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131.
Regarding biological products, increasing awareness of potential side effects have placed great importance not only at protein
purity regarding other proteins but on the removal of biologicals such as DNA and especially virus the importance of which
may not be known. Monoclonal antibodies (Mab) have come to be an important class of molecules obtained from hybridoma cells,
i.e., nonrecombinant cells in culture. It has been noted during the last years, that with rare exceptions hybridoma cell lines
contain retrovirus like particles. The infectious nature of the EM-visible particles has been tested for, however, in most
cases not been substantiated. In order to bring these valuable biological reagents, Mab's, to good use in man for imaging
or therapy, the remaining concern about a potential retroviral infection has to be reduced to an acceptable minimum. We describe
experimental approaches for the validation of chromatographic and ultrafiltration steps used in the production of monoclonal
antibodies to remove and inactivate murine retrovirus.
Present day biotechnological manufacturing processes have been devised incorporating a number of strategic preventive measures
that have found wide spread acceptance. They permit to answer the question: how can a potentially harmful infection by an
unknown virus be excluded.
Knowledge of the efficacy of purification steps to clear infectious model virus is fundamental to devise biotechnological
manufacturing processes yielding a purified antibody for use in man. 相似文献
132.
Summary Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD) that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at 180 copies per uninduced cell and was measured at 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III. 相似文献
133.
Carbon and nitrogen isotope ratios in different compartments of a healthy and a declining Picea abies forest in the Fichtelgebirge,NE Bavaria 总被引:4,自引:0,他引:4
Summary Natural carbon and nitrogen isotope ratios were measured in different compartments (needles and twigs of different ages and crown positions, litter, understorey vegetation, roots and soils of different horizons) on 5 plots of a healthy and on 8 plots of a declining Norway spruce (Picea abies (L.) Karst.) forest in the Fichtelgebirge (NE Bavaria, Germany), which has recently been described in detail (Oren et al. 1988a; Schulze et al. 1989). The 13C values of needles did not differ between sites or change consistently with needle age, but did decrease from the sun-to the shade-crown. This result confirms earlier conclusions from gas exchange measurements that gaseous air pollutants did no long-lasting damage in an area where such damage was expected. Twigs (13C between-25.3 and-27.8) were significantly less depleted in 13C than needles (13C between-27.3 and-29.1), and 13C in twigs increased consistently with age. The 15N values of needles ranged between-2.5 and-4.1 and varied according to stand and age. In young needles 15N decreased with needle age, but remained constant or increased in needles that were 2 or 3 years old. Needles from the healthy site were more depleted in 15N than those from the declining site. The difference between sites was greater in old needles than in young ones. This differentiation presumably reflects an earlier onset of nitrogen reallocation in needles of the declining stand. 15N values in twigs were more negative than in needles (-3.5 to-5.2) and showed age- and stand-dependent trends that were similar to the needles. 15N values of roots and soil samples increased at both stands with soil depth from-3.5 in the organic layer to +4 in the mineral soil. The 15N values of roots from the mineral soil were different from those of twigs and needles. Roots from the shallower organic layer had values similar to twigs and needles. Thus, the bulk of the assimilated nitrogen was presumably taken up by the roots from the organic layer. The problem of separation of ammonium or nitrate use by roots from different soil horizons is discussed. 相似文献
134.
An inflatable minirhizotron system for root observations with improved soil/tube contact 总被引:3,自引:1,他引:2
Commonly used minirhizotrons consisting of a transparent tube inserted into the soil seldom attain good contact between the tube and the soil, which leads to root growth occurring in a gap rather than in the soil. A new system is described involving an inflatable flexible rubber wall, made from a modified motorcycle tube. Pressure ensures a proper tube/soil contact so that the environmental circumstances for root growth along the tube more closely correspond to those in the undisturbed soil. Before the endoscope slide is introduced into the minirhizotron for taking pictures, the inflatable tube is removed, so that there is no-often opaque-wall between the endoscope and the roots. This improves the picture quality and facilitates the analysis of root images. 相似文献
135.
Many N2-fixing organisms can turn off nitrogenase activity in the presence of NH4
+ and turn it on again when the NH4
+ is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH4
+ is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genusAzospirillum possess the DRAT and DRAG systems (A. lipoferum andA. brasilense), whereasA. amazonense in the same genus lacks DRAT and DRAG.A. amazonense responds to NH4
+ but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them intoA. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function inAcetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N2 at a pH as low as 3.0.A. diazotrophicus does show a rather sluggish response to NH4
+. A level of about 10 M NH4
+ is required to switch off the system. The response to NH4
+ is influenced by the dissolved oxygen concentration (DOC) as has been reported forAzospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O2 seems optimal for the response inA. diazotrophicus. 相似文献
136.
