Protein Fractions (protein + fraction)

Distribution by Scientific Domains


Selected Abstracts


IN VITRO DIGESTIBILITY OF CHINESE TARTARY BUCKWHEAT PROTEIN FRACTIONS: THE MICROSTRUCTURE AND MOLECULAR WEIGHT DISTRIBUTION OF THEIR HYDROLYSATES

JOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2006
XIAONA GUO
ABSTRACT Our previous study showed that in vitro pepsin digestibility of Chinese tartary buckwheat protein was relatively low compared to those of other edible seeds. In vitro pepsin digestibilities of four protein fractions of tartary buckwheat, microstructure and molecular weight (MW) distributions of hydrolysates were investigated. In vitro pepsin digestion assay showed that the digestibilities of tartary buckwheat protein fractions were albumin (81.20%), globulin (79.56%), prolamin (66.99%) and glutelin (58.09%). Scanning electron microscopy showed that albumin and globulin fractions were digested by pitting from the outer surface to the inner part and were more digestible, while prolamin and glutelin fractions resisted digestion because only the outer surfaces of their protein bodies were digested and the interior was protected. MW distribution of the hydrolysates from the four protein fractions was determined by high-performance liquid chromatography. The hydrolysates of albumin mainly consisted of polypeptides with lower MW. The hydrolysates of glutelin had larger polypeptides together with small and medium-sized peptide fractions. [source]


CE-TOF MS analysis of complex protein hydrolyzates from genetically modified soybeans , A tool for foodomics

ELECTROPHORESIS, Issue 7 2010
Carolina Simó
Abstract A CE-TOF MS proteomic approach was applied for the analysis of hydrolyzates from complex soybean protein mixtures. After CE-TOF MS method development, the new approach provided the simultaneous analysis of more than 150 peptides from the soybean protein fraction soluble in ACN-water (80/20,v/v). The method is fast (about 30,min of analysis per sample) and is characterized by a relatively low running cost. The approach was used to study the substantial equivalence between a genetically modified variety of soybean compared with its traditional counterpart. No significant differences were found between the two studied soybeans based on the protein fraction studied. The capacity of the CE-TOF MS method to analyze complex mixtures of peptides in short times opens interesting possibilities in the growing Foodomics area. [source]


Multiple polypeptide forms observed in two-dimensional gels of Methylococcus capsulatus (Bath) polypeptides are generated during the separation procedure

ELECTROPHORESIS, Issue 4 2003
Frode S. Berven
Abstract We have examined two-dimensional electrophoresis (2-DE) gel maps of polypeptides from the Gram-negative bacterium Methylococcus capsulatus (Bath) and found the same widespread trains of spots as often reported in 2-DE gels of polypeptides of other Gram-negative bacteria. Some of the trains of polypeptides, both from the outer membrane and soluble protein fraction, were shown to be generated during the separation procedure of 2-DE, and not by covalent post-translational modifications. The trains were found to be regenerated when rerunning individual polypeptide spots. The polypeptides analysed giving this type of trains were all found to be classified as stable polypeptides according to the instability index of Guruprasad et al. (Protein Eng. 1990, 4, 155,161). The phenomenon most likely reflects conformational equilibria of polypeptides arising from the experimental conditions used, and is a clear drawback of the standard 2-DE procedure, making the gel picture unnecessarily complex to analyse. [source]


Effects of 4-nonylphenol on the endocrine system of the shore crab, Carcinus maenas

ENVIRONMENTAL TOXICOLOGY, Issue 3 2008
Christina M. Lye
Abstract There is a considerable body of evidence to suggest that many anthropogenic chemicals, most notably xeno-estrogens, are able to disrupt the endocrine system of vertebrates. There have been few comparable studies on the effects of exposure to these chemicals that may serve as biomarkers of endocrine disruption in aquatic invertebrate species. In addition, the evidence available is complex, conflicting, and far from conclusive. The present study aimed to investigate the impact of the xeno-estrogen 4-nonylphenol (4-NP, nominal concentrations 10,100 ,g L,1) on the regulation and functioning of the endocrine system of the shore crab Carcinus maenas. It also set out to establish whether 4-NP are causing the effects (i.e., changes of exoskeletons including secondary sexual characteristics, pheromonally mediated behavior and ecdysone levels, and the presence of vt in the male hepatopancreas) found recently in wild shore crabs (Lye et al.,2005). The study utilizes morphological (e.g., gonadosomatic and hepatosomatic indices) and hormonal (ecdysteroid moulting hormone levels and the induction of female specific proteins, vitellins) biomarkers using radioimmunoassay and an indirect enzyme linked immunosorbent assay applied to the soluble protein fraction of adult male hepatopancreatic homogenates. Exposure of C. maenas to an effective concentration as low as 1.5 ,g L,1 4-NP resulted in a reduced testis weight, increased liver weight, and altered levels of ecdysone equivalents compared to controls. Induction of vitellin-like proteins was absent in all samples tested. The ecological implications and the possible mechanisms for the action of 4-NP on the response of the shore crab to xeno-estrogen exposure are discussed. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source]


