Sequential activation of cyclin E and cyclin A gene expression by human papillomavirus type 16 E7 through sequences necessary for transformation.

To investigate E7-dependent biochemical changes which are involved in cellular transformation, we analyzed the influence of human papillomavirus type 16 (HPV-16) E7 on the expression of cell cycle regulatory proteins. Expression of E7 in established rodent fibroblasts (NIH 3T3), which was shown to be sufficient for transformation of these cells, leads to constitutive expression of the cyclin E and cyclin A genes in the absence of external growth factors. Surprisingly, expression of the cyclin D1 gene, which encodes a major regulator of G1 progression, is unaltered in E7-transformed cells. In transient transfection experiments, the cyclin A gene promoter is activated by E7 via an E2F binding site. In 14/2 cells, which were used as a model system to analyze the role of HPV-16 E7 in the transformation of primary cells, we observed rapid E7-dependent activation of cyclin E gene expression, which can be uncoupled from activation of the cyclin A gene, since the latter requires additional protein synthesis. E7-driven induction of cyclin E and cyclin A gene expression was accompanied by an increase in the associated kinase activities. Two domains of the E7 oncoprotein, which are designated cd1 and cd2, are essential for transformation of rodent fibroblasts. It is shown here that growth factor-independent expression of the cyclin E gene requires cd2 but not cd1, while activation of cyclin A gene expression requires cd1 function in addition to that of cd2. These data suggest that cyclin A gene expression is controlled by two distinct negative signals, one of which also restricts expression of the cyclin E gene. The ability of E7 to separately override each of these inhibitory signals, via cd1 and cd2, cosegregates with its ability to fully transform rodent fibroblasts. Unlike serum growth factors, E7 induces S-phase entry without activating cyclin D1 gene expression, in keeping with the finding that cyclin D1 function is not required in cells transformed by DNA tumor viruses.     

The tetraspanin CD81 regulates the expression of CD19 during B cell development in a postendoplasmic reticulum compartment

CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81(-/-) B cells express lower levels of CD19, especially cd81(-/-) small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9(-/-) B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81(-/-) B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81(-/-) pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81(-/-) B cells. The higher M(r) glycoform is significantly reduced in cd81(-/-) B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M(r) glycoform is comparably expressed in cd81(-/-) and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.     

香烟烟雾提取物对小鼠气管环上皮钙粘附素表达的影响

体外进行器官培养,通过免疫组织化学和原位杂交方法检测香烟烟雾提取物（CSE）作用48、72h,小鼠气管上皮细胞上皮钙粘附素（E－cd）表达的变化。结果表明,正常对照组E－cd分布于气管假复层纤毛柱状上皮细胞连接处的胞膜上,50％CSE作用后,膜上E－cd表达减少,随着作用时间延长,胞浆内E-cd表达明显增加（P＜0.05）,而细胞内E－cd mRNA含量无明显变化。提示,CSE对小鼠气管上皮细胞E-cd表达的调控在翻译后水平,胞膜上E－cd表达的下调与吸烟所致气道上皮细胞的损伤修复过程相关。     

吸烟小鼠呼吸道上皮细胞上皮钙粘附素表达的研究

通过免疫荧光法检测不同吸烟时间小鼠呼吸道上皮细胞上皮钙粘附素（Ｅｃａｄｈｅｒｉｎ,Ｅｃｄ）表达的动态变化,并采用显微荧光光度术进行定量分析。结果表明,首次吸烟１ｈ后呼吸道上皮细胞Ｅｃｄ表达与正常对照组比较明显下调（Ｐ＜００１）,４６Ｗ后降到最低水平。以后逐渐上调,吸烟８Ｗ后Ｅｃｄ表达恢复正常,气管上皮细胞表达高于正常（Ｐ＜００１）。提示,Ｅｃｄ表达的动态变化在呼吸道上皮细胞损伤与修复过程中起重要作用     

Moderate exercise leads to decreased expression of β1 and β2 integrins on leucocytes

