三聚氰胺对SD孕鼠及胎鼠的毒作用

为阐明三聚氰胺对孕鼠和胎鼠毒性作用,将40只SD孕鼠随机分为高剂量组(Ⅰ)、中剂量组(Ⅱ)、低剂量组(Ⅲ)及空白对照组(Ⅳ),共4组,于妊娠第6~19天,分别经口灌服800 mg/kg·d、200 mg/kg·d、20 mg/kg·d和0 mg/kg·d三聚氰胺混悬液。染毒期间,观察孕鼠中毒表现,以及不同妊娠时间体重变化;于妊娠第20天处死母鼠,检测血清尿素氮、肌酐及尿酸,胎鼠及胎盘重量等指标;观察母鼠和胎儿肾脏、胎盘组织病理变化。结果显示,在染毒期,Ⅰ、Ⅱ组母鼠出现精神差等症状;与Ⅳ组相比,Ⅰ、Ⅱ组母鼠、胎鼠和胎盘重量,血液生化指标有显著或极显著差异(P0.05或P0.01),且Ⅰ、Ⅱ组母鼠、胎鼠肾脏以及胎盘有明显病理变化;但3个剂量水平的三聚氰胺对胚胎的存活无影响,也不产生致畸作用。     

全氟辛烷磺酸盐对胚胎期大鼠雄性生殖系统的影响

目的:研究全氟辛烷磺酸盐(PFOS)对胚胎期大鼠雄性生殖系统发育的影响.方法:PFOS分别以5、10、20 mg· kg-1·d-1灌胃染毒孕12-19天的SD母鼠,染毒结束后,测量雄性胎鼠体重、身长、睾丸重量、AGD;酶联免疫吸附法检测雄性胎鼠睾丸内睾酮水平;实时荧光定量PCR法测睾丸组织类固醇激素合成急性调节蛋白(StAR)、胰岛素样因子3(INSL3)mRNA的相对表达量.结果:与对照组相比,高剂量染毒组雄性胎鼠体重、体长、AGD长度显著降低(P＜0.01),睾丸重量减轻(P＜0.05);高剂量染毒组雄性胎鼠睾丸中内睾酮水平下降(P＜0.05);高剂量染毒组雄性胎鼠睾丸组织中StAR mRNA的表达量降低(P＜0.05),而中剂量染毒组StAR mRNA和INSL3 mRNA表达量则明显升高(P＜0.01).结论:孕期染毒PFOS对雄性胚胎生殖系统的发育有毒性作用,其机制可能与PFOS影响睾酮合成和INSL3 mRNA的表达有关.     

妊娠期糖尿病孕妇胎盘中HIF-1α、ET-1及VEGF的表达及与妊娠结局的关系研究

目的:探讨妊娠期糖尿病孕妇胎盘中缺氧诱导因子-1α(HIF-1α)、内皮素-1(ET-1)及血管内皮生长因子(VEGF)的表达及与妊娠结局的关系。方法:选取2015年8月至2016年10月间济南市中心医院收治的妊娠期糖尿病患者80例。根据患者血糖控制效果分为血糖控制不良组(A组)40例和血糖控制良好组(B组)40例,另取同期于我院体检的健康孕妇40例为C组。应用免疫组化SP法检测各组胎盘中HIF-1α、ET-1及VEGF的表达情况,观察各组不良妊娠结局发生情况,并分析妊娠期糖尿病孕妇胎盘中HIF-1α、ET-1及VEGF的表达与妊娠结局相关性。结果:A组孕妇胎盘中HIF-1α、ET-1及VEGF的阳性表达率均高于B组与C组,B组HIF-1α、VEGF的阳性表达率高于C组(P0.05)。A组羊水过多、巨大儿、产后出血的发生率均高于B组与C组,胎儿窘迫、妊娠高血压的发生率高于C组,B组胎儿窘迫、羊水过多、产后出血、妊娠高血压的发生率高于C组(P0.05)。经Spearman相关性分析可得,妊娠期糖尿病孕妇胎盘中HIF-1α、ET-1及VEGF的表达与妊娠高血压、产后出血、巨大儿、羊水过多、胎儿窘迫等不良妊娠结局呈正相关(P0.05)。结论:妊娠期糖尿病孕妇胎盘中HIF-1α、ET-1及VEGF呈高表达,其表达会增加不良妊娠结局的发生率。     

