Proteomics analysis revealed changes in rat bronchoalveolar lavage fluid proteins associated with oil mist exposure

Exposure to oil mist has been associated with a variety of acute and chronic respiratory effects. Using proteomics approaches to investigate exposure-associated proteins may provide useful information to understand the mechanisms of associated respiratory effects. The aim of this study was to investigate changes in rat bronchoalveolar lavage fluid proteins associated with oil mist exposure using nano-HPLC-ESI-MS/MS. The results revealed that 29 proteins exhibited significant changes after exposure. These proteins included surfactant-associated proteins (SP-A and SP-D), inflammatory proteins (complement component 3, immunoglobulins, lysozyme, etc.), growth factors (e.g., transforming growth factor alpha (TGF-alpha)), calcium-binding proteins (calcyclin, calgranulin A, calreticulin, and calvasculin), and other proteins (e.g., cathepsin D, saposin, and intestinal trefoil factor). To further evaluate changes in protein levels, a simple quantitative strategy was developed in this study. A large decrease in protein levels of SP-A and SP-D (0.24- and 0.38-fold, respectively) following exposure was observed. In contrast, protein levels of TGF-alpha and calcium-binding proteins were significantly increased (4.46- and 1.4-1.8-fold, respectively). Due to the diverse functions of these proteins, the results might contribute to understand the mechanisms involved in lung disorders induced by oil mist exposure.     

A Dominant-negative Clathrin Mutant Differentially Affects Trafficking of Molecules with Distinct Sorting Motifs in the Class II Major Histocompatibility Complex (MHC) Pathway

The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3γ, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA–DM and the invariant chain (I chain) complexed to HLA–DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR–I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR–I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR–I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR–I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR–I chain until their intended rendezvous in late endocytic compartments.     

Discovery of a novel bicycloproline P2 bearing peptidyl alpha-ketoamide LY514962 as HCV protease inhibitor

We describe herein the design, syntheses and evaluation of a number of bicycloproline P2 bearing HCV protease inhibitors endowed with impressive enzyme potency, enzyme selectivity, cellular activity and favorable ADME profiles.     

Phosphorylated p38MAPK specific antibodies cross-react with sarkosyl-insoluble hyperphosphorylated tau proteins

Neurofibrillary tangles (NFT) accumulated in Alzheimer's diseases and related disorders contain hyperphosphorylated tau and display immunoreactivity for active forms of various kinases. To understand the role of p38MAPK (mitogen-activated protein kinase) in NFT formation, we have studied a transgenic (Tg) mouse model of tauopathy, JNPL3, that expresses P301L mutant tau, and bigenic mice, TAPP, generated by cross-breeding of JNPL3 with Tg2576 mice. Age-matched non-Tg mice (NTg), wild-type human tau Tg mice (JN25), and Tg2576 mice were used as controls. Phosphorylated p38MAPK (active form) immunoreactivity was consistently located in NFT and granulovaculolar degeneration in JNPL3 and TAPP mice older than 5 months of age. Unphosphorylated/total-p38MAPK was not detectable in spinal cord and brain sections from 2- to 11-month-old mice, even though JNPL3 mice, but not controls had an age-dependent increase of total-p38MAPK by western blotting. Spinal cord/brain extracts from mice and human with tauopathy were demonstrated to have insignificant amount of active-p38MAPK. However, they contained antiactive-p38MAPK cross-reactive proteins insoluble in sarkosyl and similar to phosphorylated tau in size. Consistently, antiactive-p38MAPK immunoprecipitates displayed tau immunoreactivity, but not total-p38MAPK, and antitau immunoprecipitates displayed active-p38MAPK immunoreactivity. Together, the results indicate that the cross-reactivity of antiactive-p38MAPK antibody with phosphorylated tau is responsible for the immunolabeling of tau-positive inclusion.     

Exercise enhances surfactant-mediated phagocytosis in bronchoalveolar macrophages

Severe exercise augments the phagocytic capability of bronchoalveolar macrophages (BAMs) in the absence of pulmonary surfactant, a lung immunity modulator in vivo. This study was to investigate whether the exercise effect on BAM phagocytosis is partially mediated by surfactant components. Male BALB/c mice (9-12 wk old) were divided into control and severe exercise groups. Mice in the exercise group received progressive treadmill running exercise until exhaustion. BAMs and lung lavage supernatant were collected under either sedentary or post-severe exercise conditions. Phagocytosis of IgG/C'-opsonized beads by BAMs was determined in the presence of lavage supernatant. Mannose, a monosaccharide competitor for the carbohydrate recognition domain of surfactant protein A (SP-A), and SP-A antibodies were applied to examine the role of SP-A in the exercise-induced facilitating effects on BAM phagocytosis. BAMs from either control or post-exercise animals had elevated phagocytosis of IgG/C'-opsonized beads when incubated with autologous lung lavage supernatant. The supernatant-mediated increase in BAM phagocytosis of IgG/C'-opsonized beads was dose-dependently inhibited by mannose or SP-A antibodies. In addition, higher concentrations of SP-A inhibitors were needed to inhibit BAM phagocytosis in post-exercise group than that in the control group. We also observed that SP-A inhibitors were ineffective in the absence of lung lavage supernatant. Furthermore, post-exercise, but not control, BAMs displayed time-dependent alterations in their membrane-bound SP-A amount during 30-min incubation with autologous lung lavage supernatant. SP-A plays a major role in the severe exercise-enhanced surfactant-mediated BAM phagocytosis.     

