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141.
Holger Hackstein Gerhard Jahn Holger Kirchner Gregor Bein 《Histochemistry and cell biology》1996,106(2):229-234
Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV)
probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant
improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization
(FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV
genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV
probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal
resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood
leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes
in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining,
we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between
HCMV and peripheral blood leukocytes at the single-cell level.
Accepted: 16 February 1996 相似文献
142.
Using electron microscopy we demonstrate that degenerating neurons and cellular debris resulting from neuronal reorganization
are phagocytosed by glial cells in the brain and nerve cord of the fruitfly Drosophila melanogaster during the first few hours following pupariation. At this stage several classes of glial cells appear to be engaged in intense
phagocytosis. In the cell body rind, neuronal cell bodies are engulfed and phagocytosed by the same glial cells that enwrap
healthy neurons in this region. In the neuropil, cellular debris in tracts and synaptic centres resulting from metamorphic
re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial cells are hypertrophied,
produce large amounts of lysosome-like bodies and contain a large number of mitochondria, condensed chromatin bodies, membranes
and other remains from neuronal degeneration in phagosomes.
Received: 23 January 1996 / Accepted in revised form: 21 May 1996 相似文献
143.
Bettina Schmidt Thomas Tradler Jens-U. Rahfeld Birgit Ludwig Bunty Jain Karlheinz Mann K. Peter Rücknagel Bernhard Janowski Angelika Schierhorn Gerhard Küllertz Jörg Hacker Gunter Fischer 《Molecular microbiology》1996,21(6):1147-1160
Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histi-dine tag and an enterokinase cleavage site exhibits 相似文献
144.
It was our goal to determine the location of the intermediate chain within the complex of cytoplasmic dynein by immunoelectron microscopy. To do so we generated two monoclonal antibodies (m74-1 and m74-2) specific for the intermediate chain. Both antibodies recognised the intermediate chain by sodium dodecyl sulphate–polyacrylamide gel electrophoresis immunoblot and ELISA assays of native and denatured proteins. When sucrose density gradient-purified cytoplasmic dynein from bovine brain was incubated with the gold-conjugated monoclonal antibodies, m74-1 and m74-2, and examined by negative staining, the gold label was found opposite the globular heads at the base of the V-shaped stalk of the motor complex. The labelling of the intermediate chain is the first mapping of a component within cytoplasmic dynein. The identification of the intermediate chain at the base of the complex supports a possible docking function of the intermediate chain. 相似文献
145.
A cell-free system capable of converting [14C]geranylgeranyl diphosphate to ent-[14C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [14C]geranylgeranyl diphosphate into ent-[14C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[14C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH
alcohol dehydrogenase
- AMO 1618
2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate
- BSA
bovine serum albumin
- DTT
dithioth-reitol
- GAn
gibberellin An
- GAPDH
NADP+-glyceraldehyde 3-phosphate dehydrogenase
- GC-MS
combined gas chromatography-mass spectrometry
- GGPP
all trans-isomer of geranyl-geranyl diphosphate
- KS
ent-kaurene synthetase
- MDH
malate dehydrogenase
- MAA
mevalonate activating activity
- SOR
shikimate oxidoreductase
We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[2H2]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft. 相似文献
146.
147.
Habitat preference, seasonal occurrence, starvation resistance, hatching eggs ofBranchipus schaefferi, and effects of predation onB. schaefferi were studied.Branchipus was only present in turbid, unvegetated ponds and absent in ponds which contain higher aquatic vegetation and theSpirogyra sp. The first individuals ofB. schaefferi appeared in April when water temperature was 10 °C and the last adults in November at a water temperature of 3.5 °C. Up to 6 reproducing generations were observed during this period. Abundance ofB. schaefferi was higher in temporary ponds than in permanent ponds. Sex ratio was close to unity for most of the year. Body size ofB. schaefferi males and females was significantly positively correlated with pond volume. Without foodB. schaefferi could survive for 1.5 to 2 days at 20 °C and 4 to 5 days at 10 °C. Hatching success of eggs decreased when eggs were dried for 7 months. Freezing of eggs had no effect on hatching success. From] the predators tested,Chaoborus sp. larvae clearly selected smallB. schaefferi; one consumed approximately 6Branchipus d–1 at a density of 6 to 12 prey 1–1. The other predators, dragonfly larvae, and larvae and adults ofTriturus alpestris selected alternative prey types, for exampleTubifex sp. and ostracods. 相似文献
148.
Anton Stefan Reiter und Gerhard Loupal 《Journal of Ornithology》1995,136(2):221-223
In July 1992, in the Austrian part of Hanság, a seventy day old young bustard was found dead in a grassland. On the left intertarsal joint a walnut sized open pock was located. Other pocks reaching pea-size were found on both legs. The diagnosis pox was established by light- and electron-microscopic examination of the lesions. A further chick of another hen, fledged in the same year, observed from a distance showed abnormal thickening of the intertarsal joint area. The consequences of pox for such a small group of Great Bustards (total for 1988–1993 15–20 birds) should be watched carefully. 相似文献
149.
Maurizio Pellecchia Gerhard Wider Hideo Iwai Kurt Wüthrich 《Journal of biomolecular NMR》1997,10(2):193-197
A novel 2D NMR experiment, 2D HE(NE)HGHH, is presented for the assignment ofarginine side chain 1H and 15N resonances inuniformly 15N-labeled proteins. Correlations between1H, 1Hand 1H are established on the basis of3J(15N,1H) heteronuclear scalarcoupling constants, and sequence-specific assignments are obtained by overlapof these fragments with 1H chemical shiftsobtained by assignment procedures starting from the polypeptide backbone.Since guanidino protons exchange quite rapidly with the bulk water, the 2DHE(NE)HGHH pulse scheme has been optimized to avoid saturation and dephasingof the water magnetization during the course of the experiment. As anillustration, arginine side chain assignments are presented for two uniformly15N-labeled proteins of 7 and 23 kDa molecular weight. 相似文献
150.
Retrofitting YACs for direct DNA transfer into plant cells 总被引:3,自引:0,他引:3
Gerhard Adam Jeffrey A. Mullen Karen L. Kindle 《The Plant journal : for cell and molecular biology》1997,11(6):1349-1358
The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures. 相似文献