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Native Form (native + form)
Selected AbstractsSulfonated molecules that bind a partially structured species of ,2 -microglobulin also influence refolding and fibrillogenesisELECTROPHORESIS, Issue 7 2008Chiara Carazzone Abstract Human ,2 -microglobulin (,2 -m) is a small amyloidogenic protein responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of ,2 -m through the binding to a small molecule, to possibly inhibit protein misfolding and amyloid fibril formation. The search of a strong ligand of this protein is extremely challenging: by using CE in affinity and refolding experiments we study the effect that previously selected sulfonated molecules have on the equilibrium between the native form and an ensemble of conformers populating the slow phase of ,2 -m folding. These data are correlated with the effect that the same molecules exert on in vitro fibrillogenesis experiments. [source] Identification of yeast aspartyl aminopeptidase gene by purifying and characterizing its product from yeast cellsFEBS JOURNAL, Issue 1 2006Ryo Yokoyama Aspartyl aminopeptidase (EC 3.4.11.21) cleaves only unblocked N-terminal acidic amino-acid residues. To date, it has been found only in mammals. We report here that aspartyl aminopeptidase activity is present in yeast. Yeast aminopeptidase is encoded by an uncharacterized gene in chromosome VIII (YHR113W, Saccharomyces Genome Database). Yeast aspartyl aminopeptidase preferentially cleaved the unblocked N-terminal acidic amino-acid residue of peptides; the optimum pH for this activity was within the neutral range. The metalloproteases inhibitors EDTA and 1.10-phenanthroline both inhibited the activity of the enzyme, whereas bestatin, an inhibitor of most aminopeptidases, did not affect enzyme activity. Gel filtration chromatography revealed that the molecular mass of the native form of yeast aspartyl aminopeptidase is ,,680 000. SDS/PAGE of purified yeast aspartyl aminopeptidase produced a single 56-kDa band, indicating that this enzyme comprises 12 identical subunits. [source] Oxidation by mushroom tyrosinase of monophenols generating slightly unstable o -quinonesFEBS JOURNAL, Issue 19 2000Lorena G. Fenoll Tyrosinase can act on monophenols because of the mixture of mettyrosinase (Em) and oxytyrosinase (Eox) that exists in the native form of the enzyme. The latter form is active on monophenols although the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex as the products of the enzymatic reaction, the o -quinones, are unstable and continue evolving to generate o -diphenols in the medium. In the case of substrates such as 4-methoxyphenol, 4-ethoxyphenol and 4- tert -butylphenol, tyrosinase generates o -quinones which become unstable with small constants of approximately < 10,3 s,1. The system evolves from an initial steady state, reached when t,0, through a transition state towards a final steady state, which is never reached because the substrate is largely consumed. The mechanisms proposed to explain the enzyme's action can be differentiated by the kinetics of the first steady state. The results suggest that tyrosinase hydroxylates monophenols to o -diphenols, generating an intermediate Em -diphenol in the process, which may oxidize the o -diphenol or release it directly into the medium. In the case of o -quinone formation, its slow instability generates o -diphenol which activates the enzymatic system yielding parabolic time recordings. [source] Type I Collagen Racemization and Isomerization and the Risk of Fracture in Postmenopausal Women: The OFELY Prospective StudyJOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2002Patrick Garnero Ph.D. Abstract The Asp1211 residue of the1209AHDGGR1214 sequence of the C-terminal cross-linking telopeptide of type I collagen (CTX) can undergo spontaneous post-translational modifications, namely, racemization and isomerization, which result in the formation of four isomers: the native form (,- L) and three age-related forms, that is, an isomerized form (,- L), a racemized form (,- D), and an isomerized/racemized (,- D) form. Previous studies have suggested that changes in the pattern of type I collagen racemization/isomerization, which can be assessed in vivo by measuring the degradation products of the CTX isoforms, may be associated with alterations of bone structure. The aim of this study was to examine prospectively the value of the different urinary CTX isoforms and their related ratio in the prediction of osteoporotic fractures in 408 healthy untreated postmenopausal women aged 50-89 years (mean, 64 years) who were part of the OFELY cohort. During a median 6.8 years follow-up, 16 incident vertebral fractures and 55 peripheral fractures were recorded in 65 women. The baseline levels of the four CTX isoforms in women