Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Patterns Research Articles

참치 가공부산물로부터 단백가수분해물 제조 및 항산화 활성 평가

This study aims to investigate the production and characteristics of protein hydrolysates derived from tuna byproducts (TP) using various proteolytic enzymes and to compare the antioxidant activity of the resulting hydrolysates. The TP were subjected to enzymatic hydrolysis using five different proteases: alcalase, bromelain, flavourzyme, neutrase, and papain, and the antioxidant activities of the hydrolysates were evaluated. Subsequent analysis of the available amino group contents and sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns indicated a high degree of hydrolysis in TP after treatment with all the enzymes, except for papain. Based on the RC50 values obtained from four different antioxidant analyses, all the hydrolysates exhibited similar antioxidant activity, except for the flavourzyme hydrolysate, which showed significantly higher scavenging activity against ABTS radicals and hydrogen peroxide than the other hydrolysates. These findings suggest that protein hydrolysates derived from TP hold promise as potential sources of natural antioxidants.

Gelatin From Cartilage of Siberian Sturgeon (Acipenser baerii): Preparation, Characterization, and Protective Function on Ultraviolet-A-Injured Human Skin Fibroblasts

For full use of the by-products during Siberian sturgeon (Acipenser baerii) processing, gelatin was extracted from the cartilages using the hot water method, and its physico-chemical properties and protective function on ultraviolet-A injured human skin fibroblasts (HFSBs) were measured. Using single-factor and orthogonal experiments, the conditions for extracting gelatin from Siberian sturgeon cartilage were optimized as extraction time of 7 h, pH 9, material-to-liquid ratio (g/ml) of 1:5, and temperature of 45°C. The prepared gelatin (TCG) with a yield of 28.8 ± 1.53% had Gly (307 residues/1,000 residues) as the major amino acid and contained a lower amount (214 residues/1,000 residues) of imino acids than that (227 residues/1,000 residues) of pigskin gelatin (PSG). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), ultraviolet spectrum, and infrared spectroscopy analysis indicated that TCG had the main spectral characteristics of fish gelatin and contained α chains (α1 and α2 chains) and β chain of type I collagen, but its structural stability was lower than that of PSG due to its low content of imino acids, which induced the smaller molecular bands observed in the SDS-PAGE pattern. TCG exhibited lower water content, gel strength, emulsion stability index, foam capacity, foam stability, and water-holding capacity but higher ash content, transmittance, emulsion activity index, and fat-binding capacity (P < 0.05). Moreover, TCG could significantly protect HFSBs against ultraviolet-A injury by enhancing the activity of superoxide dismutase, catalase, and glutathione peroxidase to scavenge excess reactive oxygen species and decrease the content of malondialdehyde. Therefore, gelatin from Siberian sturgeon cartilages could act as promising candidates when applied in health-promoting products against ultraviolet-A injury.

Changes in RAPD-DNA Markers and Plasma Protein Profile in Progesterone-Treated Chicken

The abuse of contraceptive pills as growth promoters in the poultry industry has serious health concerns. Consumption of hormonal residues in animal products results in reproductive disorders and an increased risk of cancers in humans. This study aimed to investigate the effect of treatment with progesterone hormone on the plasma proteins and RAPD-DNA markers of broiler chickens. One hundred, one-day-old broiler chicks were equally divided into 5 groups (n = 20). Four groups were treated daily with progesterone hormone either subcutaneously or orally (1.0 mg, 1.5mg/kg). The 5th group was control non-treated chicken. Sodium dodecyl sulfate polyacrylamide gel-electrophoresis (SDS-PAGE), and RAPD assays in conjunction with productive parameters such as growth rate were assessed. SDS-PAGE and RAPD-DNA patterns were significantly affected by progesterone hormone treatment. Either or oral or subcutaneous progesterone hormone treatments increased the body weight of chicks compared to control. Treatment with progesterone hormone induces effects on both DNA and plasma proteins in broiler chickens. Changes at the genomic DNA level may be due to specific modifications in RAPD patterns or arise as a consequence of hot spot DNA changes and/or mutations.

