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Background: Gastric cancer (GC) is the second most prevalent cancer worldwide and the eighth most common cause of tumor-related death in Taiwan. Helminthostachys zeylanica, a flavonoid compound, has anti-inflammatory, immunomodulatory, and anticancer effects. We examined whether an extract of H. zeylanica (E1 and E2) has potential as a treatment for GC. Methods: We investigated the effects (pro-apoptosis, pro-autophagy, and antiproliferation ability) of H. zeylanica-E2 on cell viability in healthy gastric epithelial (GES-1) and GC cells (AGS and BGC823). H. zeylanica-E2 was toxic to GC cells but had little or no toxicity to normal cells. Results: In this study, H. zeylanica-E2 induced apoptosis through caspase 3/7, Bcl-2, Bax, cyclooxygenase-2 (COX-2), and cleaved poly (ADP-ribose) polymerase pathways in GC cells. In addition, it increased autophagy by stimulating autophagy-related protein (ATG)5, ATG7, LC3-I/LC3-II, and inhibiting COX-2 activity in GC cells. We also found that H. zeylanica-E2 exhibited antiproliferation ability through cell cycle arrest in G0/G1 and G2/M and suppressed the migration of GC cells. The anticancer effects of H. zeylanica-E2 in GC cells might be mediated partly through inhibition of tumor necrosis factor-α (TNF-α)-activated proinflammatory cytosolic phospholipase A2 (cPLA2)-COX-2-prostaglandin E2 (PGE2) pathway. Conclusions: Our results suggest that H. zeylanica-E2 has potential as a novel adjunctive agent for the treatment of GC.

Fisetin is isolated from many fruits and vegetables and has been confirmed to improve airway hyperresponsiveness in asthmatic mice. However, whether fisetin reduces inflammatory response and oxidative stress in bronchial epithelial cells is unclear. Here, BEAS-2B human bronchial epithelial cells were treated with various concentrations of fisetin and then stimulated with tumor necrosis factor-α (TNF-α) or TNF-α/interleukin-4. In addition, ovalbumin-sensitized mice were treated with fisetin to detect inflammatory mediators and oxidative stress expression. Fisetin significantly reduced the levels of inflammatory cytokines and chemokines in TNF-α-stimulated BEAS-2B cells. Fisetin also attenuated intercellular adhesion molecule-1 expression in TNF-α-stimulated BEAS-2B cells, suppressing THP-1 monocyte adhesion. Furthermore, fisetin significantly suppressed airway hyperresponsiveness in the lungs and decreased eosinophil numbers in the bronchoalveolar lavage fluid of asthmatic mice. Fisetin decreased cyclooxygenase-2 expression, promoted glutathione levels, and decreased malondialdehyde levels in the lungs of asthmatic mice. Our findings indicate that fisetin is a potential immunomodulator that can improve the pathological features of asthma by decreasing oxidative stress and inflammation.

Periodontal diseases are prevalent worldwide. Lipoteichoic acid (LTA), a major component of gram-positive bacteria, may play a key role in periodontally inflammatory diseases. Carbon monoxide (CO) is a critical messenger in many biological processes. It can elicit various biological properties, especially anti-inflammatory effects. As the straight administration of CO remains difficult, CO-releasing molecules (CO-RMs) are emerging as promising alternatives. To explore the pharmacological actions and signaling pathways of CO battling LTA-induced periodontal inflammation, this study investigated the cytoprotective effects of CORM-2 against the adhesion of THP-1 monocytes to human gingival fibroblasts (HGFs) and the underlying molecular mechanism. After exposing HGFs to LTA with or without CORM-2 pretreatment, monocyte adhesion was determined. VCAM-1 and ICAM-1 expression in HGFs was measured by real-time PCR. To identify the signaling pathways of CO involved in the cytoprotective effects of CORM-2, HGFs underwent pharmacological or genetical interventions before LTA incubation. The expression and/or activity of possible regulatory molecules were determined. The release of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α, were measured using ELISA. The results showed that LTA increased cytokine production and upregulated VCAM-1 and ICAM-1 expression in HGFs, promoting monocyte adhesion. These events were dependent on TLR2/MyD88/TRAF6- and PI3K/Akt/NADPH oxidase/ROS-regulated NF-κB activation. CORM-2 inhibited LTA-induced inflammatory cascades in HGFs, in which CO seemed to be the hitman. To conclude, CO released from CORM-2 can prevent the LTA-stimulated HGFs from increasing VCAM-1 and ICAM-1 expression and promoting monocyte adhesion by inhibiting TLR2/MyD88/TRAF6 association and PI3K/Akt/NADPH oxidase/ROS signaling, both converge on the canonical NF-κB activation.

In Taiwan, the root extract of Vitis thunbergii Sieb. et Zucc. (Vitaceae, VT) is rich in stilbenes, with resveratrol (Res) and its derivatives being the most abundant. Previously, we showed that the effect of Res derivatives against tumor necrosis factor-α (TNF-α)-stimulated inflammatory responses occurs via cPLA2/COX-2/PGE2 inhibition. This study compared and explored the underlying anti-inflammatory pharmacological mechanisms. Before stimulation with TNF-α, RMCs were treated with/without pharmacological inhibitors of specific protein kinases. The expression of inflammatory mediators was determined by Western blotting, gelatin zymography, real-time PCR, and luciferase assay. Cellular and mitochondrial ROS were measured by H2DHFDA or DHE and MitoSOX™ Red staining, respectively. The RNS level was indirectly measured by Griess reagent assay. Kinase activation and association were assayed by immunoprecipitation followed by Western blotting. TNF-α binding to TNFR recruited Rac1 and p47phox, thus activating the NAPDH oxidase-dependent MAPK and NF-κB pathways. The TNF-α-induced NF-κB activation via c-Src-driven ROS was independent from the EGFR signaling pathway. The anti-inflammatory effects of Res derivatives occurred via the inhibition of ROS derived from mitochondria and NADPH oxidase; RNS derived from iNOS; and the activation of the ERK1/2, JNK1/2, and NF-κB pathways. Overall, this study provides an understanding of the various activities of Res derivatives and their pharmacological mechanisms. In the future, the application of the active molecules of VT to health foods and medicine in Taiwan may increase.

