Ginsenoside Rb1 from Panax notoginseng Suppressed TNF-α-Induced Matrix Metalloproteinase-9 via the Suppression of Double-Strand RNA-Dependent Protein Kinase (PKR)/NF-κB Pathway.

Chronic inflammation is commonly accompanied by the stimulation of matrix metalloproteinases (MMPs) production and the degradation of the extracellular matrix. The overexpression of MMP-9 (Gelatinase B) highly participates in the progression of pathetic cardiac remodeling and liver cancer metastasis. Panax notoginseng (Burkill) F. H. Chen (Sanqi), a widely used traditional Chinese medicinal herb, shows myocardial protective and anti-tumor effects. In this study, we examined the inhibitory effect of different PNG extracts on tumor necrosis factor (TNF)-α-induced MMP-9 expression in cardiac myoblast H9c2 cells. Using a bioassay-guided fractionation scheme, the most active extract was fractionated by silica gel column chromatography and high-performance liquid chromatography until an active compound was obtained. The compound was identified as Ginsenoside Rb1 by nuclear magnetic resonance. Ginsenoside Rb1 inhibited TNF-α-induced MMP-9 production in both H9c2 and liver carcinoma HepG-2 cells. Interestingly, it did not affect the MMP-2 (Gelatinase A) level and the cell proliferation of the two cell lines. The inhibitory effects of Ginsenoside Rb1 may be due to its modulation of double-strand RNA-dependent protein kinase and nuclear factor kappa B signaling pathways. The results reveal the potential use of Ginsenoside Rb1 for the treatment of inflammatory and MMP-9-related cardiac remodeling and metastasis of hepatocellular carcinomas.

Impact of the Novel Z-Acceptor Ligand Bis{(ortho-diphenylphosphino)phenyl}zinc (ZnPhos) on the Formation and Reactivity of Low-Coordinate Ru(0) Centers.

The preparation and reactivity with H2 of two Ru complexes of the novel ZnPhos ligand (ZnPhos = Zn(o-C6H4PPh2)2) are described. Ru(ZnPhos)(CO)3 (2) and Ru(ZnPhos)(IMe4)2 (4; IMe4 = 1,3,4,5-tetramethylimidazol-2-ylidene) are formed directly from the reaction of Ru(PPh3)(C6H4PPh2)2(ZnMe)2 (1) or Ru(PPh3)3HCl/LiCH2TMS/ZnMe2 with CO and IMe4, respectively. Structural and electronic structure analyses characterize both 2 and 4 as Ru(0) species in which Ru donates to the Z-type Zn center of the ZnPhos ligand; in 2, Ru adopts an octahedral coordination, while 4 displays square-pyramidal coordination with Zn in the axial position. Under photolytic conditions, 2 loses CO to give Ru(ZnPhos)(CO)2 that then adds H2 over the Ru-Zn bond to form Ru(ZnPhos)(CO)2(µ-H)2 (3). In contrast, 4 reacts directly with H2 to set up an equilibrium with Ru(ZnPhos)(IMe4)2H2 (5), the product of oxidative addition at the Ru center. DFT calculations rationalize these different outcomes in terms of the energies of the square-pyramidal Ru(ZnPhos)L2 intermediates in which Zn sits in a basal site: for L = CO, this is readily accessed and allows H2 to add across the Ru-Zn bond, but for L = IMe4, this species is kinetically inaccessible and reaction can only occur at the Ru center. This difference is related to the strong π-acceptor ability of CO compared to IMe4. Steric effects associated with the larger IMe4 ligands are not significant. Species 4 can be considered as a Ru(0)L4 species that is stabilized by the Ru→Zn interaction. As such, it is a rare example of a stable Ru(0)L4 species devoid of strong π-acceptor ligands.

Systemic effects of gut microbiota and its relationship with disease and modulation.

