Alicyclic acid metabolism in plants 3. Fate of 14C-shikimate and 14C-quinate in mung bean plants

Young mung bean plants (Phaseolus mungo) were exposed to ¹⁴C-shikimateor ¹⁴C-quinate in the light. After 8 or 23.5 hr of incubationat 25°C, radioactivities in free and bound amino acids,organic acids, soluble and insoluble carbohydrates, ether-solublefraction and lignin were determined. Shikimic and quinic acidswere separated by the combined use of paper-chromatography andcolumn chromatography. Specific activity of formed quinate orshikimate was only slightly lower than that of fed shikimateor quinate. Specific activities of phenylalanine, tyrosine andbound tryptophan were high as compared with those of non-aromaticamino acids. Discussion is focused upon the interconversionbetween shikimate and quinate, and their roles in the biosynthesisof aromatic amino acids. (Received November 15, 1968; )     

Antimelanoma Activity of Chloroquine,an Antimalarial Agent With High Affinity for Melanin

The antimalarial agent chloroquine is known for high affinity for melanin. This 4-aminoquinoline derivative was examined for anti-melanoma activity and uptake into melanoma cells. Chloroquine inhibited growth of cultured melanoma cells; the effect was much greater to a moderately pigmented cell line HMV-II than to a nonpigmented HMV-I. Treatment with chloroquine at a dose of 62 mg/kg i.p. for 12 days prolonged by 71% the life span of mice bearing B16 melanoma, while 24-day treatment at 31 mg/kg resulted in a 81% increase in life span. HMV-II cells showed a two-fold increase in up-take of chloroquine as compared with HMV-I cells. Chloroquine, 24 hr after administration to mice implanted s.c. with B16 melanoma, was selectively accumulated in the pigmented tissues, melanoma and eyes. Other nonpigmented tissues such as the liver, lung, and kidney showed rapid uptake (within 1 hr) and release. These results suggest that chloroquine is toxic to pigmented melanoma cells, the process being partly mediated by binding to melanin     

The Fate of L-Tyrosine-UL−14C in Shoot-Forming Tobacco Callus

Tobacco callus fed L-tyrosine-UL^?14C was sampled at 3-day intervals for 15 days, homogenized and studied with respect to distribution of incorporated radioactivity. The supernatant obtained by centrifuging of the homogenates at 270 g contained the bulk of the radioactivity although significant activity was also detected in the pellet. Sucrose density gradient centrifugation of the supernatant showed over 90% of the recovered label to be associated with a fraction designated as “less dense than mitochondria”, with the remainder being found in the fraction identified as “mitochondria”. During tissue culture, virtually all of the radioactivity in the fraction “less dense than mitochondria” was recovered in the supernatant obtained by centrifugation at 100,000 g. From 4 to 18% of the labeling in the 100,000 g supernatant fraction was attributable to tyrosine-containing protein, and the rest to free tyrosine and unidentified anionic constituents. The highest proportions of radio-activity in the 270 g pellet were associated with substances extractable with NaCl, pronase, 4.6 N NaOH, and acetolyzing reagent. Low but substantial labeling characterized the extracts obtained with Triton X-100 and 1 N NaOH. The final unextractable residue contained 20% of the 270 g pellet radioactivity.     

Indispensability of Iron-bound Chick Transferrin for Chick Myogenesis in Vitro

Myotrophic activity of highly purified chick transferrins (Tfs) to chick primary myogenic cells has been studied in a culture medium containing horse serum. Iron-binding to Tfs is indispensable for the activity. The removal of iron from Tfs gives rise to a complete loss of the activity and it is restored by iron-rebinding depending on the amount of bound iron. This result, combined with other physicochemical and immunological data, strongly, confirms that the myotrophic activity is exerted by the Tfs themselves, not by a contaminating material(s). It has been found that culture medium containing horse Tf which seems inadequate for the study of the biological effects of Tfs is, however, suitable for studies on chick Tfs, since horse Tf is inactive in promoting chick myogenesis. Terminal sialic acid residues are unrelated to myotrophic activity since Tfs with different numbers of residues (0, 1, and 2 moles/Tf molecule) are comprable in their activities. The mechanism of Tf action on cells and contradictions among previous papers as to the requirement of Tf for cell growth have been discussed from the viewpoint of an iron-donor with class-specificity.     

