UROTENSIN II MEDIATES ERK1/2 PHOSPHORYLATION AND PROLIFERATION IN GPR14-TRANSFECTED CELL LINES

ABSTRACT

Urotensin-II (U-II), a vasoactive cyclic neuropeptide, was recently identified as the natural ligand for the G-protein coupled receptor GPR14. The expression pattern of U-II and GPR14 are consistent with a role as a neurohormonal regulatory system in cardiovascular homeostasis. Urotensin-II induces a rapid and short-lasting rise in intracellular calcium in recombinant GPR14 expressing cells. In the present study we show that U-II induces signal transduction pathways leading to the long-lasting activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in chinese hamster ovary cells expressing human GPR14 (CHO-GPR14). Furthermore, we observed a growth-stimulating and PD98059 sensitive activity of U-II in CHO-GPR14 cells, but not CHO-K1 cells. The investigation of the GPR14 induced signal transduction pathways leading to ERK1/2 phosphorylation revealed a previously unsuspected role for G_i/o-protein coupling and showed an involvement of phospatidylinositol-3-kinase, phospholipase C and calcium channel mediated mechanisms. Our results suggest that U-II and its receptor GPR14 may be involved in long-lasting physiological effects such as cardiovascular remodeling.     

THE PHOSPHORYLATION OF NUCLEAR PROTEINS IN THE REGENERATING AND PREMALIGNANT RAT LIVER AND ITS SIGNIFICANCE FOR CELL PROLIFERATION

In liver regeneration or neoplastic transformation, phosphorylation of nuclear proteins is stimulated. In the regenerating liver all main histone fractions are involved in this process. The type of histone phosphorylated seems to be dependent on the position of the partially synchronized cells within the generation cycle. At a time when most cells are exhibiting maximum HnRNA-synthesis, histone F2a2 belongs to those fractions with highly stimulated phosphate incorporation. Phosphorylation of this fraction alone is stimulated by cyclic AMP in parallel to a stimulation of HnRNA-synthesis. The preneoplastic liver is characterized by oscillating phosphorylation and dephosphorylation reactions of nearly all histone fractions during the first days of N-nitroso-diethylamine administration. After 2 months of carcinogen feeding a 50–150% stimulation of the phosphorylation of Fl subfractions is observed. The phosphate content of the other histones, however, has returned to the original level. A series of further proteins, isolated together with the histones, show very similar phosphorylation characteristics. These proteins are mostly of non-histone origin. It is suggested that some of them are responsible for the transport of RNA with messenger properties within the cell.     

非小细胞肺癌中HMGA2的表达与细胞增殖的关系

目的探讨High mobility group A2 protein（HMGA2）的表达对非小细胞肺癌生长、转移的影响,与临床病理参数和细胞增殖的关系。方法应用免疫组化法检测38例非小细胞肺癌组织（23例鳞癌,15例腺癌）及癌旁的正常肺组织标本中HMGA2和Ki-67的表达。结果HMGA2和Ki-67在癌旁的正常肺组织中均不表达,而在肺癌组织中的表达阳性率分别为39．47％、44．74％,二者在肺癌组和癌旁的正常肺组织组之间差异有显著性（P〈0．05）。HMGA2的表达在伴淋巴结转移的肺癌组织明显高于不伴有淋巴结转移的肺癌组织（P〈0．05）,而与其他临床病理参数包括肿瘤的分化程度、肿瘤大小和TNM分期以及细胞增殖指标Ki-67之间没有相关性。结论本研究显示HMGA2在非小细胞肺癌组织中异常表达,可能与肺癌的发生和进展有关。     

