Protein-enriched Fraction Research Articles

Effect of Musca domestic maggot polypeptide extract on HUVEC dysfunction induced by early-activated macrophages

Context: Musca domestica Linn. maggot is a traditional Chinese medicine. In our previous studies, Musca domestica maggot protein-enriched fraction and polypeptide extract (molecular weight <30 kD) were found to reverse endothelial cell dysfunction in atherosclerotic lesions.Objective: This study determines whether and how M. domestica maggot polypeptide extract affects the dysfunction of human umbilical vein endothelial cells (HUVEC) induced by macrophages (Mϕ).Materials and methods: HUVEC and early-activated THP-1 Mϕ (incubated with LPS of 1 μg/ml for 2 h) were co-cultured in a Transwell system. The effects of Musca domestica maggot polypeptide extract (with increasing concentrations, i.e., 1.0, 2.5, 5.0, 10.0, 20.0, and 40.0 µg/ml) on the proliferation and migration HUVEC and their secretion of vascular endothelial growth factor (VEGF) were determined by flow cytometry, modified Boyden chamber assay, and enzyme-linked immunosorbent assay (ELISA) after incubation for 24 h.Results: Musca domestica maggot polypeptide extract decreased the proliferation of HUVEC in a concentration-dependent manner, with a 50% effective concentration (EC50) of 22.16 ± 1.48 µg/ml, and effectively inhibited HUVEC migration (EC50 = 35.15 ± 2.03 µg/ml) and VEGF secretion (EC50 = 28.64 ± 1.29 µg/ml).Discussion and conclusion: Musca domestica maggot polypeptide extract can inhibit the dysfunction of HUVEC induced by early-activated THP-1 Mϕ.

Evaluation of anti-inflammatory and analgesic activities of extracts from herb of Chelidonium majus L.

The aim of the study was to evaluate analgesic activity (“hot plate” test), anti-inflammatory activity (carrageenan-induced paw edema) and locomotor activity in rats under the influence of three fractions of Chelidonium majus herb extract: full water extract (FWE), protein enriched fraction (PEF), and non-protein fraction (NPF). Effects of the fractions on the level of chosen cytokines and their mRNA levels were also assessed using lipopolysaccharide (LPS) administration as a proinflammatory cue. All fractions and diclofenac did not affect the locomotor activity of rats in comparison with the control group. FWE and PEF three hours after administration showed statistically significant analgesic activities comparable to morphine (p < 0.05). A slight reduction in rat paw edema was observed after three (comparable with diclofenac) and six hours in the NPF group. FWE revealed a statistically significant pro-inflammatory effect after three hours in comparison with the control group. Peripheral IL-1 and IL-4 cytokine concentrations were reduced under FWE and NPF, PEF fractions. The combination of FWE, PEF and NPF together with LPS showed only the effects of LPS. We suggest that protein enriched fraction (PEF) produced centrally mediated (morphine-like) analgesic action, whereas the anti-inflammatory potential was shown only after LPS-induced inflammation. The precise mechanisms involved in the production of anti-nociceptive and anti-inflammatory responses of studied fractions are not completely understood, but they may be caused rather by the presence of protein more than alkaloids-enriched fraction. This fraction of the extract could be used as an alternative therapy for the prevention of inflammatory-related diseases in the future, but further studies are needed.

Antihepatitis B Virus Activity of a Protein-Enriched Fraction from Housefly (Musca domestica) in a Stable HBV-Producing Cell Line

Hepatitis B virus (HBV) infection remains a major public health problem. Although several vaccines and therapeutic strategies are currently being implemented to combat HBV virus, effective antiviral therapy against HBV infection has not been fully developed. Alternative strategies and new drugs to combat this disease are urged. Insects and insect derivatives are a large and unexploited source of potentially useful compounds for modern medicine. In the present study, we investigated the first anti-HBV activity of a protein-enriched fraction (PE) from the larvae of the housefly (Musca domestica) in a stable HBV-producing cell line. HBsAg and HBeAg in the culture medium were measured by enzyme-linked immunosorbent assay. HBV-DNA was quantified by fluorescent quantification PCR. HBV core protein was assayed by immunofluorescent staining. Results indicate PE treatment inhibited both HBsAg, HBeAg secretion, and HBV-DNA replication. Furthermore, PE could also suppress HBV core protein expression. PE could be a potential candidate for the development of a novel and effective drug for the treatment of HBV infection.

