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R Stracke

Publications and source records attributed to R Stracke.

At least 19 recordsLinked to original sources
PubMed2003

Generation of Arabidopsis protein chips for antibody and serum screening.

Protein array technology has emerged as a new tool to enable ordered screening of proteins for expression and molecular interactions in high throughput. Besides classical solid-phase substrates, such as micro-titre plates and membrane filters, protein arrays have recently been devised with chip-sized supports. Several applications on protein chips have been described, but to our knowledge no studies using plant protein chips were published so far. The aim of this study was to generate Arabidopsis protein chips and to demonstrate the feasibility of the protein chip technology for the investigation of antigen-antibody interactions. Therefore, Arabidopsis cDNAs encoding 95 different proteins were cloned into a GATEWAY-compatible Escherichia coli expression vector. RGS-His6-tagged recombinant proteins were purified in high throughput and robotically arrayed onto glass slides coated either with a nitrocellulose based polymer (FAST slides) or polyacrylamide (PAA slides). Using an anti-RGS-His6 antibody all proteins were detected on the chips. The detection limit was ca. 2-3.6 fmol per spot on FAST slides or 0.1-1.8 fmol per spot on PAA slides. The Arabidopsis protein chips were used for the characterisation of monoclonal antibodies or polyclonal sera. We were able to show that a monoclonal anti-TCP1 antibody and anti-MYB6 and anti-DOF11 sera bound specifically to their respective antigens and did not cross-react with the other 94 proteins including other DOF and MYB transcription factors on the chips. To enable screening of antibodies or other interacting molecules against thousands of Arabidopsis proteins in future, we generated an ordered cDNA expression library and started with high-throughput cloning of full-length cDNAs with GATEWAY technology.

Animals↗
PubMed2002

Analysis of the migration behaviour of single microtubules in electric fields.

By video contrast microscopy, individual microtubules formed from pure tubulin in the presence of taxol were studied in constant electric fields. At nearly physiological conditions, i.e., in a buffer at pH 6.8 and 120 mM ionic strength, suspended microtubules moved towards the anode with an electrophoretic mobility of approximately 2.6 x 10(-4) cm(2)/V s, corresponding to an unbalanced negative charge of 0.19 electron charges per tubulin dimer. Strikingly, this value is lower by a factor of at least 50 than that calculated from crystallographic data for the non-assembled tubulin dimer. Moreover, the taxol-stabilized microtubules had an isoelectric point of about pH 4.2 which is significantly lower than that known for the tubulin monomers. This indicates that microtubule formation is accompanied by substantial changes of charge distribution within the tubulin subunits. Constant electric fields were shown to affect also the orientation of microtubules gliding across a kinesin-coated surface at pH 6.8.

Animals↗
PubMed2001

The R2R3-MYB gene family in Arabidopsis thaliana.

MYB factors represent a family of proteins that include the conserved MYB DNA-binding domain. In contrast to animals, plants contain a MYB-protein subfamily that is characterised by the R2R3-type MYB domain. 'Classical' MYB factors, which are related to c-Myb, seem to be involved in the control of the cell cycle in animals, plants and other higher eukaryotes. Systematic screens for knockout mutations in MYB genes, followed by phenotypic analyses and the dissection of mutants with interesting phenotypes, have started to unravel the functions of the 125 R2R3-MYB genes in Arabidopsis thaliana. R2R3-type MYB genes control many aspects of plant secondary metabolism, as well as the identity and fate of plant cells.

Arabidopsis↗
PubMed2000

Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity.

DeCuevas et al. [J. Cell Biol. 116 (1992) 957-965] demonstrated by circular dichroism spectroscopy for the kinesin stalk fragment that shifting temperature from 25 to 30 degrees C caused a conformational transition. To gain insight into functional consequences of such a transition, we studied the temperature dependence of a full-length kinesin by measuring both the velocity of microtubule gliding across kinesin-coated surfaces and microtubule-promoted kinesin ATPase activity in solution. The corresponding Arrhenius plots revealed distinct breaks at 27 degrees C, corroborating the temperature-dependent conformational transition for a motility-competent full-length kinesin. Microtubules were found to glide up to 45 degrees C; at higher temperatures, kinesin was irreversibly damaged.

