Sodium Dodecyl Sulfate-polyacrylamide Electrophoresis Research Articles

Gellan gum conjugation with soy protein via Maillard-driven molecular interactions and subsequent clustering lead to conjugates with tuned technological functionality.

Soy proteins are frequently used in the food industry; however, they have rigid and compact structure with relatively poor interfacial properties and solubility. This study was therefore aimed to modify techno-functional characteristics of soy protein isolate (SPI; 0.1% w/v) by conjugating to low acyl gellan gum (LAGG; 0.1, 0.2, and 0.3% w/v), through the Maillard reaction (at 90°C for 90min). The SPI-LAGG conjugates were confirmed by changes in pH, glycation degree (DG; up to 48%), Fourier transform infrared spectroscopy, and sodium dodecyl sulphate polyacrylamide electrophoresis. The conjugates were then classified into three clusters of low, medium, and high DG, via K-means clustering method. The low DG conjugate had lower surface hydrophobicity and foaming capacity, and higher thermal stability, solubility, emulsifying properties, foam stability, and antioxidant activity compared to the other clusters. This indicated that a low DG is required to enhance the functional properties of proteins.

In Vitro Protein Digestion of Cooked Spent Commercial Laying Hen and Commercial Broilers Breast Meat.

Chicken meat from spent laying hens (SHs) has been considered as nutritive as the meat of commercial broilers (CBs) based on chemical composition. High insoluble collagen in SH meat might reduce protein digestibility and bio-accessibility compared to CB meat. This study aimed at comparing the in vitro protein digestibility of CB and SH cooked breast meat. In the first part, CB samples were digested using two static in vitro digestion methods and collected at different digestion points for determining the degree of hydrolysis (DH). The method providing a greater DH value was chosen for comparing protein digestibility between CB and SH samples. The activities of used enzymes during in vitro digestion were evaluated based on bicinchoninic acid assay 2,4,6-trinitrobenzenesulfonic acid colorimetric method, gas chromatography-mass spectrometry, and sodium dodecyl sulfate-polyacrylamide electrophoresis. Particle size distribution of solid content collected from hydrolysate was also determined. The results showed that after digestion, CB showed 1–3 mg/mL protein concentration lower, while 7–13% DH and 50–96 µmoL/g protein-free NH2 groups higher when compared to those of SH. Based on sodium dodecyl sulfate-polyacrylamide electrophoresis, CB samples exhibited greater intensity of band at MW < 15 kDa than that of SH. Regarding particle size in terms of volume weighted mean (D[4,3]), at the end of the oral phase, the end of the gastric phase, and the beginning of the intestinal phase, D[4,3] of the SH samples were 133.17 ± 2.16, 46.52 ± 2.20, and 112.96 ± 3.63 µm, respectively, which were greater than those of CB (53.28 ± 1.23, 35.59 ± 1.19, and 51.68 ± 1.25 µm). However, at the end of the intestinal phase, D[4,3] of SH and CB, which were 17.19 ± 1.69 and 17.52 ± 2.46 µm, respectively, did not significantly differ from each other. The findings suggested a greater in vitro protein digestibility of cooked CB breast meats than that of SH ones.

Analysis and Comparison of Nutrition Profiles of Canine Milk with Bovine and Caprine Milk.