Antiviral, immunomodulatory, and free radical scavenging activities of a protein-enriched fraction from the larvae of the housefly, Musca domestica.

In our previous study, protein-enriched fraction (PEF) that was isolated from the larvae of the housefly, Musca domestica L. (Diptera: Muscidae), showed excellent hepatoprotective activity as well as the potential for clinical application in therapy for liver diseases. In this study, antiviral, immunomodulatory, and free radical scavenging activities of PEF were evaluated. The antiviral results demonstrated that PEF inhibited the infection of avian influenza virus H9N2 and had a virucidal effect against the multicapsid nucleopolyhedrovirus of the alfalfa looper, Autographa californica Speyer (Lepidoptera: Noctuidae) in vitro. The mortality of silkworm larve in a PEF treatment group decreased significantly compared with a negative control. PEF showed excellent scavenging activity for 1,1-diphenyl-2-picrylhydrazyl and superoxide anion radicals, which were similar to those of ascorbic acid. The imunomodulatory results suggested that PEF could effectively improve immune function in experimental mice. Our results indicated that PEF could possibly be used for the prophylaxis and treatment of diseases caused by avian influenza virus infection. In addition, PEF with virucidal activity against insect viruses might provide useful for the development of antimicrobial breeding technology for economically important insects. As a natural product from insects, PEF could be a potential source for the discovery of potent antioxidant and immunomodulatory agents.

Short-Term Feed Deprivation Rapidly Induces the Protein Degradation Pathway in Skeletal Muscles of Young Mice

Analysis of protein kinase B (AKT) and S6 kinase1 (p70S6K) activity is widely used to assess the efficacy of interventions designed to increase or maintain skeletal muscle mass; these studies are often performed on feed-deprived mice. One problem associated with feed deprivation is that it promotes catabolism, and young or metabolically compromised mice may have less tolerance. The aim of our study was to determine the effect of various times of feed deprivation on the activity of AKT and p70S6K signaling and markers of protein catabolism in young, growing mice compared with adult mice. Young 23-d-old and adult 3-mo-old mice were feed deprived for 8, 10, and 12 h starting at 0700 h. In addition, adult mice were feed deprived for 24 h. AKT(Ser473) phosphorylation decreased by 50 and 76% from fed amounts by 10 and 12 h of feed deprivation, respectively, in young but not adult muscles. In adult muscles, feed deprivation for 24 h reduced AKT(Ser473) phosphorylation by 70%. Significant de-phosphorylation of p70S6K(Thr389) occurred in all feed-deprived young and adult mice. There was an increase in muscle RING-finger protein-1 (Murf1; 133–1245%) and muscle atrophy F-box protein or Atrogin-1 (Fbxo32; 210–2420%) mRNA in all young but not adult groups deprived of feed for 8–12 h, and there was a trend (P = 0.08) toward increased MURF1 associated with the contractile protein-enriched fraction isolated from young muscles of mice feed deprived for 12 h. This study demonstrates that skeletal muscles of young mice respond rapidly to feed deprivation by decreasing AKT activity and upregulating the protein degradation program.

Protein aggregation induced by phase separation in a pea proteins–sodium alginate–water ternary system

The physicochemical properties of a native, globular plant protein–linear anionic polysaccharide aqueous system at 20°C were investigated in conditions where biopolymers carry a net negative charge (pH 7.2, 0.1M NaCl). The pea proteins–sodium alginate mixtures showed a phase separation mainly by thermodynamic incompatibility, characterized at both the macroscopic and microscopic scale. Phase diagram was established and confocal laser scanning microscopy (CLSM) provided accurate data on the microstructure morphology of the system, regarding its phase behavior. In admixture, sodium alginate induced a protein aggregation, certainly by a local depletion of the polysaccharide. Protein aggregates were present in both single-phase and biphasic mixtures, while increasing the sodium alginate concentration provided larger and denser protein microdomains, leading to a non-equilibrium state. By phase separation, the pea protein aggregates entrapped a part of the sodium alginate phase, thus modifying the protein volume fraction. Along a tie-line, a phase inversion phenomenon was detected, from a sodium alginate to a pea proteins-continuous phase. Rheological properties of the mixed systems depended on the biopolymer composition and were modified with respect to individual components. The protein-enriched phase volume fraction modified the flow behavior to non-Newtonian for starting concentrated mixtures; a shear-thinning at high shear rates was evidenced, while mixtures with a particularly high sodium alginate concentration (>0.8 wt%) had an apparent yield stress for low shear rates. Mechanical spectra showed that both protein enrichment within microdomains in the presence of alginate and stronger protein–protein interaction with time impacted the viscoelastic properties (G′ and G″ moduli) of the whole mixture.

