Hydroxyhydroquinone or 1,2,4-benzenetriol (BT) detected in the beverages has a structure that coincides with the water-soluble form of a sesame lignan, sesamol. We previously showed that sesame antioxidants had neuroprotective abilities due to their antioxidant properties and/or inducible nitric oxide synthase (iNOS) inhibition. However, studies show that BT can induce DNA damage through the generation of reactive oxygen species (ROS). Therefore, we were interested to investigate the neuroprotective effect of BT in vitro and in vivo. The results showed that instead of enhancing free radical generation, BT dose-dependently (10-100 microM) attenuated nitrite production, iNOS mRNA and protein expression in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. BT significantly reduced LPS-induced NF-kappaB and p38 MAPK activation. It also significantly reduced the generation of ROS in H2O2-induced BV-2 cells and in H2O2-cellfree conditions. The neuroprotective effect of BT was further demonstrated in the focal cerebral ischemia model of Sprague-Dawley rat. Taken together, the inhibition of LPS-induced nitrite production might be due to the suppression of NF-kappaB, p38 MAPK signal pathway and the ROS scavenging effect. These effects might help to protect neurons from the ischemic injury.
Protein Kinase C (PKC) is a serine/threonine kinase that involved in controlling of many cellular processes such as cell proliferation and differentiation. We have observed previously that TPA (12-O-tetradecanoylphorbol 13-acetate) induces cell cycle arrest in G0/G1 phase in human hepatoma HepG2 cells. However, is there any miRNA involved in PKCalpha mediated cell growth arrest is still unknown.
We first surveyed 270 miRNA expression profiles in 20 pairs of human hepatoma tissues. We identified 11 up-regulated and 23 down-regulated miRNAs (FDR < = 0.01; fold-change > = 2) in human hepatoma tissue after Student's T-test and Mann-Whitney rank test. We then examined miRNAs expression profile in TPA treated HepG2 cells. Two miRNAs, miR-101, and miR-29c, were shown to be significantly down regulated in human hepatoma tissues and induced over 4-fold in HepG2 cells under TPA treatment.
In this study, we examined TPA regulated miRNA expression profile in human hepatoma HepG2 cells. We identified two miRNAs, 101 and 29c, were induced by TPA and down regulated in human hepatoma tissues suggest that they might play as tumor suppressor gene and in tumor formation of HCC. Since induction kinetics of miR-101 by TPA was much faster than miR-29c suggests that the induction of miR-101 may be the primary response of TPA treatment. We then further investigated how miR-101 was regulated by TPA. MiR-101 targets two subunits of PRC2 complex, enhancer of zeste homolog 2 (EZH2) and EED, and was shown to play as a tumor suppressor gene in human prostate, breast and liver cancers. The target sequence of miR-101 located in the 3' UTR of both EZH2 and EED's mRNA was identified by bioinformatic analysis and was validated by reporter luciferase activity assay. Then we showed that TPA not only up regulated miR-101 expression, but also reduced protein level of EZH2, EED and H3K27me3 in HepG2 cells. Using lenti-virus-mediated shRNA to knockdown endogenous PKCalpha expression, we observed that TPA induced growth arrest, elevation of miR-101 and reduction of EZH2, EED and H3K27me3 proteins were all PKCalpha dependent. Specific inhibitor of ERK completely blocked TPA induced miR-101 expression.
Therefore, this is the first time to show that PKCalpha and ERK pathway play important role to activate miR-101 expression, reduce PRC2 complex and H3K27me3 level. This epigenetic regulatory pathway may represent a novel mechanism of carcinogenesis and deserve further investigation.
A wide variety of up-to-date results and knowledge were presented at the 10th International AIDS Meeting, Yokohama. Epidemiologically, most interest was focused on the discovery of a new HIV subtype O, which cannot be reliably detected by currently available ELISA kits. Clinically, it is gradually appreciated that one single most important parameter is the viral load; the extent of viral load can help explain many clinical observations. Another eye-catching finding was the report of a clinical follow-up of a group of long-term nonprogressors. If the underlying operative mechanism can be elucidated, we can learn the necessary elements for halting HIV infection progression. Therapeutically, the trend has shifted to combination therapy, preferentially 3-drug combination of 2 RT inhibitors and 1 protease inhibitor. For the vaccine development, many novel vectors were introduced, but their potentials are unknown at present. The successful application of single-cell in situ PCR has changed our perception of HIV infection. This powerful technique can detect a single viral genome inside cells and revealed that a large proportion of cells already harbor HIV genomes soon after the entry of HIV into the body. A direct viral effect may fully explain subsequent T cell depletion without invoking a lot of indirect mechanisms such as apoptosis. Copyright 1995 S. Karger AG, Basel
The conserved Notch signaling pathway regulates cell fate decisions and maintains stem cells in multicellular organisms. Up-regulation of Notch signaling is observed in several types of cancer and is causally involved in proliferation and survival of cancer cells. Thus, it is of great interest to look for anti-Notch reagents for therapeutic purposes. In model animal Drosophila, Notch signaling restricts selection of sensory organ precursors (SOPs) during external sensory (ES) organ development. To look for novel genes that can suppress Notch signaling, we performed a gain-of-function modifier screen to look for genes that enhance the phenotype of ectopic ES organs induced by overexpression of phyllopod, a gene required for SOP specification.
From the gain-of-function screen, we discovered that overexpression of polished rice/tarsal-less (pri/tal) increases the numbers of ES organs as well as SOPs. pri/tal is a polycistronic gene that contains four short open reading frames encoding three 11-amino acid and one 32-amino acid peptides. Ectopic expression of the 11 amino-acid peptides recapitulates the pri/tal misexpression phenotype in ectopic ES organ formation. In situ hybridization experiment reveals that pri/tal mRNA is expressed in the SOPs of the chemosensory organs and the stretch-sensing chordotonal organs.In Drosophila wing development, the Notch signaling pathway mediates the formation of the dorsal-ventral (DV) compartmental boundary and the restriction of the vein width from the primordial veins, the proveins. We also found that pri/tal mRNA is expressed in the DV boundary and the longitudinal proveins, and overexpression of Pri/Tal peptides disrupts the DV boundary formation and helps to expand the width of the wing vein. Genetic analyses further show that a Notch loss-of-function allele strongly enhances these two phenotypes. Cut and E(spl)mβ are target genes of the Notch pathway in DV boundary formation and vein specification, respectively. We also found that overexpression of Pri/Tal peptides abolishes Cut expression and co-expression of Pri/Tal peptides with phyl strongly reduces E(spl)mβ expression.
We show for the first time that the overexpression of Pri/Tal 11-amino acid peptides disrupts multiple Notch-mediated processes and reduces Notch target gene expression in Drosophila, suggesting that these peptides have novel antagonistic activity to the Notch pathway. Thus, our discovery might provide insights into designing new therapeutic reagents for Notch-related diseases.
Nasopharyngeal carcinoma (NPC) is commonly found in Southern China and South East Asia. Epstein-Barr virus (EBV) infection is well associated with NPC and has been implicated in its pathogenesis. Moreover, various chromosome rearrangements were reported in NPC. However, the underlying mechanism of chromosome rearrangement remains unclear. Furthermore, the relationship between EBV and chromosome rearrangement with respect to the pathogenesis of NPC has not been established. We hypothesize that during virus- or stress-induced apoptosis, chromosomes are initially cleaved at the base of the chromatin loop domain structure. Upon DNA repair, cell may survive with rearranged chromosomes.
In this study, cells were seeded at various densities to induce apoptosis. Genomic DNA extracted was processed for Southern hybridization. In order to investigate the role of EBV, especially the latent membrane protein 1 (LMP1), LMP1 gene was overexpressed in NPC cells and chromosome breaks were analyzed by inverse polymerase chain (IPCR) reaction.
Southern analysis revealed that high cell density resulted in cleavage of the mixed lineage leukemia (MLL) gene within the breakpoint cluster region (bcr). This high cell density-induced cleavage was significantly reduced by caspase inhibitor, Z-DEVD-FMK. Similarly, IPCR analysis showed that LMP1 expression enhanced cleavage of the MLL bcr. Breakpoint analysis revealed that these breaks occurred within the matrix attachment region/scaffold attachment region (MAR/SAR).
Since MLL locates at 11q23, a common deletion site in NPC, our results suggest a possibility of stress- or virus-induced apoptosis in the initiation of chromosome rearrangements at 11q23. The breakpoint analysis results also support the role of chromatin structure in defining the site of chromosome rearrangement.