 Intravascular adhesion of leucocytes plays a role in the pathogenesis of acute and chronic vascular disease. Regular aerobic exercise seems to protect against vascular disease. Since leucocyte adhesion is mediated by integrins, we tested the hypothesis that surface expression of the integrin adhesive receptors LFA-1 (cd11a/cd18), MAC-1 (cd11b/cd18), gp 150/95 (cd11c/cd18), and VLA-4 (cd29/cd49) is decreased by moderate endurance exercise. Surface expression of integrins was measured by FACS analysis in 19 healthy subjects (16 males, 3 females, 36.6 ± 8.7 years, 177.1 ± 7.5 cm, 70.3 ± 8.1 kg) before and after submaximal exercise (3 h run) using monoclonal antibodies against cd11a, cd11b, cd11c, cd18, cd29 and cd49. In addition, we compared resting integrin expression in this group with a group of sedentary subjects (19 males, 6 females, 29.3 ± 5.3 years). White blood cell count increased from 5300 ml^–1 to 9740 ml^–1 during exercise (P<0.001). Nevertheless, the expression (indicated by the mean log fluorescence) of cd11a (94 ± 24 vs. 78 ± 14) and cd18 (128 ± 31 vs. 102 ± 21) on lymphocytes and of cd11a (104 ± 25 vs. 85 ± 16), cd11c (497 ± 171 vs. 408 ± 126) cd29 (109 ± 16 vs. 89 ± 16), cd49 (69± 8 vs. 54 ± 11) on monocytes was decreased after exercise (all P<0.05). In contrast, integrin expression on granulocytes was not altered by exercise. Comparison of exercising and sedentary subjects showed a significantly decreased expression of integrins in exercising subjects. Our results demonstrate that moderate exercise leads to decreased expression of integrin receptors on leucocytes. This decreased expression of adhesion molecules may result in decreased adhesion and infiltration of leucocytes into the vessel wall. This phenomenon may play a role in the beneficial effect of moderate exercise in prevention of acute and chronic vascular disease. Accepted: 18 March 1997     

CD9 tetraspanin interacts with CD36 on the surface of macrophages: a possible regulatory influence on uptake of oxidized low density lipoprotein

CD36 is a type 2 scavenger receptor with multiple functions. CD36 binding to oxidized LDL triggers signaling cascades that are required for macrophage foam cell formation, but the mechanisms by which CD36 signals remain incompletely understood. Mass spectrometry analysis of anti-CD36 immuno-precipitates from macrophages identified the tetraspanin CD9 as a CD36 interacting protein. Western blot showed that CD9 was precipitated from mouse macrophages by anti-CD36 monoclonal antibody and CD36 was likewise precipitated by anti-CD9, confirming the mass spectrometry results. Macrophages from cd36 null mice were used to demonstrate specificity. Membrane associations of the two proteins on intact cells was analyzed by confocal immunofluorescence microscopy and by a novel cross linking assay that detects proteins in close proximity (<40 nm). Functional significance was determined by assessing lipid accumulation, foam cell formation and JNK activation in wt, cd9 null and cd36 null macrophages exposed to oxLDL. OxLDL uptake, lipid accumulation, foam cell formation, and JNK phosphorylation were partially impaired in cd9 null macrophages. The present study demonstrates that CD9 associates with CD36 on the macrophage surface and may participate in macrophage signaling in response to oxidized LDL.     

E-cd在胃癌、癌旁组织及转移淋巴结中的表达

研究E-cd在胃癌、癌旁组织及转移淋巴结中的表达。用免疫组化方法检测E-cd的表达,用图像分析仪进行定量分析。结果显示胃癌及转移淋巴结中E－cd的表达明显低于癌旁组织（P＜0.001）。分化不良胃癌中E－cd的表达明显低于分化良好胃癌中的表达（P＜0.05）。结果表明E-cd可作为判断胃癌预后的指标。     

Identification of a major enzyme for the synthesis and hydrolysis of cyclic ADP-ribose in amphibian cells and evolutional conservation of the enzyme from human to invertebrate