Antioxidant activities of protein-enriched fraction from the larvae of housefly, Musca domestica

The protein-enriched fraction (PEF) was isolated and purified from the larvae of housefly, Musca domestica. This study was designed to investigate amino acid compositions, antioxidative effects and protective effects of PEF on red blood cell (RBC) hemolysis, lipid peroxidation. The effects of PEF treatment were studied on aged mice liver lipid peroxidation and antioxidant enzyme activities, which included superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Results: PEF not only inhibited H2O2 stimulated oxidative hemolysis of erythrocytes of mice, but also depressed malondialdehyde (MDA) production in mice liver homogenate by auto-oxidation and hepatic mitochondria expanded induced by Fe2+-ascorbic acid system. Compared to control group, treatments of PEF significantly increases SOD and GSH-Px activity of serum and liver homogenate in aged mice. MDA level of serum and liver homogenate decreased significantly in aged mice. In conclusion, this study demonstrates that PEF possesses antioxidative activity and might be a valuable source of natural antioxidative agents.

Diversity of Translation Start Sites May Define Increased Complexity of the Human Short ORFeome

Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048-2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5'-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5'-end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome.

Hepatoprotective effect of a protein-enriched fraction from the maggots (Musca domestica) against CCl4-induced hepatic damage in rats

The hepatoprotective potential of a protein-enriched fraction (PEF) isolated from the maggots of housefly (Musca domestica) was evaluated in rats against carbon tetrachloride (CCl4)-induced acute hepatic damage. Activities of serum aspartate aminotransferase and alanine aminotransferase increased by 4- and 13-fold induced by CCl4, were significantly inhibited by pretreatment with 50, 100 and 200 mg PEF/kg. The formation of malondialdehyde was also significantly decreased in PEF-treated group compared with CCl4-treated group. The treatments with PEF also elevated total protein levels significantly. These results were further supplemented by histopathological examination of liver sections. Hyperplasia of kupffer cells was observed after treatment with PEF (100 and 200 mg/kg). We conclude that PEF is effective in this model of liver damage.

β Subunits of Voltage-gated Sodium Channels Are Novel Substrates of β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE1) and γ-Secretase

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and gamma-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that beta subunits of voltage-gated sodium channels are sequentially processed by BACE1 and gamma-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.

Evidence That Bacterial Cyanide Oxygenase Is a Pterin-Dependent Hydroxylase

The soluble cell-free fraction (150,000g high-speed supernatants [HSS]) of Pseudomonas fluorescens NCIMB 11764 contains putative cyanide oxygenase (CNO) responsible for initiating cyanide oxidation and assimilation as a nitrogenous growth substrate. CNO activity, assayed either by cyanide-dependent O2 or NADH uptake, or by conversion of radioactive K14CN to 14CO2, was detected at micromolar concentrations (apparent half-saturation constant, 4 μM). Results demonstrating that CNO requires a protein-enriched cell fraction and a low MW redox factor (<500 Da) for which reduced biopterin could substitute are presented. The properties of CNO are consistent with those of a pterin hydroxylase.