Bitter gourd (Momordica charantia L.) is a common vegetable in Asia that has been used in traditional medicine for the treatment of Diabetes. PPARs are ligand-dependent transcription factors that belong to the steroid hormone nuclear receptor family and control lipid and glucose homeostasis in the body. We previously reported that the ethyl acetate (EA) extract of bitter gourd activated peroxisome proliferator receptors (PPARs) alpha and gamma. To identify the active compound that activated PPARalpha, wild bitter gourd EA extract was partitioned between n-hexane and 90% methanol/10% H(2)O, and the n-hexane soluble fraction was further separated by silica gel column chromatography and finally by preparative HPLC. A transactivation assay employing a clone of CHOK1 cells stably transfected with a (UAS)(4)-tk-alkaline phosphatase reporter and a chimeric receptor of GAL4-rPPARalpha LBD was used to track the active component. Based on Mass, NMR, and IR spectroscopy, 9cis, 11trans, 13trans-conjugated linolenic acid (9c, 11t, 13t-CLN) was identified as a PPARalpha activator in wild bitter gourd. The isolated 9c, 11t, 13t-CLN rich fraction also significantly induced acyl CoA oxidase (ACO) activity in a peroxisome proliferator-responsive murine hepatoma cell line, H4IIEC3, implying that 9c, 11t, 13t-CLN was able to act on a natural PPARalpha signaling pathway as well. The content of 9c, 11t, 13t-CLN was estimated to be about 7.1 g/kg of our dried wild bitter gourd sample. The concentration of 9c, 11t, 13t-CLN and activation activity in the hydrolyzed EA extract of the seeds was higher than that of the flesh. The potential health benefits of 9c, 11t, 13t-CLN through the PPARalpha regulated mechanism are worthy to be further characterized in in vivo studies.
In this study, we examined the tissue-specific expression of two electroneutral Na/HCO(3) cotransporter (NBCn1) variants that differ from each other by the presence of the N-terminal 123 amino acids (cassette II). A rat Northern blot with the probe to nucleotides encoding cassette II detected a 9 kb NBCn1 mRNA strongly in the heart and weakly in skeletal muscles, but absent from most of the tissues including kidney, brain, and pancreas. In the rat heart, PCR with primers flanking cassette II preferentially amplified a DNA fragment that lacked cassette II. However, in the human heart, PCR preferentially amplified a fragment that contained cassette II. This larger PCR product was found virtually in all regions of the human cardiovascular system with strong amplification in the apex, atrium, and atrioventricular nodes. These findings indicate that the variant containing cassette II is almost absent in tissues including brain, kidney, and pancreas, where NBCn1 has been extensively examined.
The association between ischemic stroke and 2 single nucleotide polymorphisms (SNPs) on chromosome 12p13, rs12425791 and rs11833579 appears inconsistent across different samples. These SNPs are close to the ninjurin2 gene which may alter the risk of stroke by affecting brain response to ischemic injury. The purpose of this study was to investigate the association between these two SNPs and ischemic stroke risk, as well as prognostic outcomes in a Taiwanese sample.
We examined the relations of these two SNPs to the odds of new-onset ischemic stroke, ischemic stroke subtypes, and to the one year risk of stroke-related death or recurrent stroke following initial stroke in a case-control study. A total of 765 consecutive patients who had first-ever ischemic stroke were compared to 977 stroke-free, age-matched controls. SNPs were genotyped by Taqman fluorescent allelic discrimination assay. The association between ischemic stroke and SNPs were analyzed by multivariate logistic regression. Cox proportional hazard model was used to assess the effect of individual SNPs on stroke-related mortality or recurrent stroke.
There was no significant association between SNP rs12425791 and rs11833579 and ischemic stroke after multiple testing corrections. However, the marginal significant association was observed between SNP rs12425791 and large artery atherosclerosis under recessive model (OR, 2.30; 95%CI, 1.22-4.34; q-value = 0.062). Among the 765 ischemic stroke patients, 59 died or developed a recurrent stroke. After adjustment for age, sex, vascular risk factors and baseline stroke severity, Cox proportional hazard analysis indicated that the hazard ratios were 2.76 (95%CI, 1.34-5.68; q-value, 0.02) and 2.15 (95%CI, 1.15-4.02; q-value, 0.03) for individuals with homozygous variant allele of rs12425791 and rs11833579, respectively.
This is a precedent study that found genetic variants of rs12425791 and rs11833579 on chromosome 12p13 are independent predictors of stroke-related mortality or stroke recurrence in patients with incident ischemic stroke in Taiwan. Further study is needed to explore the details of the physiological function and the molecular mechanisms underlying the association of this genetic locus with ischemic stroke.
Interleukin-1 (IL-1) has been implicated in the regulation of the expression of various matrix metalloproteinases (MMPs) in many mesenchymal cell types, but its role in liver myofibroblasts (MFs) has not been elucidated. A myofibroblast-like cell line, MG2, was derived from an isolate of rat hepatic stellate cells (HSCs). These cells expressed desmin, vimentin, smooth muscle alpha-actin, and fibulin-2. Using a recombinant IL-1alpha at 5 ng/ml, it was shown that IL-1alpha would upregulate, while IL-1Ra, an IL-1 receptor antagonist, would down-regulate the expression of IL-1alpha mRNA in MG2 cells, indicating the presence of an autostimulatory loop of IL-1alpha in these cells. Besides, a paracrine source of IL-1 may be produced from Kupffer cells, as we showed primarily cultured Kupffer cells responded much more remarkably than MG2 cells to lipopolysaccharide stimuli to produce both IL-1alpha and IL-1beta. Recombinant IL-1alpha upregulated the expression of both MMP-9 and -13, and the induction of MMP-13 but not MMP-9 could be inhibited by SB203580, an inhibitor of p38. Similarly, in primarily cultured human liver MFs, upregulation of MMP-1 by IL-1alpha was also shown to be inhibited by SB203580. All of these data suggested that, during liver inflammation, IL-1 produced by an autocrine model from MFs or by a paracrine model from Kupffer cells might play a crucial role in the remodeling of liver fibrosis through an either p38-dependent or p38-independent pathway to regulate the expression of various MMPs by liver MFs.
The true identity of Hodgkin's mononuclear cells and Reed-Sternberg (H-RS) cells has been a subject of controversy for decades. Those who believe that Hodgkin's disease (HD) is a heterogeneous disease may consider it to constitute lymphomas of various origins. However, this theory seems incompatible with the finding of similar phenotypic, biologic, and immunologic properties among most HD. We believe that, in the majority of cases, HD, except for LP and some LD-type HD, is a homogeneous disease despite differences in the degree of fibrosis and/or cellular reaction. The heterogeneity in cellular reactions is a result of secretion of various cytokines by H-RS cells, which may or may not be influenced by the presence of EBV. H-RS cells, and anaplastic large cell lymphoma (ALCL) cells as well, can express a combination of cytokines and cytokine receptors that is not seen in other types of lymphomas. The unique cytokine/receptor profile (e.g. the expression of c-kit-R/CD117), along with various properties associated with H-RS/ALCL cells, leads to a hypothesis that H-RS/ALCL cells are related to similar lymphohematopoietic progenitor cells with different etiologies and somewhat limited differentiation capacity. A number of H-RS cells may differentiate with limited capacity along the B-cell pathway and may be infected by EBV, which further complicates the biologic and immunologic properties of these cells. The majority of H-RS cells may also, however, differentiate along the antigen-presenting dendritic cell pathway, as indicated by the abundant expression of restin, CD15, CD40, CD54, CD58, CD80, and CD86. The majority of ALCL cells clearly differentiate to T cells, but some may acquire B-cell or histiocyte phenotypes. The progenitor cell hypothesis may explain (1) the variable expression of CD117, CD43, and CD34 as well as the absence of CD27, CD45 and CD45RA in H-RS cells; (2) the inconsistent and irregular patterns of phenotype and genotype and the various, often very limited, degrees of differentiation among these two types of lymphoma cells; (3) the existence of secondary HD or ALCL associated with rare types of lymphomas or leukemias, or vice versa; (4) the absence of recombinase and of the B-specific transcription factors BSAP; and (5) the frequent expression of IL-7 and IL-9 in H-RS cells. Copyright 1996 S. Karger AG, Basel
The eukaryotic protein degradation pathway involves the ubiquitin (Ub) modification of substrates targeted for degradation by the 26S proteasome. The addition of Ub, a process called ubiquitination, is mediated by enzymes including the E3 Ub ligases which transfer the Ub to targeted substrates. A major type of E3 Ub ligases, the SCF (Skp-Cullin-F-box) complex, is composed of four major components: Skp1, Cul1/Cdc53, Roc1/Rbx1/Hrt1, and an F-box protein. The F-box component of the SCF machineries is responsible for recognizing different substrates for ubiquitination. Interaction with components of the SCF complex is mediated through the F-box motif of the F-box protein while it associates with phosphorylated substrates through its second protein-protein interaction motif such as Trp-Asp (WD) repeats or leucine-rich repeats (LRRs). By targeting diverse substrates, F-box proteins exert controls over stability of proteins and regulate the mechanisms for a wide-range of cellular processes. Here we discuss the importance of F-box proteins by providing a general overview and examples of how F-box proteins function in various cellular settings such as tissue development, cell proliferation, and cell death, in the modeling organism Drosophila.