CHEMICAL CHANGES IN LESPEDEZA ASSOCIATED WITH INDUCED POLYPLOIDY

Hanson , C. H. (U.S.D.A., Beltsville, Md.), W. A. Cope and R. M. Brinkley . Chemical changes in Lespedeza associated with induced polyploidy. Amer. Jour. Bot. 46(1): 36-39. 1959.— Changes in the quantities of 5 chemical constituents which were associated with induced polyploidy were studied using leaves of the following polyploids: Autotetraploids—Lespedeza cuneata, L. latissima, L. stipulacea, and L. striata; amphidiploids—L. hedysaroides × L. cuneata and L. latissima × L. cuneata. L. stipulacea and L. striata were annuals with low concentrations of tannin; the other species were perennials and high in tannin content. The polyploids were generally lower in crude protein, P₂O₅, and CaO, and higher in K₂O and total tannin. The greatest and most significant shift was in total tannin. Induced polyploids of Lespedeza did not appear promising as forage plants.     

葡萄糖转运蛋白-1在胰腺癌组织中的表达及其与细胞增殖和凋亡的相关性研究

目的探讨胰腺癌组织中葡萄糖转运蛋白-1(Glut-1)的表达及其与细胞增殖、凋亡的关系。方法采用免疫组化链霉抗生物素蛋白-过氧化物酶连接法(SP法)检测15例正常胰腺组织和49例胰腺癌组织中Glut-1、增殖细胞核抗原(PCNA)蛋白的表达,并计算增殖指数(PI)。采用原位末端标记法(TUNEL)检测胰腺癌凋亡细胞,计算凋亡指数(AI)。结果Glut-1在正常胰腺组织中不表达,而在胰腺癌组织中阳性表达率为73.47%(36/49),胰腺癌组织中Glut-1的阳性表达率明显高于正常胰腺组织(P<0.01)。正常胰腺组织及癌组织中的AI分别为0.41%±0.13%和5.93%±4.18%,两者比较有显著性差异(P<0.05)。而两组中的增殖指数分别为6.25%±2.59%和32.54%±14.69%,胰腺癌组织的细胞增殖程度明显高于正常胰腺组织(P<0.05)。随胰腺癌细胞Glut-1蛋白表达水平升高,肿瘤细胞增殖活性相应增加,而凋亡则相应减少。Glut-1表达与胰腺癌细胞增殖呈正相关性(r=0.726,P<0.01),与凋亡程度无相关性(r=-0.102,P>0.05)。结论Glut-1在胰腺癌组织中表达增高,Glut-1与细胞的增殖密切相关,可能在胰腺癌的发生发展中起重要作用。     

CHANGES IN SOIL AGGREGATION ASSOCIATED WITH TALLGRASS PRAIRIE RESTORATION

To investigate the dynamics of soil aggregation associated with the restoration of cultivated soil to tallgrass prairie, changes in soil aggregation and aboveground production were compared in a corn field, restored prairie plantings of various ages (second, fifth, eighth, and eleventh growing season), and an uncultivated prairie remnant. The restored prairie was also compared with a long-term (fourteenth growing season) ungrazed pasture dominated by Eurasian grasses. All plots were located on similar soils. The regression model, Y = 95.8 - 56.2/X (R² = 0.93), best described the relatively rapid recovery of water-stable soil aggregates >0.2 mm diameter with time (in years) since cultivation. Similar models were also found to describe changes in the percentages of aggregates > 1 and > 2 mm diameter. Aggregates > 0.2 and > 2 mm diameter were more closely associated with prairie graminoids than with other vegetation categories. However, time without disturbance may be a more important factor in soil aggregate formation than vegetation type, but it was difficult to separate the effects of these two factors in this study. The percentage of aggregates > 0.2 mm diameter was found to be significantly higher (P = 0.0553) in the oldest restored prairie than in ungrazed pasture although the former had been cultivated more recently. This suggests that C₄ prairie graminoids may confer some advantage over introduced C₃ Eurasian grasses for the development of water-stable aggregates in soils of the Prairie Peninsula.     

