Striated myofibrils in anti-myosin stained,isolated chicken gizzard smooth muscle cells

Summary Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM.Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 m. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5±0.2 m. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gröschel-Stewart.     

Double-immunofluorescent staining of isolated smooth muscle cells

Summary Contractile proteins have been co-localized by double-immunofluorescent staining in several types of cultured cells. Since freshly isolated smooth muscle cells are more representative of the organization within smooth muscle cells in the intact tissue than cultured cells, the present study was undertaken to determine the feasibility of using double-staining techniques in freshly isolated cells. A new method of purifying -actinin from chicken gizzards was used to provide antigen for raising anti--actinin. Fluorescein isothiocyanate-labelled anti--actinin (FAA) was used in conjunction with tetramethyl rhodamine isothiocyanate-labelled anti-myosin (TRAM) Ouchterlony gels against myosin, tropomyosin, actin, and -actinin showed that antimyosin reacted only with myosin, anti--actinin only with -actinin. Anti--actinin stained only the Z-line of isolated chicken skeletal muscle myofibrils. FAA stained bright, discrete patches or strips on the plasma membrane, while TRAM was excluded from these areas. FAA stained myofibrils faintly in a striated pattern, while TRAM stained myofibrils heavily with less evident striations. Evidence for extramyofibrillar localization of -actinin within the cytoplasm was inconclusive. Although antibodies were quite specific in their labelling, resolution with double-staining was subject to the same limitations described for single labelling of whole cells (Bagby and Pepe 1978). Double-staining of whole cells is just as feasible as single-staining. Indeed, having a definite marker for myofibrils (TRAM) makes the localization of -actinin much easier to interpret.     

The fine structure of myocytes in the sponges microciona prolifera (Ellis and Solander) and tedania ignis (Duchassaing and Michelotti)

Myocytes are long, fusiform cells found in the osculum and other contractile areas of many sponges. Myocytes in the oscular sphincter of Tedania ignis and the osculum and dermal membrane of Microciona prolifera were studied with light- and electron-microscopes to compare their structure to that of muscles. Salient points of similarity between myocytes and smooth muscles were their long, fusiform shape, their red color after staining with Mallory's triple stain, and the presence of filaments running longitudinally in the cytoplasm. Microciona myocytes have thick filaments of 150–250 Å diameter and thin filament of 50–70 Å diameter, and in transverse sections the thin filaments occasionally appear as a ring of dots around a thick filament. Longitudinal sections of Tedania myocytes show only one type of filament, which varies from 100 Å to 200–300 Å diameter in thick regions of the filament. Although transverse sections show light material around the dense filaments, a distinct pattern of thick and thin filaments is not seen in Tedania. Due to infrequent contacts between cells, the large extra-cellular space observed with the electron microscope (49% in Tedania, 57% in Microciona), and the absence of nerves, each myocyte probably acts as an independent contractile unit.     

重庆地区人类免疫缺陷病毒携带者的微孢子虫感染情况调查

目前我国关于微孢子虫感染人的研究相对较少,更没有关于重庆地区人类免疫缺陷病毒(HIV)携带者感染微孢子虫的数据统计。对重庆市公共卫生医疗救治中心收治的22例HIV抗体阳性,但尚未接受抗病毒治疗的患者进行研究。将22例患者粪便样本分别提取总DNA,通过聚合酶链反应(PCR)法和DNA测序等技术对微孢子虫在患者中的感染情况进行检测,并对微孢子虫种类进行鉴定。同时对22例患者的免疫细胞亚群进行计数和分析。结果显示,微孢子虫的感染率为36.3%(8/22),主要由脑炎微孢子虫属(Encephalitozoon spp)的海伦脑炎微孢子虫(E. hellem)和肠脑炎微孢子虫(E. intestinalis)引起。22例患者的免疫细胞亚群分析显示,平均淋巴细胞数0.93±0.13×10⁹/L,CD4^＋T细胞数132±22 个/mL,CD8^＋T细胞数495±91 个/mL,CD4/CD8细胞比值0.37±0.1,表明患者免疫功能受到严重抑制。进一步将微孢子虫感染患者与未感染患者的免疫细胞亚群进行比较发现,感染患者淋巴细胞数为0.51±0.1×10⁹/L,未感染患者为1.17±0.17 ×10⁹/L,两者具有显著差异(P<0.05);感染患者CD4^＋T细胞数为71±27 个/mL,未感染患者为167±28 个/mL,两者具有显著差异(P<0.05);感染患者CD8^＋T细胞数为209±35 个/mL,未感染患者为658±123 个/mL,两者具有显著差异(P<0.05),以上表明微孢子虫感染组的免疫功能受损情况更严重。本研究结果提示微孢子虫的机会性感染与患者免疫功能受损情况密切相关,脑炎微孢子虫属在重庆地区有较高的感染率,这为进一步阐明微孢子虫与宿主相互作用关系奠定基础,也为公共卫生健康管理提供重要参考。     

