Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aim of the present work was to find a suitable method for the introduction of thiol functions on the surface of poly(DL-lactic acid) (PLA) nanoparticles. Three different approaches were investigated. The modification of the surface involves the activation of PLA carboxylic acid groups followed by the attack of a nucleophile such as cysteine (method #1) or cystamine (method #2 and #3) that provide thiol functions via an amide bond. For the conjugation with cystamine, a second reaction step is required to expose the sulfhydryl function of cystamine that is otherwise protected in a disulfide bond. 1,4-Dithio-DL-threitol (DTT) (method #2) or Tris(2-carboxyethyl)-phosphine hydrochloride (method #3) were evaluated for their ability to reduce this linkage. Method #1 allowed a maximum of 8.5+/-2.8 mmol of thiol functions per mol of PLA to be attached on the surface of the nanoparticles. Method #2 allowed the introduction of a greater number of thiol functions (up to 190+/-15 mmol per mol of PLA). However, this latter method has a major drawback: DTT interacts strongly with the nanoparticle matrix during the reduction step. Method #3 has the advantage over method #2 in that it allowed a significant number of thiol functions to be covalently bound to the particles (up to 107.6+/-0.6 mmol per mol of PLA) without the problem of undesired interaction between DTT and the nanoparticle matrix. The introduction of thiol groups onto the surface of PLA nanoparticles is possible with all three suggested methods. The method #3 provides a straight forward approach for the substitution of carboxylic acid groups with a high number of activated sulfhydryl at the surface of PLA nanoparticles.
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PMID:Surface modification of poly(lactic acid) nanoparticles by covalent attachment of thiol groups by means of three methods. 1252 60

Venom toxin-specific antibodies offer a more rational treatment of snake envenoming than conventional antivenom. Here, we describe novel cDNAs encoding phospholipase A(2) (PLA(2)) isoforms from venom gland RNA of Echis pyramidum leakeyi (Epl), Echis sochureki (Es) and Echis ocellatus (Eo). The deduced amino acid sequences of these cDNAs encoded proteins with high overall sequence identity to the viper group II PLA(2) protein family, including the 14 cysteine residues capable of forming seven disulphide bonds that characterize this group of PLA(2) enzymes. Comparison of the PLA(2) sequences from Echis with those from related vipers failed to make significant geographic, taxonomic or PLA(2)-function distinctions between these Echis PLA(2) isoforms. However, their deduced hydrophilicity profiles revealed a conserved tertiary structure that we will exploit, by epidermal DNA immunization, to generate PLA(2)-neutralizing antibodies with polyspecific potential.
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PMID:Molecular cloning of phospholipases A(2) from venom glands of Echis carpet vipers. 1287 67

As plasminogen activator inhibitor-1 (PAI-1), the physiological inhibitor of tissue-type plasminogen activator, is considered to be an important risk factor in several (patho)physiological conditions, many research activities focus on attempts to inhibit this serpin. The approach illustrated in the current study focuses on elucidating important interaction sites allowing the inhibition of PAI-1. Since monoclonal antibodies are in most cases not ideal for therapeutic use, the question of whether smaller molecules exert comparable effects is a hot issue. To answer this question, Cys residues were introduced in PAI-1 at positions previously identified as determining the epitope of a PAI-1-inhibiting antibody, MA-8H9D4, resulting in PAI-1-R300C, PAI-1-Q303C, and PAI-1-D305C. Subsequently, low molecular mass sulfhydryl-specific reagents (i.e. BODIPY 530/550 IA (molecular mass 626 Da) and BODIPY FL C(1)-IA (molecular mass 417 Da)) were allowed to react covalently with the cysteine. The functional distribution (inhibitory versus substrate) toward tissue-type plasminogen activator was determined for the labeled and the unlabeled samples. Labeling at position 300 leads to a 1.7- and 2.2-fold increase in SI value for BODIPY 530/550 IA and BODIPY FL C(1)-IA, respectively. Labeling at position 303 results in a 3.3- and 1.9-fold increase of the SI value for the large and the small label, respectively. At position 305, the SI values are 3.1-fold increased for both labels. The effect (on SI and on serpin activity) of the manipulations at these positions is in good agreement with the effect exerted by MA-8H9D4. In conclusion, our study provides proof of concept for the proposed approach in evaluating whether targeting a functional epitope with a small synthetic compound may be a feasible strategy in rational drug design.
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PMID:Site-directed targeting of plasminogen activator inhibitor-1 as an example for a novel approach in rational drug design. 1498 11