妊娠期糖尿病与胎盘PPARg及FATP-3表达水平与新生儿体脂的关系

妊娠期糖尿病(gestational diabetes mellitus,GDM)是在妊娠期间发生或首次发现的糖代谢异常,属于糖尿病的一个独立类型,对GDM的形成和发展机理尚未完全明确。本研究试图探讨GDM与胎盘PPARγ水平的关系,以及对新生儿体脂的影响,以进一步阐明GDM的形成机理。我们选取常规产检的孕妇292例并将其分为两组,GDM孕妇86例(GDM组),正常孕妇206例(对照组)。本研究测量了两组新生儿体格特征,采用RT-PCR、Western blotting、凝胶电泳法分别检测胎盘PPAR、FATP-3和CD36蛋白。研究表明,GDM组新生儿总脂肪质量和体脂含量分别为(0.67±0.21)kg和(18.50±3.28)%,明显高于对照组新生儿(p0.05);GDM组胎盘PPAR m RNA及蛋白相对表达量分别为(0.433±0.098)和(0.892±0.112),明显低于对照组胎盘(p0.05);GDM组胎盘FATP-3蛋白相对表达量为(0.563±0.080),明显低于对照组胎盘(p0.05);胎盘PPAR m RNA及蛋白相对表达量与新生儿体脂含量呈负相关(r=-0.573和-0.691,p0.05),与胎盘FATP-3蛋白表达呈正相关(r=0.463,p0.05)。初步认为,GDM产妇胎盘PPAR表达水平降低,其与脂肪酸转运蛋白FATP-3有一定关联,这可能导致新生儿体脂含量升高,值得进一步研究。     

妊娠糖尿病对仔鼠肺成熟的影响及吡格列酮的干预作用

摘要 目的：探究妊娠糖尿病（GDM）对仔鼠肺成熟的影响及吡格列酮对肺发育的干预作用。方法：将30只SD孕鼠分为对照组、GDM组和GDM+吡格列酮组（GDM+P组）,每组10只。GDM组和GDM+P组孕鼠通过腹腔注射链脲霉素（STZ,45 mg/kg）和高脂饮食饲养构建GDM孕鼠模型,GDM+P组大鼠建模后灌胃10 mg/kg的吡格列酮,对照组和GDM组孕鼠每天灌胃等体积生理盐水。分娩后,检测各组仔鼠的血糖和血浆胰岛素水平以及胎肺组织中的总磷脂量。通过苏木精伊红（HE）染色、油红O染色和透射电镜观察胎肺组织结构和形态变化。通过RT-PCR和Western blot检测胎肺组织中SP-A、SP-B、SIRT1和PPARγ的表达。结果：GDM组仔鼠的血糖水平与对照组无显著差异（P>0.05）,胰岛素水平明显高于对照组（P<0.05）。与对照组相比,GDM组仔鼠胎肺组织中的总磷脂含量降低（P<0.05）;胎肺组织中肺泡Ⅱ型上皮细胞(AECⅡ)数量和脂滴明显减少。与对照组相比,GDM组仔鼠胎肺组织中的SP-A、SP-B、SIRT1和PPARγ的mRNA和蛋白相对表达水平均降低（P<0.05）。吡格列酮干预显著逆转了GDM对仔鼠胰岛素、胎肺组织结构和形态变化的影响;GDM+P组仔鼠胎肺组织中的SP-A、SP-B、SIRT1和PPARγ的mRNA和蛋白相对表达水平相较GDM组均升高（P<0.05）。结论：GDM母鼠所生仔鼠存在肺发育延迟,吡格列酮干预可有效促进仔鼠的肺成熟。     

孕期大鼠暴露PFOS对胎鼠肝脏毒性的影响

目的:研究在孕期暴露PFOS对胎鼠的肝脏毒性的影响.方法:将孕期为12天的16只SD雌性大鼠,随机分为4组给予不同剂量的PFOS[0(对照),5,10,20 mg·kg-1],连续灌胃7天,在GD19天时对母鼠和胎鼠的体重、胎鼠肝脏的生化指标、母鼠血清的生化指标进行了相应的检测.结果:与对照组相比,母鼠体重在20 mg·kg-1组显著下降(P＜0.001);胎鼠的体重和体长在20mg·kg-1组显著下降(P＜0.001);胎鼠的肝脏重量降低,呈剂量依赖性,并伴有肝细胞浊肿、变性甚至坏死;10 mg· kg-1组胎鼠肝脏中的酶活性(ALT、AST、GGT和ALP等)显著升高(P＜0.001);母鼠血清的大部分生化指标未发生明显变化.结论:孕期大鼠暴露在PFOS的环境下会严重损伤胎鼠的肝脏功能.     

