New N-arylsulfonyl-N-alkoxyaminoacetohydroxamic acids as selective inhibitors of gelatinase A (MMP-2)

New N-arylsulfonyl-substituted alkoxyaminoaceto hydroxamic acid derivatives of types 8 and 10 designed as oxa-analogues of known sulfonamide-based MMPi of types 2 and 7 were synthesized and tested for their inhibitory activities on some matrix metalloproteinases. The combination of a biphenylsulfonamide group with oxyamino oxygen in the pharmacophoric central skeleton of sulfonamide-based MMPi obtained in the new sulfonamides 10 seems to be able to give selectivity for MMP-2 over MMP-1. The most potent derivative of this type, 10a, shows similar anti-invasive properties to the analogue reference drug CGS27023A, 2, in an in vitro model of invasion on matrigel, carried out on cellular lines of fibrosarcoma HT1080 (tumoural cells over-expressing MMP-2 and MMP-9).     

Augmentation of bovine airway smooth muscle responsiveness to carbachol, KCl, and histamine by the isoprostane 8-iso-PGE2

Isoprostanes are generated during periods of oxidative stress, which characterize diseases such as asthma and cystic fibrosis. They also elicit functional responses and may therefore contribute to the pathology of these diseases. We set out to examine the effects of isoprostanes on airway responsiveness to cholinergic stimulation. Muscle bath techniques were employed using isolated bovine tracheal smooth muscle. 8-Isoprostaglandin E2 (8-iso-PGE2) increased tone directly on its own, although the magnitude of this response, even at the highest concentration tested, was only a fraction of that evoked by KCl or carbachol. More importantly, though, pretreatment of the tissues with 8-iso-PGE2 (10 microM) markedly augmented responses to submaximal and even subthreshold concentrations of KCl, carbachol, or histamine, whereas maximal responses to these agents were unaffected by the isoprostane. The augmentative effect on cholinergic responsiveness was mimicked by PGE2 (0.1 microM) and by the FP agonists PGF2 (0.1 microM) and fluprostenol (0.1 microM), but not by the EP3 agonist sulprostone (0.1 microM) or the TP agonist U-46619 (0.1 microM). Antagonists of EP1 receptors (AH-6809 and SC-19920, 10 microM) and TP receptors (ICI-192605, 1 microM) had no effect on 8-iso-PGE2-induced augmentation of cholinergic responsiveness. We conclude that 8-iso-PGE2 induces nonspecific airway smooth muscle hyperresponsiveness through a non-TP non-EP prostanoid receptor.     

LIMK1 regulates Golgi dynamics, traffic of Golgi-derived vesicles, and process extension in primary cultured neurons

In this study, we examined the subcellular distribution and functions of LIMK1 in developing neurons. Confocal microscopy, subcellular fractionation, and expression of several epitope-tagged LIMK1 constructs revealed that LIMK1 is enriched in the Golgi apparatus and growth cones, with the LIM domain required for Golgi localization and the PDZ domain for its presence at neuritic tips. Overexpression of wild-type LIMK1 suppresses the formation of trans-Golgi derived tubules, and prevents cytochalasin D-induced Golgi fragmentation, whereas that of a kinase-defective mutant has the opposite effect. Transfection of wild-type LIMK1 accelerates axon formation and enhances the accumulation of Par3/Par6, insulin-like growth factor (IGF)1 receptors, and neural cell adhesion molecule (NCAM) at growth cones, while inhibiting the Golgi export of synaptophysin-containing vesicles. These effects were dependent on the Golgi localization of LIMK1, paralleled by an increase in cofilin phosphorylation and phalloidin staining in the region of the Golgi apparatus, and prevented by coexpression of constitutive active cofilin. The long-term overexpression of LIMK1 produces growth cone collapse and axon retraction, an effect that is dependent on its growth cone localization. Together, our results suggest an important role for LIMK1 in axon formation that is related with its ability to regulate Golgi dynamics, membrane traffic, and actin cytoskeletal organization.     

In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.     

