α_1肾上腺素能受体介导去甲肾上腺素促大鼠培养心肌细胞蛋白质合成效应

在无血清和含１．０％、５．０％血清培养的新生大鼠心肌细胞标本上,去甲肾上腺素（ＮＥ,２．０μｍｏｌ／Ｌ）使细胞蛋白质含量（Ｌｏｗｒｙ法）分别比相应对照组增加４０％、２６％、１９％（Ｐ均＜０．０１）;细胞３Ｈ－亮氨酸参入量与ＮＥ浓度呈正性剂量依赖性,最大效应浓度为２０．０μｍｏｌ／Ｌ;无血清培养体系中,０．２、２．０、２０μｍｏｌ／Ｌ的ＮＥ使参入量分别比对照增加１７．８％、３５３％、３７．７％;１．０％血清培养体系中分别比对照增加１６．２％、２７．９％、３１．６％（Ｐ均＜０．０５）;哌唑嗪（２．０μｍｏｌ／Ｌ）可阻断ＮＥ的促蛋白质合成作用,心得安（２．０μｍｏｌ／Ｌ）则无此效应。提示在无血清或低浓度血清培养体系中,ＮＥ可促进心肌细胞蛋白质合成,增加细胞蛋白质含量,该作用可能是通过α１肾上腺素能受体介导。     

10株水痘带状疱疹病毒的分离鉴定与生物学特性

用ＶｅｒｏＥ６细胞从１２例水痘及带状疱疹病人水疱液中分离到１０株病毒,分离阳性率为８３．３％。１０株病毒均具有使感染细胞圆缩、融合、脱落等典型的ＶＺＶ局灶性细胞病变（ＣＰＥ）特点,ＣＰＥ随着传代次数增加而加快。用带状疱疹恢复期病人血清作间接免疫荧光染色镜检,可见典型的感染细胞核内荧光块。１０株病毒感染细胞制成的抗原片,检测５例带状疱疹病人急性期及恢复期血清,荧光抗体滴度均有４倍以上增高。ＶＺＶ对Ｖｅｒｏ细胞敏感;对沙鼠肾,胎兔肾,胎豚鼠肾、肺、脾原代单层细胞不敏感;对乳小白鼠不敏感。毒种在－２０℃保存一周内死亡;但在３０％脱脂牛奶、２０％小牛血清及１０％山梨醇Ｅａｇｌｅ’ｓ液中,液体或真空冷冻干燥－１００℃可保存二年以上。     

α肾上腺素能受体介导去甲肾上腺素促大鼠培养心肌细胞蛋白…

在无血清和含１．０％、５．０％血清培养的新生大鼠心肌细胞标本上,去甲肾上腺素（ＮＥ,２．０μｍｏ１／Ｌ）使细胞蛋白质含量（Ｌｏｗｒｙ法）分别比相应对照组增加４０％、２６％、１９％（Ｐ均＜０．０１）;细胞＾３Ｈ－亮氨酸参入量与ＮＥ浓度呈正性剂量依赖性,最大效应浓度为２０．０μｍｏ１／Ｌ;无血清培养体系中,０．２．２．０、２０μｍｏ１／Ｌ的ＮＥＷ使参入量分别比对照增加１７．８％、３５．３％、３７．７％     

不同温度下锐劲特对受马拉硫磷抑制的麦穗鱼AChE活性恢复的影响

研究了１５～１７℃、２０～２２℃下,０．０１ｍｇ／Ｌ锐劲特对被１ｍｇ／Ｌ马拉硫磷抑制的麦穗鱼（Ｐｓｅｕｄｏｒａｓｂｏｒａ ｐａｒｖａ）脑乙酰胆碱酯酶（ＡＣｈＥ）活性恢复的影响。结果表明：在１５～１７℃,经４８ｈ抑制,ＡＣｈＥ被抑制了４０％,恢复１４４ｈ后,处理组与无锐劲特对照都恢复到了空白对照的８０％以上。在此温度下锐劲特对ＡＣｈＥ活性恢复的影响不明显;而在２０～２２℃下,经相同的处理时间,ＡＣｈＥ被抑制了７０％,恢复２４０ｈ后,处理组与无锐劲特对照都只恢复到了对照的７０％。其间于１４４、１９２ｈ,ＡＣｈＥ活性差异达显著水平（Ｐ〈０．０５）,说明了在此温度下锐劲特对ＡＣｈＥ活性恢复的影响。文中还讨论了恢复研究的意义。     