Quercetin is a flavonoid polyphenolic compound present in fruits and vegetables that has proven anti-inflammatory activity. The goal of the present investigation was to investigate the effects of quercetin on tumor necrosis factor-α (TNF-α)-induced inflammatory responses via the expression of ICAM-1 and MMP-9 in human retinal pigment epithelial cells (ARPE-19 cells). Real-time PCR, gelatin zymography, and Western blot analysis showed that TNF-α induced the expression of ICAM-1 and MMP-9 protein and mRNA in a time-dependent manner. These effects were attenuated by pretreatment of ARPE-19 cells with quercetin. Quercetin inhibited the TNF-α-induced phosphorylation of PKCδ, JNK1/2, ERK1/2. Quercetin, rottlerin, SP600125 and U0126 attenuated TNF-α-stimulated c-Jun phosphorylation and AP-1-Luc activity. Pretreatment with quercetin, rottlerin, SP600125, or Bay 11-7082 attenuated TNF-α-induced NF-κB (p65) phosphorylation, translocation and RelA/p65-Luc activity. TNF-α significantly increased MMP-9 promoter activity and THP-1 cell adherence, and these effects were attenuated by pretreatment with quercetin, rottlerin, SP600125, U0126, tanshinone IIA or Bay 11-7082. These results suggest that quercetin attenuates TNF-α-induced ICAM-1 and MMP-9 expression in ARPE-19 cells via the MEK1/2-ERK1/2 and PKCδ-JNK1/2-c-Jun or NF-κB pathways.

Introduction Resveratrol has been reported to alleviate inflammatory responses and oxidative stress in mesangial cells and in several types of renal injury in animal models. Previously, the active resveratrol derivatives from the roots of Vitis thunbergii Sieb. & Zucc. (Vitaceae) were shown to have significant anti-platelet and anti-oxidative activities. However, the anti-inflammatory mechanisms of these resveratrol derivatives in rat mesangial cells (RMCs) have not been clarified fully. Methods The protective mechanisms of resveratrol derivatives involved in tumor necrosis factor-α (TNF-α)-induced inflammatory responses were assessed by Western blot analysis, real-time PCR, and RT–PCR. The involvement of various signaling molecules in these responses was investigated using selective pharmacological inhibitors. Results Nontoxic concentrations of the resveratrol derivatives significantly attenuated cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2) expression in RMCs challenged by TNF-α. These resveratrol derivatives inhibited TNF-α-activated ERK1/2 and JNK1/2 without affecting p38 phosphorylation. Next, we demonstrated that TNF-α induced NF-κB activation, translocation, and promoter activity, which was inhibited by pretreatment with resveratrol derivatives in RMCs. Conclusion The protective mechanisms of resveratrol derivatives against TNF-α-stimulated inflammatory responses via cPLA2/COX-2/PGE2 inhibition was caused by the attenuation of the JNK1/2, ERK1/2, and NF-κB signaling pathways in RMCs.

Among adolescent girls, overweight or obesity has both physical and psychological involvement. We conducted a randomized controlled trial of moxibustion using a moxa burner. Fifty-four eligible girls aged 15–18 years with a body mass index (BMI) greater than 25.3 were enrolled in the study. The girls were randomly allocated to the treatment (n = 27) and control (n = 27) groups. The girls underwent treatment three times per week for 8 weeks (24 treatments). Moxibustion was applied to the RN12, RN6, ST25, ST36, and SP6 acupoints. Physical assessments were BMI, waist-to-hip ratio (WHR), and body fat ratio (BFR). Psychological outcomes were measured using the Rosenberg Self-Esteem Scale (RSE). Data were collected at the beginning of the study (baseline), week 4, and week 8. Of the 54 participants, 46 completed the trial. The difference in mean BMI from baseline between the two groups was 0.097 (p=0.655) at week 4 and −0.794 (p=0.001) at week 8. The mean WHR of the treatment group was significantly reduced compared with baseline, with a −0.011 (p=0.017) and −0.035 (p<0.001) mean change at weeks 4 and 8, respectively. The mean BFR was slightly reduced (−0.253;p=0.474) at week 4 compared with baseline in the treatment group. At week 8, it was significantly reduced (−2.068;p<0.001) from baseline in the treatment group. The mean RSE in the treatment group showed no significant increase from baseline at week 4 (0.155 points,p=0.803), but it improved significantly from baseline at week 8 (1.606 points,p=0.021) compared to that in the control group. No obvious adverse effect was reported during this study. Moxibustion using a moxa burner may be an effective and safe intervention for overweight adolescent girls, having both physical and psychological benefits.

Inflammation is a hallmark of many metabolic diseases. We previously showed that ferrocene-appended 1H-1,2,3-triazole hybrids inhibit nitric oxide (NO) production in in vitro models of lipopolysaccharide-induced inflammation in the BV-2 cell. In the present study, we explored the viability, anti-inflammatory, and antioxidant potential of ferrocene-1H-1,2,3-triazole hybrids using biochemical assays in rat mesangial cells (RMCs). We found that, among all the ferrocene-1H-1,2,3-triazole hybrids, X2&ndash, X4 exhibited an antioxidant effect on mitochondrial free radicals. Among all the studied compounds, X4 demonstrated the best anti-inflammatory effect on RMCs. These results were supplemented by in silico studies including molecular docking with human cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2) enzymes as well as absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling. Besides, two new crystal structures of the compounds have also been reported. In addition, combining the results from the inducible nitric oxide synthase (iNOS), cPLA2, COX-2, and matrix metalloproteinase-9 (MMP-9) enzymatic activity analysis and NO production also confirmed this argument. Overall, the results of this study will be a valuable addition to the growing body of work on biological activities of triazole-based compounds.

Resveratrol was shown to exert anti-inflammatory effects in experimental models of psoriasis. Several natural oligomers of resveratrol have been extracted from plants. We investigated the antipsoriatic activity of topical administration of resveratrol oligomers and explored the effect of the number of resveratrol subunits on skin absorption to establish the structure-permeation relationship (SPR). Three oligomers, e-viniferin (dimer), ampelopsin C (trimer) and vitisin A (tetramer), extracted from Vitis thunbergii root were compared to the resveratrol glycoside polydatin. Delivery to porcine skin was assessed in vitro using the Franz cell. Keratinocytes activated with imiquimod (IMQ) were utilized to evaluate cytokine/chemokine inhibition. Topical application of resveratrol and oligomers was characterized in vivo by assessing cutaneous absorption, skin physiology, proinflammatory mediator expression, and histopathology in IMQ-treated mice. Skin deposition decreased as the molecular size and lipophilicity of the permeants increased. Resveratrol exhibited highest absorption, followed by e-viniferin. The monomers resveratrol and polydatin exhibited higher flux across skin than the larger oligomers. In silico modeling revealed the permeants that strongly interacted with stratum corneum (SC) lipids exhibited lower transport to viable skin and the receptor compartment. In vitro, resveratrol and its derivatives had comparable ability to inhibit IMQ-induced IL-1β, IL-6, and CXCL8 secretion in activated keratinocytes. In vivo, topically applied e-viniferin accumulated at higher levels than resveratrol (0.067 versus 0.029 nmol/mg) in psoriasis-like mouse skin with impaired barrier capacity. Topical e-viniferin alleviated psoriasiform symptoms and reduced IL-23 secretion (by 58% vs. 37%) more effectively than resveratrol. e-Viniferin has potential as an anti-inflammatory agent to prevent or treat psoriasis.