Pet foods are gaining ground in China. Canine milk substitute formulations are based on their milk chemistry. This study aimed to analyze and compare the differences in proteins, fatty acids, minerals, and basic chemical composition between canine, bovine, and caprine milk. Canine milk contains higher contents of protein (6.62–17.34%), fat (8.92–14.31%), and ash (1.11–1.81%), and a lower content of lactose (1.56–3.92%) compared to bovine and caprine milk. The protein profiles of canine, bovine, and caprine milk were similar as confirmed by sodium dodecyl sulphate-polyacrylamide electrophoresis gel (SDS-PAGE). The quantification of proteins in canine, bovine, and caprine milk were significantly different when analyzed by inverse high-performance liquid chromatography. Canine milk showed higher contents of monounsaturated fatty acids (29.71–32.95% of total fatty acids) and polyunsaturated fatty acids (16.83–20.56% of total fatty acids), but a lower proportion of saturated fatty acids (47.57–53.18% of total fatty acids) than bovine and caprine milk. The essential fatty acids ARA and DHA were also found in canine milk in the ranges of 0.82–1.77% and 0.12–0.43% of total fatty acids, respectively. Canine milk had higher levels of Mg, K, Na, Fe, and Zn than those in bovine and caprine milk. The nutrient profile of canine milk was different from that of bovine and caprine milk. There were differences in nutritional compositions of the milk samples from four breeds, and Rottweiler milk had the highest nutritional content. The data of this study may provide useful information about the nutritional needs of puppies during their first months of life and the basic information for formulations of puppy milk substitutes.

Effect of tear fluid sampling and processing on total protein quantity and electrophoretic pattern.

Human tears contain more than 1500 proteins that could be diagnostically relevant. To date, numerous candidates on a biomarker of protein origin were identified for ocular and systemic diseases. However, the suitable sampling method is still the subject of discussion. To address the need for a description of sampling methods properties for possible clinical analyses, we studied a total protein concentration and electrophoretic pattern of tear fluid collected by capillary tubes, Schirmer strips, cellulose microsponges, and flushing. The total protein concentration was 4.339 μg/μL ± 1.905 μg/μL, 0.967 μg/μL ± 0.117 μg/μL, 0.022 μg/μL ± 0.016 μg/μL, and 0.008 μg/μL ± 0.006 μg/μ for the capillary tubes, Schirmer strips, flushing, and cellulose microsponges, respectively. Sodium dodecyl sulfate polyacrylamide electrophoresis showed the different patterns of tear proteins obtained by the above-mentioned sampling methods. These differences could originate from the use of a bigger amount of extraction reagent that was not used in the case of capillary tubes, and retention of the proteins by strips and sponges. Taken together, capillary tubes, Schirmer strips, cellulose microsponges, and flushing represent sensitive and convenient sampling methods for tear fluid collection. For the isolation of proteins from strips and sponges, and for the flushing, less than 100 μL of a reagent should be used to ensure the sufficient concentration of the biomarkers in a trace amount.

The effect of lipopolysaccharide from uropathogenic Escherichia coli on the immune system, testis tissue, and spermatozoa of BALB/c mice.

ObjectiveUropathogenic Escherichia coli is known to cause urinary tract infections, and the endotoxin (lipopolysaccharide [LPS]) of this bacterium may cause deficiencies of sperm quality and morphology. In the present study, the effects of LPS on mouse sperm were studied, and the levels of interleukin (IL)-17A and possible changes in testis tissue were evaluated.MethodsLPS of uropathogenic E. coli was extracted using the methanol-chloroform method, followed confirmation using sodium dodecyl sulfate-polyacrylamide electrophoresis. Purified LPS (100 µg/kg) or phosphate-buffered saline was injected intraperitoneally into BALB/c mice for 7 days consecutively in the test and control groups, mice were sacrificed on days 3, 7, and 42 after the first injection. Blood was tested for levels of IL-17A using the enzyme-linked immunosorbent assay method. Testis tissue and sperm were collected from each mouse and were studied according to standard protocols.ResultsThe mean sperm count and motility significantly decreased (p=0.03) at 3, 7, and 42 days after the injections. The level of IL-17A in the test groups increased, but not significantly (p=0.8, p=0.11, and p=0.15, respectively). Microscopic studies showed no obvious changes in the morphology of the testis tissue; however, significant changes were observed in the cellular parenchyma on day 42.ConclusionLPS can stimulate the immune system to produce proinflammatory cytokines, resulting in an immune response in the testis and ultimately leading to deficiency in sperm parameters and testis tissue damage. In addition, the presence of LPS could significantly impair sperm parameters, as shown by the finding of decreased motility.