The gut microbiota makes up the majority of the human bacterial population, and although the gut microbiota resides in the intestines, it is able to exert systemic effects. Therefore, many diseases and conditions could be impacted by the gut microbiota when its composition is imbalanced, otherwise known as dysbiosis. However, apart from understanding the illnesses, we must also try to understand the intestinal flora itself to move forward and develop potential treatments.

A role for c-Myc in regulating anti-mycobacterial responses.

c-Myc (Myc) is a well known transcription factor that regulates many essential cellular processes; however, its role in modulating immunity is not known. Here, we showed different species of mycobacteria can induce Myc expression via ERK1/2 and JNK activation. Unexpectedly, the induced Myc is localized in the cytoplasm but not in the nucleus. This induced Myc expression is associated with the induction of TNF-α and IL-6 and with the suppression of intracellular mycobacterial growth. To delineate the underlying mechanisms, we demonstrated that Myc enhances IRAK1 degradation, leading to specific activations of ERK1/2 and p38 MAPK but not Akt, and reduces IκBα protein recovery upon degradation. Hence, our findings may provide insights into a potential role for Myc in regulating the antimicrobial responses.

A role for interleukin-17A in modulating intracellular survival of Mycobacterium bovis bacillus Calmette-Guérin in murine macrophages.

Interleukin 17A IL-17A is a crucial immunomodulator in various chronic immunological diseases including rheumatoid arthritis and inflammatory bowel disease. The cytokine has also been demonstrated to control the pathogenesis of the Mycobacterium tuberculosis by dysregulating production of cytokines and chemokines and promoting granuloma formation. Whether IL-17A regulates innate defence mechanisms of macrophages in response to mycobacterial infection remains to be elucidated. In the current report, we investigated the effects of IL-17A on modulating the intracellular survival of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in RAW264.7 murine macrophages. We observed that IL-17A pre-treatment for 24 hr was able to synergistically enhance BCG-induced nitric oxide (NO) production and inducible nitric oxide synthase expression in dose- and time-dependent manners. We further delineated the mechanisms involved in this synergistic reaction. IL-17A was found to specifically enhanced BCG-induced phosphorylation of Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. By using a specific JNK inhibitor (SP600125), we found that the production of NO in BCG-infected macrophages was significantly suppressed. Taken together, we confirmed the involvement of the JNK pathway in IL-17A-enhanced NO production in BCG-infected macrophages. We further demonstrated that IL-17A significantly enhanced the clearance of intracellular BCG by macrophages through an NO-dependent killing mechanism. In conclusion, our study revealed an anti-mycobacterial role of IL-17A through priming the macrophages to produce NO in response to mycobacterial infection.

HIV-1 transactivator protein induction of suppressor of cytokine signaling-2 contributes to dysregulation of IFN{gamma} signaling.

HIV infection remains a worldwide threat. HIV-1 transactivator protein Tat is one of the retroviral proteins identified as a key immunomodulator in AIDS pathogenesis. Although the primary function of Tat is to regulate HIV-1 replication in the infected cell, it also dysregulates cytokine production resulting in perturbation of the host immune response and enhancement of the retrovirus survival. Because interferon-gamma (IFNgamma) is a pleiotropic cytokine with potent antiviral and immunoregulatory effects, we investigated whether Tat interferes with the IFNgamma signal transduction in primary monocytes. We demonstrated that Tat impaired the IFNgamma-receptor signaling pathway at the level of STAT1 activation, possibly via Tat-dependent induction of suppressor of cytokine signaling-2 (SOCS-2) activity. We delineated the inhibitory role of SOCS-2 in IFNgamma signaling pathway by overexpression of exogenous SOCS-2 in HEK293 cell. The results showed that SOCS-2 suppressed the IFNgamma-activated STAT1 phosphorylation and consequent IFNgamma-regulated transcription of specific genes. To confirm the role of SOCS2 in the Tat-induced process, we demonstrated that SOCS-2 siRNA in human blood monocytes abrogated the Tat-dependent inhibition of IFNgamma signaling. Our data suggested a possible mechanism implicating the role of SOCS-2 in mediating HIV-1-induced immune evasion and dysregulation of IFNgamma signaling in primary human monocytes.