Snail1基因表达在妊娠期糖尿病大鼠氧化应激及肝脏损伤的作用机制

摘要 目的：探讨Snail1基因表达在妊娠期糖尿病大鼠氧化应激及肝脏损伤的作用机制。方法：健康成年C57BL/6雌性大鼠32只作为研究对象。采用高脂喂养联合小剂量链脲佐菌素注射的方式构建妊娠期糖尿病大鼠模型。将所有大鼠分为正常妊娠组,正常妊娠+Snail1过表达组,妊娠期糖尿病组,妊娠期糖尿病+Snail1过表达组。采用qRT-PCR检测大鼠Snail1的mRNA表达水平,并检测大鼠妊娠期体重、氧化应激指标、炎症指标和肝功能指标。结果：与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的孕鼠体重、胎鼠体重、胎盘重量、Snail1 mRNA,明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组孕鼠体重、胎鼠体重、胎盘重量、Snail1 mRNA均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）;与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的FBG、FINS、HOMA-IR明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组FBG、FINS、HOMA-IR均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）;与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的ROS、GSH-Px、MDA明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组ROS、GSH-Px、MDA均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）;与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的TNF-α、IL-1β、IL-18明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组TNF-α、IL-1β、IL-18均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）;与正常妊娠组相比,正常妊娠+Snail1过表达组、妊娠期糖尿病组、妊娠期糖尿病+Snail1过表达组的GPT、GOT、ALP明显更高（P＜0.05）,且妊娠期糖尿病+Snail1过表达组GPT、GOT、ALP均显著高于正常妊娠+Snail1过表达组和妊娠期糖尿病组（P＜0.05）。结论：妊娠期糖尿病大鼠Snail1基因的表达上调可加重糖脂代谢紊乱,并激活下游氧化应激和炎症反应,进而加重大鼠肝脏损伤。     

阿霉素肾病大鼠表皮生长因子及其受体的表达

目的研究阿霉素肾病大鼠肾组织中表皮生长因子(EGF)及其受体EGFR的表达分布以及表达量与尿蛋白之间关系。方法选择第5天、14天、28天作为动态观察的时点,同期设立正常对照。采用荧光定量RT-PCR、免疫组织化学及计算机图像定量分析EGF mRNA以及EGF、EGFR蛋白在肾组织的表达,同时测定24 h尿蛋白定量。WT1和EGFR双重免疫组化确定EGFR在肾小球内确切细胞定位。结果阿霉素注射后第5天,EGFmRNA即较正常增高,28 d明显增高并高于5 d和14 d。正常对照组EGF阳性细胞主要分布于远曲小管和髓袢,阿霉素组EGF还在集合管和近曲小管上表达;EGF阳性表达范围和强度随尿蛋白增加而增加;EGFmRNA表达量以及EGF在肾小管中的表达强度与24 h尿蛋白量呈正相关。肾小管上皮细胞广泛表达EGFR,阿霉素组EGFR在小管表达均高于正常,但组间各时点差异无显著性;随尿蛋白增加EGFR在肾小球内表达逐渐增多。EGFR在肾小球和肾小管中的表达强度均与24 h尿蛋白量呈正相关。WT1和EGFR双重免疫组化显示阿霉素肾病组EGFR可在足突细胞上表达,正常组则无。结论阿霉素肾病大鼠的肾小球脏层上皮有EGFR的表达。EGF/EGFR可能参与了阿霉素肾病的发病过程以及蛋白尿的形成。     

