Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A versatile gene-fusion technique for immobilizing and visualizing biologically active enzymes which includes from the N to C-termini, an affinity histidine tag, the green fluorescent protein (GFP), a proteolytic enzyme (enterokinase, EK) cleavage site and the enzyme of interest, were developed. Specifically, the organophosphorus hydrolase was bound to the affinity (His(6))-reporter(GFP)-EK fusion elements. Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. In the case of immobilized OPH, paraoxon was rapidly degraded when pumped through a packed column. In reaction mixtures containing CHES buffer at pH 6.9, a continual decay in OPH activity was observed and importantly, this was monitored by GFP fluorescence. This decay in activity was fully restored, along with fluorescence, upon washing with PBS buffer. Many subsequent experiments were performed at varied pH and in different background buffer solutions. In all cases when there was OPH activity there was also marked fluorescence from the GFP fusion partner. Likewise, when OPH activity was lost, so was GFP fluorescence and, importantly, both were regenerated when washed in the presence of the kosmotropic salt, phosphate. Recently, Waldo et al. (1999) showed that GFP fluorescence from whole cells indicated the extent of proper folding of normally aggregated proteins designed via directed evolution. The present work demonstrates an application wherein GFP fluorescence indicates stability and activity of its fusion partner.
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PMID:GFP-visualized immobilized enzymes: degradation of paraoxon via organophosphorus hydrolase in a packed column. 1175 28

The effect of pH on the kinetics of the bioelectrocatalytic reduction of H(2)O(2) catalysed by horseradish peroxidase (HRP) has been studied at -50 mV vs. Agmid R:AgCl on HRP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Native HRP (nHRP) and a nonglycosylated recombinant form containing a six-histidine tag at the C-terminus, C(His)rHRP, produced by genetic engineering of nonglycosylated recombinant HRP using an E. coli expression system, have been used for adsorptive modification of Au electrodes. A favourable adsorption of C(His)rHRP on pre-oxidized Au from a protein solution at pH 6.0 provided a high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct (mediator-less) electron transfer (ET) between Au and the active site of HRP. The heterogeneous ET rate constant, k(s), calculated from experimental data on direct ET, on mediated ET in the presence of catechol as well as from microbalance data, increased more than 30 times when changing from nHRP to C(His)rHRP. For both forms of HRP, the increasing efficiency of bioelectrocatalysis with increasing [H(3)O(+)] was observed. The values of the apparent k(s) between C(His)rHRP and Au changed from a value of 12+/-2 s(-1) in PBS at pH 8.0 to a value of 434+/-62 s(-1) at pH 6.0; a similar k(s)-pH dependence was also observed for nHRP, providing the possibility to consider the reaction mechanism involving the participation of a proton in the rate-determining step of the charge transfer.
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PMID:Effect of pH on direct electron transfer in the system gold electrode-recombinant horseradish peroxidase. 1178 47

A His-tagged coiled coil stem loop peptide with stable secondary structure was designed and biosynthesized. A series of oligopeptides related to the EBV envelope glycoprotein 350/220 N-terminal nonapeptide as potential CD21 receptor-binding epitopes were engineered into the loop region of the peptide scaffold. It was shown that these peptides had a stable alpha-helical coiled coil structure and assumed a monomeric form in PBS. Biorecognition of the epitopes was studied by immobilizing the epitope-containing peptides on complexed Ni2+-containing surfaces through His-Ni2+ chelation and incubating with purified soluble CD21 receptor or CD21+ cells. The results showed that the potential epitopes bound to CD21 and CD21+ cells at different affinities depending on oligopeptide structures. This approach allows for the evaluation of epitope biorecognizabilities and the selection of optimal oligopeptides among sequences for use as targeting moieties in the design of new lymphoma-targeting polymeric drug carriers.
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PMID:Presentation of epitopes on genetically engineered peptides and selection of lymphoma-targeting moieties based on epitope biorecognition. 1200 10

Ultrasonically induced cell damage and active oxygen generation with 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl(IX)-6-7-diaspartic acid (ATX-S10) were compared in the same in vitro insonation setup. Sarcoma 180 cells suspended in air-saturated PBS were exposed to ultrasound at 2 MHz for up to 60 s in the presence and absence of ATX-S10. The viability was determined by Trypan blue exclusion test. Ultrasonically induced active oxygen generation in the presence and absence of ATX-S10 in air-saturated aqueous solutions of 50 mM 2,2,6,6-tetramethyl-4-piperidone was detected by electron spin resonance (ESR). Significant enhancement of the rates of both ultrasonically induced cell damage and nitroxide generation was demonstrated with 40-160 microM ATX-S10. Both rates correlated very well. The enhancement of both rates with ATX-S10 was suppressed by 10 mM histidine. These results suggest that ultrasonically generated active oxygen plays a primary role in the ultrasonically induced cell damage in the presence of ATX-S10.
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PMID:Ultrasonically induced cell damage and active oxygen generation by 4-formyloximeetylidene-3-hydroxyl-2-vinyl-deuterio-porphynyl(IX)-6-7-diaspartic acid: on the mechanism of sonodynamic activation. 1259 87

