猕猴桃实生苗再生体系的建立

以成熟饱满的美味猕猴桃种子发芽获得实生苗,分别以实生苗的茎段、叶柄和叶片为材料建立了再生体系。结果表明:种子以2.5mg/L的赤霉素(GA3)处理8h较适合;以培养基发芽较为适合;茎段、叶柄和叶片的愈伤组织的诱导率均为100%,且茎段、叶柄比叶片容易脱分化,但叶片的平均出苗率最高。再生苗在移栽5d后,开始长出新叶,10d后就能完全适应外界环境。     

Secretion and degradation of parathormone as a function of intracellular maturation of hormone pools

The biosynthesis, processing, and secretion of parthormone and the effect of calcium on these processes were measured in dispersed porcine parthyroid cells incubated with [(35)S]methionine. Proparathormone was detected at 10 min, the earliest time measured, and was rapidly and apparently quantitatively converted to parathormone. The half-life of the prohomormone pool was 15 min. Secretion of parathormone was detected by 20 min. In pulse-chase experiments there was a period between 20 and 40 min during which the wave of newly-synthesized parathormone was secreted. After 40 min during little additional radioactive hormone was secreted, but dibutyryl cyclic AMP, an agent that can mobilize stored parathormone, when added to the incubation mixtures enhanced radioactive parathormone secretion but only after 60 min, although it increased net hormone secretion as determined by radioimmunoassay to the same extent at all times studied. When the ionized calcium concentration of the medium was lowered, more radioactive hormone was secreted at all times but the effect was greatest on that hormone that was synthesized less than 60 min previously ; however, net hormone secretion in contrast to radioactive hormone was enhanced equally at all intervals. These data could mean that the refractoriness to secretion of parathormone 40-60 min of age was related to maturation of secretory container preparatory to storage. Low calcium (0.5 mM) stimulated hormone secretion up to fivefold compared to high calcium (3.0 mM) but did not affect synthesis of parathormone or proparathormne or conversion of the latter to hormone. During processing at least 70 percent of the intracellular parathormone was lost, presumably through proteolysis and this degradation was greater at high calcium. These data have been interpreted in light of the concept that two secretable pools of parathormone exist within the parathyroid.     

重组人尿激酶原的临床研究概况

尿激酶原或尿激酶型纤溶酶原激活剂具有特异性溶解血栓作用,引起人们的极大兴趣。西方国家1986年开始进行临床研究,A-74187或Sp2/0表达的糖基化尿激酶原以及大肠杆菌表达的非糖基化尿激酶原治疗急性心肌梗死阻塞相关血管开通率为70%~80%。德国Grünenthal公司用大肠杆菌表达的非糖基化尿激酶原(Saruplase)治疗急性心肌梗死研究采用20 mg推注,60 mg/60 min滴注,分别与重组组织型纤溶酶原激活剂、尿激酶、链激酶进行比较,并做了1698名心肌梗死患者的开放性临床试验,梗塞相关血管的开通率达到70%~80%。Saruplase与链激酶在104个临床研究中心,入组3089名急性心肌梗死患者进行大规模临床试验。结果表明,Saruplase对阻塞相关血管开通率、30天死亡率、出血合并症等副作用与链激酶没有明显差异,而出血性中风发生率高于链激酶,欧盟未批准Saru plase上市。国产重组人尿激酶原也进行了探索研究,用于治疗急性心肌梗死给药剂量为30~60 mg,给药时间为30或60 min,阻塞相关血管开通率为63.4%~80.0%,而尿激酶为52.2%~66.7%(平均58.0%)。此外,国外用重组人尿激酶原治疗深层静脉血栓和缺血性中风进行了探索研究,但病例数较少,尚须进一步研究。     

Subunit Structure of Glucoamylase of Saccharomyces diastaticus

Extracellular glucoamylase produced by a starch-fermenting yeast, Saccharomyces diastaticus 5106-9A, was purified. The enzyme was found to be heterogeneous in molecular weight, ranging from approximately 80K to 66K as estimated by gel filtration, and consisted of two subunits, H and Y. The molecular weight of subunit H was heterogeneous and was determined to be approximately 68K, 59K, and 53K by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of subunit Y was 14K, estimated by the same gel. the molecular weight of the deglycosylated form of subunit H was 41K, suggesting that the heterogeneity of the enzyme was due to glycosyl moieties of subunit H. Subunits H and Y were separated by gel filtration in the presence of sodium dodecyl sulfate. Subunit Y seemed to be hydrophobic, since it was insoluble in an aqueous buffer without detergent.     