who subsequently had a fracture were compared with those of the 343 women who did not fracture. At baseline, women with fractures had increased levels of ratios of native ,- L -CTX to age-related isoforms (,- L, ,- D, and ,- D) compared with controls (p < 0.01). In logistic regression analysis after adjustment for age, prevalent fractures, and physical activity, women with levels of ,- L/,- L, ,- L/,- D, and ,- L/,- D -CTX ratios in the highest quartile had a 1.5- to 2-fold increased risk of fractures compared with women with levels in the three lowest quartiles with relative risk (RR) and 95% CI of 2.0 (1.2-3.5), 1.8 (1.02-2.7), and 1.5 (0.9-2.7), respectively. Adjustment of ,- L/,- L and ,- L/,- D -CTX ratios by the level of bone turnover assessed by serum bone alkaline phosphatase (ALP)- or femoral neck bone mineral density (BMD) decreased slightly the RR, which remained significant for the ,- L/,- L -CTX ratio (RR [95%] CI, 1.8 [1.1-3.2] after adjustment for bone ALP, 1.8 [1.03-3.1] after adjustment for BMD, and 1.7 [0.95-2.9] after adjustment for both bone ALP and BMD). Women with both high ,- L/,- L -CTX ratio and high bone ALP had a 50% higher risk of fracture than women with either one of these two risk factors. Similarly, women with both increased CTX ratio and low femoral neck BMD (T score < ,2.5) had a higher risk of fracture with an RR (95% CI) of 4.5 (2.0-10.1). In conclusion, increased urinary ratio between native and age-related forms of CTX, reflecting decreased degree of type I collagen racemization/isomerization, is associated with increased fracture risk independently of BMD and partly of bone turnover rate. This suggests that alterations of type I collagen isomerization/racemization that can be detected by changes in urinary CTX ratios may be associated with increased skeletal fragility. [source] Oestrogen Receptor , is Essential for Female-Directed Chemo-Investigatory Behaviour but is not Required for the Pheromone-Induced Luteinizing Hormone Surge in Male MiceJOURNAL OF NEUROENDOCRINOLOGY, Issue 2 2000S. R. Wersinger The expression of normal masculine sexual behaviour requires testosterone. Testosterone can bind to androgen receptors, either in its native form, or after reduction to other androgen metabolites. In addition, testosterone can be aromatized to oestrogen, and bind to oestrogen receptor , and/or ,. Male copulatory behaviour is deficient in mice lacking functional oestrogen receptor , gene (ER,KO mice). We sought to determine which aspect(s) of masculine sexual behaviour is compromised in the ER,KOs. Specifically, we asked whether ER,KO males have reduced motivation and/or an inability to recognize oestrous females. We found significant differences between mice of different genotypes in the amount of chemo-investigatory behaviour displayed and in the target of their investigation. Wild-type males spent more time investigating ovariectomized, oestradiol-treated females, than either males, or ovariectomized females that had not received hormone priming. ER,KO males spent little time investigating any of the stimulus mice and showed no preferences. To test the hypothesis that this lack of chemo-investigatory behaviour is due to the inability of ER,KO males to detect and respond to female pheromones, we exposed males to chemosensory cues (soiled bedding) from females. Males resided in clean, or female-soiled, cage bedding for 60 min. Next, blood was collected and plasma luteinizing hormone (LH) assayed. We also assessed Fos-like immunoreactivity (Fos-ir) in several neural regions involved in processing chemosensory cues. Despite the fact that male ER,KOs spend little time engaged in chemo-investigation of females, their neuroendocrine responses to female-soiled bedding were similar to those seen in wild-type males. Our data suggest that the normal coupling between the neuroendocrine response to females and the generation of sexual behaviour is disrupted in ER,KO mice. Responses to female pheromones do not require ER,. However, normal male sexual performance requires the ER, gene. [source] Recombinant Human Lactoferrin is Effective in the Treatment of Helicobacter felis -infected MiceJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2000E. J. DIAL Recombinant human lactoferrin possesses in-vitro antibiotic and anti-inflammatory activity similar to the native form. It was tested for in-vivo activity in mice infected with the gastritis-inducing bacterium Helicobacter felis. A two-week course of treatment with lactoferrin was sufficient to partially reverse both infection-induced gastritis and the infection rate, and fully reverse gastric surface hydro-phobicity changes. A comparison of lactoferrin with amoxicillin and standard triple therapy revealed no differences in infection rate. These results show that recombinant