TNF-α, NF-KBp65, PAI-1 在妊娠期糖尿病孕妇血清及胎盘组织中的表达 变化及其意义

目的:探究TNF-α、NF-KBp65以及PAI-1在妊娠期糖尿病孕妇血清和胎盘组织中的表达变化及其临床意义。方法:选取自47例定期产检并且住院分娩的妊娠期糖尿病孕妇作为观察组,再选取同期分娩的47例正常孕妇作为对照组,比较两组孕妇血清和胎盘组织中TNF-α、NF-KBp65和PAI-1的表达水平,并分析血清中TNF-α、NF-KBp65、PAI-1表达水平的相关性。结果:观察组孕妇血浆的TNF-α和PAI-1表达水平明显较高,差异具有统计学意义(P均0.001)。妊娠期糖尿病患者血浆中PAI-1表达水平和TNF-α表达水平呈正相关关系(r=0.843,P0.001)。观察组孕妇的空腹-OGTT、1h-OGTT、2h-OGTT、空腹胰岛素、空腹血糖以及胰岛素抵抗指数均明显高于对照组(P均0.001)。胰岛素抵抗指数与妊娠期糖尿病患者血浆中TNF-α的表达水平相关性最高(Beta=0.769);口服葡萄糖耐受实验中1-h的血糖水平与PAI的表达水平相关性最高(Beta=0.637)。观察组孕妇的TNF-α、NF-KBp65和PAI-1的阳性率显著高于对照组,差异具有统计学意义(P0.001,=0.039,0.007)。结论:血清和胎盘组织中的TNF-α、NF-KBp65和PAI-1可能参与了妊娠期糖尿病的发生与发展。     

大鼠脊髓损伤后表皮生长因子受体在脊髓的表达特点

目的研究大鼠脊髓损伤(spinal cord injury,SCI)后表皮生长因子受体(epidermal growth factor re-ceptor,EGFR)在脊髓的表达特点及意义。方法健康成年雄性SD大鼠,随机分为4组(每组10只):假手术组,SCI术后3 d、7 d和14 d组。应用Basso Beattie Bresnahan(BBB)评分观察大鼠行为学改变;逆转录-聚合酶链反应(RT-PCR)检测损伤段脊髓组织中EGFR mRNA表达水平;免疫组织化学方法观察损伤段脊髓灰质中EGFR蛋白表达情况;并对EGFRmRNA及蛋白表达情况与BBB评分进行相关性分析。结果行为学观察发现大鼠脊髓损伤后下肢神经功能逐步恢复;RT-PCR结果显示EGFR mRNA在假手术组大鼠脊髓中微量表达,SCI术后3 d表达显著升高,随后趋于下降,14 d时仍高于假手术组(P<0.01);免疫组织化学染色显示损伤段脊髓灰质中EGFR阳性细胞数在损伤后3 d显著高于假手术组(P<0.01),随后趋于下降,但14 d时仍高于假手术组(P<0.01);EGFR mRNA及蛋白的表达均与BBB评分呈显著负相关(r=-0.956,P<0.05;r=-0.966,P<0.05)。结论EGFR在大鼠脊髓损伤后具有时相分布特点,且与动物行为呈负相关,提示其表达可能阻碍损伤后的神经功能恢复。     

Acute Regulation of the Epidermal Growth Factor Receptor in Response to Nerve Growth Factor

PC12 cells possess specific receptors for both nerve growth factor and epidermal growth factor, and by an unknown mechanism, nerve growth factor is able to attenuate the propagation of a mitogenic response to epidermal growth factor. The differentiation response of PC12 cells to nerve growth factor, therefore, predominates over the proliferative response to epidermal growth factor. We have observed that the addition of nerve growth factor to PC12 cells rapidly produces a decrease in surface 125I-epidermal growth factor binding capacity. Unlike previously described nerve growth factor effects on 125I-epidermal growth factor binding capacity, which required several days of nerve growth factor exposure, the decreases we report occur within minutes of nerve growth factor addition: A 50% decrease in 125I-epidermal growth factor binding capacity is evident at 10 min. This rapid nerve growth factor response is concentration dependent; inhibition of 125I-epidermal growth factor binding is detectable at nerve growth factor levels as low as 0.2 ng/ml and is maximal at approximately 50 ng/ml, consistent with known ranges of biological activity. No demonstrable differences in the rate of epidermal growth factor receptor synthesis or degradation were observed in cells acutely exposed to nerve growth factor. Scatchard analysis revealed that acute nerve growth factor treatment decreased the number of both high- and low-affinity 125I-epidermal growth factor binding sites, while the receptor affinity remained unchanged. We have also investigated the involvement of various potential intracellular mediators of nerve growth factor action and of known intracellular modulatory systems of the epidermal growth factor receptor for their capacity to participate in this nerve growth factor activity.     

Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

Specific epidermal growth factor receptors on porcine thyroid cell membranes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.     