Chronic arsenic poisoning is a world public health issue. Long-term exposure to inorganic arsenic (As) from drinking water has been documented to induce cancers in lung, urinary bladder, kidney, liver and skin in a dose-response relationship. Oxidative stress, chromosomal abnormality and altered growth factors are possible modes of action in arsenic carcinogenesis. Arsenic tends to accumulate in the skin. Skin hyperpigmentation and hyperkeratosis have long been known to be the hallmark signs of chronic As exposure. There are significant associations between these dermatological lesions and risk of skin cancer. The most common arsenic-induced skin cancers are Bowen's disease (carcinoma in situ), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Arsenic-induced Bowen's disease (As-BD) is able to transform into invasive BCC and SCC. Individuals with As-BD are considered for more aggressive cancer screening in the lung and urinary bladder. As-BD provides an excellent model for studying the early stages of chemical carcinogenesis in human beings. Arsenic exposure is associated with G2/M cell cycle arrest and DNA aneuploidy in both cultured keratinocytes and As-BD lesions. These cellular abnormalities relate to the p53 dysfunction induced by arsenic. The characteristic clinical figures of arsenic-induced skin cancer are: (i) occurrence on sun-protected areas of the body; (ii) multiple and recrudescent lesions. Both As and UVB are able to induce skin cancer. Arsenic treatment enhances the cytotoxicity, mutagenicity and clastogenicity of UV in mammalian cells. Both As and UVB induce apoptosis in keratinocytes by caspase-9 and caspase-8 signaling, respectively. Combined UVB and As treatments resulted in the antiproliferative and proapoptotic effects by stimulating both caspase pathways in the keratinocytes. UVB irradiation inhibited mutant p53 and ki-67 expression, as well as increased in the number of apoptotic cells in As-BD lesions which resulted in an inhibitory effect on proliferation. As-UVB interaction provides a reasonable explanation for the rare occurrences of arsenical cancer in the sun-exposed skin. The multiple and recurrent skin lesions are associated with cellular immune dysfunction in chronic arsenism. A decrease in peripheral CD4+ cells was noticed in the inhabitants of arsenic exposure areas. There was a decrease in the number of Langerhans cells in As-BD lesion which results in an impaired immune function on the lesional sites. Since CD4+ cells are the target cell affected by As, the interaction between CD4+ cells and epidermal keratinocytes under As affection might be closely linked to the pathogenesis of multiple occurrence of arsenic-induced skin cancer. In this review, we provide and discuss the pathomechanisms of arsenic skin cancer and the relationship to its characteristic figures. Such information is critical for understanding the molecular mechanism for arsenic carcinogenesis in other internal organs.
Skin is the largest organ in the body, and is directly exposed to extrinsic assaults. As such, the skin plays a central role in host defense and the cutaneous immune system is able to elicit specific local inflammatory and systemic immune responses against harmful stimuli. 12-O-tetradecanoylphorbol-13-acetate (TPA) can stimulate acute and chronic inflammation and tumor promotion in skin. TPA-induced dermatitis is thus a useful in vivo pharmacological platform for drug discovery. In this study, the inhibitory effect of briarane-type diterpenes (BrDs) from marine coral Briareum excavatum on TPA-induced dermatitis and dendritic cell (DC) function was explored.
Evans blue dye exudation was used to determine vascular permeability. H&E-stained skin section was used to determine the formation of edema in mouse abdominal skin. We also used immunohistochemistry staining and western blot assays to evaluate the activation of specific inflammation makers and key mediators of signaling pathway in the mouse skin. Furthermore, mouse bone marrow DCs were used to determine the relationship between the chemical structure of BrDs and their regulation of DC function.
BrD1 remarkably suppressed TPA-induced vascular permeability and edema in skin. At the biochemical level, BrD1 inhibited TPA-induced expression of cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloproteinase-9, the key indicators of cutaneous inflammation. This inhibition was apparently mediated by interference with the Akt/NF-κB-mediated signaling network. BrD1 also inhibited TNF-α and IL-6 expression in LPS-stimulated BMDCs. The 8, 17-epoxide of BrDs played a crucial role in the inhibition of IL-6 expression, and replacement of the C-12 hydroxyl group with longer esters in BrDs gradually decreased this inhibitory activity.
Our results suggest that BrDs warrant further investigation as natural immunomodulatory agents for control of inflammatory skin diseases.
Most types of normal cells require integrin-mediated attachment to extracellular matrix to be able to respond to growth factor stimulation for proliferation and survival. Therefore, a consensus that integrins are close collaborators with growth factors in signal transduction has gradually emerged. Some integrins and growth factor receptors appear to be normally in relatively close proximity, which can be induced to form complexes upon cell adhesion or growth factor stimulation. Moreover, since integrins and growth factor receptors share many common elements in their signaling pathways, it is clear tzhat there are many opportunities for integrin signals to modulate growth factor signals and vice versa. Increasing evidence indicates that integrins can crosstalk with receptor tyrosine kinases in a cell- and integrin-type-dependent manner through a variety of specific mechanisms. This review is intended specifically for summarizing recent progress uncovering how the hepatocyte growth factor receptor c-Met coordinates with integrins to transmit signals.
Tumor necrosis factor-alpha (TNF-alpha) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide, alpha-N-phthalidoglutarimide, (C(13)H(10)N(2))(4), has been shown to have immunomodulatory and anti-inflammatory properties, possibly mediated through its anti-TNF-alpha effect. In this study, we investigated the in vitro and in vivo effects of thalidomide on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-beta1 (TGF-beta1) or TNF-alpha. The inhibitory effects of thalidomide on the NFkappaB signaling cascade and fibrosis markers including alpha-smooth muscle actin (alpha-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats, which were randomly assigned to 1 of 4 groups: vehicle (0.7% carboxyl methyl cellulose, CMC), thalidomide (40 mg/kg), thalidomide (200 mg/kg), or silymarin (50 mg/kg), each given by gavage twice daily for 3 weeks starting after 1 week of DMN administration. Thalidomide (100-800 nM) concentration-dependently inhibited NFkappaB transcriptional activity induced by TNF-alpha, including IKKalpha expression and IkappaBalpha phosphorylation in HSC-T6 cells. In addition, thalidomide also suppressed TGF-beta1-induced alpha-SMA expression and collagen deposition in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats receiving high dose of thalidomide (0.89 +/- 0.20) were significantly reduced in comparison with those of DMN-treated rats receiving vehicle (1.56 +/- 0.18). Hepatic collagen contents of DMN rats were also significantly reduced by either thalidomide or silymarin treatment. Immunohistochemical double staining results showed that alpha-SMA- and NFkappaB-positive cells were decreased in the livers from DMN rats receiving either thalidomide or silymarin treatment. In addition, real-time PCR analysis indicated that hepatic mRNA expressions of TGF-beta1, alpha-SMA, collagen 1alpha2, TNF-alpha and iNOS genes were attenuated by thalidomide treatment. In conclusion, our results showed that thalidomide inhibited activation of HSC-T6 cells by TNF-alpha and ameliorated liver fibrosis in DMN-intoxicated rats.
Ischemic postconditioning (IPost) has aroused much attention since 2003 when it was firstly reported. The role of microRNAs (miRNAs or miRs) in IPost has rarely been reported. The present study was undertaken to investigate whether miRNAs were involved in the protective effect of IPost against myocardial ischemia-reperfusion (IR) injury and the probable mechanisms involved.
Thirty SD rats weighing 250-300 g were equally randomized to three groups: Control group, where the rats were treated with thoracotomy only; IR group, where the rats were treated with ischemia for 60 min and reperfusion for 180 min; and IPost group, where the rats were treated with 3 cycles of transient IR just before reperfusion. The extent of myocardial infarction, LDH and CK activities were measured immediately after treatment. Myocardial apoptosis was detected by TUNEL assay. The myocardial tissue was collected after IR or IPost stimulation to evaluate the miRNAs expression level by miRNA-microarray and quantitative real-time RT-PCR. Real-time PCR was conducted to identify changes in mRNA expression of apoptosis-related genes such as Bcl-2, Bax and Caspase-9 (CASP9), and Western blot was used to compare the protein expression level of CASP9 in the three groups. The miRNA mimics and anti-miRNA oligonucleotides (AMO) were transferred into the cultured neonatal cardiomyocytes and myocardium before they were treated with IR. The effect of miRNAs on apoptosis was determined by flow cytometry and TUNEL assay. CASP9, as one of the candidate target of miR-133a, was compared during IR after the miR-133a mimic or AMO-133a was transferred into the myocardium.
