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Evaluation of [Co(gly)3]- as a 35Cl- NMR Shift Reagent for Cellular Studies

Diven, Conrad F.; Wang, Fei; Abukhdeir, Abde M.; Salah, Wajeeh; Layden, Brian T.; Geraldes, Carlos F. G. C.; Freitas, Duarte Mota de
Fonte: American Chemical Society Publicador: American Chemical Society
Tipo: Artigo de Revista Científica
ENG
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45.89%
We studied the efficacy of the tris-glycinatocobaltate(II) complex ([Co(gly)3]-) as a shift reagent (SR) for chloride by 35Cl NMR spectroscopy and compared to that of Co2+(aq). Due to the relatively low thermodynamic stability of [Co(gly)3]-, a 1:3 Co(II)/gly stoichiometric solution at physiological pH is approximately a 2:1 mixture of [Co(gly)2(H2O)2] and [Co(gly)(H2O)4]+. This SR was found to be stable up to higher pH values than Co2+(aq), better preventing Co(OH)2 formation at alkaline pH. No significant differences in the 35Cl- NMR chemical shift induced by Co(II)/gly or Co2+(aq) were observed in the presence of physiological concentrations of either Ca2+ or Mg2+, or of either Na+ or K+. Although Co2+(aq) was almost twice as effective as Co(II)/gly in shifting the 35Cl- NMR resonance at the same high ρ ([SR]/[Cl-]) value and low ionic strength, Co2+(aq) showed a significant decrease (p < 0.05) in the 35Cl- chemical shift at higher ionic strength. Line widths at half-height were significantly (p < 0.05) less for Co(II)/gly than for Co2+(aq) at ρ values in the range 0.066−0.40. Intracellular chloride was clearly detectable by 35Cl NMR spectroscopy in human skin fibroblast cells suspended in medium containing 40 mM Co(II)/gly SR. We determined that...

Acanthoscurrin Fragment 101-132: Total Synthesis at 60 degrees C of a Novel Difficult Sequence

REMUZGO, Cesar; ANDRADE, Gustavo F. S.; TEMPERINI, Marcia L. A.; MIRANDA, M. Teresa M.
Fonte: JOHN WILEY & SONS INC Publicador: JOHN WILEY & SONS INC
Tipo: Artigo de Revista Científica
ENG
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35.7%
Glycine-rich proteins (GRP), serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated front hemocytes-of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs` glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113) GGGGYGGGYG(123)GGy(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was mode when CLEAR amide resin (Fmoc chemistry) was 32 used, as it allowed for obtaining fragment (G(113)-K(132) NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon...

Dark-light: model for nightblindness from the human rhodopsin Gly-90-->Asp mutation.

Sieving, P A; Richards, J E; Naarendorp, F; Bingham, E L; Scott, K; Alpern, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 31/01/1995 EN
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A human rhodopsin mutation, Gly-90-->Asp (Gly90Asp), cosegregated with an unusual trait of congenital nightblindness in 22 at-risk members of a large autosomal dominant kindred. Although rhodopsin mutations typically are associated with retinal degeneration, Gly90Asp-affected subjects up to age 33 did not show clinical retinal changes. Absolute threshold for visual perception was elevated nearly 3 logarithmic units in 7 individuals tested (ages 11-64), indicating greatly compromised rod threshold signaling. However, in vivo rhodopsin density was normal. Although the 38-year-old proband could not perceive dim lights, his rod increment threshold function was normal on brighter backgrounds. The impaired rod vision for dim but not bright backgrounds is consistent with a mechanism of increased basal "dark-light" from thermal isomerization equivalent to an increase of > 10(4) over that of wild-type rhodopsin. The Gly90Asp mutation on the second transmembrane helix places an extra negative charge in the opsin pocket; this could contribute to partial deprotonation of the retinal Schiff base and thereby increase photoreceptor noise. In vitro evidence had suggested that transducin is activated by the Gly90Asp mutation in the absence of both the retinal chromophore and light...

Effects of interferon gamma and major histocompatibility complex-encoded subunits on peptidase activities of human multicatalytic proteases.