人神经干细胞的体外生物学特性

本实验利用有丝分裂因子,体外诱导生成人神 经干细胞(NSCs),观察其生长特性并进行鉴定。取胎龄10-22周的大脑半球,分散细胞后种于添加表皮生长因子(EGF,20ng/ml)和/或碱性成纤维生长因子(bFGF,20ng/ml)的培养基中。利用免疫组织化学方法鉴定分化后的细胞类型。同时,进行细胞克隆分析、传代培养及端粒酶活性检测。结果显示:NSCs呈悬浮生长的干细胞球,其特异性抗原nestin阳性。NSCs具有增殖能力,可连续传代而不丢失其增殖和多分化潜能的干细胞特性。撤除EGF和bFGF的作用,细胞停止分裂,并分化为神经元、星形胶质细胞和少突胶质细胞。克隆分析显示NSCs生长呈密度依赖性。人NSCs表达较低的端粒酶水平,并随培养时间延长而下调。研究表明,利用有丝分裂因子,可在体外成功诱导生成人NSCs,其生长,分化受内外源因素的调节,相关的机制还有待阐明。     

Model analysis of difference between EGF pathway and FGF pathway

The difference in time course of Ras and mitogen activated protein kinase (MAPK) cascade by different growth factors is considered to be the cause of different cellular responses. We have developed the computer simulation of Ras-MAPK signal transduction pathway containing newly identified negative feedback system, Sprouty, and adaptor molecules. Unexpectedly, negative feedback system did not profoundly affect time course of MAPK activation. We propose the key role of fibroblast growth factor receptor substrate 2 (FRS2) in NGF/FGF pathway for sustained MAPK activation. More Grb2-SOS complexes were recruited to the plasma membrane by binding to membrane-bound FRS2 in FGF pathway than in EGF pathway and caused sustained activation of ERK. The EGF pathway with high concentration of EGF receptor also induced sustained MAPK activation, which is consistent with the results in the PC12 cell overexpressing the EGF receptors. The simulated time courses of FRS2 knock-out cells were consistent with those of the reported experimental results.     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

pp42/44MAP Kinase Is a Component of the Neurogenic Pathway Utilized by Nerve Growth Factor in PC12 Cells

Nerve growth factor-stimulated mitogen-activated protein kinase (pp42/44MAP) kinase was characterized by sequential column chromatography on DEAE-Sephacel, phenyl-Sepharose CL4B, and S-200. The kinase displayed an apparent molecular mass of 42 kDa and reacted with an antiphosphotyrosine antibody. Peptide mapping of myelin basic protein revealed the presence of one phosphopeptide that was phosphorylated on Thr-97. pp42/44MAP kinase activity was dependent on Mg2+ and inhibited by K252a both in vitro and in vivo. Nerve growth factor-stimulated kinase activation was diminished by down-regulation of protein kinase C with 200 nM 12-phorbol 13-myristate acetate or with staurosporine (1 nM), a protein kinase C inhibitor. Genistein, a protein tyrosine kinase inhibitor, blocked nerve growth factor-mediated neurite extension as well as diminished activation of pp42/44MAP kinase. Our data demonstrate that activation of this kinase system by nerve growth factor displays a requirement for both protein kinase C as well as protein tyrosine kinase. In addition, other agents that are capable of promoting neurite outgrowth in PC12 cells, such as fibroblast growth factor or dibutyryl cyclic AMP, do so independently of activating this kinase system.     

Stimulation of tyrosine phosphorylation in lectin treated human lymphocytes

Large increases in tyrosine phosphorylation have been detected in subcellular matrixes isolated from lectin treated human lymphocytes. In lectin stimulated cells proteins of molecular weight 105, 75, 58 and 35 kDa contained phosphotyrosine (P-tyr) whereas non-stimulated cells had no 105 and low levels of P-tyr in proteins of 75, 58 and 35 kDa. In stimulated cells increased tyrosine kinase activity was also shown using gastrin as substrate. In both stimulated and non-stimulated cells the 58 kDa phosphoprotein was the most heavily labelled, after partial proteolysis of the 58 kDa different phosphopeptides were generated. A peptide with a sequence analogous to the autophosphorylated tyrosine site of pp60src inhibited tyrosine phosphorylation in stimulated cells. The lymphocyte system provides a useful tool to study normal tyrosine protein kinases and their role in cellular proliferation.