A coiling-inducing factor was isolated from tendrils of Bryonia dioica Jacq. and identified by infrared, 1H-, 13C-nuclear magnetic resonance and mass spectrometry as -linolenic acid. When applied to detached tendrils, exogenous -linolenic acid, but not linoleic acid or oleic acid, induced tendril coiling. Further investigations showed that metabolites of -linolenic acid, jasmonic acid and, even more so, methyljasmonate, are highly effective inducers of tendril coiling in B. dioica. Methyljasmonate was most active when administered by air and, in atmospheric concentrations as low as 40–80 nM, induced a full free-coiling response with kinetics similar to mechanical stimulation. Even atmospheric levels as low as 4–5 nM methyljasmonate were still found to be significantly active. Methyljasmonate could be one of the endogenous chemical signals produced in mechanically stimulated parts of a tendril and, being highly volatile, act as a diffusible gaseous mediator spreading through the intracellular spaces to trigger free coiling of tendrils.Abbreviations EI-MS
electron impact-mass spectrometry
- HPLC
high-performance liquid chromatography
- IAA
indole-3-acetic acid
- NMR
nuclear magnetic resonance
- TFA
trifluoroacetic acid
We are indebted to the Deutsche Forschungsgemeinschaft, Bonn and the Fonds der Chemischen Industrie, Frankfurt (literature provision) for their support and to Dr. C. Brückner, Halle, for jasmonic-acid determinations. 相似文献
137.
The relative contributions made by the l-arginine/agmatine/N-carbamoylputrescine/putrescine and the l-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, dl--difluoromethylarginine or dl--difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of dl--difluoromethylornithine.Abbreviations ADC
arginine decarboxylase
- DFMA
dl--dif-luoromethylarginine
- DFMO
dl--difluoromethylornithine
- MPO
N-methylputrescine oxidase
- ODC
ornithine decarboxylase
- PMT
putrescine N-methyltransferase
We are indebted to Dr. E.W.H. Bohme of Merrell Dow Research Laboratories (Cincinnati, Ohio, USA) for kind gifts of DFMO and DFMA and to Dr. M.J.C. Rhodes for helpful advice and discussion. 相似文献
138.
Summary The distribution of two seed proteins, namely conglutin and a legumin-like globulin, in developing and mature seeds ofLupinus albus L. has been examined by immunocytochemistry and the concomitant modifications of their constituent polypeptides followed by SDS-PAGE. Both proteins were found within vacuolar protein bodies in various tissues of the cotyledons, although with some differences in the distribution patterns. The legumin-like protein was found to be deposited within the large storage parenchyma cells of the cotyledons in a manner similar to that reported for other storage proteins; little or no immunolabelling was associated with the cotyledonary epidermal and vascular parenchyma cells. In contrast conglutin was present in all cell types.A precursor of the legumin-like protein accumulated transiently in the developing cotyledon, but was subsequently modified by proteolytic cleavage. The onset of such modification was concomitant with a transition in the predominant vacuolar forms within the storage parenchyma cells. No precursor molecules of conglutin have been detected in this study, thus indicating that this protein is deposited in the protein bodies in its mature form.Abbreviations LM
light microscopy
- EM
electron microscopy
- DAF
days after flowering
- SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel electrophoresis
- GAR
goat antirabbit antiserum 相似文献
139.
P. Masojć M. D. Gale 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1991,82(6):771-776
Summary Rye -Amy1, -Amy2, and -Amy3 genes were studied in the cross between inbred lines using wheat -amylase cDNA probes. The -Amy1 and -Amy2 probes uncovered considerable restriction fragment length polymorphism, whereas the -Amy3 region was much more conserved. The numbers of restriction fragments found and the F2 segregation data suggest that there are three -Amy1 genes, two or three -Amy2 genes, and three -Amy3 genes in rye. These conclusions were supported by a simultaneous study of -amylase isozyme polymorphism. The F2 data showed the three individual -Amy1 genes to span a distance of 3cM at the locus on chromosome 6RL. The genes were mapped relative to other RFLP markers on 6RL. On chromosome 7RL two -Amy2 genes were shown to be separated by 5 cM. Linkage data within -Amy3 on 5RL were not obtained since RFLP could be detected at only one of the genes. 相似文献
140.
S. Hinnisdaels L. Bariller A. Mouras V. Sidorov J. Del-Favero J. Veuskens I. Negrutiu M. Jacobs 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1991,82(5):609-614
Summary Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt60,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via gamma-fusion. 相似文献