Antinociceptive and anti-inflammatory effects of electroacupuncture on experimental arthritis of the rat temporomandibular joint

This study investigated the antinociceptive and anti-inflammatory effects of electroacupuncture (EA) on zymosan-induced acute arthritis of the rat temporomandibular joint (TMJ). Male Wistar rats were injected with saline or zymosan (control group; 2?mg) into the left TMJ. Low frequency EA (10?Hz, 30?min) was performed at acupoints (LI4, LI11, ST36, ST44) or sham points 2?h after or 1?h before zymosan administration. Mechanical hypernociception was accessed by the electronic Von Frey method after zymosan administration. Rats were sacrificed 6?h after zymosan administration and the joint was removed for histopathological analysis, myeloperoxidase activity assessment, vascular permeability observations, and immunohistochemical verification of inflammatory mediators. The results showed that EA inhibited zymosan-induced hypernociception, compared with the control group and with the sham group (p?< 0.05). The results showed that EA inhibited inflammatory parameters such as neutrophil migration, vascular permeability, and tumour necrosis factor α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) expression in the TMJ compared with the sham group (p < 0.05). Histopathological analysis showed that EA significantly inhibited edema and periarticular infiltration (p?< 0.05) compared with the control and sham groups. EA at acupoints produced antinociceptive and anti-inflammatory effects on zymosan-induced arthritis in the rat TMJ.     

DDT concentrations in surficial sediments of three estuarine systems in Portugal

Levels of p,p-DDT and its metabolites in surficial sediments of Tagus and Sado Estuary and lagoon Ria Formosa varied one or two orders of magnitude. In general, p,-DDE and p,-DDD were the dominant metabolites and p,-DDT was a minor component. While concentrations of p,-DDT, p,-DDD and p,-DDE were hardly related to loss on ignition, concentrations of total DDT (tDDT) in surficial sediments of each studied system were positively correlated to that parameter. Higher contaminated samples were excluded from these relationships: a few sediment samples from the Tagus outlet channel, most of the sediments from the upper Sado Estuary, and some particulate matter samples collected in sediment traps in the adjoining area to the Ria Formosa. Concentration of tDDT appears, thus, to be related to sedimentary organic matter of these estuarine systems except near the actual sources of pollution. On the basis of the relationship slopes, one may conclude that lower Tagus Estuary is more contaminated than Ria Formosa and lower Sado Estuary.     

Three-phase partitioning as an efficient method for extraction/concentration of immunoreactive excreted-secreted proteins of Corynebacterium pseudotuberculosis

The three-phase partitioning (TPP) technique was used upstream to isolate/concentrate secreted proteins from Corynebacterium pseudotuberculosis cultured in a complex liquid medium. Several parameters of the TPP technique (15, 30, or 60% ammonium sulfate concentration; 4.0, 5.5, or 7.0 pH; and primary (n) or tertiary (t)-butanol solvent isomer) were varied to determine the optimal recovery of serologically and cellularly immunoreactive extracted proteins. A TPP extraction made with 30% ammonium sulfate and an initial pH of 4.0 gave the best humoral and cellular immunoreactivity of caseous lymphadenitis infected goats. In particular, two immunogenic secreted (16 and 125 kDa) proteins, which had not been found by other extraction methods, were identified.     

Modeling protein complexes with BiGGER

This article describes the method and results of our participation in the Critical Assessment of PRediction of Interactions (CAPRI) experiment, using the protein docking program BiGGER (Bimolecular complex Generation with Global Evaluation and Ranking) (Palma et al., Proteins 2000;39:372-384). Of five target complexes (CAPRI targets 2, 4, 5, 6, and 7), only one was successfully predicted (target 6), but BiGGER generated reasonable models for targets 4, 5, and 7, which could have been identified if additional biochemical information had been available.     

Mitochondrial apoptotic cell death and moderate superoxide generation upon selective activation of non-desensitizing AMPA receptors in hippocampal cultures

In the present work we investigated the effect of selective stimulation of non-desensitizing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in the intracellular processes leading to hippocampal neuronal death and production of reactive oxygen species (ROS). Activation of AMPA receptors in the presence of cyclothiazide (CYZ), a blocker of AMPA receptor desensitization, resulted in the death of approximately 25% of neurones, which was prevented by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline (NBQX), an AMPA-preferring receptor antagonist. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) protected the neurones from necrotic death induced by AMPA or NMDA receptor activation. Neurodegeneration caused by selective activation of non-desensitizing AMPA receptors, in the presence of AMPA, CYZ and MK-801, significantly decreased the number of Co2+-positive neurones, used as a cytochemical marker of Ca2+-permeable AMPA receptors, but maintained intracellular ATP/ADP. The AMPA-mediated apoptotic cell death involved mitochondrial cytochrome c release and the activation of caspases-1 and -3, which was prevented by NBQX. Interestingly, although selective activation of AMPA receptors was not associated with production of intracellular peroxides, a moderate increase in superoxide production was observed upon exposure to antimycin A (AA). Furthermore, increased activity of Mn- superoxide dismutase (SOD) was observed on selective activation of non-desensitizing AMPA receptors. Taken together, these data make important contributions to the elucidation of the downstream pathways activated in AMPA receptor-mediated excitotoxicity in cultured rat hippocampal neurones.     

Myosin V motor proteins: marching stepwise towards a mechanism

Mammalian myosin V motors transport cargo processively along actin filaments. Recent biophysical and structural studies have led to a detailed understanding of the mechanism of myosin V, making it perhaps the best understood cytoskeletal motor. In addition to describing the mechanism, this review will illustrate how "dynamic" single molecule measurements can synergize with "static" protein structural studies to produce amazingly clear information on the workings of a nanometer-scale machine.     

ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility

Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.     

The Na+/H+ antiporter of the thermohalophilic bacterium Rhodothermus marinus

In the thermohalophilic bacterium Rhodothermus marinus, the NADH:quinone oxidoreductase (complex I) is encoded by two single genes and two operons, one of which contains the genes for five complex I subunits, nqo10-nqo14, a pterin carbinolamine dehydratase, and a putative single subunit Na+/H+ antiporter. Here we report that the latter encodes indeed a functional Na+/H+ antiporter, which is able to confer resistance to Na+, but not to Li+ to an Escherichia coli strain defective in Na+/H+ antiporters. In addition, an extensive amino acid sequence comparison with several single subunit Na+/H+ antiporters from different groups, namely NhaA, NhaB, NhaC, and NhaD, suggests that this might be the first member of a new type of Na+/H+ antiporters, which we propose to call NhaE.