Biological significance of metals partitioned to subcellular fractions within earthworms (Aporrectodea caliginosa),

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2006
Martina G. Vijver
Abstract Metal ions in excess of metabolic requirements are potentially toxic and must be removed from the vicinity of important biological molecules to protect organisms from adverse effects. Correspondingly, metals are sequestrated in various forms, defining the accumulation pattern and the magnitude of steady-state levels reached. To investigate the subcellular fractions over which Ca, Mg, Fe, Cu, Zn, Cd, Pb, Ni, and As are distributed, earthworms (Aporrectodea caliginosa) collected from the field were analyzed by isolating metal-rich granules and tissue fragments from intracellular microsomal and cytosolic fractions (i.e., heat-stable proteins and heat-denatured proteins). The fractions showed metal-specific binding capacity. Cadmium was mainly retrieved from the protein fractions. Copper was equally distributed over the protein fraction and the fraction comprising tissue fragments, cell membranes, and intact cells. Zinc, Ca, Mg, and As were mainly found in this fraction as well. Lead, Fe, and Ni were mainly isolated from the granular fraction. To study accumulation kinetics in the different fractions, three experiments were conducted in which earthworms were exposed to metal-spiked soil and a soil contaminated by anthropogenic inputs and, indigenous earthworms were exposed to field soils. Although kinetics showed variation, linear uptake and steady-state types of accumulation patterns could be understood according to subcellular compartmentalization. For risk assessment purposes, subcellular distribution of metals might allow for a more precise estimate of effects than total body burden. Identification of subcellular partitioning appears useful in determining the biological significance of steady-state levels reached in animals. [source]


Urinary pheromones promote ERK/Akt phosphorylation, regeneration and survival of vomeronasal (V2R) neurons

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006
Jing Xia
Abstract The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are continually replaced throughout the lifetime of the mouse. Moreover, active signalling of V2Rs via the transient receptor potential 2(TRPC2) channel is necessary for regeneration of receptors, as the TRPC2 null mutant mouse showed a 75% reduction of V2Rs by the age of two months. Here we describe V2R mediated signalling in a neuronal line established from vomeronasal stem cells taken from postnatal female mice. Cells were immunoreactive for G,o and V2R, whereas V1R and G,i immunoreactivity could not be detected. Biological ligands (dilute urine and its protein fractions) were found to increase proliferation and survival of these neurons. Dilute mouse urine but not artificial urine also induced ERK, Akt and CREB signalling in a dose dependent way. The volatile fraction of male mouse urine alone was without effect while the fraction containing peptides (> 5 kDa) also stimulated ERK and Akt phosphorylation. The ERK, Akt and CREB phosphorylation response was sensitive to pertussis toxin, confirming the involvement of V2R linked G,o. Dilute mouse urine or its high molecular weight protein fraction increased survival and proliferation of these neurons. Hence, urinary pheromones, which signal important social information via mature neurons, also promote survival and proliferation of their regenerating precursors. These data show that regenerating V2Rs respond to urine and the urinary peptides by activation of the Ras-ERK and PI3-Akt pathways, which appear to be important for vomeronasal neural survival and proliferation. [source]


Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin

IMMUNOLOGY, Issue 2 2003
Claudio Rhyner
Summary Coeliac disease (CD), a gastrointestinal illness characterized by intestinal malabsorption, results from gluten intolerance accompanied with immunological responses towards gliadin, an ethanol-soluble protein fraction of wheat and other cereals. The role of gliadin in eliciting immune responses in CD is still partly unclear; however, the occurrence of anti-gliadin in the sera of patients suffering from CD correlates well with clinical symptoms. In this work we report the construction of isotype-specific, phage-displayed scFv libraries from peripheral blood lymphocytes of a patient with CD and from a healthy control individual. VH and VL chains were amplified by reverse transcription,polymerase chain reaction (RT,PCR) using a set of oligonucleotides recognizing all human variable gene families. The three scFv libraries (IgA, IgG and IgM) were selectively enriched for gliadin-binding phage. After four rounds of affinity selection, polyclonal enrichment of gliadin-binding phage was observed in all libraries from the CD patient but in none from the healthy donor. Phagemid particles generated from single clones were demonstrated to be gliadin-specific, as shown by strongly positive enzyme-linked immunosorbent assay (ELISA) and BiaCore signals. The VH and VL chains from samples of these monoclonal isotype-specific phage were sequenced to identify the most common variable regions used by the immune system to elicit antibody responses against gliadin. [source]


A teratocyte gene from a parasitic wasp that is associated with inhibition of insect growth and development inhibits host protein synthesis

