Development of Novel EE/Alginate Polyelectrolyte Complex Nanoparticles for Lysozyme Delivery: Physicochemical Properties and In Vitro Safety.

The number of biologic drugs has increased in the pharmaceutical industry due to their high therapeutic efficacy and selectivity. As such, safe and biocompatible delivery systems to improve their stability and efficacy are needed. Here, we developed novel cationic polymethacrylate-alginate (EE-alginate) pNPs for the biologic drug model lysozyme (Lys).

Preparation of a novel lipid-core micelle using a low-energy emulsification method.

High-energy methods for the manufacturing of nanomedicines are widely used; however, interest in low-energy methods is increasing due to their simplicity, better control over the process, and energy-saving characteristics during upscaling. Here, we developed a novel lipid-core micelle (LCM) as a nanocarrier to encapsulate a poorly water-soluble drug, nifedipine (NFD), by hot-melt emulsification, a low-energy method. LCMs are self-assembling colloidal particles composed of a hydrophobic core and a hydrophilic shell.

Nanomedicine and nanotoxicology: the pros and cons for neurodegeneration and brain cancer.

Current strategies for brain diseases are mostly symptomatic and noncurative. Nanotechnology has the potential to facilitate the transport of drugs across the blood-brain barrier and to enhance their pharmacokinetic profile. However, to reach clinical application, an understanding of nanoneurotoxicity in terms of oxidative stress and inflammation is required.

Kininogens: More than cysteine protease inhibitors and kinin precursors.

Two kininogens are found in mammalian sera: HK (high molecular weight kininogen) and LK (low molecular weight kininogen) with the exception of the rat which encompasses a third kininogen, T-Kininogen (TK). Kininogens are multifunctional glycosylated molecules related to cystatins (clan IH, family I25). They harbor three cystatin domains but only two of them are tight-binding inhibitors of cysteine cathepsins.

A comprehensive nomenclature for serine proteases with homology to tissue kallikreins.

The human kallikrein locus on chromosome 19q13.3-13.4 contains kallikrein 1--the tissue kallikrein--and 14 related serine proteases.

Mu-calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation.

Mu- and m-calpain are cysteine proteases requiring micro- and millimolar Ca2+ concentrations for their activation in vitro. Among other mechanisms, interaction of calpains with membrane phospholipids has been proposed to facilitate their activation by nanomolar [Ca2+] in living cells. Here the interaction of non-autolysing, C115A active-site mutated heterodimeric human mu-calpain with phospholipid bilayers was studied in vitro using protein-to-lipid fluorescence resonance energy transfer and surface plasmon resonance.

Histochemical assessment of early myocardial infarction using 2,3,5-triphenyltetrazolium chloride in blood-perfused porcine hearts.

Introduction: Although the chemical mechanism of the triphenyltetrazolium (TTC) reaction, for macroscopic detection of myocardial infarction, has been described previously, literature reports on correct tissue preparation and the use of this technique in intact large animals are lacking.

Methods: We investigated the special requirements for TTC staining in blood-perfused porcine hearts, validated the various handling steps and provided detailed information for precise and easy use of this histochemical method. The left anterior descending coronary artery was occluded for 45 min followed by 6 h of reperfusion in an open chest preparation using anesthetised domestic pigs.

A Kunitz-type glycosylated elastase inhibitor with one disulfide bridge.

A glycosylated Bauhinia rufa elastase inhibitor (gBrEI) was purified and characterized using acetone precipitation, affinity chromatography on concanavalin A-Sepharose, ion-exchange chromatography on a HiTrap Q column, size exclusion chromatography on a Superdex 200 column and reverse-phase chromatography on a C18 column. gBrEI inhibited pancreatic porcine elastase with an equilibrium dissociation constant (K(i)) of 6.18 x 10(-8) M, but it did not inhibit human neutrophil elastase, bovine trypsin, human plasma kallikrein or porcine pancreatic kallikrein.

Calpain inhibition reduces infarct size and improves global hemodynamics and left ventricular contractility in a porcine myocardial ischemia/reperfusion model.

Calpains, a family of Ca2+-dependent cysteine proteases, are activated during myocardial ischemia and reperfusion. This study investigates the cardioprotective effects of calpain inhibition on infarct size and global hemodynamics in an ischemia/reperfusion model in pigs, using the calpain inhibitor A-705253. The left anterior descending coronary artery was occluded for 45 min and reperfused for 6 h.

Reduction of myocardial infarction by calpain inhibitors A-705239 and A-705253 in isolated perfused rabbit hearts.

Two novel calpain inhibitors (A-705239 and A-705253) were studied in isolated perfused rabbit hearts subjected to 60-min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid in various final concentrations from the beginning of the experiments before the coronary artery was blocked. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were carried out.

Electrostatic interactions of domain III stabilize the inactive conformation of mu-calpain.

The ubiquitous mu- and m-calpains are Ca2+-dependent cysteine proteases. They are activated via rearrangement of the catalytic domain II induced by cooperative binding of Ca2+ to several sites of the molecule. Based on the crystallographic structures, a cluster of acidic residues in domain III, the acidic loop, has been proposed to function as part of an electrostatic switch in the activation process.

Evaluation of phage display system and leech-derived tryptase inhibitor as a tool for understanding the serine proteinase specificities.

A small combinatorial library of LDTI mutants (5.2 x 10(4)) restricted to the P1-P4' positions of the reactive site was displayed on the pCANTAB 5E phagemid, and LDTI fusion phages were produced and selected for potent neutrophil elastase and plasmin inhibitors. Strong fusion phage binders were analyzed by ELISA on enzyme-coated microtiter plates and the positive phages had their DNA sequenced.

Expression levels strongly affect ligand-induced sequestration of B2 bradykinin receptors in transfected cells.

Transfection of cells with expression vectors is one of the most important tools used to assess the effects of receptor mutations on ligand-induced receptor sequestration. Most transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels that may also depend on the mutations made. It is, therefore, important to determine how the regulation of the receptors depends on their numbers per cell.

Subcellular localization and in vivo subunit interactions of ubiquitous mu-calpain.

Ubiquitously expressed calpains are Ca(2+)-dependent, intracellular cysteine proteases comprising a large catalytic subunit (domains DI-DIV) and a noncovalently bound small regulatory subunit (domains DV and DVI). It is unclear whether Ca(2+)-induced calpain activation is followed by subunit dissociation or not. Here, we have applied advanced fluorescence microscopy techniques to study calpain subunit interactions in living cells using recombinant calpain subunits or domains fused to enhanced cyan and enhanced yellow fluorescent reporter proteins.

Synthesis of antisense oligonucleotides conjugated to a multivalent carbohydrate cluster for cellular targeting.

Carrier-mediated delivery holds great promise for significantly improving the cellular uptake and therefore the therapeutic efficacy of antisense oligonucleotides in vivo. A multivalent carbohydrate recognition motif for the asialoglycoprotein receptor has been designed for tissue- and cell-specific delivery of antisense drugs to parenchymal liver cells. To combine low molecular weight with high receptor affinity, the synthetic ligand contains three galactosyl residues attached to a cholane scaffold via epsilon-aminocapramide linkers.

Mast cell tryptase degrades HDL and blocks its function as an acceptor of cellular cholesterol.

Objective: In human atherosclerotic lesions, degranulated mast cells are found in the vicinity of macrophage foam cells. Mast cell granules contain tryptase, a tetrameric serine protease requiring glycosaminoglycans for stabilization. No endogenous inhibitors have been described for tryptase, and the physiological functions of the enzyme are poorly understood.