scholarly journals Antagonists of Hsp16.3, a Low-Molecular-Weight Mycobacterial Chaperone and Virulence Factor, Derived from Phage-Displayed Peptide Libraries

2005 ◽  
Vol 71 (11) ◽  
pp. 7334-7344 ◽  
Author(s):  
Abhik Saha ◽  
Archna Sharma ◽  
Amlanjyoti Dhar ◽  
Bhabatarak Bhattacharyya ◽  
Siddhartha Roy ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Close Association ◽  
Peptide Libraries ◽  
Eye Lens ◽  
Low Molecular Weight ◽  
Lens Protein ◽  
Dependent Manner ◽  
Random Peptide ◽  
Eye Lens Protein

ABSTRACT The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.

Dermatan Sulfate Is a More Potent Inhibitor of Clot-bound Thrombin than Unfractionated and Low Molecular Weight Heparins

1994 ◽  
Vol 71 (05) ◽  
pp. 576-580 ◽  
Author(s):  
P Bendayan ◽  
H Boccalon ◽  
D Dupouy ◽  
B Boneu
Keyword(s):  
Molecular Weight ◽  
Dermatan Sulfate ◽  
Potent Inhibitor ◽  
Final Concentration ◽  
Coagulation Cascade ◽  
Low Molecular Weight ◽  
Dependent Manner ◽  
Low Molecular Weight Heparins ◽  
Experimental Conditions

SummaryClot-bound thrombin proteolyses fibrinogen and amplifies the coagulation cascade at its close vicinity, thereby ensuring the growth of fibrin-rich thrombus. The present study compares the ability of various glycosaminoglycans (GAGs) to inhibit these 2 properties. Unfractionated heparin (UH), 3 low molecular weight heparins (LMWHs) with increasing antifactor Xa/antifactor Ha ratio, the synthetic pentasaccharide (PS), devoid of antifactor Ha activity, and dermatan sulfate (DS), a catalyst of thrombin inhibition by heparin cofactor II, were selected on the basis of their different properties. Proteolysis of fibrinogen by clot-bound thrombin was evaluated by measuring fibrinopeptide A (FPA) generation after an incubation of standardized washed clots in plasma for 120 min in absence or in presence of increasing concentrations of heparins or of DS. The results were compared to those obtained when free a-thrombin (0.4 nM) was added to plasma in the same experimental conditions. On the basis of equivalent antithrombin units, UH and LMWHs gave identical results. To inhibit by 70% fibrinogen proteolysis induced by clot-bound thrombin (IC 70), 5- to 9-fold higher concentrations of UH or of LMWHs were required in comparison with those required to inhibit free thrombin. For DS, only a 1.3 times higher concentration was required. PS (final concentration 1 anti Xa U • ml-1) was devoid of any inhibitory effect. The amplification of the coagulation cascade induced by dot-bound thrombin was evaluated by measuring the shortening of whole blood clotting time (WBCT) resulting from the incubation of washed clots in native blood. In absence of GAG, clot-bound thrombin reduced WBCT from 18 ± 2 min to 9 ± 1 min. Each GAG prolonged WBCT in a dose-dependent manner but these prolongations were smaller in presence of washed clots. The most potent agent to suppress the shortening of WBCT was DS. LMWH and UH were less effective and PS (final concentration 1 anti Xa U/ml) was almost ineffective. Therefore, in these in vitro experiments, DS is a more potent inhibitor of clot-bound thrombin than heparin. Whether or not these observations are relevant for the treatment of established deep-vein thrombosis requires comparative clinical studies.

scholarly journals Inhibition of advanced glycation end-product formation on eye lens protein by rutin

2011 ◽  
Vol 107 (7) ◽  
pp. 941-949 ◽  
Author(s):  
P. Muthenna ◽  
C. Akileshwari ◽  
Megha Saraswat ◽  
G. Bhanuprakash Reddy
Keyword(s):  
Product Formation ◽  
Protein Glycation ◽  
Eye Lens ◽  
Lens Protein ◽  
Advanced Glycation ◽  
Mechanism Of Inhibition ◽  
Eye Lens Protein ◽  
Herbs And Spices