Incorporation effect of inulin and microbial transglutaminase on the gel properties of silver carp (Hypophthalmichthys molitrix) surimi

Surimi-based products with elasticity and specific shapes have received considerable notice in recent years. To ascertain the effects of inulin, microbial transglutaminase (MTGase) and the combination of inulin/MTGase on gel formation of silver carp surimi, the gel strength, textural properties, water-holding capacity (WHC), dynamic rheological measurements, Raman spectroscopy, light microscopy of gel structure and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of gels from different groups were analyzed. The results showed that addition of inulin, MTGase and MTGase + inulin improved gel strength (increased by 7.7%, 26.8% and 38.56% compared with control group), textural properties and WHC. Raman spectroscopy proven that MTGase + inulin promoted the conversion of α-helixes to β-sheets and β-turns compared with other experimental and control groups. Pure surimi gelation contained a loose and non-heterogeneous network via light microscopy. With the addition of MTGase and inulin, the microstructure of surimi gelation became more compact and homogeneous, particularly the MTGase + inulin groups. Moreover, The SDS-PAGE pattern further disclosed that the MTGase + inulin had a weakened myosin heavy chain band, indicating that inulin could enhance the cross-linking effects of MTGase on myosin heavy chains. These data demonstrated inulin incorporating with MTGase may be a new and effective strategy to improve the gel properties of fish surimi.

Species identification of a causative moray eel meat by SDS-PAGE

Attempts were made to identify the species of food poisoning moray eel. Besides the causative processed moray eel meat, eight other raw commercial moray eel meats including Gymnothorax favagineus, G.fimbriatus, G. flavimarginatus, G. meleagris, G. pseudothyrsoideus, G. undulates, G. albimarginatus and G. javanicus were heated at 100°C for 30 min and then assayed using the electrophoresis method of sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE patterns for eight processed commercial moray eel meats showed species-specific protein bands < 30 kD. The species of causative processed moray eel implicated in food poisoning case was identified as G. javanicus in comparison to the 2% SDS and 8 M urea extracted protein revealed by SDS-PAGE patterns.

A New Method of Gelatin Modified Collagen and Viscoelastic Study of Gelatin-Collagen Composite Hydrogel

Pure collagen materials are expensive with poor mechanical properties, which need modifications in most cases. As the degradation product of collagen, gelatin is cheap, degradable and biocompatible, but few literatures have reported the research about gelatin-collagen composite materials. This is because gelatin and collagen have different soluble temperatures—gelatin is soluble in hot water (≥30 °C) and swells in cold water. However, a low temperature (2–10 °C) is required to prepare and store collagen solution, and neutral collagen solution denatures quickly above the room temperature. In this study, gelatin was ground into powders and swelled in neutral bovine tendon pepsin-soluble collagen solution (BPSC) to form a homogeneous gelatin-collagen mixture, in light of the swelling characteristics of gelatin in cold water. The assembly properties and gel properties of this composite material were further studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) test results showed that the bovine tendon collagen had typical type-I collagen structural characterizations with two α chains of about 100 kDa and one β chain of about 200 kDa; while the SDS-PAGE pattern of gelatin displayed bands continuously distributed from 30 to 200 kDa. Amino acid composition analysis test indicated that the content of polar amino acids and the sum of acidic and base amino acids for gelatin were higher than that of BPSC. Studies on gel properties demonstrated that gelatin-collagen mixed solution had collagen-like assembly characteristics and assembly kinetics. The moduli of the assembled gel at 35 °C were equivalent to that of pure bovine tendon collagen system; moreover, the system moduli didn’t change with time with elastic moduli (G′) of about 40 Pa. However, at 25 °C, the moduli of gelatin-collagen composite hydrogel increased with the extension of time, its G′ increased about 18 times within 8 h, and the ratio of elastic modulus to viscous modulus (G″) increased 4.6 times, showing a significant aging effect of structural strength. Meanwhile, the mechanical strength of the composite hydrogel was also regulated by temperature—the gel was highly elastic (G′≈3,000 Pa, G′>>G″) at a low temperature (5 °C); as the temperature rose, the system moduli gradually decreased and the elastic gel transformed into waterlike fluid at 50 °C little by little. What’s more, gelatin-collagen composite hydrogel also had reversible sol-gel performances and self-healing capability similar to the gelatin hydrogel. This novel preparation method for preparing composite materials and the resultant composite hydrogel are expected to be used in the fields of natural food gels, injectable hydrogels, cell scaffolds, drug sustained-release materials and so on, and improve and promote the processing performances, price and large-scale production of collagen-based materials.

