Methyl jasmonate inhibits lipopolysaccharide-induced inflammatory cytokine production via mitogen-activated protein kinase and nuclear factor-κB pathways in RAW 264.7 cells.

Methyl jasmonate is an important signaling molecule involved in plant defense as well as in the regulation of plant growth and development. Despite its various functions in plants, its effects on animal cells have not been widely studied and no report has been issued on the molecular aspects of its anti-inflammatory effect. In the present study, we investigated the in vitro anti-inflammatory properties of methyl jasmonate in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Methyl jasmonate treatment effectively inhibited LPS-induced production of pro-inflammatory mediators (nitric oxide and prostaglandin E2) and cytokines (tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6) in a concentration-dependent manner. Furthermore, it attenuated the LPS-induced activation of nuclear factor-κB (NF-κB) by suppressing the degradation of the inhibitor of κB-α (IκB-α). Additionally, methyl jasmonate dose-dependently blocked the phosphorylation of mitogen-activated protein kinases (MAPKs), i.e., p38 kinase, extracellular signal-regulated kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK), in these cells. These results suggest that methyl jasmonate attenuated the LPS-induced release of pro-inflammatory mediators and cytokines by suppressing the activation of MAPK (JNK, ERK and p38) and NF-κB signaling. This study not only demonstrated that methyl jasmonate exerts anti-inflammatory activities in macrophages but also revealed its potential as a candidate for the treatment of various inflammation-associated diseases.

Potent inhibition by ropivacaine of metastatic colon cancer SW620 cell invasion and NaV1.5 channel function.

BACKGROUND: Metastatic breast and colon cancer cells express neonatal and adult splice variants of NaV1.5 voltage-activated Na(+) channels (VASCs). Block of VASCs inhibits cell invasion. Local anaesthetics used during surgical tumour excision inhibit VASC activity on nociceptive neurones providing regional anaesthesia. Inhibition of VASCs on circulating metastatic cancer cells may also be beneficial during the perioperative period. However, ropivacaine, frequently used to provide analgesia during tumour resection, has not been tested on colon cancer cell VASC function or invasion. METHODS: We used reverse transcription-polymerase chain reaction and sequencing to identify NaV1.5 variants in the SW620 metastatic colon cancer cell line. Recombinant adult and neonatal NaV1.5 variants were expressed in human embryonic kidney cells. Voltage-clamp recordings and invasion assays were used to examine the effects of ropivacaine on recombinant NaV1.5 channels and the metastatic potential of SW620 cells, respectively. RESULTS: SW620 cells expressed adult and neonatal NaV1.5 variants, which had similar steady-state inactivation profiles, but distinctive activation curves with the neonatal variant having a V1/2 of activation 7.8 mV more depolarized than the adult variant. Ropivacaine caused a concentration-dependent block of both NaV1.5 variants, with IC50 values of 2.5 and 3.9 µM, respectively. However, the reduction in available steady-state current was selective for neonatal NaV1.5 channels. Ropivacaine inhibited SW620 invasion, with a potency similar to that of inhibition of NaV1.5 channels (3.8 µM). CONCLUSIONS: Ropivacaine is a potent inhibitor of both NaV1.5 channel activity and metastatic colon cancer cell invasion, which may be beneficial during surgical colon cancer excision.

Anti-inflammatory effect of pachymic acid promotes odontoblastic differentiation via HO-1 in dental pulp cells.

OBJECTIVES: Heme oxygenase-1 (HO-1) is contributed to odontoblast differentiation in human dental pulp cells (HDPCs). In this study, pachymic acid from mushroom Formitopsis niagra is examined to determine whether it affects pulpal inflammation and promotes odontogenesis via HO-1 gene expression. MATERIALS AND METHODS: The HDPCs were given H2O2 for inflammation. The anti-inflammatory character and odontoblast differentiation by pachymic acid were analyzed by Western blotting, alkaline phosphatase activity, and alizarin red S staining. To understand the mechanism of pachymic acid via HO-1 induction, the cells were treated with zinc protoporphyrin IX (ZnPP: HO-1 inhibitor). RESULTS: H2O2 induced pulp inflammation and disturbed odontoblast differentiation. However, the HDPCs treated with pachymic acid affected anti-inflammatory effect and induction of odontoblast differentiation through increasing HO-1 expression. In addition, pachymic acid has potent cytoprotection and mineralization under H2O2 treatment. Furthermore, pachymic acid significantly suppressed nuclear factor-kappa B (NF-κB) translocation into nucleus and induced NE-E2-related factor-2 (Nrf2) translocation into nucleus. Overall, NF-κB and Nrf2 translocation were regulated by the HO-1 pathway. CONCLUSIONS: The pachymic acid showed anti-inflammatory function and odontoblast differentiation via HO-1 pathway. These results suggested that pachymic acid may be applicable for prevention of oral inflammation or to improve dentin mineralization against several stresses.