Introduction Exposure to airborne particulate matter (PM) increases the proportion of oral inflammatory diseases. During the formation of inflammatory conditions, the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome activation plays an important regulator. Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by heme oxygenase-1 (HO-1), has been shown to own cytoprotective effects including anti-inflammation and antioxidant. Here, we determined the novel mechanisms of carbon monoxide releasing molecule-2 (CORM-2) on PM-induced inflammatory responses in human oral keratinocytes (HOKs). Methods The effects of CORM-2 on the expression of various inflammatory proteins induced by PM were determined by Western blot, real-time PCR, promoter assay, and ELISA. The involvement of signaling molecules in these responses was studied by using the selective pharmacological inhibitors and siRNAs. Results We proved that PM enhanced C-reactive protein (CRP) levels, NLRP3 inflammasome and caspase-1 activation, and IL-1β release, which were reduced by preincubation with CORM-2. Transfection with PKCα siRNA and preincubation with the ROS scavenger (N-acetyl-cysteine, NAC), an inhibitor of NADPH oxidase (diphenyleneiodonium, DPI), or the mitochondria-specific superoxide scavenger (MitoTEMPO) inhibited PM-mediated inflammatory responses. In addition, PM-regulated PKCα and NADPH oxidase activation as well as NADPH oxidase- and mitochondria-derived ROS generation were inhibited by CORM-2, but not inactivate CORM-2 (iCORM-2) pretreatment. At the end, we confirmed that CORM-2 improved PM-induced inflammatory responses via the induction of Nrf2 activation and HO-1 expression. Conclusion We suggest that CORM-2 inhibits PM-induced inflammatory responses in HOKs via the inhibition of PKCα/ROS/NLRP3 inflammasome activation combined with the induction of Nrf2/HO-1 expression.

Staphylococcus aureus (S. aureus) can lead to many life-threatening diseases. It has the ability to invade normal endovascular tissue. The molecular mechanisms and pathological changes of endothelial cells after S. aureus infection are of interest, but the basic understanding of how S. aureus destroys this barrier is not clear. Here, we showed that S. aureus enhanced COX-2 expression and prostaglandin E2 (PGE2) secretion in human aortic endothelial cells (HAECs). In addition, S. aureus induced PGE2/interleukin-6 (IL-6)/matrix metallopeptidase-9 (MMP-9)-dependent cell migration. S. aureus-induced COX-2, IL-6, and MMP-9 levels were inhibited by transfection with siRNA of Toll-like receptor 2 (TLR2), p38, p42, p44, p50, or p65. S. aureus also induced p38 MAPK, ATF2, ERK1/2, and NF-κB p65 activation. Interestingly, we proved that S. aureus decreased intracellular generation of reactive oxygen species (ROS), which suggests that the inhibition of ROS production promoted inflammatory responses. Finally, we showed that S. aureus enhanced a variety of biomarkers of inflammation in cardiovascular diseases. However, the free radical scavenger (MCI-186) or antioxidant (N-acetyl-L-cysteine, NAC) markedly enhanced S. aureus-induced COX-2 mRNA levels in the aorta tissues. Taken together, these findings established that S. aureus promoted aorta inflammation via activation of p38 MAPK, ERK1/2, and NF-κB and inhibition of ROS generation.

Quercetin, a bioflavonoid derived from vegetables and fruits, exerts anti-inflammatory effects in various diseases. Our previous study revealed that quercetin could suppress the expression of matrix metalloprotease-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1) to achieve anti-inflammatory effects in tumor necrosis factor-&alpha, (TNF-&alpha, )-stimulated human retinal pigment epithelial (ARPE-19) cells. The present study explored whether quercetin can inhibit the interleukin-1&beta, (IL-1&beta, )-induced production of inflammatory cytokines and chemokines in ARPE-19 cells. Prior to stimulation by IL-1&beta, ARPE-19 cells were pretreated with quercetin at various concentrations (2.5&ndash, 20 &micro, M). The results showed that quercetin could dose-dependently decrease the mRNA and protein levels of ICAM-1, IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1). It also attenuated the adherence of the human monocytic leukemia cell line THP-1 to IL-1&beta, stimulated ARPE-19 cells. We also demonstrated that quercetin inhibited signaling pathways related to the inflammatory process, including phosphorylation of mitogen-activated protein kinases (MAPKs), inhibitor of nuclear factor &kappa, B kinase (IKK)&alpha, /&beta, c-Jun, cAMP response element-binding protein (CREB), activating transcription factor 2 (ATF2) and nuclear factor (NF)-&kappa, B p65, and blocked the translocation of NF-&kappa, B p65 into the nucleus. Furthermore, MAPK inhibitors including an extracellular signal-regulated kinase (ERK) 1/2 inhibitor (U0126), a p38 inhibitor (SB202190) and a c-Jun N-terminal kinase (JNK) inhibitor (SP600125) decreased the expression of soluble ICAM-1 (sICAM-1), but not ICAM-1. U0126 and SB202190 could inhibit the expression of IL-6, IL-8 and MCP-1, but SP600125 could not. An NF-&kappa, B inhibitor (Bay 11-7082) also reduced the expression of ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1. Taken together, these results provide evidence that quercetin protects ARPE-19 cells from the IL-1&beta, stimulated increase in ICAM-1, sICAM-1, IL-6, IL-8 and MCP-1 production by blocking the activation of MAPK and NF-&kappa, B signaling pathways to ameliorate the inflammatory response.

