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C Kruse

Publications and source records attributed to C Kruse.

At least 19 recordsLinked to original sources
PubMed2006

Derivation of oocyte-like cells from a clonal pancreatic stem cell line.

Adult pancreatic stem cells (PSCs) are able to differentiate spontaneously in vitro into various somatic cell types. Stem cells isolated from rat pancreas show extensive self-renewal ability and grow in highly viable long-term cultures. Additionally, these cells express typical stem cell markers such as Oct-4, nestin and SSEA-1. Although differentiation potential is slightly decreasing in long-term cultures, it is possible to keep cell lines up to passage 140. Clonal cell lines could be established from different passages and showed similar characteristics. Remarkably, one clonal cell line, generated from passage 75, showed deviant properties during further culture. Clonal cells formed aggregates, which built tissue-like structures in suspension culture. These generated 3D aggregates produced permanently new cells at the outside margin. Released cells had remarkable size, and closer examination by light microscopy analysis revealed oocyte-like morphology. A comparison of the gene expression patterns between primary cultures of passages 8 and 75, the clonal cell line and the produced oocyte-like cells (OLCs) from tissue-like structures demonstrated some differences. Expression of various germ cell markers, such as Vasa, growth differentiation marker 9 and SSEA-1, increased in the clonal cell line, and OLCs showed additionally expression of meiosis-specific markers SCP3 and DMC1. We here present a first pilot study investigating the putative germ line potential of adult PSCs.

Animals↗
PubMed2006

Autonomously contracting human cardiomyocytes generated from adult pancreatic stem cells and enhanced in co-cultures with myocardial biopsies.

Myocardial regeneration with artificially applied cardiomyocytes is emerging as a promising issue of significant scientific and clinical impact. Nevertheless the source of cells for human cardiomyocyte differentiation especially from adult tissue is still unclear. We hypothesized that human pancreatic stem cells may differentiate into cardiomyocyte-like cells and may increase in number when co-cultured with myocardial tissue. Adult stem cells were harvested from pancreatic tissue of patients undergoing operative procedures including the pancreas. The cells were selected, cultured and passaged. To promote self-differentiation into cardiomyocytes, human pancreatic stem cells were co-cultered with biopsies of human myocardium. After co-culture and breeding, cells were phenotyped as well with respect to RNA, protein and cardiomyocyte specificity at the electron-microscopic level.Pancreatic stem cells have already differentiated spontaneously into cardiomyocyte-like cells performing netlike cell clusters with rare but distinct multilocular cellular autonomous contractions with a frequency of about 20 beats per minute. The number of contracting areas however could be enhanced by co-culture with human myocardial biopsies. On RNA and protein levels as well as in electron-microscopy, evidence for cardiomyocyte specificity is shown. To the best of our knowledge this is the first report demonstrating the feasibility of generating autonomously contracting cardiomyocyte-like cells from adult human pancreatic stem cells and their enhancement by myocardial co-culture. This procedure might prove to be an alternative source and method for myocardial regenerative medicine.

Adult↗
PubMed2005

[Intestinal malperfusion in critical care patients].

Due to the bowel's poor tolerance of hypoxia, intestinal malperfusion presents as a grave disease with high mortality. The intensivist is confronted with this condition in association with other underlying diseases, in the course of surgery, during application of medication or associated with invasive therapy. In a critical care setting, the non-occlusive mesenteric ischemia (NOMI) is of increasing importance. Since critical care patients often lack clinical symptoms, special attention is required and one main factor of the patient's prognosis is early diagnosis. This review summarizes pathophysiology and diagnostic aspects and the range of therapeutic and preventive measures.

Blood Transfusion↗
PubMed2004

A study of HLA-DR and -DQ alleles in 588 patients and 562 controls confirms that HLA-DRB1*03 is associated with recurrent miscarriage.

BACKGROUND: Previous studies have demonstrated an association between recurrent miscarriage (RM) and the maternal HLA-DRB1*01 and -DRB1*03 alleles. The primary aim of the present study was to confirm or reject the hypothesis about this association in a larger case-control study. METHODS: HLA-DRB1, -DQA1 and -DQB1 genotyping was carried out by the PCR-sequence-specific primer (SSP) method in 354 patients with unexplained RM and 202 fertile controls. These results were combined with the results from a previous study of 234 RM patients and 360 controls. RESULTS: The prevalence of patients with HLA-DRB1*03 was significantly increased compared with controls [odds ratio (OR) = 1.4, 95% confidence interval (CI) = 1.1-1.9, P = 0.01, P corrected for the number of comparisons (Pc) = 0.02]. In patients with at least four previous miscarriages or with secondary RM, the association became even stronger (OR = 1.8, 95% CI = 1.3-2.5, P = 0.0005, Pc = 0.004; and OR = 1.8, 95% CI = 1.3-2.5, P = 0.0007, Pc = 0.006, respectively). There was no significant difference between patients and controls with regard to HLA-DRB1*01. CONCLUSION: The HLA-DRB1*03 allele or genes in linkage disequilibrium with it confer susceptibility to RM.