INSECT MOLECULAR BIOLOGY, Issue 5 2003
D. L. Dahlman
Abstract After parasitization, some wasps induce hosts prematurely to initiate metamorphic development that is then suspended in a postwandering, prepupal state. Following egression of the parasite larva, the host remains in this developmentally arrested state until death. Teratocytes, cells released at egg hatch from extra-embryonic serosal membranes of some wasp parasites, inhibit growth and development when injected into host larvae independent of other parasite factors (e.g. venom, polydnavirus). Synthesis of some developmentally regulated, abundantly expressed Heliothis virescens host proteins is inhibited in hosts parasitized by Microplitis croceipes and by teratocyte injection. A cDNA encoding a 13.9 kDa protein (TSP14) that inhibited protein synthesis, growth and development was isolated from a protein fraction secreted by teratocytes. TSP14 appears to be responsible, in part, for the teratocyte-mediated inhibition of host growth and development. Interestingly, this cDNA encoded a cysteine-rich amino acid motif similar to that described from Campoletis sonorensis polydnavirus, a mutualistic virus that enables wasp parasitization of lepidopteran larvae. Moreover, TSP14 inhibited protein synthesis in a dose-dependent manner in rabbit reticulocyte lysate and wheat germ extract translation systems. We hypothesize that some wasp parasites inhibit translation as a general means to regulate and redirect lepidopteran host physiology to support endoparasite development. [source]


The effect of wheat ,-amylase inhibitors incorporated into wheat-based artificial diets on development of Sitophilus granarius L., Tribolium confusum Duv., and Ephestia kuehniella Zell

JOURNAL OF APPLIED ENTOMOLOGY, Issue 4 2002
J. R. Warchalewski
Artificial grain kernels made from ground wheat grain, commercial wheat starch and wheat proteinaceous ,-amylase inhibitors were used as diets for adults of the granary weevil (Sitophilus granarius L). For the confused flour beetle (Tribolium confusum Duv.) and the Mediterranean flour moth (Ephesitia kuehniella Zell.), a friable mixture of the diets was used. The results of feeding trials showed that the survival of S. granarius adults was not correlated with the soluble proteins extracted from wheat and amylolytic activity located in this protein fraction. On the other hand, the weight of dust (the index of feeding intensity) produced during feeding depended on the presence of ,-amylase and trypsin inhibitors in wheat-based diets. Ephesitia kuehniella larvae did not develop at all on a diet consisting of 50% wheat starch and 50% crude ,-amylase inhibitors from wheat. The same diet lengthened the development time of T. confusum larvae by 15.1 days. These results attest to the existence of a specific native enzymatic apparatus in the alimentary canals of these three grain pests. However, the highly active insect ,-amylase inhibitors appear to have a limited influence on the developmental parameters studied although some reduction of insects populations might be expected. [source]


Urinary protein fraction in healthy subjects using cellulose acetate membrane electrophoresis followed by colloidal silver staining

JOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 4 2004
Ryoko Machii
Abstract We previously reported a rapid and highly sensitive colloidal silver staining solution suitable for the cellulose acetate membrane. This method was useful for detecting even very small amounts of urinary protein. In the present study, we examined urinary protein fractions in healthy subjects, using cellulose acetate membrane electrophoresis (CAE) with a highly sensitive colloidal silver staining, in an attempt to determine the clinical relevance of urinary protein fractions. Sixty unconcentrated spot urine specimens were analyzed by CAE and calculated by densitometry. All of the samples were separated into five fractions by CAE. The mean±1 SD of the percentage of five fractions was 28.37±8.51 in albumin, 4.30±4.19 in ,1 -globulin, 14.41±6.14 in ,2 -globulin, 19.45±7.10 in ,-globulin, and 33.46±8.24 in ,-globulin. The albumin/globulin (A/G) ratio was 0.41±0.17. These six items and the concentrations of total protein, albumin, and ,- N -acetyl- D -glucosaminidase (NAG) did not significantly differ between males and females. NAG is the marker of tubulointerstitial nephropathy. The results suggest that there are no gender-dependent differences in the urinary protein fractions of healthy subjects. J. Clin. Lab. Anal. 18:231,236, 2004. © 2004 Wiley-Liss, Inc. [source]


Soluble Albumin and Biological Value of Protein in Cocoa (Theobroma cacao L.) Beans as a Function of Roasting Time

JOURNAL OF FOOD SCIENCE, Issue 4 2005
Luis Abecia-Soria
ABSTRACT: An association has been identified between the extent of roasting and the amount of extractable protein from the cocoa bean, its nutritive value, and the sensorial quality of the liquor. Cocoa nibs from fermented seeds (Theobroma cacao L.) were precision-roasted at 150°C for 0, 30, 34, 38, 42, and 46 min and the protein fraction extracted. From the beginning of roasting, until minute 38, about 87% of the proteins were extractable, but the extractability substantially decreased to 72.7% at 42 min and to 65.3% at 46 min. Both total soluble protein determination and albumin concentration of the roasted nibs diminished slightly until minute 38, when acceptable sensory characteristics were obtained for the liquor. Both total nitrogen and capillary-electrophoretic separation and quantification of the albumin showed that the amounts of extractable protein in this fraction consistently followed a cyclic pattern until minute 42, irreversibly decreasing thereafter. Biological evaluation of the protein from the cocoa nibs roasted for the various times showed that at the point that the sensory rating approached that of a commercial liquor, the albumin content and nutritive value were still high. The findings suggest that with moderate, uniform roasting it may not be necessary to sacrifice the protein's biological value for the sensorial attributes of chocolate in a standard commercial roast. [source]