Identification of a 53 kDa protein, as a new high molecular weight allergen from Fraxinusexcelsior (Ash) pollen

Fraxinus excelsior (Ash) is a common tree and is important cause of winter–spring pollinosis in many temperate regions in the world. In this study, a high molecular weight allergen from ash pollen was identified. In all, 20 individuals allergic to ash participated in the study. Characterization and immunoreactivity of ash pollen proteins was performed using sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE), two-dimensional (2D) gel electrophoresis and immunoblotting. Immunoglobulin E (IgE)-binding proteins with apparent molecular mass ranging from 9 to 110 kDa were detected in ash pollen extract. Serum IgE of 7 (35%) patients reacted with a 53-kDa protein band. Analysis of 2D immunoblots showed several IgE-binding proteins. Moreover, mass spectrometry analysis indicated that the 53-kDa allergen was homologous to calreticulin. We defined a novel allergen from F. excelsior pollen with a molecular weight of about 53 kDa. This allergen could be considered as an important high molecular weight allergenic protein for further studies on cross-reactivity and development of diagnostic and therapeutic approaches.

Increasing the Enzyme Loading Capacity of Porous Supports by a Layer-by-Layer Immobilization Strategy Using PEI as Glue

A new strategy to increase the enzyme-loading capacity of porous supports was investigated. Lipase from Pseudomonas fluorescens (PFL) was immobilized on octyl-agarose (OA) beads and treated with polyethyleneimine (PEI). Then, PFL was immobilized on the previous PFL layer. Next, the biocatalyst was coated with PEI and a third layer of PFL was added. Sodium dodecyl sulfate polyacrylamide electrophoresis showed that the amount of PFL proportionally increased with each enzyme layer; however, the effects on biocatalyst activity were not as clear. Hydrolyzing 50 mM of triacetin at 25 °C, the activity of the three-layer biocatalyst was even lower than that of the bi-layer one; on the contrary its activity was higher when the activity was measured at 4 °C in the presence of 30% acetonitrile (that reduced the activity and thus the relevance of the substrate diffusion limitations). That is, the advantage of the multilayer formation depends on the specific activity of the enzyme and on the diffusion limitations of the substrate. When octyl agarose (OA)-PFL-PEI-PFL preparation was treated with glutaraldehyde, the activity was reduced, although the enzyme stability increased and the immobilization of the last PFL layer offered results similar to the one obtained using the three-layer preparation without glutaraldehyde modification (90%).

Expression of Surfactant Proteins in the Human Canaliculus: Evidence and Potential Insights Into the Tear Flow Dynamics

To investigate the presence and distribution patterns of 6 surfactant proteins (SPs) in the human lacrimal canaliculus. The study was performed on fresh frozen cadaveric samples of canaliculi. Immunohistochemical labeling was performed for assessing the presence and distribution of SP: SP-A, SP-B, SP-C, SP-D, SP-G/SFTA2, and SP-H/SFTA3. Immunofluorescence double staining was performed using the respective fluorescein-conjugated antibodies and the results were scored as positive or negative and the distribution pattern within the canalicular system was assessed. Western blot analysis was performed on the protein content which was resolved by reducing 15% sodium dodecyl sulfate-polyacrylamide electrophoresis and bands were studied following staining with primary and secondary antibodies. Human lung tissues were used as controls. Fluorescence double staining with 4,6-diamidino 2-pheynlindole and SPs showed strong immunostaining for SP-A, SP-B, SP-C, SP-D, and SP-H/SFTA3. The positive immunofluorescence was noticed across all the layers of the epithelium but not the subepithelial structures. The expression was noted on the surfaces and superficial cytoplasm of the superficial and deep epithelial cells. There was no expression of SP-G/SFTA2 across the canalicular system. Western blot analysis of the proteins confirmed and concurred with the immunofluorescence findings. This study provides a proof of principle for the presence of SPs known from lungs in the canalicular system and hypothesizes their possible functions and also their potential role in the tear flow dynamics between the ocular surface and the lacrimal drainage system.