human lactoferrin is effective in a mouse model of Helicobacter infection, and support further testing of this promising agent for this application. [source] Effect of crosslinking on the elasticity of polyelectrolyte multilayer films measured by colloidal probe AFMMICROSCOPY RESEARCH AND TECHNIQUE, Issue 2 2006Grégory Francius Abstract A homemade colloidal probe atomic force microscope was used to perform nanoindentation with a spherical probe of 5 ,m in diameter, at different approach velocities in order to extract the Young's modulus, E0, of poly(L-lysine)/hyaluronan (PLL/HA) films. This parameter is of prime importance to control cellular adhesion. The films were either kept in their native form or cross-linked with a mixture of 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) and N -hydrosulfosuccinimide (sulfo-NHS), where the EDC concentration was varied from 1 up to 100 mg mL,1 (approximately from 5 to 500 mM). A model based on Hertz mechanics was used to account for the interactions between film and probe. It is shown that the Young's modulus varies with the approach velocity for the native (PLL/HA) films, whereas for cross-linked ones, E0 is independent from the velocity over the whole range investigated. It is found that for native films, E0 takes a value of 3 kPa at low approach velocities, a velocity domain that should be relevant in cellular adhesion processes. The Young's modulus increases with the EDC concentration used to cross-link the films and levels off at a value of about 400 kPa for EDC concentrations exceeding 40 mg mL,1. Thus, it is possible by crosslinking PLL/HA films to control their elastic properties with the aim to alter their behavior as to the cellular adhesion. Microsc. Res. Tech. 69:84,92, 2006. © 2006 Wiley-Liss, Inc. [source] Molecular weight of guar gum affects short-chain fatty acid profile in model intestinal fermentationMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 10 2006Maria L. Stewart Abstract Dietary fiber exerts many beneficial physiological effects; however, not all types of dietary fiber display the same effects. Partially hydrolyzed guar gum (PHGG), a lower molecular weight form of guar gum, is more easily incorporated into food, but may have less pronounced physiological effects than the native form. The aim of this study was to identify differences in intestinal fermentability based on the molecular weight of guar gum. Guar gum of four molecular masses (15, 20, 400, and 1100 kDa) was fermented using a batch in vitro fermentation system. Human fecal inoculum was the source of microbes. The 400-kDa fraction produced the greatest concentrations of total short-chain fatty acid (SCFA) at 8 h and the highest amounts of butyrate at 24 h. At 24 h, the 400-kDa fraction produced more total SCFA and propionate than the 15 kDa, but was not different than 20 kDa or 1100 kDa fractions. The molecular weight of guar gum was positively correlated with acetate production and negatively correlated with propionate production. This study concludes that 400-kDa guar gum may be optimal for intestinal fermentability. In conclusion, the molecular weight of guar gum affects in vitro fermentability and should be considered when adding to a food or beverage. [source] Photophysics in Motionally constrained Bioenvironment: Interactions of Norharmane with Bovine Serum Albumin,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2005Arabinda Mallick ABSTRACT Steady-state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (,G) for the probe-protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein-bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water-dioxane mixture with varying composition. [source] Crystal structure of Mycobacterium tuberculosis LrpA, a leucine-responsive global regulator associated with starvation responsePROTEIN SCIENCE, Issue 1 2008Manchi C.M. Reddy Abstract The bacterial leucine-responsive regulatory protein (Lrp) is a global transcriptional regulator that controls the expression of many genes during starvation and the transition to stationary phase. The Mycobacterium tuberculosis gene Rv3291c encodes a 150-amino acid protein (designated here as Mtb LrpA) with homology with Escherichia coli Lrp. The crystal structure of the native form of Mtb LrpA was solved at 2.1 Å. The Mtb LrpA structure shows an N-terminal DNA-binding domain with a helix-turn-helix (HTH) motif, and a C-terminal regulatory domain. In comparison to the complex of E. coli AsnC with asparagine, the effector-binding pocket (including loop 100,106) in LrpA appears to be largely preserved, with hydrophobic substitutions consistent with its specificity for leucine. The effector-binding pocket is formed at the interface between adjacent dimers, with an opening to the core of the octamer as in AsnC, and an additional substrate-access