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K Fischer
Publications and source records attributed to K Fischer.
[Determination of systolic left ventricular time interval using the doppler technique. Analysis of aortic flow spectrum with transthoracic echocardiography].
UNLABELLED: In 10 healthy volunteers (age 21-28 years; mean age 24.8 years +/- 1.9 years) systolic left ventricular time intervals (STI) were determined by analysis of aortic Doppler flow pattern. This method was compared with conventional calculation of STI, using electrocardiogram, carotid pulse curve and phonocardiography, which were registered in synchronicity to the Doppler examination. Both, CW- and PW Doppler echocardiography constantly showed slightly lower values than the conventional method. This referred to basic measurements as well as to measurements after the application of isoprenaline and quinidine. The PW Doppler method underestimated the conventional method concerning the electromechanic systole (QS2) by on average 3.98%, the left ventricular ejection period (LVET) by on average 2.24% and the pre-ejection period (PEP) by on average 7.3%. Using CW-Doppler-method, QS2 was on average 5.31% and LVET was on average 6.67% smaller than the values determined by conventional method, whereas PEP was overestimated by on average 1.69%. The study documented that positive and negative inotropic pharmacological effects were measured reliably by the Doppler-echocardiographic method. Isoprenaline caused a significant shortening of frequency corrected QS2 (QS2c) from -55 +/- 17 to -85 +/- 20 ms (p < 0.05) using the PW-Doppler method; frequency corrected PEP (PEPc) was shortened from -40 +/- 14 to -67 +/- 14 ms (p < 0.05). The CW Doppler method also showed a statistically significant reduction of QS2c (from -64 +/- 18 to -89 +/- 25 ms; p < 0.05) and PEPc (from -37 +/- 16 to -64 +/- 12; p < 0.05). Likewise, the conventional method demonstrated statistically significant shortening of QS2c and PEPc after application of isoprenaline. LVETc did not change in a state of positive inotropy, no matter which method was used for determination. The negative inotropic effect of quinidine, measured by PW-Doppler, resulted in a prolongation of QS2c from -55 +/- 16 to -32 +/- 24 ms (p < 0.05) and of LVETc from -20 +/- 11 to +6 +/- 17 ms (p < 0.05). Using CW Doppler method, quinidine led to a lengthening of QS2c from -63 +/- 16 to -42 +/- 22 (p < 0.05) and of LVETc from -33 +/- 11 to -12 +/- 8 ms (p < 0.05). The conventional method also demonstrated a statistically significant increase of QS2c and LVETc. None of the 3 methods in question showed a statistically significant alteration of PEPc in the negative inotropic state. CONCLUSIONS: Doppler-echocardiographic analysis of aortic flow pattern constitutes a new method for the measuring of systolic time intervals. Basic values as well as changes due to positive or negative inotropic effects are reliably determined, in comparison with conventional methods the measurements are slightly lower.
Efficacy of current molecular cytogenetic protocols for the diagnosis of chromosome aberrations in tumor specimens.
Molecular cytogenetics provides a powerful link between molecular genetic analysis and chromosome morphology, allowing one to pinpoint structurally aberrant chromosome regions on the molecular level. Fluorescence in situ hybridization with selected DNA probes allows the design of efficient and sensitive tools for the diagnosis of chromosomal aberrations present in tumor cells. Comparative genomic hybridization (CGH) allows the identification of chromosomal imbalances in a comprehensive manner, and is applied to solid tumors and hematological malignancies in order to (i) identify clonal differences within a specimen, (ii) contribute to tumor classifications, (iii) identify recurrent chromosomal gains and losses as starting points for the characterization and isolation of pathogenetically relevant genes, such as proto-oncogenes and tumor suppressor genes respectively, (iv) identify imbalances of prognostic relevance, (v) detect high-copy-number amplification and other markers of genetic instability, and (vi) analyze chromosomal imbalances during tumor progression.
Sorting of nuclear-encoded chloroplast membrane proteins to the envelope and the thylakoid membrane.