Abortion, Habitual↗
PubMed2003

Evidence for posttranscriptional regulation of the multi K homology domain protein vigilin by a small peptide encoded in the 5' leader sequence.

Vigilin, a K homology (KH) protein has been found in all eukaryotic species studied. It has a unique structure of 14-15 consecutively arranged KH domains which apparently mediate RNA-protein binding. Cloning and sequencing of the mouse vigilin cDNA confirmed that the amino acid sequences of vertebrate vigilins are highly conserved and contain conserved sequence motifs of nuclear import and export sequences. The human and murine vigilin mRNAs carry two alternatively spliced 5' exons. In the 5' leader region of one of the splice variants, variant 1A, we found an upstream open reading frame (uORF) highly conserved between mouse and human. Here we present for the first time evidence that a 13 amino acid long peptide encoded by this uORF is an inhibitor of vigilin expression operating on a posttranscriptional level. We propose that the two structurally different 5' leader sequences of the human vigilin mRNA are involved in the regulation of vigilin biosynthesis.

5' Untranslated Regions↗
PubMed2003

The multi-KH protein vigilin associates with free and membrane-bound ribosomes.

The-multi-KH domain protein vigilin has been identified by ex vivo experiments as both a tRNA- and/or mRNA-binding protein. We show here that in vitro under conditions previously shown to allow tRNA binding, recombinant vigilin also binds to selected mRNA species and ribosomal RNA. An in vivo link of vigilin to mRNA and rRNA was elucidated by several approaches. (i) Coexpression/costimulation of vigilin was found with many other proteins independently of whether their mRNA was translated on free or membrane-bound ribosomes. (ii) A close codistribution of vigilin with free ribosomes was seen in the cytoplasm while nucleoli were a major organelle of vigilin accumulation in the nucleus. (iii) Furthermore, free and membrane-bound ribosomes can be enriched for vigilin which suggests that this binding does not depend on the class of mRNA translated. Therefore, we suggest that vigilin does not distinguish between free or membrane-bound ribosomes but is generally necessary for the localization of mRNAs to actively translating ribosomes.

Carrier Proteins↗
PubMed2003

Prospective, serial investigations of in-vitro lymphocyte cytokine production, CD62L expression and proliferative response to microbial antigens in women with recurrent miscarriage.

BACKGROUND: Lymphocytes from pregnant women with unexplained recurrent miscarriage (RM) may be characterized by a T-helper type 1-dominated cytokine production and a higher proliferative response to microbial recall antigens compared with normal pregnant women. METHODS: Serial blood samples were taken from 14 women with RM (at least three previous consecutive miscarriages) during the first 14 weeks of pregnancy, and one blood sample was taken from 15 control women in gestational weeks 7-8. Of the 14 pregnant RM patients, four produced a live birth and 10 miscarried. Lymphocytes were in-vitro-stimulated by mitogens, allogeneic cells and microbial antigens, and the production of a series of cytokines, the proliferative responses and lymphocytic expression of CD62L (which may be a marker of T-helper type 2 lymphocytes) were measured. RESULTS: Repeated measurements of cytokine production were reproducible during the first trimester. The proliferative responses to herpes simplex and tetanus antigens were increased, and the ratio of CD62L-/CD62L+ expressing CD4+CD45RO+ lymphocytes was decreased in patients compared with controls (P = 0.01, P < 0.01 and P < 0.01 respectively). CONCLUSION: The results of the in-vitro assays used were reproducible in serial testing during pregnancy. The importance of CD62L expression on lymphocytes for RM and the relevance of the maternal response to microbial antigens during pregnancy should be further explored.

Abortion, Habitual↗
PubMed2002

HLA-G polymorphisms in couples with recurrent spontaneous abortions.

