HOMOLOGOUS REGULATION OF MELANOCORTIN-1 RECEPTOR (MC1R) EXPRESSION IN MELANOMA TUMOR CELLS IN VIVO

ABSTRACT

As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist α-melanocyte-stimulating hormone (α-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B16F1 cells, a single injection of 50 to 500?µg α-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16–24?h. By continuous administration of α-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.     

十二指肠上皮细胞转铁蛋白受体表达调控的免疫组织化学观察与铁吸收机制的探讨

转铁蛋白受体（ＴｆＲ）在细胞的铁转运方面起重要作用。本研究用免疫组织化学法比较铁缺乏（甲组）、铁过剩（乙组）与对照组（丙组）Ｗｉｓｔａｒ系大鼠活体十二指肠上皮细胞ＴｆＲ的表达调控及其在铁吸收过程中的意义。结果表明：甲组十二指肠上皮细胞内ＴｆＲ表达明显强于雨组,乙组ＴｆＲ表达最弱。可见ＴｆＲ的表达在一定范围内随体内铁含量的增高或降低而减弱或增强,受铁状态的负调节。丙组ＴｆＲ主要位于细胞基底部,游离面ＴｆＲ表达不明显。甲组细胞ＴｆＲ表达增强,基底部可见线状ＴｆＲ高强度表达。乙组虽ＴｆＲ表达明显减弱,但在细胞基底部仍可见线状ＴｆＲ表达。提示十二指肠上皮细胞基底部有ＴｆＲ介导的铁转运,而铁从上皮的游离面进入细胞内并非由ＴｆＲ介导。三组动物小肠粘膜固有层中均可见ＴｆＲ阳性的巨噬细胞。在铁过剩组、此巨噬细胞数量增加,且ＴｆＲ表达未受明显抑制。认为铁过剩时,粘膜固有层的巨噬细胞内可贮存过量的铁,减少其对实质细胞、组织的损伤。     

花椰菜花叶病毒(CaMV)的基因表达调控

花椰菜花叶病毒（CaMV）是一种具有重要经济价值和生物学意义的植物双链DNA病毒,它有7个开放阅读框（ORF）,其中6个呆各自编码一种蛋白产物。35S启动子区域含有3个转录因子专一的结合位点;RNA多腺苷化位点具有AAUAAA特征序列,它和其上游序列对35SRNA的加工和翻译有影响作用;下游ORFI在转录激活因子存在时可被翻译。由此表明,CaMV的表达调控表现在不同调控机制工作的基础之上。     

斑马鱼HO1基因的表达特征及功能研究

实验对血红素加氧酶1（HO1）在斑马鱼发育中的功能进行了研究。多重序列比对结果显示,斑马鱼HO1与哺乳类、鸟类及其他鱼类的HO1氨基酸序列的总体相似性为44.1%86.8%,血红素结合标签相似性为87.5%95.8%。对斑马鱼早期胚胎和成鱼各组织进行RT-PCR检测,结果显示HO1转录本母源存在,HO1 mRNA的表达水平在尾芽期前较低,到咽囊期迅速上升并稳定在较高水平。HO1基因在斑马鱼成鱼多个组织中均有表达,在肝脏、脾、鳃、肾中的表达量较高。WISH结果显示,HO1基因在斑马鱼胚胎的卵黄合胞层、眼和血液中的表达量较高。利用超表达和基因敲降技术发现,注射HO1 mRNA使HO1基因过表达对斑马鱼早期胚胎发育无明显影响。注射HO1 MO使HO1基因表达抑制可导致斑马鱼胚胎出现发育迟缓、围心腔水肿、尾部消失等不同程度的畸形。HO1 MO导致的斑马鱼胚胎发育异常可被HO1 mRNA回复。利用Real-Time PCR研究发现,HO1基因表达抑制可导致IGF1表达量显著下降,IGFBP1表达量显著升高。这些结果表明斑马鱼HO1基因可通过调节IGF信号途径调控胚胎的正常发育。       

斑马鱼 HO1 基因的表达特征及功能研究简

实验对血红素加氧酶1(HO1)在斑马鱼发育中的功能进行了研究。多重序列比对结果显示,斑马鱼HO1与哺乳类、鸟类及其他鱼类的HO1氨基酸序列的总体相似性为44.1%—86.8%,血红素结合标签相似性为87.5%—95.8%。对斑马鱼早期胚胎和成鱼各组织进行RT-PCR检测,结果显示HO1转录本母源存在,HO1mRNA的表达水平在尾芽期前较低,到咽囊期迅速上升并稳定在较高水平。HO1基因在斑马鱼成鱼多个组织中均有表达,在肝脏、脾、鳃、肾中的表达量较高。WISH结果显示,HO1基因在斑马鱼胚胎的卵黄合胞层、眼和血液中的表达量较高。利用超表达和基因敲降技术发现,注射HO1 mRNA使HO1基因过表达对斑马鱼早期胚胎发育无明显影响。注射HO1 MO使HO1基因表达抑制可导致斑马鱼胚胎出现发育迟缓、围心腔水肿、尾部消失等不同程度的畸形。HO1 MO导致的斑马鱼胚胎发育异常可被HO1 mRNA回复。利用Real-Time PCR研究发现,HO1基因表达抑制可导致IGF1表达量显著下降,IGFBP1表达量显著升高。这些结果表明斑马鱼HO1基因可通过调节IGF信号途径调控胚胎的正常发育。     