无血清培养鹌鹑胚胎骨骼肌细胞的光镜与电镜观察

用无血清、无胚胎抽提液培养液（Ｅａｇｌｅ’ｓ，ＭＥＭ）培养孵育８ｄ的鹌鹑胚胎骨骼肌细胞，接种后圆形的生肌细胞可发育、分化形成线状的肌管，历时４—６ｄ。对培养７２ｈ肌细胞苏木精－伊红染色光镜照相和电镜观察证明肌细胞在本实验条件下能分化形成肌细胞特有的横纹结构和肌丝结构。     

长角血蜱幼虫中的蜕皮甾类(英文)

本文用放射免疫分析和高效液相色谱法对长角血蜱幼虫中的蜕皮甾类进行了测定。　蜕皮甾类存在于吸血和饱血幼虫,并且随生理时期而变化。吸血期和饱血后前３天,激素含量低（小于 １４． ３５ｐｇ ＥＥ／只）;饱血后５天迅速上升（２９．８７ｐｇ ＥＥ／只）;饱血后７天达到高峰（５６． ０４ｐｇ ＥＥ／只）;高峰后又下降到低水平（１９．３８ ｐｇＥＥ／只）。高峰期的蜕皮甾类主要为两种成分,２０Ｅ和Ｅ（２０Ｅ∶Ｅ＝３．０７∶１）。２０Ｅ可能是幼虫发育中起主要作用的激素。     

蛇毒蛋白Echistatin的C端突变基因的构建，表达及其活性研究

通过ＰＣＲ定点突变的技术,将蛇毒蛋白Ｅｃｈｉｓｔａｔｉｎ基因的Ｃ端进行了突变（Ａｌａ４８→Ａｒｇ４８→,Ｔｈｒ４９→Ｖａｌ４９）,模拟纤维蛋白Ｎ端的四肽（Ｇｌｙ－Ｐｒｏ－Ａｒｇ－Ｖａｌ）,以期增加Ｅｃｓ（Ｅｃｈｉｓｔａｔｉｎ）的活性。突变的基因重组到表达质粒ｐＪＣ２６４上,经ＩＰＴＧ诱导,以ＣｈｅＹ－Ｅｃｓ融合蛋白方式进行了表达,表达量占菌体总蛋白的１５～２０％。ＳｅｐｈａｄｅｘＧ－７５初步纯化该融合蛋白,然后用ＣＮＢｒ裂解,透析,冻干,反相ＨＰＬＣ纯化Ｃ端突变体Ｅｃｓ蛇毒蛋白,Ｎ端十个氨基酸分析与天然的相符,在ＰＲＰ（ｐｌａｔｅｌｅｔ－ｒｉｃｈｐｌａｓｍａ）测活体系中,１０μｍｏｌ／Ｌ的ＡＤＰ诱导,Ｃ端突变体Ｅｃｓ抑制血小板凝聚的活性约为野生型４倍。得到了Ｅｃｓ的Ｃ端突变后使Ｅｃｓ抑制血小板凝聚的活性提高的结果。     

蛇毒蛋白Echistatin的C端突变基因的构建,表达及其活性研究

通过ＰＣＲ定点突变的技术,将蛇毒蛋白Ｅｃｈｉｓｔａｔｉｎ基因的Ｃ端进行了突变（Ａｌａ４８→Ａｒｇ４８→,Ｔｈｒ４９→Ｖａｌ４９）,模拟纤维蛋白Ｎ端的四肽（Ｇｌｙ－Ｐｒｏ－Ａｒｇ－Ｖａｌ）,以期增加Ｅｃｓ（Ｅｃｈｉｓｔａｔｉｎ）的活性,突变的基因重组到表达质粒ｐＪＣ２６４上,经ＩＰＴＧ诱导,以ＣｈｅＹ－Ｅｃｓ融合蛋白方式进行了表达,表达量占菌体总蛋白的１５￣２０％,Ｓｅｐｈａｄｅｘ Ｇ－７５初步纯化     