Production, purification and characterization of an acid/alkali and thermo tolerant cellulase from Schizophyllum commune NAIMCC-F-03379 and its application in hydrolysis of lignocellulosic wastes

A cellulase producing fungus Schizophyllum commune NAIMCC-F-03379 was isolated from decomposed leaf sample of Lantana camera. The nutritional components (wheat bran, magnesium sulphate and calcium chloride concentrations) and physical parameters (temperature and pH) were optimised by response surface methodology for enhanced cellulase production by S. commune NAIMCC-F-03379. The optimized medium contained: 1% (w/v) wheat bran, 0.3 g/L MgSO4, 0.8–1.0 g/L CaCl2, optimum temperature and pH were 25 °C and 5 respectively. Under optimum condition, 5.35-fold increase in CMCase and 6.62-fold increase in FPase activity was obtained as compared to un-optimized condition. Crude cellulase enzyme was subjected to different purification techniques and comparative evaluation of their efficiency was performed. The aqueous two-phase system using polyethylene glycol 8000/MnSO4 system showed maximum purification with 10.4-fold increase in activity, 79.5% yield and 0.5 partition coefficient. The cellulase enzyme obtained from S. commune NAIMCC-F-03379 has shown high stability i.e. more than 55% relative activity after 12 h of incubation over wide range of temperature (25–65 °C) and pH (3–10). The molecular weight of the cellulase enzyme was estimated as ~ 60 kDa by using sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) and zymography. Km and Vmax value of cellulase on carboxy-methyl cellulose were obtained as 0.0909 mg/mL and 45.45 μmol/min mg respectively. Rice straw and wheat bran were subjected to hydrolysis using cellulase and cellulase–xylanase cocktail and analysed by thin layer chromatography and high performance liquid chromatography (HPLC). The HPLC analysis showed glucose concentration of 1.162 mg/mL after enzymatic hydrolysis of rice straw.

Structural and interfacial characteristics of oil bodies in coconuts (Cocos nucifera L.)

This study investigated the structural characteristics of oil bodies from mature coconut (Cocos nucifera L.) fruit. The ultrastructure and the distribution of oil bodies in coconut endosperm were investigated using cryo-scanning electron microscopy. The interfacial characteristics of the oil bodies in suspensions isolated using two different protocols were studied using confocal laser scanning microscopy (CLSM), and the oleosins stabilizing the oil bodies were characterized using sodium dodecyl sulfate polyacrylamide electrophoresis. The oil bodies were found to be preferentially accumulated in endosperm tissues away from the inner endosperm and had a polydisperse size distribution, both intracellularly and in suspensions. The CLSM of oil bodies revealed uniform distribution of proteins and phospholipids at the interface along with glycolipids. Six different proteins were found to be associated with oil bodies some of which were disulfide-linked. This work provides new insights into the structure of coconut oil bodies and mechanisms for their stabilization.

Partial characterization of digestive proteases in adults of bigclaw river shrimp Macrobrachium carcinus

The present research was focused to characterize the digestive proteases in Macrobrachium carcinus adults using biochemical and electrophoretic techniques. Our results showed that the alkaline proteolytic activity from males and females did not show significant differences ( P > 0.05) between them, the optimum pH for digestive proteases is 8, and is very stable to changes in alkaline pH (8 and 10). The optimum temperature for alkaline proteases is 45oC and is stable from 25 to 45oC. The activity was totally inhibited with phenylmethylsulfonyl fluoride (PMSF), additionally the inhibition with trypsin soybean inhibitor 1 (SBT1) and tosyl-lysine-methyl ketone (TLCK) indicate high effect over serine proteases. Eight active bands were found using sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) zymogram (range 17.8-94.0 kDa), which were partially inhibited with ovalbumin (Ovo), SBT1, phenanthroline (Phen), tosyl-phenylalanine-methyl ketone (TPCK), TLCK and ethylenediaminetetraacetic acid (EDTA), indicating an omnivorous digestive capacity, which remarks that the mainly alkaline protease in M. carcinus hepatopancreas are trypsin like enzymes.