PHOTOPERIODIC REGULATION OF MT1 AND MT2 MELATONIN RECEPTOR EXPRESSION IN SPLEEN AND THYMUS OF A TROPICAL RODENT FUNAMBULUS PENNANTI DURING REPRODUCTIVELY ACTIVE AND INACTIVE PHASES

Photoperiodic regulation of melatonin receptor types on target tissues, such as lymphatic organs, has never been explored for any seasonal breeder. In the present study, we accessed the high affinity membrane melatonin receptors MT1 and MT2 expression dynamics in lymphoid organs (i.e., spleen and thymus) of a seasonally breeding rodent Funambulus pennanti during two major reproductive phases (i.e., active and inactive), when the internal hormonal (melatonin and gonadal steroid) as well as the ecological conditions were entirely different. Photoperiod regulates circulatory melatonin level; hence, we noted the effect of different photoperiodic regimes (long; 16L:8D and short; 10L:14D photoperiod) equivalent to summer and winter daylength on membrane melatonin receptor MT1 and MT2 expression in spleen and thymus. We have correlated the melatonin receptor expression with two major hormones varying seasonally (i.e., melatonin and testosterone) also being responsible for modulation of immunity of a seasonal breeder. Differential immunoreactivity of MT1 and MT2 receptor in spleen and thymus of F. pennanti suggests an involvement of both the receptor types in signal transduction of photoperiod for seasonal immunomodulation, because in the tropical zone, a slight difference (1:45–2?h) in daylength may change reproductive physiology and immunity of animals for adaptation. Our above suggestion receives strong support from the experiment of photoperiodic exposure on MT1 and MT2 expression at the translational level, where long daylength decreased the circulatory melatonin level and melatonin receptor expression in both lymphatic tissues. On the other hand, under short daylength, expression of MT1 and MT2 receptor increased in both spleen and thymus along with concomitant increase in circulatory melatonin level. Differential hormonal level of melatonin and gonadal hormones during reproductively active and inactive phase and its direct relation with melatonin receptor expression dynamics in lymphoid organs could be responsible for seasonal adjustment of immunity and reproduction. (Author correspondence: chaldar2001@yahoo.com)     

DURATIONS OF GAMETE MOTILITY AND CONJUGATION ABILITY OF ULVA COMPRESSA (ULVOPHYCEAE)1

The present study was designed to develop a technique for crossing and to gain insight into how sexual reproduction contributes to the maintenance of local populations of Ulva compressa L. To examine the durations of gamete motility and conjugation ability, freshly released gametes were incubated for various periods of time prior to mixing both mating types. The conjugation ability of the gametes gradually declined after being released from the thalli when the gametes were incubated without mixing with the opposite mating type. The ability to conjugate decreased by half after 6 h, although most of the gametes remained motile. The gametes released 4 h later had the same level of conjugation ability when mixed immediately after releasing. When the mature thalli were wrapped in a moist paper towel to prevent gametes from being released, the gametes were preservable for 7 h without a significant decrease in their conjugation ability. Conjugation occurred soon after mixing gametes of both mating types and reached a plateau after 30 s. However, conjugation rates did not exceed a rate of ～70%, even though freshly released gametes were used. Interestingly, a portion of the gametes newly conjugated 30 min after mixing both mating types, and conjugation rates reached a second plateau at ～90%. Gametes with delayed conjugation are provided some period of time that allows them to be transported away and increases their chances of mating with more distant populations, thus contributing to the maintenance of genetic variation.     