A Lipoprotein-derived Antimicrobial Factor from Hen-egg Yolk is Active Against Streptococcus Species

JOURNAL OF FOOD SCIENCE, Issue 8 2002
D. Brady
ABSTRACT: Oral administration of hen-egg yolk provides protection against specific pathogens. We examined the antibacterial activity of fractionated egg yolk against 2 pathogenic Streptococcus strains, using an in vitro assay. A water-soluble protein fraction (WSPF) of egg yolk consistently inhibited the growth of S. mutans by 25%. The WSPF treated with pancreatin demonstrated > 80% inhibition of bacterial growth. Growth of S. sanguis was completely inhibited. Gel filtration and ion exchange chromatography established that anti-Streptococcal activity resided with lipoproteins. Antibacterial activity was released by crude lipase or a combination of lipase and protease treatment of egg lipoproteins. Thus, hen-egg yolk lipoproteins are important molecules for lipid-mediated antimicrobial activity. [source]


Characterization of in vitro and in vivo metabolic pathways of the investigational anticancer agent, 2-methoxyestradiol

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2007
Nehal J. Lakhani
Abstract The aim of this study was to characterize the metabolic pathways of 2-methoxyestradiol (2ME2), an investigational anticancer drug. In vitro metabolism studies were performed by incubation of 2ME2 with human liver microsomes under various conditions and metabolite identification was performed using liquid chromatography-tandem mass spectrometry. In microsomal mixtures, four major oxidative metabolites and two glucuronic acid conjugates were observed originating from 2ME2. Human liver S9 protein fraction was used to screen for in vitro sulfation but no prominent conjugates were observed. The total hepatic clearance as estimated using the well-stirred model was approximately 712 mL/min. In vivo metabolism, assessed using 24-h collections of urine from cancer patients treated with 2ME2 revealed that <0.01% of the total administered dose of 2ME2 is excreted unchanged in urine and about 1% excreted as glucuronides. Collectively, this suggests that glucuronidation and subsequent urinary excretion are elimination pathways for 2ME2. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 1821,1831, 2007 [source]


Milk production of dairy goats fed diets with different legume seeds: Effects of amino acid composition of the rumen undegradable protein fraction

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 13 2008
E Ramos Morales
Abstract BACKGROUND: To establish the effect of the nature of four different legume seeds, lupins (L), faba beans (FB), bitter vetch (BV) and vetch (V) on the production and composition of goat milk, we studied the ruminal degradation of these legumes, and the amino acid (AA) composition of the seeds and that of the undegradable fractions of the protein sources. Four isonitrogenous and isoenergetic diets were designed, and in each case 30% of the protein was supplied by one of the different legume seeds. A group of eight Granadina goats was used to study the utilisation of these diets for milk production. The goats were allocated to a replicated 4 × 4 Latin square. RESULTS: Ruminal fermentation modified the AA profile of the protein of the legume seeds which was different in each case. However, milk production remained unchanged irrespective of the diet consumed. On the contrary, and despite the high level of degradability of the protein in the different legumes, the concentration of total protein, casein (CN) and overall ,S1 -CN and ,S2 -CN in milk depended on the protein source used. CONCLUSION: The concentration of total protein in milk, as well as its composition, was apparently determined by the AA composition of the corresponding fraction of the rumen-undegradable protein. Copyright © 2008 Society of Chemical Industry [source]


In vitro determination of digestible and unavailable protein in edible seaweeds

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 15 2002
Isabel Goñi
Abstract Edible seaweeds are considered a complementary source of food protein for human and animal nutrition. The physiological effects of seaweed protein depend on the degree of enzymatic digestion of protein in the small intestine and bacterial fermentation in the large intestine. The objective of this work was to estimate total, digestible, fermentable and unavailable protein in some red and brown seaweeds. Brown seaweeds Fucus vesiculosus, Laminaria digitata and Undaria pinnatifida and red seaweeds Chondrus crispus and Porphyra tenera were treated with pepsin and pancreatin to separate digestible protein. Residues containing indigestible protein were inoculated for 24,h with rat caecal droppings, and protein contents were evaluated in the non-fermented residue. Protein content in the seaweeds ranged from 8.9 to 25% of dry matter. Digestible protein was the major protein fraction (69%) only in P tenera; in the other seaweeds, this fraction ranged from 15 to 45%. Significant amounts of unavailable protein were found in all samples (2,24%). The distribution of total protein among the three fractions, ie digestible, fermentable and unavailable protein, could yield information about the physiological and metabolic consequences of the intake of seaweed proteins. © 2002 Society of Chemical Industry [source]


Chemical composition and ruminal degradability of lucerne (Medicago sativa) products

JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 15 2001
Arif F Mustafa
Abstract A study was conducted to determine the chemical composition and in situ nutrient ruminal degradability of three lucerne products. These were dehydrated pellets, sun-cured pellets and cubes. Results of the chemical analysis showed that sun-cured pellets had the highest (P,<,0.05) neutral and acid detergent fibre as well as total carbohydrate levels, followed by cubes and dehydrated pellets respectively. Crude protein (CP) content was highest (P,<,0.05) for dehydrated pellets (204.3,g,kg,1), intermediate for sun-cured pellets (160.0,g,kg,1) and lowest for cubes (153.2,g,kg,1). Intermediately degradable CP (buffer-insoluble CP minus neutral detergent-insoluble CP) was the main protein fraction in the three products and was higher (P,<,0.05) in cubes than in dehydrated and sun-cured pellets. Estimated net energy of lactation was highest (P,<,0.05) for dehydrated pellets (5.9,MJ,kg,1), intermediate for cubes (5.23,MJ,kg,1) and lowest (P,<,0.05) for sun-cured pellets (5.15,MJ,kg,1). Results of the in situ experiment indicated that dehydrated pellets had higher (P,<,0.05) ruminal protein degradability than sun-cured pellets and cubes. The estimated ruminal escape protein values for dehydrated pellets, sun-cured pellets and cubes were 361, 420 and 498,g,kg,1 CP respectively. It was concluded that differences in chemical composition and ruminal degradability among the three lucerne products were mainly due to differences in stage of maturity. It was also concluded that the dehydration process failed to increase the ruminal escape protein value of dehydrated pellets relative to sun-cured pellets and cubes. © 2001 Society of Chemical Industry [source]


Performance of Juvenile Coho Salmon Oncorhynchus kisutch Fed Diets Containing Meals from Fish Wastes, Deboned Fish Wastes, or Skin-and-Bone By-Product as the Protein Ingredient

JOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 1 2001
Cindra K. Rathbone
The suitability of meals derived from fish processing wastes as the protein fraction in practical diets for hatchery-reared coho salmon was investigated. The study compared the performance of coho salmon fed diets containing three products: a skin-and-bone meal (SB), a deboned meal (DM), and a whole-fish meal (WM) made directly from the fish wastes. A commercial trout diet (CO) was fed to a fourth treatment group. Diets were fed at 3% of body weight per day to juvenile coho salmon for 12 wk. Survival (> 94%) was not significantly different among treatment groups. Average fish weight, feed conversion ratio, whole body proximate and mineral composition, and protein and phosphorus retention were compared. There were no significant differences after 12 wk of feeding in fish weight between WM, DM, and CO, but SB had significantly lower weight and whole body lipid, and significantly higher ash. Compared to WM, DM had a significantly lower feed conversion ratio and higher retention of protein and phosphorus, but these indices were not significantly different from CO. It is concluded that DM is a potentially superior protein ingredient compared to WM, while specific characteristics of SB will limit its use as a protein source in feeds for salmonids. However, SB may prove to be a suitable mineral supplement when added at a low level. Utilization of fish processing wastes in salmonid diets could be a commercially viable alternative to direct disposal of processing wastes. [source]


Expression of a novel T-complex testis expressed 5 (Tctex5) in mouse testis, epididymis, and spermatozoa

MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 9 2007
Y.B. Han
Abstract Expression of T-complex testis expressed 5 (Tctex5), an orthologue of protein phosphatase-1 inhibitor-3 (PPP1R11), was enhanced in mouse testis and was also expressed in epididymis and spermatozoa. There were three transcripts of Tctex5 including one brain specific and two common transcripts dominant in mouse testis. Tctex5 protein isoforms (75, 52, 32, 25, and 14.3 kDa) were identified. Isoforms of 75 and 52 kDa were spermatogenic-specific and were found in protein fraction containing nuclei, mitochondria, and flagellum accessory, and also in protein fraction containing mainly membranes. Tctex5 was localized in nuclei of pachytene spermatocytes, round spermatocytes, cytoplasm of Sertoli cells in testis; cilia, secretion bodies and nuclei of epithelial cells and interstitium smooth muscle cells in epididymis; and head and principal piece of tail in epididymal spermatozoa. The results suggested that Tctex5 might be a specific protein phosphatase-1 inhibitor in sperm; various Tctex5 transcripts and isoforms and cellular locations imply its different roles in spermatogenesis. Nuclei-type isoforms (75 and 52 kDa) might take part in nucleus remodeling during spermatogenesis whilst membrane-type isoform (52 kDa) might be responsible for dephosphorylation of proteins during capacitation. The other isoforms might play general roles for all kinds of cell types. Mol. Reprod. Dev. 74: 1132,1140, 2007. © 2007 Wiley-Liss, Inc. [source]


Membranous expression of glucose transporter-1 protein (GLUT-1) in embryonal neoplasms of the central nervous system

NEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 1 2000
M. Loda
The human erythrocyte GLUT-1 is a transmembrane protein which facilitates transport of glucose in the cell in an energy-independent fashion. Neuroectodermal stem cells show strong membrane immunoreactivitry with this marker at early developmental stages in rodents. Membranous expression by undifferentiated neuroectodermal cells gradually decreases while GLUT-1 becomes confined to the endothelial cells, when these acquire blood,brain barrier function. We thus sought to determine whether GLUT-1 expression was limited to embryonal neoplasms of the central nervous system (CNS) which are presumably derived from developmentally arrested neuroectodermal stem cells. Archival material of 40 primary CNS neoplasms were examined for immunoreactivity with anti-GLUT-1. This included both non-embryonal neoplasms (18 astrocytic tumours, one ependymoma and three oligodendroglioma) and embryonal neoplasms (12 cerebellar medulloblastomas, four supratentorial PNETs and two atypical teratoid/rhabdoid tumours (AT/RhT)). In addition, cell lines and nude mice xenografts derived from both undifferentiated and differentiated tumours were assessed for GLUT-1 immunoreactivity by both immunohistochemistry and Western blotting. All embryonal tumours, MBs and PNET xenografts consistently showed GLUT-1 membrane staining. Non-embryonal neoplasms were negative except for vascular staining. Membrane protein fraction of embryonal tumours cell lines immunoreacted by immunoblot with GLUT-1, whereas the glioblastoma cell line was negative. Expression of GLUT-1 supports the stem cell nature of the cells of origin of MBs, supratentorial PNET and AT/RhTs. As a result, GLUT-1 is a useful marker to define the embryonal nature of CNS neoplasms. [source]


The protein fraction of Phyllanthus niruri plays a protective role against acetaminophen induced hepatic disorder via its antioxidant properties

PHYTOTHERAPY RESEARCH, Issue 7 2006
Rajesh Bhattacharjee
Abstract The aim of this study was to investigate the hepatoprotective action of the protein fraction of Phyllanthus niruri against acetaminophen (APAP) hepatotoxicity. The partially purified protein fraction of P. niruri was injected intraperitoneally in mice either prior to (preventive) or after the induction of toxicity (curative). Levels of different liver marker enzymes in serum and different antioxidant enzymes, as well as lipid peroxidation in total liver homogenates were measured in normal, control (toxicity induced) and P. niruri protein fraction-treated mice. P. niruri significantly reduced the elevated glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) levels in the sera of toxicity induced mice, compared with the control group. Lipid peroxidation levels were also reduced in mice treated with P. niruri protein fraction compared with the APAP treated control group. Among the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione,S-transferase (GST) levels were restored to almost normal levels compared with the control group. P. niruri treatment also enhanced reduced hepatic glutathione (GSH) levels caused by APAP administration. The results demonstrated that the protein fraction of P. niruri protected liver tissues against oxidative stress in mice, probably acting by increasing antioxidative defense. Copyright © 2006 John Wiley & Sons, Ltd. [source]


Sialylation enhancement of CTLA4-Ig fusion protein in Chinese hamster ovary cells by dexamethasone

BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010
Ying Jing
Abstract The importance of glycoprotein sialic acid levels is well known, as increased levels have been shown to increase in vivo serum half-life profiles. Here we demonstrate for the first time that dexamethasone (DEX) was capable of improving the sialylation of a CTLA4-Ig fusion protein produced by Chinese hamster ovary (CHO) cells. DEX was shown to enhance the intracellular addition of sialic acid by sialyltransferases as well as reduce extracellular removal of sialic acid by sialidase cleavage. We illustrated that DEX addition resulted in increased expression of the glycosyltransferases ,2,3-sialyltransferase (,2,3-ST) and ,1,4-galactosyltransferase (,1,4-GT) in CHO cells. Based upon our previous results showing DEX addition increased culture cell viability, we confirmed here that cultures treated with DEX also resulted in decreased sialidase activity. Addition of the glucocorticoid receptor (GR) antagonist mifepristone (RU-486) was capable of blocking the increase in sialylation by DEX which further supports that DEX affected sialylation as well as provides evidence that the sialylation enhancement effects of DEX on recombinant CHO cells occurred through the GR. Finally, the effects of DEX on increasing sialylation were then confirmed in 5-L controlled bioreactors. Addition of 1,µM DEX to the bioreactors on day 2 resulted in harvests with average increases of 16.2% for total sialic acid content and 15.8% in the protein fraction with N-linked sialylation. DEX was found to be a simple and effective method for increasing sialylation of this CTLA4-Ig fusion protein expressed in CHO cells. Biotechnol. Bioeng. 2010;107: 488,496. © 2010 Wiley Periodicals, Inc. [source]


Identification of two pistachio allergens, Pis v 1 and Pis v 2, belonging to the 2S albumin and 11S globulin family