Cyclic ADP-ribose (cADPR), a metabolite of NAD(+), is known to function as a second messenger for intracellular Ca(2+) mobilization in various vertebrate and invertebrate tissues. In this study, we isolated two Xenopus laevis cDNAs (frog cd38 and cd157 cDNAs) homologous to the one encoding the human cADPR-metabolizing enzyme CD38. Frog CD38 and CD157 are 298-amino acid proteins with 35.9 and 27.2 % identity to human CD38 and CD157, respectively. Transfection of expression vectors for frog CD38 and CD157 into COS-7 cells revealed that frog CD38 had NAD(+) glycohydrolase, ADP-ribosyl cyclase (ARC), and cADPR hydrolase activities, and that frog CD157 had no enzymatic activity under physiological conditions. In addition, when recombinant CD38 and frog brain homogenate were electrophoresed on an SDS-polyacrylamide gel, ARC of the brain homogenate migrated to the same position in the gel as that of frog CD38, suggesting that frog CD38 is the major enzyme responsible for cADPR metabolism in amphibian cells. The frog cd38 gene consists of eight exons and is ubiquitously expressed in various tissues. These findings provide evidence for the existence of the CD38-cADPR signaling system in frog cells and suggest that the CD38-cADPR signaling system is conserved during vertebrate evolution.     

Upper thermal tolerance of larval Arctic lamprey (Lethenteron camtschaticum)

Ichthyological Research - The population of Arctic lamprey (Lethenteron camtschaticum) is declining at the southern limit of its distribution in the Northern Hemisphere. Rising river water...     

Double-stranded RNA induces galectin-9 in vascular endothelial cells: involvement of TLR3, PI3K, and IRF3 pathway

Galectin-9 is a member of the galectin family, which induces various biological reactions such as chemotaxis of eosinophils and apoptosis of T cells. We previously reported that polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA (dsRNA), induces the expression of galectin-9 in human umbilical vein endothelial cells (HUVECs). In the present study, we addressed the possible involvement of two potential receptors for dsRNA, Toll-like receptor (TLR) 3 and retinoic acid-inducible gene-I (RIG-I), in the expression of galectin-9 in HUVECs. Poly IC-induced galectin-9 expression was almost completely suppressed by RNA interference (RNAi) against TLR3, but not against RIG-I. LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited the induction of galectin-9 by poly IC. RNAi against interferon regulatory factor 3 (IRF3) also inhibited poly IC-induced galectin-9 expression. We conclude that TLR3, PI3K, and IRF3 are involved in the poly IC-induced galectin-9 expression in HUVECs.     

Expression of peroxisome proliferator-activated receptors in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) depending on gender and developmental stage

Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors involved in embryo development and differentiation of several tissues in mammals. The aim of the present study was to investigate the possible differential expression of the three PPAR subtypes (PPAR, PPAR, and PPAR) in relation to gender and developmental stage in zebrafish. For this purpose PPAR expression was assessed by immunohistochemistry in 7-day-old larvae, 1-month-old juveniles, and 1-year-old adults. Additionally, the activity of peroxisomal acyl-CoA oxidase (AOX), a gene regulated by PPARs, and the volume density of catalase-immunolabeled liver peroxisomes (V_VP) was examined. No significant gender-related differences were detected in the tissue distribution of the three PPAR subtypes or in peroxisomal AOX activity and V_VP. The percentage of PPAR-positive hepatocytes was significantly higher in females than in males suggesting a specific regulatory role of this subtype in female zebrafish. The three PPAR subtypes were already expressed at the larval stage, with a similar tissue distribution pattern to that found in adults. For all stages, PPAR and PPAR were expressed at higher levels than PPAR, and PPAR immunolabeling was stronger in juveniles than in larval or adult stages. The percentages of hepatocyte nuclei immunolabeled for PPARs was higher in early developmental stages than in adults, similarly to AOX activity and V_VP. In conclusion, our results indicate that PPAR expression, the activity of its target gene AOX, and peroxisomal biogenesis are developmentally modulated in zebrafish.     

The zinc-binding motif of TRPM7 acts as an oxidative stress sensor to regulate its channel activity