Casticin has been isolated from Vitex trifolia and found to have anti-inflammatory and anti-tumor properties. We also previously discovered that casticin can reduce pro-inflammatory cytokines and ICAM-1 expression in inflammatory pulmonary epithelial cells. In the present study, we evaluated whether casticin reduced airway hyper-responsiveness (AHR), airway inflammation, and oxidative stress in the lungs of a murine asthma model and alleviated inflammatory and oxidative responses in tracheal epithelial cells. Female BALB/c mice were randomly divided into five groups: normal controls, ovalbumin (OVA)-induced asthma, and OVA-induced asthma treated with intraperitoneal injection of casticin (5 or 10 mg/kg) or prednisolone (5 mg/kg). Casticin reduced AHR, goblet cell hyperplasia, and oxidative responses in the lungs of mice with asthma. Mechanistic studies revealed that casticin attenuated the levels of Th2 cytokine in bronchoalveolar lavage fluids and regulated the expression of Th2 cytokine and chemokine genes in the lung. Casticin also significantly regulated oxidative stress and reduced inflammation in the lungs of mice with asthma. Consequently, inflammatory tracheal epithelial BEAS-2B cells treated with casticin had significantly suppressed levels of pro-inflammatory cytokines and eotaxin, and reduced THP-1 monocyte cell adherence to BEAS-2B cells via suppressed ICAM-1 expression. Thus, casticin is a powerful immunomodulator, ameliorating pathological changes by suppressing Th2 cytokine expression in mice with asthma.

Staphylococcus aureus (S. aureus) can lead to many life-threatening diseases. It has the ability to invade normal endovascular tissue. The molecular mechanisms and pathological changes of endothelial cells after S. aureus infection are of interest, but the basic understanding of how S. aureus destroys this barrier is not clear. Here, we showed that S. aureus enhanced COX-2 expression and prostaglandin E

Objectives To modify a chitosan membrane (CM) by cross-linking the chitosan with genipin, a naturally occurring cross-linker extracted from Gardenia jasminoides fructus, with the aim of developing a new cell culture support and to observe the phenotypes of cultured human corneal epithelial cells (HCECs) on genipin-cross-linked chitosan membrane (GCM). Methods We tested the cross-linking characteristics and mechanical strength of the GCM. CMs modified by cross-linking with different concentrations of genipin were prepared to investigate the rate of membrane degradation. The biocompatibility of the GCMs was investigated by determining the viability of HCECs cultured on them in vitro. The morphology of the HCECs cultured on CM or GCM was analyzed by confocal microscopy and scanning electron microscopy (SEM). Immunocytochemical staining was conducted to determine the phenotypes of the cultured cells. Results The fixation index of the GCM was 31 ± 3% after treatment of CM with 0.5 mM genipin. A stress–strain test showed that the GCM could tolerate three times the mechanical force of noncross-linked CM. The biodegradation rate of GCM was much slower than for CM. A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that cell viability was not affected by cross-linking with 5.0 mM genipin. SEM showed that the cultured HCECs adhered to and grew well on the surface of the GCM. Immunocytochemical staining showed keratin 3 (K3) and connexin 43 (Cx-43) immunoreactive HCECs on the GCM and their proliferative ability was not significantly affected by strong immunoreactivity of Ki-67 and p63 markers. Conclusions GCM has potential as a scaffold for corneal epithelium in ocular surface surgery and greater mechanical strength and slower degradation than unmodified CM.

Multidrug resistance in cancer cells arises from altered drug permeability of the cell. We previously reported activation of the Wnt pathway in ABCB1-overexpressed human uterus sarcoma drug-resistant MES-SA/Dx5 cells through active β-catenin and associated transactivation activities, and upregulation of Wnt-targeting genes. In this study, Wnt5A was found to be significantly upregulated in MES-SA/Dx5 and MCF7/ADR2 cells, suggesting an important role for the Wnt5A signaling pathway in cancer drug resistance. Higher cAMP response elements and Tcf/Lef transcription activities were shown in the drug-resistant cancer cells. However, expression of Wnt target genes and CRE activities was downregulated in Wnt5A shRNA stably-transfected MES-SA/Dx5 cells. Cell viability of the drug-resistant cancer cells was also reduced by doxorubicin treatment and Wnt5A shRNA transfection, or by Wnt5A depletion. The in vitro data were supported by immunohistochemical analysis of 24 paired breast cancer biopsies obtained pre- and post-chemotherapeutic treatment. Wnt5A, VEGF and/or ABCB1 were significantly overexpressed after treatment, consistent with clinical chemoresistance. Taken together, the Wnt5A signaling pathway was shown to contribute to regulating the drug-resistance protein ABCB1 and β-catenin-related genes in antagonizing the toxic effects of doxorubicin in the MDR cell lines and in clinical breast cancer samples.

This study aimed at improving the understanding of speech characteristics of fricatives produced by five-year-old Mandarin-acquiring children with cerebral palsy (CP). Productions from nine CP children and nine gender-and-age-matched typically developing (TD) children were collected and analyzed. Results from transcription indicated that the CP group had lower production accuracy rates for all the five fricatives in Mandarin Chinese. Additionally, when the CP children failed to articulate the target fricative segments, they tended to delete them or convert them into non-continuant segments. Results from acoustic analyses indicated that the M2 values of the labiodental [f] and the M1 and M2 values of the alveolar [s] were higher among the CP children. The experimental results revealed that: (1) Observable differences were available once the age of the groups was properly controlled and acoustical measurements were adopted; (2) the lack of finer-grained speech motor control abilities among CP children were reflected in the M1 and M2 values; (3) for segments at the anterior places, the clinical group failed to extend the articulatory gestures to the desirable positions. It is suggested that future studies focusing on different age groups and children with different native languages would help to approach the nature of articulatory barriers among individuals with CP.

Keywords: Adenine; Aminoimidazole carboxamide ribonucleotide; AMP-activated protein kinase; Dermal papilla; Senescence

Acne is the over growth of the commensal bacteria Propionibacterium acnes (P. acnes) on human skin. Lauric acid (LA) has been investigated as an effective candidate to suppress the activity of P. acnes. Although LA is nearly insoluble in water, dimethyl sulfoxide (DMSO) has been reported to effectively solubilize LA. However, the toxicity of DMSO can limit the use of LA on the skin. In this study, LA-loaded poly(ɛ-caprolactone)-poly(ethylene glycol)-poly(ɛ-caprolactone) micelles (PCL-PEG-PCL) were developed to improve the bactericidal effect of free LA on P. acnes. The block copolymers mPEG-PCL and PCL-PEG-PCL with different molecular weights were synthesized and characterized using 1H Nuclear Magnetic Resonance spectroscopy (1H NMR), Fourier-transform infrared spectroscopy (FT-IR), Gel Permeation Chromatography (GPC), and Differential Scanning Calorimetry (DSC). In the presence of LA, mPEG-PCL diblock copolymers did not self-assemble into nano-sized micelles. On the contrary, the average particle sizes of the PCL-PEG-PCL micelles ranged from 50–198 nm for blank micelles and 27–89 nm for LA-loaded micelles. The drug loading content increased as the molecular weight of PCL-PEG-PCL polymer increased. Additionally, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of free LA were 20 and 80 μg/mL, respectively. The MICs and MBCs of the micelles decreased to 10 and 40 μg/mL, respectively. This study demonstrated that the LA-loaded micelles are a potential treatment for acne.