刺梨GL2同源基因的克隆、系谱树和表达分析

为了观察刺梨果实的果刺细胞学发育过程,该研究以刺梨‘贵农5号''的cDNA为模板,通过RACE克隆获得刺梨中与拟南芥表皮毛形成GL2的同源基因RrGL2,并对该基因进行生物信息学分析和表达分析。结果表明:(1)刺结构在花芽形成早期基部内的细胞首先不断分裂,向外继续发育,中部的细胞变细、变长形成“针”状结构,顶部的细胞逐渐木质化使刺变硬,形成果刺。(2)通过RACE扩增得到RrGL2的cDNA全长2 292 bp,编码763 aa氨基酸。(3)RrGL2具有Homeodomain同源结构域和StAR磷脂酰胆碱转移蛋白的结构域,RrGL2与其他物种编码的GL2氨基酸同源性高度相似,并且系谱树分析揭示刺梨RrGL2和野草莓的GL2密切相关。(4)qRT-PCR分析表明,RrGL2在茎和果实中的表达水平高于其他组织,在花后7周果刺中的表达最高,是3周和5周果刺中的7.87倍和2.10倍。综上结果发现RrGL2的功能与果刺的形成发育密切相关,该研究为刺梨中刺形成的分子机制和育种提供了理论基础。     

桂林岩溶石山常绿落叶阔叶混交林乔木层优势物种生态位研究

采用样方法对不同资源位群落进行调查,通过冗余分析（RDA）结合Monte Carlo随机置换检验筛选出对桂林岩溶石山常绿落叶阔叶混交林乔木层优势物种分布影响显著的土壤资源轴（土壤有机质、土壤水溶钙、土壤速效氮、土壤全氮、土壤pH值和土壤厚度）。利用Levins生态位宽度指数和Pianka生态位重叠指数对桂林岩溶石山常绿落叶阔叶混交林乔木层14个优势物种在土壤有机质、土壤水溶钙、土壤速效氮、土壤全氮、土壤pH值和土壤厚度6个土壤资源轴上的生态位特征进行研究。结果表明：（1）在土壤有机质和土壤水溶钙资源轴上,生态位宽度最大值为常绿物种檵木（0.89、0.94）;在土壤pH值资源轴上,生态位宽度最大值为常绿物种粗糠柴（0.96）;在土壤速效氮、土壤全氮和土壤厚度资源轴上,生态位宽度最大值为落叶物种南酸枣（0.87、0.99、0.97）。（2）14个优势物种在6个土壤资源轴上,生态位重叠均值由大到小依次为土壤厚度 > 土壤全氮 > 土壤水溶钙 > 土壤速效氮 > 土壤pH值 > 土壤有机质,且均表现为常绿物种与常绿物种形成的种对生态位重叠均值（0.84） > 常绿物种与落叶物种（0.74） > 落叶物种与落叶物种（0.66）。（3）综合6个土壤资源轴,生态位重叠值≥0.5的种对高达480对,占总种对数的87.91%,暗示桂林岩溶石山常绿落叶阔叶混交林乔木层优势物种在资源匮乏且资源异质性高的特殊生境中存在激烈竞争,且乔木层种间关系相对不稳定。     

青藏高原草地产量与草畜平衡变化

深入了解青藏高原草地生产力与草畜平衡状态的变化,是青藏高原生态屏障建设和生态环境保护的基础。利用遥感植被指数和叶面积指数产品,基于VIP生态水文过程模型,模拟分析了青藏高原2000—2018年间草地生产力的时空格局,并结合同期农牧业统计资料,分析了青藏高原县域尺度草畜平衡状态的变化。结果表明:青藏高原草地多年平均净初级生产力(NPP)为158.4 g C·m^-2·a^-1;近20年来草地生产力上升趋势明显,显著上升面积的比例为44.7%。气候变暖、降水量增加、草本植物生长期延长和大气CO₂浓度升高是青藏高原草地生产力提高的主要驱动因素。基于草场产量估算的青藏高原平均理论载畜量为1.17 SU·hm^-2,年增长率为0.011 SU·hm^-2。2000年以来青藏高原草地超载情况总体趋于好转,严重超载县的面积比例已降至20%以下,其中超载程度较严重的地区,畜牧业的维持和发展主要依靠作物秸秆补饲。研究结果可为区域农牧业结构调整和生态环境保护提供科学依据。     

