昆虫杆状病毒晚期启动子在大肠杆菌中启动CAT基因表达

将油桐尺蠖（Ｂｕｚｕｒａｓｕｐｐｒｅｓｓａｒｉａ）核多角体病毒晚期基因──多角体蛋白基因启动子及５′端编码区,以两种不同方式置于缺乏启动子的氯霉素乙酰基转移酶（ＣＡＴ）基因上游,使其分别终止在不同翻译终止位点,其宿主菌具有明显不同的氯霉素抗性,最高达２００ｍｇ／Ｌ　ＬＢ培养基以上,表明昆虫病毒启动子能启动原核基因表达。对多角体蛋白基因启动子能在大肠杆菌中有效工作的原因进行了讨论。     

毛叶芋兰的生物学特性和栽培技术

药用植物毛叶芋兰一般每年仅从地下块茎或根状茎顶端长出一片新叶,地下部分有真菌共生,地上部生活期短,平均为１６９天。花芽５月中旬出土,６月中旬果熟,花果期约３０天。毛叶芋兰通常生长于以漫射光为主,或短时直射光,土壤潮湿疏松,透水性良好,ｐＨ４．４～６．５的灌木林下。栽培时,应根据毛叶芋兰生境条件选择植地,并注意防止积水和虫害。     

t-PA cDNA在CHO细胞中的高效稳定表达

我们曾报道t-PA mRNA非翻译区序列对其表达有明显的抑制作用,在此基础上,通过对5′-UTR及3′-UTR的改造,使t-PA在COS-7细胞中的表达水平提高30倍左右。将t-PA表达质粒用电击法转染中国仓鼠卵巢细胞二氢叶酸还原酶缺陷株(CHO-dhfr),经过混合加压及筛选,在CHO细胞中高效表达了t-PA,表达水平达到5000～6000 IU/10~6细胞/24hr。重组t-PA具有与天然t-PA相同的分子量及酶活性。经过8个月连续传代,表达水平未下降,表明细胞株是稳定的,其主要指标均符合工程细胞株的要求。     

Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G-protein,inositol 1,4,5-trisphosphate—sensitive Ca2+ pool,and Ca2+ channels

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca²⁺]_i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca²⁺ ([Ca²⁺]_e). [Ca²⁺]_i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca²⁺]_e. However, in the absence of [Ca²⁺]_e, the [Ca²⁺]_i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca²⁺ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5-trisphosphate (IP₃) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT-induced increase in [Ca²⁺]_i and IP₃ formation. U-73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca²⁺]_i by OT dose dependently. U-73122 (3 μM) also abolished the OT-induced IP₃ formation. Thapsigargin (2 μM), an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, did not increase [Ca²⁺]_i. However, it did time-dependently inhibit the OT-induced increase in [Ca²⁺]_i. Nimodipine (1 μM), a Voltage-dependent Ca²⁺ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La³⁺ (1 μM), a nonspecific Ca²⁺ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca²⁺ Current (I_ca) by 40% with no apparent changes in the current-voltage relationship. The OT-induced increase in I_ca reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX-insensitive G-protein–PLC-IP₃ signal transduction, resulting in an increase in [Ca²⁺]_i; (2) the OT-induced increase in [Ca²⁺]_i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca²⁺ release from the IP₃-sensitive pool, and to a lesser extent by Ca²⁺ influx, whereas the plateau is from increased Ca²⁺ influx; and (3) the influx is via VDCC and receptor-operated Ca²⁺ channels. © 1995 Wiley-Liss, Inc.     

Application of the multiple antigenic peptides (MAP) strategy to the production of prophormone convertases antibodies: Synthesis,characterization and use of 8-branched immunogenic peptides

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediatley following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84–99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, ¹H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventioanl method of peptide–carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the ease of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.     

Comparison of arbitrarily primed polymerase chain reaction and ribotyping for subtyping Actinobacillus actinomycetemcomitans

This study investigated the compatibility of arbitrarily primed polymerase chain reaction (AP-PCR) and ribotyping in the characterization of Actinobacillus actinomycetemcomitans , a major pathogen in the mixed anaerobic microflora of human periodontitis. AP-PCR was performed directly on lysed bacterial colonies using a random-sequence 10-base oligonucleotide primer. Ribotyping was carried out by using purified bacterial chromosomal DNA digested with BglI. DNA fragments were separated electrophoretically, blotted onto a nylon membrane and hybridized with the plasmid pKK3535 containing the rRNA operon of Escherichia coli. The two genetic methods were evaluated on isolates from single individuals and from family members. Twelve AP-PCR types and 47 ribotypes were distinguished among 76 A. actinomycetemcomitans isolates of different serotypes. AP-PCR typing and ribotyping gave compatible results in 18 of 20 comparisons. Although AP-PCR detected less genetic heterogeneity in A. actinomycetemcomitans than ribotyping, the rapid and relatively simple AP-PCR technique seems to be sufficiently discriminative to be used in large scale epidemiological studies which preclude the application of the more laborious ribotyping technique.     

根瘤菌资源数据库（RRDB）的建立

在总结我室多年对根瘤菌资源调查与分类研究的基础上,建立了根瘤菌资源数据库（ＲＲＤＢ）。此库依据根瘤菌研究特点而设计,包括：基本信息、采集信息、保藏信息、回接信息、参考文献、性状信息、菌株说明等七个子库。目前收录了来自国内２１个省或地区及国外部分研究单位提供的;并经全面性状分析与分类研究的２８６个菌株的信息,每个菌株设有寄主来源、固氮酶活性、碳源、氮源的利用等３２１个数据项。该库具有数据维护、查询检索、数据统计、输出打印等功能,引入并连接了聚类分析软件包－ＭＩＮＴＳ系统,可以满足当前研究的需要。     

马铃薯Y病毒组病毒高产量提取方法的建立

本文报道了高产量提取芜菁花叶病毒（ＴｕＭＶ）、莴苣花叶病毒（ＬＭＶ）、芋花叶病毒（ＤＭＶ）和大豆花叶病毒（ＳＭＶ）的提取方法。本方法通过使用高盐浓度的磷酸盐缓冲液以及在缓冲液中加入氯化镁和脲,并用ＴｒｉｔｏｎＸ－１００作为澄清剂,替代常规使用的氯仿和正丁醇,成功的提取到了大量病毒粒子,上述四种病毒提取的得率分别是ＴｕＭＶ为１７３．３ｍｇ／ｋｇ病叶,ＬＭＶ为９６ｍｇ／ｋｇ病叶,ＳＭＶ为１９９．２ｍｇ／ｋｇ病叶,ＤＭＶ为１７６．６ｍｇ／ｋｇ病叶。