Quality Monitoring of Porous Zein Scaffolds: A Novel Biomaterial

Abstract Our previous studies have shown that zein has good biocompatibility and good mechanical properties. The first product from a porous scaffold of zein, a resorbable bone substitute, has passed the biological evaluation of medical devices (ISO 10993) by the China Food and Drug Administration. However, Class III medical devices need quality monitoring before being placed on the market, and such monitoring includes quality control of raw materials, choice of sterilization method, and evaluation of biocompatibility. In this paper, we investigated four sources of zein through amino acid analysis (AAA) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in order to monitor the composition and purity, and control the quality of raw materials. We studied the effect of three kinds of sterilization method on a porous zein scaffold by SDS-PAGE. We also compared the changes in SDS-PAGE patterns when irradiated with different doses of gamma radiation. We found that polymerization or breakage did not occur on peptide chains of zein during gamma-ray (γ-ray) sterilization in the range of 20–30 kGy, which suggested that γ-ray sterilization is suitable for porous zein scaffolds. Regarding cell compatibility, we found a difference between using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and a cell-counting kit-8 (CCK-8) assay to assess cell proliferation on zein film, and concluded that the CCK-8 assay is more suitable, due to its low background optical density.

Effect of pulsed electric field on structural properties of protein in solid state

There are few studies applying pulsed electric field (PEF) on solid foods in databases. Egg white protein (EWP) was subjected to PEF for elucidating mechanism of changes in molecular structure of enzymes and protein components for solid foods. Determination of free and total sulfhydryl groups, ultraviolet (UV) spectroscopy, infrared (IR) spectroscopy, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to compare EWP before and after PEF treatment. The results showed that the amount of free sulfhydryl increased while that of total sulfhydryl decreased after PEF exertion. PEF did not change the SDS-PAGE patterns visibly and no shift in maximum absorption wavelength but intensity could be observed in UV spectra of EWP. IR spectra of EWP were subjected to Gaussian multi-component peak modeling for molecular secondary structure analyses. Either the voltage of the PEF (P < 0.01) or the residence time of the sample (P < 0.05) significantly decreased the relative portion of α-helices while increased that of β-sheets in EWP, and impact could also be seen in respect of contents of β-turns and random coils in amide I region of EWP and its fractions. The results are of reference values to potential applications of PEF on solid foods.

Quality characteristics of fermented cheese analogs produced using roasted soy flour and different fermentation times with rice straw

To improve the nutritional and physiological functionality of cheese analogs, fermented cheese analogs treated with Flavourzyme® were developed and the effects of roasting soy flour and fermentation time (1, 2, and 3 days) with rice straw on their quality characteristics, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoflavone content, and antioxidant activity were examined. Heat coagulation was the highest for the unroasted and roasted soy flour groups fermented for 1 day, with the unroasted group having higher values than the roasted group, regardless of fermentation time. The daidzein and genistein contents were the highest in the soy flour and roasted soy flour groups fermented for 2 days and 1 day, respectively. The antioxidant activity and heat extension were the highest for the soy flour group and roasted soy flour group fermented for 1 day, respectively. In the SDS-PAGE patterns, protein subunits of P26 in roasted soy flour groups were consistently decomposed by increasing the fermentation time. The yield and pH in both groups decreased as fermentation time increased. The adhesiveness of the soy flour group was highly increased by fermentation, and the cohesiveness, springiness, and appearance of the roasted soy flour group were improved by fermentation. The optimum fermentation time for the fermented cheese analogs was 1 day, as higher yields, heat coagulation, heat extension, lightness, dE, cohesiveness, and springiness were obtained. Between the two groups fermented for 1 day, the soy flour group showed better heat coagulation, genistein content, antioxidant activity, cohesiveness, springiness, and adhesiveness; however, the roasted soy flour group showed better yield and heat extension.

