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M Köhler

Publications and source records attributed to M Köhler.

At least 73 records · Page 4Linked to original sources
PubMed1999

High-throughput extraction, amplification, and detection (HEAD) of HCV-RNA in individual blood donations.

BACKGROUND: High-throughput nucleic acid amplification techniques (NATs) are required for the detection of viral genomes in individual blood donations and might be helpful in any virological laboratory. OBJECTIVE: To develop and automate a method for the detection of hepatitis C virus RNA in individual blood donations, compatible with the time schedule of routine blood bank screening an product release. STUDY DESIGN: The viral RNA was isolated with the use of target specific capture oligonucleotides and magnetic beads. This extraction method was combined with reverse transcription/amplification (RT/PCR) and fluorescence detection. We adapted our method on a pipetting robot and pipetted all steps in a single room. When the pipetting was completed, microtiter plates were heat-sealed with foils and placed into a thermocycler. Positive reactions were detected with a fluorescent dye in a second room. Aerosols were avoided with programmed slow pipetting steps and with a special device constructed for the removal of the used disposable tips. During a 7 month period, we used this method in routine testing of individual donations prior to the release of all blood components. RESULTS: The total number of 11,700 individual donations including platelet concentrates were analysed. We tested up to 192 specimens in one run within 7 h. The frequency of cross-contamination using the automated procedure was 0.1%. Five specimens have been found repeatedly reactive for HCV-RNA, four of these were anti-HCV positive, one sample from a repeat donor was negative in anti-HCV assays. A seroconversion was detectable at his next presentation, 6 months later. CONCLUSION: In this pilot study, we demonstrate that automated HCV-RT-PCR testing is practicable for individual donations in high-throughput. Additionally, the described PCR approach could easily be adapted to the detection of other viral genomes by the use of specific primers.

Automation↗
PubMed1999

Application of RHD and RHCE genotyping for correct blood group determination in chronically transfused patients.

BACKGROUND: In chronically transfused patients, conventional blood group typing may be impossible because of mixed-field agglutination. STUDY DESIGN AND METHODS: In 27 patients with congenital anemia and lifelong transfusion history, genotyping for D, RHD, and RHCE was performed with polymerase chain reactions. These results were compared with the blood group typing results documented in the medical record. RESULTS: Two of 27 cases had been typed D-negative by serologic tests and D-positive by genotyping. In 20 patients, the CDE formula had been determined serologically according to the medical record; 4 of these patients were Cc by serologic tests and C/C by genotyping. One patient typed ee by serologic tests, and genotyping revealed heterozygosity (E/e). CONCLUSION: In patients with a lifelong transfusion history, serologic blood group determination may be impossible, and pretransfusion test results are not always available or reliable. In whites, Rh-matched transfusions are possible with genotyping. The genetic background of the RH genes has to be elucidated in other ethnic groups, such as in black patients with sickle cell disease, before genotyping can be applied without restriction.

ABO Blood-Group System↗
PubMed1999

A prospective, randomized, sequential, crossover trial of large-volume versus normal-volume leukapheresis procedures: effect on progenitor cells and engraftment.

BACKGROUND: The influence of leukapheresis size on the number of harvested peripheral blood progenitor cells is still unclear. A prospective randomized crossover trial was thus performed, to evaluate the effect of large-volume leukapheresis (LVL) versus normal-volume leukapheresis (NVL) on progenitor cells and engraftment in 26 patients with breast cancer and 15 patients with non-Hodgkin's lymphoma who were eligible for peripheral blood progenitor cell transplantation. STUDY DESIGN AND METHODS: Patients were randomly assigned to undergo either LVL on Day 1 and on Day 2 or vice versa. The number of progenitor cells was evaluated in the harvest and before and after leukapheresis in the peripheral blood. RESULTS: The number of harvested CD34+ cells (4.8 x 10(6) vs. 3.4 x 10(6)/kg body weight, p < 0.001) and colony-forming units-granulocyte-macrophage (3.1 x 10(5) vs. 2.4 x 10(5)/kg body weight, p = 0.0026) was significantly higher for LVL procedures than for NVL procedures. The median extraction efficacy, defined as the difference between the yield in the harvest and the decrease in the total number of CD34+ cells in peripheral blood during leukapheresis, was significantly (p < 0.0001) higher for LVL than for NVL (2.6 x 10(8) and 8 x 10(7), respectively). In patients with breast cancer, the median amount of CD34+ cells in the harvest and the median extraction efficacy were higher for LVL than for NVL (p < 0.0001). This was not found for patients with non-Hodgkin's lymphoma. CONCLUSION: LVL results in a higher yield of CD34+ cells and colony-forming units-granulocyte-macrophage than NVL, but only in patients with breast cancer and with high numbers of CD34+ cells in the peripheral blood before leukapheresis.