Mucopolysaccharidosis IVA: characterization of a common mutation found in Finnish patients with attenuated phenotype

Mucopolysaccharidosis IVA (MPS IVA) is caused by the deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase encoded by the GALNS gene on chromosome 16. We describe, in detail, the clinical phenotype of five patients from three unrelated Finnish families and have characterized the disease-causing mutations in GALNS. Genotypes of the patients are D60N/A291T, D60N/W230X, and D60N/1374delT. Mutation 1374delT introduces premature termination of GALNS. Cells over-expressing the novel mutation W230X and A291T had no residual GALNS activity, whereas D60N gave 12.2% residual activity compared with the wild type. Co-transfection of D60N/A291T and D60N/W230X showed 5.5% and 6.7% of wild type activity, respectively. The precursor proteins of D60N and A291T were observed at 55 kDa and 57 kDa, respectively, whereas there was no detectable band in cells over-expressing W230X. At 55 degrees C, the mutant protein showed lower thermostability than the wild type protein at pH 3.8 and 7.0. The tertiary structural model of the GALNS protein revealed that aspartic acid at position 60 is located on the surface of the molecule, away from the active site. This makes it unlikely that the enzymatic function of the protein with D60N is severely impaired. On the other hand, A291 and W230 are localized near the active site. The molecular characteristics of the D60N mutation explain the attenuated clinical phenotype of the patients.     

The Oligomeric states of Haloarcula marismortui malate dehydrogenase are modulated by solvent components as shown by crystallographic and biochemical studies

The three-dimensional crystal structure of the (R207S, R292S) mutant of malate dehydrogenase from Haloarcula marismortui was solved at 1.95A resolution in order to determine the role of salt bridges and solvent ions in halophilic adaptation and quaternary structure stability. The mutations, located at the dimer-dimer interface, disrupt two inter-dimeric salt bridge clusters that are essential for wild-type tetramer stabilisation. Previous experiments in solution, performed on the double mutant, had shown a tetrameric structure in 4M NaCl, which dissociated into active dimers in 2M NaCl. In order to establish if the active dimeric form is a product of the mutation, or if it also exists in the wild-type protein, complementary studies were performed on the wild-type enzyme by analytical centrifugation and small angle neutron scattering experiments. They showed the existence of active dimers in NaF, KF, Na(2)SO(4), even in the absence of NADH, and in the presence of NADH at concentrations of NaCl below 0.3M. The crystal structure shows a tetramer that, in the absence of the salt bridge clusters, appears to be stabilized by a network of ordered water molecules and by Cl(-) binding at the dimer-dimer interface. The double mutant and wild-type dimer folds are essentially identical (the r.m.s. deviation between equivalent C(alpha) positions is 0.39A). Chloride ions are also observed at the monomer-monomer interfaces of the mutant, contributing to the stability of each dimer against low salt dissociation. Our results support the hypothesis that extensive binding of water and salt is an important feature of adaptation to a halophilic environment.     

Neutrophil restraint by green tea: inhibition of inflammation,associated angiogenesis,and pulmonary fibrosis

Neutrophils play an essential role in host defense and inflammation, but the latter may trigger and sustain the pathogenesis of a range of acute and chronic diseases. Green tea has been claimed to exert anti-inflammatory properties through unknown molecular mechanisms. We have previously shown that the most abundant catechin of green tea, (-)epigallocatechin-3-gallate (EGCG), strongly inhibits neutrophil elastase. Here we show that 1) micromolar EGCG represses reactive oxygen species activity and inhibits apoptosis of activated neutrophils, and 2) dramatically inhibits chemokine-induced neutrophil chemotaxis in vitro; 3) both oral EGCG and green tea extract block neutrophil-mediated angiogenesis in vivo in an inflammatory angiogenesis model, and 4) oral administration of green tea extract enhances resolution in a pulmonary inflammation model, significantly reducing consequent fibrosis. These results provide molecular and cellular insights into the claimed beneficial properties of green tea and indicate that EGCG is a potent anti-inflammatory compound with therapeutic potential.