channel opening to the outer surface of the octamer. Using electrophoretic mobility shift assays, purified Mtb LrpA protein was shown to form a protein,DNA complex with the lat promoter, demonstrating that the lat operon is a direct target of LrpA. Using computational analysis, a putative motif is identified in this region that is also present upstream of other operons differentially regulated under starvation. This study provides insights into the potential role of LrpA as a global regulator in the transition of M. tuberculosis to a persistent state. [source] Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complexPROTEIN SCIENCE, Issue 1 2002Silvano Geremia Abstract The three-dimensional structures of the native cytochrome c2 from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 Å and in the reduced state at 1.95 Å resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c2 with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 310 -helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c2 with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles , and , of approximately ,140 and ,130°, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 Å resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c2 of the rare six-residue insertion in the helix 310 conformation that increases Met loop flexibility. [source] Direct tandem mass spectrometric analysis of amino acids in dried blood spots without chemical derivatization for neonatal screeningRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2003Kornél Nagy Neonatal screening performed by electrospray tandem mass spectrometry is a powerful technique in clinical diagnostics. In the present paper an alternative to the widely accepted method involving butylation has been developed. In the new method butylation is not required, and multiple reaction monitoring (MRM) was used instead of constant neutral loss scanning. The method was optimized for detection of 23 L-amino acids in their native form. Quantitation was based on isotope-labeled internal standards, calibration curves were linear from 0 to 500,,mol/L, and detection limits were in the range 2,42,,mol/L. The utility of the present technique is illustrated in the case of one neonate suffering from citrullinaemia. Copyright © 2003 John Wiley & Sons, Ltd. [source] Structure of native laccase from Trametes hirsuta at 1.8,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2009Konstantin M. Polyakov This paper describes the structural analysis of the native form of laccase from Trametes hirsuta at 1.8,Å resolution. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8,Å resolution native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given. [source] Crystallization and preliminary crystallographic studies of CorC, a magnesium-ion transporterACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Ning Zhang CorC is a magnesium transporter that is involved in the Mg2+ -efflux function of the CorA transporter system, an Mg2+ channel, from Shigella flexneri. Native CorC was purified and crystallized in the native form and in a ligand-free form and diffraction data sets were collected to 2.9 and 3.4,Å resolution, respectively. The native CorC crystals belonged to space group P212121, with unit-cell parameters a = 64.31, b = 74.44, c = 132.78,Å. The ligand-free CorC crystals belonged to space group P3121/P3221, with unit-cell parameters a = b = 71.89, c = 125.96,Å. The CorC,ATP complex has also been crystallized and the crystals belonged to space group P2 or P21. [source] Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase from Thermoanaerobacter sp.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010Annelies Van Hoorebeke Disaccharide phosphorylases are attractive enzymatic platforms for tailor-made sugar synthesis owing to their ability to catalyze both the synthesis and the breakdown of disaccharides. Trehalose phosphorylase from Thermoanaerobacter sp. (TP) is a glycoside hydrolase family 65 enzyme which catalyzes the reversible breakdown of trehalose [d -glucopyranosyl-,(1,1),- d -glucopyranose] to ,- d -glucose 1-phosphate and d -glucose. Recombinant purified protein was produced in Escherichia coli and crystallized in space group P212121. Crystals of recombinant TP were obtained in their native form and were soaked with glucose, with n -octyl-,- d -glucoside and with trehalose. The crystals presented a number of challenges including an unusually large unit cell, with a c axis measuring 420,Å, and variable diffraction quality. Crystal-dehydration protocols led to improvements in diffraction quality that were often dramatic, typically from 7,8 to 3,4,Å resolution. The structure of recombinant TP was determined by molecular replacement to 2.8,Å resolution, thus establishing a starting point for investigating the structural