Shiang-Jiuun Chen

The SmeYZ Efflux Pump of Stenotrophomonas maltophilia Contributes to Drug Resistance, Virulence-Related Characteristics, and Virulence in Mice

ABSTRACT The resistance-nodulation-division (RND)-type efflux pump is one of the causes of the multidrug resistance of Stenotrophomonas maltophilia. The roles of the RND-type efflux pump in physiological functions and virulence, in addition to antibiotic extrusion, have attracted much attention. In this study, the contributions of the constitutively expressed SmeYZ efflux pump to drug resistance, virulence-related characteristics, and virulence were evaluated. S. maltophilia KJ is a clinical isolate of multidrug resistance. The smeYZ isogenic deletion mutant, KJΔYZ, was constructed by a gene replacement strategy. The antimicrobial susceptibility, virulence-related physiological characteristics, susceptibility to human serum and neutrophils, and in vivo virulence between KJ and KJΔYZ were comparatively assessed. The SmeYZ efflux pump contributed resistance to aminoglycosides and trimethoprim-sulfamethoxazole. Inactivation of smeYZ resulted in attenuation of oxidative stress susceptibility, swimming, flagella formation, biofilm formation, and secreted protease activity. Furthermore, loss of SmeYZ increased susceptibility to human serum and neutrophils and decreased in vivo virulence in a murine model. These findings suggest the possibility of attenuation of the resistance and virulence of S. maltophilia with inhibitors of the SmeYZ efflux pump.

Possible involvement of glutathione and p53 in trichloroethylene- and perchloroethylene-induced lipid peroxidation and apoptosis in human lung cancer cells

Trichloroethylene (TCE) and perchloroethylene (PERC) are volatile organic compounds (VOCs) that are primarily inhaled through the respiratory system. The aim of this study was to elucidate the role of glutathione (GSH) and p53 in TCE- and PERC-induced lung toxicity. Human lung adenocarcinoma cells NCI-H460 (p53-wild-type) have constitutively lower levels of GSH than NCI-H1299 (p53-null) cells. The results showed that exposure to vapor TCE and PERC produced a dose-dependent and more pronounced accumulation of H(2)O(2) in p53-WT H460 than p53-null H1299 cells. The accumulation of H(2)O(2) was accompanied by severe cellular damage, as indicated by the significant increase of lipid peroxidation and apoptosis in p53-WT H460 cells, but not p53-null H1299 cells. Cotreatment of p53-WT H460 cells with free radical scavengers, such as D-mannitol, uric acid, and sodium selenite, significantly attenuated the TCE- or PERC-induced lipid peroxidation. In contrast, depletion of GSH in p53-null H1299 cells enhanced TCE- or PERC-induced lipid peroxidation. The levels of p53 and Bax proteins were elevated, while Bcl-2 protein was downregulated in TCE- or PERC-treated p53-WT H460 cells. Activity of caspase 3, the apoptotic executioner, was also significantly enhanced in TCE- or PERC-treated cells. These data suggest that, in human lung cancer cells, GSH plays a vital role in the protection of TCE- and PERC-induced oxidative stress and apoptosis, which may be mediated through a p53-dependent pathway.

Recovery of heat shock-triggered released apoplastic Ca2+ accompanied by pectin methylesterase activity is required for thermotolerance in soybean seedlings

Synthesis of heat shock proteins (HSPs) in response to heat shock (HS) is essential for thermotolerance. The effect of a Ca2+ chelator, EGTA, was investigated before a lethal HS treatment in soybean (Glycine max) seedlings with acquired thermotolerance induced by preheating. Such seedlings became non-thermotolerant with EGTA treatment. The addition of Ca2+, Sr2+ or Ba2+ to the EGTA-treated samples rescued the seedlings from death by preventing the increased cellular leakage of electrolytes, amino acids, and sugars caused by EGTA. It was confirmed that EGTA did not affect HSP accumulation and physiological functions but interfered with the recovery of HS-released Ca2+ concentration which was required for thermotolerance. Pectin methylesterase (PME, EC 3.1.1.11), a cell wall remodelling enzyme, was activated in response to HS, and its elevated activity caused an increased level of demethylesterified pectin which was related to the recovery of the HS-released Ca2+ concentration. Thus, the recovery of HS-released Ca2+ in Ca2+-pectate reconstitution through PME activity is required for cell wall remodelling during HS in soybean which, in turn, retains plasma membrane integrity and co-ordinates with HSPs to confer thermotolerance.