翻压山黧豆绿肥与氮肥减施对水稻生长及其养分吸收与产量的影响

为探明种植翻压山黧豆绿肥与减施氮肥下的水稻生产潜力,通过3年田间定位试验,设置冬闲+不施肥(NF)、山黧豆绿肥(GM)、冬闲+常规氮肥(100%N,CK)、山黧豆绿肥+80%常规氮肥(GM+80%N)、山黧豆绿肥+70%常规氮肥(GM+70%N)、山黧豆绿肥+60%常规氮肥(GM+60%N)6个处理,研究不同处理对水稻生长、养分吸收及产量的影响。结果表明：(1)与CK相比,翻压山黧豆绿肥并减施氮肥处理均能够显著提升水稻株高、增加水稻分蘖数、提高水稻干物质积累量,其中以GM+70%N施肥处理提升效果最为明显。(2)GM+70%N施肥处理下,不同生育时期水稻株高、有效分蘖数分别较对照常规施肥(100%N)提升了13.32%~ 15.73%和33.98%~59.47%,水稻干物质积累量提高了23.19%~144.18%,且随着生育时期的推进增加速率依次降低。(3)种植翻压山黧豆绿肥并减施氮肥处理下水稻产量均有所提高,其中GM+70%N和GM+80%N处理显著提高,增产分别达13.84%,7.25%,且GM+70%N处理下水稻植株和籽粒养分吸收更为全面。研究发现,种植翻压山黧豆并适量减施氮肥能有效促进水稻生长和养分的吸收积累,显著提高水稻产量,说明翻压山黧豆绿肥可替代稻田30%~40%的氮肥施入量,并可在避免水稻旺长的同时实现水稻高产,是四川水稻种植较好的耕作措施。     

硅素对水稻幼苗镉积累及抗胁迫应答的调节效应

施硅(Si)可以显著缓解镉(Cd)胁迫对水稻生长发育的毒害效应。本研究通过水培分根试验,研究了Si对水稻幼苗Cd积累及胁迫应答的调节效应。结果表明: Cd胁迫下水稻幼苗的生物量显著降低,加Si可以显著缓解Cd对水稻幼苗生长的抑制效应。水稻幼苗对Cd的吸收、转运和积累明显受到Si的影响,单侧根系Cd胁迫下加Si(Si-Cd+Si,Si-Cd)使根系对Cd的滞留系数达83.3%～83.6%,限制了Cd从根向地上部转移。单侧根系Cd胁迫下非胁迫侧加Si(Si-Cd)处理的植株对Cd的吸收和累积明显增加,尤其是根中Cd的积累量较单侧根系Cd胁迫下无Si(CK-Cd)处理增加了48.2%;而单侧根系Cd胁迫下双侧加Si(Si-Cd+Si)处理则显著降低了根和地上部对Cd的吸收,较CK-Cd处理分别降低了36.7%和54.9%。双侧Cd胁迫下单侧加Si(Cd-Cd+Si)则使根和地上部对Cd的吸收量显著减少,较双侧根Cd胁迫(Cd-Cd)处理分别降低了57.8%和46.5%。Cd胁迫下水稻幼苗根中含较高浓度的Si,加Si则使Cd胁迫下根和地上部积累更多的Si。加Si也影响了水稻幼苗对其他金属元素如钙(Ca)、镁(Mg)、锰(Mn)的吸收,Cd-Cd+Si处理显著增加了根系和地上部的Ca、Mg浓度,但Mn浓度的变化则因Cd胁迫程度而表现不同。加Si对Cd胁迫下根系超氧化物歧化酶(SOD)和过氧化物酶(POD)活性有一定的影响,尤其是Si-Cd处理的胁迫侧POD和非胁迫侧SOD活性显著上升,有利于清除Cd胁迫产生的氧自由基。总之,Si对Cd胁迫下水稻幼苗生长、Cd和Si等的吸收及根系的抗氧化反应有一定的调节效应,植株体内较高的Si浓度有利于增强植株对Cd的耐受性。     

斑马鱼Fbxl5基因多克隆抗体的制备与检测

Fbxl5为F-box基因家族的一员,目前研究发现其与心脏的发育有关。为了在斑马鱼模型中进一步研究该基因的功能,有必要制备其多克隆抗体。通过桥式PCR扩增出亲水性和特异性均好的斑马鱼Fbxl5基因片段,将其克隆入表达载体pET-28a,转化至大肠杆菌Rosseta中。用IPTG诱导Fbxl5重组质粒得到His-Fbxl5的融合蛋白。这个融合蛋白用Ni-IDA凝胶柱亲和纯化,将纯化的His-Fbxl5融合蛋白免疫新西兰大白兔制备多克隆抗体。使用Western Blot检测,获得了Fbxl5原核表达重组融合蛋白及高效价的特异性兔抗Fbxl5多克隆抗体。随后检测了Fbxl5在斑马鱼胚胎和成体组织中蛋白的表达,通过基因芯片分析在Fbxl5-MO和Std胚胎样品的mRNA含量差异.所得结果显示获得了较高效价和特异性好的斑马鱼Fbxl5多克隆抗体,为Fbxl5功能的进一步研究奠定了基础。