112株志贺菌菌群分布和药敏特点分析

目的研究本地区2001年至2005年志贺菌菌群分布及其药敏特点,以指导临床合理抗菌治疗。方法经大便培养筛选志贺菌,用生化和血清学方法鉴定菌群和血清型,采用K-B法检测病原菌耐药性。结果在112例细菌性痢疾患者中,男女比例相似,年龄分布以婴幼儿最高,临床表现不典型者较多,菌群分布以福氏志贺菌最多,F2b为优势血清型,对抗菌药物敏感性差异有显著性。结论近5年来本地区细菌性痢疾患者发病特点有年龄差异,菌群仍以福氏志贺菌为主,血清型以F2b为主,第3代头孢菌素是治疗细菌性痢疾最佳的抗菌药物。     

The origin of transfer of P307

The DNA fragment carrying the oriT region from the enterotoxin plasmid P307 was isolated and its polynucleotide sequence was determined. Using Southern hybridization assays with a synthetic oligonucleotide probe, the oriT region was identified on a 7.9-kb EcoRI fragment from P307. By ligating the fragment with the cloning vector pUC119, plasmid pAG10 was obtained. The physical map of the insert was determined and oriT was located on a 540-bp BglII/SalI fragment. After this fragment was subcloned into sequencing phages, the polynucleotide sequence was established. Part of the sequence proved to be almost identical to segments of the oriT regions of the plasmids F and R1; another neighboring region was very different among all three sequences. The polynucleotide sequence proximal to traM is highly similar to that of F but different from that of R1.     

Effect of Copper on Growth of Yeast

The effect of copper was tested on the growth of many strains of yeast. Plate culture on density gradient agar of copper was used for estimating the growth response to copper. Growth in many strains was more strongly inhibited by the copper-aquo complex than by the copper-amino acid complex. Debaryomyces hansenii IFO 023 was found a suitable strain for the present study, because it was not resistant, not producing H₂S, and copper absorption by this strain was similar to that of the resistant strain. Growth of yeast cells in medium containing copper was affected by pH and concentration of amino acid in medium. Absorption of copper into intact cells was almost saturated for the initial few minutes. It was also affected by the addition of amino acid to copper solution. Our results indicated that the growth response of yeast to copper was closely related to copper absorption into cells. About 60 percent of copper absorbed into cells was distributed in the soluble fraction of the cell homogenate which was obtained by centrifugation at 105,000 g for 60 min.     

猪圆环病毒2型病毒样颗粒的高效组装技术研究*

目的:探索猪圆环病毒2型(PCV2)病毒样颗粒(VLPs)的高效组装技术,提高VLPs的稳定性。方法:利用大肠杆菌表达PCV2 Cap蛋白自组装为VLPs,分析不同离子强度下VLPs的稳定性。利用切向流技术添加尿素,降低pH,可使VLPs解组装,利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。结果:PCV2 Cap蛋白自组装VLPs在150mmol/L NaCl下稳定性较差,而在500mmol/L NaCl下可提高VLPs的稳定性,但仍较易发生聚集,核酸含量均较高。在150mmol/L NaCl、300mmol/L尿素和pH 5.5的缓冲体系条件下,能够使VLPs解组装。经25%~50%饱和硫酸铵(V/V)分级沉淀粗纯,阴离子交换层析500mmol/L NaCl下洗脱获得精纯Cap蛋白,蛋白质纯度≥95%,并能够有效去除核酸。通过切向流技术去除体系中的尿素,并将NaCl浓度提高至1mol/L、pH提高至8.0,改变蛋白质表面静电荷分布,实现VLPs的高效、均一再组装,组装效率≥99%,回收率为65.85%,并明显提高VLPs的稳定性,能够稳定保存6个月以上。结论:利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。     

Isolation and characterization of a cysteine protease of freesia corms

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The Mr of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-p-NAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.     