IPost reduced the IR-induced infarct size of the left ventricle, and decreased CK and LDH levels. TUNEL assay showed that myocardial apoptosis was attenuated by IPost compared with IR. MiRNA-microarray and RT-PCR showed that myocardial-specific miR-1 and miR-133a were down-regulated by IR, and up-regulated by IPost compared with IR. Furthermore, IPost up-regulated the mRNA expression of Bcl-2, down-regulated that of Bax and CASP9. Western blot showed that IPost also down-regulated the CASP9 protein expression compared with IR. The results of flow cytometry and TUNEL assay showed that up-regulation of miR-1 and miR-133a decreased apoptosis of cardiomyocytes. MiR-133a mimic down-regulated CASP9 protein expression and attenuated IR-induced apoptosis.
MiRNAs are associated with the protective effect of IPost against myocardial IR injury. IPost can up-regulate miR-1 and miR-133a, and decrease apoptosis of cardiomyocyte. Myocardial-specific miR-1 and miR-133a may play an important role in IPost protection by regulating apoptosis-related genes. MiR-133a may attenuate apoptosis of myocardiocytes by targeting CASP9.
The metabolic fate of ethanol into the phospholipid pool of calf pulmonary artery endothelial cells was studied. [14C]-ethanol was incorporated into various endothelial cell phospholipids including phosphatidylethanol (PEth), which may represent a substantial fraction in microdomains of membrane phospholipids. The incorporation into phospholipids was reduced in the presence of pyrazole and cyanamide, inhibitors of ethanol metabolism. Wortmannin, the phosphatidylinositol 3-kinase inhibitor, increased [14C]-PEth formation. [3H]-acetate was also incorporated into endothelial cell phospholipids but in a different pattern. Distribution of [3H]-acetate and [14C]-ethanol into the fatty acyl moiety versus the glycerophosphoryl backbone of the phospholipids was also different. Stimulation of the endothelial cells with ATP increased [3H]-acetate incorporation into platelet-activating factor (PAF) and ethanol decreased it. Ethanol exposure increased ATP-stimulated [3H]-acetate incorporation into sphingomyelin. However, ATP had no effect on the incorporation of [14C]-ethanol into any phospholipids. The results suggest that the two precursors contribute to a separate acetate pool and that the sphingomyelin cycle may be sensitized in ethanol-treated cells. Thus, metabolic conversions of ethanol into lipids and the effect of ethanol on specific lipid mediators, e.g PAF, PEth and sphingomyelin, may be critical determinants in the altered responses of the endothelium in alcoholism.
The Solanum nigrum Linne (SNL) has been traditionally used as a herbal agent in folk medicine for various cancers in Korea. We found that the SNL glycoprotein consists of carbohydrate (69.74%) and protein content (30.26%), which has mainly the hydrophobic amino acids containing glycine and proline. With respect to its characters, we evaluated the apoptotic effects of glycoprotein isolated from SNL in human cervical cancer cell. In the activity of the apoptotic related proteins [cytochrome c, caspase 8, 3 and poly (ADP-ribose) polymerase (PARP)], the results showed that SNL glycoprotein (50 microg/ml) has a stimulatory effect on cytochrome c release into cytosol, caspase 8, 3 activation and PARP cleavage in HeLa cells. To verify the possible mechanism for apoptotic activity of SNL glycoprotein in HeLa cells, the binding activities of transcription factors (NF-kappaB and AP-1) and nitric oxide (NO) production was evaluated. The activities of NF-kappaB and AP-1 significantly decreased after SNL glycoprotein (50 microg/ml) treatment for 4 h, compare to the control. Interestingly, there was no difference of the DNA binding activity between NF-kappaB and AP-1. Also, nitric oxide (NO) production was significantly declined at 50 microg/ml SNL glycoprotein for 4 h. Therefore, we speculated that SNL glycoprotein exhibits inhibitory effect on HeLa cells via apoptosis, and it may be a potential candidate in field of anticancer drug discovery.
MiR-155 has emerged as an "oncomiR", which is the most significantly up-regulated miRNA in breast cancer. However, the mechanisms of miR-155 functions as an oncomiR are mainly unknown. In this study, the aims were to investigate the effects of miR-155 on cell proliferation, cell cycle, and cell apoptosis of ERalpha (+) breast cancer cells and to verify whether TP53INP1 (tumor protein 53-induced nuclear protein 1) is a target of miR-155, and tried to explore the mechanisms of miR-155 in this process.
The expression of miR-155 is significantly higher in MCF-7 cells compared with MDA-MB-231 cells. Ectopic expression of TP53INP1 inhibits growth of MCF-7 cells by inducing cell apoptosis and inhibiting cell cycle progression. Overexpression of miR-155 increases cell proliferation and suppress cell apoptosis, whereas abrogating expression of miR-155 suppress cell proliferation and promotes cell apoptosis of MCF-7 cells. In addition, miR-155 negatively regulates TP53INP1 mRNA expression and the protein expression of TP53INP1, cleaved-caspase-3, -8, -9, and p21, and luciferase reporter reveals that TP53INP1 is targeted by miR-155.
TP53INP1 is the direct target of miR-155. MiR-155, which is overexpressed in MCF-7 cells, contributes to proliferation of MCF-7 cells possibly through down-regulating target TP53INP1.
Human papillomavirus (HPV) E6 and E7 are consistently expressed and are responsible for the malignant transformation of HPV-associated lesions. Thus, E6 and E7 represent ideal targets for therapeutic HPV vaccine development. We have previously used the gene gun approach to test several intracellular targeting and intercellular spreading strategies targeting HPV-16 E7. These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat shock protein 70 (HSP70), calreticulin (CRT) and herpes simplex virus type 1 (HSV-1) VP22 proteins. All of these strategies have been shown to be capable of enhancing E7-DNA vaccine potency. In the current study, we have characterized DNA vaccines employing these intracellular targeting or intercellular spreading strategies targeting HPV-16 E6 for their ability to generate E6-specific CD8+ T cell immune responses and antitumor effects against an E6-expressing tumor cell line, TC-1, in C57BL/6 mice. We found that all the intracellular targeting strategies (CRT, LAMP-1, HSP70) as well as the intercellular spreading strategy (VP22) were able to enhance E6 DNA vaccine potency, although the orientation of HSP70 linked to E6 antigen in the E6 DNA vaccine appears to be important for the HSP70 strategy to work. The enhanced E6-specific CD8+ T cell immune response in vaccinated mice also translated into potent antitumor effects against TC-1 tumor cells. Our data indicate that all of the intracellular targeting and intercellular spreading strategies that have been shown to enhance E7 DNA vaccine potency were also able to enhance E6 DNA vaccine potency.
Low power laser irradiation (LPLI) promotes proliferation of multiple cells, which (especially red and near infrared light) is mainly through the activation of mitochondrial respiratory chain and the initiation of cellular signaling. Recently, the signaling proteins involved in LPLI-induced proliferation merit special attention, some of which are regulated by mitochondrial signaling. Hepatocyte growth factor receptor (c-Met), a member of tyrosine protein kinase receptors (TPKR), is phosphorylated during LPLI-induced proliferation, but tumor necrosis factor alpha (TNF-alpha) receptor has not been affected. Activated TPKR could activate its downstream signaling elements, like Ras/Raf/MEK/ERK, PI3K/Akt/eIF4E, PI3K/Akt/eNOS and PLC-gamma/PKC pathways. Other two pathways, DeltaPsim/ATP/cAMP/JNK/AP-1 and ROS/Src, are also involved in LPLI-induced proliferation. LPLI-induced cell cycle progression can be regulated by the activation or elevated expressions of cell cycle-specific proteins. Furthermore, LPLI induces the synthesis or release of many molecules, like growth factors, interleukins, inflammatory cytokines and others, which are related to promotive effects of LPLI.
To investigate the expression of human papillomavirus type 16 (HPV-16) E5 protein in squamous neoplastic changes in the uterine cervix, the specific E5 antibody was generated and used to identify the expression of E5 protein in 40 cases of HPV-16-positive tissues and 5 previously identified HPV-negative normal cervical tissues. The results revealed that E5 protein was primarily expressed in the lower third of the epithelium in low-grade squamous intraepithelial lesions (SILs) and throughout the whole epithelium in high-grade SILs. In invasive squamous carcinoma, 60% of HPV-16-infected cancers which contained the episomal viral genome had the E5 gene, and could express E5 protein which was located throughout the whole epithelium. Previously, we documented the expression of type I growth factor receptors [ERBB1/EGFR (epidermal growth factor receptor), ERBB2, ERBB3 and ERBB4] in the full range of cervical neoplasias by immunohistochemistry assay. Hence, in this study, we extensively analyzed the correlation between the expression of E5 protein and the expression of type I growth factor receptors. Among 40 HPV-16- infected cervical neoplasias, we found that the expression of E5 protein was significantly correlated with either the expression of the ERBB1 or the ERBB4 receptor.