The spinach triose phosphate/phosphate translocator and the 37-kDa protein are both integral components of the chloroplast inner envelope membrane. They are synthesized in the cytosol with N-terminal extensions, the transit peptides, that are different in structural terms from those of imported stromal or thylakoid proteins. In order to determine if these N-terminal extensions are essential for the correct localization to the envelope membrane, they were linked to the mature parts of thylakoid membrane proteins, the light-harvesting chlorophyll a/b binding protein and the CF0II-subunit of the thylakoid ATP synthase, respectively. In addition, the transit peptide of the CF0II-subunit that contains signals for the transport across both the envelope and the thylakoid membrane was fused to the mature parts of both envelope membrane proteins. The chimeric proteins were imported into isolated spinach chloroplasts, and the intraorganellar routing of the proteins was analyzed. The results obtained show that the N-terminal extensions of both envelope membrane proteins possess a stroma-targeting function only and that the information for the integration into the envelope membrane is contained in the mature parts of the proteins. At least part of the integration signal is provided by hydrophobic domains in the mature sequences since the removal of such a hydrophobic segment from the 37-kDa protein leads to missorting of the protein to the stroma and the thylakoid membrane.
Heterogeneity of deletions involving RB-1 and the D13S25 locus in B-cell chronic lymphocytic leukemia revealed by fluorescence in situ hybridization.
Recently, the D13S25 locus, which is in close proximity to the retinoblastoma gene (RB-1) on chromosome band 13q14, was discussed to play a role in the pathogenesis of B-CLL. In the present study, we isolated two overlapping genomic DNA clones (termed c13S25) containing the D13S25 DNA segment and used them as probes to analyze 85 B-CLL cases by fluorescence in situ hybridization; of the 55 cases with two RB-1 copies, 13 exhibited hemizygous (n = 7) or homozygous (n = 6) deletion of D13S25. Of 29 cases with hemizygous deletion of RB-1, all but two also showed loss of D13S25 (hemizygous, n = 25; homozygous, n = 2). One case had a homozygous deletion of both loci. We conclude that deletion of D13S25 occurs in a substantial number of B-CLL without deletion of RB-1. However, in some cases there is deletion of RB-1 without loss of D13S25, suggesting that D13S25 is not the locus of the putative tumor suppressor gene. According to our data, such a gene is most likely located within the genomic region between D13S25 and RB-1.
Comparative genomic hybridization in chronic B-cell leukemias shows a high incidence of chromosomal gains and losses.
In chronic B-cell leukemias, fluorescence in situ hybridization has greatly improved the ability to detect certain chromosomal aberrations, as cells in all phases of the cell cycle are analyzed. To obtain a comprehensive view of chromosomal gains and losses, we applied the recently developed technique of comparative genomic hybridization (CGH) to 28 patients with chronic B-cell leukemias. CGH results were compared with those obtained by chromosome banding analysis and interphase cytogenetics. In 19 of the 28 cases, chromosomal imbalances were detected, including amplified DNA sequences in three instances. The most common aberrations included gains of chromosomal material on 8q and 12 as well as losses of 6q, 11q, 13q, and 17p. In 13 cases, CGH revealed chromosomal gains and losses not detected by banding analysis. In 8 of these 13 cases, discrepancies were further investigated using other methods, and in all instances, the CGH findings were confirmed. A limitation of detecting small deleted regions by CGH was found in one example of 18p. In conclusion, our data show that the results of banding analysis in chronic B-cell leukemias often do not reflect the chromosomal changes in the predominant cell clone. This may be one explanation for the as yet poor correlation between cytogenetic findings and clinical course in this group of neoplasms.
Characterization of the superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg.
A gene (sod) encoding superoxide dismutase (SOD) was isolated from the strictly anaerobic archaeon Methanobacterium thermoautotrophicum Marburg. Its identify was confirmed by functional complementation of an Escherichia coli mutant strain lacking SOD activity and by DNA sequence analysis of a cloned fragment. Upstream of sod, separated by a 5-bp intergenic region, lies the open reading frame orfk which potentially codes for a protein of 209 amino acid residues. The amino acid sequence for this presumptive product had a similarity coefficient of 55.5% to a subunit of the alkyl hydroperoxide reductase (encoded by the ahpC gene) from Salmonella typhimurium.