and mechanistic determinants of the disaccharide phosphorylase activity. To the best of our knowledge, this is the first crystal structure determination of an inverting trehalose phosphorylase. [source] Production of a recombinant cholera toxin B subunit-insulin B chain peptide hybrid protein by Brevibacillus choshinensis expression system as a nasal vaccine against autoimmune diabetesBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2005Yoshikazu Yuki Abstract Mucosally induced tolerance is an attractive strategy for preventing or reducing autoimmune diseases. Here, we produced a recombinant CTB fusion protein linked with autoantigen T cell epitope of insulin B chain peptide 9,23 (C19S) at levels up to 200 mg/L culture media in Brevibacillus choshinensis secretion-expression system. Receptor-competitive assay showed that the CTB-insulin peptide binds to GM1 receptor almost equivalent degree as the native form of CTB. Non-obese diabetes (NOD) mice that spontaneously develop an insulin-dependent diabetes were nasally immunized with CTB-insulin peptide (5 µg) for three times. The nasal treatment significantly reduced the development of insulin-dependent diabetes and peptide specific DTH responses after systemic immunization with the insulin peptide B 9,23(C19S) in CFA. Nasal administration of as high as 50 µg of the peptide alone demonstrated a similar level of the disease inhibition. In contrast, all mice given 5 µg of the insulin peptide alone or 5 µg of insulin peptide with 25 µg of the free form of CTB did not lead to the suppression of diabetes development and DTH responses. Because molecular weight of the insulin peptide is about one tenth of that of the CTB-insulin peptide, the results demonstrate that the recombinant hybrid of autoantigen and CTB increased its tolerogenic potential for nasal administration by up 100-fold on molar base of autoantigen peptide. Taken together, nasally-induced tolerance by administration of the recombinant B.choshinensis -derived hybrid protein of CTB and autoantigen T cell-epitope peptide could be useful mucosal immunetherapy for the control of T cell-mediated autoimmune diseases. © 2005 Wiley Periodicals, Inc. [source] Expression system for recombinant human growth hormone production from Bacillus subtilisBIOTECHNOLOGY PROGRESS, Issue 1 2009Tunçer H. Özdamar Abstract We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid-gene of two DNA fragments, i.e., signal (pre- ) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg -promoter in B. subtilis. Recombinant-hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N-terminal sequence and mass spectrometry analyses of rhGH confirm the mature hGH sequence, and indicate that the signal peptide was properly processed by B. subtilis signal-peptidase. The highest rhGH concentration was obtained at t = 32 h as CrhGH = 70 mg L,1 with a product yield on substrate YrhGH/S = 9 g kg,1, in a glucose based defined medium. Fermentation characteristics and influence of hGH gene on the rhGH production were investigated by comparing B. subtilis carrying pMK4::pre(subC)::hGH with that of carrying merely pMK4. Excreted organic-acid concentrations were higher by B. subtilis carrying pMK4::pre(subC)::hGH, whereas excreted amino-acid concentrations were higher by B. subtilis carrying pMK4. The approach developed is expected to be applicable to the design of expression systems for heterologous protein production from Bacillus species. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Molecularly Imprinted Polymer-Assisted Refolding of LysozymeBIOTECHNOLOGY PROGRESS, Issue 5 2007Mitsuru Haruki For production of active proteins using heterologous expression systems, refolding of proteins from inclusion bodies often creates a bottleneck due to its poor yield. In this study, we show that molecularly imprinted polymer (MIP) toward native lysozyme promotes the folding of chemically denatured lysozyme. The MIP, which was prepared with 1 M acrylamide, 1 M methacrylic acid, 1 M 2-(dimethylamino)ethyl methacrylate, and 5 mg/mL lysozyme, successfully promoted the refolding of lysozyme, whereas the non-imprinted polymer did not. The refolding yield of 90% was achieved when 15 mg of the MIP was added to 0.3 mg of the unfolded lysozyme. The parallel relationship between the refolding yield and the binding capacity of the MIP suggests that MIP promotes refolding through shifting the folding equilibrium toward the native form by binding the refolded protein. [source] Comparative enzymology of native and recombinant house dust mite allergen Der p 1ALLERGY, Issue 3 2009J. Zhang Background:, The cysteine peptidase activity of group 1 house dust mite allergens is important for their allergenicity and may offer new therapeutic targets for allergy treatment. Hitherto, the design of specific inhibitors has