Involvement of sphingomyelinase in insulin-induced phosphatidylinositol 3-kinase activation.
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Animals↗
Biomedical subjects
X Shi
Publications and source records attributed to X Shi.
Altered expression and localization of the tight junction protein ZO-1 in primary and metastatic pancreatic cancer.
INTRODUCTION: ZO-1 is a tight junction membrane protein that plays a critical role in cell-cell interaction, proliferation, and differ entiation. AIM: To localize and evaluate the expression of ZO-1 in the normal human pancreas, in pancreatic ductal adenocarcinoma (PDAC), and in chronic pancreatitis (CP). METHODOLOGY AND RESULTS: Northern and Western blot analysis revealed ZO-1 expression in all six tested pancreatic cancer cell lines. Expression of ZO-1 mRNA was increased sixfold in PDAC samples in comparison with normal samples (p = 0.04). Confocal microscopy revealed the presence of ZO-1 in the apical and apicolateral areas of ductular cells in the normal pancreas. Similarly, in CP, ZO-1 was localized at apical and apicolateral areas of small proliferating ductular cells and large metaplastic ducts. In PDAC, however, ZO-1 expression was observed irrespective of whether the cancer cells formed duct-like structures or exhibited a diffuse infiltrating pattern. Metastatic pancreatic cancer cells within lymph nodes displayed variable staining patterns, ranging from apical and apicolateral to a diffuse membranous staining. CONCLUSION: These observations suggest that ZO-1 is overexpressed in PDAC and raise the possibility that this overexpression may confer a metastatic advantage to pancreatic cancer cells.
Adult↗
Vagal cardiac function and arterial blood pressure stability.
This study was designed to investigate the importance of vagal cardiac modulation in arterial blood pressure (ABP) stability before and after glycopyrrolate or atropine treatment. Changes in R-R interval (RRI) and ABP were assessed in 10 healthy young (age, 22 +/- 1.8 yr) volunteers during graded lower body negative pressure (LBNP) before and after muscarinic cholinergic (MC) blockade. Transient hypertension was induced by phenylephrine (1 microg/kg body wt), whereas systemic hypotension was induced by bilateral thigh cuff deflation after a 3-min suprasystolic occlusion. Power spectral densities of systolic [systolic blood pressure (SBP)] and diastolic ABP variability were examined. Both antimuscarinic agents elicited tachycardia similarly without significantly affecting baseline ABP. The increase in SBP after phenylephrine injection (+14 +/- 2 mmHg) was significantly augmented with atropine (+26 +/- 2 mmHg) or glycopyrrolate (+27 +/- 3 mmHg) and associated with a diminished reflex bradycardia. The decrease in SBP after cuff deflation (-9.2 +/- 1.2 mmHg) was significantly greater after atropine (-15 +/- 1 mmHg) or glycopyrrolate (-14 +/- 1 mmHg), with abolished reflex tachycardia. LBNP significantly decreased both SBP and RRI. However, after antimuscarinic agents, the reduction in SBP was greater (P < 0.05) and was associated with less tachycardia. Antimuscarinic agents reduced (P < 0.05) the low-frequency (LF; 0.04-0.12 Hz) power of ABP variability at rest. The LF SBP oscillation was significantly augmented during LBNP, which was accentuated (P < 0.05) after antimuscarinic agents and was correlated (r = -0.79) with the decrease in SBP. We conclude that antimuscarinic agents compromised ABP stability by diminishing baroreflex sensitivity, reflecting the importance of vagal cardiac function in hemodynamic homeostasis. The difference between atropine and glycopyrrolate was not significant.
Adrenergic alpha-Agonists↗
Interleukin-10-mediated inhibition of free radical generation in macrophages.
Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines that are known to be transcriptionally regulated by nuclear factor-kappaB (NF-kappaB). Although still controversial, IL-10 has been shown to inhibit NF-kappaB activation through a process that involves proteolytic degradation of inhibitory subunit IkappaB-alpha. What is not known, however, is the mechanism by which IL-10 exerts its effect on IkappaB-alpha degradation. The present study investigates the possible role of reactive oxygen species (ROS) and their inhibition by IL-10 in NF-kappaB activation and IkappaB-alpha degradation in macrophages. Treatment of the cells with lipopolysaccharide (LPS) caused activation of NF-kappaB and rapid proteolysis of IkappaB-alpha as determined by the electrophoretic mobility shift assay, gene transfection, and Western blot. IL-10 pretreatment inhibited both NF-kappaB activation and IkappaB-alpha degradation. Both of these processes were also inhibited by ROS scavengers, catalase (H(2)O(2) scavenger), and sodium formate (.OH scavenger) but were minimally affected by superoxide dismutase (O scavenger). These results suggests that.OH radicals, formed by an H(2)O(2)-dependent, metal-catalyzed Fenton reaction, play a major role in this process. Electron spin resonance studies confirmed the formation of.OH radicals in LPS-treated cells. Addition of IL-10 inhibited both IkappaB-alpha degradation and generation of.OH radicals in response to LPS stimulation. These results demonstrate, for the first time, direct evidence for the role of IL-10 in ROS-dependent NF-kappaB activation.
Animals↗
Pancreatic cancer: factors regulating tumor development, maintenance and metastasis.
Pancreatic cancer has one of the poorest prognoses of all gastrointestinal malignancies. Today, it is the fourth or fifth leading cause of cancer-related deaths in Western industrialized countries, and the incidence has been increasing throughout the past decades. Insensitivity to growth-inhibitory and apoptotic signals as well as self-sufficiency of growth-promoting factors are hallmarks of the pathogenesis of this malignancy. In pancreatic cancer, a variety of growth factors and their receptors are expressed at increased levels. For example, the concomitant presence of the epidermal growth factor (EGF) receptor and its ligand EGF is associated with enhanced tumor aggressiveness and shorter survival following tumor resection. Furthermore, a number of other growth factors and their receptors, such as nerve growth factor and its receptor, are overexpressed in pancreatic cancer and contribute to its malignant phenotype. Besides factors which directly promote cell proliferation, a variety of other factors such as galectins are upregulated, which influences the tumor environment and the invasiveness of pancreatic cancer cells. In addition, tumor suppressor genes such as KAI1 are expressed at reduced levels, thereby enhancing the ability of pancreatic cells to form metastases. A complex disturbance of factors is present in pancreatic cancer, resulting in a distinct growth advantage which clinically results in rapid tumor progression and poor patient survival.
Genes, Tumor Suppressor↗
Cell surface beta-1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling.
ZP3 is a protein in the mammalian egg coat (zona pellucida) that binds sperm and stimulates acrosomal exocytosis, enabling sperm to penetrate the zona pellucida. The nature of the ZP3 receptor/s on sperm is a matter of considerable debate, but most evidence suggests that ZP3 binds to beta-1,4-galactosyltransferase-I (GalTase) on the sperm surface. It has been suggested that ZP3 induces the acrosome reaction by crosslinking GalTase, activating a heterotrimeric G protein. In this regard, acrosomal exocytosis is sensitive to pertussis toxin and the GalTase cytoplasmic domain can precipitate G(i) from sperm lysates. Sperm from mice that overexpress GalTase bind more soluble ZP3 and show accelerated G protein activation, whereas sperm from mice with a targeted deletion in GalTase have markedly less ability to bind soluble ZP3, undergo the ZP3-induced acrosome reaction, and penetrate the zona pellucida. We have examined the ability of GalTase to function as a ZP3 receptor and to activate heterotrimeric G proteins using Xenopus laevis oocytes as a heterologous expression system. Oocytes that express GalTase bound ZP3 but did not bind other zona pellucida glycoproteins. After oocyte maturation, ZP3 or GalTase antibodies were able to trigger cortical granule exocytosis and activation of GalTase-expressing eggs. Pertussis toxin inhibited GalTase-induced egg activation. Consistent with G protein activation, both ZP3 and anti-GalTase antibodies increased GTP-gamma[(35)S] binding as well as GTPase activity in membranes from eggs expressing GalTase. Finally, mutagenesis of a putative G protein activation motif within the GalTase cytoplasmic domain eliminated G protein activation in response to ZP3 or anti-GalTase antibodies. These results demonstrate directly that GalTase functions as a ZP3 receptor and following aggregation, is capable of activating pertussis toxin-sensitive G proteins leading to exocytosis.