Metformin activation of AMPK-dependent pathways is neuroprotective in human neural stem cells against Amyloid-beta-induced mitochondrial dysfunction

Alzheimers disease (AD) is the general consequence of dementia and is diagnostic neuropathology by the cumulation of amyloid-beta (Aβ) protein aggregates, which are thought to promote mitochondrial dysfunction processes leading to neurodegeneration. AMP-activated protein kinase (AMPK), a critical regulator of energy homeostasis and a major player in lipid and glucose metabolism, is potentially implied in the mitochondrial deficiency of AD. Metformin, one of the widespread used anti- metabolic disease drugs, use its actions in part by stimulation of AMPK. While the mechanisms of AD are well established, the neuronal roles for AMPK in AD are still not well understood. In the present study, human neural stem cells (hNSCs) exposed to Aβ had significantly reduced cell viability, which correlated with decreased AMPK, neuroprotective genes (Bcl-2 and CREB) and mitochondria associated genes (PGC1α, NRF-1 and Tfam) expressions, as well as increased activation of caspase 3/9 activity and cytosolic cytochrome c. Co-treatment with metformin distinct abolished the Aβ-caused actions in hNSCs. Metformin also significantly rescued hNSCs from Aβ-mediated mitochondrial deficiency (lower D-loop level, mitochondrial mass, maximal respiratory function, COX activity, and mitochondrial membrane potential). Importantly, co-treatment with metformin significantly restored fragmented mitochondria to almost normal morphology in the hNSCs with Aβ. These findings extend our understanding of the central role of AMPK in Aβ-related neuronal impairment. Thus, a better understanding of AMPK might assist in both the recognition of its critical effects and the implementation of new therapeutic strategies in the treatment of AD.

Rosiglitazone activation of PPARγ-dependent signaling is neuroprotective in mutant huntingtin expressing cells

Peroxisome proliferator-activated receptor gamma (PPARγ) is a crucial transcription factor for neuroprotection in several brain diseases. Using a mouse model of Huntingtons Disease (HD), we recently showed that PPARγ not only played a major function in preventing HD, but also oral intake of a PPARγ agonist (thiazolidinedione, TZD) significantly reduced the formation of mutant Huntingtin (mHtt) aggregates in the brain (e.g., cortex and striatum). The molecular mechanisms by which PPARγ exerts its HD neuroprotective effects remain unresolved. We investigated whether the PPARγ agonist (rosiglitazone) mediates neuroprotection in the mHtt expressing neuroblastoma cell line (N2A). Here we show that rosiglitazone upregulated the endogenous expression of PPARγ, its downstream target genes (including PGC1α, NRF-1 and Tfam) and mitochondrial function in mHtt expressing N2A cells. Rosiglitazone treatment also significantly reduced mHtt aggregates that included ubiquitin (Ub) and heat shock factor 1 (HSF1), as assessed by a filter-retardation assay, and increased the levels of the functional ubiquitin-proteasome system (UPS), HSF1 and heat shock protein 27/70 (HSP27/70) in N2A cells. Moreover, rosiglitazone treatment normalized endoplasmic reticulum (ER) stress sensors Bip, CHOP and ASK1, and significantly increased N2A cell survival. Taken together, these findings unveil new insights into the mechanisms by which activation of PPARγ signaling protects against the HD-mediated neuronal impairment. Further, our data also support the concept that PPARγ may be a novel therapeutic target for treating HD.

Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish.

During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish. The association of different layers relies on E-cadherin based cellular junctions, whose stability can be affected by actin turnover. Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO). Knockdown of cfl1 interfered with epibolic movement of deep cell layer (DEL) but not in the enveloping layer (EVL) and the defect could be specifically rescued by overexpression of cfl1. It appeared that the uncoordinated movements of DEL and EVL were regulated by the differential expression of cfl1 in the DEL, but not EVL as shown by in situ hybridization. The dissociation of DEL and EVL was further evident by the loss of adhesion between layers by using transmission electronic and confocal microscopy analyses. cfl1 morphants also exhibited abnormal convergent extension, cellular migration and actin filaments, but not involution of hypoblast. The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays. These results suggest that proper actin turnover mediated by Cfl1 is essential for adhesion between DEL and EVL and cell movements during gastrulation in zebrafish.

GROWTH STRAINS AND RELATED WOOD STRUCTURES IN THE LEANING TRUNKS AND BRANCHES OF TROCHODENDRON ARALIOIDES - A VESSEL-LESS DICOTYLEDON

Leaning trunks and branches of Trochodendron aralioides Sieb. & Zucc., a primitive vessel-less dicotyledon, show increased radial growth and gelatinous fibers on the upper side similar to the features found in dicotyledons with vessels. The patterns of peripheral longitudinal growth strain are variable among trees but similar at different heights within the same leaning trunk. Growth strains on the lower side of the trunks are very small but they are relatively large on the lower side of the branches. Growth stress in the branches is partly affected by the gravitational bending stress, which would be exerted mostly on the lower side. Large spring back strains of branches are associated with large surface strains. Both the microfibril angle (MFA) and the percentage area of gelatinous fiber show positive relationships with the measured strains. The MFA of the S2 wall layer in tracheids in the opposite wood is 24.6 ± 2.2°, whereas the MFA of gelatinous layer in the tension wood is only 14.2 ± 2.7°. The difference of MFA between the gelatinous fibers and the opposite wood is one of the factors accounting for the large contracting force for reorientation.