OPTICAL AND ELECTRON MICROSCOPIC CHANGES IN ULTRAVIOLET-IRRADIATED CHROMOSOME SEGMENTS

Chromosome segments of urodele cells lose some substance after irradiation with about 10^-1 ergs/µ² of heterochromatic ultraviolet light. These segments stain faintly or negatively with the Feulgen and pyronine-methyl-green methods and weakly with the Alfert-Gesch-wind stain for basic protein. In the living cells, Perry found in these chromosome segments a decrease of 50 to 60 per cent in absorption at 2400, 2600, and 2800 A, i.e., in the region of intense chromosomal absorption that is maximal at 2600 A. Apparently the material lost contains DNA (?DNP) and we call the process DNA-steresis. In such cells, fixed in neutral formalin in Tyrode''s solution and stained with phosphotungstic acid, electron microscopy shows that the unirradiated parts of the chromosomes consist of (a) a homogeneous or finely fibrillar material (component-A) filling the meshes of (b) an irregular network with bars 40 to 300 A in diameter, some of which continue into a similar interchromosomal network. DNA-steretic portions of the chromosomes consist mainly of this network and only small amounts of component-A, which presumably contains the DNA. We have not been able to demonstrate DNA-steresis with the electron microscope after primary fixation with OsO⁴ or KMnO⁴. Structural changes due to DNA-steresis are compared with certain nuclear changes in the mitotic cycle.     

PHYSIOLOGICAL AND HERITABLE CHANGES IN CYCLIC AMP LEVELS ASSOCIATED WITH CHANGES IN FLAGELLAR FORMATION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Synchronously dividing cells of Chlamydomonas reinhardtii Dang. (Chlorophyta) produce a single peak of cyclic adenosine monophosphate (cAMP), about sevenfold above the basal level, at the time of onset of the flagellar shortening that precedes mitosis. Cultures of a spontaneous palmelloid variant (which forms flagella-less cell clusters) of C. reinhardtii produce up to 15 times more cAMP per gram fresh weight of cells than do cultures of normal C. reinhardtii. Revertants from the palmelloid phenotype to the normal phenotype exhibit the low levels of cAMP characteristic of normal C. reinhardtii. Thus, elevation of cAMP level and decreased ability to form or maintain flagella are closely related phenomena. We propose that flagellar assembly/disassembly is regulated by endogenous cAMP in C. reinhardtii.     

掌叶半夏有效提取物对宫颈癌细胞株增殖的抑制作用

目的既往的临床研究证实中药掌叶半夏可有效治疗宫颈癌。在此基础上,课题组观察了中药掌叶半夏有效提取物(Pinellia Extract,PE)对宫颈癌细胞株CaSki和HeLa细胞增殖的作用,先前采用MTT法检测细胞活性以及倒置相差显微镜观察细胞形态的变化,初步显示了PE对CaSki和HeLa细胞的增殖有抑制作用;本文将对PE的增殖抑制作用进行进一步的研究。方法采用扫描电镜和透射电镜观察PE作用后CaSki和HeLa细胞表面及内部超微结构的变化,In-cell Western blot检测不同浓度PE作用15min后CaSki和HeLa细胞磷酸化ERK的表达。结果0.15mg/mlPE作用24h后CaSki和HeLa细胞表面结构及内部结构均发生了显著的变化,提示细胞增殖受到抑制;不同浓度PE作用15min后CaSki和HeLa细胞磷酸化ERK的表达随PE浓度的增加逐渐下降。结论PE对CaSki和HeLa宫颈癌细胞株的增殖有明显抑制作用,表现在细胞表面及内部超微结构的变化;增殖抑制作用之一是阻断ERK的磷酸化。实验结果为进一步研究PE的抗宫颈癌作用机制以及新药的研制开发奠定了一定的理论依据和实验基础。     

人肺鳞癌和腺癌组织中c-erbB-2癌基因蛋白表达与细胞增殖及预后的关系

用S－P免疫组织化学方法,用c－erbB－2多克隆抗体及PCNA单克隆抗体对50例肺鳞癌和30例腺癌组织石蜡包埋标本进行检测。结果显示：c－erbB－2染色总阳性率为46．25％,鳞癌阳性率为40％,腺癌阳性率为56．67％,两者之间无显著性差异。伴有淋巴结转移的肺癌组织中c－erbB－2阳性表达率较高（66．67％）,并且与肺癌预后有关。PCNA表达阳性级别与患者的术后生存期呈负相关（P＜0．05）,与c－erbB－2表达阳性级别呈正相关（P＜0．05）。本研究提示,用免疫组织化学方法检查,c－erbB－2蛋白和PCNA共同表达可作为临床判断肺鳞癌和腺癌预后的较好指标之一。     