Amino Acid Motifs↗
Characterization of residues and sequences of the carbohydrate recognition domain required for cell surface localization and ligand binding of human lectin-like oxidized LDL receptor.
Lectin-like oxidized low-density lipoprotein receptor (LOX-1) has been cloned from human aortic endothelial cells, and has a sequence identical to that from human lung. Previous studies showed that human LOX-1 can recognize modified LDL, apoptotic cells and bacteria. To further explore the relationship between the structure and function of LOX-1, a mutagenesis study was carried out. Our results showed that the carbohydrate recognition domain (CRD) was the ligand-binding domain of human LOX-1. We also investigated the sequences and residues in CRD that were essential for protein cell surface localization and ligand binding. LOX-1s carrying a mutation on each of six Cys in CRD resulted in a variety of N-glycosylation and failed to be transported to the cell surface. This was strong evidence for the involvement of all six Cys in the intrachain disulfide bonds required for proper folding, processing and transport of LOX-1. The C-terminal sequence (KANLRAQ) was also essential for protein folding and transport, while the four final residues (LRAQ) were involved in maintaining receptor function. Both positive charged (R208, R209, H226, R229 and R231) and non-charged hydrophilic (Q193, S198, S199 and N210) residues were involved in ligand binding, suggesting that ligand recognition of LOX-1 is not merely dependent on the interaction of positively charged residues with negatively charged ligands.
Amino Acid Sequence↗
Expression of the cytoplasmic domain of NodC as an active form in Drosophila S2 cells.
NodC, a membrane protein that catalyzes the synthesis of the chitin oligosaccharide chain, was successfully produced in a soluble form. The truncated NodC gene encoding only the cytoplasmic domain that deletes the hydrophobic N-terminus expressed both cytoplasmic and secreted proteins in Drosophila Schneider 2 cells. The expressed protein maintained the ability to synthesize chitin oligosaccharides, primarily (GlcNAc)4, similar to the native membrane-bound NodC. This evidence suggests that only the large hydrophilic loop of NodC is efficient for enzymatic activity. Moreover, immobilizing the soluble NodC to a solid phase has no effect on the enzymatic activity. This, anchoring NodC is not necessary for its activity.
Journal Article↗
Laboratory evaluations of a toothbrush with diamond-shaped filaments for stiffness and efficacy.
The purpose of these studies was to evaluate four toothbrushes with a similar flat profile for stiffness and efficacy in removing artificial plaque deposits from interproximal areas, or depth of removal from broad tooth surfaces, and to relate these findings to bristle stiffness. The test toothbrush had 5-mil diameter, elongated diamond-shaped bristles (D). Three toothbrushes contained 5-mil (5), 6-mil (6), or 7-mil (7) diameter round bristles. Stiffness studies were performed using the ISO 8627 method. In the efficacy studies, toothbrushing was conducted with a vertical or horizontal brushing motion on simulated anterior or posterior teeth using a brushing weight of 250 g. The bristles were placed at a 90 degrees angle to the tooth surface, and brushing was performed for 15 seconds at two strokes per second with 50 mm strokes. Each of the toothbrush designs was tested 24 times. Interproximal access efficacy (IAE) was recorded as the maximum width of simulated plaque deposit removed. Depth of deposit removal (DDR) was determined using a color comparative scale, where 0 = no deposit change and 4 = maximum removal. The averages of wet and dry stiffness measurements were found to be 7 > D > 6 > 5; all values were in the range accepted as soft texture. The means for total IAE were ranked D > 5 > 6 > 7, and total DDR mean rankings were D > 7 > 6 > 5. For the round bristles, stiffness was inversely related to IAE and paralleled the DDR rankings. Using ANOVA, the D and 5-mil toothbrushes had significantly higher (p < 0.05) IAE means compared to the 6- or 7-mil products. The D toothbrush had significantly higher (p < 0.05) DDR means than the 5-mil product. The diamond-shaped bristles in these flat profile products resulted in improved depth of deposit removal compared to the same design with 5-mil round bristles.