Animals↗
PubMed2000

Speeding up kinesin-driven microtubule gliding in vitro by variation of cofactor composition and physicochemical parameters.

So far, there has been a discrepancy between the velocities of kinesin-dependent microtubule motility measured in vitro and within cells. By changing ATP, Mg(2+), and kinesin concentrations, pH and ionic strength, we tried to find conditions that favour microtubule gliding across kinesin-covered glass surfaces. For porcine brain kinesin, we found that raising the molar Mg(2+)/ATP ratio can substantially elevate gliding velocity. Gliding became also faster after temperature elevation or lowering the number of kinesin molecules bound to the glass surface. The highest mean gliding velocity (1.8 microm/s+/-0.09 microm/s), approaching velocities measured for anterograde transport in vivo, was achieved by combination of favourable factors (2.5 m m ATP, 12.5 m m Mg(2+), 37 degrees C, 450 kinesin molecules/microm(2)).

Adenosine Diphosphate↗
PubMed1997

Multi-functional acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene family: indication for plastidic localization of at least one isoform.

Three genes coding for different multifunctional acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) isoenzymes from Brassica napus were isolated and divided into two major classes according to structural features in their 5' regions: class I comprises two genes with an additional coding exon of approximately 300 bp at the 5' end, and class II is represented by one gene carrying an intron of 586 bp in its 5' untranslated region. Fusion of the peptide sequence encoded by the additional first exon of a class I ACCase gene to the jellyfish Aequorea victoria green fluorescent protein (GFP) and transient expression in tobacco protoplasts targeted GFP to the chloroplasts. In contrast to the deduced primary structure of the biotin carboxylase domain encoded by the class I gene, the corresponding amino acid sequence of the class II ACCase shows higher identity with that of the Arabidopsis ACCase, both lacking a transit peptide. The Arabidopsis ACCase has been proposed to be a cytosolic isoenzyme. These observations indicate that the two classes of ACCase genes encode plastidic and cytosolic isoforms of multi-functional, eukaryotic type, respectively, and that B. napus contains at least one multi-functional ACCase besides the multi-subunit, prokaryotic type located in plastids. Southern blot analysis of genomic DNA from B. napus, Brassica rapa, and Brassica oleracea, the ancestors of amphidiploid rapeseed, using a fragment of a multi-functional ACCase gene as a probe revealed that ACCase is encoded by a multi-gene family of at least five members.

Acetyl-CoA Carboxylase↗
PubMed1996

Signal sequence trap to clone cDNAs encoding secreted or membrane-associated plant proteins.

A method was developed for rapid cloning of plant cDNAs encoding proteins with membrane-spanning domains. A novel expression vector was constructed for expression of plant cDNA libraries in COS cells. Fusion proteins were expressed containing at their N-terminus an endoplasmic reticulum (ER) signal peptide. After entry into the ER these proteins could traffic via the default pathway to the plasma membrane. Trapping at the cell surface occurred when the protein contained one or more membrane-spanning domains. A simple color-based immunoscreening procedure allowed the isolation of cDNAs after only two rounds of COS cell transfection and screening. Several cDNA clones encoding proteins with putative membrane-spanning domains were isolated. Among them were cytochrome b5 and full-length cDNA clones encoding putative secretory proteins targeted to the ER membrane by their N-terminal signal peptide.

Amino Acid Sequence↗
PubMed1995

Antigenic properties and immunoelectron microscopic localization of Mycobacterium fortuitum beta-lactamase.

Mycobacterium fortuitum is a fast-growing Mycobacterium species which produces a beta-lactamase involved in the intrinsic resistance of the microorganism to beta-lactam antibiotics. An anti-beta-lactamase serum against the purified enzyme was raised in rabbits. Antibody binding was specific for native beta-lactamase, and enzyme activity was partially inhibited by the serum; furthermore, cross-reactions with denatured class A beta-lactamases were observed. This serum was used as a probe in immunogold labeling for the localization of the cell-bound beta-lactamase in both the low-level producer ATCC 19542 (parental strain) and the overproducer mutant D316. By the combination of preembedding immunogold labeling and replica technique, it was shown that the beta-lactamase was uniformly distributed on the whole external cell surface, where it appeared to be associated with a Tween 80-removable capsule-like material. Compared with the parental strain, a much higher level of expression of surface enzyme was observed in strain D316. Surface labeling was more intense in the stationary phase of growth than in exponentially growing cells. The data obtained are interpreted in the context of the intrinsic resistance of M. fortuitum to beta-lactam antibiotics.