Endogenous fibrinolysis inhibitors may be involved in t-PA resistance, decreasing stroke thrombolysis benefits. We aim to determine the impact of pretreatment levels of plasminogen activator inhibitor (PAI-1), lipoprotein(a), thrombin-activatable fibrinolysis inhibitor (TAFI) and homocysteine on arterial recanalization and outcome. Forty-four consecutive patients with acute proximal middle cerebral artery occlusion were studied, including assessment of transcranial Doppler artery patency. The neurological status was determined by NIH Stroke Scale (NIHSS) and long-term outcome with modified Rankin Scale (mRS). Patients who recanalized after t-PA infusion had lower PAI-1 levels than those who remained occluded. Similarly, patients who achieved dramatic clinical recovery at 12 hours exhibited significantly lower PAI-1 levels as those independent (mRS< or =2) at third month. We observed a trend towards lower lipoprotein p(a) in patients who achieved recanalization at 1 hour, whereas no relation was found between TAFI or homo-cysteine levels and recanalization. After a regression model was applied the only independent predictor of thrombolysis resistance was baseline PAI-1>34 ng/ml, such that high PAI-1 levels interfere with tPA-induced recanalization in stroke, predicting a higher susceptibility towards clot-lysis resistance and poor out-come.
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PMID:Admission fibrinolytic profile predicts clot lysis resistance in stroke patients treated with tissue plasminogen activator. 1517 1

Exposure of renal proximal tubule cells to oxalate may play an important role in cell proliferation, but the signaling pathways involved in this effect have not been elucidated. Thus the present study was performed to examine the effect of oxalate on (3)H-labeled thymidine incorporation and its related signal pathway in primary cultured rabbit renal proximal tubule cells (PTCs). The effects of oxalate on [(3)H]thymidine incorporation, lactate dehydrogenase (LDH) release, Trypan blue exclusion, H(2)O(2) release, activation of mitogen-activated protein kinases (MAPKs), and (3)H-labeled arachidonic acid (AA) release were examined in primary cultured renal PTCs. Oxalate inhibited [(3)H]thymidine incorporation in a time- and dose-dependent manner. However, its analogs did not affect [(3)H]thymidine incorporation. Oxalate (1 mM) significantly increased H(2)O(2) release, which was blocked by N-acetyl-l-cysteine (NAC) and catalase (antioxidants). Oxalate significantly increased p38 MAPK and stress-activated protein kinase (SAPK)/c-Jun NH(2)-terminal kinase (JNK) activity, not p44/42 MAPK. Oxalate stimulated [(3)H]AA release and translocation of cytosolic phospholipase A(2) (cPLA(2)) from the cytosolic fraction to the membrane fraction. Indeed, oxalate significantly increased prostaglandin E(2) (PGE(2)) production compared with control. Oxalate-induced inhibition of [(3)H]thymidine incorporation and increase of [(3)H]AA release were prevented by antioxidants (NAC), a p38 MAPK inhibitor (SB-203580), a SAPK/JNK inhibitor (SP-600125), or PLA(2) inhibitors [mepacrine and arachidonyl trifluoromethyl ketone (AACOCF(3))], but not by a p44/42 MAPK inhibitor (PD-98059). These findings suggest that oxalate inhibits renal PTC proliferation via oxidative stress, p38 MAPK/JNK, and cPLA(2) signaling pathways.
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PMID:Oxalate inhibits renal proximal tubule cell proliferation via oxidative stress, p38 MAPK/JNK, and cPLA2 signaling pathways. 1522 3