Flower-inducing and -inhibiting activities of Phloem Exudate from Cotyledons of Pharbitis Seedlings

The flower-inducing and -inhibiting activities of phloem exudate (PE) prepared from cotyledons of Pharbitis seedlings were examined, using apex cultures in vitro from Pharbitis as a bioassay system.The PE was prepared from photoperiodically-induced cotyledons (SD-PE). The SD-PE was subjected to the following fractionations: When the SD-PE was extracted with CHCl₃ and then ethyl acetate, the inducing activity was located in the final aqueous fraction. The activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). The diffusate was fractionated by ion exchange chromatography, and flower-inducing activity was found in the fraction adsorbed onto anion exchange resin. When the fraction was applied to a Sep-Pak C18 cartridge, the activity eluted with 25% MeOH. As a result of the above fractionation, activity was increased about 30-fold.The nature of the flower-inhibiting activity of the PE taken from cotyledons exposed to continuous-light conditions was examined (CL-PE). The inhibiting activity was decreased as the cotyledons were exposed to longer dark periods; it appeared to be heat-stable. The CL-PE also inhibited flowering in Lemna. The CL-PE was subjected to the following fractionations: When the CL-PE was extracted with CHCl₃ and ethyl acetate, activity was located in the final aqueous fraction. Activity was localized in the diffusate when the aqueous fraction was dialyzed (molecular weight cut off was 10,000). When the diffusate was fractionated by ion exchange chromatography, the activity was found in the flow-through fraction. When the fraction was applied to a hydroxyapatite cartridge, the activity eluted with 25 mM sodium phosphate buffer. When the fraction was re-dialyzed (molecular weight cut off was 1,000), the diffusate contained the activity. As a result of the above fractionation, activity was increased about 10-fold.     

Role of acetate in sporogenesis of Bacillus cereus

Nakata, H. M. (Washington State University, Pullman). Role of acetate in sporogenesis of Bacillus cereus. J. Bacteriol. 91:784-788. 1966.-The distribution of radioactivity associated initially with acetate-2-C(14) was followed during sporogenesis of Bacillus cereus strain T. This was accomplished by replacing cells committed to sporulation into a chemically defined sporulation medium. It was observed that 65 to 70% of the initial radioactivity was incorporated into poly-beta-hydroxybutyrate, whereas 20 to 25% was found in other cellular constituents. Virtually no radioactivity was lost as C(14)O(2) during the first 5 to 6 hr after replacement. Then, a gradual evolution of C(14)O(2) coincident with poly-beta-hydroxybutyrate degradation, was observed until about the ninth hour. By this time, the polymer was essentially depleted, and the first spore structures were observed in stained preparations. The total amount of radioactivity lost as C(14)O(2) was 20 to 25%. The major portion of products derived from poly-beta-hydroxybutyrate was incorporated into the spores. As much as 17% of the radioactivity associated with the spores was found in dipicolinic acid. More than 50% was located in spore proteins, 20 to 25% in the hot 5% trichloroacetic acid-soluble fraction, 4 to 5% in the lipid fraction, and 15 to 20% in the cold 5% trichloroacetic acid-soluble fraction. These data, accounting for 70 to 75% of the initial radioactivity, confirmed the hypothesis that the major role of acetate, and subsequently of poly-beta-hydroxybutyrate, in sporulation of B. cereus T is to provide carbon precursors and energy for sporogenesis.     

破色期番茄果实均一化cDNA沉默文库的构建和功能基因筛选模型的初步建立

番茄果实的成熟是由多基因精细调控的一个过程．利用破色期番茄果实,根据复性动力学原理在mRNA水平进行均一化操作使高丰度和低丰度的mRNA丰度接近．然后把均一化之后mRNA反转录得到cDNA,再与基因沉默载体pTRV重组,最后把构建好的载体通过电转化的方法转入到GV3101农杆菌中,从而建立起破色期番茄果实均一化cDNA沉默文库．通过番茄果实中病毒诱导基因沉默技术,对cDNA沉默文库进行初步筛选,从而确定功能基因筛选模型．在模型建立阶段,以番茄红素合成途径相关的PDS基因作为内标基因,在100个混合农杆菌样中,成功筛选到了PDS基因．     

Isolation and properties of beta-lactamase of the cephalosporinase type from cells of Enterobacter aerogenes 6803

beta-Lastamase with the molecular weight of 32500 was isolated from the cells of clinical strain 6803 of Enterobacter aerogenes and purified. By the substrate profile determined microiodometrically beta-lactamase was classified as belonging to the cephalosporinase type. The activity of the electrophoretically homogenous enzyme was equal to 430 microM a minute per mg protein with respect to benzylpenicillin. The Km for benzylpenicillin, dicloxacillin, cephaloridin and cephalothin was 6.5410(-5), 3 X 10(-4), 2.1 X 10(-5) and 5.7 X 10(-5) M, respectively. The isoelectric point of the enzyme equal to 5.45 was estimated with the method of preparative isoelectrofocusing. The presence of the serine residue or residues was shown with the use of selective reagents applied to the functionally important groups. With the method of circular dichroism the ratio of alpha- and beta-structures in the enzyme molecule was determined, the slow hydrolysis of cephazolin was demonstrated and the values of Km and Kcat for this process were estimated.     