Transferrin Receptor on Chick Fibroblast Cell Surface and the Binding Affinity in Relevance to the Growth Promoting Activity of Transferrin

We examined the transferrin (Tf) receptor of chick skin fibroblasts using chick ¹²⁵I-Tf. When the cells were incubated with ¹²⁵I-Tf on ice, most of the cell-associated ¹²⁵I-Tf was found on the cell surface; on the other hand, a large part of it was located inside the cells when incubated at 37°C. By equilibrium binding assay, the number of Tf receptors per cell was determined as 6.7 × 10³. Dissociation constant was estimated to be 2.6 × 10⁻⁸ M.
The binding of ¹²⁵I-Tf was competitively inhibited by the addition of chick unlabeled Tf. Weaker inhibition was observed when bovine Tf was used as a competitor. Horse Tf had no effect on the binding of chick Tf. This agrees well qualitatively with chick cell growth-promoting activites of these Tfs.
Removal of Fe from Tf affected the affinity for its receptors. About 5- to 10-fold higher concentrations of chick apo–Tf was needed to achieve the same degree of inhibition of ¹²⁵I-Tf binding as that made by chick Fe-Tf.     

亚热带人工林下植被根际土壤酶化学计量特征

为探讨林下植被根际土壤酶化学计量特征及其对林分类型和季节的响应, 该研究以江西省泰和县千烟洲试验站典型人工杉木(Cunninghamia lanceolata)、马尾松(Pinus massoniana)和湿地松(Pinus elliottii)林林下优势灌草檵木(Loropetalum chinense)、杨桐(Adinandra millettii)、格药柃(Eurya muricata)、狗脊蕨(Woodwardia japonica)和暗鳞鳞毛蕨(Dryopteris atrata)为对象, 在植被生长初期(4月)和旺盛期(7月)测定优势灌草根际土壤与碳(C)循环相关的β-1,4-葡萄糖苷酶(BG)、与氮(N)循环相关的β-1,4-N-乙酰葡糖氨糖苷酶(NAG)和亮氨酸氨基肽酶(LAP)、与磷(P)循环相关的酸性磷酸酶(AP)活性、酶化学计量比及土壤理化性质。结果发现: (1)根际土壤与C和N循环相关的酶活性以及BG:AP (酶C:P)在不同林下植被之间存在显著差异, 而与P循环相关的酶活性差异不显著。林分类型和取样季节显著影响BG:(NAG+LAP)(酶C:N), 且林下植被类型、林分类型和取样季节交互影响酶C:P。主成分分析表明, 根际土壤酶的活性及计量比在不同林下植被(檵木不同于格药柃, 且二者显著区别于其他物种)、林分类型(杉木林区别于马尾松、湿地松林)和取样季节之间均存在显著差异。土壤硝态氮(NO₃ ^--N)、铵态氮(NH₄ ⁺-N)、可溶性有机碳(DOC)含量和碳氮比(C:N)是影响林下植被根际土壤酶的活性及化学计量比的主要因素。(2)标准主轴回归分析表明, 林下植被根际土壤lg(BG)、lg(NAG+LAP)和lg(AP)之间存在显著线性关系, lgBG:lg(NAG+LAP):lgAP (酶C:N:P)约为1:1:1.3, 酶C:P及(NAG+LAP):AP (酶N:P)分别为0.14和0.15。AP远大于BG和NAG+LAP的活性, 导致lg(BG)和lg(NAG+LAP)与lg(AP)的回归斜率极显著偏离1。说明林下植被根际土壤酶的活性及计量比受植被种类、林分类型及取样季节影响, 且基质有效性在其中发挥重要作用。相较于C循环和N循环, 微生物会分配更多资源用于P循环相关酶的生产, 暗示亚热带人工林林下植被根际土壤微生物生长和活性更易受P限制。     