Ustrell, V; Pratt, G; Rechsteiner, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 17/01/1995 EN
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35.71%
We have examined several peptidase activities of human multicatalytic protease (MCP) purified from the lymphoblastoid cell line 721.45 and a deletion mutant derivative, 721.174, lacking MCP subunits encoded in the major histocompatibility complex (MHC) class II region. Wild-type lymphoblast MCP hydrolyzed a specific peptide, glutaryl-Gly-Gly-Phe-4-methylcoumaryl-7-amide (-MCA), several times faster than the mutant enzyme did, suggesting that MHC-encoded subunits may provide this activity. Contrary to a recent report [Driscoll, J., Brown, M. G., Finley, D. & Monaco, J J. (1993) Nature (London) 365, 262-264], we did not detect significant aminopeptidase associated with lymphoblast MCPs. Our results also differ markedly from those of Gaczynska et al. [Gaczynska, M., Rock, K. L. & Goldberg, A L. (1993) Nature (London) 365, 264-267], who reported that gamma interferon (IFN-gamma) alters the peptidase activities of lymphoblast MCPs. We found that IFN-gamma did not produce significant differences in the peptidase activities of purified MCPs. Moreover, our measurements of Vmax and Km for succinyl-Leu-Leu-Val-Tyr-MCA hydrolysis differ 600-fold and 15-fold, respectively, from those reported by Gaczynska et al. On balance, the findings presented here do not support the idea that IFN-gamma induces major changes in the peptidase activity of purified MCPs.

The human gastrin/cholecystokinin type B receptor gene: alternative splice donor site in exon 4 generates two variant mRNAs.

Song, I; Brown, D R; Wiltshire, R N; Gantz, I; Trent, J M; Yamada, T
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1993 EN
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35.81%
Gastrin and its carboxyl-terminal homolog cholecystokinin (CCK) exert a variety of biological actions in the brain and gastrointestinal tract that are mediated in part through one or more G protein-coupled receptors which exhibit similar affinity for both peptides. Genomic clones encoding a human gastrin/CCKB receptor were isolated by screening a human EMBL phage library with a partial-length DNA fragment which was based on the nucleotide sequence of the canine gastrin receptor. The gene contained a 1356-bp open reading frame consisting of five exons interrupted by 4 introns and was assigned to human chromosome 11p15.4. A region of exon 4, which encodes a portion of the putative third intracellular loop, appears to be alternatively spliced to yield two different mRNAs, one containing (452 amino acids; long isoform) and the other lacking (447 amino acids; short isoform) the pentapeptide sequence Gly-Gly-Ala-Gly-Pro. The two receptor isoforms may contribute to functional differences in gastrin- and CCK-mediated signal transduction.

Complete amino acid sequence and homologies of human erythrocyte membrane protein band 4.2.

Korsgren, C; Lawler, J; Lambert, S; Speicher, D; Cohen, C M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1990 EN
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35.63%
The complete amino acid sequence for human erythrocyte band 4.2 has been derived from the nucleotide sequence of a full-length 2.35-kilobase (kb) cDNA. The 2.35-kb cDNA was isolated from a human reticulocyte cDNA library made in the expression vector lambda gt11. Of the 2348 base pairs (bp), 2073 bp encode 691 amino acids representing 76.9 kDa (the SDS/PAGE molecular mass is 72 kDa). RNA blot analysis of human reticulocyte total RNA gives a message size for band 4.2 of 2.4 kb. The amino acid sequence of band 4.2 has homology with two closely related Ca2(+)-dependent cross-linking proteins, guinea pig liver transglutaminase (protein-glutamine gamma-glutamyltransferase; protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) (32% identity in a 446-amino acid overlap) and the a subunit of human coagulation factor XIII (27% identity in a 639-amino acid overlap), a transglutaminase that forms intermolecular gamma-glutamyl-epsilon-lysine bonds between fibrin molecules. The region of greatest identity includes a 49-amino acid stretch of band 4.2, which is 69% and 51% identical with guinea pig liver transglutaminase and the a subunit of factor XIII, respectively, within the regions that contain the active sites of these enzymes. Significantly...