The etiology of a fraction of recurrent spontaneous abortions (RSA) may involve immunological mechanisms. Aberrant profiles of Th1 and Th2 cytokines have been observed which are not present in uncomplicated pregnancies. Studies of classical HLA class I and II antigens in relation to RSA have not been conclusive. Furthermore, these antigens are not expressed in the placenta with the exception of HLA-C. However, HLA-G is expressed on especially invasive cytotrophoblasts and exists in both membrane and soluble forms. HLA-G may be involved in materno-fetal tolerance. Therefore, 61 RSA couples (with three or more spontaneous abortions) and 47 fertile control couples were HLA-G genotyped by direct DNA sequencing and analyzed for specific polymorphisms. No statistically significant differences were observed in the distribution of HLA-G alleles between controls and RSA couples, however, 15% of the RSA women carried the HLA-G*0106 allele compared to 2% of the control women. The 14 bp deletion polymorphism in exon 8 was investigated separately. There were a greater number of heterozygotes for the 14 bp polymorphism in the group of fertile control women than expected, according to Hardy-Weinberg equilibrium. Furthermore, the HLA-G alleles without the 14 bp sequence were prominent in the RSA males in contrast to the RSA women in whom alleles including the 14 bp sequence were frequently observed, especially as homozygotes. These results are discussed in relation to two hypotheses concerning HLA-G and RSA. A hypothesis of HLA-G histo-incompatibility between fetus/placenta and the mother was not supported by the data. Another hypothesis concerned certain HLA-G alleles associated with an altered expression profile of HLA-G isoforms or reduced expression of certain HLA-G isoforms.

Abortion, Habitual↗
PubMed2001

Tumor suppressor p53 protein is a new target for the metastasis-associated Mts1/S100A4 protein: functional consequences of their interaction.

A physical and functional interaction between the Ca(2+)-binding protein Mts1 (S100A4) and the tumor suppressor p53 protein is shown here for the first time. We demonstrate that Mts1 binds to the extreme end of the C-terminal regulatory domain of p53 by several in vitro and in vivo approaches: co-immunoprecipitation, affinity chromatography, and far Western blot analysis. The Mts1 protein in vitro inhibits phosphorylation of the full-length p53 and its C-terminal peptide by protein kinase C but not by casein kinase II. The Mts1 binding to p53 interferes with the DNA binding activity of p53 in vitro and reporter gene transactivation in vivo, and this has a regulatory function. A differential modulation of the p53 target gene (p21/WAF, bax, thrombospondin-1, and mdm-2) transcription was observed upon Mts1 induction in tet-inducible cell lines expressing wild type p53. Mts1 cooperates with wild type p53 in apoptosis induction. Our data imply that the ability of Mts1 to enhance p53-dependent apoptosis might accelerate the loss of wild type p53 function in tumors. In this way, Mts1 can contribute to the development of a more aggressive phenotype during tumor progression.

Animals↗
PubMed2001

Estradiol has a direct impact on the exocrine pancreas as demonstrated by enzyme and vigilin expression.

BACKGROUND: Estrogen receptors have been found in the exocrine pancreas; however, the exact role of estrogen in pancreatic enzyme synthesis and secretion remains to be elucidated. Vigilin, a multi-KH domain protein, is part of a tRNA-containing ribonucleoprotein complex and may be a suitable marker for stimulation of the translational machinery. In the present study, we investigated the influence of estradiol and compared it to CCK on the expression of vigilin, trypsin and amylase in rat pancreatic acini. METHODS: Acini were isolated and incubated with CCK or estradiol. The change in amylase and trypsin levels in the medium and in cell extracts were determined using a photometric method. The change in vigilin mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. RESULTS: Treatment of isolated exocrine pancreatic cells with estradiol caused stimulation of amylase and trypsin production and inhibition of secretion, while treatment with CCK showed only a minor effect on enzyme production and resulted mainly in a stimulation of secretion. Further we found an increase in vigilin mRNA and protein expression in acini stimulated with both CCK-8 and estradiol. CONCLUSION: Our data suggest that estradiol may play a role in inducing exocrine enzyme production but not secretion, and that vigilin, as a marker for translational activity, is stimulated in parallel to the pancreatic enzymes: amylase and trypsin.

Amylases↗
PubMed2000

She2p, a novel RNA-binding protein tethers ASH1 mRNA to the Myo4p myosin motor via She3p.