Comparison of centrifugal and other methods for standardization of extraction of nematodes from soil

The extraction of many genera of nematodes from four different soil types by sedimentation, mobility, sieving and centrifuging was compared. All methods were found to extract similar numbers of nematodes. Centrifuging was the most versatile technique and the best for clay soils. Baerman sieving was generally poor and particularly so for sands and for the genus Tylen-chorhynchus. Rotylenchus was the only genus of those identified that was extracted equally well by all techniques. Centrifugal extraction was selected as the routine method for surveys as it was effective on all soil types, was less likely to be subject to variation and, although poor for Longidorus, extracted a wider spectrum of nematodes.     

Structural studies of fc receptors. II. The effect of dissociation and reassociation of phospholipids on the activity of Fc gamma receptors of macrophages

The receptor for the Fc portion of immunoglobulin G (Fc receptor) of guinea pig macrophages was solubilized with a detergent and partially delipidated to the point where the ligand binding activity was essentially lost. Delipidation of the Fc receptor was done by fractionating the macrophage lysate by gel filtration in the presence of detergent. The elution behavior of Fc receptor-detergent complex and phospholipid-detergent mixed micelles varied depending on the kind of detergents used for membrane solubilization and for gel filtration. Separation of phospholipids from Fc receptor was best achieved when octylglucoside-solubilized fraction was chromatographed on Sepharose CL-6B in the presence of deoxycholate; the phospholipid peak emerged at Kav = 0.55 and the Fc receptor at Kav = 0.45. The fraction of Kav = 0.45 showed only a marginal activity when the activity was measured after removal of detergents, but activity was clearly shown when phospholipid fraction was added to this fraction prior to removal of the detergents. Reappearance of the Fc receptor activity was shown to be due to association of phospholipids with the Fc receptor. Three kinds of phospholipids with different polar head groups examined, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine, were all able to reconstitute active Fc receptor, although phosphatidylethanolamine was somewhat less effective than the others. Thus, our study demonstrated the amphipathic nature of the Fc receptor, the binding of which is dependent on the interaction with phospholipids.     

Studies on the mechanism of castanospermine inhibition of alpha- and beta-glucosidases

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is an indolizidine alkaloid that was isolated from the Australian plant, Castanospermum australe. This alkaloid was found to be a potent inhibitor of lysosomal alpha- and beta-glucosidases. In this report, the mechanism of inhibition of amyloglucosidase (an exo-1,4-alpha-glucosidase) and almond emulsin beta-glucosidase was examined. Castanospermine proved to be a competitive inhibitor of amyloglucosidase at both pH 4.5 and 6.0 when assayed with the p-nitrophenyl-alpha-D-glucoside. It was also a competitive inhibitor of almond emulsin beta-glucosidase at pH 6.5, but in this case previous studies had shown that inhibition was of the mixed type at pH 4.5 to 5.0. Th pH of the incubation mixture had a marked effect on the inhibition. Thus, in all cases, castanospermine was a much better inhibitor at pH 6.0 to 6.5 than it was at lower pH values. The pK for castanospermine was found to be 6.09, indicating that the alkaloid was probably more active in the unprotonated form. This was also suggested by the fact that the N-oxide of castanospermine, while still a competitive inhibitor, was 50 to 100 times less active than was castanospermine, and its activity was not markedly altered by pH. These results probably explain why castanospermine is a good inhibitor of the glycoprotein processing enzyme, glucosidase I, since this is a neutral enzyme.     

A comparative study of three bacteriocins of Bacteroides fragilis

Three different bacteriocins produced by strains of Bacteroides fragilis were compared in terms of their production kinetics, physico-chemical nature, and action on macromolecular synthesis in a common indicator strain. Bacteriocin 78/438 was produced during the logarithmic growth phase, was thermolabile and stable between pH 5 and 9. It was susceptible to trypsin and pepsin, and affected DNA, RNA and protein syntheses in susceptible cells. Bacteriocin A49 was produced during the stationary growth phase, was thermolabile and stable between pH 7 and 9. This bacteriocin was also susceptible to trypsin and pepsin, but only RNA synthesis was affected in the indicator strain. Bacteriocin A55 differed markedly from both 78/438 and A49, and was found to be predominantly cell-bound, resistant to inactivation by high temperatures and stable over a wide pH range of 2 to 12. It was susceptible to trypsin but resistant to pepsin. A55 had a delayed effect on macromolecular synthesis with DNA synthesis being inhibited after 60 min. With all three bacteriocins, killing of the indicator strain followed single hit kinetics with the interaction of bacteriocin and target cell occurring in two stages. Killing by bacteriocin A55 was much slower than the other two and this may be related to its effect on macromolecular synthesis. The killing action of all three bacteriocins was dependent on the growth phase of the susceptible cells.     