Adult↗
PubMed1999

Adenylosuccinase deficiency: possibly underdiagnosed encephalopathy with variable clinical features.

Adenylosuccinase deficiency is an autosomal recessive inherited defect of purine synthesis. In enzyme deficient patients, two normally undetectable compounds, succinylaminoimidazole carboxamide riboside and succinyladenosine, accumulate in urine, cerebrospinal fluid and, to a minor extent, in plasma. Analysing 150 highly selected urine specimens from patients with unidentified neurogenerative disorders we discovered the first two German cases of adenylosuccinase deficiency. The deficiency causes moderate to severe mental retardation, often accompanied by epileptic seizures and/or autistic features, and is occasionally associated with growth retardation and muscular hypotonia. Of the two German cases we present here, one patient fits into the clinical picture outlined by previous reports. The other patient, however, shows a pattern of symptoms so far undescribed: severe early infantile epileptic encephalopathy with reduced myelination. On mutation analysis this patient is the first to reveal a 39 base pair deletion in the adenylosuccinase gene in contrast to the point mutations detected in previous cases. Adenylosuccinase deficiency may be an underdiagnosed metabolic disorder with variable expression. This should be taken into consideration in patients with unclassified neurological conditions.

Adenylosuccinate Lyase↗
PubMed1999

A novel testicular haploid expressed gene (THEG) involved in mouse spermatid-sertoli cell interaction.

The successful production of male gametes requires extensive and precise interactions between germ cells and surrounding testicular somatic cells. We have made use of the mRNA differential display technique to isolate genes involved in germ cell-Sertoli cell interaction. We have identified five differential cDNA bands by comparing RNA from Sertoli cells, spermatids and spermatid-Sertoli cell cocultures. One of the isolated cDNA fragments detected a 1. 4-kilobase (kb) testis- and spermatid-specific transcript (designated as THEG: testicular haploid expressed gene). Northern blot analysis on RNA from spermatids and spermatid-Sertoli cell cocultures demonstrated that Sertoli cells are required for the continued expression of THEG in spermatids. We found two alternatively spliced transcripts for the THEG gene with 1437 base pairs (bp) and 1375 bp by using reverse transcription-polymerase chain reaction. The two open reading frames of 376 amino acids and 181 amino acids coded for putative nuclear proteins. The gene is approximately 10 kb pairs in size, contains 8 exons, and was mapped on mouse chromosome 10 to region B5-C1. Comparison of the two cDNA sequences with the genomic sequence indicated that the smaller transcript lacks exon 4. The differential gene expression of THEG in spermatid-Sertoli cell coculture supports the relevance of germ cell-Sertoli cell interaction for gene regulation during spermatogenesis.

Alternative Splicing↗
PubMed1999

Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import.

Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway. In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfamilies, exist in higher eucaryotes. We describe here a novel member of the human family, importin alpha7. To analyze specific functions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xenopus and Saccharomyces cerevisiae. Binding affinity studies showed that all importin alpha proteins from humans or Xenopus bind their import receptor (importin beta) and their export receptor (CAS) with only marginal differences. Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin alpha proteins. When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin alpha3, whereas most of the other substrates were imported by all importin alpha proteins with similar efficiencies. However, strikingly different substrate preferences of the various importin alpha proteins were revealed when two substrates were offered simultaneously.

