28 resultados para biologically active molecules


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Neutrophilic lung inflammation is an essential component of host defense against diverse eukaryotic and prokaryotic pathogens, but in chronic inflammatory lung diseases, such as chronic obstructive lung disease (COPD), severe asthma, cystic fibrosis, and bronchiolitis, it may damage the host. Glucocorticosteroids are widely used in these conditions and in their infectious exacerbations; however, the clinical efficacy of steroids is disputed. In this study, we used a proteomic approach to identify molecules contributing to neutrophilic inflammation induced by transnasal administration of lipopolysaccharide (LPS) that were also resistant to the potent glucocorticosteroid dexamethasone (Dex). We confirmed that Dex was biologically active at both the transcript (suppression of GM-CSF and TNFalpha transcripts) and protein levels (induction of lipocortin) and used 2D-PAGE/MALDI-TOF to generate global expression profiles, identifying six LPS-induced proteins that were Dex resistant. Of these, S100A8, a candidate neutrophil chemotactic factor, was profiled in detail. Steroid refractory S100A8 expression was highly abundant, transcriptionally regulated, secreted into lung lavage fluid and immunohistochemically localized to tissue infiltrating neutrophils. However, in marked contrast to other vascular beds, neutralizing antibodies to S100A8 had only a weak anti-neutrophil recruitment effect and antibodies against the related S100A9 were ineffective. These data highlight the need for extensive in vivo profiling of proteomically identified candidate molecules and demonstrates that S100A8, despite its abundance, resistance to steroids and known chemotactic activity, is unlikely to be an important determinant of LPS-induced neutrophilic lung inflammation in vivo.

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Enzymatically active Delta(5)-3-ketosteroid isomerase (KSI) protein with a C-terminus his(6)-tag was produced following insoluble expression using Escherichia coli. A simple, integrated process was used to extract and purify the target protein. Chemical extraction was shown to be as effective as homogenization at releasing the inclusion body proteins from the bacteria] cells, with complete release taking less than 20 min. An expanded bed adsorption (EBA) column utilizing immobilized metal affinity chromatography (IMAC) was then used to purify the denatured KSI-(His(6)) protein directly from the chemical extract. This integrated process greatly simplifies the recovery and purification of inclusion body proteins by removing the need for mechanical cell disruption, repeated inclusion body centrifugation, and difficult clarification operations. The integrated chemical extraction and EBA process achieved a very high purity (99%) and recovery (89%) of the KSI-(His(6)), with efficient utilization of the adsorbent matrix (9.74 mg KSI-(His(6))/mL adsorbent). Following purification the protein was refolded by dilution to obtain the biologically active protein. Seventy-nine percent of the expressed KSI-(His(6)) protein was recovered as enzymatically active protein with the described extraction, purification, and refolding process. In addition to demonstrating the operation of this intensified inclusion body process, a plate-based concentration assay detecting KSI-(His(6)) is validated. The intensified process in this work requires minimal optimization for recovering novel his-tagged proteins, and further improves the economic advantage of E. coli as a host organism. (c) 2006 Wiley Periodicals, Inc.

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Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five(TM)) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth ( growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five(TM) cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium ( LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures.

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The incorporation of organic matter ( OM) in soils that are able to rapidly sorb applied phosphorus ( P) fertiliser reportedly increases P availability to plants. This effect has commonly been ascribed to competition between the decomposition products of OM and P for soil sorption sites resulting in increased soil solution P concentrations. The evidence for competitive inhibition of P sorption by dissolved organic carbon compounds, derived from the breakdown of OM, includes studies on the competition between P and (i) low molecular weight organic acids (LOAs), (ii) humic and fulvic acids, and (iii) OM leachates in soils with a high P sorption capacity. These studies, however, have often used LOAs at 1 - 100 mM, concentrations much higher than those in soils ( generally < 0.05 mM). The transience of LOAs in biologically active soils further suggests that neither their concentration nor their persistence would have a practical benefit in increasing P phytoavailability. Higher molecular weight compounds such as humic and fulvic acids also competitively inhibit P sorption; however, little consideration has been given to the potential of these compounds to increase the amount of P sorbed through metal - chelate linkages. We suggest that the magnitude of the inhibition of P sorption by the decomposition products of OM leachate is negligible at rates equivalent to those of OM applied in the field. Incubation of OM in soil has also commonly been reported as reducing P sorption in soil. However, we consider that the reported decreases in P sorption ( as measured by P in the soil solution) are not related to competition from the decomposition products of OM breakdown, but are the result of P release from the OM that was not accounted for when calculating the reduction in P sorption.