Effect of three epsilon-aminocaproylaminoacids with a significant antifibrinolytic activity on amidolytic activity of tissue plasminogen activator (t-PA), urokinase and kallikrein was examined. epsilon-Aminocaproyl-S-benzyl)-L-cysteine and epsilon-aminocaproyl-L-norleucine were weak inhibitors of kallikrein. Weak activation of t-PA activity was observed at high concentration of the tested compounds. Only one of the examined dipeptides was a weak inhibitor of amidolytic activity of urokinase.
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PMID:Effects of epsilon-aminocaproiloaminoacids on the amidolytic activity of tissue plasminogen activator, urokinase and kallikrein. 1525 61

Plasminogen activator inhibitor type-1 (PAI-1), the primary regulator of plasminogen activator - urokinase (uPA) plays a crucial role in the cell adhesion and migration and in angiogenesis. We had previously demonstrated that PAI-1 - endothelial cell interplay is critical for the formation of new blood vessels and the process is mostly conducted via uPA- anti-proteinase interaction. In the present study we wished to further examine the role of PAI-1 in the sprout formation, representing the first step of new capillary vessels development by evaluating the effect of PAI-1 on the sprout area. We addressed the issue by assessing the influence of cysteine-mutated PAI-1 proteins characterized by a prolonged half-life time (hD beta T - T(1/2)= 63.59 h and beta T-T(1/2)= 6931.47 h), and therefore more stable anti-uPA activity, on the appearance of newly formed sprouts. We found that both CysPAI-1 proteins significantly diminished the mean sprout area in a concentration-dependent fashion. The inhibitory effect present in the two examined endothelial cells systems of different origin and functional characteristics - human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells (HLMVEC) cultures - was noticeably greater for HLMVEC -high urokinase-producers. Moreover, the inhibition rate was significantly greater for the beta T mutant than that for the hD beta T PAI-1 mutant in all examined doses (P<0.002), proving a key role of anti-proteinase activity for this effect. We concluded that, apart from the total sprout length, as it had repeatedly been demonstrated before, also affects the sprout area in the in vitro sprout formation angiogenesis assay. This effect was achieved mainly via PAI-1's antiproteinase activity.
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PMID:Plasminogen activator inhibitor type-1 controls the process of the in vitro sprout formation. 1561 93

Accelerated evolution of toxins is a unique feature of venoms, with the toxins evolving via the birth-and-death mode of molecular evolution. The venoms of sea snakes, however, are remarkably simple in comparison to those of land snakes, which contain highly complex venoms. Aipysurus eydouxii (Marbled sea snake) is a particularly unique sea snake, feeding exclusively upon fish eggs. Secondary to this ecological change, the fangs have been lost and the venom glands greatly atrophied. We recently showed that the only neurotoxin (a three-finger toxin) gene found in the sea snake A. eydouxii has a dinucleotide deletion, resulting in the loss of neurotoxic activity. During these studies, we isolated and identified a number of cDNA clones encoding isozymes of phospholipase A(2) (PLA(2)) toxins from its venom gland. Sixteen unique PLA(2) clones were sequenced from the cDNA library and TA cloning of reverse transcription-polymerase chain reaction products. Phylogenetic analysis of these clones revealed that less diversification of the PLA(2) toxins has occurred in the A. eydouxii venom gland in comparison to equivalent terrestrial and other marine snakes. As there is no longer a positive selection pressure acting upon the venom, mutations have accumulated in the toxin-coding regions that would have otherwise had a deleterious effect upon the ability to use the venom for prey capture. Such mutations include substitutions of highly conserved residues; in one clone, the active site His(48) is replaced by Arg, and in two other clones, highly conserved cysteine residues are replaced. These mutations significantly affect the functional and structural properties of these PLA(2) enzymes, respectively. Thus, in A. eydouxii, the loss of the main neurotoxin is accompanied by a much slower rate of molecular evolution of the PLA(2) toxins as a consequence of the snake's shift in ecological niche. This is the first case of decelerated evolution of toxins in snake venom.
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PMID:Putting the brakes on snake venom evolution: the unique molecular evolutionary patterns of Aipysurus eydouxii (Marbled sea snake) phospholipase A2 toxins. 1563 56