Acetyltransferases↗
PubMed1999

RanBP3 contains an unusual nuclear localization signal that is imported preferentially by importin-alpha3.

The full range of sequences that constitute nuclear localization signals (NLSs) remains to be established. Even though the sequence of the classical NLS contains polybasic residues that are recognized by importin-alpha, this import receptor can also bind cargo that contains no recognizable signal, such as STAT1. The situation is further complicated by the existence of six mammalian importin-alpha family members. We report the identification of an unusual type of NLS in human Ran binding protein 3 (RanBP3) that binds preferentially to importin-alpha3. RanBP3 contains a variant Ran binding domain most similar to that found in the yeast protein Yrb2p. Anti-RanBP3 immunofluorescence is predominantly nuclear. Microinjection of glutathione S-transferase-green fluorescent protein-RanBP3 fusions demonstrated that a region at the N terminus is essential and sufficient for nuclear localization. Deletion analysis further mapped the signal sequence to residues 40 to 57. This signal resembles the NLSs of c-Myc and Pho4p. However, several residues essential for import via the c-Myc NLS are unnecessary in the RanBP3 NLS. RanBP3 NLS-mediated import was blocked by competitive inhibitors of importin-alpha or importin-beta or by the absence of importin-alpha. Binding assays using recombinant importin-alpha1, -alpha3, -alpha4, -alpha5, and -alpha7 revealed a preferential interaction of the RanBP3 NLS with importin-alpha3 and -alpha4, in contrast to the simian virus 40 T-antigen NLS, which interacted to similar extents with all of the isoforms. Nuclear import of the RanBP3 NLS was most efficient in the presence of importin-alpha3. These results demonstrate that members of the importin-alpha family possess distinct preferences for certain NLS sequences and that the NLS consensus sequence is broader than was hitherto suspected.

Animals↗
PubMed1998

Changes in cytoplasmic ATP concentration parallels changes in ATP-regulated K+-channel activity in insulin-secreting cells.

Changes in cytoplasmic ATP concentration were monitored in intact insulin-producing cells and correlated to changes in the activity of ATP-sensitive K+-channels (KATP channels). Luciferase was introduced into HIT M2.2 cells and whole pancreatic islets by transient expression of firefly (Photinus pyralis) luciferase cDNA. In transfected cells, extracellular addition of luciferin increased the luminescence signal to a maximum within 50-120 s. Addition of 1 microM of the mitochondrial uncoupler FCCP decreased the luminescence, an effect partly reversed upon withdrawal of the compound. High concentrations of glucose increased cytoplasmic free ATP concentration. Changes in the luminescence signal were accompanied by changes in activity of the ATP-sensitive K+-channel. Transfection per se did not deteriorate cell function, as verified by experiments showing similar changes in cytoplasmic free Ca2+-concentration, [Ca2+li, in both transfected and non-transfected cells. By measuring the cytoplasmic ATP concentration and KATP channel activity under similar experimental conditions, it was possible to establish, for the first time, a direct relationship between these two parameters. This indeed suggests that the cytoplasmic ATP concentration has a crucial role in the regulation of KATP channel activity under physiological conditions.

Adenosine Triphosphate↗
PubMed1998

Glucose and tolbutamide induce apoptosis in pancreatic beta-cells. A process dependent on intracellular Ca2+ concentration.

High concentrations of glucose are considered to be toxic for the pancreatic beta-cell. However, the mechanisms underlying beta-cell dysfunction and resulting cell death are not fully characterized. In the present study we have demonstrated that incubation of pancreatic islets and beta-cells from ob/ob mice and Wistar rats with glucose induced a process of apoptotic beta-cell death, as shown by DNA laddering, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, and by using DNA-staining dye HOECHST 33342. The obtained results show that the percentage of apoptotic cells was dependent on glucose concentration, being minimal at 11 mM glucose. At a concentration of 100 microM, aurintricarboxylic acid, an inhibitor of endonuclease activity, almost completely inhibited apoptosis triggered by 17 mM glucose. We have also shown that long term incubation with 100 microM sulfonylurea, tolbutamide, triggered apoptosis in pancreatic beta-cells. The process of beta-cell death induced by high glucose concentration and tolbutamide were Ca2+-dependent, because introduction to the culture medium of 50 microM D-600 or 200 microM diazoxide, which blocked glucose- and tolbutamide-induced [Ca2+]i increase, inhibited apoptosis. Thus, this study shows for the first time that high glucose concentrations and tolbutamide induce apoptosis in pancreatic beta-cells, and that this process is Ca2+-dependent.