ECOLOGICAL CONSIDERATIONS OF DIATOM CELL MOTILITY. I. CHARACTERIZATION OF MOTILITY AND ADHESION IN FOUR DIATOM SPECIES1

To better determine the ecological role of motility in pennate diatoms, we quantitatively characterized several motility and adhesion properties of four species of motile pennate diatoms (Craticula sp., Pinnularia sp., Nitzschia sp., and Stauroneis sp.) isolated from the same freshwater pond. Using computer-assisted video microscopy, we measured speed, size/shape, functional adhesion, path curvature, and light sensitivity for these species, each of which shows a distinctive set of motile behaviors. The average speeds of Stauroneis, Pinnularia, Nitzschia, and Craticula cells are 4.6, 5.3, 10.4, and 10.0 μm · s^?1, respectively. Craticula and Nitzschia cells move in a relatively straight path (<4 degrees rotation per 100 μm movement), Stauroneis exhibits minor rotation (about 7 degrees per 100 μm movement), and Pinnularia rotates considerably during movement (about 22 degrees per 100 μm moved). Functional adhesion (as measured by the release rate of attached cells from the underside of an inverted coverslip) shows a half time for cell release of approximately 50 min for Craticula, 192 min for Pinnularia, and >1 day for Nitzschia and Stauroneis. Direction reversal at light/dark boundaries, which appears to be the main contributor to diatom Phototaxis, is most responsive for Craticula, Pinnularia, and Nitzschia at wavelengths around 500 nm. Craticula and Nitzschia cells are the most sensitive in the photophobic response, with over 60% of these cells responding to a 30-1x light/dark boundary at 500 nm, whereas Pinnularia cells are only moderately responsive at this irradiance, showing a maximal response of approximately 30% of cells at 450 nm. Stauroneis cells, in contrast, had a maximal photosensitive response at 700 nm, suggesting that this cell type may use a different response mechanism than the other three cell types. In addition, Craticula and Pinnularia show a net movement out of the light spot when illuminated at 650 nm, whereas Stauroneis shows a net movement out of the light spot when illuminated at 450 nm. Such quantitative characterizations of species-specific responses to environmental stimuli should give us a firm foundation for future studies analyzing the behavior of interspecies diatom competition for limited light or nutrient resources.     

OLISTHODISCUS LUTEUS (CHRYSOPHYCEAE) I. EFFECTS OF SALINITY AND TEMPERATURE ON GROWTH. MOTILITY AND SURVIVALS1, 2

The effect of salinity and temperature on Olisthodiscus luteus Carter has been examined to across the relative importance of these factory on dynamics of natural population. A salinity range 2–50% was observed with increased tolerance to low salinity (<5%.) at higher temperature (20–30°C). Slinities at 4–5%. Had densities of 10³ cells/ml^?1, and growth >0.5 division day^?1 at temperature of 15–30°C higher salinities (5–50%.) variable but distinct optima for density, growth and motility were observed 5, 10 and 30°C. Density and motility showed no clear optima from 10–10%.15–25°C where maximum growth rates >1.0 division/day^?1 were common. Temperature increased from (0.5–1.9 division. Day^?1) and increases of three orders of magnitude (10^2?10³) for maximum densities. Temperature optima 20°C for growth 5–35%. And 25°C for >40%. were observed. The implications of these findings to natural populations of O. luleus are discussed.     

大鼠卵巢促黄体激素受体膜外微区(1-341)在昆虫细胞中的表达及其性质的初步研究

促黄体激素/人绒毛膜促性腺激素受体(LH/hCG receptor)N端膜外微区341个氨基酸残基(简称R341)与激素有很高的亲和力。本文报道编码R341的cDNA在昆虫细胞中的表达及其产物性质的初步研究。SDS-PAGE银染和免疫印迹结果显示,表达产物呈两条带:分子量为38.5Kd的主带和分子量为40.0Kd的次带。配基结合免疫印迹和~(125)IhCG结合印迹分析表明,表达产物R341具有与配基专一性结合的生物活力。竞争性配基结合曲线和Scatchard分析结果显示,重组受体R341和配基hCG有较高的亲和力,与hCG反应的Kd为5.68×10~(-10)mol/L。     

CYTARABINE TREATMENT OF HUMAN T-LYMPHOID CELLS INDUCES DECREASED HIV-1 RECEPTOR EXPRESSION AND REDUCED HIV-1 SUSCEPTIBILITY

Continuous cultivation of T-lymphoid C8166 cells in the presence of pharmacological relevant concentration of cytarabine (Ara-C) results in significantly decreased expression of CD4 and CXCR4 molecules, the major cellular receptor and co-receptor of T-lymphotropic HIV-1 isolates. This change in receptor expression leads to decreased susceptibility of Ara-C resistant cells to HIV-1 infection demonstrated by reduced binding and penetration of HI-virus.     