CLINICAL & EXPERIMENTAL ALLERGY, Issue 6 2009
K. Ahn
Summary Background IgE-mediated allergic reactions to pistachio appear to be occurring more frequently; however, little is known about its allergenic proteins. Objective We attempted to identify pistachio allergens and to clone the encoding genes. Methods Pistachio proteins were extracted and separated by SDS-PAGE. Immunolabelling was performed with sera from 28 pistachio-allergic individuals. Proteins of interest were further analysed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS). In parallel, a cDNA library was generated from immature pistachios and screened with primers designed on the basis of internal sequences and peptide spectra. Full-length cDNA clones were isolated from the library and sequenced. Recombinant proteins were expressed and tested with sera from pistachio-allergic patients. Results Nineteen out of 28 patients (68%) showed IgE binding to a 7 kDa protein fraction, while 14 (50%) showed specific IgE to a 32 kDa protein fraction. Analysis by Edman sequencing and MS/MS revealed that these proteins were homologue to the cashew nut allergens Ana o 3 and Ana o 2, respectively. Screening of the pistachio cDNA library resulted in isolation of novel protein cDNAs. Open-reading frame translation provided the complete amino acid sequences of two new allergenic pistachio proteins. Recombinant proteins were recognized by six out of six selected patients. Therefore, these new allergens were named Pis v 1 and Pis v 2 by the Allergen Nomenclature Subcommittee. Conclusion Novel allergens in pistachio, Pis v 1 and Pis v 2, which belong to 2S albumin and 11S globulin family, respectively, were isolated and the genes encoding these allergens were identified. [source]


Heat shock protein translocation and expression response is attenuated in response to repeated eccentric exercise

ACTA PHYSIOLOGICA, Issue 3 2009
K. Vissing
Abstract Aim:, This study hypothesized that heat shock protein (HSP) translocation and upregulation is more probable to occur after eccentric exercise than after concentric exercise or repeated eccentric exercise. Methods:, Fourteen young, healthy, untrained male subjects completed two bench-stepping exercise bouts with 8 weeks between bouts, and were compared with a control group (n = 6). Muscle biopsies collected from m. vastus lateralis of both legs prior to and at 3 h, 24 h and 7 days after exercise were quantified for mRNA levels and/or for HSP27, ,,-crystallin and inducible HSP70 content in cytosolic and cytoskeletal protein fractions. Results:, The first bout of exercise reduced muscle strength and increased muscle soreness predominantly in the eccentric leg (P < 0.05). These responses were attenuated after the repeated eccentric exercise bout (P < 0.05), suggesting a repeated bout adaptation. Increases in inducible HSP70 and HSP27 protein content in cytoskeletal fractions were observed exclusively after eccentric exercise (P < 0.05). For HSP27, an approx. 10-fold upregulation after first-bout eccentric exercise was attenuated to a an approximately fourfold upregulation after the repeated eccentric exercise bout. mRNA levels for HSP70, HSP27 and ,,-crystallin were upregulated within approximately two to fourfold ranges at time points 3 and 24 h post-exercise (P < 0.05). This upregulation was induced exclusively by eccentric exercise but with a tendency to attenuated expression 3 h after the repeated eccentric exercise bout. Conclusion:, Our results show that HSP translocation and expression responses are induced by muscle damaging exercise, and suggest that such HSP responses are closely related to the extent of muscle damage. [source]


Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampus

ELECTROPHORESIS, Issue 11 2010
Seok Heo
Abstract Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate,aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. ModiroÔ v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease. [source]


Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics

ENVIRONMENTAL MICROBIOLOGY, Issue 12 2003
Catalina Arevalo-Ferro
Summary The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N -acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem. [source]


Subcellular distribution of zinc in Daphnia magna and implication for toxicity

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2010
Wen-Xiong Wang
Abstract We examined the subcellular partitioning of zinc (Zn) in Daphnia magna both under acute and chronic exposures. In the acute Zn toxicity tests, the daphnids were exposed to different Zn concentrations for 48,h or to one lethal concentration (1,000,µg/L) for different durations (time to death for up to 47,h). Significant mortality of daphnids was observed when the newly accumulated Zn concentration reached a threshold level of approximately 40,µg/g wet weight (or 320,µg/g dry wt), approximately 3.5 times higher than the background tissue concentration (92,µg/g dry wt). Chronic exposure (14 d) to Zn resulted in nonobservable effect on survivorship and growth at newly accumulated tissue concentration of over 40,µg/g wet weight. With increasing Zn acute exposure, more Zn was partitioned into the cellular debris fraction, indicating that this fraction was presumably the first targeted site of binding for Zn upon entering the animals. The importance of other subcellular fractions either decreased accordingly or remained comparable. We found that the metal-sensitive fraction (Zn distribution in the organelles and heat-denatured proteins) did not predict the acute Zn toxicity in Daphnia. During chronic exposure, however, no major change of the subcellular partitioning of Zn with increasing Zn exposure was documented. Zinc was mainly found in the organelles and heat-stable protein fractions during chronic exposure, suggesting that any subcellular repartitioning occurred primarily during acute exposure. Metallothioneins were induced upon chronic Zn exposure, but its induction evidently lagged behind the Zn accumulation. Our present study showed that the subcellular fractionation approach could not be readily used to predict the acute and chronic toxicities of Zn in Daphnia. A tissue-based Zn accumulation approach with a threshold Zn tissue concentration was better in predicting acute Zn toxicity. Environ. Toxicol. Chem. 2010; 29:1841,1848. © 2010 SETAC [source]


Biological significance of metals partitioned to subcellular fractions within earthworms (Aporrectodea caliginosa),