Hypothalamic neuronal histamine is involved in the central regulation of energy expenditure through the activation of sympathetic nerves innervating brown adipose tissue (BAT). The present study examined the effect of L-histidine, a precursor of neuronal histamine, on BAT sympathetic nerve activity in rats. Infusion of histamine at a dose of 1 nmol/rat into the third cerebroventricle significantly increased BAT sympathetic nerve activity as compared with the effect of phosphate buffered saline (P < 0.05). Intraperitoneal (i.p.) injection of L-histidine (0.3 mmol/rat) also significantly increased BAT sympathetic nerve activity as compared with the effect of PBS (P < 0.05). Pretreatment with an i.p. bolus injection of 224 micromol/kg alpha-fluoromethylhistidine, a suicide inhibitor of the histamine synthesizing enzyme histidine decarboxylase, blocked the stimulatory effect of l-histidine on BAT sympathetic nerve activity. These results indicate that L-histidine regulates BAT sympathetic nerve activity through its conversion into neuronal histamine in the hypothalamus.
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PMID:L-histidine stimulates sympathetic nerve activity to brown adipose tissue in rats. 1519 56

Gastrin/CCK-2 receptors are overexpressed in a number of tumors such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). Recently [D-Glu1]-minigastrin (MG) has been radiolabeled with 131I, 111In, and 90Y and evaluated in patients. This study describes the labeling and evaluation of MG with technetium-99m using two different labeling approaches: HYNIC as bifunctional coupling agent and (Nalpha-His)Ac as tridentate ligand for 99mTc(CO3) labeling. Labeling was perfomed at high specific activities using Tricine and EDDA as coligands for HYNIC-MG and [99mTc(OH2)3(CO)3]+ for (Nalpha-His)Ac-MG. Stability experiments were carried out by reversed phase HPLC analysis in PBS, serum, histidine, and cysteine solutions, as well as rat liver and kidney homogenates. Receptor binding and internalization experiments were performed using CCK-2 receptor positive AR42J rat pancreatic tumor cells. Biodistribution experiments were carried out in nude mice carrying AR42J tumors by injection of 99mTc-labeled peptide with or without coinjection of 50 microg of minigastrin I human (MGh). HYNIC-MG and (Nalpha-His)Ac-MG could be radiolabeled at high specific activities (>1 Ci/micromol). For HYNIC-MG, high labeling yields (>95%) were achieved using Tricine and EDDA as coligands. Stability experiments of all 99mTc-labeled conjugates revealed a high stability of the label in PBS and serum as well as toward challenge with histidine and cysteine. Incubation in kidney homogenates resulted in a rapid degradation of all conjugates with <10% intact peptide after 60 min at 37 degrees C, with no considerable differences between the radiolabeled conjugates; a somewhat lower degradation rate was seen in liver homogenates. Protein binding varied considerably with lowest levels for 99mTc-EDDA/HYNIC-MG. Competition experiments of unlabeled conjugates on AR42J membranes versus [125I-Tyr12]-gastrin I showed high CCK-2 receptor affinity for all conjugates under study. Internalization behavior was very rapid for all radiolabeled conjugates in the order of 99mTc-(Nalpha-His)Ac-MG > 99mTc-EDDA/HYNIC-MG > 99mTc-Tricine/HYNIC-MG. In tumor-bearing nude mice the highest tumor-uptake was observed with 99mTc-EDDA/HYNIC-MG (8.1%ID/g) followed by 99mTc-Tricine/HYNIC-MG (2.2%ID/g) and 99mTc-(Nalpha-His)Ac-MG (1.2%ID/g) which correlated with kidney uptake (101.0%ID/g, 53.8%ID/g, 1.8%ID/g respectively). In this series of compounds 99mTc-EDDA/HYNIC-MG with its very high tumor/organ ratios except for kidneys seems to be the most promising agent to target CCK-2 receptors. Despite promising properties concerning receptor binding, internalization, and in vitro stability, 99mTc-(Nalpha-His)Ac-MG showed low tumor uptake in vivo.
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PMID:99mTc-labeling and in vitro and in vivo evaluation of HYNIC- and (Nalpha-His)acetic acid-modified [D-Glu1]-minigastrin. 1526 75

This study was aimed to further investigate the function of platelet collagen receptor-glycoprotein VI and to screen its specific inhibitor. The extracellular domain of platelet glycoprotein VI (GPVI) in E. coli was expressed by recombinant technology, the extracellular domain cDNA of GPVI was amplified from pBluescript KS(-)-GPVI plasmid by PCR. Proved by sequencing, the expression vector pET-20b(+)-GPVI was constructed, which was then transformed into E. coli (BL21(DE3)pLysS) and induced by IPTG. The recombinant GPVI was purified on Ni-NTA resin column and renatured in PBS containing GSH and GSSG. The anti-penta His McAb and anti-GPVI polyclonal antibody were used to identify the recombinant GPVI in Western blotting. Collagen binding test was conducted to investigate the biological activity of recombinant GPVI. The results showed that the recombinant GPVI was expressed in E. coli and successfully purified, which was confirmed to be similar to the native GPVI in Western blotting. The recombinant GPVI can bind the type I collagen in dose-dependent manner. In conclusion, the recombinant GPVI can be achieved in E. coli and restore its native characteristics after renaturation.
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PMID:[Expression of platelet collagen receptor-glycoprotein VI fragment in E. coli and its biological activities]. 1585 97

