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K Pfeifer

Publications and source records attributed to K Pfeifer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Motor performance in different dynamic tests in knee rehabilitation.

Functional dynamic tests are increasingly used in rehabilitation after injuries of the lower extremities. In these tests quantitative measures (e.g. time, height, distance) are mainly used as parameters, whereas the quality of the neuromuscular performance is poorly evaluated. In the present study the neuromuscular performance of leg muscles in different motor tasks was investigated 10-16 months after ACL-reconstruction. In 39 subjects with arthroscopically assisted ACL-reconstruction and 20 controls, isometric knee extensor strength, thigh circumference, knee stability, subjective knee function and the neuromuscular performance (by kinematic data and EMG) during three dynamic tests (stair descending, one-legged drop jump, one-legged cyclic hops) were measured. During the strength measurements a superimposed twitch technique was used for the detection of neuromuscular inhibition. The results demonstrate a significantly reduced Lysholm-Score and a distinct strength deficit, but no neuromuscular inhibition and no differences in knee stability in the operated leg. Besides reduced motor abilities of the injured leg, significant differences of the neuromuscular performance could be detected. It was evident that different test conditions revealed different persisting changes of the neuromuscular performance, which could not be detected by kinematic parameters alone.

Adult↗
PubMed1998

Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle.

Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are disorganized compared with those of Rpe65+/+ and Rpe65+/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/- mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.

Animals↗
PubMed1998

Imprinting of mouse Kvlqt1 is developmentally regulated.

Mouse distal chromosome 7 contains a cluster of at least five imprinted genes. The syntenic region in humans, at 11p15.5, has been implicated in several genetic disorders. Consistent with the imprinted status of the genes in the region, Beckwith-Wiedemann syndrome (BWS) and Wilms tumor are each associated with loss of maternal information. Also mapping to 11p15.5 are long QT and Jervell and Lange-Nielsen (JLN) syndromes. In contrast to BWS and Wilms tumor, these syndromes do not show any parent of origin bias. Recently positional cloning has identified KVLQT1 as the 11p15.5 gene responsible for increased susceptibility to long QT and JLN syndromes. Other studies associate KVLQT1 with BWS. Human KVLQT1 is paternally imprinted in embryos. In this study we present a contig and transcript map of distal mouse 7 and we physically and genetically map mouse Kvlqt1 to the region. Mouse Kvlqt1 is strongly expressed in heart, lung, gut, kidney and uterus. While its early developmental expression is maternal in origin, the paternal allele becomes increasingly active during development. Late juvenile and adult animals show complete biallelism, suggesting an explanation for the lack of parent of origin bias in JLN and long QT.

Aging↗
PubMed1998

Expression of interleukin-6 and interleukin-6 receptors in human granulosa lutein cells.

Cytokines are important regulators of reproductive functions. Significant amounts of interleukin-6 (IL-6) have been detected in the serum and ascites of patients with ovarian hyperstimulation syndrome (OHSS). These findings suggest the involvement of IL-6 as a mediator in the pathogenesis of OHSS. This study was performed to analyse IL-6 and IL-6 receptor (IL-6-R) expression in human granulosa lutein cells (GC). GC were cultured after isolation from follicular fluid. IL-6 concentrations in follicular fluid and serum from individual patients and GC supernatants were measured by enzyme-linked immunosorbent assay. We found detectable concentrations of IL-6 in serum and follicular fluid of all patients. Expression of IL-6 in GC was shown immunocytochemically. IL-6 mRNA was detected in GC by in-situ hybridization. Gene expression for IL-6 and IL-6-R in GC was demonstrated using reverse transcription-polymerase chain reaction. IL-6 significantly inhibited human chorionic gonadotrophin (HCG)-induced progesterone secretion of GC. The results of our study suggest that IL-6 is expressed in HGC and that this cytokine is able to modulate GC function via its specific receptor. This is the first report that describes the precence of IL-6-R in human granulosa lutein cells.

Cells, Cultured↗
PubMed1996

The structural H19 gene is required for transgene imprinting.

