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)

The literature reported DPP-IV substrate specificity includes oligopeptides with a penultimate Pro, Hyp or Ala residue. Bovine GRF is a substrate for DPP-IV and is rapidly degraded by the enzyme via removal of its N-terminal Tyr-Ala. Incubation of selected GRF analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series with a porcine-kidney-derived DPP-IV in PBS (pH 7.4) resulted in cleavage at the X2-Asp3 bond. The extent of enzymatic hydrolysis varied with X2 as reflected in the following relative cleavage rates: Ala2 (100%), Ser2 (4%), Thr2 (2.5%), Val2 (0.53%), Ile2 (0%). These cleavages were sequestered when similar experiments were performed in the presence of the DPP-IV-specific inhibitor N-epsilon-(p-NO2-benzyloxycarbonyl)-Lys-Pro-OH. A side reaction, buffer-induced deamidation of Asn8, contributed less than 5% of the total substrate degradation. Although our finding qualitatively extends the DPP-IV substrate specificity to also include N-terminal X-Ser, X-Thr and X-Val sequences, quantitatively, relatively fast cleavages of the GRFs with Ala2 make the latter preferred substrates for DPP-IV. The data presented here indicates that the observed GRF(3-29) fragment formation upon incubation of Ser2- and Thr2-substituted bGRF analogs in bovine plasma could have been DPP-IV-related.
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PMID:Dipeptidyl peptidase IV (DPP-IV) from pig kidney cleaves analogs of bovine growth hormone-releasing factor (bGRF) modified at position 2 with Ser, Thr or Val. Extended DPP-IV substrate specificity? 810 71

8-Epi PGF2alpha, a potent vasocontrictor, is a specific product of non-enzymatic peroxidation of arachidonic acid. It seems likely that similar products could arise from other polyunsaturated fatty acids (PUFAs) and might be useful biomarkers of their peroxidation in vivo. This was investigated using eicosapentaenoic acid (EPA). EPA liposomes (1 mg/ml PBS) were exposed at 37 degrees C to either 2,2'-azobis-(2-amidinopropane) dichloride (AAPH) or copper ions at final concentrations of 1 mM and 10 microM, respectively. Sample processing involved solid-phase extraction on a C18-followed by an NH2 cartridge. After conversion to pentafluorobenzyl ester/trimethylsilyl derivatives, F3-isoprostanes were analysed by negative ion-chemical ionisation mass spectrometry (GC-MS/NICI) using tetradeuterated PGF2alpha (PGF2-d4) as the internal standard. Quantitative analysis was carried out by selected ion monitoring of the carboxylated anion [M-180] at m/z 567 and 573 for the PGF3-like compounds and PGF2-d4, respectively. EPA oxidised by AAPH or by copper ions gave rise to a family of F3-isoprostanes with 8-epi PGF3alpha as a minor product. Formation of F3-isoprostanes correlated well with other indices of lipid peroxidation (hydroperoxides and thiobarbituric acid reactive substances). The possibility of analysing specific lipid peroxidation products from individual fatty acids should facilitate nutritional and biomedical studies.
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PMID:Evidence for the formation of F3-isoprostanes during peroxidation of eicosapentaenoic acid. 924 Apr 62

alpha 1-Acid glycoproteins (AAGs) have a structure resembling beta-adrenergic receptors and bind several basic drugs in plasma. Chromatographic columns were prepared by linking epsilon-NH2 groups of AAG lysines to a Sepharose 4B support, in order to purify by affinity chromatography adrenergic drugs of possible use in animal production. Loading capacities, binding efficiency, memory effects and matrix interferences from urine samples were studied. The method developed involves sample application in buffered media (pH 7.4), washing with 5 ml of PBS, and elution with 4 ml of 1% v/v acetic acid. Under these conditions no memory effect was observed. Loading capacity is correlated with the physiological plasma binding rate (PB) of the drug. For clenbuterol (PB 50%) and anilino-like related drugs, 5 mg of AAG were able to bind about 15 x 10(-6) g of drug, with a 100% recovery from the column. Repeatability and reproducibility, expressed as RSD, were 4.2 and 5.4%, respectively. The calculated AAG: drug molar ratio was 4.5:1, indicating 22% of the AAG bound to the column retained drug affinity. Among phenolic-like agonists, salbutamol (PB 5%), fenoterol and isoxsuprine hardly interacted, whereas nylidrin, ritodrine and bamethan showed more effective binding. We also checked binding of other drugs of possible use in veterinary medicine. Application of the AAG column to spiked bovine urine revealed a mean recovery of 97.8%; no matrix interferences were observed.
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PMID:Alpha 1-acid glycoprotein affinity columns for the clean-up of adrenergic drugs. 1043 25