β-internexin is a microtubule-associated protein identical to the 70-kDa heat-shock cognate protein and the clathrin uncoating ATPase

Abstract We have examined the relationship of the ubiquitous 68-70-kDa cytoskeletal-associated protein beta-internexin (Napolitano, E. W., Pachter, J. S., Chin, S. S. M., and Liem, R. K. H. (1985) J. Cell Biol. 101, 1323-1331) to heat-shock cognate 70 (hsc70), the major constitutive member of the mammalian heat-shock protein 70 (hsp70) family of stress proteins. We purify beta-internexin from rat brain microtubules and confirm its identity with hsc70 and the clathrin-uncoating ATPase by the following criteria: 1) The partial sequence of a cyanogen bromide-derived peptide from beta-internexin matches the inferred amino acid sequence of the cDNA clone pRC62 encoding hsc70 from rat brain (O'Malley, K., Mauron, A., Barchas, J. D., and Kedes, L. (1985) Mol. Cell. Biol. 5, 3476-3483). 2) Mixing experiments followed by two-dimensional gel analyses reveal the precise co-migration of beta-internexin, the clathrin-uncoating ATPase, and the in vitro translation product of cDNA clone pHSP-4 encoding rat brain hsc70. 3) beta-Internexin is recognized by a monoclonal antibody reactive against the class of hsp70 proteins. 4) beta-Internexin purified from a microtubule-associated protein-enriched fraction of rat brain by virtue of high affinity binding to ATP-agarose possesses clathrin cage-specific ATPase activity.

Antigens and allergens of Aspergillus fumigatus: II. Their further identification and partial characterization of a major allergen (Ag 3)

These further studies demonstrated that most antigens and allergens detected by allergic bronchopulmonary aspergillosis (ABPA) sera were recognized by rabbit antiserum, and a major allergenic component, Ag 3, as previously demonstrated by self-crossed radioimmunoelectrophoresis, was identified in the conventional system. Affinocrossed immunoelectrophoresis with concanavalin A indicated that for the patients with ABPA, although the major antigens contained α- d-manno- or α- d-glucopyranoside terminal residues, the major allergenic components did not. Fractionation of the salt-precipitated, protein-enriched fraction by gel filtration on Sephacryl S-200, monitored by fused-rocket immunoelectrophoresis/fused-rocket radioimmunoelectrophoresis with both the ABPA serum pool and specific rabbit antisera, revealed that most of the antigens were in a molecular weight range >43 kd, whereas the allergens spread from as low as 10 to >100 kd. The major allergen, Ag 3, was identified and demonstrated to have a molecular weight of approximately 24 kd. This component does not bind to concanavalin A and is also very heat labile.

Selective removal of heparan sulfate chains from proteoheparan sulfate with a commercial preparation of heparitinase

Procedures employing the commercial preparation of heparitinase were developed for isolating a protein-enriched core molecule from proteoheparan sulfate by selective removal of the heparan sulfate chains. Treatment of proteoheparan sulfate with the enzyme preparation caused seriously extensive degradation owing to the presence of proteolytic activity in the enzyme preparation. This effect could be avoided by using a series of protease inhibitors which prevented proteolytic degradation with less significant effect on the heparitinase activity. Application of the procedures to a purified preparation from the Engelbreth-Holm-Swarm tumor yielded a single protein-enriched core fraction with a molecular weight of approximately 450,000, as ascertained by sodium dodceyl sulfate-gel electrophoresis.

The occurrence of three different proteoglycan species in chick embryo cartilage. Isolation and characterization of a second proteoglycan (PG-Lb) and its precursor form.

Three different molecular species of proteoglycan (designated PG-H, PG-Lb, and PG-Lt) have been isolated from chick embryo epiphyseal cartilage. PG-H is a major proteoglycan of the tissue and identical, or nearly identical, with so-called cartilage-characteristic proteoglycan previously described in mammalian and avian cartilages. The third proteoglycan, PG-Lt, differs from the other two in containing disulfide-bonded collagenous polypeptides (Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 258, 9323-9331). The second proteoglycan, PG-Lb, consists of a core protein with Mr congruent to 52,000 dermatan sulfate copolymer chains with glucuronic acid/iduronic acid residues. Upon chondroitinase ABC digestion, the proteoglycan yields a protein-enriched core fraction of Mr congruent to 43,000. Its amino acid composition, tryptic peptide profile, and immunochemical properties indicate that PG-Lb is distinctly different from PG-H and PG-Lt in core protein structure. PG-Lb shows no specific binding with hyaluronic acid. Pulse-chase experiments with [3H]serine indicate that PG-Lb is first synthesized as a precursor form (pro-PG-Lb) that can be distinguished from PG-Lb by the production of a core molecule of Mr congruent to 52,000 after chondroitinase ABC digestion. This core molecule is labeled when [2-3H]mannose is used as a precursor, suggesting that it contains a glycoprotein type oligosaccharide. Since the core molecule from pro-PG-Lb is significantly larger in molecular weight than that from PG-Lb, the conversion of pro-PG-Lb to PG-Lb should involve scission of the polypeptide or possibly removal of mannose-containing oligosaccharide chain.