The product of the H19 gene is an untranslated RNA that is expressed exclusively from the maternal chromosome during mammalian development. The H19 gene and its 5'-flanking sequence are required for the genomic imprinting of two paternally expressed genes, Ins-2 (encodes insulin-2) and Igf-2 (encodes insulin-like growth factor-2), that lie 90 and 115 kb 5' to the H19 gene, respectively. In this report, the role of the H19 gene in its own imprinting is investigated by introducing a Mus spretus H19 gene into heterologous locations in the mouse genome. Multiple copies of the transgene were sufficient for its paternal silencing and DNA methylation. Replacing the H19 structural gene with a luciferase reporter gene resulted in loss of imprinting of the transgene. That is, high expression and low levels of DNA methylation were observed upon both paternal and maternal inheritance. The removal of 701 bp at the 5' end of the structural gene resulted in a similar loss of paternal-specific DNA methylation, arguing that those sequences are required for both the establishment and maintenance of the sperm-specific gametic mark. The M. spretus H19 transgene could not rescue the loss of Igf-2 imprinting in trans in H19 deletion mice, implying a cis requirement for the H19 gene. In contrast to a previous report in which overexpression of a marked H19 gene was a prenatal lethal, expression of the M. spretus transgene had no deleterious effect, leading to the conclusion that the 20-base insertion in the marked gene created a neomorphic mutation.

Animals↗
PubMed1994

Long-term genome stability and minimal genotypic and phenotypic alterations in HPV16 E7-, but not E6-, immortalized human uroepithelial cells.

Parameters of genome instability and morphological alterations associated with cell transformation were studied in an isogeneic set of clonal human uroepithelial cell (HUC) lines immortalized by the human papilloma virus 16 (HPV16) E6 and/or E7 gene(s). HPV16 E6 binds p53, leading to rapid degradation of p53, whereas E7 binds and alters pRb and other proteins. We report that two independent E7-immortalized HUC lines showed minimal phenotypic or genotypic alterations, except that both lines contained amplification of 20q DNA sequences and a greater polyploidization at an early passage. The E7-immortalized HUC line resembled normal HUC lines, except that they failed to senesce. In contrast, the E6-immortalized HUC lines were morphologically altered, contained numerous random chromosome aberrations, and showed unstable evolving karyotypes with passage in culture. No amplified DNA sequences were detected in E6-immortalized HUC lines. Instead, clonal losses of chromosome regions (i.e., -3p, -6q, -9p), putatively containing tumor suppressor or senescence genes, accompanied the E6-HUC immortalization event. E6-immortalized HUC lines showed transformed phenotypes similar to E6/E7-HUC lines. The difference in genome stability between E6- and E7-immortalized HUC was highly significant statistically (p-value < 10(-6). Thus, the HPV16 E7 gene led to HUC immortalization by a pathway that blocked cellular senescence, but did not disrupt genome stability. These results implicate p53 loss, but not pRb alteration, in genome destabilization.

Base Sequence↗
PubMed1993

Kinetics of expression of prion protein in uninfected and scrapie-infected N2a mouse neuroblastoma cells.

The scrapie prion protein, PrPSc, is formed from its isoform, the cellular PrPc. There is evidence available indicating that PrPSc is a necessary component of the infectious prion particle to cause a series of transmissible spongiform encephalopathies. We have used immunocytochemistry and RNA blotting techniques to investigate if infection with prions results in an increased PrP gene expression. For the experiments we used N2a cells which had been infected with prions (ScN2a cells). We demonstrated by confocal laser scanning microscopy that PrP-protein was present in the nucleus (predominantly in the nucleoli) of ScN2a cells. Analysis of the PrP-mRNA levels both in N2a- and in ScN2a cells using cDNA encoding PrPc revealed no marked alteration of the mRNA steady state level between the two cell strains. Likewise, in run-off experiments no changes in either PrP-specific transcription or in general transcriptional activity were found. The half-life of PrP-mRNA was found to be identical in both cell strains (7h). Taken together, these results show that PrPSc and/or PrPc is present in the nucleus (nucleoli) of ScN2a cells but does not display an effect on the expression of the PrP gene.

Animals↗
PubMed1993

Evidence for age-dependent impairment of antiviral 2',5'-oligoadenylate synthetase/ribonuclease L-system in tissues of rat.