Formation of advanced glycation end products (AGE) plays a key role in the several pathophysiologies associated with ageing and diabetes, such as arthritis, atherosclerosis, chronic renal insufficiency, Alzheimer's disease, nephropathy, neuropathy and cataract. This raises the possibility of inhibition of AGE formation as one of the approaches to prevent or arrest the progression of diabetic complications. Previously, we have reported that some common dietary sources such as fruits, vegetables, herbs and spices have the potential to inhibit AGE formation. Flavonoids are abundantly found in fruits, vegetables, herbs and spices, and rutin is one of the commonly found dietary flavonols. In the present study, we have demonstrated the antiglycating potential and mechanism of action of rutin using goat eye lens proteins as model proteins. Under in vitro conditions, rutin inhibited glycation as assessed by SDS-PAGE, AGE-fluorescence, boronate affinity chromatography and immunodetection of specific AGE. Further, we provided insight into the mechanism of inhibition of protein glycation that rutin not only scavenges free-radicals directly but also chelates the metal ions by forming complexes with them and thereby partly inhibiting post-Amadori formation. These findings indicate the potential of rutin to prevent and/or inhibit protein glycation and the prospects for controlling AGE-mediated diabetic pathological conditions in vivo.

scholarly journals Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging

2021 ◽  
Author(s):  
Brenna Norton-Baker ◽  
Pedram Mehrabi ◽  
Ashley O. Kwok ◽  
Kyle W. Roskamp ◽  
Marc A. Spague-Piercy ◽  
...  
Keyword(s):  
Structural Changes ◽  
Accelerated Aging ◽  
Redox Activity ◽  
Human Lens ◽  
Eye Lens ◽  
Lens Protein ◽  
Wild Type ◽  
Eye Lens Protein ◽  
Over Time

Cataract disease, a clouding of the eye lens due to precipitation of lens proteins, affects millions of people every year worldwide. The proteins that comprise the lens, the crystallins, show extensive post-translational modifications (PTMs) in aged and cataractous lenses, most commonly deamidation and oxidation. Although surface-exposed glutamines and asparagines show the highest rates of deamidation, multiple modifications can accumulate over time in these long-lived proteins, even for buried residues. Both deamidation and oxidation have been shown to promote crystallin aggregation in vitro; however, it is not clear precisely how these modified crystallins contribute to insolubilization. Here, we report six novel crystal structures of a major human lens protein, γS-crystallin (γS): one of the wild-type in a monomeric state, and five of deamidated γS variants, ranging from three to nine deamidation sites, after varying degrees of sample aging. Consistent with previous work that focused on single- to triple-site deamidation, the deamidation mutations do not appear to drastically change the fold of γS; however, increasing deamidation leads to accelerated oxidation and disulfide bond formation. Successive addition of deamidated sites progressively destabilized protein structure as evaluated by differential scanning fluorimetry. Light scattering showed the deamidated variants display an increased propensity for aggregation compared to the wild-type protein. The results suggest the deamidated variants are useful as models for accelerated aging; the structural changes observed over time provide support for redox activity of γS-crystallin in the human lens.

In Vitro Characterization of the Neutralization of Unfractionated Heparin and Low Molecular Weight Heparin by Novel Salicylamide Derivatives.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1869-1869
Author(s):  
Walter Jeske ◽  
Aleah Brubaker ◽  
Dahui Liu ◽  
Trevor Young ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Anticoagulant Activity ◽  
Protamine Sulfate ◽  
In Vitro Assays ◽  
Low Molecular Weight ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Salicylamide Derivatives