植物油对虫霉菌液体培养与保存的作用*

在萨氏营养液（ＳＤＢ）中加入用 ０．１％蔗糖脂肪酸酯－１５（ＳＥ１５）乳化的０．５０％芝麻油,可使在ＳＤＢ中生长慢而结块的新蚜虫疠霉、安徽虫瘟霉、飞虱虫疠毒和根虫瘟霉等四种虫霉的五个菌株形成均匀分散的菌丝体和能稳定转接的菌液。在ＳＤＢ中加入乳化芝麻油 （ ０．２５％～１．５％）、菜籽油（ ０．５０％～１．５％）和色拉油（ ８０％花生油和 ２０％菜籽油,０．７５％～１．５％）,能明显提高新蚜虫疠霉Ｆ９８０１８的生物量;而在ＳＤＢ中加入０．５０％乳化芝麻油,可使液培菌丝产孢量增加近一倍;在ＳＤＢ中加入的三种植物油（０．７５％）,在Ｆ９８０１８生长４ｄ后,含量分别下降了８５％～９４％,但其中脂肪酸相对组成基本不变,表明该菌生长能充分利用植物油中各脂肪酸。用萨氏培养基（ ＳＤＡＹ）、加入 ０．５０％乳化芝麻油的 ＳＤＡＹ（ ＯＳ－ＳＤＡＹ）和牛奶蛋黄培养基（ＳＥＭＡ）培养安徽虫瘟霉Ｆ９７０２８和新蚜虫疠霉Ｆ９８０２８,两菌株在ＳＥＭＡ上生长最快;在ＯＳ－ＳＤＡＹ以较慢速度正常生长;而在ＳＤＡＹ上,两菌株或不能生长,或在第二次培养时出现明显退化。用ＳＥＭＡ和ＯＳ－ＳＤＡＹ在３℃下来保存上述四种虫霉的七个菌株,在ＯＳ－ＳＤＡＹ上?     

米曲霉Aspergilus oryzae发酵生产曲酸的研究

分离到Ａｓｐｅｒｇｉｌｕｓｏｒｙｚａｅ１３个菌株,其曲酸产量变化幅度１６６—４８６ｍｇ／ｍｌ,从中选出４个高产菌株。在１％酵母提取物和１５％蔗糖培养液中３０℃发酵培养,８—１０天菌体生长量和曲酸产量达到最大值,随后曲酸产量迅速下降。蔗糖浓度对菌体生长和曲酸产量影响甚大,最适蔗糖浓度为１５％。天冬氨酸、甘氨酸、赖氨酸、谷氨酸、吡哆醇、叶酸和抗坏血酸有利于菌体生长并显著提高曲酸产量。将在ＹＥＳ培养液中培养１０天的菌体重新悬浮于含１５％蔗糖的ＹＥＳ培养液或０２Ｍ磷酸缓冲液（ｐＨ６５）中８—１０天曲酸产量仍可达到４５ｍｇ／ｍｌ以上。低温条件下制备的培养８—１０天的Ａｏｒｙｚａｅ菌体匀浆反应系统仅有痕量曲酸形成。     

天然心房肽对实验性心功能与心肌酶的影响

以天然心房肽灌流豚鼠离体工作心脏,药后１分钟心率开始减慢,左室压及其微分开始升高;５分钟后逐渐恢复,至１０分钟恢复正常。体外培养大鼠心肌细胞,培养液中乳酸脱氢酶含量在药后２小时减少至４８．５±６．８ｕ,为药前的４２．７％（Ｐ＜０．０１）,药后８小时仍明显低于药前水平。为心房肽的新药开发提示了新思路。     