Fv-4: Identification of the Defect in Env and the Mechanism of Resistance to Ecotropic Murine Leukemia Virus

Taylor, Gwen M.; Gao, Yi; Sanders, David Avram
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2001 EN
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35.84%
Mice expressing the Fv-4 gene are resistant to infection by ecotropic murine leukemia viruses (MuLVs). The Fv-4 gene encodes an envelope (Env) protein whose putative receptor-binding domain resembles that of ecotropic MuLV Env protein. Resistance to ecotropic MuLVs appears to result from viral interference involving binding of the endogenously expressed Fv-4 env-encoded protein to the ecotropic receptor, although the immune system also plays a role in resistance. The Fv-4 env-encoded protein is processed normally and can be incorporated into virus particles but is unable to promote viral entry. Among the many sequence variations between the transmembrane (TM) subunit of the Fv-4 env-encoded protein and the TM subunits of other MuLV Env proteins, there is a substitution of an arginine residue in the Fv-4 env-encoded protein for a glycine residue (gly-491 in Moloney MuLV Env) that is otherwise conserved in all of the other MuLVs. This residue is present in the MuLV TM fusion peptide sequence. In this study, gly-491 of Moloney MuLV Env has been replaced with other residues and a mutant Env bearing a substitution for gly-487 was also created. G491R recapitulates the Fv-4 Env phenotype in cell culture, indicating that this substitution is sufficient for creation of an Env protein that can establish the interference-mediated resistance to ecotropic viruses produced by the Fv-4 gene. Analysis of the mutant MuLV Env proteins also has implications for an understanding of the role of conserved glycine residues in fusion peptides and for the engineering of organismal resistance to retroviruses.

DNA gyrase gyrA mutations in quinolone-resistant clinical isolates of Pseudomonas aeruginosa.

Yonezawa, M; Takahata, M; Matsubara, N; Watanabe, Y; Narita, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1995 EN
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35.74%
The mutations in the quinolone resistance-determining region of the gyrA gene from clinical isolates of Pseudomonas aeruginosa were determined by DNA sequencing. The strains were isolated in 1989 and 1993. No mutations were detected in the clinical isolates in 1989, while five types of mutations were identified in the isolates in 1993. These mutations were as follows: group 1, a Thr residue to an Ile residue at position 83 (Thr-83-Ile); group 2, Asp-87-Asn; group 3, Thr-83-Ile and Asp-87-Gly; group 4, Thr-83-Ile and Asp-87-Asn; group 5, Thr-83-Ile and Asp-87-His. Three types of double mutations (groups 3, 4, and 5) have not been described previously. These mutations were homologous to the Ser-83-Leu, Asp-87-Asn, and Asp-87-Gly changes observed in Escherichia coli. Thus, DNA gyrase A subunit mutations are implicated in resistance to quinolones in P. aeruginosa as well as E. coli.

Biochemical and mutational analysis of a gingipain-like peptidase activity from Prevotella ruminicola B(1)4 and its role in ammonia production by ruminal bacteria.

Madeira, H M; Peng, L; Morrison, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1997 EN
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35.84%
A chemical mutagenesis protocol was used with the ruminal bacterium Prevotella ruminicola strain B(1)4 to generate mutant strains defective in peptidase activity. Compared with the wild-type parent strain, the isolated mutants possessed 1/10 of the enzyme activity responsible for cleavage of glycine-arginine-4-methoxy-beta-naphthylamide (Gly-Arg-MNA). A concomitant loss in activity against arginine-arginine-4-methoxy-beta-naphthylamide (Arg-Arg-MNA) was also observed. Both activities were similarly affected by various proteinase inhibitors, suggesting that the same enzyme is responsible for the Arg-Arg-MNA peptidase and Gly-Arg-MNA peptidase activities. Growth rates of wild-type and mutant strains grown in batch culture with various nitrogen sources did not differ. However, a role for the Gly-Arg-MNA peptidase activity was demonstrated in coculture experiments with gram-positive, ammonia-producing ruminal bacteria. The rate and extent of ammonia production were reduced by approximately 25% in cocultures containing the mutants when compared with that of wild-type-containing cultures. These reductions could not be accounted for simply by the decrease in ammonia production by the mutant strain alone. To our knowledge, this paper reports the first successful use of chemical mutagenesis with ruminal microorganisms.

Activation of superoxide formation and lysozyme release in human neutrophils by the synthetic lipopeptide Pam3Cys-Ser-(Lys)4. Involvement of guanine-nucleotide-binding proteins and synergism with chemotactic peptides.

Seifert, R; Schultz, G; Richter-Freund, M; Metzger, J; Wiesmüller, K H; Jung, G; Bessler, W G; Hauschildt, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/05/1990 EN
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35.77%
Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins...

Catalytic mechanism of active-site serine beta-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad.

Dubus, A; Wilkin, J M; Raquet, X; Normark, S; Frère, J M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/07/1994 EN
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35.7%
The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C.