MXD1 regulates the H9N2 and H1N1 influenza A virus-induced chemokine expression and their replications in human macrophage.

Human infection with influenza A/Hong Kong/156/97 (H5N1) avian influenza virus is associated with a high mortality rate of 60%. This virus is originated from influenza A/Quail/Hong Kong/G1/97 (H9N2/G1) avian influenza virus. Since the 1990s, four lineages of H9N2 viruses have been circulating in poultry and cause occasional infection in humans in different countries. Due to its zoonotic and genetic reassortment potential, H9N2/G1 and H5N1 viruses are believed to be the next pandemic candidates. Previous reports, including ours, showed that the virulence of avian virus strains correlates with their ability to dysregulate cytokine expression, including TNF-α, CXCL10, and related chemokines in the virus-infected cells. However, the transcriptional factors required for this cytokine dysregulation remains undefined. In light of our previous report showing the unconventional role of MYC, an onco-transcriptional factor, for regulating the antibacterial responses, we hypothesize that the influenza virus-induced cytokine productions may be governed by MYC/MAX/MXD1 network members. Here, we demonstrated that the influenza A/Hong Kong/54/98 (H1N1)- or H9N2/G1 virus-induced CXCL10 expressions can be significantly attenuated by knocking down the MXD1 expression in primary human blood macrophages. Indeed, only the MXD1 expression was up-regulated by both H1N1 and H9N2/G1 viruses, but not other MYC/MAX/MXD1 members. The MXD1 expression and the CXCL10 hyperinduction were dependent on MEK1/2 activation. By using EMSAs, we revealed that MXD1 directly binds to the CXCL10 promoter-derived oligonucleotides upon infection of both viruses. Furthermore, silencing of MXD1 decreased the replication of H9N2 but not H1N1 viruses. Our results provide a new insight into the role of MXD1 for the pathogenicity of avian influenza viruses.

The functional role of surface molecules on extracellular vesicles in cancer, autoimmune diseases, and coagulopathy.

Extracellular vesicles (EVs) are nanosized particles that have emerged as mediators for intercellular communication in physiologic and pathologic conditions. EVs carry signaling information on their bilipid membrane as well as cargo within, allowing them to perform a wide range of biologic processes and contribute to pathophysiologic roles in a wide range of diseases, including cancer, autoimmune diseases and coagulopathy. This review will specifically address the function of surface molecules on EVs under normal and diseased conditions, as well as their potential to emerge as therapeutic targets in clinical settings, and the importance of further research on the surface topography of EVs.

Bioactivity-guided identification and cell signaling technology to delineate the immunomodulatory effects of Panax ginseng on human promonocytic U937 cells.

BACKGROUND: Ginseng is believed to have beneficial effects against human diseases, and its active components, ginsenosides, may play critical roles in its diverse physiological actions. However, the mechanisms underlying ginseng's effects remain to be investigated. We hypothesize some biological effects of ginseng are due to its anti-inflammatory effects. METHODS: Human promonocytic U937 cells were used to investigate the immunomodulatory effects of ginseng following TNF-alpha treatment. A global gene expression profile was obtained by using genechip analysis, and specific cytokine expression was measured by quantitative RT-PCR and ELISA. HPLC was used to define the composition of ginsenosides in 70% ethanol-water extracts of ginseng. Activation of signalling kinases was examined by Western blot analysis. RESULTS: Seventy percent ethanol-water extracts of ginseng significantly inhibited the transcription and secretion of CXCL-10 following TNF-alpha stimulation. Nine ginsenosides including Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3 and Rh1 were identified in our extract by HPLC. Seven out of nine ginsenosides could significantly inhibit TNF-alpha-induced CXCL-10 expression in U937 cells and give comparable inhibition of CXCL-10 transcription to those with the extract. However, the CXCL-10 suppressive effect of individual ginsenosides was less than that of the crude extract or the mixture of ginsenosides. The CXCL-10 suppression can be correlated with the inactivation of ERK1/2 pathways by ginseng. CONCLUSION: We showed ginseng suppressed part of the TNF-alpha-inducible cytokines and signalling proteins in promonocytic cells, suggesting that it exerts its anti-inflammatory property targeting at different levels of TNF-alpha activity. The anti-inflammatory role of ginseng may be due to the combined effects of ginsenosides, contributing in part to the diverse actions of ginseng in humans.