Matrix metalloproteinase-9 (MMP-9) plays an important role in the outgrowth of expanded human limbal epithelial cells on intact amniotic membranes (AM). The mechanisms of MMP-9 expression and cell outgrowth remain unknown. Here, we demonstrated that MMP-9 is preferentially expressed at the leading edge of limbal epithelial outgrowth. Treatment with the inhibitors of PI3-K (LY294002), Akt (SH-5), MEK1/2 (U0126), and JNK1/2 (SP600125) attenuated the outgrowth area, indicating that PI3-K/Akt, p42/p44 MAPK, and JNK1/2 are involved in the outgrowth of intact AM-expanded limbal epithelial cells. However, MMP-9 expression at both transcriptional and translational levels was attenuated by treatment with SP600125, LY294002, or SH-5, not by U0126 and SB202190, suggesting that JNK1/2 and PI3-K/Akt participate in MMP-9 expression. Moreover, NF-κB phosphorylation and nuclear translocation was especially noted at the leading edge, which was attenuated by treatment with SP600125 or LY294002. Helenalin, a selective NF-κB inhibitor, reduced both the limbal epithelial outgrowth and MMP-9 expression. Finally, the data reveal that PI3-K/Akt is an upstream component of the JNK1/2 pathway in MMP-9 expression. Thus, both MAPKs and PI3-K/Akt are required for limbal epithelial outgrowth on intact AM, only the PI3-K/Akt/JNK is essential for MMP-9 expression mediated through activation of transcriptional factor NF-κB in this model.

Nano Pd–Au particles have been electrochemically fabricated utilizing ionic liquid as green electrolyte (1-Butyl-3-methylimidazolium tetrafluoroborate). Nano Pd–Au particles modified glassy carbon electrode (GCE) and indium tin oxide coated glass electrodes were examined using atomic force microscopy, field emission scanning electron microscope and X-ray diffraction studies. Electrodeposited nano Pd–Au particles' average diameter was found as 33 nm. Nano Pd–Au particle modified GCE was electrochemically active and stable in various pH solutions. The proposed nano particle modified GCE reduces the over potential and shows the well defined oxidation peaks for the detection of epinephrine and simultaneous determination of dopamine and uric acid (in pH 7.0 phosphate buffer solution) using cyclic voltammetry and differential pulse voltammetry.

Preanodized screen printed carbon electrode (SPCE) has been utilized for the detection of propofol. Here the preanodized SPCE possess the specific functional groups which help the detection and determination of propofol. The proposed SPCE shows a clear oxidation peak for the detection of propofol in pH 7.0 phosphate buffer solutions. Interestingly, it shows a well-defined individual oxidation peak for the detection of propofol in the presence interferences (mixture of ascorbic acid, dopamine, and uric acid). This type of pretreated SPCE successfully enhances the electrooxidation current and overcomes the interference effects and clearly exhibits the signals for the propofol detection using cyclic voltammetry and flow injection analysis techniques. The preanodized SPCE shows the electrooxidation signals for the propofol detection in the linear range of 0.09 to 0.90 μM, respectively. Further, the sensitivity of the proposed electrode for the propofol detection is found to be 3.6 μA μM−1.

Matrix metalloproteinases (MMPs), in particular MMP-9, is induced by cytokines including IL-1 beta and contributes to airway injury and remodeling. However, the mechanisms underlying IL-1 beta-induced MMP-9 expression and cell migration in human A549 cells remain unclear. Here, we report that the IL-1 beta-induced MMP-9 gene expression was mediated through the activation of p42/p44 MAPK, p38 MAPK, and JNK1/2 in A549 cells, determined by zymographic, RT-PCR, and Western blotting. The involvement of MAPKs in the IL-1beta-induced responses was further ensured by transfection with siRNA of MEK1, p42, p38, or JNK2. Moreover, the IL-1 beta-induced MMP-9 gene expression was also mediated through the translocation of NF-kappaB (p65) into the nucleus and the degradation of I kappaB alpha. In addition, the IL-1 beta-induced c-Jun phosphorylation was reduced by pretreatment with U0126 or SP600125. IL-1 beta stimulated the transcriptional activity of wild-type MMP-9 promoter in A549 cells, which was inhibited by U0126, SB203580, SP600125, and helenalin. In contrast, IL-1 beta had no effect on the cells transfected with a NF-kappaB-mutated MMP-9 promoter construct, suggesting that NF-kappaB is required for this response. Finally, the IL-1 beta-induced MMP-9 expression led to cell migration which was attenuated by pretreatment with U0126, SB203580, SP600125, helenalin, or MMP-2/9 inhibitor. These results suggested that in A549 cells, the activation of p42/p44 MAPK, p38 MAPK, JNK1/2, NF-kappaB, and AP-1 are essential for the IL-1 beta-induced MMP-9 gene expression and cell migration.

Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-alpha (TNF-alpha) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-alpha in human A549 cells remain unclear. Here, we showed that TNF-alpha induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-kappaB (helenalin), and transfection with dominant negative mutants of ERK2 (DeltaERK) and JNK (DeltaJNK), and siRNAs for MEK1, p42 and JNK2. TNF-alpha-stimulated phosphorylation of p42/p44 MAPK and JNK were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of DeltaERK and DeltaJNK. Furthermore, the involvement of NF-kappaB in TNF-alpha-induced MMP-9 production was consistent with that TNF-alpha-stimulated degradation of IkappaB-alpha and translocation of NF-kappaB into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-kappaB was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-alpha in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-alpha-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-kappaB MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-alpha-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-kappaB are essential for TNF-alpha-induced MMP-9 gene expression.