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Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class 1) found in fungi and most bacteria, and a long form (Class 11) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class 11 KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.

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Harmful algal blooms (HABs) have increased in abundance and severity in recent decades. Whereas the implications for human impacts and intoxication resulting from blooms have been extensively studied, the ecological implications of these microalgae are less well understood. Many HAB species produce biologically active, secondary metabolites and the fate of these toxins through the foodweb is generally not well understood unless it culminates in extensive fish mortalities or human poisonings. This review focusses on one HAB species, the cyanobacterium Lyngbya majuscula, and presents a hypothetical role for its involvement in fibro-papillornatosis (FP), a neoplastic disease of marine turtles. FP is expressed as benign tumours that grow both internally and externally on marine turtles, preventing vision, movement and organ function. The aetiology of FP is currently not conclusively understood, but virus material has been associated with tumours and previous studies have suggested a role for naturally produced tumour promoters. In this review, we present a hypothesis regarding the involvement of L. majuscula in FP, either through direct intoxication and action of tumour-promoting compounds or indirectly by causing seagrass loss and compromised immune function, thus leaving the turtles more susceptible to disease.

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The complement system is an innate immune defense mechanism that protects the host from infection and injury. Complement activation results in the formation of anaphylatoxins, including the biologically active protein C5a. This anaphylatoxin is a potent chemotactic agent for immune and inflammatory cells and induces cell activation. In situations of excessive or uncontrolled complement activation, the overproduction of C5a can cause deleterious effects to the host, and this process is implicated in the pathogenesis of numerous immunoinflammatory disease states, including rheumatoid arthritis, psoriasis, inflammatory bowel disease, ischemia-reperfusion injuries and others. The presence of C5a in a wide variety of condition's has prompted many groups to examine the potential of inhibiting this complement activation product, with the aim of controlling these diseases and reducing the pathologic process. However, to date there is no clinically available specific C5a inhibitor and development of this new drug class is still in a relatively early stage, although limited phase I and phase II human clinical trials have been undertaken in the last few years with selected agents. In this review, examination of the current evidence supporting a specific role of C5a in selected disease states and an overview of potential therapeutic C5a inhibitors will enable the critical evaluation of the potential for C5a as a therapeutic target.

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We show that the quantum decoherence of Forster resonant energy transfer between two optically active molecules can be described by a spin-boson model. This allows us to give quantitative criteria that are necessary for coherent quantum oscillations of excitations between the chromophores. Experimental tests of our results should be possible with flourescent resonant energy transfer (FRET) spectroscopy. Although we focus on the case of protein-pigment complexes our results are also relevant to quantum dots and organic molecules in a dielectric medium. (c) 2006 Elsevier B.V. All rights reserved.

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Cadmium (Cd) is a metal toxin of continuing worldwide concern. Daily intake of Cd, albeit in small quantities, is associated with a number of adverse health effects which are attributable to distinct pathological changes in a variety of tissues and organs. In the present review, we focus on its renal tubular effects in people who have been exposed environmentally to Cd at levels below the provisional tolerable intake level set for the toxin. We highlight the data linking such low-level Cd intake with tubular injury, altered abundance of cytochromes P450 (CYPs) in the kidney and an expression of a hypertensive phenotype. We provide updated knowledge on renal and vascular effects of the eicosanoids 20-hydroxyeicosatetraenoic acid (20-HETE) and eicosatrienoic acids (EETs), which are biologically active metabolites from arachidonate metabolism mediated by certain CYPs in the kidney. We note the ability of Cd to elicit oxidative stress and to alter metal homeostasis notably of zinc which may lead to augmentation of the defense mechanisms involving induction of the antioxidant enzyme heme oxygenase-1 (HO-1) and the metal binding protein metallothionein (MT) in the kidney. We hypothesize that renal Cd accumulation triggers the host responses mediated by HO-I and MT in an attempt to protect the kidney against injurious oxidative stress and to resist a rise in blood pressure levels. This hypothesis predicts that individuals with less active HO-1 (caused by the HO-1 genetic polymorphisms) are more likely to have renal injury and express a hypertensive phenotype following chronic ingestion of low-level Cd, compared with those having more active HO-1. Future analytical and molecular epidemiologic research should pave the way to the utility of induction of heme oxygenases together with dietary antioxidants in reducing the risk of kidney injury and hypertension in susceptible people.