During pathologic vessel remodeling, vascular smooth muscle cells (VSMCs) embedded within the collagen-rich matrix of the artery wall mobilize uncharacterized proteolytic systems to infiltrate the subendothelial space and generate neointimal lesions. Although the VSMC-derived serine proteinases, plasminogen activator and plasminogen, the cysteine proteinases, cathepsins L, S, and K, and the matrix metalloproteinases MMP-2 and MMP-9 have each been linked to pathologic matrix-remodeling states in vitro and in vivo, the role that these or other proteinases play in allowing VSMCs to negotiate the three-dimensional (3-D) cross-linked extracellular matrix of the arterial wall remains undefined. Herein, we demonstrate that VSMCs proteolytically remodel and invade collagenous barriers independently of plasmin, cathepsins L, S, or K, MMP-2, or MMP-9. Instead, we identify the membrane-anchored matrix metalloproteinase, MT1-MMP, as the key pericellular collagenolysin that controls the ability of VSMCs to degrade and infiltrate 3-D barriers of interstitial collagen, including the arterial wall. Furthermore, genetic deletion of the proteinase affords mice with a protected status against neointimal hyperplasia and lumen narrowing in vivo. These studies suggest that therapeutic interventions designed to target MT1-MMP could prove beneficial in a range of human vascular disease states associated with the destructive remodeling of the vessel wall extracellular matrix.
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PMID:MT1-matrix metalloproteinase directs arterial wall invasion and neointima formation by vascular smooth muscle cells. 1614 77

To resolve the bleeding risk associated with thrombolytic therapy, we have designed an approach, termed ATTEMPTS (Antibody Targeted Triggered Electrically Modified Prodrug Type Strategy), to deliver t-PA to the clot site in an inactive form and then trigger its conversion to the active form, so that it would selectively activate the clot bound plasminogen while alleviating the bleeding risk. This delivery system was composed of a large protein complex, consisting of two components: (i) a heparin-modified, negatively charged fibrin-targeting antibody; and (ii) a cationic peptide-modified, positively charged t-PA. Both in vitro and in vivo studies have confirmed the feasibility of this targeted drug delivery approach. A site-specific thrombolysis was observed in animals, without concomitant depletion of the coagulation factors -- the phenomenon in conventional thrombolytic therapy that contributes to the bleeding risk. Despite promise, the chemical conjugation method employed previously in the preparation of the cationic peptide-modified t-PA also revealed several major shortcomings. The primary drawback was that the number of the cationic peptides and the location at which these peptides were attached to a t-PA molecule could not be regulated by using the chemical conjugation method. As a consequence, the resultant modified t-PA possessed a wide range of heparin-binding strength, rendering the inhibition of t-PA activity by heparin binding ineffective. In this paper, we present a new strategy in producing the desired modified t-PA, utilizing the genetic engineering approach. A computer simulation-guided rational design strategy was adopted to identify the most desirable site in t-PA (i.e. the 37-loop) for incorporation of the heparin-binding peptide sequence. By altering the amino acid composition via mutation at three locations, i.e. Ser(300) to Cys, Gly(302) to Arg, and Glu(303) to Arg, a highly cationic nanomer sequence consisting of (297)KHRRCPRRR(304) and possessing a well-demonstrated heparin-binding domain was established within the 37-loop. To ensure the binding of heparin to this specifically modified domain, a cysteine residue (i.e. Cys(300)) was created to allow for site-specific conjugation of an additional heparin-binding peptide (i.e. the LMWP peptide previously developed in our laboratory) to this domain via the chemical conjugation method. In vitro fibrinolysis assays showed that both the t-PA mutant and the LMWP-attached t-PA mutant exhibited a fibrinolytic potency similar to that of the wild type t-PA. Inhibition studies using small chromogenic substrates demonstrated that the activity of mutant tPA-LMWP could be significantly inhibited by heparin binding. In conclusion, using computer simulation and molecular biology approaches, a mutated t-PA that meets the needs of the ATTEMPTS system, in providing a safe thrombolytic therapy, could be readily prepared.
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PMID:Construction and characterization of a t-PA mutant for use in ATTEMPTS: a drug delivery system for achieving targeted thrombolysis. 1626 60


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