Advantages and limitations in the use of hairy root cultures for the production of tropane alkaloids: Use of anti-auxins in the maintenance of normal root morphology

Summary Brugmansia candida hairy roots, obtained by infection withAgrobacterium rhizogenes LBA 9402, exhibit, after subculturing in liquid media, a tendency towards dedifferentiation. It has been found that the following strategies can be applied to inhibit this dedifferentiation and preserve normal root morphology: (a) lowering both the mineral and sucrose concentration in the media employed so as to diminish osmotic stress (a condition to which these roots appear to be particularly susceptible); (b) employing antiauxins in appropriate concentrations; and (c) maintaining the hairy roots on solid media prior to use in production processes in liquid media. The first strategy suggested does not favor alkaloid productivity, but in this case a two-step method could be attempted: biomass with normal root morphology could be obtained in a first stage using low sucrose concentrations, and in a second stage, sucrose could be increased in order to achieve higher productivity. In all the clones ofB. candida obtained, alkaloid production was biased towards scopolamine.     

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.     

PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations

PGE₂ involvement in experimental Trypanosoma cruzi infection depends on the lethal capacity of the parasite subpopulation used. Mice acutely infected with non-lethal K98 displayed an enhancement in PGE₂ serum levels during the acute period, while those infected with lethal T. cruzi subpopulations (RA or K98-2) showed levels not different from normal mice. The enhancement detected in K98 group could be related both to an increased number of CD8+ T cell number and to enhanced PGE₂ release per cell by CD8+; values of PGE₂ release by adherent cells were not altered in this group. Treatment with cyclooxygenase inhibitors enhanced mortality rates of mice infected with K98, and administration of 16,16-dimethyl PGE₂ (dPGE) reversed this effect. However, mice infected with RA did not reduce their mortality rates by administration of diverse doses of dPGE. These findings suggest that PGE₂ could play a role in resistance in mice infected with K98.     

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。     

一种分离培养和观察微生物的简易方法

本文介绍了一种简易的分离、培养和观察微生物的方法（双载片法）,适用于好氧菌及耐氧性厌氧菌。试验表明,此法的优点是：适于连续地观察和研究微生物的个体生长,了解其生活史;能快速简便地测定微生物的死亡率;能有效地分离纯种;为耐氧性厌氧菌的研究提供了一种便利的方法。     

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.     

Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen.

The ability of simian virus 40-encoded large T antigen to disrupt the growth control of a variety of cell types is related to its ability to interfere with certain cellular proteins, such as p53 and the retinoblastoma susceptibility gene product (pRB). We have used wild-type and mutant forms of T antigen in transgenic mice to dissect the roles of pRB, p53, and other cellular proteins in tumorigenesis of different cell types. In this study, using a cell-specific promoter to target expression specifically to brain epithelium (the choroid plexus) and to B and T lymphoid cells, we characterize the tumorigenic capacity of a T-antigen fragment that comprises only the amino-terminal 121 residues. This fragment (dl1137) retains the ability to interact with pRB and p107 but lacks the p53-binding domain. While loss of the p53-binding region results in loss of the capacity to induce lymphoid abnormalities, dl1137 retains the ability to induce choroid plexus tumors that are histologically indistinguishable from those induced by wild-type T antigen. Tumors induced by dl1137 develop much more slowly, however, reaching an end point at around 8 months of age rather than at 1 to 2 months. Analysis of tumor progression indicates that tumor induction by dl1137 does not require secondary genetic or epigenetic events. Rather, the tumor growth rate is significantly slowed, indicating that the T-antigen C-terminal region contributes to tumor progression in this cell type. In contrast, the pRB-binding region appears essential for tumorigenesis as mutation of residue 107, known to disrupt pRB and p107 binding to wild-type T antigen, abolishes the ability of the dl1137 protein to induce growth abnormalities in the brain.     

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.