alpha-chymotrypsin and thiazolyl-blue

Hyaluronic acid-L-cysteine conjugate, a novel thiolated polymer, was synthesized and characterized for mucoadhensive drug delivery. L-Cysteine was covalently attached to hyaluronic acid via the formation of an amide bond. Adhesion studies on the mucosa indicated a 4.82-fold increase in the adhesion force of the obtained conjugate (containing 210.58 micromol thiol groups per gram polymer) versus unmodified hyaluronic acid. The results of a peptidase inhibition study revealed that the inhibitory effect of hyaluronic acid toward trypsin and chymotrypsin was significantly improved compared to hyaluronic acid. Permeation studies utilizing a MDCK cell monolayer system demonstrated a sustained drug release. Based on these features the novel thiolated polymer might represent a promising multifunctional excipient for various drug delivery systems.

Topics: Animals; Cell Line; Cell Membrane Permeability; Cell Survival; Chymotrypsin; Coloring Agents; Cysteine; Delayed-Action Preparations; Dogs; Drug Delivery Systems; Excipients; Hyaluronic Acid; Intestines; Mucous Membrane; Protease Inhibitors; Sulfhydryl Compounds; Tetrazolium Salts; Thiazoles; Tissue Adhesives; Trypsin Inhibitors

The aim of this work was to evaluate the role of ubiquitin-proteasome system (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson's disease (PD) cellular models.. We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patient population we evaluated the aSN levels in the plasma and the influence of several demographic characteristics in the above mentioned determinations.. We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a downregulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomer levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients.. aSN oligomers are ubiquitinated and we identified a ubiquitin-dependent clearance insufficiency with the accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Analysis of Variance; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Chymotrypsin; Citrate (si)-Synthase; Electron Transport Complex I; Female; Green Fluorescent Proteins; Humans; Immunoprecipitation; Lactic Acid; Leukocytes, Mononuclear; Male; Middle Aged; Mitochondria; Neuroblastoma; Parkinson Disease; Plasma; Proteasome Endopeptidase Complex; RNA, Small Interfering; Statistics as Topic; Tetrazolium Salts; Thiazoles; Transfection; Ubiquitin; Ubiquitination; Up-Regulation

Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC(50) value of 13.8 microM, which was less potent than copper(II) chloride (IC(50) 5.3 microM). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells.

Topics: Animals; Apoptosis; Blotting, Western; Calcium-Binding Proteins; Calpain; Caspase 3; Cell Line, Tumor; Chymotrypsin; Copper; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Female; Humans; In Situ Nick-End Labeling; Kinetics; Proteasome Inhibitors; Pyrrolidines; Rabbits; Tetrazolium Salts; Thiazoles; Thiocarbamates; Time Factors; Zinc

Activated microglia have been observed in various neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, and multiple sclerosis. They may exacerbate neuronal damage by secreting various toxic molecules. The list of candidate toxins includes proteases. Since it is currently not known which, if any, proteases are involved in human microglia neurotoxicity, we studied the effects of a panel of protease inhibitors on the toxicity of cell-free supernatants of stimulated human microglia and THP-1 monocytic cells to human SH-SY5Y cells. Five structurally distinct inhibitors that are known to inhibit chymotrypsin-like proteases were partially protective. They included chymostatin, AEBSF (Pefabloc SC), alpha1-antichymotrypsin, bromoenol lactone, and 3,4-dichloroisocoumarin. The data suggest that certain protease inhibitors could inhibit microglial-mediated toxicity. They might represent a novel class of drugs with benefit in diseases where overactivity of microglia contributes to the pathogenesis.

Topics: Cell Line; Cell Survival; Cells, Cultured; Chymotrypsin; Humans; Hydrogen Peroxide; Interferon-gamma; L-Lactate Dehydrogenase; Microglia; Monocytes; Neurotoxicity Syndromes; Serine Proteinase Inhibitors; Stimulation, Chemical; Subcellular Fractions; Temporal Lobe; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha

During inflammation, microglial cells go through phenotypic and functional changes that include the production and release of large amounts of oxygen and nitrogen radicals. As such, activated microglia are subject to heightened oxidative stress. The multicatalytic proteasome clears oxidized and damaged proteins from cells, and has been shown to be an important aspect of the microglial compensatory response to activation. The female sex steroid estrogen is both cytoprotective and anti-inflammatory, and has been shown to affect microglial signaling in particular. To determine if estrogen might affect the proteasome in microglial cells, we examined the effects of 17 beta-estradiol treatment on proteasome activity in N9 microglial cells. Specifically, we measured ATP-dependent and ATP-independent chymotrypsin-like, trypsin-like, and peptidyl glutamyl peptide hydrolase (PGPH)-like activities in response to both 17 beta-estradiol and interferon gamma. Data indicate that estrogen, but not interferon gamma, significantly increases ATP-dependent chymotrypsin-like and PGPH-like activity. Furthermore, this effect was blocked by the p44/42 MAPK inhibitor PD98059. Hence, these data demonstrate that through the MAPK pathway, estrogen can upregulate proteasome activity, suggesting a possible mechanism for estrogen's cytoprotective effects.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Cell Line; Chymotrypsin; Dose-Response Relationship, Drug; Drug Interactions; Endopeptidases; Enzyme Inhibitors; Estradiol; Estrogens; Interferon-gamma; Leupeptins; Mice; Microglia; Protease Inhibitors; Proteasome Endopeptidase Complex; Receptors, Estradiol; Tetrazolium Salts; Thiazoles; Time Factors