PHOTOTACTIC MOTILITY OF SYNECHOCYSTIS SP. UNIWG (CYANOBACTERIA) FROM BRACKISH ENVIRONMENT1

Although type IV pilus has been implicated in the phototactic motility of some unicellular cyanobacteria, its regulatory mechanism and the effect of environmental factors on motility are still unknown. Equally important is the ability of cyanobacterial cells to anchor themselves to an environment that is conducive for survival. We compared the motility of a newly isolated unicellular brackish cyanobacterium, Synechocystis sp. UNIWG, with the morphologically and phylogenetically similar freshwater cyanobacterium Synechocystis sp. PCC6803 under different environmental conditions. The phototactic motility of Synechocystis sp. UNIWG on semisolid BG‐11 medium with various concentrations of nitrogen source was significantly faster than that of Synechocystis PCC6803. Interestingly, the cell surface of Synechocystis sp. UNIWG showed the presence of rigid spicules when grown in liquid BG‐11, a phenomenon that was absent in Synechocystis PCC6803. Negative staining of Synechocystis sp. UNIWG revealed the presence of two distinct pilus morphotypes, which resembled type IV pili and thin pili of Synechocystis PCC6803. This finding suggested a similar pattern of phototactic motility in both strains. However, the rigid spicules on Synechocystis sp. UNIWG seem to be more of a hindrance during type IV motility. It was determined that the spicules were degraded when the cells moved, such as under prolonged darkness and/or depletion of nitrogen source, indicating that the function of the spicules is to attach the cell to an environment that is conducive for its survival. Thus, Synechocystis sp. UNIWG shows phototaxis regulation that is more complex than Synechocystis PCC6803.     

STRUCTURE AND FUNCTION OF THE BRISTLES OF PEDIASTRUM BORYANUM (CHLOROPHYTA)1

Tufts of long delicate bristles were detected on mature colonies of Pediastrum boryanum (Turp.) Meneghini. The bristles are not uniform in length and can exceed 100 μm. Bristle number per tuft can exceed eighty. Ultrastructure studies revealed that tufts are an aggregate of hexagonal tubules arranged in staggered rows. Each hexagonal tubule has an inner diameter of 6 nm and is made up of six interconnected subunits 4 nm in diameter that are shared by adjoining tubules. The bristles can be easily removed from mature colonies with a vortex stirrer. Removal of bristles from mature colonies in culture results in the loss of buoyancy and subsequent settling to the bottom of the flask.     

乙型肝炎病毒核心启动子及其上游顺序对其基因表达的调控

构建了线性化的,基本核心启动子开始并有不同长度Ｃｐ上游顺序的,含完整转录单元的ＨＢＶ基因组克隆,以研究Ｃｐ及其上游顺序对ＨＢＶ基因表达的调控及对ＨＢＶ子代ＤＮＡ复制的影响。发现从基本核心启动子Ｃｐ开始的ＨＢＶ轨录单元能够有效地起始３．５ｋｂ ｍＲＮＡ转录。Ｃｐ上游ＥＮⅡ顺序对子代ＤＮＡ复制有极强的刺激作用,而ＥＮⅡ更上游顺序地ＥＮⅡ的刺激作用又有所抑制,证明Ｃｐ的转录受其上游顺序的正负双重调控。     

佐剂型类风湿关节炎模型改良与DDR2表达的研究

目的 建立更加简便实用的类风湿关节炎模型,为类风湿关节炎发病机制的研究提供良好的实验材料;观察盘状结构域受体2(Discoidin Domain Receptor2,DDR2)在类风湿模型动物早期的表达,为探讨DDR2与类风湿关节炎滑膜细胞损伤的关系提供依据。方法 放射影像学、酶联免疫检测和免疫组织化学。结果 从病理学、影像学、血清学及临床特征观察模型的改良情况,符合类风湿关节炎临床特征的个体达到85％以上,且建模时间明显缩短;免疫组化显示,DDR2免疫反应阳性产物定位于关节囊滑膜细胞和滑膜下层细胞的胞膜和胞浆。结论 经改良的佐剂型类风湿关节炎模型优于常规的动物模型;类风湿关节炎关节囊滑膜和滑膜下层细胞可特异性表达DDR2。     

间隙连接蛋白基因connexin43、ER、PR的表达在子宫平滑肌瘤中相关性研究

研究细胞间隙连接蛋白基因43（connexin43,CX43）及其蛋白、雌激素受体（estrogen receptor,ER）、孕激素受体（progesterone receptor,PR）在子宫平滑肌瘤中的表达,从核酸及蛋白水平探讨在子宫平滑肌瘤发生中的相关关系。应用核酸原位杂交技术和SP免疫组织化学法,研究37例子宫平滑肌瘤、20例正常子宫平滑肌组织中cx43mRNA及其蛋白、ER、PR的表达规律。结果显示,cx43mRNA及其蛋白、ER、PR在子宫平滑肌瘤中的表达明显高于在子宫平滑肌组织中的水平,差异有显著性（P＜0.05）。cx43mRNA及其蛋白在子宫平滑肌瘤中的过度表达,是子宫平滑肌瘤发生过程的重要事件,与ER、PR水平升高呈现一致性,对进一步揭示子宫平滑肌瘤的复杂分子机制、寻求可靠的早期标志有重要意义。