Association between non-vascularised bone graft failure and compartment of the defect in mandibular reconstruction: a systematic review and meta-analysis.

Controversy exists regarding the influence of the graft placement site in the mandible on the success of non-vascularised bone grafts. In this study, we examine the association between the compartment of the mandibular defect and the bone graft failure rate. A systematic literature review and meta-analysis was performed using MEDLINE, Embase, and Cochrane databases. Failure rates according to the compartment of mandibular defect were extracted and analysed by meta-analysis. The Newcastle-Ottawa Scale was used to assess the quality of the studies, and publication bias was evaluated using funnel plots. The search strategy identified 27 publications. After screening, five were selected for review. Based on the result of comparison among these five, we found no significant statistical association between the bone graft failure rate and compartment of mandibular defect, although further investigation of prospective randomised cohort studies is required.

Effect of epidural saline washout on regression of sensory and motor block after epidural anaesthesia with 2% lidocaine and fentanyl in elderly patients.

Seventy elderly males received lumbar epidural anaesthesia with 12 ml of 2% lidocaine containing fentanyl 50 mug. At the end of transurethral surgery, the washout group (n = 33) received an epidural bolus of 30 ml saline while the control group (n = 34) did not. Mean (SD) times to 1-grade (17.2 (11.9) vs 32.7 (11.3) min) and 2-grade regression (23.8 (12.2) vs 56.0 (23.9) min) of motor block, 3-dermatomal sensory regression (31.4 (11.6) vs 42.2 (14.4) min for cold and 30.8 (15.6) vs 40.6 (14.2) min for pinprick), and regression to S1 (57.7 (16.1) vs 76.2 (20.2) min for cold and 56.8 (17.3) vs 69.2 (16.2) min for pinprick) were significantly shorter in the washout group than the control group. There were no differences in postoperative pain scores and side effects between the two groups. We concluded that epidural washout facilitates regression of both motor and sensory block following epidural anaesthesia without reducing the postoperative analgesic benefit.

Non-tuberculous Mycobacterium infection after transfer of autologous fat to the face: a rare case.

Autologous fat has long been used as a filler in the face, and has recently gained popularity in plastic surgery with a wound infection rate of 1% - 5%. The incidence of mycobacterial infections has increased over recent decades, which is attributed in part to the increased popularity of these procedures.2 Infections by non-tuberculosis mycobacteria often cause chronic inflammation and progressive infection that may eventually manifest themselves as severe scars, fistulas, and hollows, and irregular facial contours. However, few cases of mycobacterial infection have been reported to have been caused by plastic surgery. We present a rare case of non-tuberculosis mycobacterial infection after transfer of autologous fat to the face.

Virus- and interferon-induced loss of inhibitory M2 muscarinic receptor function and gene expression in cultured airway parasympathetic neurons.

Viral infections increase vagally mediated reflex bronchoconstriction. Decreased function of inhibitory M2 muscarinic receptors on the parasympathetic nerve endings is likely to contribute to increased acetylcholine release. In this study, we used cultured airway parasympathetic neurons to determine the effects of parainfluenza virus and of interferon (IFN)-gamma on acetylcholine release, inhibitory M2 receptor function, and M2 receptor gene expression. In control cultures, electrically stimulated acetylcholine release increased when the inhibitory M2 receptors were blocked using atropine (10(-)5 M) and decreased when these receptors were stimulated using methacholine (10(-)5 M). Acetylcholine release was increased by viral infection and by treatment with IFN-gamma (300 U/ml). In these cells, atropine did not further potentiate, nor did methacholine inhibit, acetylcholine release, suggesting decreased inhibitory M2 receptor function and/or expression. Using a competitive reverse transcription-polymerase chain reaction method, we demonstrated that M2 receptor gene expression was decreased by more that an order of magnitude both by virus infection and by treatment with IFN. Thus, viral infections may increase vagally mediated bronchoconstriction both by directly inhibiting M2 receptor gene expression and by causing release of IFN-gamma which inhibits M2 receptor gene expression.

Silencing of Wnt signaling and activation of multiple metabolic pathways in response to thyroid hormone-stimulated cell proliferation.