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2006
Martina G. Vijver
Abstract Metal ions in excess of metabolic requirements are potentially toxic and must be removed from the vicinity of important biological molecules to protect organisms from adverse effects. Correspondingly, metals are sequestrated in various forms, defining the accumulation pattern and the magnitude of steady-state levels reached. To investigate the subcellular fractions over which Ca, Mg, Fe, Cu, Zn, Cd, Pb, Ni, and As are distributed, earthworms (Aporrectodea caliginosa) collected from the field were analyzed by isolating metal-rich granules and tissue fragments from intracellular microsomal and cytosolic fractions (i.e., heat-stable proteins and heat-denatured proteins). The fractions showed metal-specific binding capacity. Cadmium was mainly retrieved from the protein fractions. Copper was equally distributed over the protein fraction and the fraction comprising tissue fragments, cell membranes, and intact cells. Zinc, Ca, Mg, and As were mainly found in this fraction as well. Lead, Fe, and Ni were mainly isolated from the granular fraction. To study accumulation kinetics in the different fractions, three experiments were conducted in which earthworms were exposed to metal-spiked soil and a soil contaminated by anthropogenic inputs and, indigenous earthworms were exposed to field soils. Although kinetics showed variation, linear uptake and steady-state types of accumulation patterns could be understood according to subcellular compartmentalization. For risk assessment purposes, subcellular distribution of metals might allow for a more precise estimate of effects than total body burden. Identification of subcellular partitioning appears useful in determining the biological significance of steady-state levels reached in animals. [source]


Urinary pheromones promote ERK/Akt phosphorylation, regeneration and survival of vomeronasal (V2R) neurons

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006
Jing Xia
Abstract The G protein-coupled pheromone receptor neurons (V1R and V2R) of the vomeronasal organ (VNO) are continually replaced throughout the lifetime of the mouse. Moreover, active signalling of V2Rs via the transient receptor potential 2(TRPC2) channel is necessary for regeneration of receptors, as the TRPC2 null mutant mouse showed a 75% reduction of V2Rs by the age of two months. Here we describe V2R mediated signalling in a neuronal line established from vomeronasal stem cells taken from postnatal female mice. Cells were immunoreactive for G,o and V2R, whereas V1R and G,i immunoreactivity could not be detected. Biological ligands (dilute urine and its protein fractions) were found to increase proliferation and survival of these neurons. Dilute mouse urine but not artificial urine also induced ERK, Akt and CREB signalling in a dose dependent way. The volatile fraction of male mouse urine alone was without effect while the fraction containing peptides (> 5 kDa) also stimulated ERK and Akt phosphorylation. The ERK, Akt and CREB phosphorylation response was sensitive to pertussis toxin, confirming the involvement of V2R linked G,o. Dilute mouse urine or its high molecular weight protein fraction increased survival and proliferation of these neurons. Hence, urinary pheromones, which signal important social information via mature neurons, also promote survival and proliferation of their regenerating precursors. These data show that regenerating V2Rs respond to urine and the urinary peptides by activation of the Ras-ERK and PI3-Akt pathways, which appear to be important for vomeronasal neural survival and proliferation. [source]


Fat and protein contents, acidity and somatic cell counts in bulk milk of Holstein cows in the Khorasan Razavi Province, Iran

INTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 1 2009
MASOUD NAJAF NAJAFI
Relationships between total bulk milk somatic cell score (SCS) and milk fat and protein contents and acidity were investigated in the Khorasan Razavi Province, a region that contributes 6.83% of total milk production in Iran. A total of 1476 samples were analysed. Data were obtained by randomly collecting 123 samples of bulk tank milk from 41 dairy farms during April 2006 to March 2007, every month. Milk was analysed for titratable acidity, protein and fat contents and somatic cell counts (direct microscopic cell count and with Somatos, Russia). Microscopic and Somatos somatic cell counts were comparable. Results showed that the season of raw milk production did not have a significant effect on acidity. Milk fat content increased gradually from spring to winter and there were significant differences (P < 0.05) between spring and other seasons. Higher levels of milk protein fractions were observed during the autumn and winter than in other seasons. The highest total bulk milk somatic cell counts were observed in July. Total bulk milk SCS had significant effects (P < 0.05) on acidity and fat and protein contents. Moreover, the level of acidity and fat in milk decreased with increasing SCS. A significant positive relationship was observed between total bulk milk SCS and the protein content of milk. Elevated SCS were associated with lowered milk quality in Holsteins in the Khorasan Razavi Province. [source]


Processing of extended shelf life milk using microfiltration

INTERNATIONAL JOURNAL OF DAIRY TECHNOLOGY, Issue 4 2006
WOLFGANG HOFFMANN
Extended shelf life (ESL) milk was processed with integrated microfiltration (pore size 1.4 µm). The germ-enriched retentate was not used for the final whole milk. Microfiltration led only to a negligible change in the content of the main components of the ESL product compared with the source milk. The total protein was only slightly decreased (0.02,0.03%) and the ratio of the protein fractions was unchanged within the measurement accuracy. The furosine content of the isolated fat globuline membrane fraction could be used as a diagnostic to prove cream had been subjected to high-temperature treatment. The shelf life of the ESL milk was distinctly prolonged compared to HTST-pasteurized milk. [source]