Dental Plaque↗
[Cloning and sequence analysis of light chain gene of human antibody against HBV pre-S2].
OBJECTIVE: To obtain the gene of light chain of human antibody against HBV pre-S2. METHODS: A human antibody against HBV pre-S2 displayed on phage was obtained from the constructed human immunoglobulin phage displaying library through four rounds panning of "adherence-elution-amplification" with Pre-S2 (120aa-145aa) short peptide synthesized and help phage. The positive clone of anti-pre-s2 was characterized by ELISA competitive inhibition assay. The plasmid from the positive clone was amplified by PCR with designed oligonucleotide primers of kaph light chain. The light chain fragment was amplified with PCR and plasmid pUC18 digested by restriction endonuclease SacI & XbaI, respectively, then it was subcloned into the plasmid pUC18 and was transformed into E.coli XL1-blue sensitized by CaCl2. The recombinant plasmid pUC18-kaph was sequenced. RESULTS: The competitive inhibition rate of the positive clone of anti-pre-S2 was 65% at 1:50 dilution. Sequence was compared with data in gene bank through internet and analyzed with Blast. According to the sequence and ELISA competitive inhibition assay the integral gene of kaph light chain of human antibody against HBV-PreS2 was obtained. Its variable region located on 1-328bp including three hypervariable regions (82-105bp, 145~177bp, 274~309bp) and four frame regions (1-81bp, 106-144bp, 178-273bp, 310-328bp). Its constant region was located on 329-645bp. CONCLUSIONS: The HBV pre-S2 kaph light chain is screened from phage antibody displaying library by antigen-antibody special response.
Base Sequence↗
Reactivity of free radicals on hydroxylated quartz surface and its implications for pathogenicity experimental and quantum mechanical study.
We studied the adsorption of hydroxyl radicals and superoxide anion radicals on a hydroxylated alpha-quartz surface using cluster and periodic slab models by means of density functional calculations. Models of two hydroxylated alpha-quartz surfaces--(0001) and (0111)--have been used in the simulations. The hydroxyl radical adsorbs readily on both surfaces. The subsurface Si-O bonds are weakened during the adsorption resulting in surface layer destabilization. This destabilization leads directly to surface disintegration in the case of *OH/(0111) adsorption. The product of the surface disintegration and reconstruction is a surface terminated by silanol groups (Si-OH) and siloxyl radicals (Si-O*). The model calculations suggest that adsorption of *OH on a hydroxylated quartz surface transforms a chemically inert, aged, silanol terminated surface to a very reactive, silicon-based radical terminated surface. The activated surface may then cause oxidative damage to the adsorbed biomaterial. The superoxide anion radical adsorbs on both surfaces, but the adsorption products are only weakly bonded to the surface. The calculated energy barrier for the O2*- activated subsurface Si-O bond dissociation is 10 kcal/mol, which is higher than for the *OH activated process (4 kcal/mol). The calculated weaker bonding to the surface and higher activation energy barrier suggest that the superoxide anion radical will be less efficient in reactivation of an aged, hydroxylated quartz surface than the hydroxyl radical. The importance of the specific geometry of the surface silicon atoms on the surface reactivity and adsorption properties is also discussed. The theoretical predictions are supported experimentally using chemiluminescence to monitor reactivation of the aged silica surface by superoxide anion radicals.