The 2',5'-oligoadenylate system (2-5A system) has an essential role in the establishment of the antiviral state of cells exposed to virus infection. The effects of 2-5A are mediated by a 2-5A-dependent ribonuclease (RNase L) which cleaves viral RNA. A study of 2-5A metabolism in different tissues of rats of different age (newborn: 1-day-old; young adult: 2- to 3-month-old; middle-aged adult: 12-month-old; and old: 32- to 33-month-old) revealed that the activities of the 2-5A metabolic enzymes alter during aging and development. We demonstrate that soluble 2-5A synthetase activity strongly increases after birth, reaching maximal levels in young adult and middle-aged adult animals and then significantly decreases with age; the age-dependent decrease was found also for the nuclear matrix-associated enzyme. In contrast, the activity of 2',3'-exoribonuclease which inactivates 2-5A increases by 3-fold with age. The decrease in 2-5A synthetase activity and increase in 2-5A nuclease activity were found to result in a decrease in the cellular 2-5A content with age. The RNase L which is activated by 2-5A also changes age-dependently. The amount and activity of this enzyme were determined in cross-linking experiments, in nitrocellulose binding assays and in the ribosomal RNA cleavage assay. The livers of old rats displayed a 5- to 6-fold decrease in RNase L activity compared to the adult animal groups, whilst the amount of the enzyme did not change significantly during aging with the exception of a drop by 30% in the nuclear matrix fraction. From these results we conclude that the antiviral activity of the 2-5A system is impaired in old cells with the consequences that virus production cannot be efficiently suppressed.

2',5'-Oligoadenylate Synthetase↗
PubMed1993

S-type lectins occur also in invertebrates: high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium.

The marine sponge Geodia cydonium contains several lectins. The main component, called lectin-1, is composed of three to four identical subunits. The subunits of the lectins were cloned from a cDNA library; two clones were obtained. From the deduced aa sequence of one clone, LECT-1, a mol. wt of 15,313 Da is calculated; this value is in good agreement with mass spectrometric analysis of 15,453 +/- 25 Da. The sequence of another clone, LECT-2, was analysed and the aa sequence was deduced (15,433 Da). The two subunits have a framework sequence of 38 conserved aa which are characteristic for the carbohydrate-binding site of vertebrate S-type lectins. Clustering of lectin sequences of various species following their pairwise comparison establishes a dendrogram, which reveals that the sponge lectin could be considered as the ancestor for vertebrate S-type lectins.

Amino Acid Sequence↗
PubMed1993

Cloning of the polyubiquitin cDNA from the marine sponge Geodia cydonium and its preferential expression during reaggregation of cells.

Ubiquitination of proteins is a critical step in the controlled degradation process of many polypeptides. Here we show that sponges, the simplest multicellular group of eukaryotic organisms, are also equipped with the ubiquitin pathway. The polyubiquitin cDNA was isolated and characterized from the marine sponge Geodia cydonium. The open reading frame contains six ubiquitin moieties, which are lined up head to tail without spacers. A comparison of the predicted amino acid sequence of the six sponge ubiquitin-coding units with those from other organisms revealed a high degree of homology (> 93%). The ubiquitin gene is expressed to almost the same extent in the two main compartments of the sponge, the cortex and the medulla. However, only in the cortex are detectable amounts of the ubiquitin protein synthesized. The ubiquitin protein isolated from the sponge organism was found to initiate protein degradation in the heterologous reticulocyte system in the same manner as bovine ubiquitin. In vitro studies with dissociated sponge cells revealed that the homologous aggregation factor causes (i) a strong increase in the steady-state level of mRNA coding for ubiquitin and (ii) a drastic increase in ubiquitin protein synthesis, while the homologous lectin failed to display that effect in isolated cells. These data suggest that ubiquitin may play a role in sponge morphogenesis.

Adenosine Triphosphate↗
PubMed1992

Localization of Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 in interphase and mitotic Burkitt lymphoma cells.