Diagnosis and monitoring of chromosome aberrations in hematological malignancies by fluorescence in situ hybridization.
Besides its application in biological research, fluorescence in situ hybridization (FISH) is increasingly used for the cytogenetic analysis of human malignancies. Compared to conventional cytogenetic analysis, FISH allows delineation of specific numerical and structural chromosome aberrations in interphase cells (interphase cytogenetics). We have developed sets of genomic DNA probes for the identification of chromosome aberrations associated with chronic lymphocytic leukemia (CLL), chronic myeloid leukemias (CML), and acute myeloid leukemia (AML). In CLL, interphase cytogenetics will greatly contribute to the evaluation of the true incidence of specific chromosome aberrations and will provide the basis for more accurate correlations with the clinical outcome. The Philadelphia chromosome can be detected by FISH with high specificity and sensitivity in both CML and acute lymphoblastic leukemia. In CML, it can be used to better assess the cytogenetic remission status following therapy with interferon-alpha. Finally, in AML interphase cytogenetics provides a rapid and reliable technique for the identification of chromosome aberrations which are one of the most important prognostic factors in this disease. With the design of complex DNA probe sets and the development of digital microscopy and automated image analysis, it will be possible to use such disease-specific probe sets for monitoring residual disease following chemotherapy.
[Enlarged lymph nodes].
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Parasitism and chromosome dynamics in protozoan parasites: is there a connection?
Genomic plasticity is a hallmark of many protozoan parasites, including Plasmodium spp, Trypanosoma spp, Leishmania ssp and Giardia lamblia. Strikingly, there is a common theme regarding the structural basis of this karyotype variability. Chromosomes are compartmentalized into conserved central domains and polymorphic chromosome ends. Since antigen-encoding genes frequently reside in telomere-proximal domains, it is tempting to speculate that the genetic flexibility of chromosome ends has been recruited as a tool in immune evasion strategies by some parasitic protozoa.
The N-terminal extension of the ADP/ATP translocator is not involved in targeting to plant mitochondria in vivo.
The mitochondrial ADP/ATP translocator, also called adenine nucleotide translocase (ANT), is synthesized in plants with an N-terminal extension which is cleaved upon import into mitochondria. In contrast, the homologous proteins of mammals or fungi do not contain such a transient amino terminal presequence. To investigate whether the N-terminal extension is needed for correct intracellular sorting in vivo, translational fusions were constructed of the translocator cDNA--with and without presequence--with the beta-glucuronidase (gus) reporter gene. The distribution of reporter enzymatic activity in the subcellular compartments of transgenic plants and transformed yeast cells was subsequently analysed. The results show that: (i) the plant translocator presequence is not necessary for the correct localization of the ANT to the mitochondria; (ii) the mitochondrial targeting information contained in the mature part of the protein is sufficient to overcome, to some extent, the presence of plastid transit peptides; and (iii) the presequence alone is not able to target a passenger protein to mitochondria in vivo.
Subunit E of the vacuolar H(+)-ATPase of Hordeum vulgare L.: cDNA cloning, expression and immunological analysis.
A tonoplast protein of 31 kDa apparent molecular mass (TpP 31) was isolated from two-dimensional gels. Amino acid sequences were determined from LysC endoproteinase-peptide fragments. Using degenerate oligonucleotides, a corresponding cDNA clone of 1034 bp was isolated from a barley leaf cDNA library. It encodes for subunit E of the vacuolar H(+)-ATPase, the first one identified in plants so far. The open reading frame extends over 681 bp, encoding a gene product of 227 amino acids and a calculated molecular weight of 26,228 g mol-1. Northern and Western blot analysis indicates constitutive expression of subunit E in all plant organs with only small effects of salt stress. Localization of TpP 31 at the tonoplast was confirmed in fractions of purified vacuolar membrane obtained by free-flow electrophoresis. Immunoprecipitation of newly synthesized 35S-labelled membrane proteins with anti-TpP 31 gave two additional bands with apparent molecular masses of about 53 and 62 kDa. Gel filtration after mild solubilization showed co-purification of TpP 31 with the 55 kDa subunit of the H(+)-ATPase. Both results provide evidence beyond the sequence homology that TpP 31 is a structural component of the vacuolar H(+)-ATPase.