Antigens, Bacterial↗
PubMed1990

Effect of different fixatives on Con A surface receptors of mouse peritoneal macrophages.

The effects of glutaraldehyde, formaldehyde, or osmium tetroxide fixation on the number of labeled Con A surface receptors on mouse peritoneal macrophages were compared. Gold-labeled Con A receptors were found to be isolatedly arranged and evenly distributed on cell surfaces independent of the fixative used. Only cells preincubated with Con A and subsequently fixed by osmium tetroxide showed arrangement of labeled receptors in clusters. Significant differences were found in the number of Con A receptors per cell depending on the fixative used. The fluorescence intensity of FITC-Con A staining was detected spectrophotometrically, the characteristic X-rays of gold-labeled Con A receptors were determined by means of electron beam-induced X-ray microanalysis. The experimental results obtained both at light and electron microscopic level pointed to formaldehyde being the best fixative also for this purpose.

Animals↗
PubMed1989

[Use of direct calorimetry for continuously following the course of experimental infections in the mouse].

To obtain information about early symptoms of infection measurements of heat production were performed in experimentally infected animals. As a model Mengo virus-infected mice were used. Construction and physical properties of the flow calorimeter device are described. Fever was not detected by method used. However, deviations from normal behaviour of the animals were demonstrated as a result of the relationship between motoric activity and heat produced. Furthermore, the method may be particularly useful for the determination of the exact time of death, which is of interest for mathematical modelling of survival data (RECKNAGEL et al. 1986; GUTHKE et al. 1987; SUHNEL und VECKENSTEDT 1989).

Animals↗
PubMed1987

Dynamic model of the pathogenesis of Mengo virus infection in mice.

A mathematical model of the pathogenesis of experimental Mengo virus infection in mice has been developed and fitted using kinetic data of both virus multiplication in different organs and mortality. The behaviour of the model proved to be bistable. In contrast to the widely accepted hypothesis that an acutely virus-infected host dies when virus replication has attained a critical level in the main target organ, the present results showed the following: the maximum virus titre in brain, the main target organ, has been reached already 24 hr post infection (p.i.) but the animals began to die since 60 hr. Hence, it was postulated and confirmed by a good model fit to the experimental data that the so-called AUC (area under the curve) of the virus multiplication kinetics may be a critical quantity. From this finding a hypothesis was deduced assuming that in the presence of high amounts of the virus the antiviral effect of IFN wanes with time. Since this process accounts for death, it may be a potential target of antiviral therapy.

Animals↗
PubMed1986

Semi-quantitative determination of IgG-binding structures on bacteria by direct fluorescence technique.

A simple semiquantitative method for determination of IgG-binding structures on bacteria by direct fluorescence technique is described. The fluorescence of bacterial bound IgG-FITC-conjugate was measured in a paste-like bacterial sediment by using a fluorescence microscope photometer unit. For this purpose sharply centrifuged IgG-FITC conjugate treated bacteria, from which the washing fluid was carefully removed, were transferred to a glass slide and fluorescence was measured at the contact layer of the adhered drop on the inverted slide. The measured fluorescence intensity area was found to be correlated with the amount of bound IgG-FITC/cell, if bacteria had been incubated with an excess of fluorescein labeled IgG. The IgG-binding of different streptococcal strains was compared with the average IgG-binding of strain Cowan I resulting from 13 different cultivations. For strain Cowan I 9.4 X 10(4) IgG-molecules were estimated to bind on one staphylococcal cell. For a screening of IgG-binding bacterial strains the method did not demand a standardization of bacteria by cell counting.

Cell Membrane↗
PubMed1986

Critical point drying by use of distilled CO2.

For avoiding specimen contamination with particles or liquid residues from CO2-supply cylinders during critical point drying the carbon dioxide is taken from the gaseous phase. Condensation takes place in the water cooled pipe between supply cylinder and CPD-apparatus. A sufficient flow of liquid CO2 is guaranteed, all solid and nonvolatile liquid contaminations are eliminated.