Proton NMR and CD solution conformation determination and opioid receptor binding studies of a dynorphin A(1-17) model peptide

The opioid peptide dynorphin A(1-17) contains a peptide segment in residues 7-15 with the potential to form an amphiphilic beta-strand. This amphiphilic structure may, like the amphiphilic alpha-helices found in many other peptide hormones, be an important determinant of its interactions with membranes and receptors. In order to investigate and characterize these interactions, we have synthesized a 17-residue dynorphin analogue (YGGFLKKVKPKVKVKSS) that incorporates a peptide model of this amphiphilic secondary structure with minimized homology (25%) relative to the native sequence. This peptide exhibits the full biological potency of dynorphin in assays of kappa-opioid receptor binding, and is more selective for this type of opioid receptor than the natural peptide. The conformation of the model peptide in aqueous solution has been investigated in detail by NMR spectroscopy. The values of the NH-CH alpha coupling constants together with rotating frame NOEs indicate the presence of an amphiphilic structure together with some beta-strand structure in residues 7-15, and demonstrate that a peptide model that stabilizes this structure in aqueous solution and enhances kappa-opioid receptor selectivity can be successfully designed using using alternating lysine and valine residues.     

Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium

The distribution of ANF was studied in the heart of the frog (Rana ridibunda) using indirect immunofluorescence. ANF-like immunoreactivity was localized mainly in the right and left atrium, most of cardiocytes being intensively labelled. At the electron microscopic level, all secretory granules present in atrial cardiocytes contained ANF immunoreactive material. Using a specific radioimmunoassay, we found higher concentrations of ANF in the left atrium (208 +/- 25 ng/mg protein) than in the right atrium (120 +/- 16 ng/mg protein) whilst in the rat, the right atrium contains the highest ANF concentration. The concentration of ANF in the ventricle was 10 times lower than in the whole atrium (32 +/- 4 ng/mg protein). Sephadex G-50 gel filtration of atrial extracts showed that ANF-like immunoreactivity eluted in three peaks. Most of the immunoreactivity corresponded to high molecular weight material eluting at the void volume while 20% of the material co-eluted with synthetic (Arg 101-Tyr 126) ANF. These results indicate that frog cardiocytes synthetize a peptide which is immunologically and biochemically related to mammalian ANF.     

不同时间采集的小牛血清对CHO-C28细胞培养及表达的影响

通过研究小牛血清对CHO-C28细胞培养及HBsAg表达的影响,探讨小牛血清的不同采集时间对血清质量的影响。采集出生后4、8、12h(未进食)小牛的血清,对CHO-C28细胞进行传代、换液培养,并检测乙肝表面抗原(HBsAg)表达量。结果可见:①在细胞传代4次时,4h采集的血清细胞生长状态良好,8h采集的血清细胞出现明显的衰老,12h采集的血清细胞大面积死亡;②在细胞维持换液方面,4h采集的血清可维持细胞换液25次以上,8h采集的血清可勉强维持20次,12h采集的血清维持细胞换液10次时已大部分死亡;③乙肝表面抗原(HBsAg)表达量的检测结果,同批培养上清,4h采集的血清培养细胞表达量最高。可见,小牛出生后采集血清时间越早越好。     

Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis

This study evaluated the influence of temperature on the immune responses and hematological parameters in channel catfish Ictalurus punctatus immunized via intraperitoneal injection with live theronts of Ichthyophthirius multifiliis. Fish were distributed in 18 aquaria and received 9 treatments: 4 groups of fish were vaccinated with live theronts and maintained at constant temperature 15 °C, 20 °C, 25 °C and 30 °C; 3 groups of fish vaccinated and subjected to cycling temperature regime from 15-25 °C, 20-25 °C and 20-30 °C, changed 5 °C each day; 2 groups of fish were not vaccinated and served as controls at 25 °C, one with Ich challenge and the other without challenge. Non vaccinated fish and those vaccinated at 15 °C or 15-25 °C did not show anti-Ich antibodies in the serum 14 and 21 days post-immunization. The antibody levels were significantly higher from fish vaccinated at 25 °C, 30 °C, 20-25 °C and 20-30 °C compared to fish at 15 °C, 20 °C and 15-25 °C both 14 and 21 days post-immunization. At constant water temperature, fish vaccinated at 15 °C showed significantly higher mortality rate (67.8%, P < 0.05) than those vaccinated at 20 °C, 25 °C, and 30 °C (0-10.7% mortalities). At cycling water temperature, fish vaccinated at 15-25 °C showed significantly higher mortality rate (67.8%) than those vaccinated at 20-25 °C and 20-30 °C (P < 0.05). Twenty days after immunization fish vaccinated at 30 °C and 20-30 °C showed significant increase in the red blood cells, white blood cells, thrombocytes and monocytes. Six days after challenge with I. multifiliis theronts the fish showed decreased white blood cells, thrombocytes and monocytes. This study suggests that vaccinated catfish were severely impacted by low temperature, either at 15 °C constant temperature or at 15-25 °C cycling temperature. The fish showed no anti-Ich antibodies and suffered high mortality similar to non vaccinated control fish.     

Amphibian cells in culture. I. Nutritional studies.

Nutritional requirements of amphibian cells in culture were studied for the purpose of modifying a minimal medium in which frog cells could proliferate and which could be used for obtaining drug-resistant and auxotrophic variants. The serum, purine, CO2, and amino acid requirements for ICR 2A (a Rana pipiens haploid cell strain) have been investigated employing two different media: L-15, a nonbicarbonate, amino acid-buffered medium and Eagle's MEM, a bicarbonate-buffered medium. In this paper we present evidence to support the following conclusions: (1) With L-15 as the base medium, 10% fetal calf serum (FCS) supports optimal cell growth during exponential phase. Calf serum, whole, dialyzed, or heat-inactivated, cannot substitute for FCS and, in fact, is inhibitory. (2) Purines are required by ICR 2A cells only if grown in a nonbicarbonate-buffered medium, since the cells under these conditions cannot produce enough endogenous CO2 to support de novo purine synthesis. (3) In addition to the amino acids considered essential for mammalian cells in culture, ICR 2A cells depend upon exogenous asparagine. Glutamine and/or aspartic acid cannot replace the asparagine requirement. However, ICR 2A cells do utilized exogenous glutamine as an oxidative substrate.     

Dead cell counts during serum cultivation are underestimated by the fluorescent live/dead assay

The live/dead fluorescent assay provides a quick method for assessing the proportion of live and dead cells in cell culture systems or tissues and is widely used. Dead cells are detected by the fluorescence produced when propidium iodide (PI) binds to DNA; PI and similar molecules are excluded from live cells but can penetrate dead cells because of their loss of membrane integrity. Here we investigated the effect of serum in the culture medium on the reliability of the method. We assessed viability of chondrocytes with/without serum using both a live/dead assay kit and also trypan blue staining. We found that after 2 days of culture, the DNA-binding dye PI could no longer detect dead cells if serum was present but they were readily detected in serum-free medium or if an inhibitor to DNase I was added to the serum-containing medium. Dead cells could be detected by trypan blue staining in all cultures. Hence dead cells are no longer detected as the DNase I present in serum degrades their DNA. DNA-binding dyes may thus not give a reliable estimate of the number of dead cells in systems that have been cultured in the presence of serum for several days.     

Purification of Gekko Small Peptide Fraction and Its Effect of Inducing Apoptosis of EC 9706 Esophageal Cancer Cells by Inhibiting PI3K/Akt/GLUT1 Signaling Pathway