Potential Correlation between Tumor Aggressiveness and Protein Expression Patterns of Nipple Aspirate Fluid (NAF) Revealed by Gel-Based Proteomic Analysis

Breast cancer is the leading cause of cancer related deaths in women. Most breast cancers stem from mammary ductal cells that secrete nipple aspirate fluid (NAF), a biological sample that contains proteins associated with the tumor microenvironment. In this study, NAF samples from both breasts of 7 Brazilian patients with unilateral breast cancer were analyzed. These samples were systematically compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional fluorescence difference gel electrophoresis (2D-DIGE); substantial qualitative individual differences were observed. In general, when NAF samples were compared from both breasts within the same patient their electrophoretic patterns were very similar, regardless of their cancer status. A comparison of all patients identified 2 main NAF protein profiles. The HomEP, homogeneous expression profile, was characterized by typical SDS-PAGE and 2D-DIGE protein patterns that were observed in patients with a good breast cancer prognosis and were similar to previous Type I NAF classifications that used one-dimensional electrophoresis. The HetEP, heterogeneous expression profile, was characterized by distinct protein patterns that have not been reported in previous studies and have been primarily observed in breast cancer patients with a poor prognosis. The NAF samples were rich in metal-dependent proteolytic enzymes, as visualized by SDS-PAGE zymography. They varied qualitatively with respect to their gelatinolytic band distribution. However, there were no correlations between these characteristics and the pathologic features of these tumors. A comparative analysis of NAF samples taken from each breast in a single patient showed conserved zymographic patterns. In conclusion, the present study highlights important distinctions in the protein content of individual NAF samples and provides insight into the composition of the tumor microenvironment. These data reinforce breast cancer as a heterogeneous disease with a diverse natural history, which is becoming increasingly evident through other recent studies.

Effect of organic salts on setting gels and their corresponding acids on kamaboko gels prepared from squid Todarodes pacificus mantle muscle

The present study evaluated the quality of setting gels prepared by adding sodium acetate, sodium gluconate, sodium citrate, and sodium succinate to Todarodes pacificus mantle muscle meat, and of the acid-induced kamaboko gels produced by soaking the setting gels in their corresponding acid solutions. The breaking strength was the lowest for the setting gel treated with sodium acetate. The pH values of the acid-induced kamaboko gels decreased when soaked in acid, whereas their moisture contents, whitenesses, and breaking strengths increased. The citric acid-induced kamaboko gel had the highest whiteness index and breaking strength among the gels. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) patterns did not show large differences among the gels, except for a low-intensity polymer band in the gluconic acid-induced kamaboko gel. Sodium citrate combined with citric acid yielded the best-quality acid-induced kamaboko gel among all the combinations, so this condition was used to study the effect of organic salt levels. The breaking strength increased when a sodium citrate level of up to 3% was used for the sodium citrate-treated setting gel, and 10% for the citric acid-induced kamaboko gel. The SDS-PAGE patterns did not differ among the sodium citrate levels. Egg white and microbial transglutaminase were necessary for citric acid-induced kamaboko gel production.

Physicochemical properties and molecular weight characterisation of porcine dermal collagen digested under varying conditions with clostridium histolytic collagenase

AbstractPorcine dermal collagen is successfully digested by clostridium histolyticun collagenases (CHC) at varying compositions and conditions. Collagen fragments thus prepared, with significantly low molecular weight but visible denaturation temperatures and/or triple helix structures, may be useful for clinical applications. As evidenced by Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and gel permeation chromatography (GPC) analyses, relatively high molecular weights of α1, α2 subunit, β1 ,β2 dimmers and γ trimmers of the collagen fragments gradually disappear as their incubation temperature and/or time values increase. The time values corresponding to the disappearance of sigmoid plots in the denaturation curves, denaturation endotherm in DSC thermograms and γ bands in SDS-PAGE patterns of digested collagen specimens are the same and reduce significantly as their incubation temperatures increase. Fourier transform infra-red analyses results suggest that the percentages of preserved triple helix structures present in collagen molecules of digested collagen specimens also reduce significantly with increasing the incubation temperature and time values. Possible reasons accounting for the degradation, denaturation and physicochemical properties of digested porcine dermal collagens are explored in this study.

Preparation of Artificial Antigen and Egg Yolk-derived Immunoglobulin (IgY) of Citrinin for Enzyme-Linked Immunosorbent Assay

To prepare artificial antigens and anti-citrinin egg yolk-derived immunoglobulin (IgY) to build an enzyme-linked immunosorbent assay (ELISA) for citrinin (CTN). CTN was conjugated with bovine serum albumin (BSA), ovalbumin (OVA) with formaldehyde condensation method to prepare artificial antigens and identified by ultraviolet (UV) spectrometry and Infrared (IR) spectrometry. Artificial antigens for CTN and anti-CTN IgY were purified with polyethylene glycol two-step precipitation method and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ELISA with IgY was established. Cross-reactivity of IgY with various structural similarities to CTN and possible co-occurrence with CTN in agricultural commodities were studied. UV and IR absorption spectra suggested that CTN was correlated with the carrier protein of BSA or OVA. SDS-PAGE patterns showed that the anti-CTN IgY was almost pure with a molecular weight of approximate 100 KD. The indirect competitive ELISA showed that the detection limit of CTN was 10 ng x mL(-1), with a good linearity ranging 20-640 ng x mL(-1). Artificial antigens of CTN can be successfully synthesized. The established ELISA can be used to determine CTN- contaminated samples.