To investigate the transcriptional program underlying thyroid hormone (T3)-induced cell proliferation, cDNA microarrays were used to survey the temporal expression profiles of 4,400 genes. Of 358 responsive genes identified, 88% had not previously been reported to be transcriptionally or functionally modulated by T3. Partitioning the genes into functional classes revealed the activation of multiple pathways, including glucose metabolism, biosynthesis, transcriptional regulation, protein degradation, and detoxification in T3-induced cell proliferation. Clustering the genes by temporal expression patterns provided further insight into the dynamics of T3 response pathways. Of particular significance was the finding that T3 rapidly repressed the expression of key regulators of the Wnt signaling pathway and suppressed the transcriptional downstream elements of the beta-catenin-T-cell factor complex. This was confirmed biochemically, as beta-catenin protein levels also decreased, leading to a decrease in the transcriptional activity of a beta-catenin-responsive promoter. These results indicate that T3-induced cell proliferation is accompanied by a complex coordinated transcriptional reprogramming of many genes in different pathways and that early silencing of the Wnt pathway may be critical to this event.

Differential expression patterns of IRF3 and IRF7 in pediatric lymphoid disorders.

Interferon regulatory factors (IRFs) are multifunctional transcriptional factors. To define the role of IRFs in lymphoid disorders, we determined the expression patterns of IRF3 and IRF7 by immunohistochemistry in 5 normal lymph nodes, 12 reactive hyperplastic lymph nodes, and 27 pediatric lymphomas. IRF3 was prominently expressed in the nuclei of the histiocytes, and was expressed very weakly in the cytoplasm of most of the lymphocytes of the normal lymph nodes. However, IRF7 was expressed strongly in the nuclei of over 50% of the lymphocytes throughout the normal lymph nodes, but the histiocytes and fibroblasts were spared. In the reactive hyperplastic lymph nodes, the number of IRF3- and IRF7- positive cells in the nuclei was elevated. In the lymphomas, the number of IRF3-positive cells in the nucleus appeared to have decreased, and the cells were scattered throughout the lymphoma tissue in no specific pattern. However, in most cases the number of IRF7-positive cells was elevated. These results suggested that IRF3 was activated principally in the histiocytes and T cells under inflammatory conditions, but IRF3 activation was attenuated in cases of lymphoma. However, the number of IRF7-positive cells was found to be elevated in the reactive hyperplastic lymph nodes and pediatric lymphoma.

Crossover clinical trial to determine the effect of manual acupuncture at Siguan points (bilateral LI4 and LR3) on intestinal motility in healthy subjects.

This study examined whether manual acupuncture at the Siguan points (bilateral points LI4 and LR3) affects intestinal motility in healthy human subjects. Twenty healthy male subjects were randomly assigned either to real acupuncture (RA) at Siguan points or sham acupuncture (SA) groups in a crossover manner. All subjects underwent two experimental sessions; the RA group in the first session was treated with SA in the second session after a 2-week washout period, and vice versa. Each subject took 20 radio-markers and was treated with acupuncture 0, 12, 24, and 36 hours after radio-marker intake. Radiographs were taken at 6, 12.5, 24.5, and 48 hours, and the effect of acupuncture on intestinal motility was evaluated based on the distribution of the radio-markers in the ileum, ascending colon, transverse colon, descending colon, sigmoid/ rectum, and outside the body. Defecating habit was monitored during the trial, and complete blood counts were checked before and after the two acupuncture sessions. The RA and SA results showed extremely similar distributions of the radio-markers in these five regions of the alimentary canal and outside the body in radiographs taken at four different times, verifying that there was no effect of manual acupuncture at the Siguan points on intestinal motility, at least in healthy human subjects.

Total antioxidant status and oxygen free radicals during hepatic regeneration.

OBJECTIVES: The damage induced by oxygen free radicals (OFRs) is caused by an imbalance of the production of versus the antioxidant defenses against OFRs. METHODS: To understand hepatic damage induced by oxygen free radicals after hepatectomy in rats, total antioxidant status and total production of oxygen free radicals were serially measured in regeneration liver. At 1, 2, 3, 7, and 10 days after hepatectomy of Sprague-Dawley rats, blood was obtained into a capillary tube from a tail vein. Total antioxidant status and total production of oxygen free radicals were measured using the Randox kit, a colorimetric method, and the Free Radical Analytical System. We also measured the amount of malonyldialdehyde, which provides an indirect index of oxidative injury. RESULTS: The level of malonyldialdehyde after hepatectomy was higher compared with that before hepatectomy. The level of total oxygen free radicals after hepatectomy was higher compared with that before hepatectomy. Total antioxidant status after hepatectomy was lower compared with that before hepatectomy. CONCLUSIONS: The results suggested that the damage by OFRs to the regenerating liver was caused by increased production of OFRs and decreased antioxidant defense against OFRs.