Adhesion to fibronectin primes eosinophils via alpha 4 beta 1 (VLA-4).

Anwar, A R; Walsh, G M; Cromwell, O; Kay, A B; Wardlaw, A J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1994 EN
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35.68%
Human peripheral blood eosinophils adhered specifically to microtitre plates coated with plasma fibronectin (Fn) in a dose- and time-dependent fashion. Adhesion was optimal at 60 min at a concentration of 100 micrograms/ml. Adherence to Fn was up-regulated by platelet-activating factor (PAF; optimum concentration of 10(-6) M) and was significantly inhibited by a polyclonal anti-Fn antibody (P < 0.05). The following evidence suggested that eosinophil adhesion to Fn was mediated by alpha 4 beta 1: (1) eosinophil adherence to Fn was not inhibited by an Arg-Gly-Asp-Ser (RGDS) synthetic peptide; (2) there was a dose-dependent adherence of eosinophils to microtitre plates coated with the 40,000 MW proteolytic fragment of Fn that contains the CS-1 alpha 4 beta 1 binding region, whereas adherence to the 120,000 MW chymotryptic fragment of Fn, which contains the RGD-dependent binding site, was weak and only observed at high concentrations (> 250 micrograms/ml); (3) significant inhibition of eosinophil adherence to Fn was achieved by monoclonal antibodies (mAb) against the alpha chain of VLA-4 but not by a mAb against CD45 or a mouse myeloma antibody as negative controls. After adhesion to Fn, eosinophils were investigated for their capacity to release leukotriene C4 in response to stimulation with a suboptimal concentration of calcium ionophore (2 x 10(-6) M). Significant enhancement of release was detected with Fn-coated plates but not with the control bovine serum albumin (BSA) (P < 0.01). Furthermore...

In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV.

Potter, S. M.; Henzel, W. J.; Aswad, D. W.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /10/1993 EN
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35.83%
We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus...

Phenotypic and Enzymatic Comparative Analysis of the Novel KPC Variant KPC-5 and Its Evolutionary Variants, KPC-2 and KPC-4▿

Wolter, Daniel J.; Kurpiel, Philip M.; Woodford, Neil; Palepou, Marie-France I.; Goering, Richard V.; Hanson, Nancy D.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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35.63%
A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated blaKPC-5, was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of blaKPC-5 showed significant differences from the flanking regions of other blaKPC variants. Comparison of amino acid sequences with those of other KPC enzymes revealed that KPC-5 was an intermediate between KPC-2 and KPC-4, differing from KPC-2 by a single amino acid substitution (Pro103→Arg), while KPC-4 contained Pro103→Arg plus an additional amino acid change (Val239→Gly). Transformation studies with an Escherichia coli recipient strain showed differences in the properties of the KPC variants. KPC-4 and KPC-5 both had pIs of 7.65, in contrast with the pI of 6.7 for KPC-2. KPC-2 transformants were less susceptible to the carbapenems than KPC-4 and KPC-5 transformants. These data correlated with higher rates of imipenem hydrolysis for KPC-2 than for KPC-4 and KPC-5. However, KPC-4 and KPC-5 transformants had higher ceftazidime MICs, and the enzymes from these transformants had slightly better hydrolysis of this drug than KPC-2. KPC-4 and KPC-5 were more sensitive than KPC-2 to inhibition by clavulanic acid in both susceptibility testing and hydrolysis assays. Thus...

Anionic Amino Acids near the Pro-α-defensin N Terminus Mediate Inhibition of Bactericidal Activity in Mouse Pro-cryptdin-4*S⃞

Figueredo, Sharel M.; Weeks, Colby S.; Young, Steven K.; Ouellette, André J.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 13/03/2009 EN
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35.67%
In mouse Paneth cells, α-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser43 ↓ Ile44 in mouse pro-Crp4-(20–92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20–43) are responsible for blocking bactericidal activity and membrane disruption. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20–43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp20, Asp26, Glu27, and Glu28), mid (Glu32 and Glu33), and proximal (Glu37, Glu38, and Asp39) clusters in pro-Crp4-(20–92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity. Substitution of the mid and proximal Asp and Glu clusters with Gly produced additive effects with respect to the induction of both bactericidal activity and membrane permeabilization of live Escherichia coli ML35 cells. In contrast...

Thymosin-beta(4) changes the conformation and dynamics of actin monomers.