The presence of a cholinergic vasodilator innervation to cerebral circulation is well established. Despite its high endogenous concentration in cerebral blood vessels, acetylcholine (ACh) is not the transmitter for vasodilation. This finding has led to the discovery that nitric oxide (NO), which is coreleased with ACh and neural peptides such as vasoactive intestinal polypeptide (VIP) from the respective cholinergic-nitrergic (nitric oxidergic) nerves and the VIPergic-nitrergic nerves, is the primary transmitter in relaxing smooth muscle. ACh and VIP act presynaptically to inhibit and facilitate, respectively, the release of NO. Release of NO from cerebral vascular endothelial cells is also well established. A similar system for recycling L-citrulline to L-arginine for synthesizing more NO has been demonstrated in both cerebral perivascular nerves and endothelial cells. Neuronal and endothelial NO appears to play an important role in controlling cerebral vascular tone and circulation in health and disease.
The HPV oncoproteins E6 and E7 are consistently expressed in HPV-associated cancer cells and are responsible for their malignant transformation. Therefore, HPV E6 and E7 are ideal target antigens for developing vaccines and immunotherapeutic strategies against HPV-associated neoplasms. Recently, it has been demonstrated that codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8+ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Our data indicated that transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8+ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications.
There are three apolipoprotein E (apoE) isoforms involved in human lipid homeostasis. In the present study, truncated apoE2-, apoE3- and apoE4-(72-166) peptides that are tailored to lack domain interactions are expressed and elucidated the structural and functional consequences.
Circular dichroism analyses indicated that their secondary structure is still well organized. Analytical ultracentrifugation analyses demonstrated that apoE-(72-166) produces more complicated species in PBS. All three isoforms were significantly dissociated in the presence of dihexanoylphosphatidylcholine. Dimyristoylphosphatidylcholine turbidity clearance assay showed that apoE4-(72-166) maintains the highest lipid-binding capacity. Finally, only apoE4-(72-166) still maintained significant LDL receptor binding ability.
Overall, apoE4-(72-166) peptides displayed a higher lipid-binding and comparable receptor-binding ability as to full-length apoE. These findings provide the explanation of diverged functionality of truncated apoE isoforms.
Using RNase protection analysis, we found a novel C to G mutation at nucleotide position 3093 of mitochondrial DNA (mtDNA) in a previously reported 35-year-old woman exhibiting clinical features of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome together with diabetes mellitus, hyperthyroidism and cardiomyopathy. The patient also had an A3243G mutation in the tRNA(Leu(UUR)) gene and a 260-base pair duplication in the D-loop of mtDNA. The fibroblasts of the patient were cultured and used for the construction of cybrids using cytoplasmic transfer of the patient's mtDNA to the mtDNA-less rho(0) cells. RNA isolated from the cybrids was subjected to RNase protection analysis, and a C3093G transversion at the 16S rRNA gene and a MELAS-associated A3243G mutation of mtDNA were detected. The novel C3093G mutation together with the A3243G transition were found in muscle biopsies, hair follicles and blood cells of this patient and also in her skin fibroblasts and cybrids. The proportion of the C3093G mutant mtDNA in muscle biopsies of the patient was 51%. In contrast, the mutation was not detected in three sons of the proband. To characterize the impact of the mtDNA mutation-associated defects on mitochondrial function, we determined the respiratory enzyme activities of the primary culture of fibroblasts established from the proband, her mother and her three sons. The proportions of mtDNA with the C3093G transversion and the A3243G transition in the fibroblasts of the proband were 45 and 58%, respectively. However, the fibroblasts of the proband's mother and children harbored lower levels of mtDNA with the A3243G mutation but did not contain the C3093G mutation. The complex I activity in the proband's fibroblasts was decreased to 47% of the control but those of the fibroblasts of the mother and three sons of the proband were not significantly changed. These findings suggest that the C3093G transversion together with the A3243G transition of mtDNA impaired the respiratory function of mitochondria and caused the atypical MELAS syndrome associated with diabetes mellitus, hyperthyroidism and cardiomyopathy in this patient.
Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by cortico-striatal dysfunction and loss of glutamate uptake. At 7 weeks of age, R6/2 mice, which model an aggressive form of juvenile HD, show a glutamate-uptake deficit in striatum that can be reversed by treatment with ceftriaxone, a beta-lactam antibiotic that increases GLT1 expression. Only at advanced ages (> 11 weeks), however, do R6/2 mice show an actual loss of striatal GLT1. Here, we tested whether ceftriaxone can reverse the decline in GLT1 expression that occurs in older R6/2s.
Western blots were used to assess GLT1 expression in both striatum and cerebral cortex in R6/2 and corresponding wild-type (WT) mice at 9 and 13 weeks of age. Mice were euthanized for immunoblotting 24 hr after five consecutive days of once daily injections (ip) of ceftriaxone (200 mg/kg) or saline vehicle. Despite a significant GLT1 reduction in saline-treated R6/2 mice relative to WT at 13, but not 9, weeks of age, ceftriaxone treatment increased cortical and striatal GLT1 expression relative to saline in all tested mice.
The ability of ceftriaxone to up-regulate GLT1 in R6/2 mice at an age when GLT1 expression is significantly reduced suggests that the mechanism for increasing GLT1 expression is still functional. Thus, ceftriaxone could be effective in modulating glutamate transmission even in late-stage HD.
Epidemiological studies demonstrate that the incidence and mortality rates of colorectal cancer in women are lower than in men. However, it is unknown if 17β-estradiol treatment is sufficient to inhibit prostaglandin E2 (PGE2)-induced cellular motility in human colon cancer cells.
We analyzed the protein expression of urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), matrix metallopeptidases (MMPs), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinases (TIMPs), and the cellular motility in PGE2-stimulated human LoVo cells. 17β-Estradiol and the inhibitors including LY294002 (Akt activation inhibitor), U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), QNZ (NFκB inhibitor) and ICI 182 780 were further used to explore the inhibitory effects of 17β-estradiol on PGE2-induced LoVo cell motility. Student's t-test was used to analyze the difference between the two groups.
Upregulation of urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA) and matrix metallopeptidases (MMPs) is reported to associate with the development of cancer cell mobility, metastasis, and subsequent malignant tumor. After administration of inhibitors including LY294002, U0126, SB203580, SP600125 or QNZ, we found that PGE2 treatment up-regulated uPA and MMP-9 expression via JNK1/2 signaling pathway, thus promoting cellular motility in human LoVo cancer cells. However, PGE2 treatment showed no effects on regulating expression of tPA, MMP-2, plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1, -2, -3 and -4 (TIMP-1, -2, -3 and -4). We further observed that 17β-estradiol treatment inhibited PGE2-induced uPA, MMP-9 and cellular motility by suppressing activation of JNK1/2 in human LoVo cancer cells.
Collectively, these results suggest that 17β-estradiol treatment significantly inhibits PGE2-induced motility of human LoVo colon cancer cells.
Taurine demonstrates multiple cellular functions including a central role as a neurotransmitter, as a trophic factor in CNS development, in maintaining the structural integrity of the membrane, in regulating calcium transport and homeostasis, as an osmolyte, as a neuromodulator and as a neuroprotectant. The neurotransmitter properties of taurine are illustrated by its ability to elicit neuronal hyperpolarization, the presence of specific taurine synthesizing enzyme and receptors in the CNS and the presence of a taurine transporter system. Taurine exerts its neuroprotective functions against the glutamate induced excitotoxicity by reducing the glutamate-induced increase of intracellular calcium level, by shifting the ratio of Bcl-2 and Bad ratio in favor of cell survival and by reducing the ER stress. The presence of metabotropic taurine receptors which are negatively coupled to phospholipase C (PLC) signaling pathway through inhibitory G proteins is proposed, and the evidence supporting this notion is also presented.
The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin) on human retinal pigment epithelial cells is investigated.
MTT and flow cytometry were used to study the antiproliferative effects of the 17-AAG treatment of ARPE-19 cells. 2D gel electrophoresis (2-DE) and mass spectrometry were applied to detect the altered expression of proteins, which was verified by real-time PCR. Gene Ontology analysis and Ingenuity Pathway Analysis (IPA) were utilized to analyze the signaling pathways, cellular location, function, and network connections of the identified proteins. And SOD assay was employed to confirm the analysis.
17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis. Proteomic analysis revealed that the expression of 94 proteins was altered by a factor of more than 1.5 following exposure to 17-AAG. Of these 94, 87 proteins were identified. Real-time PCR results indicated that Hsp90 and Hsp70, which were not identified by proteomic analysis, were both upregulated upon 17-AAG treatment. IPA revealed that most of the proteins have functions that are related to oxidative stress, as verified by SOD assay, while canonical pathway analysis revealed glycolysis/gluconeogenesis.
17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis, and possibly by oxidative stress.