多重RT-PCR用于临床检测三种胃肠炎病毒的研究

轮状病毒、诺瓦克病毒和星状病毒是引起病毒性胃肠炎的主要病原因子。研究采用JV12/JV13、P1/P2和Mon340/Mon348三对引物,建立了同时检测这3种病毒的多重RT-PCR技术,并应用于128份临床粪便样本的检测,检出轮状病毒62份(48.44%),诺瓦克病毒8份(6.25%),星状病毒11份(8.59%)。在灵敏度试验中,轮状病毒的检测灵敏度为5pg/mL、诺瓦克病毒和星状病毒的检测灵敏度均为50pg/mL。该研究所建立3种常见胃肠炎病毒的多重RT-PCR方法具有特异性强、灵敏度高的特点,可用于临床病原诊断和溯源。     

组胺H1受体功能及调节机制研究进展

组胺1型受体(H1R]受体作为组胺最主要的受体亚型,广泛分布于中枢和外周神经末梢,随着各类新型H1受体桔抗剂的发现和基因敲除动物的应用,H1R的功能研究及其活化调节机制研究也不断深入.组胺通过H1R参与调节机体多种重要的生理病理功能,如参与炎症反应、疼痛反应、血管调节、认知功能、睡眠清醒节律、饮食节律和肥胖等.H1R活化可激活磷脂酶C(PLC),PLC水解1,4,5-磷脂酰二磷酸盐产生甘油二酯(DAG)和肌醇三磷酸(IP3),后者激活细胞内Ca2+通道,活化氮氧化物合成酶,最终生成NO和鸟苷酸环化酶(cGMP),并引起钾通道开放,导致超极化;也可激活磷脂酶A2(PLA2)形成花生四烯酸(AA).H1R可通过活化其基因转录水平进行上调.本文就近十年来国外相关进展情况做一综述.     

一种展示SARS冠状病毒受体结合区的重组枯草杆菌芽孢的制备及免疫原性分析

目的：利用枯草杆菌芽孢呈递技术制备表达SARS冠状病毒S蛋白受体结合区（RBD）的重组芽孢。方法：将枯草杆菌 CotB 基因构建到基因组整合质粒pDG1664中,再将 RBD 基因连接到 CotB 基因的下游,构建成重组质粒pDG1664-CotB-RBD,通过同源重组整合到PY-79枯草杆菌基因组中;利用红霉素抗性筛选重组菌并进行PCR和DNA测序鉴定,Western印迹鉴定重组菌芽孢表面RBD蛋白的表达情况;用表达RBD的重组芽孢以口服方式免疫小鼠,通过ELISA和流式细胞术检测重组芽孢的免疫原性。结果：制备出枯草杆菌基因组整合了RBD抗原基因的重组菌株RS1931,形成的重组芽孢表达相对分子质量约62×103的CotB-RBD融合蛋白;重组芽孢免疫的小鼠血清RBD抗原特异性IgG抗体滴度在末次免疫后2周可达1∶10880,重组芽孢初免后18周的小鼠脾细胞中IFN-γ＋CD4^＋、IL-4＋CD4^＋和IFN-γ＋CD8^＋T细胞比例上调,表明重组芽孢经口服免疫产生良好的体液免疫和细胞免疫应答。结论：针对SARS冠状病毒S蛋白RBD建立了枯草杆菌芽孢呈递技术方法,制备出在枯草杆菌芽孢表面稳定表达外源RBD蛋白的重组株,获得的重组芽孢具有良好的免疫原性,为开发芽孢呈递型SARS疫苗奠定了基础。