The subcellular distribution of the small Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 has been investigated by using a high-resolution in situ hybridization technique. The distribution patterns in Raji cells of fluorescent oligodeoxynucleotides complementary to each RNA were detected by confocal laser scanning microscopy. Both RNAs were found in the cytoplasm as well as in the nuclei of interphase cells. In contrast, use of the same technique indicated an exclusively nuclear location for cellular U2 RNA. In the cytoplasm distribution of the EBERs was similar to that of the double-stranded RNA-dependent protein kinase, to which these RNAs can bind, and was coincident with the rough endoplasmic reticulum. In cells undergoing mitosis the EBERs became localized around the chromosomes, whereas the protein kinase remained uniformly distributed in the cytoplasm. A cytoplasmic location for EBER-1 and EBER-2 in interphase cells is consistent with the evidence for a role for these small RNAs in translational control.

Base Sequence↗
PubMed1992

Association of AUUUA-binding protein with A+U-rich mRNA during nucleo-cytoplasmic transport.

Resealed nuclear envelope (NE) vesicles from rat liver containing entrapped exogenous RNA were used to study the effect of adenosine+uridine binding factor (AUBF), present in cytosolic cell extracts, on ATP-dependent transport of A+U-rich RNA (AU+RNA) and A+U-free RNA (AU-RNA) across the NE. This factor specifically binds to A+U-rich sequences present in the 3' untranslated regions of lymphokine and cytokine mRNAs, containing overlapping AUUUA boxes (granulocyte-macrophage colony stimulating factor, interleukin-3). Addition of AUBF to the extravesicular compartment markedly increased the efflux of the in vitro transcribed, capped and polyadenylated AU+ RNAs. Export of entrapped AU- control RNA, such as beta-globin RNA, was not affected by AUBF, in contrast to chimeric AU+ beta-globin RNA containing the A+U-rich sequence of human interferon-alpha mRNA (6 reiterated AUUUA motifs). Competition experiments revealed that AUBF enhances the affinity of poly(A)-containing AU+ RNAs to the NE poly(A)-binding component (poly(A)-recognizing mRNA carrier p106), and thereby accelerates nuclear export of these RNAs. We could demonstrate that AUBF added to the extravesicular space forms stable complexes with polyadenylated AU+ RNA with relative molecular masses of about 45,000, 62,000 and 70,000 inside the vesicles or during ATP-dependent export. In addition we determined that AUBF may affect mRNA stability by protecting A+U-rich RNA against degradation by trans-acting, nuclear matrix-associated and A+U-specific endoribonuclease V.

Adenosine↗
PubMed1992

Accumulation of transcripts coding for prion protein in human astrocytes during infection with human immunodeficiency virus.

The abnormal isoforms of the normal cellular prion protein (PrP), also termed Scrapie-associated fibril protein, are assumed to be one causative factor of spongiform encephalopathies. The mRNA of PrP contains stem-loop structures which are very similar to the human immunodeficiency virus-1 (HIV-1) cis-acting sequence TAR within the LTR; both structures contain the pentanucleotide CUGGG in the loop, and the uridine- and adenine-bulge in the stem. In this study, using purified HIV-encoded trans-activator, Tat, and HIV-1 TAR-RNA or PrP-mRNA containing the stem-loop structure, we demonstrate by use of gel-retardation and filter binding assays that Tat binds to TAR- and PrP-RNA with the dissociation constants of 2.9 or 37.0 nM, respectively, at a molar ratio of 0.7 mol of Tat to 1 mol of RNA fragment. The Tat-RNA (TAR or PrP) complexes bind to protein(s) in the nuclear matrix, isolated from human astrocytes (glial fibrillary acidic protein positive brain cells). Infection of astrocytes with HIV-1 resulted in an increased level of PrP mRNA. The data presented led us to assume that certain sequences in the PrP mRNA might be targets for proteins acting in trans.

Astrocytes↗
PubMed1992

Purification of a glucose-binding protein from rat liver nuclei. Evidence for a role in targeting of nuclear mRNP to nuclear pore complex.