Matrix degrading enzymes are implicated in several disease processes such as abdominal aortic aneurysms and emphysema, however, monitoring proteolytic activity in a single assay is not well-established. Numerous assays have been developed to measure matrix degrading enzymes, which use artificial substrates or substrates derived from natural substrate protein. We have recently developed an assay for elastolytic activity based on the detection of primary amines, using trinitrobenzene sulfonic acid (TNBSA), following the digestion of succinylated elastin. The assay is also versatile enough to allow the detection of other proteases through the use of succinylated substrate specific for given protease. In order to improve the sensitivity and versatility of the assay we have refined the buffer conditions (PBS pH 7.2/1 mM CaCl2) to provide a 60 % increase in sensitivity to elastolytic activity and also formulated a substrate mixture containing succinylated elastin, collagen and gelatin. The combination of a substrate mixture and an optimum buffer will allow a spectrum of enzymes to be detected in a single reaction, providing a more complete picture of total proteolytic activity in a biological sample. This assay may also provide a tool to use proteolytic activity as a marker to monitor pathologic conditions involving matrix turn-over.
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PMID:Single microassay for matrix degrading enzymes. 1128 68

Two kit preparations of the organometallic precursor [(188)Re(H(2)O)(3)(CO)(3)](+) in aqueous media are presented. Method A uses gaseous carbon monoxide and amine borane (BH(3).NH(3)) as the reducing agent. In method B CO(g) is replaced by K(2)[H(3)BCO(2)] that releases carbon monoxide during hydrolysis. Both procedures afford the desired precursor in yields >85% after 10 min at 60 degrees C. HPLC and TLC analyses revealed 7 +/- 3% of unreacted (188)ReO(4)(-) and <5% of colloidal (188)ReO(2). Solutions of up to 14 GBq/mL Re-188 have been successfully carbonylated with these two methods. The syntheses of two tailor-made bifunctional ligand systems for the precursor [(188)Re(H(2)O)(3)(CO)(3)](+) are presented. The tridentate chelates consist of a bis[imidazol-2-yl]methylamine or an iminodiacetic acid moiety, respectively. Both types of ligand systems have been prepared with alkyl spacers of different length and a pendent primary amino or carboxylic acid functionality, enabling the amidic linkage to biomolecules. The tridentate coordination of the ligands to the rhenium-tricarbonyl core could be elucidated on the macroscopic level by X-ray structure analyses and 1D and 2D NMR experiments of two representative model complexes. On the nca level, the ligands allow labeling yields >95% with [(188)Re(H(2)O)(3)(CO)(3)](+) under mild reaction conditions (PBS buffer, 60 degrees C, 60 min) at ligand concentrations between 5 x 10(-4) M and 5 x 10(-5) M. Thus, specific activities of 22-220 GBq pe micromol of ligand could be achieved. Incubation of the corresponding Re-188 complexes in human serum at 37 degrees C revealed stabilities between 80 +/- 4% and 45 +/- 10% at 24 h, respectively, and 63 +/- 3% and 34 +/- 3% at 48 h postincubation in human serum depending on the chelating system. Decomposition product was mainly (188)ReO(4)(-). The routine kit-preparation of the precursor [(188)Re(H(2)O)(3)(CO)(3)](+) in combination with tailor-made ligand systems enables the organometallic labeling of biomolecules with unprecedented high specific activities.
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PMID:Steps toward high specific activity labeling of biomolecules for therapeutic application: preparation of precursor [(188)Re(H(2)O)(3)(CO)(3)](+) and synthesis of tailor-made bifunctional ligand systems. 1212 Nov 30