CDKN2 gene deletion is not found in chronic lymphoid leukaemias of B- and T-cell origin but is frequent in acute lymphoblastic leukaemia.
Homozygous deletions of the cyclin-dependent kinase 4 (CDK4) inhibitor gene CDKN2 (p16, MTS1) have been demonstrated to occur frequently in human cancer cell lines of different origin. However, in most primary tumours the frequencies of CDKN2 deletions are not well defined. We studied primary samples of 100 patients with lymphoid leukaemias [B-lineage acute lymphoblastic leukaemia (ALL), n = 23; T-ALL, n = 7; B-cell chronic lymphocytic (B-CLL) or prolymphocytic (B-PLL) leukaemia, n = 50; T-CLL/T-PLL, n = 20] using fluorescence in situ hybridization (FISH) with eight overlapping cosmid clones covering the region on chromosome band 9p21 containing CDKN2. We did not observe any CDKN2 deletions in the 70 patients with chronic lymphoid leukaemias of B- or T-cell origin. Of the 23 patients with B-lineage ALL, one (4%) exhibited a CDKN2 deletion: in this patient, two clones were detected, one exhibiting a hemizygous and the other a homozygous deletion. On chromosome banding analysis, four patients with B-lineage ALL had a 9p aberration, whereas all CDKN2 copies were retained. In contrast, six of the seven (86%) patients with T-ALL exhibited CDKN2 deletions (homozygous, n = 4; hemizygous, n = 2). We conclude that hemizygous or homozygous deletions of the CDKN2 gene occur at high frequency in T-ALL and at low frequency in B-lineage ALL, supporting the role of this gene as a tumour suppressor, especially in T-ALL. However, from our data there is no evidence that CDKN2 is involved in the pathogenesis of chronic lymphoid leukaemias of B- or T-cell origin.
[Flow cytometry detection of erythrocyte antigens and antibodies. Technical aspects and new clinical applications].
BACKGROUND: Although hemagglutination techniques have proved worthwhile since many years in immunohematology, they also have several disadvantages. They are manual and subjective visual methods, which make it difficult to quantitate red cell antibodies or surface antigens. Flow cytometric analysis overcomes these limitations because of its ability to analyze individual populations of cells by sensitive, reproducible, and objective methods. MATERIALS AND METHODS: Washed red cells from regular blood donors and patients were analyzed natively and after treatment with enzymes (sialidase, protease) or pneumococcal polysaccharides, using monoclonal Rh antibodies, human 7s-immunoglobulin, and FITC-labeled anti-human IgG or FITC anti-T lectin. The fluorescence intensity of single red cells was determined in the Ortho Cytoron Absolute flow cytometer. RESULTS: We determined the optimal test conditions and normal values by investigation of 50 blood donors. The fluorescence intensity of untreated red cells proved to be constant and therefore was used to adjust the instrument. Furthermore, the data of experimental adsorptions of red cells with pneumococcal antigens, sialidase (Vibrio cholerae) and protease (papain) as well as data from patients suffering from chronic HBV infection and autoimmune hemolytic anemia and acute pancreatitis are presented. A special software program was developed for statistical analysis and graphical presentation of the raw data. The computer program permits to analyze results from different experiments or from different dates and depicts them comparatively in overlay histograms, which may be useful in a serial study of changes of the antibody concentration or antigen expression. CONCLUSION: The flow cytometric analysis of red cells proves to be a simple, rapid, reproducible, and objective method for antigen and antibody quantitation. Furthermore, this technique may be a useful new tool for the investigation of acute, infection-associated hemolytic anemia.
Occipitocervical fusion with posterior plate and screw instrumentation. A long-term follow-up study.