Identification of c-myc responsive genes using rat cDNA microarray.

c-Myc functions through direct activation or repression of transcription. Using cDNA microarray analysis, we have identified c-Myc-responsive genes by comparing gene expression profiles between c-myc null and c-myc wild-type rat fibroblast cells and between c-myc null and c-myc null cells reconstituted with c-myc. From a panel of 4400 cDNA elements, we found 198 genes responsive to c-myc when comparing wild-type or reconstituted cells with the null cells. The plurality of the named c-Myc-responsive genes that were up-regulated, including 30 ribosomal protein genes, are involved in macromolecular synthesis and metabolism, suggesting a major role of c-Myc in the regulation of protein synthetic and metabolic pathways. When ectopically overexpressed, c-Myc induced a different and smaller set of c-Myc-responsive genes as compared with the physiologically expressed c-Myc condition. Thus, these results from expression profiling suggest a new primary function for c-Myc and raise the possibility that the physiological and transforming functions of c-myc may be separable.

Identification of tumor markers in models of human colorectal cancer using a 19,200-element complementary DNA microarray.

Metastasis represents a crucial transition in disease development and progression and has a profound impact on survival for a wide variety of cancers. Cell line models of metastasis have played an important role in developing our understanding of the metastatic process. We used a 19,200-element human cDNA microarray to profile transcription in three paired cell-line models of colorectal tumor metastasis. By correlating expression patterns across these cell lines, we have identified 176 genes that appear to be differentially expressed (greater than 2-fold) in all highly metastatic cell lines relative to their reference. An analysis of these genes reiterates much of our understanding of the metastatic process and suggests additional genes, many of previously uncharacterized function, that may be causatively involved in, or at least prognostic of, metastasis. Northern analysis of a limited number of these genes validates the observed pattern of expression and suggests that further investigation and functional characterization of the identified genes is warranted.

Photoprotective Effect of Carpomitra costata Extract against Ultraviolet B-Induced Oxidative Damage in Human Keratinocytes.

Natural marine products show various biological properties such as antiphotoaging, antioxidant, anticancer, and anti-inflammation. This study evaluated the protective effects of the brown alga Carpomitra costata (Stackhouse) Batters (Sporochnaceae) against ultraviolet B (UVB)-provoked damage in human HaCaT keratinocytes. C. costata extract (CCE) effectively reduced superoxide anion, hydroxyl radical, and UVB-stimulated intracellular reactive oxygen species (ROS) levels. CCE also restored the expression and activity of UVB-suppressed antioxidant enzymes. Furthermore, CCE decreased UVB-triggered oxidative damage to cellular components including DNA, protein, and lipid and defended the cells against mitochondrial membrane depolarization-medicated apoptosis. The results of this study indicate that CCE can safeguard human keratinocytes against UVB-induced cellular damage via a potent antioxidant mechanism. CCE may find utility as part of a therapeutic arsenal against the damaging effects of UVB radiation on the skin.

Heterogeneity of chondrocyte mitochondria. A study of the Ca2+ concentration and density banding characteristics of normal and rachitic cartilage.

Previous morphological studies of the mineralizing epiphysis suggested that some mitochondria were concerned with Ca2+ accumulation while others were associated with cellular energetics and metabolism. To determine if there was mitochondrial heterogeneity in chondrocytes of the epiphyseal growth plate, mitochondria were isolated from four different regions of the plate and subjected to continuous sucrose gradient centrifugation. Centrifugation of the organelles in a narrow density sucrose gradient (1.5--2.0 M) in the presence of inhibitors of Ca2+ transport (ruthenium red and 5,5'-dithiobis-(2-nitrobenzoic acid)) revealed that considerable heterogeneity existed. In the least calcified zone 20% of the mitochondria formed a low density band of low Ca2+ concentration (309 nmol/mg protein). Organelles isolated from more calcified tissue zones showed a concomitant increase in Ca2+ concentration (up to 5700 nmol/mg protein) as well as an increase in the total percentage of mitochondria sedimenting in 2.0 M sucrose. The banding patterns of mitochondria isolated from rachitic and hypertrophic cartilage were similar. In addition, similarities were also noted in the Ca2+ concentration and the cytochrome oxidase activities of mitochondria of these tissues. During recovery from the rachitic condition, there was a change in the density centrifugation characteristics of this tissue and a substantial increase was noted in the proportion of mitochondria sedimenting in 2.0 M sucrose. The Ca2+ concentration of mitochondria of this rapidly calcifying tissue suggested that the critical Ca2+ concentration necessary for initiation of the calcification mechanism was 4 mumol/mg protein.

cDNA sequencing: a means of understanding cellular physiology.