Effects of Gamma Irradiation on Chemical Composition, Antinutritional Factors, Ruminal Degradation and In vitro Protein Digestibility of Full-fat Soybean

The aim of this study was to evaluate the effects of gamma irradiation (y-irradiation) at doses of 15, 30 and 45 kGy on chemical composition, anti-nutritional factors, ruminal dry matter (DM) and crude protein (CP) degradibility, in vitro CP digestibility and to monitor the fate of true proteins of full-fat soybean (SB) in the rumen. Nylon bags of untreated or γ-irradiated SB were suspended in the rumens of three ruminally-fistulated bulls for up to 48 h and resulting data were fitted to a nonlinear degradation model to calculate degradation parameters of DM and CP. Proteins of untreated and treated SB bag residues were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Digestibility of rumen undegraded CP was estimated using the three-step in vitro procedure. The chemical composition of raw and irradiated soybeans was similar. Results showed that phytic acid in γ-irradiated SB at dose of 30 kGy was eliminated completely. The trypsin inhibitor activity of 15, 30 and 45 kGy γ-irradiated SB was decreased (p<0.01) by 18.4, 55.5 and 63.5%, respectively. From in sacco results, γ-irradiation decreased (p<0.05) the washout fractions of DM and CP at doses of 30 and 45 kGy, but increased (p<0.05) the potentially degradable fractions. Gamma irradiation at doses of 15, 30 and 45 kGy decreased (p<0.05) effective degradability of CP at a rumen outflow rate of 0.05 h -1 by 4.4, 14.4 and 26.5%, respectively. On the contrary, digestibility of ruminally undegraded CP of irradiated SB at doses of 30 and 45 kGy was improved (p<0.05) by 12 and 28%, respectively. Electrophoretic analysis of untreated soybean proteins incubated in the rumen revealed that β-conglycinin subunits had disappeared at 2 h of incubation time, whereas the subunits of glycinin were more resistant to degradation until 16 h of incubation. From the SDS-PAGE patterns, acidic subunits of 15, 30 and 45 kGy γ-irradiated SB disappeared after 8, 8 and 16 h of incubation, respectively, while the basic subunits of glycinin were not degraded completely until 24, 48 and 48 h of incubation, respectively. It was concluded that γ-irradiated soybean proteins at doses higher than 15 kGy could be effectively protected from ruminal degradation.

PREPARATION AND CHARACTERIZATION OF PEPSIN-SOLUBILIZED TYPE I COLLAGEN FROM THE SCALES OF SNAKEHEAD (OPHIOCEPHALUS ARGUS)

ABSTRACT Pepsin-solubilized collagen prepared from the scales of snakehead (Ophiocephalus argus) was separated into two fractions, major and minor, by NaCl precipitation. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition, and secondary structure showed that the major collagen was typical type I collagen; in contrast, the minor collagen might be classified as type V collagen from the SDS-PAGE patterns and precipitation properties by NaCl. A sharp decrease in solubility of type I collagen was observed at the NaCl concentration of 40 g/L. The maximum and the minimum solubilities of collagen were observed at pH 3 and 8, respectively. Peptide maps of type I collagen digested by trypsin and V8 protease were different from those of calfskin and fish skin collagens. The imino acid content of type I collagen was lower than those of mammalian collagens and so did its denaturation temperature that was 30.3C obtained by viscosity measurement. PRACTICAL APPLICATIONS Collagen has been widely utilized as a material for foods, cosmetics, and pharmaceuticals. However, the use of collagen-derived products from land animals (e.g., bovine and pig) has been called into question because of foot-and-mouth disease crisis etc. Aquatic animal offals, which are readily available and inexpensive, seem to be safe sources for extraction of collagen. This work reports on preparation and characterization of collagen from snakehead scales, which will have potential in supplementing the skins and bones of land animals as an important collagen resource for use in functional food, biomedical, and cosmetic industries.