Characteristics of Vibrio parahaemolyticus O3:K6 from Asia

A variety of serovars of the food-borne pathogen Vibrio parahaemolyticus normally cause infection. Since 1996, the O3:K6 strains of this pathogen have caused pandemics in many Asian countries, including Taiwan. For a better understanding of these pandemic strains, the recently isolated clinical O3:K6 strains from India, Japan, Korea, and Taiwan were examined in terms of pulsed-field gel electrophoresis (PFGE) typing and other biological characteristics. After PFGE and cluster analysis, all the O3:K6 strains were grouped into two unrelated groups. The recently isolated O3:K6 strains were all in one group, consisting of eight closely related patterns, with I1(81%) and I5(13%) being the most frequent patterns. Pattern I1 was the major one for strains from Japan, Korea, and Taiwan. All recently isolated O3:K6 strains carried the thermostable direct hemolysin (tdh) gene. No significant difference was observed between recently isolated O3:K6 strains and either non-O3:K6 reference strains or old O3:K6 strains isolated before 1996 with respect to antibiotic susceptibility, the level of thermostable direct hemolysin, and the susceptibility to environmental stresses. Results in this study confirmed that the recently isolated O3:K6 strains of V. parahaemolyticus are genetically close to each other, while the other biological traits examined were usually strain dependent, and no unique trait was found in the recently isolated O3:K6 strains.     

Search for Novel Remedies to Augment Radiation Resistance of Inhabitants of Fukushima and Chernobyl Disasters: Identifying DNA Repair Protein XRCC4 Inhibitors

Abstract

Two nuclear plant disasters occurring within a span of 25 years threaten health and genome integrity both in Fukushima and Chernobyl. Search for remedies capable of enhancing DNA repair efficiency and radiation resistance in humans appears to be a urgent problem for now. XRCC4 is an important enhancer in promoting repair pathway triggered by DNA double-strand break (DSB). In the context of radiation therapy, active XRCC4 could reduce DSB-mediated apoptotic effect on cancer cells. Hence, developing XRCC4 inhibitors could possibly enhance radiotherapy outcomes. In this study, we screened traditional Chinese medicine (TCM) database, TCM Database@Taiwan, and have identified three potent inhibitor agents against XRCC4. Through molecular dynamics simulation, we have determined that the protein-ligand interactions were focused at Lys188 on chain A and Lys187 on chain B. Intriguingly, the hydrogen bonds for all three ligands fluctuated frequently but were held at close approximation. The pi-cation interactions and ionic interactions mediated by o-hydroxyphenyl and carboxyl functional groups respectively have been demonstrated to play critical roles in stabilizing binding conformations. Based on these results, we reported the identification of potential radiotherapy enhancers from TCM. We further characterized the key binding elements for inhibiting the XRCC4 activities.     

NO signaling in exercise training-induced anti-apoptotic effects in human neutrophils

Short-lived neutrophils play a predominant role in innate immunity, the effects of exercise training on neutrophil survival is unclear. In this study, we investigated the underlying mechanisms of training effects on human neutrophil apoptosis. Healthy male subjects were trained on a cycling ergometer for 8 weeks and followed by 4 weeks of detraining. Blood neutrophils were collected before exercise, after training, and after detraining. Comparing with pre-exercise specimens, neutrophils collected after training showed reduced apoptosis rate, which partially returned after detraining. Various intracellular proteins, including iNOS, Mcl-1, A1, Grp78, and IL-8, were upregulated by training, and they remained high after detraining. Upregulated iNOS was closely correlated with these anti-apoptotic molecules in neutrophils. Furthermore, the possible mechanism by which iNOS suppressed apoptosis was explored. Neutrophil apoptosis was accelerated by blocking and retarded by stimulating the endogenous iNOS activity. As an anti-apoptosis mediator of NO signaling, the Mcl-1 level dropped by depletion of the major NO downstream molecule cGMP and such loss of Mcl-1 was avoidable when supplying exogenous NO. Upon activation of NO-cGMP signaling, neutrophils held increased Mcl-1 expression and delayed apoptosis. Collectively, our results suggested that exercise training may retard neutrophil apoptosis by upregulating the iNOS-NO-cGMP-Mcl-1 pathway.