In Vitro Gibberellin A4 Binding to Extracts of Cucumber Hypocotyls

Cucumber hypocotyls were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [(3)H]-gibberellin(4) (GA(4)) to soluble macromolecular components present in the cytosol was demonstrated at 0 C by Sephadex chromatography. Binding assays performed with cytosol that had been preheated or incubated with protease, DNase, RNase, or phospholipase A or C indicated that heat and protease treatments disrupted the binding, which suggests that binding occurred to a protein. Equilibrium dialysis of a protein-enriched fraction prepared by ammonium sulfate precipitation also indicated binding of [(3)H]GA(4) to macromolecular components. [(3)H]GA(4) binding was pH-sensitive, saturable, reversible, and significantly affected by biologically active gibberellins, but not by inactive gibberellins or other plant hormones such as indoleacetic acid, abscisic acid, or kinetin. Thin layer chromatography indicated that [(3)H]GA(4), and not a metabolite, was the species bound. A kinetic analysis indicated that specific binding of [(3)H]GA(4) was due to a single class of binding sites having an estimated K(d) of 10(-7) molar and a concentration of 0.8 x 10(-12) moles gram(-1) fresh weight or 0.4 x 10(-12) moles milligram(-1) soluble protein.

Structural studies on ascophyllan and the fucose-containing complexes from the brown alga Ascophyllum nodosum

The two major “complexes” isolated from a dilute-acid extract of Ascophyllum nodosum were degraded by mild, acid hydrolysis to a fucan portion and an ascophyllan-like portion. Protein was enriched in the ascophyllan-like fraction. A time-course study of the acid hydrolysis, together with data on digestion by pronase and hydrolysis by base indicated that the complexes were formed by a fucan backbone to which varyious numbers of ascophyllan-like molecules were attached by an acid-labile linkage. The primary uronic acid in the ascophyllan-like portion of the complex was mannuronic acid, whereas the free ascophyllan isolated in this study contained a mixture of guluronic, mannuronic, and glucuronic acids*. In each of these uronic acid-rich materials, hydrolysis by base suggested a uronic acid backbone having relatively long, fucose-containing, side chains. Hydroxyproline was present in the complex, but did not appear to be part of the major carbohydrate-protein linkages. A protein-enriched fraction was obtained by treatment of the ascophyllan-like portion of the complex with oxalic acid. Amino acid analysis, before and after treatment with mild base suggested that serine and threonine were involved in the linkage of protein to carbohydrate.

The proteinaceous, gum polysaccharide from Azadirachta indica A. Juss.

The gum exuded by Azadirachta indica trees contains 35% of proteinaceous material. The most abundant amino acid is aspartic acid; there are also considerable proportions of serine and threonine, and at least 2% of amino sugars. Attempts to deproteinise the polysaccharide material were unsuccessful. Fractionation of the gum exudate on agarose columns gave a protein-depleted fraction of high molecular weight and a protein-enriched fraction of lower molecular weight. Studies involving molecular-sieve chromatography showed a close correspondence in protein and carbohydrate content, and it appears that the proportion of proteinaceous component that is free or only loosely associated with carbohydrate is small. The carbohydrate component is much more complex than was indicated by earlier investigators. In addition to galactose and arabinose (major components), mannose, xylose, fucose, and rhamnose are also present. The uronic acid content (28%) is higher than previously believed, and a relatively high methoxyl content has been found. The major aldobiouronic acid, previously undetected, is 4- O-(4- O-methyl-α- D-glucopyranosyluronic acid)- D-galactose, with 4- O-(α- D-glucopyranosyluronic acid)- D-galactose also present. The exudate from Azadirachta indica is therefore a complex material having several features that are unusual in gum chemistry.