Electrophoretic and HPLC methods for comparative study of the protein fractions of malts, worts and beers produced from Scarlett and Prestige barley ( Hordeum vulgare L.) varieties

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reversed-phase high performance liquid chromatography (RP-HPLC) methods were used for studying the protein fractions (hordeins; albumins and other soluble proteins) of Scarlett and Prestige barley malts and to follow changes of the protein profile of worts and beers from these two malt varieties. Similar industrial brewing conditions were applied for both varieties. Statistical analyses of RP-HPLC data showed that hordeins were exposed to a proteolytic process during germination, which reduced its content and originated less hydrophobic peptides. In contrast, albumins and other soluble proteins increased during the germination process. Some malt water-soluble proteins result from the hordein proteolysis. Quantitative differences were observed between the protein fractions of the two malt varieties. SDS-PAGE patterns indicate that most of the components present in the worts were also detectable in final beers. However, chemometric analysis of the HPLC data showed quantitative differences between Scarlett and Prestige worts quantitative protein profiles. Scarlett wort contained more protein than Prestige worts. However, final beer samples presented a quantitative protein profile more similar than the respective worts. The optimized methodologies can be successfully used to compare the protein fractions of malts produced from two barley varieties, to follow the evolution of protein fraction during germination and the evolution of protein fraction content of worts and beers.

기능성 어육단백질의 젤화 특성과 산업적 응용-1. 가열변성 중 화학결합에 미치는 pH의 영향

The effect of pH on surface hydrophobicity, sulfhydryl group, infrared spectrum, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) pattern and enthalpy was investigated in recovered protein from mackerel and frozen blackspotted croaker by alkaline processing. Hydrophobic residue in myofibrillar protein exposed to the surface of protein, and hydrophobic interaction were the highest around 6. The surface hydrophobicity was different between myofibrillar protein and myofibrillar protein including sarcoplasmic protein (recovered protein). The peak at 1636 c was increased with pH, and the recovered protein was unfolded in alkali pH. Difference of surface and total sulfhydryl group at pH 7.0 and 10 was comparative high, and decrease of surface sulfhydryl group indicated formation of S-S bonds. Mackerel and frozen blackspotted croaker in alkaline pH showed bands of polymerized myosin heavy chain on SDS-PAGE pattern. The transition temperatures of recovered protein were 33.1, 44.3 and 65.5. Gelation of recovered protein from alkali processing was estimated by increase of -sheet structure by pH treatment, S-S bonds by oxidation of surface sulfhydryl group in heating, polymerization of myosin heavy chain in order.r.

Assessing the end-point temperature of heated fish and shellfish meats

: Attempts have been made to assess the end-point temperature (EPT) of heated fish and shellfish meats by using the coagulation method together with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and enzyme activity determination. Unfrozen and frozen fish and shellfish meats were heat-treated at different selected temperatures with 0, 15 or 30 min holding times. Proteins were extracted with NaCl solution. The coagulation method was able to determine EPT of heated fish and shellfish meats between 60 and 67°C. SDS-PAGE patterns of the filtrates from heated meats were closely related to the results of the coagulation method and enzyme activity determination. Two proteins responsible for producing coagulum of fish meat extracts seem to be lactate dehydrogenase (LDH) and glyceraldehyde phosphate dehydrogenase (GAPDH). End-point temperatures determined by these methods were not significantly different between unfrozen and frozen samples. On the contrary, a highly thermostable protein with a molecular mass of 35 kDa was detected in heated shellfish meats up to 108°C. In scallop adductor muscle, this highly thermostable protein was found to be the tropomyosin subunit from its amino acid composition and their partial sequences. Tropomyosin could be used as an EPT indicator up to 108°C for heated shellfish meats.