Adsorption↗
Reactive oxygen species and molecular mechanism of silica-induced lung injury.
Silica particles are considered to be fibrogenic and carcinogenic agents, but the mechanisms of disease initiation and progression are not fully understood. This article summarizes the literature on the generation of reactive oxygen species (ROS) directly from interaction of silica with aqueous medium and from silica-stimulated cells. This article also discusses the role of ROS in silica-induced lung injury, with particular focus on the silica-induced NF-kappaB activation, including the molecular mechanisms of its regulation, its possible attenuation, and its relationship to silica-induced generation of cyclooxygenase II and TNF-alpha.
Air Pollutants, Occupational↗
Effect of Cr(VI) exposure on sperm quality: human and animal studies.
The semen status of male workers occupationally exposed to hexavalent chromium(VI) was investigated. Sperm counts from exposed workers were 47.05+/-2.13 x 10(6)/ml and those from control group 88.96+/-3.40 x 10(6)/ml. Sperm motility decreased from 81.92+/-0.41% for the control group to 69.71+/-0.93% for the exposed workers. The levels of zinc, lactate dehydrogenase (LDH), and lactate dehydrogenase C4 isoenzyme (LDH-x) in seminal plasma for the exposed workers were 1.48+/-0.07 micromol/ml, 1.05+/-0.02 x 10(3) U, and 0.47+/-0.01 x 10(3) U, respectively, which were significantly lower than those of 5.72+/-0.15 micromol/ml, 1.49+/-0.02 x 10(3) U, and 0.78+/-0.15 x 10(3) U for the control group, respectively. Follicle stimulating hormone (FSH) (7.34+/-0.34 x 10(-3) IU/ml) in serum from the exposed workers was significantly higher than that (2.41+/-0.08 x 10(-3) IU/ml) from the control group. On the other hand, there were no significant differences in semen volume, semen liquefaction time, luteinizing hormone (LH) level in serum, and Cr concentration in both serum and seminal plasma between the exposed workers and the control group. Feeding Cr(VI) to rats significantly reduced the epididymal sperm counts from 87.40+/-3.85 x 10(6)/g epididymis in control group to 21.40+/-1.20 x 10(6)/g epididymis at a CrO(3) dose of 10 mg/kg body weight and to 17.48+/-1.04 x 10(6)/g epididymis at a CrO(3) dose of 20 mg/kg body weight. Exposure of rats to Cr(VI) also significantly increased the sperm abnormality from 2.75+/-0.06% in the control group to 6.68+/-0.32% in the exposed group at a CrO(3) dose of 10 mg/kg body and to 7.6+/-0.15% at a CrO(3) dose of 20 mg/kg body weight. In exposed rats, there was visible disruption in germ cell arrangement near the walls of the seminiferous tubules. The diameters of seminiferous tubules in exposed rats were smaller. These results suggest that occupational exposure to chromium(VI) leads to alteration of semen status and may affect the reproductive success of exposed workers.
Animals↗
[Studies on mimotopes of hepatitis C virus E1 protein].
OBJECTIVE: To study the B-cell epitope of E1 protein of hepatitis C virus. METHODS: By induction of IPTG, the E.coli M15 strains harboring the pQE30-HCVe118 expressed truncated C-terminal HCV E1 protein (Pte1). The proteins were purified with preparative electrophoreses system, which captured anti-E1 IgG in HCV (+) sera. By applying the antibodies as selective molecular 12 mers random peptide libraries were panned, and positive clones were obtained by ELISA. Amino acid sequences of display peptide were compared with that of HCV E1 protein. RESULTS: The purified HCV E1 proteins could react specifically with partly anti-HCV sera by ELISA. Among 10 phage display peptides, 6, 6, 2 were the most homologous to HCV E1 protein at position 320-336aa, 251-263aa, 225-248, respectively. CONCLUSIONS: There exist multiple B-cell epitopes in HCV E1 protein. At least one preponderant epitope is mapped at residues 320-336 of HCV E1 protein.