Zinc finger protein 179 (Znf179), also known as ring finger protein 112 (Rnf112), is a member of the RING finger protein family and plays an important role in neuronal differentiation. To investigate novel mechanisms of Znf179 regulation and function, we performed a yeast two-hybrid screen to identify Znf179-interacting proteins.
Using a yeast two-hybrid screen, we have identified promyelocytic leukemia zinc finger (Plzf) as a specific interacting protein of Znf179. Further analysis showed that the region containing the first two zinc fingers of Plzf is critical for its interaction with Znf179. Although the transcriptional regulatory activity of Plzf was not affected by Znf179 in the Gal4-dependent transcription assay system, the cellular localization of Znf179 was changed from cytoplasm to nucleus when Plzf was co-expressed. We also found that Znf179 interacted with Plzf and regulated Plzf protein expression.
Our results showed that Znf179 interacted with Plzf, resulting in its translocation from cytoplasm to the nucleus and increase of Plzf protein abundance. Although the precise nature and role of the Znf179-Plzf interaction remain to be elucidated, both of these two genes are involved in the regulation of neurogenesis. Our finding provides further research direction for studying the molecular functions of Znf179.
It is well documented that 17β-estradiol (E2) exerts a cardiovascular protective effect. A possible role of E2 in the regulation of endothelin-1 (ET-1) production has been reported. However, the complex mechanisms by which E2 inhibits ET-1 expression are not completely understood. The aims of this study were to examine whether E 2 may alter angiotensin II (Ang II)-induced cell proliferation and ET-1 gene expression and to identify the putative underlying signaling pathways in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with E2, then stimulated with Ang II, and [ 3H]thymidine incorporation and ET-1 gene expression were examined. The effect of E2 on Ang-II-induced extracellular signal-regulated kinase (ERK) phosphorylation was tested to elucidate the intracellular mechanism of E2 in proliferation and ET-1 gene expression. Ang II increased DNA synthesis which was inhibited with E2 (1-100 nM). E2, but not 17α-estradiol, inhibited the Ang-II-induced ET-1 gene expression as revealed by Northern blotting and promoter activity assay. This effect was prevented by coincubation with the estrogen receptor antagonist ICI 182,780 (1 μM). E2 also inhibited Ang-II-increased intracellular reactive oxygen species (ROS) as measured by a redox-sensitive fluorescent dye, 2′,7′-dichlorofluorescin diacetate, and ERK phosphorylation. Furthermore, E2 and antioxidants, such as N-acetyl cysteine and diphenylene iodonium, decreased Ang-II-induced cell proliferation, ET-1 promoter activity, ET-1 mRNA, ERK phosphorylation, and activator protein-1-mediated reporter activity. In summary, our results suggest that E2 inhibits Ang-II-induced cell proliferation and ET-1 gene expression, partially by interfering with the ERK pathway via attenuation of ROS generation. Thus, this study provides important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of estrogen on the cardiovascular system. Copyright
In 1889 Dr. John Bland-Sutton, a prominent London surgeon, was consulted about fatal rickets in over 20 successive litters of lion cubs born at the London Zoo. He evaluated the diet and found the cause of rickets to be nutritional in origin. He recommended that goat meat with crushed bones and cod-liver oil be added to the lean horsemeat diet of the cubs and their mothers. Rickets were reversed, the cubs survived, and subsequent litters thrived. Thirty years later, in classic controlled studies conducted in puppies and young rats, the definitive role of calcium, phosphate and vitamin D in prevention and therapy of rickets was elucidated. Further studies led to identifying the structural features of vitamin D.
Although the Bland-Sutton diet provided calcium and phosphate from bones and vitamins A and D from cod-liver oil, some other benefits of this diet were not recognized. Taurine-conjugated bile salts, necessary for intestinal absorption of fat-soluble vitamins, were provided in the oil cold-pressed from cod liver. Unlike canine and rodent species, felines are unable to synthesize taurine, yet conjugate bile acids exclusively with taurine; hence, it must be provided in the diet. The now famous Bland-Sutton “experiment of nature,” fatal rickets in lion cubs, was cured by addition of minerals and vitamin D. Taurine-conjugated bile salts undoubtedly permitted absorption of vitamins A and D, thus preventing the occurrence of metabolic bone disease and rickets.
The effective therapies for oral cancer patients of stage III and IV are generally surgical excision and radiation combined with adjuvant chemotherapy using 5-Fu and Cisplatin. However, the five-year survival rate is still less than 30% in Taiwan. Therefore, evaluation of effective drugs for oral cancer treatment is an important issue. Many studies indicated that aurora kinases (A, B and C) were potential targets for cancer therapies. Reversine was proved to be a novel aurora kinases inhibitor with lower toxicity recently. In this study, the potentiality for reversine as an anticancer agent in oral squamous cell carcinoma (OSCC) was evaluated.
Effects of reversine on cell growth, cell cycle progress, apoptosis, and autophagy were evaluated mainly by cell counting, flow cytometry, immunoblot, and immunofluorescence.
The results demonstrated that reversine significantly suppressed the proliferation of two OSCC cell lines (OC2 and OCSL) and markedly rendered cell cycle arrest at G2/M stage. Reversine also induced cell death via both caspase-dependent and -independent apoptosis. In addition, reversine could inhibit Akt/mTORC1 signaling pathway, accounting for its ability to induce autophagy.
Taken together, reversine suppresses growth of OSCC via multiple mechanisms, which may be a unique advantage for developing novel therapeutic regimens for treatment of oral cancer in the future.
Colorectal carcinoma is a common and often fatal disease in which methods of early detection and monitoring are essential. The present study was conducted for measuring serum levels of nucleosomes, carcinoembryonic antigen (CEA) and CA 19-9 in patients newly diagnosed with colorectal carcinoma and confirmed by clinicopathological study.
Thirty subjects were included in the current study: six normal subjects as a control group with mean age (45.6 ± 7.9) and twenty four colorectal carcinoma patients with mean age (46.9 ± 15.6), which were classified pathologically according to the degree of malignant cell differentiation into well differentiated (group I), moderately differentiated (group II) and poorly differentiated (group III). Fasting venous blood samples were collected preoperative.
The results revealed a significant increase in serum level of nucleosomes in patients with poorly differentiated tumors versus patients with well differentiated tumors (p = 0.041). The levels of CEA and CA19-9 showed no significant increase (p = 0.569 and 0.450, respectively).
In conclusion, serum level of nucleosomes provides a highly sensitive and specific apoptotic marker for colorectal carcinoma.
The pharmacological effects of ethanol are complex and widespread without a well-defined target. Since glutamatergic and GABAergic innervation are both dense and diffuse and account for more than 80% of the neuronal circuitry in the human brain, alterations in glutamatergic and GABAergic function could affect the function of all neurotransmitter systems. Here, we review recent progress in glutamatergic and GABAergic systems with a special focus on their roles in alcohol dependence and alcohol withdrawal-induced seizures. In particular, NMDA-receptors appear to play a central role in alcohol dependence and alcohol-induced neurological disorders. Hence, NMDA receptor antagonists may have multiple functions in treating alcoholism and other addictions and they may become important therapeutics for numerous disorders including epilepsy, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's chorea, anxiety, neurotoxicity, ischemic stroke, and chronic pain. One of the new family of NMDA receptor antagonists, such as DETC-MESO, which regulate the redox site of NMDA receptors, may prove to be the drug of choice for treating alcoholism as well as many neurological diseases.
MiR-1 (microRNA-1) has been used as a positive control in some microRNA experiments. We found that miR-1 transfection of nasopharyngeal carcinoma cells reveals a typical apoptotic process as shown by time-lapse microscopy so we investigated the mechanisms of miR-1 inducing apoptosis.
To confirm that miR-1 induces apoptosis, we used Annexin V and TUNEL staining and caspase assay. To determine that miR-1 directly targets genes that involve in apoptosis, we analyzed microRNA and pathway databases, and cDNA expression microarrays from miR-1 transfected cells. To demonstrate candidate miR-1 targeted genes, we used qRT-PCR analysis and luciferase reporter vector assays. To assess the miR-1 target gene PTMA (prothymosin alpha, ProTalpha) involves in apoptosis, we used PTMA siRNA to knock down PTMA.
Annexin V and TUNEL staining and caspase assay confirm that miR-1 induces nasopharyngeal carcinoma cell apoptosis. MiR-1 transfection of HeLa, Cal-27, KYSE30 and NPC-TW06 cell lines which express low levels of endogenous miR-1 also induces apoptosis. However, miR-1 transfection of cell lines such as SW620, HepG2, HEK-293T, SAS and PC-13 which express high levels of endogenous miR-1 does not result in apoptosis. MiR-1 directly targets PTMA gene. PTMA siRNA and miR-1 accelerate the apoptotic process in cells treated with apoptosis inducers.