In previous study, interleukin-1beta (IL-1beta) has been shown to induce ICAM-1 expression through MAPKs and NF-kappaB in A549 cells. In addition to these pathways, transactivation of non-receptor tyrosine kinase (Src), PDGF receptors (PDGFRs), and phosphatidylinositol 3-kinase (PI3K)/Akt has been implicated in the expression of inflammatory genes. Here, we further investigated whether these different mechanisms participating in IL-1beta-induced ICAM-1 expression in A549 cells. We initially observed that IL-1beta-induced ICAM-1 promoter activity was attenuated by the inhibitors of Src (PP1), PDGFR (AG1296), PI3-K (LY294002 and wortmannin), and Akt (SH-5), revealed by reporter gene assay, Western blotting, and RT-PCR analyses. The involvement of Src and PI3-K/Akt in IL-1beta-induced ICAM-1 expression was significantly attenuated by transfection of A549 cells with dominant negative plasmids of Src, p85 and Akt, respectively. Src, PDGFR, and PI3K/Akt mediated the effects of IL-1beta because pretreatment with PP1, AG1296, and wortmannin also abrogated IL-1beta-stimulated Src, PDGFR, and Akt phosphorylation, respectively. Moreover, pretreatment with p300 inhibitor (curcumin) also blocked ICAM-1 expression. We further confirmed that p300 was associated with ICAM-1 promoter which was dynamically linked to histone H4 acetylation stimulated by IL-1beta, determined by chromatin immunoprecipitation assay. Association of p300 and histone-H4 to ICAM-1 promoter was inhibited by LY294002. Up-regulation of ICAM-1 enhanced the adhesion of neutrophils onto A549 cell monolayer exposed to IL-1beta, which was inhibited by PP1, AG1296, LY294002, wortmannin, and helenalin. These results suggested that Akt phosphorylation mediated through transactivation of Src/PDGFR promotes the transcriptional p300 activity and eventually leads to ICAM-1 expression induced by IL-1beta.

Background Sebaceous cell carcinoma of the eyelid is a malignant tumor. However, the pathoetiology of sebaceous cell carcinoma is not clear. Retinoic acid (RA) signaling is essential for skin epidermal differentiation including the eyelids. In this study, we investigate the expression of β-catenin, RA-binding proteins and RA receptors in sebaceous cell carcinoma of the eyelid and try to estimate their influence on its pathoetiology. Methods Retrospective, noncomparative, consecutive interventional case series. Sixteen cases of eyelid sebaceous gland carcinoma who received tumor excision at our hospital between 2001 and 2011 were included. Immunohistochemical staining for β-catenin, cellular retinoic acid binding protein 1 (CRABP1), cellular retinoic acid binding protein 2 (CRABP2), fatty acid-binding protein 5 (FABP5), retinoic acid receptors (RAR-α, −β, −γ), and retinoid X receptors (RXR-α, −β, −γ) was performed on tissue samples obtained from tumor excision. Results Of the 16 sebaceous cell carcinoma cases reviewed, six were male and 10 female. The mean follow-up period was 6.7 ± 3.66 years (range, 0.3–13 years). Of these 16 cases, the expression of β-catenin was significantly increased in sebaceous cell carcinoma cases. CRABP1 was similarly expressed in the sebaceous cell carcinoma and control groups. CRABP2 and FABP5 were expressed in hair follicles of lid skin in both groups, whereas the CRABP2 and FABP5 were aberrantly expressed in the tumor cells of the sebaceous glands. Notably, the expression of retinoic acid receptor (RAR-β) and retinoid X receptors (RXR-β, −γ) was significantly upregulated in sebaceous cell carcinoma of the eyelids. Conclusions Our findings indicate that retinoic acid signaling is related to the pathogenesis of sebaceous cell carcinoma of the eyelids.

Stem cells of the corneal epithelium have been found to be located exclusively at the anatomical junction between the cornea and the conjunctiva, the limbus. Ex vivo expanded limbal epithelial cells on amniotic membrane (AM) are capable of restoring the corneal surface with limbal stem cell deficiency. Recent studies indicate that intact AM preserves the limbal epithelial phenotype and that distinct epithelial morphology is noted among various culture matrix. However, the factors in response to the interaction between limbal epithelial cells and AM were not well understood. Using Annexin V-fluorescein isothiocyanate staining, we found that human limbal epithelial cells expanded on intact human AM demonstrated fewer apoptotic cells as compared with those on plastic dishes. To identify the anti-apoptotic factors, we performed cDNA microarray analysis and showed that interleukin-1 receptor antagonist (IL-1RA) was overexpressed in cultures on intact AM, which was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (Q-PCR) and enzyme-linked immunosorbent assay. In addition, we also noted that the phenomenon of apoptosis detected in cultures on plastic dishes could be reversed by adding recombinant IL-1RA protein into the media, whereas apoptosis of limbal epithelial cells cultivated on intact AM could be induced by exogenous neutralizing IL-1RA neutralizing antibody. These results demonstrated that intact human AM may prevent cultured human limbal epithelial cells from undergoing apoptosis. IL-1RA might be a candidate mediator to exert as an anti-apoptotic molecule during the interaction between human limbal epithelial cells and intact human AM.

This paper illustrates applications of the Web-based collaborative visualization (WCV) in distributed product development. Three software prototypes were developed using the WCV technologies. The first one enables the end users to configure individual parts of 3D assembly in a regular browser, and thus provides an effective tool to collect the customer's voices in e-commerce. The second system realizes mass customization for car interior design. Online ergonomic evaluation is conducted based on the interactions between a digital human mimicking the user and the design model. Finally, an Internet-based design review system is implemented. It enables synchronous communications between a designer and people involved in the change process with no access to CAD. These prototypes demonstrate the practicality, flexibility, and versatility of the WCV in integration with other proprietary software tools using a variety of platforms like ASP framework, Java-based technology, and Windows C/C++ applications. This work shows that the WCV is an interfacing technology, which facilitates 3D information sharing for most product-centric activities in a simple and cost-effective manner.

Although euryhaline teleosts can adapt to environmental fluctuation of salinity, their energy source for responding to changes in salinity and osmolarity remains unclear. This study examines the cellular localization of creatine kinase (CK) expression in branchia of tilapia ( Oreochromis mossambicus). Western blot analysis of muscle-type CK (MM form) revealed a high association with salinity changes, but BB and MB forms of CK in the gills of fish adapted to seawater did not change. With the use of immunocytochemistry, three CK isoforms (MM, MB, and BB) were localized in mitochondria-rich (MR) cells and other epithelial cells of tilapia gills. In addition, staining intensity of MM-form CK in MR cells increased after seawater transfer, whereas BB and MB forms did not significantly change. To our knowledge, this work presents the first evidence of CK expression in MR cells of tilapia gills, highlighting the potential role of CK in providing energy for ion transport.

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.