The activity of the TRPM7 channel is negatively regulated by intracellular Mg²⁺. We previously reported that oxidative stress enhances the inhibition of TRPM7 by intracellular Mg²⁺. Here, we aimed to clarify the mechanism underlying TRPM7 inhibition by hydrogen peroxide (H₂O₂). Site-directed mutagenesis of full-length TRPM7 revealed that none of the cysteines other than C1809 and C1813 within the zinc-binding motif of the TRPM7 kinase domain were involved in the H₂O₂-induced TRPM7 inhibition. Mutation of C1809 or C1813 prevented expression of full-length TRPM7 on the plasma membrane. We therefore developed an assay to functionally reconstitute full-length TRPM7 by coexpressing the TRPM7 channel domain (M7cd) and the TRPM7 kinase domain (M7kd) as separate proteins in HEK293 cells. When M7cd was expressed alone, the current was inhibited by intracellular Mg²⁺ more strongly than that of full-length TRPM7 and was insensitive to oxidative stress. Coexpression of M7cd and M7kd attenuated the inhibition by intracellular Mg²⁺ and restored sensitivity to oxidative stress, indicating successful reconstitution of a full-length TRPM7-like current. We observed a similar effect when M7cd was coexpressed with the kinase-inactive mutant M7kd-K1645R, suggesting that the kinase activity is not essential for the reconstitution. However, coexpression of M7cd and M7kd carrying a mutation at either C1809 or C1813 failed to restore the full-length TRPM7-like current. No reconstitution was observed when using M7kd carrying a mutation at H1750 and H1807, which are involved in the zinc-binding motif formation with C1809 and C1813. These data suggest that the zinc-binding motif is essential for the intracellular Mg²⁺-dependent regulation of the TRPM7 channel activity by its kinase domain and that the cysteines in the zinc-binding motif play a role in the oxidative stress response of TRPM7.     

Cannabinoid receptor 1 modulates the autophagic flux independent of mTOR‐ and BECLIN1‐complex

Cannabinoid Receptor 1 (CB1) has been initially described as the receptor for Delta‐9‐Tetrahydrocannabinol in the central nervous system (CNS), mediating retrograde synaptic signaling of the endocannabinoid system. Beside its expression in various CNS regions, CB1 is ubiquituous in peripheral tissues, where it mediates, among other activities, the cell's energy homeostasis. We sought to examine the role of CB1 in the context of the evolutionarily conserved autophagic machinery, a main constituent of the regulation of the intracellular energy status. Manipulating CB1 by siRNA knockdown in mammalian cells caused an elevated autophagic flux, while the expression of autophagy‐related genes remained unaltered. Pharmacological inhibition of CB1 activity using Rimonabant likewise caused an elevated autophagic flux, which was independent of the mammalian target of rapamycin complex 1, a major switch in the control of canonical autophagy. In addition, knocking down coiled‐coil myosin‐like BCL2‐interacting protein 1, the key‐protein of the second canonical autophagy control complex, was insufficient to reduce the elevated autophagic flux induced by Rimonabant. Interestingly, lysosomal activity is not altered, suggesting a specific effect of CB1 on the regulation of autophagic flux. We conclude that CB1 activity affects the autophagic flux independently of the two major canonic regulation complexes controlling autophagic vesicle formation.

     

Reduced Expression of Galectin-9 Contributes to a Poor Outcome in Colon Cancer by Inhibiting NK Cell Chemotaxis Partially through the Rho/ROCK1 Signaling Pathway

Galectin-9 is a widely expressed protein that is involved in immune regulation and tumorpathogenesis and serves as a marker of a poor prognosis in various types of cancers. However, the clinical impact and the precise mechanism by which this protein contributes to colon tumor progression are unclear. In the present study, we detected the expression of galectin-9 and CD56 cells using immunohistochemistry. Spearman''s rank correlation was used to clarify the association between galectin-9 expression and natural killer (NK) cell infiltration. The influence of galectin-9 on NK-92 cell migration was evaluated in vitro using transwell chemotaxis assays. The role of rh-galectin-9 in F-actin polarization in NK-92 cells was investigated using laser scanning confocal microscopy. We showed that galectin-9 was expressed in 101 (78.91%) colon tumor tissues and that was expressed at lower levels in these tissues than in para-tumor tissues. Low levels of galectin-9 expression were positively correlated with a poor histological grade and lymph node metastasis (P<0.05). A Kaplan-Meier method and Cox proportional hazards regression analysis showed that overall survival was longer in patients with high galectin-9 expression in an 8-year follow-up (P<0.05). Spearman''s rank correlation indicated that there was a linear correlation between galectin-9 expression and CD56⁺ NK cell infiltration (R² = 0.658; P<0.0001). Galectin-9 stimulated migration in human NK-92 cells by affecting F-actin polarization through the Rho/ROCK1 signaling pathway. These results suggest that galectin-9 expression potentially represents a novel mechanism for tumors to escape immune surveillance in colon tumors.     

Downregulation of CD9 in Keratinocyte Contributes to Cell Migration via Upregulation of Matrix Metalloproteinase-9

Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.