A nuclear carbohydrate-binding protein with a molecular mass of 67 kDa (CBP67), which is specific for glucose residues, was purified to essential homogeneity from rat liver nuclear extracts. This protein could also be isolated from nuclear ribonucleoprotein (RNP) complexes by extraction in the presence of 0.6 M or 2 M NaCl, but it was absent in polysomal RNP complex. The binding of the purified protein, which has an isoelectric point of 7.3, to glucose-containing glycoconjugates depends on the presence of Ca2+ and Mg2+. Using closed nuclear envelope vesicles as a system to study nuclear transport of RNA, it was shown that both entrapped polysomal mRNA and nuclear RNA precursors are readily exported from the vesicles in an ATP-dependent manner. The transport was unidirectional and strongly promoted by the poly(A) segment attached to these RNAs. In contrast, nuclear RNP complexes entrapped into the vesicles together with glucose-conjugated bovine serum albumin or nucleoplasmin, or bird nest glycoprotein, were not exported into the extravesicular space. However, transport of nuclear RNP complexes could be achieved in the presence of glucose or after co-addition of a glucose-recognizing lectin from Pellina semitubulosa. In Western blots, radioiodinated CBP67 binds to an 80-kDa polypeptide both in isolated rat liver nuclear envelopes and pore-complex laminae. From these results we postulate that CBP67 may direct nuclear RNP complexes to the nuclear pore.

Animals↗
PubMed1992

Immunological and biological identification of tumour necrosis-like factor in sponges: endotoxin that mediates necrosis formation in xenografts.

Xenografts of the sponge Geodia cydonium in its closely related species G. rovinjensis resulted in a rapid rejection of the graft within a period of 5 days. We identified an immunoreactive tumour necrosis factor (TNF)-like activity in the xenograft (Mr of 30,000) two days after grafting. In-vivo injection of 5 micrograms human recombinant TNF-alpha induced cytotoxicity in sponge cells in the same pattern and time course as during natural xenograft rejection. Anti-TNF-alpha polyclonals were found to react with xenograft extracts, by Western blot analysis, as from day 2 after grafting. Using ELISA we detected the TNF-like activity from day 2 after grafting with peak levels at days 4 and 5, where the amount was 0.72 ng/micrograms tissue DNA. By day 1, gp27 (inhibitory aggregation factor) is already formed in the xenograft. In-vitro experiments on isolated G. cydonium cells showed that addition of purified gp27 induced the production of the TNF-like activity (up to 13.5 ng/ml). Evidence is presented that gp27 is a product of the gp180 lectin receptor. We conclude that gp27 induces TNF-like factor production, resulting in destruction and dissolution of the xenograft after 5 days.

Animals↗
PubMed1991

Formation of a small ribonucleoprotein particle between Tat protein and trans-acting response element in human immunodeficiency virus-infected cells.

The trans-acting response element (TAR) within the long terminal repeat of human immunodeficiency virus (HIV) is present in all 5' termini of HIV mRNAs and is recognized by the viral Tat protein. Now we describe that the 59-nucleotide-long TAR-RNA exists as a ribonucleoprotein particle in polysomal and heterogeneous nuclear RNP fractions of HIV-1-infected HeLa-T4+ cells. Applying an immunoprecipitation technique this Tat.TAR complex could be isolated from total cell extracts as well as from polysomal or heterogeneous nuclear RNP fractions. The chain length and the identity of the TAR-RNA were established by RNase protection assays while the Tat protein was confirmed by Western blotting technique. The TAR-RNA in this complex was sequenced and found to comprise nucleotides +2 to +61 and hence includes the 3-nucleotide bulge (nucleotides +23 to +25) and the loop sequence of the TAR stem-and-loop structure. The Tat.TAR complex is present in cells at low abundance (12.5 x 10(3) copies/cell). In contrast to the TAR-containing mRNAs, which decay very rapidly after incubation of cells with actinomycin D (half-life of approximately 120 min) the half-life of TAR in the Tat.TAR complex is greater than 180 min. Alignment studies revealed that TAR-RNA (positive strand) has a potential binding ability to the U5 region within the long terminal repeat (DNA negative strand; nucleotides +107 to +147); a complementary binding with a continuous homology of 16 nucleotides was identified. It is proposed that the Tat.TAR complex functions as a small ribonucleoprotein particle during transcription initiation of HIV mRNA.

Base Sequence↗