Animals↗
PubMed1985

Effects of antifertility estrogens and progestins on the endometrial surface of early pregnant rats. A scanning electron microscopic study.

Two estrogens, mestranol and 3-methoxy-14 beta, 15 beta-methyleneestra-1,3,5(10)triene-17 beta-ol (STS 593), and two progestins, levonorgestrel and 17 alpha-cyanomethyl-17 beta-hydroxyestra-4,9(10)-diene-3-one (STS 557), all having antifertility properties in rodents, were examined for their effects on the uterine luminal surface of early pregnant rats. Scanning electron microscopic studies showed that there are clear qualitative differences between the effects of estrogens and progestins on both morphology and microvillous pattern of the endometrial surface while STS 557 showed intermediate effects. The results are discussed with respect to the anti-implantation activity of steroids.

Animals↗
PubMed1983

Interceptive activities of STS 557 in rabbits, mice and rats.

STS 557 (17 alpha-cyanomethyl-17 beta-hydroxy-estra-4, 9-dien-3-one) is a interceptive agent in rabbits, mice and rats. In rats, it also shows post-implantational pregnancyterminating activity. In rabbits treated orally before mating or after ovulation for three consecutive days, it inhibited pregnancy largely at total doses of 0.08 mg/kg in the former and completely with 8.0 mg/kg in the latter case. A single subcutaneous dose of 40 mg/kg given on day 1 of pregnancy inhibited completely nidation in rats. In inhibition of pregnancy in rats could also be realized when the dose of 40 mg/kg was distributed on several days and given subcutaneously at daily doses of 5.0 mg/kg on days 1--8 of pregnancy, and even only daily doses of 2 mg STS 557/kg were needed in this respect, if animals which showed no living conceptuses were scored as "non-pregnant". STS 557 was effective in terminating pregnancy in rats, too, when given subcutaneously after implantation for 4 days at daily doses of 50 mg/kg, beginning on day 5 or on day 8 of pregnancy. The nidation inhibiting effects of STS 557 in rabbits treated pre-coitally, and in mice as well as in rats treated post-coitally were more marked than those of levonorgestrel. The interceptive and post-implantational inhibiting activity of STS 557 may be based on its antiprogestagenic properties. Luteolysis in the nidation phase can be excluded as shown by radioimmunoassay of progesterone in rats. The antigestagenic effects on the endometrium in rats were evident in the diamine oxidase assay. The acceleration of tubal egg transport, the morphological changes of the endometrium shown by scanning electron microscopy, and the effects on blastocyst transfer, all investigated in rats, suggest peripheral mechanisms of action.

Amine Oxidase (Copper-Containing)↗
PubMed1981

The use of a simple method to avoid cell shrinkage during SEM preparation.

In the course of preparing specimens for scanning electron microscopy, both glutaraldehyde and OsO4-fixed cells exhibit a considerable shrinkage with a reduction of the mean cellular diameter of about 45% after critical point drying. However, if cells are successively treated with glutaraldehyde, OsO4, tannic acid and uranyl acetate solutions, cellular shrinkage of only 5% is observed.

Animals↗
PubMed1980

Ultrastructural quantitative stereology on 'mixed' cell populations: problems and possibilities.

Contrary to homogeneous tissues, "mixed" tissues or cell suspensions are composed of different cell individuals. They have been characterized as heterogeneous cell populations (composed of cells of different cytogenetical source) and heteromorphous or inhomogeneous cell populations (of cells of the same type but of different individual functional state). Certain problems may arise during ultrastructural morphometrical investigations of heteromorphous populations. Because of the different size of planes of sectioning of individual cells, morphometrical results should be treated by means of modifications of the t-test [4]. Furthermore, the unambiguous classification of individual cell profiles necessitates a conscious selection of cell profiles containing a nuclear profile. This non-random sampling approach leads to systematic errors of computed parameters for the whole cell as the nuclear volume fraction and the specific cell surface area which should be corrected. Besides correction procedures derived from regular geometrical models we have presented correction methods for cells with non-spherical nucleus and a cell surface with marked surface projections [13, 21]. On the other hand, on heteromorphous cell populations, more detailed information about functional events can be gained, in comparison the customary stereological mean values, by means of frequency distributions of morphometrical data of individual cell profiles as well as by correlation of data of different cell organelles.

Animals↗