MiR-1303 Regulates Mycobacteria Induced Autophagy by Targeting Atg2B.

MicroRNAs are emerging post-transcriptional regulators of gene expressions in both innate immunity and adaptive immunity. In mycobacteria infection, autophagy plays an important role in innate defense mechanism and is tightly regulated by the autophagy-related proteins. Here, we show that Atg2B is involved in the regulation of mycobacteria-induced autophagy. MiR-1303, which function is not defined yet, is found to negatively regulate mycobacteria-induced Atg2B protein production, ultimately down-regulate mycobacteria-induced autophagy. MiR-1303 production is shown to be upregulated during BCG infection and its production is regulated by PI3K and NFκB. It is also demonstrated that miR-1303 targets putative target sites on Atg2B and possibly represses its translation.

Mechanisms for HIV Tat upregulation of IL-10 and other cytokine expression: kinase signaling and PKR-mediated immune response.

HIV Tat has been known to have multiple regulatory roles including replication of HIV and modulation of cellular kinases. We investigated whether signaling kinase PKR plays a critical role in mediating Tat-induced cytokine dysregulation. We showed Tat induction of IL-10 dysregulation is associated with PKR activation. To examine the mechanism involved, inhibition of PKR activity abrogated the Tat-induced cytokine induction. We next identified that the MAP kinases including ERK-1/2 and p38 are downstream of PKR in these Tat-induced pathways. Thus, PKR may play a critical role in mediating the subversive effects of HIV Tat resulting in IL-10 induction.

Protein kinase C inhibits transcription from the RNA polymerase III promoter of the human c-myc gene.

The c-myc promoter has a unique characteristic showing both RNA polymerase II (pol II) and RNA polymerase III (pol III) activities. Previous studies demonstrated that activating PKC results in upregulation of c-myc expression from its pol II promoter. However, how PKC activation affects expression from the pol III promoter of the c-myc gene is not well understood. This study examines the effect of PKC on the pol III transcription from the c-myc gene by using an in vitro system. We report the inhibition of the c-myc pol III transcript by activating PKC. Further, either a phosphocellulose fraction of HeLa whole cell extract (WCE) enriched for transcription factor TF IIIB, or recombinant TATA-box binding protein could restore the inhibited c-myc pol III transcription under conditions that activate PKC. A role has been proposed for the c-myc pol III transcript in the regulation of c-myc gene expression. Therefore, this report discusses the significance of the downregulation of c-myc expression from its pol III promoter and the possible interplay between the pol II and pol III promoters of this gene.

Elevation of internal 6-methyladenine mRNA methyltransferase activity after cellular transformation.

A comparison of internal 6-methyladenine mRNA methyltransferase activity in a variety of cell types demonstrated an 8-15-fold increase as a result of cellular transformation. Utilizing adenovirus transformed rat embryo cells, it was found that the increase in methyltransferase activity was concomitant with or occurred rapidly after transformation. An 80-fold increase in activity was observed in the cells isolated from the transformed foci and remained elevated through subsequent passages. The relationship between methyltransferase activity and tumor formation was also investigated. High level expression of the avian ski oncogene in mouse L cells causes a reversion of the transformed phenotype to a non-transformed state, and resulted in a 47% reduction in the specific activity of the methyltransferase as compared with mock transfected cells.

Multicenter study of noninvasive monitoring systems as alternatives to invasive monitoring of acutely ill emergency patients.