The exogenous expression of miR-1 induces apoptosis in a number of cell lines. This is a model of microRNA-induced cell apoptosis. The PTMA is one of miR-1 target genes which involve in miR-1 inducing apoptosis. The apoptotic inducers including actinomycin D, camptothecin and etoposide are also the chemotherapeutic drugs in clinical cancer therapy and PTMA siRNA can accelerate apoptotic progression in cells treated with those apoptosis inducers. Therefore PTMA siRNA may have potential applications as an adjuvant in cancer chemotherapy.
The genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS3 and NS2-3 proteases. Nonstructural protein 4A (NS4A) is a cofactor of the NS3 serine protease and has been demonstrated to inhibit protein synthesis. In this study, GST pull-down assay was performed to examine potential cellular factors that interact with the NS4A protein and are involved in the pathogenesis of HCV. A trypsin digestion followed by LC-MS/MS analysis revealed that one of the GST-NS4A-interacting proteins to be eukaryotic elongation factor 1A (eEF1A). Both the N-terminal domain of NS4A from amino acid residues 1-20, and the central domain from residues 21-34 interacted with eEF1A, but the central domain was the key player involved in the NS4A-mediated translation inhibition. NS4A(21-34) diminished both cap-dependent and HCV IRES-mediated translation in a dose-dependent manner. The translation inhibitory effect of NS4A(21-34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. Taken together, NS4A inhibits host and viral translation through interacting with eEF1A, implying a possible mechanism by which NS4A is involved in the pathogenesis and chronic infection of HCV.
High aluminum (Al) content in certain infant formula raises the concern of possible Al toxicity on brain development of neonates during their vulnerable period of growing. Results of in vivo study showed that Al content of brain tissues reached to 74 μM when oral intake up to 1110 μM, 10 times of that in the hi-Al infant formula.
Utilizing a cultured neuron cells in vitro model, we have assessed Al influence on neuronal specific gene expression alteration by immunoblot and immunohistochemistry and neural proliferation rate changes by MTT assay.
Microscopic images showed that the neurite outgrowth of hippocampal neurons increased along with the Al dosages (37, 74 μM Al (AlCl3)). MTT results also indicated that Al increased neural cell viability. On the other hand, the immunocytochemistry staining suggested that the protein expressions of NMDAR 1A and NMDAR 2A/B decreased with the Al dosages (p < 0.05).
Treated hippocampal neurons with 37 and 74 μM of Al for 14 days increased neural cell viability, but hampered NMDAR 1A and NMDAR 2A/B expressions. It was suggested that Al exposure might alter the development of hippocampal neurons in neonatal rats.
A three-dimensional molecular model of the transmembrane domain of the 5-HT(1A) receptor (5-HT(1A)R) is presented in the context of a general strategy for modeling the macromolecular structure of a guanine nucleotide binding, regulatory protein coupled receptor (GPCR). The model of the 5-HT(1A)R rests on the definition of the putative residues of the ligand-binding site guided by criteria based on specific models proposed from structure-activity studies and on published results of modifications of GPCRs using methods of molecular biology. The resulting requirements for matching recognition sites in the agonist-binding pocket define the molecular details of the interaction between the agonist 5-HT and the human 5-HT(1A)R that includes: (1) the interaction between the protonated amine moiety and the conserved negative Asp-116, located in TMH 3; (2) the hydrogen bond between the hydroxyl group and Thr-199, located in TMH 5; and (3) the interaction complex between the aromatic ring portion of the ligand and the neutral form of His-192, located in TMH 5. Results from quantum mechanical calculations of the interaction between an agonist and the proposed recognition pocket of the 5-HT(1A)R model suggest a trigger of the receptor activation mechanism resulting from ligand binding. The antagonist-binding pocket of the human 5-HT(1A)R is inferred from the interaction sites of pindolol with the receptor model: (1) the ionic interaction between the protonated amine of the ligand and the side chain of the conserved Asp-116, located in TMH 3; and (2) the hydrogen bonds between the ether oxygen and the hydroxyl group of the ligand and Asn-385, located in TMH 7. Use of the model is proposed to facilitate the identification of the structural elements of agonists and antagonists that are key for their specific functions, in order to achieve the design of new compounds with predetermined pharmacological properties. Copyright 1996 S. Karger AG, Basel
Immunotherapy with vaccines is attractive for the treatment of cancer. This study is aimed at determining the effect of recombinant Salmonella (SL3261)-based 4-1BB ligand (4-1BBL) vaccine on the development of colorectal cancers and the potential immune mechanisms in rats.
In comparison with that in the PBS group, similar levels of 4-1BBL expression, the frequency of T cells, IFN-γ responses, and comparable numbers of tumors were detected in the SL3261 and SL3261C groups of rats. In contrast, significantly fewer numbers of tumors, increased levels of 4-1BBL expression in the spleens and colorectal tissues, higher frequency of peripheral blood and splenic CD3+CD25+ T cells, and stronger splenic T cell IFN-γ responses were detected in the SL3261R group of rats.
Our results indicated that vaccination with recombinant attenuated Salmonella harboring the 4-1BBL gene efficiently enhanced T cell immunity and inhibited the development of carcinogen-induced colorectal cancers in rats.
High glucose induced lipid synthesis leads to β cell glucolipotoxicity. Sterol regulatory element binding protein-1c (SREBP-1c) is reported to be partially involved in this process. Insulin induced gene-1 (Insig-1) is an important upstream regulator of Insig-1-SREBPs cleavage activating protein (SCAP)-SREBP-1c pathway. Insig-1 effectively blocks the transcription of SREBP-1c, preventing the activation of the genes for lipid biosynthesis. In this study, we aimed to investigate whether Insig-1 protects β cells against glucolipotoxicity.
An Insig-1 stable cell line was generated by overexpression of Insig-1 in INS-1 cells. The expression of Insig-1 was evaluated by RT-PCR and Western blotting, then, cells were then treated with standard (11.2 mM) or high (25.0 mM) glucose for 0 h, 24 h and 72 h. Cell viability, apoptosis, glucose stimulated insulin secretion (GSIS), lipid metabolism and mRNA expression of insulin secretion relevant genes such as IRS-2, PDX-1, GLUT-2, Insulin and UCP-2 were evaluated.
We found that Insig-1 suppressed the high glucose induced SREBP-1c mRNA and protein expression. Our results also showed that Insig-1 overexpression protected β cells from ER stress-induced apoptosis by regulating the proteins expressed in the IRE1α pathway, such as p-IRE1α, p-JNK, CHOP and BCL-2. In addition, Insig-1 up-regulated the expression of IRS-2, PDX-1, GLUT-2 and Insulin, down-regulated the expression of UCP-2 and improved glucose stimulated insulin secretion (GSIS). Finally, we found that Insig-1 inhibited the lipid accumulation and free fatty acid (FFA) synthesis in a time-dependent manner.
There results suggest that Insig-1 may play a critical role in protecting β cells against glucolipotoxicity by regulating the expression of SREBP-1c.
Sequencing of HIV-1(GER) long terminal repeat (LTR) has demonstrated, for the first time in an HIV-1 primary isolate, a TAR duplication referred to as TAR1 (nucleotides +1 through +68) and TAR2 (nucleotides +69 through +136). This TAR duplication is stable during replication of HIV-1(GER) isolate in CEM cells. Analysis of LTR-CAT reporter constructs demonstrated that under Tat transactivation the HIV-1(GER)/LTR (containing TAR1 and TAR2) was expressed at a higher level than a similar construct (HIV-1(GER)DeltaTAR) containing a single TAR sequence. Among the two transcription initiation sites found in the HIV-1(GER)/LTR, only the most 5' start site was shown to be functionally active. The predicted secondary structure of the 5'-end mRNAs of HIV-1(GER) suggests it may fold into a double TAR hairpin which resembles that of HIV-2. Finally, HIV-1(GER) Tat protein shows primary sequence similarity with Tat proteins from other isolates of HIV-1 and is apparently unrelated to HIV-2 Tat proteins. This work provides the first evidence of a TAR sequence duplication in HIV-1 which increases the efficiency of transactivation by Tat. Copyright 1996 S. Karger AG, Basel
The use of beta-blockers has emerged as a beneficial treatment for cardiac hypertrophy. Hypoxia-inducible factor-1alpha (HIF-1alpha) is tightly regulated in the ventricular myocardium. However, the expression of HIF-1alpha in cardiac hypertrophy due to pressure overload and after treatment with beta-blocker is little known. To evaluate the effect of carvedilol on both myocardial HIF-1alpha expression and cardiac hypertrophy, infra-renal aortic banding was performed for 4 weeks in adult Sprague-Dawley rats to induce cardiac hypertrophy. Carvedilol at 50 mg/kg body weight per day after surgery was given. Heart weight and the ratio of heart weight and body weight increased significantly after aortic banding for 4 weeks in the absence of drug treatment. Mean arterial pressure increased from 80 +/- 9 mmHg in the sham group to 94 +/-5 mmHg (p < 0.001) in the banding group. Echocardiography showed concentric hypertrophy after aortic banding. Mean arterial pressure decreased after treatment with carvedilol. The increased wall thickness and heart weight was reversed to normal by carvedilol. Western blot showed that HIF-1alpha, vascular endothelial growth factor (VEGF) and brain natriuretic peptide (BNP) proteins were up-regulated and nerve growth factor-beta (NGF-beta) down-regulated in the banding group. Treatment with valsartan, doxazosin, or N-acetylcysteine did not significantly affect HIF-1alpha and VEGF proteins expression in the banding groups. Real-time polymerase chain reaction showed that mRNA of HIF-1alpha, VEGF and BNP increased and mRNA of NGF-beta decreased in the banding group. Treatment with carvedilol reversed both protein and mRNA of HIF-1alpha, VEGF, BNP, and NGF-beta to the baseline values. Increased immunohistochemical labeling of HIF-1alpha, VEGF, and BNP in the ventricular myocardium was observed in the banding group and carvedilol again normalized the labeling. In conclusion, HIF-1alpha, VEGF, and BNP mRNA and protein expression were up-regulated, while NGF-beta mRNA and protein was downregulated in the rat model of pressure-overloaded cardiac hypertrophy. Treatment with carvedilol is associated with a reversal of abnormal regulation of HIF-1alpha, VEGF, BNP, and NGF-beta in the hypertrophic myocardium.