STUDY DESIGN: Thirty-two patients at one institution underwent occipitocervical fusions with posterior plate and screw instrumentation. The average follow-up was greater than 4 years (50 months). METHODS: AO plates and screws were used and in more than 50% of the cases, the Magerl transarticular C1-C2 screw technique enhanced the occipitocervical instrumentation. In nine patients, cement was used and thus are excluded in evaluation of fusion results. All 23 patients attained solid fusions. No pseudarthrosis occurred. The average time to fusion was 13 weeks. Halos or traction immobilization was not used postoperatively. The average time of the simple orthosis wear was 11 weeks. Patients were out of bed on an average of the second postoperative day with a range of 1-4 days postoperatively. Reduction of the atlantoaxial joint was required in 10 of the 23 patients. At follow-up, nine remain reduced. RESULTS: In one patient, the atlantodens interval approximated the preoperative distance and radiographs demonstrated one transarticular C1-C2 screw was not placed satisfactorily. The average operative time was 172 minutes, and the average blood loss was 956 cc. The neurologic status of the patients improved or remained the same. No patient deteriorated neurologically. A total of 78 occipital screws were placed. No complications resulted from any of these screws. One intraoperative complication occurred secondary to massive bleeding after a transarticular screw hole was drilled. Bone wax was placed over the drill hole and the bleeding ceased. No postoperative problems occurred in this patient. Most specifically, no central nervous system sequela was evident. CONCLUSIONS: The conclusions from this study are that posterior occipitocervical fusion can be performed very safely with plate and screw instrumentation. An extremely high fusion rate can be expected with minimal complications and minimal postoperative immobilization. This technique, however, is technically demanding.
Porins from plants. Molecular cloning and functional characterization of two new members of the porin family.
Porins are voltage-gated diffusion pores found in all eukaryotic kingdoms. Here we describe, for the first time, the identification and characterization of two cDNAs encoding porins from plants. Peptide sequences obtained from a 30-kDa protein of envelope membranes from pea root plastids allowed the isolation of two cDNA clones from pea and maize. On the protein level, both proteins are homologous by 58%. Sequence comparison against the Swiss-Prot sequence data base revealed a homology of about 25% to mitochondrial porins from fungi and human. Computer-aided predictions of the secondary structure of the plant porins revealed the presence of 16 antiparallel beta-strands that are also found in mitochondrial porins. Porins from non-green plastids and from the outer mitochondrial membrane were reconstituted into planar lipid bilayers. The proteins showed high pore-forming activities and similar single-channel conductances. In vitro translated porin was preferentially imported only into non-green plastids but not into chloroplasts. To our knowledge, this is the first example of selective import of a plastid protein into different types of plastids. This finding is in line with the observation that an immunoreactive 30-kDa band was only found in non-green plastids and mitochondria but not in chloroplasts. We conclude that mitochondria and non-green plastids possess homologous porin proteins, whereas chloroplasts are characterized by a different type of porin.
The 24 kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning, in vivo expression and import pathway of a protein with unusual properties.
The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane. Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template. This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence. The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da. It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues. Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca. 25 kDa) is due to its abnormal amino acid composition. Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane. Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.
Cloning and in vivo expression of functional triose phosphate/phosphate translocators from C3- and C4-plants: evidence for the putative participation of specific amino acid residues in the recognition of phosphoenolpyruvate.
The primary sequences of the chloroplast triose phosphate/phosphate translocator precursor proteins from C4-plants (maize mesophyll cells and Flaveria trinervia) and from the C3-type Flaveria pringlei were determined. The mature parts of these translocators possess 83-94% identical amino acid residues. The C4-translocator protein can be correctly targeted to C3-type chloroplasts and inserted into the envelope membrane. Expression of the mature parts of these chloroplast translocators (cTPT) in transformed yeast cells and subsequent reconstitution of the functional proteins reveals the difference between the recombinant translocator proteins from the two cell types with respect to the transport of phosphoenolpyruvate. Comparison of the cTPT sequences from F. pringlei and F. trinervia in combination with computer-aided molecular modelling of the substrate translocation pore leads to the suggestion, that only minor exchanges of amino acid residues between the C3- and C4-translocator proteins are sufficient to extend their substrate specificities to recognize also phosphoenolpyruvate.