BACKGROUND: Recent reports showed lack of effectiveness of pulmonary artery catheterization in critically ill medical patients and relatively late-stage surgical patients with organ failure. Since invasive monitoring requires critical care environments, the early hemodynamic patterns may have been missed. Ideally, early noninvasive hemodynamic monitoring systems, if reliable, could be used as the "front end" of invasive monitoring to supply more complete descriptions of circulatory pathophysiology. OBJECTIVES: To evaluate the accuracy and reliability of noninvasive hemodynamic monitoring consisting of a new bioimpedance method for estimating cardiac output combined with arterial BP, pulse oximetry, and transcutaneous PO2 and PCO2; we compared this system of noninvasive monitoring with simultaneous invasive measurements to evaluate circulatory deficiencies in acutely ill patients shortly after hospital admission where invasive monitoring was not readily available. We also preliminarily explored early differences in temporal hemodynamic patterns of survivors and nonsurvivors. DESIGN AND SETTING: Prospective comparison of simultaneous invasive and noninvasive measurements of circulatory function with retrospective analysis of data in university-run county hospitals, university hospitals and affiliated teaching hospitals, and a community private hospital. PATIENTS: We studied 680 patients, including 139 severely injured or hemorrhaging patients in the emergency department (ED), 129 medical (nontrauma) patients on admission to the ED, 274 high-risk surgical patients intraoperatively, and 138 patients recently admitted to the ICU. RESULTS: A new noninvasive impedance device provided cardiac output estimations under conditions in which invasive thermodilution measurements were not usually applied. There were 2,192 simultaneous bioimpedance and thermodilution cardiac index measurements; the correlation coefficient, r = 0.85, r2 = 0.73, p < 0.001. The precision and bias was -0.124+/-0.75 L/min/m2. Both invasive and noninvasive monitoring systems provide similar information and identified episodes of hypotension, low cardiac index, arterial hemoglobin desaturation, low transcutaneous O2, high transcutaneous CO2, and low oxygen consumption before and during initial resuscitation. The limitations of noninvasive systems were described. CONCLUSIONS: Noninvasive monitoring systems gave continuous displays of physiologic data that provided information allowing early recognition of low flow and poor tissue perfusion that were more pronounced in the nonsurvivors. Noninvasive systems may be acceptable alternatives where invasive monitoring is not available.

Effect of trabeculectomy on pulsatile ocular blood flow.

Trabeculectomy, despite producing an effective reduction in intraocular pressure, may not prevent continued visual field loss. This may be because of the presence of other factors in the pathogenesis of glaucoma. Vascular factors have been suggested as being particularly important. To study the effect of trabeculectomy on ocular blood flow the technique of ocular pulse analysis has been used to derive a measure of pulsatile ocular blood flow in 17 patients (average age 65.6 (SD 1.8) years) undergoing trabeculectomy. A significant increase in pulsatile ocular blood flow of 29% was observed in the group as a whole in the standing position following operation but in some individuals blood flow changed only slightly despite a large reduction in intraocular pressure. The significance of these findings in relation to the prognosis of visual field preservation following trabeculectomy is discussed.

Pulsatile ocular blood flow in patients with low tension glaucoma.

Measurements of the intraocular pressure (IOP) pulse and pulsatile ocular blood flow (POBF) have been made in 22 patients with bilateral low tension glaucoma (LTG) and 29 healthy subjects matched as closely as possible for age, refractive error, IOP, systemic pulse pressure, and heart rate. Recordings were made in both the standing and supine positions. The amplitude of the intraocular pressure pulse was significantly lower in patients with LTG (1.2, SEM 0.1 mmHg standing, and 1.3, SEM 0.1 mmHg lying) than in healthy subjects (1.9, SEM 0.1 mmHg standing, and 2.0 SEM 0.1 mmHg lying): p less than 0.001 standing and p less than 0.002 lying. Measurement of POBF also showed a significant reduction between the healthy subjects (428 (31) SEM microliters/min standing and 345 (28) SEM microliters/min lying) and subjects with LTG (301 (27) SEM microliters/min standing and 249 (24) SEM microliters/min lying), p less than 0.005 standing and p less than 0.02 lying. This represents a difference of approximately 30% between the two groups in either posture. A close non-parametric correlation existed between the level of IOP and the POBF (r = 0.75, p less than 0.001 standing, and r = -0.55, p less than 0.02 lying). Such a correlation was not present in the healthy subjects. A reduction in POBF occurred in both groups on assuming the supine posture (healthy subjects 83 (16) SEM microliters/min, LTG subjects 52 (17) SEM microliters/min). These figures represent reductions of 19% and 17% respectively in comparison with the standing value. The results lend further confirmation to the hypothesis that vascular factors are associated with low tension glaucoma.