Adenosine 5′-monophosphate-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis via modulating metabolism of glucose, lipid, and protein. In addition to energy modulation, AMPK has been demonstrated to associate with several important cellular events including inflammation. The results showed that ENERGI-F704 identified from bamboo shoot extract was nontoxic in concentrations up to 80 μM and dose-dependently induced phosphorylation of AMPK (Thr-172) in microglia BV2 cells. Our findings also showed that the treatment of BV2 with ENERGI-F704 ameliorated the LPS-induced elevation of IL-6 and TNF-αproduction. In addition, ENERGI-F704 reduced increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) via downregulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), respectively. Moreover, ENERGI-F704 decreased activated nuclear translocation and protein level of NF-κB. Inhibition of AMPK with compound C restored decreased NF-κB translocation by ENERGI-F704. In conclusion, ENERGI-F704 exerts inhibitory activity on LPS-induced inflammation through manipulating AMPK signaling and exhibits a potential therapeutic agent for neuroinflammatory disease.

The importance of the Bcl-2 family proteins in normal vertebrate embryogenesis is being recognized; however, their regulatory mechanism is poorly understood. We report here the cloning and characterization of a novel zebrafish Bcl-2 family protein, zfBLP1. The zfBLP1 cDNA is 1942 nucleotides long, encoding a polypeptide of 238 amino acids. The primary sequence of zfBLP1 shares 50% identity to human Bcl-XL, and contains all four conserved BH domains of the Bcl-2 family proteins. Primary sequence analysis identified a consensus ER retention signal at the C-terminal end of zfBLP1. Northern blot analysis indicated that there were two major and two minor zfBLP1 mRNA species expressed during embryonic development. Among the two major mRNA species, the short one, approx. 3 kb in size, was expressed throughout embryonic development, while the long one, approx. 7 kb long, was not detectable until the gastrula stage. These results suggest that zfBLP1 is a novel Bcl-2 family protein under complicated regulations, and is likely to play an important role in zebrafish oogenesis and embryogenesis.

BACKGROUND AND PURPOSE Interleukin (IL)-1β-induced matrix metalloproteinase (MMP-9) expression is regulated by mitogen activated protein kinases (MAPKs) and NF-κB. IL-1β also stimulates transactivation of growth factor receptors and phosphatidylinositol 3-kinase (PI3K)/Akt., leading to the expression of inflammatory proteins. Here, we investigated whether these transactivation mechanisms participated in IL-1β-induced MMP-9 expression in A549 cells. EXPERIMENTAL APPROACH A549 cells were treated with/without pharmacological inhibitors and neutralizing antibody or transfected with dominant negative mutants and siRNA of particular protein kinases before stimulation with IL-1β. Cell migration was measured by in vitro scratch assay. Expression and enzymatic activity of MMP-9 were analysed by Western blot and gelatin zymography. Transcriptional activity of MMP-9 was analysed by RT-PCR, chromatin immunoprecipitation and promoter assays. KEY RESULTS Inhibition of MMP-9 expression by inhibitors of Src (PP1), platelet-derived growth factor (PDGF) receptor and epithelial growth factor (EGF) receptor or transfection with siRNA for Src and Akt prevented IL-1β-induced migration of A549 cells. These tyrosine kinases were involved through phosphorylation of Src, PDGF, or EGF receptors (EGFRs) via the formation of Src/PDGFR or Src/EGFR complexes, attenuated by PP1. IL-1β-induced MMP-9 expression through EGFR transactivation was diminished by inhibitors of MMPs and heparin-binding EGF-like factor (HB-EGF), or a neutralizing HB-EGF antibody. IL-1β-stimulated activation and translocation of Akt and NF-κB (p65); the recruitment of activated NF-κB (p65) to the MMP-9 promoter region was attenuated by LY294002. CONCLUSIONS AND IMPLICATIONS IL-1β-induced MMP-9 expression and cell migration was mediated through c-Src-dependent transactivation of EGFR/PDGFR/PI3K/Akt linking to the NF-κB pathway in A549 cells.

Breakdown of the extracellular matrix (ECM) is accomplished by the concerted action of several proteases, including the urokinase plasminogen-activator (uPA) system and matrix metalloproteinases (MMPs), which is crucial for cancer invasion and metastasis. Several reports have shown that the levels of IL-1 beta and MMPs in plasma of the patients with lung cancer are significantly elevated and link to the invasion of tumor cells. Therefore, we investigated whether IL-1 beta-induced expression of uPA participated in lung cancer progression. In this study, IL-1 beta significantly induced uPA expression and activity via PKC alpha-dependent JNK1/2 and NIK cascades, linking to IKK alpha/beta activation, p65 translocation and transcription activity, using pharmacological inhibitors and transfection with dominant negative mutants and siRNAs. IL-1 beta-induced uPA protein and mRNA expression in a time- and concentration-dependent manner, which was inhibited by pretreatment with the inhibitors of JNK1/2 (SP600125), PKC (Ro31-8220, Go6976), or NF-kappaB (helenalin), and transfection with dominant negative mutants of PKC alpha, NIK, and IKK beta, and siRNAs of JNK1/2 and p65. IL-1 beta stimulated PKC alpha translocation to plasma membrane leading to phosphorylation of JNK1/2, which was attenuated by PKC inhibitors and transfection with shRNAs of JNK1/2, but not by helenalin. In addition, IL-1beta stimulated p65 phosphorylation and translocation into nucleus concomitant with I kappaB alpha phosphorylation and I kappaB alpha degradation, which was mediated via activation of PKC alpha-dependent JNK1/2-NIK/IKK beta cascade. These results demonstrated that in A549 cells, activation of p50/p65 heterodimer through sequential activation of PKC alpha-JNK-NIK-IKK beta-NF-kappaB was required for IL-1 beta-induced uPA expression associated with migration of tumor cells.

This paper presents new applications of the Web-based 3D visualization technology in collaborative product development. Three software prototypes are developed with the technology for non-designer to interact with product model without the need of CAD tools. The first system allows the end customer to configure individual components of 3D assembly with a regular Web browser. It provides a simple approach to collecting the customer’s voices. Next, an integration framework is implemented that enables synchronous communications between CAD and on the shelf software tool of 3D visualization. Peer-to-peer design collaboration is thus realized. Finally, this research proposes a computation mechanism for online modification of design features based on a STL model. A Web 3D catalog is developed for customization of real industrial parts using the proposed mechanism. The implementation results of these prototypes indicate that Web-based 3D visualization is an effective interfacing technology to facilitate 3D information sharing for most product-centric activities in a simple and cost-effective manner.