A developed antibody–drug conjugate rituximab-vcMMAE shows a potent cytotoxic activity against CD20-positive cell line

Rituximab is a chimeric monoclonal antibody directed against B-lymphocyte specific antigen CD20, which is used for the treatment of B-cell malignancies. However, the effectiveness of rituximab is limited partly due to treatment resistance. The aim of this study was to develop rituximab-based antibody drug conjugate (ADC) to enhance rituximab activity. In this study, monomethyl auristatin E (MMAE) was covalently conjugated to dithiothreitol -reduced rituximab via a valine-citrulline peptide linker (rituximab-vcMMAE). The conjugates were then characterized by using nonreducing sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and cell-based enzyme-linked immunosorbent assay (ELISA). The cytotoxic activity of the ADC was evaluated against Raji (human B-cell lymphoma; CD20-positive) and MOLT-4 (T lymphoblast; acute lymphoblastic leukemia; CD20-negative) cell lines. In addition, the colony formation assay was used to identify the propagation ability of ADC-treated cells in vitro. Results from nonreducing SDS-PAGE revealed various species of rituximab-MC-Val-Cit-PABC-MMAE (rituximab-vcMMAE), as compared with unconjugated rituximab. The binding capacity of rituximab-vcMMAE to the CD20-positive cell was similar to that of the parental rituximab. Most importantly, our results revealed that rituximab-vcMMAE was highly potent against the CD20-positive cell line, but not against the CD20-negative cell. At the same time, rituximab-vcMMAE was able to inhibit colony formation in CD20-positive cells. These data indicate that rituximab-vcMMAE may be a highly effective and selective therapy for the treatment of B-cell lymphoma.

Aminopeptidase Modified Hydrolytic Enzymes to Improve the Efficiency of Sugar Production from Alkaline Pretreated Switchgrass

Cellulases and β-glucosidases (βGSD) are enzymes commonly used in the biofuel industry. In this study, smaller-sized variants were generated with aminopeptidase such that high catalytic capabilities were retained. Under the defined experimental conditions, the degree of hydrolysis was greater using cellulase substrates, compared to βGSD, based on ortho phthaldialdehyde (OPA) assay data (44% versus 15%). Proteolysis of cellulases was also evident based on sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) protein banding patterns seen after peptidase treatment. Residual cellulase activity was retained after peptidase hydrolysis (67% to 73%) based on standard filter paper assays. Peptidase treated cellulases and βGSD were then utilized for hydrolysis of alkaline-pretreated switchgrass (Panicum virgatum). Interestingly, the efficiency of the reaction, defined as milligrams of sugar produced per filter paper unit, was higher using truncated cellulases for bioprocessing reactions (~14%), especially in the absence of sodium azide. Conversely, incubation of βGSD with peptidase revealed minimal proteolysis with low impact on the efficiency of hydrolysis.

The effect of protein-to-alginate ratio on in vitro gastric digestion of nanoparticulated whey protein

The in vitro digestion of particles composed of nanoparticulated whey protein (unheated and heated for 85 °C for 10 min) and two types of alginate [low (40 kDa) and high (280 kDa) molecular mass] at ratios of 20:1 and 2:1 were evaluated under simulated gastric conditions at pH 3.0. The in vitro digestibility of nanoparticulated whey protein without addition of alginate was used as a control. The in vitro digestibility was characterised in terms of the amount of soluble peptide released in the supernatant after trichloroacetic acid precipitation, sodium dodecylsulphate-polyacrylamide electrophoresis and liquid chromatography-mass spectrometry. In addition, the zeta potential values of all mixtures at pH 3.0 were measured. Under simulated gastric conditions the nanoparticulated whey protein and alginate at a ratio of 2:1 showed the highest protection of whey proteins against pepsin digestion. Peptides derived from β-lactoglobulin 55–66 and 109–123 regions were resistant to 120 min of in vitro gastric digestion.