Effect of squint surgery on pupillary diameter.

AIM: To investigate the effect that squint surgery has on pupillary diameter. METHODS: The effect of squint surgery on pupil size was investigated in 19 patients. RESULTS: A significant mydriasis in the operated eye was observed when compared with the unoperated eye. This was independent of the number or type of extraocular muscles operated upon. CONCLUSION: It is hypothesised that this change in pupillary diameter results from the release of neurotransmitters from tissues damaged during surgery.

Infectious crystalline keratopathy.

We present a patient who developed a crystalline keratopathy after a penetrating keratoplasty. This rare complication can be caused by bacterial infection, and the patient responded to the appropriate antibiotic therapy. The literature is reviewed and possible causes and mechanisms of the crystalline appearance are discussed.

Identification of a region on the adenovirus E1A gene responsible for induction by phorbol ester tumor promoter.

12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment induces human adenovirus (Ad) early region 1A (E1A) messenger ribonucleic acid expression in infected or Ad-transformed cells. Here, we report that deletion analysis has identified a TPA-responsive element (TRE) in the E1A enhancer region. Deletion analysis indicates that the TRE is located upstream of the E1A cap site between nucleotides -237 and -47. Incubation of extracts from TPA-treated cells with radioactively labeled deoxyribonucleic acid (DNA) fragments containing the TRE (-237 to -47) form specific DNA-protein complexes as demonstrated by gel shift analysis and Southwestern blotting. These experiments provide evidence that novel protein-DNA complexes are formed on a region of the E1A promoter required for TPA-enhanced expression. We speculate that these DNA-binding proteins may interact with the TRE and play a critical role in the mechanism through which TPA upregulates transcription from the Ad E1A gene.

Isolation and identification of anti-inflammatory constituents from Ligusticum chuanxiong and their underlying mechanisms of action on microglia.

Stroke is the third most common cause of death worldwide. Recent findings showed that the severity of cerebrovascular diseases including ischemic stroke correlates with inflammation mediated responses in the neural cells. During ischemia, inflammatory mediators including tumor necrosis factor-alpha (TNF-α) and nitric oxide are produced by microglia, which play a central role in the pathogenesis of the disease. Ligusticum chuanxiong (LCX) is a commonly used traditional Chinese medicine (TCM) for empiric treatment of cerebrovascular and cardiovascular diseases for many centuries. By applying a bioactivity-guided fractionation scheme, two compounds with inhibition on neuroinflammation were isolated from LCX. Using chromatographic and spectrometric methods, they were identified to be senkyunolide A and Z-ligustilide. They could inhibit the production of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV-2 microglial cells and human peripheral blood monocyte derived macrophages. In addition, both compounds protected Neuro-2a cells from neuroinflammatory toxicity induced by the conditioned culture media produced by LPS-stimulated BV-2 cells. The underlying mechanisms of action of senkyunolide A were further delineated. Its inhibitory effects were shown to be independent of the phosphorylation of mitogen-activated protein kinases (MAPK) and translocation of nuclear factor kappa B (NF-κB). However, senkyunolide A could increase the degradation of TNF-α mRNA and reduce its half life by 43%. In conclusion, bioactivity-guided fractionation is an effective way of isolating bioactive compounds from medicinal herbs. In addition, senkyunolide A and Z-ligustilide isolated from LCX may be considered as potential complementary drug candidates for treating inflammatory processes associated with cerebrovascular diseases.