PREDICTABILITY AND IRREVERSIBILITY OF GENETIC CHANGES ASSOCIATED WITH FLOWER COLOR EVOLUTION IN PENSTEMON BARBATUS

Two outstanding questions in evolutionary biology are whether, and how often, the genetic basis of phenotypic evolution is predictable; and whether genetic change constrains evolutionary reversibility. We address these questions by studying the genetic basis of red flower color in Penstemon barbatus. The production of red flowers often involves the inactivation of one or both of two anthocyanin pathway genes, Flavonoid 3′,5′‐hydroxylase (F3′5′h) and Flavonoid 3′‐hydroxylase (F3′h). We used gene expression and enzyme function assays to determine that redundant inactivating mutations to F3′5′h underlie the evolution of red flowers in P. barbatus. Comparison of our results to previously characterized shifts from blue to red flowers suggests that the genetic change associated with the evolution of red flowers is predictable: when it involves elimination of F3′5′H activity, functional inactivation or deletion of this gene tends to occur; however, when it involves elimination of F3′H activity, tissue‐specific regulatory substitutions occur and the gene is not functionally inactivated. This pattern is consistent with emerging data from physiological experiments indicating that F3′h may have pleiotropic effects and is thus subject to purifying selection. The multiple, redundant inactivating mutations to F3′5′h suggest that reversal to blue‐purple flowers in this group would be unlikely.     

茉莉酸甲酯、水杨酸和一氧化氮诱导怀槐悬浮细胞合成异黄酮及细胞结构变化

本文对比研究了茉莉酸甲酯(MeJA)、水杨酸(SA)和一氧化氮(NO)三种激发子对怀槐悬浮培养物异黄酮合成及细胞结构变化的影响。结果表明,在三种激发子的作用下怀槐细胞异黄酮合成量显著提高:200μmol/L MeJA、100μmol/L SA及50μmol／L SNP处理培养细胞9d后,异黄酮含量分别为同期对照的417．18％、185．45％和222．45％。同时细胞内发现染色很深的电子致密小体(EDB),其数量随着异黄酮含量的升高而增加,亦在第九天达到最多,与异黄酮积累呈现正相关性。推测激发子可能诱导植物细胞结构变化来响应次生代谢产物的合成。     

OXIDATIVE PHOSPHORYLATION AND ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA IN THE INTACT ASCITES TUMOR CELL

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90° light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox → condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90° light-scattering increase, which also occurs at this time, showed a t ½ of only 25 sec which agreed temporally with a slower orthodox → maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox → condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.     

METABOLIC CHANGES ASSOCIATED WITH ISO-OSMOTIC REGULATION IN BRAIN CORTEX SLICES

Abstract— The metabolism of single, first cortical rat brain slices was studied in response to incubation in media of various osmolalities. There was an inverse relationship between osmolality and the magnitude of the increase in tissue water content, and a direct relationship between osmolality and inulin space. Brain sodium varied directly with the media sodium, but hyperosmolal sucrose and glucose resulted in a drop in brain sodium. Brain potassium was constant in hyperosmolal sodium media, but it fell in hypo-osmolal media and hyperosmolal sucrose and glucose media.
Hypo-osmolality depressed the oxidation of [¹⁴C]glucose to ¹⁴CO₂ whereas hyperosmolality obtained with sodium, sucrose, or glucose stimulated glucose oxidation. Lactate production was enhanced only by sodium hyperosmolality. Hypo-osmolality caused a 70% drop in phosphocreatine and a minor decrease in energy charge potential, analagous to the effects of hypoxia. Hyperosmolality had no effect on energy metabolism.
The total amino acid nitrogen released into the media was suppressed by hypo-osmolality but was increased by hyperosmolal incubation. Hyperosmolality also increased production of ammonia fourfold.
The time course of the change in tissue osmolality and ion content following incubation in hyperosmolal sodium media showed the tissue osmolality reached the media osmolality within 5 min, but it took 30 min for the tissue sodium to reach equilibrium with the media sodium. This indicates that unidentified or'idiogenic osmoles'are induced transiently by hyperosmolality associated with changes in amino acid and ammonia metabolism.     