Bradykinin (BK) is released into the tear-film in ocular allergic patients. BK has been shown to exert mitogenic effects on several cell types. However, the mechanisms underlying its action on corneal keratocytes (CKs) were largely unknown. This study was to investigate the mitogenic effect of BK on rabbit CKs linked to activation of p42/p44 mitogen-activated protein kinase (MAPK), assessed by [3H]thymidine incorporation and Western blotting analysis, respectively. BK stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner. Pretreatment with pertussis toxin attenuated the BK-induced responses. BK-stimulated responses were attenuated by inhibitors of selective B2 receptor (Hoe 140), phosphatidylinositol (PI)-PLC (U73122), an intracellular Ca2+chelator (BAPTA/AM), PKC (GF109203X), tyrosine kinase (genistein), and MEK1/2 (PD98059). BK also stimulated translocation of p42/p44 MAPK into nucleus and led to expression of c-fos and c-jun in CKs. These results demonstrate that in CKs, BK-stimulated phosphorylation of p42/p44 MAPK is mediated through the activation of BK B2 receptors and leads to cell proliferation.

Freshwater (FW) teleosts are capable of acclimating to seawater (SW) following such a transfer from FW. However, their osmo-regulating mechanisms are still unclear, particularly those in the brain. The present study was conducted to examine acute changes that occur in brain Na+-K+-ATPase activity, creatine kinase (CK) activity, creatine, creatinine contents, and ATP levels of tilapia (Oreochromis mossambicus) in response to this transition. After transfer to SW (25 ppt), the Na+-K+-ATPase activity was maintained for 8 hr at higher levels than that in FW. In contrast, in 35 ppt SW, Na+-K+-ATPase was maintained at a even higher level than in FW for the first 2 hr. Brain Na+-K+-ATPase contents in both the 25 and 35 ppt SW groups were significantly elevated within 1 and 0.5 hr after transfer from FW, respectively. Interestingly, brain CK activities and content (homodimer of the B subunit [BB] form) in both the 25 and 35 ppt SW groups were significantly elevated within 1 hr after transfer from FW. The ATP contents in 35 ppt SW increased abruptly within 0.5 hr, and then gradually decreased during the next 2 hr. Unlike the 35 ppt group that declined in ATP contents, the 25 ppt group leveled off within 24 hr. The elevations in CK activity and creatine levels after transfer from FW to SW imply that abrupt salinity changes alter phosphocreatine/CK ratio. Such changes are needed to satisfy the increases in the energetic requirement of the cotransport mechanisms mediating osmoregulation

The importance of the Bcl-2 family proteins in normal vertebrate embryogenesis is being recognized; however, their regulatory mechanism is poorly understood. To elucidate the embryonic roles of Bcl-2 family proteins, we cloned and characterized the first zebrafish Bcl-2 family protein, zfMcl-1a. Zebrafish Mcl-1a shows the highest homology to rat Mcl-1 and contains several conserved BH domains of the Bcl-2 family proteins. It also contains a nuclear localization signal (NLS). Using EGFP reporter analysis, we verified the nuclear localization of zfMcl-1a. Deletion of the NLS resulted in distribution of the fusion protein in the cytoplasm. Northern blot analysis indicated that zfMcl-1a mRNA is 1.5 kb and was expressed in oocytes and throughout embryonic development. Notably, the expression of zfMcl-1a transcript was significantly downregulated during gastrulation. These results suggest that zfMcl-1a is a novel nuclear Bcl-2 family protein and is likely to play an important role in zebrafish oogenesis and embryogenesis.

PURPOSE: Matrix metalloproteases (MMPs)-mediated extracellular matrix (ECM) degradation potentially releases cryptic motility factors involved in somatic stem cell migration and epithelial outgrowth. The authors previously demonstrated that MMP-9 is upregulated in limbal epithelial cells cultivated on amniotic membrane (AM). Here, the authors further investigated whether plasminogen activator (PA)/plasmin regulates MMP-9 activity in this model implicated in the processing of laminin 5 (Ln5), a component of amniotic basement membrane. METHODS: Limbal epithelial cells migrated from limbal explants were expanded on intact AM. The activities and proteins of uPA and MMP-9 in limbal epithelial cells were determined by fibrin and gelatin zymography, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Specific pharmacological inhibitors including MMPs inhibitor GM6001, MMP-2/-9 inhibitor, and uPA inhibitor B428 were used to determine whether the PA/plasmin/MMP-9 axis induces cell growth via Ln5 in this model. RESULTS: These data showed that MMP-9 activity was attenuated by a selective uPA inhibitor, B428. Furthermore, MMP-9 activity was enhanced by exogenous addition or pre-incubation with plasmin. These results demonstrated that PA/plasmin regulates MMP-9 expression. An interesting proteolytic fragment of Ln5 gamma 2-chain was suppressed by pretreatment with GM6001, B428, or neutralizing antibodies of MMP-9 and uPA, indicating that Ln5 gamma 2-chain is processed by uPA/MMP-9. Moreover, the extent of limbal outgrowth was also retarded by B428. CONCLUSIONS: This study suggested that MMP-9 activity was upregulated by PA/plasmin, which in turn processed Ln5 gamma 2-chain to facilitate limbal outgrowth on intact AM.

PURPOSE To investigate the expression and pivotal role of matrix metalloproteinase (MMP)-9 in the ex vivo expansion of human limbal explants with or without amniotic membrane (AM). METHODS Corneoscleral buttons were cultured on intact, denuded AM or plastic dishes for 3 weeks. To determine the role of MMP-9 in cell migration, either the MMP inhibitor GM6001 or an MMP-9 antibody was used. Expression of MMP-9 was determined by gelatin zymography, reverse transcription-polymerase chain reaction, and immunohistochemical staining. RESULTS The expression of MMP-9 in all culture conditions increased in a time-dependent manner. However, the active form of MMP-9 emerged only in cultures on both intact and denuded AM from the second week. The averaged corrected ratio of MMP-9 expression in cultures on intact AM versus those on denuded AM or plastic dishes was 2.76 +/- 0.69- or 4.25 +/- 0.30-fold, respectively, when total RNA was used as an internal control. MMP-9 transcripts were upregulated in cultures on intact AM compared with the other two culture conditions. Immunohistochemical staining demonstrated that the MMP-9 protein was located on the limbal epithelial cells. Upregulation of MMP-9 associated with cell migration was significantly attenuated by both GM6001 and MMP-9 antibody, consistent with the inhibition of MMP-9 activity, as determined by gelatin zymography. In contrast, the sizes of limbal outgrowth were not different between the control and MMP-9 antibody-treated plastic dishes. CONCLUSIONS These results demonstrated that MMP-9 not only was upregulated, it was also involved in the outgrowth of limbal epithelial cells. These results suggest that cell-cell matrix interaction is involved in the expansion of limbal epithelial cells on intact AM, and MMP-9 may be a key element.