Abstract Introduction: Unfractionated heparin and the low molecular weight heparins (LMWHs) are commonly used in the treatment of acute coronary syndromes, as prophylaxis against deep vein thrombosis and pulmonary embolism and to prevent clotting during interventional and surgical procedures. The neutralization of unfractionated heparin is critical following the completion of coronary bypass surgery to avoid excessive blood loss. Unfractionated heparin can be neutralized by protamine sulfate, a highly cationic peptide that binds to heparin in a charge-dependent manner. However, the use of protamine can be associated with serious side-effects such as hypotension, bronchoconstriction, or pulmonary hypertension, possibly due to the release of histamine. Additionally, large doses of protamine can produce an anticoagulant effect. This study characterizes the ability of a series of low molecular weight, homogeneous, synthetic, polycationic salicylamide derivatives (PolyMedix, Radnor, PA) to neutralize the anticoagulant actions of unfractionated heparin and enoxaparin. Methods: Human plasma was supplemented with unfractionated heparin or enoxaparin (Sanofi-Aventis, Paris, France) at a concentration of 10 μg/ml. Protamine sulfate or one of six of the structurally distinct salicylamide derivatives was added to aliquots of heparinized plasma to achieve final concentrations of 50, 25 and 12.5 μg/ml. The supplemented plasmas were immediately analyzed using clotting (aPTT, Heptest, thrombin time) and amidolytic (anti-Xa, anti-IIa) assays. Results: Using the in vitro assays, protamine sulfate was shown to concentration-dependently neutralize the actions of unfractionated heparin in all of the assays. Two of the salicylamide derivatives tested produced an effect comparable to protamine, while three derivatives exhibited a relatively stronger neutralization of unfractionated heparin. The extent of neutralization measured by anti-Xa and anti-IIa assays was also greater with the derivatives. While residual anti-Xa and anti-IIa activities (20% and 10%, respectively) were observed even with a 5-fold gravimetric excess of protamine, complete neutralization was observed with the salicylamide derivatives. Protamine is known to be less effective at neutralizing LMWHs. In this study, the anticoagulant activity of enoxaparin as measured by aPTT and Heptest was neutralized approximately 50% by protamine even at a 5:1 protamine to enoxaparin ratio. The derivatives were able to completely neutralize the anticoagulant effects of enoxaparin. A similar pattern was observed with the amidolytic assays. While protamine was unable to neutralize the anti-Xa activity of enoxaparin, 5 of the 6 salicylamide derivatives concentration-dependently inhibited the anti-Xa activity. Discussion: These studies demonstrate that the PolyMedix series of salicylamide derivatives can effectively neutralize the anticoagulant and anti-protease actions of unfractionated heparin and LMWHs such as enoxaparin. Initial results suggest that such agents are more effective than protamine at neutralizing other LMWHs. Future studies are designed to characterize the compounds’ PK/PD profiles. These results warrant further studies on the neutralization profile of PolyMedix series of salicylamide derivatives in animal models of bleeding and thrombosis.

The Effects of Standard and Low Molecular Weight Heparin on Bone Nodule Formation In Vitro

1998 ◽  
Vol 80 (09) ◽  
pp. 413-417 ◽  
Author(s):  
Mohit Bhandari ◽  
Jack Hirsh ◽  
Jeffrey Weitz ◽  
Edward Young ◽  
Thomas Venner ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Nodule Formation ◽  
Low Molecular Weight ◽  
Dependent Manner ◽  
Bone Nodule ◽  
Osteoblast Function ◽  
Alp Activity ◽  
Bone Nodule Formation

SummaryPreviously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.