This study aimed to isolate and purify a cytotoxic extraction from Gekko japonicus, identify its components and determine its cytotoxic activity in vitro. We isolated and identified the most potent cytotoxic Gekko small peptide LH-20-15. The identification and analysis of peptide sequences of LH-20-15 were performed by de novo peptide sequencing, and two new peptides were found. LH-20-15 significantly inhibited the proliferation of human esophageal squamous carcinoma EC 9706 cells in a dose-dependent manner. Furthermore, LH-20-15 induced apoptosis in esophageal cancer cells by activating the mitochondrial apoptotic pathway. Further research showed that LH-20-15 inhibited the PI3 K/Akt/GLUT1 signaling pathway. In conclusion, LH-20-15 from Gekko japonicus is a peptide mixture and may inhibit EC 9706 cell proliferation and induce apoptosis by activating the mitochondrial apoptotic pathway. It also regulates glucose metabolism by targeting the PI3 K/Akt/GLUT1 signaling pathway. These small peptides could be new sources of natural cytotoxic ingredients against esophageal cancer with potential drug values.     

Modification of MCDB 110 medium to support prolonged growth and consistent high cloning efficiency of diploid human fibroblasts

In preparation for studies on the growth factor requirements of normal and transformed human fibroblasts, we have developed a serum-free medium that supports vigorous long-term serial subculture of diploid human fibroblasts and allows them to form large-sized colonies with high efficiency (40 to 60%) when plated at cloning density (2 to 5 cells/cm2). This medium, which is a modification of Ham's MCDB 110 base medium with its serum replacement supplements, is relatively easy to prepare and the cost of the serum replacements is approximately the same as that of fetal bovine serum supplied at 10%. The ingredients of "Supplement B" of MCDB 110 medium were added in an ethanol solution, rather than in the form of liposomes, and were combined with bovine serum albumin (0.5%), a lipid carrier. Gelatin and fetuin were included as attachment factors instead of polylysine. Bioassays indicated that none of the ingredients in the medium were contaminated with either epidermal growth factor or platelet-derived growth factor. In this modified serum-free medium, which we have designated McM+SR1, diploid human fibroblasts grew for 21 days at the same rate as in the base medium, McM, supplemented with 10% FBS (i.e., 21 population doublings). During the next 20 days, they underwent 15 population doublings which was 75% of the rate of cells growing in the medium containing serum.     

Spontaneous development of protective anti-T cell receptor autoimmunity targeted against a natural EAE-regulatory idiotope located within the 39-59 region of the TCR-V beta 8.2 chain.

The development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats is mediated by V beta 8.2+ T cells specific for myelin basic protein. One consequence of this biased expression of V beta 8.2 is the spontaneous development of regulatory T cells and antibodies against residues 39-59 of the V beta 8.2 sequence. Moreover, a synthetic V beta 8.2-39-59 peptide could induce protection against and speed recovery from EAE. T cells and antibodies specific for V beta 8.2-39-59 could transfer protection from EAE. Recently, we reported that the protective T cell epitope is subsumed within the V beta 8-44-54 sequence. We now report that protection induced by V beta 8-44-54 lasted at least 102 days and produced "split tolerance," enhancing anti-myelin basic protein antibody titers but reducing anti-myelin basic protein T cell frequency. The shorter V beta 8-44-54 peptide induced a distinct set of antibodies that did not cross-react with the longer V beta 8.2-39-59 peptide, although both specificities could stain V beta 8.2+ T cells and were equally protective against EAE. However, the V beta 8.2-39-59 peptide, but not the V beta 8-44-54 peptide, would appear to represent the natural idiotope: antibodies to V beta 8.2-39-59 that develop spontaneously during EAE could be boosted to higher titers only by the V beta 8.2-39-59, but not by other TCR peptides from the V beta 8.2 sequence, including V beta 8-44-54 that contains the functional T cell epitope. These results suggest that natural processing of the TCR V beta-chain favors the formation of a peptide that resembles the V beta 8.2-39-59 sequence. The B cell epitope present on the V beta 8-44-54 sequence was evident only in the absence of residues 39-43 and 55-59, suggesting that the two peptides possess distinct conformations. However, the V beta 8-44-54 B cell epitope is most likely expressed on the V beta 8.2+ T cells, either as a low affinity determinant on the intact TCR alpha/beta heterodimer or as a cryptic epitope bound in the cleft of surface MHC molecules.