Animals↗
PubMed1998

Proteins in electric fields and pressure fields: basic aspects.

This paper emphasizes the basic aspects of the interactions of chromoproteins at low temperatures with external pressure fields and electric fields. We discuss how the respective spectral properties can be modified and what we can learn from the spectral changes about the thermodynamic, electrostatic, functional and structural properties of proteins. A few examples are discussed in more detail.

Electricity↗
PubMed1998

Proteins in electric fields and pressure fields: experimental results.

Experimental results obtained by Stark effect and pressure tuning optical spectroscopy are discussed with the emphasis on studies aimed at unraveling the coupling of prosthetic groups to proteins. A comparative, detailed analysis is given concerning the coupling of the heme group to the apoprotein in various heme proteins based on spectral hole burning data. Electrochromism and electric dichroism experiments related to the coupling problem are also discussed in the context of other protein systems.

Bacteriorhodopsins↗
PubMed1998

Short-term regulation of insulin gene transcription by glucose.

Whereas short-term regulation of insulin biosynthesis at the level of translation is well accepted, glucose-dependent transcriptional control is still believed to be a long-term effect occurring after more than 2 hr of glucose stimulation. Because pancreatic beta cells are exposed to elevated glucose levels for minutes rather than hours after food uptake, we hypothesized the existence of a short-term transcriptional control. By studying the dynamics of newly synthesized (prepro)insulin RNA and by employing on-line monitoring of gene expression in single, insulin-producing cells, we were able to provide convincing evidence that insulin gene transcription indeed is affected by glucose within minutes. Exposure of insulinoma cells and isolated pancreatic islets to elevated glucose for only 15 min resulted in a 2- to 5-fold elevation in (prepro)insulin mRNA levels within 60-90 min. Similarly, insulin promoter-driven green fluorescent protein expression in single insulin-producing cells was significantly enhanced after transient glucose stimulation. Thus, short-term signaling, such as that involved in insulin secretion, also may regulate insulin gene transcription.

Animals↗
PubMed1998

RHD genotyping in weak D phenotypes by multiple polymerase chain reactions.

BACKGROUND: Weak D phenotypes involve a quantitative variation of D. The genomic basis in weak D has been disputed, however. STUDY DESIGN AND METHODS: Five sequence-specific polymerase chain reactions (SSP-PCRs) on exons 2, 5, and 7 of the RHD gene were evaluated in 248 white and 98 Japanese blood donors and compared with the results obtained by amplification of intron 4 and serology. All methods and SSP-PCR testing on the 3' non-coding region of the RHD gene were applied to the genotyping of 94 DNA samples derived from individuals expressing weak D phenotypes. RESULTS: Concordant results were obtained with all genotyping and phenotyping methods in testing 201 D-positive and 145 D-negative donors. Four of 94 weak D samples were typed as D-negative by amplification of intron 4 and SSP-PCR on exon 5. Phenotyping with monoclonal antibodies revealed a DVI category in one of these cases and DFR phenotype in three of these cases. One weak D sample, which reacted like normal D-positive cells with all applied monoclonal antibodies, was typed falsely negative by SSP-PCR on exon 5 because of a point mutation at nucleotide 667 (T-->G) that resulted in a Phe223Val amino acid substitution. In this individual, heterozygosity was found at two other amino acid positions (Glu233Gln and Val238Met) by restriction fragment length polymorphism analysis. CONCLUSION: Genetic diversity in weak D phenotypes is rare. Only 1 of 90 true weak D phenotypes (1.1%) had a genetic variation in testing on seven gene regions of the RHD gene.

Asian People↗
PubMed1998

'Eye-witness'.

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Adult↗