外阴硬化性苔癣组织P53、PCNA表达、DNA含量及与细胞增殖的关系

探讨外阴硬化性苔癣组织中的 P5 3、PCNA表达 ,DNA含量与细胞增殖的关系。免疫组化方法测定 2 0例外阴硬化性苔癣组织和 10例正常外阴皮肤中 P5 3、 PCNA蛋白表达 ;图像分析技术检测两组基底层细胞核形态及 DNA含量。结果显示 ,外阴硬化苔癣组 P5 3阳性表达率为 40 % ,与正常皮肤比较 P<0 .0 5 ,PCNA阳性表达率为 70 % ,与正常皮肤比较 P>0 .0 5 ,阳性表达主要分布于棘层、颗粒层 ;基底细胞核显著变小和 DNA含量降低 (P<0 .0 5 )。结果表明外阴硬化性苔癣组织中存在细胞增殖异常     

CELL PROLIFERATION AND SPECIALIZATION DURING ENDOCHONDRAL OSTEOGENESIS IN YOUNG RATS

Endochondral osteogenesis was studied autoradiographically in ribs and tibiae of 32 Long-Evans rats injected with 1 µc/gm H³-thymidine at 6 days of age and sacrificed at intervals between 1 hour and 2 weeks later. Proliferation and specialization of bone cells were studied by analyses of (a) the percentage of mitoses which were labeled, (b) the percentage of labeled nuclei in bone cells, and (c) grain counts. The following conclusions were derived: The various types of bone cells represent different functional states of the same cell. Cell division is usually restricted to cells in the morphologically unspecialized "osteoprogenitor" state. Specialized bone cells arise by modulation of osteoprogenitor cells. The average cell generation time is shortest in the metaphysis, longest in the periosteum, and intermediate in the endosteum. The average duration of DNA synthesis is relatively constant (about 8 hours). With increasing length of generation time there is a slight increase in G₂ + mitosis, but the major change is a lengthening of G₁. After dividing, cells in the osteoprogenitor state may remain within the progenitor pool or undergo modulation of cell type, specializing as osteoblasts or becoming incorporated in osteoclasts.     

食管鳞癌Cyclin D1与细胞增殖关系的研究及意义

目的　探讨食管鳞癌细胞中细胞周期素D1(CyclinD1)表达与细胞增殖的关系及意义。方法　应用免疫组织化学和流式细胞术检测 48例食管鳞癌组织中CyclinD1蛋白表达及DNA倍体、S期细胞比值、G2 /M期细胞比值。结果　 48例食管鳞癌中CyclinD1阳性表达率为 45 8%(2 2 / 48) ;DNA异倍体检出率为 5 0 %(2 4/ 48) ,CyclinD1阳性表达的标本中DNA异倍体的检出率为 6 8 2 %(15 / 2 2 ) ,显著高于CyclinD1阴性表达的标本 (33 3%,9/ 2 7,P <0 0 5 )。CyclinD1表达阳性的肿瘤中细胞周期G2 /M期的比值也明显高于CyclinD1表达阴性的肿瘤 (分别为 5 98± 4 87和 4 12± 2 70 ,P<0 0 5 )。结论　从观察结果推测CyclinD1的表达可以加速肿瘤细胞增殖。分析食管鳞癌CyclinD1、DNA倍体及不同时相的细胞增殖有助于帮助分析和判断病人的预后。