Detergent-Compatible Purified Endoglucanase from the Agro-Industrial Residue by Trichoderma harzianum under Solid State Fermentation

A robust process of purification, characterization, and application of endoglucanase from the agro-industrial waste was performed using solid state fermentation (SSF). Trichoderma harzianum as a micro-organism and wheat straw as a growth supportive substrate were used in SSF under pre-optimized conditions. The maximum activity of 480 ± 4.22 U/mL of endoglucanase was attained when a fermentation medium was inoculated using 10% inoculum size and 3% substrate concentration with pH = 5.5 at 35 °C for an optimized fermentation period. In comparison with crude extract, enzyme was 1.83-fold purified with a specific activity of 101.05 U/mg using Sephadex-G-100 column chromatography. Sodium dodecyl sulfate (SDS) poly-acrylamide gel electrophoresis revealed that the enzyme exhibited a low molecular weight of 43 kDa. The purified enzyme displayed maximum activity at pH = 6 and a temperature of 50 °C, respectively. The maximum activity (Vmax) of 156 U/mL and KM value of 63 µM were observed. Ethylenediaminetetraacetic acid (EDTA), SDS, and Hg2+ inhibited enzyme activity, while Co2+ and Mn2+ enhanced enzyme activity at 1 mM concentration. The maximum substrate affinity and specific activity of biosynthesized endoglucanase revealed that it can be potentially useful for industrial applications.

Role of ovomucoid in the gelation of a β-lactoglobulin-ovomucoid mixture

The effect of ovomucoid on gelation of β-lactoglobulin—as induced by heating and subsequent cooling—was investigated using a mixture of 5 % (w/v) ovomucoid/5 % (w/v) β-lactoglobulin and pure β-lactoglobulin solutions (5 and 10 % (w/v)) with subsequent analysis by rheological measurement, ultrasonic spectroscopy, scanning electron microscopy, and sodium dodecyl sulfate polyacrylamide electrophoresis. For the three systems, the dynamic modulus of the mixed-protein sample was smaller than that of either of the two pure β-lactoglobulin samples. Although ultrasonic-relative velocity temperature sweeps for all samples showed that the relative velocities decreased with increasing temperature, the gradient values differed. Namely, the decrease for the mixed-protein sample (12 m/s) was intermediate between those of the pure β-lactoglobulin systems. Ultrasonic attenuations of all samples increased with increasing temperature, and the absolute attenuation value of the mixed-protein sample was also intermediate between those of the two pure β-lactoglobulin samples. Electrophoresis performed with or without 2-mercaptoethanol suggested that ovomucoid forms an aggregate with β-lactoglobulin via intermolecular disulfide bonds. Together, these results suggest that ovomucoid has a synergistic effect on β-lactoglobulin gelation despite the great heat stability.

High Milk-Clotting Activity Expressed by the Newly Isolated Paenibacillus spp. Strain BD3526.

Paenibacillus spp. BD3526, a bacterium exhibiting a protein hydrolysis circle surrounded with an obvious precipitation zone on skim milk agar, was isolated from raw yak (Bos grunniens) milk collected in Tibet, China. Phylogenetic analysis based on 16S rRNA and whole genome sequence comparison indicated the isolate belong to the genus Paenibacillus. The strain BD3526 demonstrated strong ability to produce protease with milk clotting activity (MCA) in wheat bran broth. The protease with MCA was predominantly accumulated during the late-exponential phase of growth. The proteolytic activity (PA) of the BD3526 protease was 1.33-fold higher than that of the commercial R. miehei coagulant. A maximum MCA (6470 ± 281 SU mL−1) of the strain BD3526 was reached under optimal cultivation conditions. The protease with MCA was precipitated from the cultivated supernatant of wheat bran broth with ammonium sulfate and purified by anion-exchange chromatography. The molecular weight of the protease with MCA was determined as 35 kDa by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and gelatin zymography. The cleavage site of the BD3526 protease with MCA in κ-casein was located at the Met106–Ala107 bond, as determined by mass spectrometry analysis.