A calcium-dependent lectin (chiletin) was isolated from oyster haemolymph by mannose elution from Sepharose CL-6B followed by anion exchange chromatography. Chiletin was predominantly composed of 12 and 24 kDa bands when examined with SDS-PAGE under reducing and non-reducing conditions, respectively. Larger molecular weight bands of 36 and 50 kDa were also variably present under reducing conditions. The NH2-terminal sequence of the 24 kDa band was determined and was not homologous to any known protein from the databases searched. Isolated chiletin was composed of multiple isomers approximately 12 kDa in size and ranging in pI from 5.2 to 6.0. Rabbit antiserum was raised to a synthetic peptide coupled to keyhole limpet hemocyanin and the size of the chiletin subunits was confirmed by Western blot. Two and five different conformational aggregates of chiletin were resolved in oyster haemolymph using size exclusion chromatography in 8 M urea and PBS, respectively. The largest aggregate obtained from size exclusion in 8 M urea was estimated to be greater than 640 kDa. The ability of whole haemolymph and isolated chiletin to agglutinate sheep red blood cells was inhibited by galactose and mannose. Chiletin was identified by immunohistochemistry to be most consistently present in the auricle, followed by the digestive gland, however staining was seen sporadically in haemocytes, gastrointestinal epithelium and interstitial connective tissue cells.
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PMID:Isolation and partial characterization of a calcium-dependent lectin (chiletin) from the haemolymph of the flat oyster, Ostrea chilensis. 1531 12

Layer-by-layer deposited anticoagulant multilayer films were prepared on ammonia plasma treated poly (vinyl chloride) (PVC). Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) and contact angle results revealed the presence of -NH2 on the ammonia plasma treated PVC surfaces and the layer-by-layer self-assembly process. The stability of multilayer film was studied with the radio labeled method. The remainder bovine serum albumin (BSA) in cross-linked 5(heparin/BSA) multilayer films dipped in phosphate buffered saline (PBS, pH 7.4) was more than 90% in 40 days. The static platelet adhesion result indicated the anticoagulant multilayer films deposited on the plasma treated PVC reduced platelet adhesion drastically and no thrombus forming. The plasma recalcification time revealed that the multilayer modified surfaces greatly prolonged the plasma recalcification time. Such an easy processing and shape-independent method may have good potential for surface modification of cardiovascular devices.
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PMID:Fabrication of thromboresistant multilayer thin film on plasma treated poly (vinyl chloride) surface. 1596 2

Thermoresponsive and injectable semi-interpenetrating polymer networks (sIPNs) containing a biospecific cell-adhesive signal and proteolytically degradable domains were developed as a synthetic equivalent of the extracellular matrix (ECM). The sIPNs synthesized define a modular hydrogel ECM where different properties of the matrix can be manipulated independently, thus creating a system where parametric analysis of the effect of hydrogel properties on cell proliferation and differentiation is possible. sIPNs composed of poly(N-isopropylacrylamide-co-acrylic acid) [p(NIPAAm-co-AAc)] and RGD-grafted poly(acrylic acid) linear chains [p(AAc)-g-RGD] were synthesized with peptide crosslinkers containing a matrix metalloproteinase-13 (MMP-13, collagenase-3) degradable domain. The lower critical solution temperature (LCST) of peptide-crosslinked p(NIPAAm-co-AAc) sIPNs was not influenced by the addition of either linear p(AAc) or peptide-modified p(AAc) chains ( approximately 34 degrees C) in PBS. Degradation of peptide-crosslinked hydrogels and sIPNs was enzyme specific and concentration dependent. Exposure of rat calvarial osteoblast (RCO) culture to the degradation products from the peptide-crosslinked hydrogels did not significantly affect cell viability. Migration of RCOs into the sIPNs was dependent upon the presence of both a cell-adhesive RGD peptide (Ac-CGGNGEPRGDTYRAY-NH2) and proteolytically-degradable crosslinks; however, there was greater dependence on the latter. The sIPNs synthesized are versatile materials for assessing cell fate in synthetic ECM constructs in vitro and tissue regeneration in vivo.
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PMID:Synthetic MMP-13 degradable ECMs based on poly(N-isopropylacrylamide-co-acrylic acid) semi-interpenetrating polymer networks. I. Degradation and cell migration. 1604 78