been impeded because the availability of pure, fully active allergens has limited the implementation of drug screening campaigns. Similarly, investigation of the mechanisms by which peptidase allergens promote sensitization has also been restricted. Our aim was to compare the enzymology of recombinant and native forms of Der p 1 to establish if an easily expressed recombinant form of Der p 1 could be used as a drug discovery tool. Methods:, Enzymatic activity of natural and recombinant Der p 1 was compared fluorimetrically using a novel specific substrate (ADZ 50,059) and a novel specific active site titrant (ADZ 50,000). The effect of recombinant Der p 1 prodomain on the catalytic activity of both Der p 1 preparations was also examined. Results:, Although differing substantially in molecular weight, the enzymological properties of recombinant and native Der p 1 were indistinguishable. Our data show clearly by experiment that, in contrast to some suggestions, Der p 1 is not an enzyme of bifunctional mechanism. Conclusion:, The catalytic activity of Der p 1 is tolerant of glycosylation differences that occur at N150 when the protein is expressed in Pichia pastoris. This suggests that this recombinant protein may be suitable for drug design studies and in the elucidation of how peptidase activity promotes sensitization to peptidase and nonpeptidase bystander allergens. [source] Feral pigs facilitate hyperpredation by golden eagles and indirectly cause the decline of the island foxANIMAL CONSERVATION, Issue 4 2001Gary W. Roemer Introduced species can compete with, prey upon or transmit disease to native forms, resulting in devastation of indigenous communities. A more subtle but equally severe effect of exotic species is as a supplemental food source for predators that allows them to increase in abundance and then overexploit native prey species. Here we show that the introduction of feral pigs (Sus scrofa) to the California Channel Islands has sustained an unnaturally large breeding population of golden eagles (Aquila chrysaetos), a native predator. The resulting increase in predation on the island fox (Urocyon littoralis) has caused the near extirpation of three subspecies of this endemic carnivore. Foxes evolved on the islands over the past 20,000 years, pigs were introduced in the 1850s and golden eagles, historically, were only transient visitors. Although these three species have been sympatric for the past 150 years, this predator-prey interaction is a recent phenomenon, occurring within the last decade. We hypothesize that this interaction ultimately stems from human-induced perturbations to the island, mainland and surrounding marine environments. [source] Low frequency resonance Raman spectra of isolated , and , subunits of hemoglobin and their deuterated analoguesBIOPOLYMERS, Issue 5 2006Edyta Podstawka Abstract In an attempt to gain further insight into the nature of the low frequency vibrational modes of hemoglobin and its isolated subunits, a comprehensive study of several different isotopically labeled analogues has been undertaken and is reported herein. Specifically, the resonance Raman spectra, between 200 and 500 cm,1, are reported for the deoxy and ligated (CO and O2) forms of the isolated , and , subunits containing the natural abundance or various deuterated analogues of protoheme. The deuterated protoheme analogues studied include the 1,3,5,8-C2H3 -protoheme (d12- protoheme), the 1,3-C2H3 -protoheme (1,3- d6-protoheme), the 5,8-C2H3 -protoheme (5,8- d6-protoheme), and the meso-C2H4 -protoheme (d4-protoheme). The entire set of acquired spectra has been analyzed using a deconvolution procedure to help correlate the shifted modes with their counterparts in the spectra of the native forms. Interestingly, modes previously associated with so-called vinyl bending modes or propionate deformation modes are shown to be quite sensitive to deuteration of the peripheral methyl groups of the macrocycle, shifting by up to 12,15 cm,1, revealing their complex nature. Of special interest is the fact that shifts observed for the 1,3- d6- and 5,8- d6-protoheme analogues confirm the fact that certain modes are associated with a given portion of the macrocycle; i.e., only certain modes shift upon deuteration of the 1 and 3 methyl groups, while others shift upon deuteration of the 5 and 8 methyl groups. Compared with the spectra previously reported for the corresponding myoglobin derivatives, the data reported here reveal the appearance of several additional features that imply splitting of modes associated with the propionate groups or that are indicative of greater distortion of the heme prosthetic groups. © 2006 Wiley Periodicals, Inc. Biopolymers 83: 455,466, 2006 This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] | |||||||||||||||||||||||||