De La Cruz, E M; Ostap, E M; Brundage, R A; Reddy, K S; Sweeney, H L; Safer, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/2000 EN
Relevância na Pesquisa
35.67%
Thymosin-beta(4) (Tbeta(4)) binds actin monomers stoichiometrically and maintains the bulk of the actin monomer pool in metazoan cells. Tbeta(4) binding quenches the fluorescence of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) conjugated to Cys(374) of actin monomers. The K(d) of the actin-Tbeta(4) complex depends on the cation and nucleotide bound to actin but is not affected by the AEDANS probe. The different stabilities are determined primarily by the rates of dissociation. At 25 degrees C, the free energy of Tbeta(4) binding MgATP-actin is primarily enthalpic in origin but entropic for CaATP-actin. Binding is coupled to the dissociation of bound water molecules, which is greater for CaATP-actin than MgATP-actin monomers. Proteolysis of MgATP-actin, but not CaATP-actin, at Gly(46) on subdomain 2 is >12 times faster when Tbeta(4) is bound. The C terminus of Tbeta(4) contacts actin near this cleavage site, at His(40). By tritium exchange, Tbeta(4) slows the exchange rate of approximately eight rapidly exchanging amide protons on actin. We conclude that Tbeta(4) changes the conformation and structural dynamics ("breathing") of actin monomers. The conformational change may reflect the unique ability of Tbeta(4) to sequester actin monomers and inhibit nucleotide exchange.

Structure of chymopapain M the late-eluted chymopapain deduced by comparative modelling techniques and active-centre characteristics determined by pH-dependent kinetics of catalysis and reactions with time-dependent inhibitors: the Cys-25/His-159 ion-pair is insufficient for catalytic competence in both chymopapain M and papain.

Thomas, M P; Topham, C M; Kowlessur, D; Mellor, G W; Thomas, E W; Whitford, D; Brocklehurst, K
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/06/1994 EN
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35.71%
Chymopapain M, the monothiol cysteine proteinase component of the chymopapain band eluted after chymopapains A and B in cation-exchange chromatography, was isolated from the dried latex of Carica papaya and characterized by kinetic and chromatographic analysis. This late-eluted chymopapain is probably a component of the cysteine proteinase fraction of papaya latex discovered by Schack [(1967) Compt. Rend. Trav. Lab. Carlsberg 36, 67-83], named papaya peptidase B by Lynn [(1979) Biochim. Biophys. Acta 569, 193-201] and partially characterized by Polgár [(1981) Biochim. Biophys. Acta 658, 262-269] and is the enzyme with unusual specificity characteristics (papaya proteinase IV) that Buttle, Kembhavi, Sharp, Shute, Rich and Barrett [Biochem. J. (1989) 261, 469-476] claimed to be a previously undetected cysteine proteinase eluted from a cation-exchange column near to the early-eluted chymopapains. A study of the time-dependent chromatographic consequences of thiol-dependent proteolysis of the components of papaya latex is reported. Chymopapain M was isolated by (i) affinity chromatography followed by separation from papain using cation-exchange f.p.l.c. on a Mono S HR5/5 column and (ii) cation-exchange chromatography followed by an unusual variant of covalent chromatography by thiol-disulphide interchange. The existence in chymopapain M of a nucleophilic interactive Cys/His catalytic-site system analogous to those in papain (EC 3.4.22.2) and other cysteine proteinases was deduced from the characteristics shape of the pH-second-order rate constant (k) profiles for its reactions with 2...

A Novel Pathogenesis-Related Class 10 Protein Gly m 4l, Increases Resistance upon Phytophthora sojae Infection in Soybean (Glycine max [L.] Merr.)