Effects of Low Molecular Weight Heparin and Unfragmented Heparin on Induction of Osteoporosis in Rats

1990 ◽  
Vol 63 (03) ◽  
pp. 505-509 ◽  
Author(s):  
Thomas Mätzsch ◽  
David Bergqvist ◽  
Ulla Hedner ◽  
Bo Nilsson ◽  
Per Østergaar
Keyword(s):  
Molecular Weight ◽  
Bone Mineral ◽  
Low Molecular Weight Heparin ◽  
Zinc Content ◽  
Low Molecular Weight ◽  
Dependent Manner ◽  
Bone Mineral Mass ◽  
Bone Ash ◽  
Dose Dependent ◽  
Mineral Mass

SummaryA comparison between the effect of low molecular weight heparin (LMWH) and unfragmented heparin (UH) on induction of osteoporosis was made in 60 rats treated with either UH (2 IU/ g b w), LMWH in 2 doses (2 Xal U/g or 0.4 Xal U/g) or placebo (saline) for 34 days. Studied variables were: bone mineral mass in femora; fragility of humera; zinc and calcium levels in serum and bone ash and albumin in plasma. A significant reduction in bone mineral mass was found in all heparin-treated rats. There was no difference between UH and LMWH in this respect. The effect was dose-dependent in LMWH-treated animals. The zinc contents in bone ash were decreased in all heparin-treated rats as compared with controls. No recognizable pattern was seen in alterations of zinc or calcium in serum. The fragility of the humera, tested as breaking strength did not differ between treatment groups and controls. In conclusion, if dosed according to similar factor Xa inhibitory activities, LMWH induces osteoporosis to the same extent as UH and in a dose-dependent manner. The zinc content in bone ash was decreased after heparin treatment, irrespective of type of heparin given.

Effects of Unfractionated and Low Molecular Weight Heparins on Platelet Thromboxane Biosynthesis “In Vivo”

1994 ◽  
Vol 72 (06) ◽  
pp. 942-946 ◽  
Author(s):  
Raffaele Landolfi ◽  
Erica De Candia ◽  
Bianca Rocca ◽  
Giovanni Ciabattoni ◽  
Armando Antinori ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Low Molecular Weight Heparin ◽  
Primary Source ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
Heparin Administration ◽  
To Receive

SummarySeveral “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.

The Effect of Dextran of Various Molecular Weight on the Coagulation in Dogs

1961 ◽  
Vol 06 (01) ◽  
pp. 015-024 ◽  
Author(s):  
Sven Erik Bergentz ◽  
Oddvar Eiken ◽  
Inga Marie Nilsson
Keyword(s):  
Molecular Weight ◽  
Body Weight ◽  
High Molecular Weight ◽  
Bleeding Time ◽  
Factor Vii ◽  
Low Molecular Weight ◽  
Factor V ◽  
Coagulation Mechanism ◽  
Coagulation Defects

Summary1. Infusions of low molecular weight dextran (Mw = 42 000) to dogs in doses of 1—1.5 g per kg body weight did not produce any significant changes in the coagulation mechanism.2. Infusions of high molecular weight dextran (Mw = 1 000 000) to dogs in doses of 1—1.5 g per kg body weight produced severe defects in the coagulation mechanism, namely prolongation of bleeding time and coagulation time, thrombocytopenia, pathological prothrombin consumption, decrease of fibrinogen, prothrombin and factor VII, factor V and AHG.3. Heparin treatment of the dogs was found to prevent the decrease of fibrinogen, prothrombin and factor VII, and factor V otherwise occurring after injection of high molecular weight dextran. Thrombocytopenia was not prevented.4. In in vitro experiments an interaction between fibrinogen and dextran of high and low molecular weight was found to take place in systems comprising pure fibrinogen. No such interaction occurred in the presence of plasma.5. It is concluded that the coagulation defects induced by infusions of high molecular weight dextran are due to intravascular coagulation.

Neutralization of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin by Protamine Sulfate in Rats

1986 ◽  
Vol 56 (03) ◽  
pp. 318-322 ◽  
Author(s):  
V Diness ◽  
P B Østergaard
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Thrombus Formation ◽  
Experimental Models ◽  
Protamine Sulfate ◽  
Low Molecular Weight ◽  
Antithrombotic Effect ◽  
Higher Doses

SummaryThe neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized.In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.

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