A modern approach to selectivity of proteinuria and tubulointerstitial damage in nephrotic syndrome

The selectivity of proteinuria, introduced in clinical nephrology in 1960 and useful in predicting steroid responsiveness in nephrotic syndrome, found little place in clinical practice in subsequent decades, since its assessment did not appear to help predict histologic diagnosis or determine prognosis. The amount of proteinuria and the degree of tubulointerstitial damage appeared to be better predictors of functional outcome. A correlation between them has been found, referred to some toxicity of proteinuria on tubular cells, but so far no single feature or component of proteinuria has been identified as being responsible for this toxicity. We evaluated 89 patients with nephrotic syndrome [9 with minimal change disease (MCD), 29 with primary focal segmental glomerulosclerosis (FSGS), and 51 with idiopathic membranous glomerulonephritis (MGN)] to determine if the selectivity of proteinuria was associated with tubulointerstitial damage. A semiquantitative grading of histologic lesions and qualitative evaluation of the "tubular" component of proteinuria expressed as a pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and as fractional excretion of the low molecular weight (LMW) protein alpha1-microglobulin (FE alpha1m) were used. A second aim of the study was to assess the predictive value on functional outcome [remission or progression to chronic renal failure (CRF)] and response to therapy of the selectivity of proteinuria, considered alone and in combination with FE alpha1m. Proteinuria was classified as highly selective [selectivity index (SI) < or = 0.10, N = 15], moderately selective (SI > or = 0.11 < or = 0.20, N = 34), or nonselective (SI > or = 0.21, N = 40). A significant relationship was found between the SI and the histologic degree of tubulointerstitial damage (score 0 to 1 vs. score > or =2, P = 0.000), severity of the tubular component of proteinuria (mixed SDS-PAGE pattern with LMW proteins not lower than 23 kD vs. mixed pattern with LMW proteins up to 20 to 10 kD, P = 0.000), and FE alpha1m (values below vs. above a defined cut-off, P = 0.000). The functional outcome was evaluated in 60 patients with baseline normal renal function (serum creatinine 0.97 +/- 0.19 mg/dL). The patients with high, moderate, or nonselective proteinuria had 100, 50, and 29% of complete or partial remission (P = 0.0001) and 0, 25, and 35% of progression to CRF, respectively (P = 0.050). In 45 patients with moderately selective (N = 28) and nonselective (N = 17) proteinuria, according to some arbitrary cutoffs for FE alpha1m (MGN, < or = vs. > 0. 240% of creatinine clearance; FSGS and MCD, < or = vs. > 0.350%), the remission rate was 62 versus 6% in patients with FE alpha1m below or above the cutoffs (P = 0.0001), and progression to CRF was 7 and 69%, respectively (P = 0.0001). The response to therapy (complete or partial remission at the last observation), evaluated retrospectively in 40 patients, was 100, 67, and 33% in high, moderate, and nonselective proteinuria (P = 0.0002); in 30 patients with moderate and nonselective proteinuria, according to an FE alpha1m value that was < or = or > the cutoffs, the response rate was 75 versus 10% (P = 0.001). There is a significant relationship between selectivity of proteinuria and tubulointerstitial damage. Moreover, the selectivity of proteinuria has a predictive value on functional outcome. When proteinuria is highly selective, the tubulointerstitial damage is rather infrequent, and 100% of patients develop clinical remission. When proteinuria is moderately selective or nonselective, increasing numbers of patients develop tubulointerstitial damage; in these patients, the functional outcome and response to therapy is partly dependent on tubulointerstitial involvement, and the best predictor of functional outcome is the combination of SI and FE alpha1m.

Structural properties of lipopolysaccharides from Rickettsia typhi and Rickettsia prowazekii and their chemical similarity to the lipopolysaccharide from Proteus vulgaris OX19 used in the Weil-Felix test.

The lipopolysaccharides (LPSs) isolated from typhus group (TG) rickettsiae Rickettsia typhi and Rickettsia prowazekii were characterized by chemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. LPSs from two species of TG rickettsiae contained glucose, 3-deoxy-D-manno-octulosonic acid, glucosamine, quinovosamine, phosphate, and fatty acids (beta-hydroxylmyristic acid and heneicosanoic acid) but not heptose. The O-polysaccharides of these LPSs were composed of glucose, glucosamine, quinovosamine, and phosphorylated hexosamine. Resolution of these LPSs by their apparent molecular masses by SDS-PAGE showed that they have a common ladder-like pattern. Based on the results of chemical composition and SDS-PAGE pattern, we suggest that these LPSs act as group-specific antigens. Furthermore, glucosamine, quinovosamine, and phosphorylated hexosamine were also found in the O-polysaccharide of the LPS from Proteus vulgaris OX19 used in the Weil-Felix test, suggesting that they may represent the antigens common to LPSs from TG rickettsiae and P. vulgaris OX19.