Metformin activation of AMPK suppresses AGE-induced inflammatory response in hNSCs

ABSTRACT A growing body of evidence suggests type 2 diabetes mellitus (T2DM) is linked to neurodegenerative diseases such as Alzheimers disease (AD). Although the precise mechanisms remain unclear, T2DM may exacerbate neurodegenerative processes. AMP‐activated protein kinase (AMPK) signaling is an evolutionary preserved pathway that is important during homeostatic energy biogenesis responses at both the cellular and whole‐body levels. Metformin, a ubiquitously prescribed anti‐diabetic drug, exerts its effects by AMPK activation. However, while the roles of AMPK as a metabolic mediator are generally well understood, its performance in neuroprotection and neurodegeneration are not yet well defined. Given hyperglycemia is accompanied by an accelerated rate of advanced glycosylation end product (AGE) formation, which is associated with the pathogenesis of diabetic neuronal impairment and, inflammatory response, clarification of the role of AMPK signaling in these processes is needed. Therefore, we tested the hypothesis that metformin, an AMPK activator, protects against diabetic AGE induced neuronal impairment in human neural stem cells (hNSCs). In the present study, hNSCs exposed to AGE had significantly reduced cell viability, which correlated with elevated inflammatory cytokine expression, such as IL‐1&agr;, IL‐1&bgr;, IL‐2, IL‐6, IL‐12 and TNF‐&agr;. Co‐treatment with metformin significantly abrogated the AGE‐mediated effects in hNSCs. In addition, metformin rescued the transcript and protein expression levels of acetyl‐CoA carboxylase (ACC) and inhibitory kappa B kinase (IKK) in AGE‐treated hNSCs. NF‐&kgr;B is a transcription factor with a key role in the expression of a variety of genes involved in inflammatory responses, and metformin did prevent the AGE‐mediated increase in NF‐&kgr;B mRNA and protein levels in the hNSCs exposed to AGE. Indeed, co‐treatment with metformin significantly restored inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) levels in AGE‐treated hNSCs. These findings extend our understanding of the central role of AMPK in AGE induced inflammatory responses, which increase the risk of neurodegeneration in diabetic patients.

Anatomical characteristics of the secondary phloem in branches of Zelkova serrata Makino

The properties and tissue compositions of reaction wood in the leaning trunks or branches of trees have been extensively investigated, but studies on the influence of the related reaction on the secondary phloem are few and incomplete, in this work, formation of the vascular and cork cambium and the secondary phloem in the upper and lower sides of branches of Zelkova serrata Makino were studied. Vascular cambium is formed first in the second internode of shoot. In the third internode, cork cambium began to initiate subepidermally, meanwhile two to four layers of grouped gelatinous fibers were first found in the cortex outside of all the primary phloem. Gelatinous fibers were also found in both the secondary xylem and secondary phloem. The leaning branches exhibited pronounced radial secondary growth promotion to the upper side, and the reaction wood formed eccentrically. The secondary phloem formed layers of conducting sieve elements alternately with gelatinous fibers. Comparing the secondary phloem in the upper side (reaction phloem) of the branches with that in the lower side (opposite phloem), there is no obvious difference in thickness. Nevertheless, it was found that in the cross sections, the gelatinous fibers formed earlier, and there were more continuous cell senates and a much larger area ratio in the upper side. Besides, the sieve tubes in the upper side of secondary phloem were longer and wider and possessed a very horizontally-orientated sieve plate between two sieve elements. These features may imply that, in branches of Zelkova serrata, the secondary phloem of the upper side (reaction phloem) may have a higher translocation efficiency.

Effect of recombinant galectin-1 on the growth of immortal rat chondrocyte on chitosan-coated PLGA scaffold

The effect of galectin-1 (GAL1) on the growth of immortal rat chondrocyte (IRC) on chitosan-modified PLGA scaffold is investigated. The experimental results showed that water absorption ratio of chitosan-modified PLGA scaffold was 70% higher than that of PLGA alone after immersion in ddH(2)O for 2 weeks, indicating that chitosan-modification significantly enhances the hydrophilicity of PLGA. The experimental results also showed that GALl efficiently and spontaneously coats the chitosan-PLGA scaffold surface to promote adhesion and growth of immortal rat chondrocyte (IRC). To investigate the effect of endogenous GAL1, the full-length GAL1 cDNAs were cloned and constructed into pcDNA3.1 vectors to generate a plasmid expressed in IRC (IRC-GAL1). The results showed that IRC-GAL1 growth was significantly higher than that of IRC on chitosan-PLGA scaffold. The GAL1-potentiated IRC growth on chitosan-PLGA scaffold was dose-dependently inhibited by TDG (specific inhibitor of GAL1 binding). These results strongly suggest that GAL1 is critical for enhancing IRC cell adhesion and growth on chitosan-PLGA scaffold. Moreover, GAL1-coating or expression tends to promote IRC cell-cell aggregation on chitosan-PLGA scaffold and significantly enhances IRC migration. These results suggest that GAL1 probably could induce tissue differentiation and facilitates cartilage reconstruction. In conclusion, the experimental results suggest that both GAL1 and chitosan are important for enhancing IRC cell adhesion and growth on PLGA scaffold, and GAL1 is a potential biomaterial for tissue engineering.