Amino Acid Motifs↗
Transactivation of RARE and GRE in the cellular response to arsenic.
Arsenic compounds are a somewhat unique class of metals, which have been considered as both carcinogens and chemotherapeutic agents for cancers. Tumor promotion effects of arsenic are believed to be associated with its transactivational activities on transcription factors, such as AP-1 and NFkappaB, while the induction of cell apoptosis and differentiation by arsenic is considered to be a mechanism for the chemotherapeutic effects of arsenic. Here, we found that exposure of cells to arsenite and arsenate leads to transactivation of retinoic acid response elements (RARE) and glucocorticoid response elements (GRE) in mouse epidermal JB6 cells. These inductions occur in a time-dependent manner. Furthermore, induction of RARE activity by arsenic was synergistically enhanced by co-treatment of cells with retinoic acid, while GRE activation by arsenic was not affected by combined treatment of cells with fluocinolone acetonide (FA). In consideration of the important role of RARE and GRE in induction of cell differentiation, we speculate that transactivation of RARE and GRE by arsenic may be involved in its induction of cell differentiation and anti-cancer activities in addition to its induction of apoptosis.
Animals↗
Carcinogenic metals and NF-kappaB activation.
Epidemiological and animal studies suggest that several metals and metal-containing compounds are potent mutagens and carcinogens. These metals include chromium, arsenic, vanadium, and nickel. During the last two decades, chemical and cellular studies have contributed enormously to our understanding of the mechanisms of metal-induced pathophysiological processes. Although each of these metals is unique in its mechanism of action, some common signaling molecules, such as reactive oxygen species (ROS), may be shared by many of these carcinogenic metals. New techniques are now available to reveal the mechanisms of carcinogenesis in precise molecular terms. In this review, we focused our attentions on metal-induced signal transduction pathways leading to the activation of NF-kappaB, a transcription factor governing the expression of most early response genes involved in a number of human diseases.
Animals↗
Cell apoptosis induced by carcinogenic metals.
Well-documented evidence suggests that environmental and occupational exposure of toxic metals or metal-containing compounds can cause a number of human diseases, including inflammation and cancer, through DNA damage, protein modifications, or lipid peroxidation. This mini-review addresses the mechanisms of cell death induced by some carcinogenic metals, including arsenic (III), chromium (VI) and vanadium (V). A possible contribution of reactive oxygen species to metal-induced cell death is also discussed.
Animals↗
Gene expression profile in response to chromium-induced cell stress in A549 cells.
Chromium (Cr) is a trace element required for life. Biological activities of Cr are complicated and remain to be fully investigated. It is known that the valence of Cr plays an important role in the biological activities of Cr. For example, Cr (VI) is classified as a metal carcinogen, but Cr (III) is widely used as a nutritional supplement. Establishment of a gene expression profile for Cr-induced cellular response is necessary to facilitate investigation of the biological activities of Cr. In the present study, a large-scale gene expression analysis was conducted using RNA of human lung epithelial cells after in vitro exposure to Cr (VI). Utilizing high-density oligonucleotide arrays representing 2400 genes, we observed that expression of 150 genes was up-regulated, and that of 70 genes were down-regulated by Cr (VI). Functional analysis of these responsive genes led to an outline of potential biological activities of Cr in six aspects. The gene expression profile reveals that Cr may involves in redox stress, calcium mobilization, energy metabolism, protein synthesis, cell cycle regulation and carcinogenesis in the cell. The results provide a critical clue for understanding molecular mechanisms of the biological activities of Cr.
Calcium↗