In this study, the reactions of N-acetyl-L-methionine (AcMet) with [{trans-PtCl(NH(3))(2)}(2)-mu-H(2)N(CH(2))(6)NH(2)](NO(3))(2) (BBR3005: 1,1/t,t 1) and its cis analog [{cis-PtCl(NH(3))(2)}(2)-mu-{H(2)N(CH(2))(6)NH(2)}]Cl(2) (1,1/c,c 2) were analyzed to determine the rate and reaction profile of chloride substitution by methionine sulfur. The reactions were studied in PBS buffer at 37 degrees C by a combination of multinuclear ((195)Pt, {(1)H-(15)N} HSQC) magnetic resonance (NMR) spectroscopy and electrospray ionization time of flight mass spectrometry (ESITOFMS). The diamine linker of the 1,1/t,t trans complex was released as a result of the trans influence of the coordinated sulfur atom, producing trans-[PtCl(AcMet)(NH(3))(2)](+) (III) and trans-[Pt(AcMet)(2)(NH(3))(2)](2+) (IV). In contrast the cis geometry of the dinuclear compound maintained the diamine bridge intact and a number of novel dinuclear platinum compounds obtained by stepwise substitution of sulfur on both platinum centers were identified. These include (charges omitted for clarity): [{cis-PtCl(NH(3))(2)}-mu-NH(2)(CH(2))(6)NH(2)-{cis-Pt(AcMet)(NH(3))(2)}] (V); [{cis-Pt(AcMet)(NH(3))(2)}(2)-mu-NH(2)(CH(2))(6)NH(2)] (VI); [{cis-PtCl(NH(3))(2)}-mu-NH(2)(CH(2))(6)NH(2)-{PtCl(AcMet)NH(3)] (VII); [{PtCl(AcMet)(NH(3))}(2)-mu-NH(2)(CH(2))(6)NH(2)] (VIII); [{trans-Pt(AcMet)(2)(NH(3))}-mu-NH(2)(CH(2))(6)NH(2)-{PtCl(AcMet)(NH(3))] (IX) and the fully substituted [{trans-Pt(AcMet)(2)(NH(3))}(2)-mu-{NH(2)(CH(2))(6)NH(2)] (X). For both compounds the reactions with methionine were slower than those with glutathione (Inorg Chem 2003, 42:5498-5506). Further, the 1,1/c,c geometry resulted in slower reaction than the trans isomer, because of steric hindrance of the bridge, as observed previously in reactions with DNA and model nucleotides.
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PMID:Effects of geometric isomerism in dinuclear antitumor platinum complexes on their interactions with N-acetyl-L-methionine. 1609 34

We cloned the streptolysin O gene from the Streptococcus pyogenes genome and tested the possibility of using it as an anticancer reagent. Transient transfection of the streptolysin O gene efficiently killed 293T cells after 12 hours of transfection as determined by lactate dehydrogenase release and propidium iodide uptake. No caspase activity was observed and necrosis was prominent during streptolysin O-induced cell death. Biochemical analysis of streptolysin O protein revealed that the deletion of only 5 amino acids from the COOH-terminal region of streptolysin O, which is essential for cholesterol binding activity, abolished its cell-killing activity, whereas the NH2-terminal region was more resilient, i.e., up to 115 amino acids could be deleted without changing its cell-killing activity. We generated a streptolysin O-expressing adenovirus and injected it into human cervical cancer cell-derived tumors grown in a nude mouse model. Twenty-one days postinjection, the average size of tumors in the streptolysin O adenovirus-injected group was 29.3% of that of the control PBS-treated group. Our results show that the genes of pore-forming toxins, like streptolysin O protein, have the potential to establish a novel class of suicide gene therapeutic reagents.
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PMID:Suicide cancer gene therapy using pore-forming toxin, streptolysin O. 1681 21


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