A close relationship exists between hypoxia-inducible factor (HIF)-1alpha and pulmonary hypertension. The present study was carried out to explore if there are temporal alterations in HIF-1alpha levels during prolonged hypoxia and after monocrotaline (MCT) treatment. First, young Wistar rats were divided into 5 groups: control, hypoxia-1, hypoxia-2, hypoxia-3 and hypoxia-4. Hypoxic rats were placed in a closed hypobaric chamber (380 mm Hg) for a 1-week (hypoxia-1), 2-week (hypoxia-2), 3-week (hypoxia-3) or 5-week (hypoxia-4) period. Second, other young Wistar rats were divided into 4 groups: control, MCT-1, MCT-2 and MCT-3. MCT-treated rats were injected subcutaneously once with MCT (60 mg/kg) for a 1-week (MCT-1), 2-week (MCT-2) or 3-week (MCT-3) period. Subsequently, pulmonary arterial pressure (Ppa) and the weight ratio of the right ventricle to the left ventricle plus the septum [RV/(LV + S)] were measured, and lungs were obtained for the determination of HIF-1alpha via Western blot analysis. Both hypoxia and MCT induced temporal increases in the Ppa, the ratio RV/(LV + S) and HIF-1alpha levels. A close relationship between the Ppa and HIF-1alpha level was found in both hypoxia- and MCT-treated animals. In addition, the PaO(2) level significantly decreased in rats 1-3 weeks after MCT treatment. These results, along with previous data in the literature, suggest that both chronic hypoxia- and MCT-induced lung hypoxia activate an increase in the production of HIF-1alpha, and result in vascular remodeling and pulmonary hypertension.
The effects of atorvastatin on SDF-1α expression under acute myocardial infarction (AMI) are still unclear. Therefore, our present study is to investigate the roles and mechanisms of atorvastatin treatment on SDF-1α expression in rats with AMI.
Male Sprague–Dawley rats were underwent permanent coronary artery ligation and randomly assigned into four groups as follow: blank control (B), atorvastatin (A), atorvastatin plus L-NAME (A+L-NAME), and atorvastatin plus AMD3100 (A+AMD3100). Rats underwent similar procedure but without ligation were used as group sham operated (S). Atorvastatin (10mg/Kg/d body weight) was administrated by gavage to rats in three atorvastatin treated groups, and L-NAME (40mg/Kg/d body weight) or AMD3100 (5mg/Kg/d body weight) was given to group A+L-NAME or A+AMD3100, respectively.
Comparing with group B, NO production, SDF-1α and CXCR4 expression were significantly up-regulated in three atorvastatin treated groups at the seventh day. However, the increments of SDF-1α and CXCR4 expression in group A+L-NAME were reduced when NO production was inhibited by L-NAME. Anti-inflammatory and anti-apoptotic effects of atorvastatin were offset either by decrease of SDF-1α and CXCR4 expression (by L-NAME) or blockage of SDF-1α coupling with CXCR4 (by AMD3100). Expression of STAT3, a cardioprotective factor mediating SDF-1α/CXCR4 axis induced cardiac protection, was up-regulated most significantly in group A. The effects of atorvastatin therapy on cardiac function were also abrogated either when SDF-1α and CXCR4 expression was diminished or the coupling of SDF-1α with CXCR4 was blocked.
SDF-1α upregulation by atorvastatin in rats with AMI was, at least partially, via the eNOS/NO dependent pathway, and SDF-1α upregulation and SDF-1α coupling with CXCR4 conferred anti-inflammatory and anti-apoptotic effects under AMI setting which we speculated that ultimately contributed to cardiac function improvement.
To investigate the mechanism how Transforming growth factor-beta(TGF-beta) represses Interleukin-1beta (IL-1beta)-induced Proteinase-Activated Receptor-2 (PAR-2) expression in human primary synovial cells (hPSCs). Human chondrocytes and hPSCs isolated from cartilages and synovium of Osteoarthritis (OA) patients were cultured with 10% fetal bovine serum media or serum free media before treatment with IL-1beta, TGF-beta1, or Connective tissue growth factor (CTGF). The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Collagen zymography was performed to assess the activity of Matrix metalloproteinases-13 (MMP-13). It was demonstrated that IL-1beta induces PAR-2 expression via p38 pathway in hPSCs. This induction can be repressed by TGF-beta and was observed to persist for at least 48 hrs, suggesting that TGF-beta inhibits PAR-2 expression through multiple pathways. First of all, TGF-beta was able to inhibit PAR-2 activity by inhibiting IL-1beta-induced p38 signal transduction and secondly the inhibition was also indirectly due to MMP-13 inactivation. Finally, TGF-beta was able to induce CTGF, and in turn CTGF represses PAR-2 expression by inhibiting IL-1beta-induced phospho-p38 level. TGF-beta could prevent OA from progression with the anabolic ability to induce CTGF production to maintain extracellular matrix (ECM) integrity and to down regulate PAR-2 expression, and the anti-catabolic ability to induce Tissue inhibitors of metalloproteinase-3 (TIMP-3) production to inhibit MMPs leading to avoid PAR-2 over-expression. Because IL-1beta-induced PAR-2 expressed in hPSCs might play a significantly important role in early phase of OA, PAR-2 repression by exogenous TGF-beta or other agents might be an ideal therapeutic target to prevent OA from progression.
Lysophosphatidic acid (LPA), a low-molecular-weight lysophospholipid enriched in platelets and mildly oxidized low-density lipoproteins, is known to regulate inflammation and atherosclerosis by binding to its cognate receptors. In this study, we reported that LPA upregulated interleukin-1 beta (IL-1 beta) expression in mouse J774A.1 macrophages. By using pharmacological inhibitors, it was suggested that G(i)/Rho activation and subsequent reactive oxygen species (ROS) production were involved in IL-1 beta induction. In addition, IL-1 beta induction by LPA was also observed in human primary macrophages. In summary, LPA is involved in the processes of inflammation by affecting macrophage behavior.
Interleukin-1beta (IL-1beta) has been recognized as a potent stimulus for the synthesis of prostaglandin (PG), which has been implicated in inflammatory responses of the airways. However, the mechanisms underlying IL-1beta-induced cyclooxygenase (COX) expression and PGE(2) synthesis via activation of p42/p44 and p38 mitogen-activated protein kinases (MAPKs) in human tracheal smooth muscle cells (HTSMCs) are not completely understood. We found that IL-1beta increased COX-2 expression and PGE(2) synthesis in time- and concentration-dependent manners. Both specific phosphatidylcholine-phospholipase C inhibitor (D609) and protein kinase C inhibitor (GF109203X) attenuated IL-1beta-induced responses in HTSMCs. IL-1beta-induced COX-2 expression and PGE(2) synthesis were also inhibited by an inhibitor of MEK1/2 (PD98059) and inhibitors of p38 MAPK (SB203580 and SB202190), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by the transient activation of p42/p44 and p38 MAPKs induced by IL-1beta. Furthermore, IL-1beta-induced activation of nuclear factor-kappaB (NF-kappaB) was inversely correlated with the degradation of IkappaB-alpha in HTSMCs. IL-1beta-induced COX-2 expression and PGE(2) synthesis were inhibited by the NF-kappaB inhibitor pyrrolidinedithiocarbamate. These findings suggest that the expression of COX-2 is correlated with the release of PGE(2) from IL-1beta-challenged HTSMCs, which is mediated, at least in part, through p42/p44 and p38 MAPKs and NF-kappaB signaling pathways in HTSMCs.