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Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays. (C) 2005 Elsevier SAS. All rights reserved.

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The cyclotide family of plant proteins is of interest because of their unique topology, which combines a head-to-tail cyclic backbone with an embedded cystine knot, and because their-remarkable chemical and biological properties make them ideal candidates as grafting templates for biologically active peptide epitopes. The present Study describes the first steps towards exploiting the cyclotide framework by synthesizing and structurally characterizing two grafted analogues of the cyclotide kalata B1. The modified peptides have polar or charged residues substituted for residues that form part of a surface-exposed hydrophobic patch that plays a significant role in the folding and biological activity of kalata B1. Both analogues retain the native cyclotide fold, but lack the undesired haemolytic activity of their parent molecule, kalata B1. This finding confirms the tolerance of the cyclotide framework to residue Substitutions and opens up possibilities for the Substitution of biologically active peptide epitopes into the framework.

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Cellular delivery involving the transfer of various drugs and bio-active molecules (peptides, proteins and DNAs, etc.) through the cell membrane into cells has attracted increasing attention because of its importance in medicine and drug delivery. This topic has been extensively reviewed. The direct delivery of drugs and biomolecules, however, is generally inefficient and suffering from problems such as enzymic degradation of DNAs. Therefore, searching for efficient and safe transport vehicles (carriers) to delivery genes or drugs into cells has been challenging yet exciting area of research. In past decades, many carriers have been developed and investigated extensively which can be generally classified into four major groups: viral carriers, organic cationic compounds, recombinant protiens and inorganic nanoparticles. Many inorganic materials, such as calcium phosphate, gold, carbon materials, silicon oxide, iron oxide and layered double hydroxide (LDH), have been studied. Inorganic nanoparticles show low toxicity and promise for controlled delivery properties, thus presenting a new alternative to viral carriers and cationic carriers. Inorganic nanoparticles generally possess versatile properties suitable for cellular delivery, including wide availability, rich functionality, good biocompatibility, potential capability of targeted delivery (e.g. selectively destroying cancer cells but sparing normal tissues) and controlled release of carried drugs. This paper reviews the latest advances in inorganic nanoparticle applications as cellular delivery carriers and highlights some key issues in efficient cellular delivery using inorganic nanoparticles. Critical proper-ties of inorganic nanoparticles, surface functionalisation (modification), uptake of biomolecules, the driving forces for delivery, and release of biomolecules will be reviewed systematically. Selected examples of promising inorganic nanoparticle delivery systems, including gold, fullerences and carbon nanotubes, LDH and various oxide nanoparticles in particular their applications for gene delivery will be discussed. The fundamental understanding of properties of inorganic nanoparticles in relation to cellular delivery efficiency as the most paramount issue will be highlighted. (c) 2005 Elsevier Ltd. All rights reserved.

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Human SULT1A1 is primarily responsible for sulfonation of xenobiotics, including the activation of promutagens, and it has been implicated in several forms of cancer. Human SULT1A3 has been shown to be the major sulfotransferase that sulfonates dopamine. These two enzymes shares 93% amino acid sequence identity and have distinct but overlapping substrate preferences. The resolution of the crystal structures of these two enzymes has enabled us to elucidate the mechanisms controlling their substrate preferences and inhibition. The presence of two p-nitrophenol (pNP) molecules in the crystal structure of SULT1A1 was postulated to explain cooperativity at low and inhibition at high substrate concentrations, respectively. In SULT1A1, substrate inhibition occurs with pNP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with pNP. We investigated how substrate inhibition occurs in these two enzymes using molecular modeling, site-directed mutagenesis, and kinetic analysis. The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition. Mutation of phenylalanine to leucine at this position in SULT1A1 results in substrate inhibition by dopamine. We also propose, based on modeling and kinetic studies, that substrate inhibition by dopamine in SULT1A3 is caused by binding of two dopamine molecules in the active site. © 2004 by The American Society for Biochemistry and Molecular Biology, Inc.