and Tau-46; and (2) significant increase in the nonphosphorylated Tau-1 epitope with resultant decreases in the ratio of total (phosphorylated plus nonphosphorylated) tau to nonphosphorylated tau and the difference of total tau minus nonphosphorylated tau. tau was phosphorylated back to the control level by 12 h and remained so at 24 and 48 h after heat shocking. Treatment of explants with cycloheximide, a protein synthesis inhibitor, did not prevent the heat shocking-induced dephosphorylation of tau. Treatment of explants with the inhibitors of protein phosphatases PP1 and PP2A, okadaic acid or calyculin A, produced hyperphosphorylated tau polypeptides, prevented the heat shocking-induced dephosphorylation of tau, and intensified the immunoreactivity of the neurofilament subunit H with the only antiphosphoneurofilament antibody that reacts with intraneuronal neurofibrillary tangles. In 35-day-old explants, in addition to the three 50-, 52-, and 60-kDa tau isoforms seen in 21-day-old explants, a 66-kDa tau polypeptide was also present.

Biochemical and nutritional changes in fish proteins during drying

AbstractBiochemical and nutritional changes in the muscle proteins of a lean marine fish Nempiterus japonicus during drying at 50, 60 and 70°C were investigated. Solubility of proteins in water, 0.6 M NaCl, 1.5 M urea, 8 M urea and 10 g litre−1 sodium dodecyl sulphate (SDS) decreased as drying progressed at all three temperatures; most of the decrease occurring in the initial 4 h of drying SDS‐polyacrylamide gel electrophoresis of 1.5 M urea, 8 M urea and SDS extracts showed that higher molecular weight (MW) protein fractions were more sensitive to drying and disappeared much earlier from electropherograms than the lower MW protein fractions. Residual solubility of proteins near the pH range of 4–6 was found to increase during drying, but solubility at acid and alkaline pH was adversely affected. Decrease of solubility by drying was more affected at acid pH, especially at higher temperatures than at alkaline pH. Sulphydryl groups registered a regular and sharp decrease with drying except at 50°C, where initially an increase was observed. Apart from disulphide and hydrophobic bonds, free amino groups also appear to be involved in denaturation reactions during drying. Pepsin digestibility of fish muscle decreased slightly during drying but a clear relationship with drying temperature was not evident. Highly significant differences in proteins between protein efficiency ratio (PER), net protein utilisation (NPU) and biological value were observed between the drying temperatures. The PER and NPU of fish dried at 60°C were significantly higher than those dried at 50 or 70°C.

Determining the authenticity of raw reformed breaded scampi ( Nephrops norvegicus) by electrophoretic techniques

A comparison was made between isoelectric focusing of aqueous extracts and sodium dodecyl sulphate (SDS) electrophoresis of SDS extracts of the ‘meat’ of raw reformed breaded scampi ( Nephrops norvegicus) as a possible means of detecting adulteration with Pacific scampi ( Metanephrops andamanicus) or tropical shrimp ( Penaeus indicus). Only SDS electrophoresis gave profiles from mixed species products in which the characteristic protein zones of the component species could be identified.

Protein glycation and fluorescent material in human atheroma

Samples of normal human aorta, atherosclerotic lesions and atheroma (necrotic ‘gruel’ from the interior of advanced lesions) were obtained at necropsy from subjects with no history of diabetes mellitus. Components of each were extracted by ethanol:diethylether (3:1) and, subsequently, by 10% sodium dodecyl sulphate (SDS). Both fractions, organic and aqueous (SDS), were assessed for their relative fluorescence (excitation: 350 nm/emission: 430 nm). The amount of early products of glycated protein was assessed by affinity chromatography in the SDS-soluble fraction. Agematched plasma samples, obtained from non-diabetic individuals, were also examined. Material from atherosclerotic lesions appeared to exhibit an inverse correlation between protein glycation and fluorescent material which was best reflected within the organic extract. This was not the case for normal aorta. A linear correlation between fluorescent material in the organic extract and SDS extract existed in the normal aorta alone. The only age-dependent change was found in normal aorta in which there was an increase in SDS-soluble fluorescence with increasing age. In plasma samples alone, protein glycation and protein fluorescence appeared to be linearly correlated. The observations are discussed in the context of the possible contribution of protein glycation to atherogenesis.