Fan, Sujie; Jiang, Liangyu; Wu, Junjiang; Dong, Lidong; Cheng, Qun; Xu, Pengfei; Zhang, Shuzhen
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 16/10/2015 EN
Relevância na Pesquisa
55.99%
Phytophthora root and stem rot of soybean, caused by Phytophthora sojae (P. sojae), is a destructive disease in many soybean planting regions worldwide. In a previous study, an expressed sequence tag (EST) homolog of the major allergen Pru ar 1 in apricot (Prunus armeniaca) was identified up-regulated in the highly resistant soybean ‘Suinong 10’ infected with P. sojae. Here, the full length of the EST was isolated using rapid amplification of cDNA ends (RACE). It showed the highest homolgy of 53.46% with Gly m 4 after comparison with the eight soybean allergen families reported and was named Gly m 4-like (Gly m 4l, GenBank accession no. HQ913577.1). The cDNA full length of Gly m 4l was 707 bp containing a 474 bp open reading frame encoding a polypeptide of 157 amino acids. Sequence analysis suggests that Gly m 4l contains a conserved ‘P-loop’ (phosphate-binding loop) motif at residues 47–55 aa and a Bet v 1 domain at residues 87–120 aa. The transcript abundance of Gly m 4l was significantly induced by P. sojae, salicylic acid (SA), NaCl, and also responded to methyl jasmonic acid (MeJA) and ethylene (ET). The recombinant Gly m 4l protein showed RNase activity and displayed directly antimicrobial activity that inhibited hyphal growth and reduced zoospore release in P. sojae. Further analyses showed that the RNase activity of the recombinant protein to degrading tRNA was significantly affected in the presence of zeatin. Over-expression of Gly m 4l in susceptible ‘Dongnong 50’ soybean showed enhanced resistance to P. sojae. These results indicated that Gly m 4l protein played an important role in the defense of soybean against P. sojae infection.

Procés per a la síntesi enzimàtica total de l'octapèptid PhAc-CCK-8

Fité i Gòdia, Mª Mercè
Fonte: Bellaterra : Universitat Autònoma de Barcelona, Publicador: Bellaterra : Universitat Autònoma de Barcelona,
Tipo: Tesis i dissertacions electròniques; info:eu-repo/semantics/doctoralThesis Formato: application/pdf; application/pdf
Publicado em //2002 CAT; CAT
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Consultable des del TDX; Títol obtingut de la portada digitalitzada; Aquesta tesi s'emmarca dins les investigacions realitzades en el Departament d'En-ginyeria Química de la Universitat Autònoma de Barcelona en col·laboració amb la Unitat de Química i Bioquímica de Proteïnes del CID-CSIC, en el camp de la síntesi enzimàtica de pèptids. El treball es centra en desenvolupar un procés per a la síntesi enzimàtica de l'octapèptid C-terminal de la CCK (CCK-8). L'interès més rellevant d'aquesta molècula és que presenta un ampli espectre d'activitat biològica i un elevat potencial terapèutic. - La introducció de la memòria s'ha distribuït en tres parts: - Diferents aspectes cabdals en la síntesi enzimàtica de pèptids. - Característiques i funcions de la CCK i CCK-8. - Un resum dels estudis previs realitzats sobre la síntesi enzimàtica del penta-pèp-tid C-terminal de la CCK-8, els quals han servit de punt de par-tida per aquest treball. - El capítol de resultats i discussió s'ha subdividit en diferents apartats. - Inicialment es presenten les investigacions de N-a protecció/desprotecció realitzades amb penicil·lina G acilasa (PGA) d'E. coli. Aquests estudis es van començar amb la N-a protecció de diferents derivats d'aminoàcids a escala analítica amb els grups protectors fenilacetil i mandelil (PhAc...

Um caso peculiar de anafilaxia a maçã e feijão-verde

Gomes,Raquel; Viana,Jorge; Carrapatoso,Isabel; Loureiro,Carlos; Borja,Bartolomé; Todo-Bom,Ana
Fonte: Sociedade Portuguesa de Alergologia e Imunologia Clínica Publicador: Sociedade Portuguesa de Alergologia e Imunologia Clínica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2015 PT
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Introdução: Na alergia alimentar é frequente a reatividade cruzada entre aeroalergénios e alergénios alimentares, tendo como exemplo a síndrome bétula -maçã. No entanto, a alergia a Rosaceas (maçã) por sensibilização a Bet v 1 é caracterizada mais frequentemente por manifestações clínicas ligeiras (síndrome de alergia oral), sendo um fenótipo típico do norte da Europa. Descrição do caso: Doente do sexo masculino, 54 anos, residente em Portugal, com antecedentes de rinite e três reações anafiláticas após ingestão de maçã e feijão -verde. Do estudo alergológico salientam-se testes cutâneos e/ou IgE específica positivos para pólen de Betula sp., maçã e feijão-verde associados a um perfil de sensibilização a Bet v 1 e Gly m 4. Discussão/Conclusão: A análise inicial, atendendo ao quadro clínico, sugere alergia a Rosaceas por sensibilização a LTP, que não se confirma, identificando -se antes sensibilização a Bet v 1 e cuja gravidade parece estar associada à cosensibilização a Gly m 4