Spherical, smooth-surfaced and mechanically stable alginate-poly(L-histidine) (PLHis) microcapsules with narrow particle size distributions were prepared by incubating calcium alginate beads in aqueous solutions of PLHis. The in vitro release characteristics, drug loading and encapsulation efficiency of the microcapsules were investigated using bovine erythrocytes hemoglobin (Hb) as a model drug. The results showed that the concentration of Ca(2+) ions had a considerable effect on the drug loading, encapsulation efficiency and in vitro release behavior of the microcapsules. When the concentration of CaCl(2) in the PLHis solution was increased from 0 to 3.0% (w/v), the drug loading and encapsulation efficiency decreased significantly from 38.0 to 4.3% and from 92.9 to 8.0%, respectively, while the total cumulative release of Hb from microcapsules in phosphate buffered saline solution (PBS, pH 6.8) decreased from 96.2 to 72.8% in 24 h. No significant protein release was observed during 70 h of incubation in hydrochloric acid solution (pH 1.2). However, under neutral conditions (PBS, pH 6.8), the Hb was completely and stably released within 24-70 h. An explosion test showed that the stability of alginate-PLHis microcapsules depended strongly on the concentration of PLHis and the calcium ions in solution. [Diagram: see text] Microscopy photo of Hb-loaded alginate-PLHis microcapsules.
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PMID:Novel alginate-poly(L-histidine) microcapsules as drug carriers: in vitro protein release and short term stability. 1588 87

The role of free radicals in protein modification and the importance of anti-4-hydroxy-2-nonenal (HNE) antibodies as marker of HNE-mediated cell toxicity has been well documented. Proteins modified by HNE in vitro, prior to immobilization on ELISA plates, have served as substrates for assaying these antibodies. We found preferential binding of HNE-modified versus unmodified proteins to ELISA plates and this prompted us to seek a more reliable assay. We report a method to HNE-modify any cysteine/histidine/lysine-containing protein or multiple antigenic peptide (MAP) following their immobilization on an ELISA plate. To a set of wells, HNE (200 microM) dissolved in PBS is added and incubated for 4 h, followed by regular ELISA. Since HNE was supplied dissolved in ethanol, PBS with appropriate amount of ethanol added was used as control. For inhibition experiments, HNE is incubated with or without inhibitors and then added to the wells. The commercial anti-HNE serum bound only to HNE-modified antigens. Sera from rabbits and mice immunized with HNE-modified 60 kDa Ro autoantigen preferentially bound the modified antigens. Modification of solid phase antigens in this manner makes assaying anti-HNE antibodies unambiguous. Lengthy dialysis procedures or the use of spin columns that lead to antigen loss becomes unnecessary for the separation of free HNE. We were able to HNE-modify various antigens (BSA, the autoantigens Ro, La and Sm/nRNP, 60 kDa Ro and Sm MAPs) using this procedure. Using MAPs, we confirmed the importance of histidine, lysine and cysteine residues in HNE modification. In addition, this method allowed identification of inhibitors of HNE-modification. We obtained 61%, 70% and 74% inhibition of HNE-modification of solid phase Ro MAP 166 substrate using BSA, Ro MAP 482 and Ro MAP 166, respectively. Glycyl-proline dipeptide and a MAP from the Sm autoantigen (PPPGMRPP) showed 0% inhibition of HNE-modification.
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PMID:In vitro modification of solid phase multiple antigenic peptides/autoantigens with 4-hydroxy-2-nonenal (HNE) provide ideal substrates for detection of anti-HNE antibodies and peptide antioxidants. 1605 45

A gene encoding poly(tetramethylene succinate), PBS, depolymerase, pbsA, has been cloned from Acidovorax delafieldii strain BS-3 chromosomal DNA. The clone expressed in Escherichia coli showed the ability to degrade both PBS and poly[(tetramethylene succinate)-co-adipate] that are kinds of biodegradable plastics. PBS depolymerase was considered to be a kind of lipase, since it also degrades olive oil. It had no apparent hydrophobic-amino-acid-rich region which exists in other known plastic-degrading enzymes. From the result of amino acid homology search, PbsA was found to have some similarities with lipases of Streptomyces sp. and Mollaxella sp. In the motif surrounding the active site Ser residue (Gly-X1-Ser-X2-Gly), PbsA was revealed to have a Trp residue in the X1 position instead of His which is most likely found in other bacterial lipases.
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PMID:Cloning and sequence analysis of poly(tetramethylene succinate) depolymerase from Acidovorax delafieldii strain BS-3. 1623 95


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