Comparative analysis of the seminal plasma proteomes of oligoasthenozoospermic and normozoospermic men

A comparative proteomic study of oligoasthenozoospermic and normozoospermic seminal plasmas was conducted to establish differences in protein expression. Oligoasthenozoospermia (when semen presents with a low concentration and reduced motility of spermatozoa) is common in male infertility. Two-dimensional protein maps from seminal plasma samples from 10 men with normozoospermia and 10 men with idiopathic oligoasthenozoospermia were obtained by isoelectric focusing followed by sodium dodecyl-sulphate polyacrylamide electrophoresis. Map images were analysed using dedicated software involving normalization, spot-to-spot volume comparison and statistical treatment of the results to establish the significance of differences between normal and oligoasthenozoospermic samples. Six out of 1028 spots showed over 1.5-fold relative intensity differences (P < 0.05, analysis of variance). Four proteins were identified by nano liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry of their tryptic peptides and database searches. Two proteins were more than three-fold under-expressed in oligoasthenozoospermia, namely epididymal secretory protein E1 and galectin-3-binding protein; the other (lipocalin-1 and a prolactin-inducible protein form) were over-expressed. The identity and differential expression of epididymal secretory protein E1 was verified by Western-blotting. The statistically significant differential expression of these four proteins in oligoasthenozoospermia compared with normozoospermia provides a molecular basis for further investigations into the pathogenic mechanisms underlying idiopathic oligoasthenozoospermia.

절단배추의 단기 저장에 대한 예열처리 효과

Kimchi cabbages were cut (3×3 cm), and were pre-heat treated at 40°C, and their physicochemical qualities and browning degrees were investigated during 8 weeks storage at 5°C. The Cut kimchi cabbages were treated at 40°C (1~8 hrs) and their protein bands profiles were determined by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). The 60, 39, 33, and 12 kDa bands considered heat shock proteins (HSPs) were expressed in the cut kimchi cabbage, and the 4-hr pre-heat treatment (HS 4) exhibited the strongest band ratio. The weight ratios and titratable acidities of the pre-heat treated cut kimchi cabbages were not changed so much after 8 weeks storage at 5°C, and the soluble solid contents of HS 4 decreased less than that of any other treatments. The browning degree of HS 4 after 8 week storage was also shown to be the least among the treatments. The polyphenol oxidase (PPO) activities of all treatments slightly rose during the over all storage period, in contrast with the decrease of total phenolic contents. The expression of HSPs was identified in the pre-heat treated cut kimchi cabbages, and HS 4 exhibited the best quality and appearance after 8 weeks storage at 5°C.

Purification, characterisation and cloning of a 2S albumin with DNase, RNase and antifungal activities from Putranjiva roxburghii.

The present study reports the characterisation of a novel ~12-kDa heterodimeric protein, designated as putrin, from the seeds of Putranjiva roxburghii. The purification of putrin to homogeneity was accomplished using DEAE-sepharose where protein was unbound, CM-sepharose and Cibacron blue 3GA where it was bound and appeared as single peak on a size-exclusion chromatography column. A 15 % sodium dodecyl sulphate polyacrylamide electrophoresis gel, under reducing condition, demonstrated that putrin is made of two polypeptide chains of approximately 4.5 and 7.5 kDa. Circular dichroism studies demonstrated the helical nature and conformational stability of protein at increasing temperatures. Putrin exhibited both RNase and DNase activities and exerted antifungal activity but possessed relatively weak translation-inhibitory activity in cell-free system. The cloning and sequence analysis revealed a 414 bp open reading frame encoding a preproprotein of 137 amino acid residues. The amino acid sequence comparisons and phylogenetic analysis of putrin showed significant homology to 2S seed storage family proteins. The results demonstrated that putrin belongs to 2S albumin family and exhibits a spectrum of biotechnologically exploitable functions.