凋亡素蛋白多克隆抗体的制备及免疫学评价

凋亡素由鸡贫血病毒中的VP3基因编码,能诱导多种肿瘤细胞发生凋亡。以真核表达载体pcDNA3.0-VP3为模板构建原核表达载体pET8a-VP3,经NdeⅠ/BamHⅠ双酶切鉴定和基因测序无误后,在IPGT诱导下表达VP3蛋白并对其进行纯化,将纯化后的VP3蛋白与弗氏完全佐剂或不完全佐剂乳化后,分别对两只新西兰大耳白兔进行皮下多点注射,间接ELISA检测免疫后血清效价,效价达到指标后第2天以心脏穿刺的方法采全血后分离抗血清。抗血清效价高的兔子进一步采用Protein A纯化总IgG,最终纯化后的抗体效价可以达到1 ∶ 243 000。用重组腺相关病毒rAAV-VP3感染细胞后对抗体的特异性进行免疫学评价。首先利用免疫荧光技术检测VP3基因在人膀胱癌细胞株T24、EJ细胞以及Vero细胞中的表达情况,观察到凋亡素在T24、EJ细胞中主要定位于细胞核,而在Vero细胞中则定位于细胞质。其次通过Western blotting检测纯化后的抗体能与细胞内腺相关病毒介导表达的凋亡素蛋白特异性结合。实验证明了制备的凋亡素蛋白多克隆抗体的有效性和特异性,为进一步阐明凋亡素抗肿瘤效应的分子机制及生物学特性奠定了基础。     

Some aspects of the regulation of the production of extracellular proteolytic enzymes by a marine bacterium

A highly proteolytic Gram-negative, rod-shaped bacterium was isolated from the gills of fresh plaice and the effect of culture conditions on the production of proteolytic enzymes was investigated. When the organism, strain SA 1, was grown in the presence of complex mixtures of proteins and amino acids, both endopeptidase and aminopeptidase activity was demonstrated in the cell-free culture medium. However, synthesis of these enzymes was not observed when the organism was grown in a mineral medium with lactate or succinate as the only carbon and energy source. Synthesis of both endopeptidase and aminopeptidase was induced by the presence of amino acids in the medium. Of the amino acids tested, l-phenylalanine was found to be the best single inducer for the production of endopeptidase. When in addition one or more different amino acids were added, endopeptidase production was found to increase with increasing complexity of the mixture, up to a maximum which was obtained with five different amino acids. Production of the aminopeptidase was optimal when l-glutamic acid was used as a single inducer. For this enzyme the amount of enzyme activity released in the medium decreased with increasing complexity of the amino acid mixture. Endopeptidase as well as aminopeptidase activity was found to accumulate in the medium at the end of the logarithmic growth phase, when the culture was no longer growing exponentially. When the stationary phase was reached, enzyme production stopped. Production of both enzymes was immediately halted upon addition of chloramphenicol and was found to be repressed by glucose and lactate. These results suggest that synthesis of proteolytic extracellular enzymes by the organism studied is controlled by an efficient regulatory mechanism, in which growth rate is an important parameter.     

稳定转染Dicer基因对HELF细胞功能的影响

目的观察正常人胚肺成纤维细胞（HELF）在稳定转染Dicer基因后增殖能力及迁移能力的变化。方法应用脂质体介导方法转染Dicer基因表达载体于HELF细胞,G418筛选,PCR检测整合情况,RT-PCR检测表达情况,Western-blot检测蛋白表达水平,确定稳定转染细胞株,MTT方法检测细胞增值能力,Transwell方法检测细胞迁移能力。结果Dicer基因稳定转染HELF细胞系建立,用RT-PCR和Western-blot方法检测到Dicer基因mRNA和蛋白表达水平都明显增强。与转染空载体的HELF细胞相比,转染Dicer基因的HELF细胞48、72、96h的MTT光吸收值明显升高（P〈0.05）,并且穿过人工重构基底膜的细胞数明显增多。结论Dicer基因稳定转染HELF细胞后,HELF细胞增殖能力和迁移能力都明显增强。