High-throughput automated sequencing has enabled researchers to examine large numbers of clones from a cDNA library as a measure of the steady-state levels of mRNA species. The past year has witnessed many new applications of this technique to allow the qualitative and quantitative comparison of the changes in transcript levels from multiple genes.

Genome-wide differentially methylated genes in prostate cancer tissues from African-American and Caucasian men.

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the ß-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between ß-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2'-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.

PPARγ Maintains Homeostasis through Autophagy Regulation in Dental Pulp.

This study investigated the relevance between pulp vitality and autophagy in aged human dental pulp cells (HDPCs) and whether peroxisome proliferator-activated receptor gamma (PPARγ) affects autophagy regulation for homeostasis in the aging progress. In vivo experiments were used in human and Sprague-Dawley rat teeth obtained from young and adult individuals. Aging- and autophagy-related molecules were determined by immunohistochemistry and hematoxylin and eosin staining. HDPCs were serially subcultured until spontaneously arrested for in vitro aging, and the replication deficiency adenovirus was introduced for PPARγ overexpression. Subsequently, the effect of PPARγ on regulation of autophagy molecules, mitochondria activity, and cell viability was assessed using Western blotting, confocal microscopy, and the MTT assay, respectively. In adult pulp tissue, autophagy molecules (autophagy protein 5, microtubule-associated protein 1A/1B light chain, and Beclin-1) were increased, but aging-related (PPARγ and heme oxygenase 1 [HO-1]) and dentinogenesis (dentin sialophosphoprotein and dentin matrix acidic phosphoprotein) molecules were decreased. In aged HDPCs, autophagy and intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 were increased, while PPARγ and HO-1 were decreased. Under stimulation with lipopolysaccharide, autophagy- and aging-related molecules were differentially expressed between young and aged cells. PPARγ induced HO-1 and autophagy molecules but reduced inflammatory molecules in aged cells. In addition, PPARγ activated strong mitochondrial activity and cell viability in aging cells. Inhibition of HO-1 by tin protoporphyrin IX exacerbated autophagy and mitochondrial activity as well as cell viability in young cells. This study indicates that PPARγ maintains pulp homeostasis through the regulation of autophagy molecules during the life span of HDPCs.

Microarray analysis of the in vivo effects of hypophysectomy and growth hormone treatment on gene expression in the rat.

Complementary DNA microarrays containing 3000 different rat genes were used to study the consequences of severe hormonal deficiency (hypophysectomy) on the gene expression patterns in heart, liver, and kidney. Hybridization signals were seen from a majority of the arrayed complementary DNAs; nonetheless, tissue-specific expression patterns could be delineated. Hypophysectomy affected the expression of genes involved in a variety of cellular functions. Between 16-29% of the detected transcripts from each tissue changed expression level as a reaction to this condition. Chronic treatment of hypophysectomized animals with human GH also caused significant changes in gene expression patterns. The study confirms previous knowledge concerning certain gene expression changes in the above-mentioned situations and provides new information regarding hypophysectomy and chronic human GH effects in the rat. Furthermore, we have identified several new genes that respond to GH treatment. Our results represent a first step toward a more global understanding of gene expression changes in states of hormonal deficiency.

Effects of cilostazol on angiographic restenosis after coronary stent placement.

This study evaluates the impact of cilostazol on post-stenting restenosis. Cilostazol is a potent antiplatelet agent with antiproliferative properties. Few data are available about the effect of cilostazol on poststenting restenosis. Four hundred nine patients (494 lesions) who were scheduled for elective stenting were randomized to receive aspirin plus ticlopidine (group I, n = 201, 240 lesions) or aspirin plus cilostazol (group II, n = 208, 254 lesions), starting 2 days before stenting. Ticlopidine was given for 1 month and cilostazol for 6 months. Follow-up angiography was performed at 6 months, and clinical evaluation at regular intervals. Baseline characteristics were similar between the 2 groups. The procedural success rate was 99.6% in group I and 100% in group II. There were no cases of stent thrombosis after stenting. Angiographic follow-up was performed in 380 of the 494 eligible lesions and the angiographic restenosis rate was 27% in group I and 22.9% in group II (p = NS). However, diffuse type in-stent restenosis was more common in group I than in group II (54.2% vs 26.8%, respectively, p <0.05). In diabetic patients, the angiographic restenosis rate was 50% in group I and 21.7% in group II (p <0.05). Clinical events during follow-up did not differ between the 2 groups. In conclusion, aspirin plus cilostazol seems to be an effective antithrombotic regimen with comparable results to aspirin plus ticlopidine, but it does not reduce the overall angiographic restenosis rate after elective coronary stenting.