RNA localization is a widespread mechanism to achieve localized protein synthesis. In budding yeast, localization of ASH1 mRNA controls daughter cell-specific accumulation of the transcriptional regulator Ash1p, which determines mating type switching. ASH1 mRNA localization depends on four independently acting sequences ('zipcodes') within the mRNA. In addition, the class V myosin Myo4p and a set of She proteins with as yet unknown function are essential for ASH1 localization. Here we show that She2p is a novel RNA-binding protein that binds specifically to ASH1 mRNA in vivo and to ASH1 RNA zip codes in vitro. She2p can interact with She3 protein via She3p's C-terminus and becomes localized to the daughter cell tip upon ASH1 expression. The N-terminal coiled-coil domain of She3p is required to form an RNA-independent complex with the heavy chain of the myosin motor protein Myo4p. She2p and She3p are the first examples of adapters for tethering a localized mRNA to the motor protein and might serve as prototypes for RNA-motor protein adapters.

DNA-Binding Proteins↗
PubMed2000

Export and transport of tRNA are coupled to a multi-protein complex.

Vigilin is a ubiquitous multi heterogeneous nuclear ribonucleoprotein (hnRNP) K homologous (KH)-domain protein. Here we demonstrate that purified recombinant human vigilin binds tRNA molecules with high affinity, although with limited specificity. Nuclear microinjection experiments revealed for the first time that the immuno-affinity-purified nuclear vigilin core complex (VCC(N)) as well as recombinant vigilin accelerate tRNA export from the nucleus in human cells. The nuclear tRNA receptor exportin-t is part of the VCC(N). Elongation factor (EF)-1alpha is enriched in VCC(N) and its cytoplasmic counterpart VCC(C), whereas EF-1beta, EF-1gamma and EF-1delta are basically confined to the VCC(C). Our results suggest further that vigilin and exportin-t might interact during tRNA export, provide evidence that the channeled tRNA cycle is already initiated in the nucleus, and illustrate that intracellular tRNA trafficking is associated with discrete changes in the composition of cellular cytoplasmic multi-protein complexes containing tRNA.

Animals↗
PubMed1999

A soluble form of the avian hepatitis B virus receptor. Biochemical characterization and functional analysis of the receptor ligand complex.

Avian hepatitis B virus infection is initiated by the specific interaction of the extracellular preS part of the large viral envelope protein with carboxypeptidase D (gp180), the primary cellular receptor. To functionally and biochemically characterize this interaction, we purified a soluble form of duck carboxypeptidase D from a baculovirus expression system, confirmed its receptor function, and investigated the contribution of different preS sequence elements to receptor binding by surface plasmon resonance analysis. We found that preS binds duck carboxypeptidase D with a 1:1 stoichiometry, thereby inducing conformational changes but not oligomerization. The association constant of the complex was determined to be 2.2 x 10(7) M-1 at 37 degreesC, pH 7.4, with an association rate of 4.0 x 10(4) M-1 s-1 and a dissociation rate of 1.9 x 10(-3) s-1, substantiating high affinity interaction of avihepadnaviruses with their receptor carboxypeptidase D. The separately expressed receptor-binding domain, comprising about 50% of preS as defined by mutational analysis, exhibits similar constants. The domain consists of an essential element, probably responsible for the initial receptor contact and a part that contributes to complex stabilization in a conformation sensitive manner. Together with previous results from cell biological studies these data provide new insights into the initial step of hepadnaviral infection.

Amino Acid Sequence↗
PubMed1998

Production of trypsin by cells of the exocrine pancreas is paralleled by the expression of the KH protein vigilin.

Vigilin, a protein with a continuous series of 14 KH motifs, forms part of a multiprotein complex containing tRNA. Several lines of evidence have suggested that vigilin expression is enhanced in those cells which were actively engaged in protein synthesis. Accordingly, we show here by immunoelectronmicroscopy a close association of vigilin with the rough endoplasmic reticulum in rat pancreatic cells. Histological examination of these cells furthermore demonstrates the highest intensity of vigilin staining in the perinuclear, intranuclear, and basolateral regions where the endoplasmic reticulum is mainly amassed. In vivo challenge of starving rats fed prior to sacrifice raised in parallel the protein levels of both trypsin and vigilin when compared to unchallenged animals and was associated with enhanced expression of the vigilin gene. In contrast, in human and rat cell lines of pancreatic tumors with a constitutively high expression of vigilin no further stimulation by cholecystokinin treatment could be achieved. Our data provide circumstantial evidence that vigilin may play a crucial role in the ability of an organ, e.g., pancreas, to cope with the physiological demand to upregulate protein synthesis.

Animals↗
PubMed1998

tRNA is entrapped in similar, but distinct, nuclear and cytoplasmic ribonucleoprotein complexes, both of which contain vigilin and elongation factor 1 alpha.

Vigilin, which is found predominantly in cells and tissues with high levels of protein biosynthesis, was isolated in its native form from human HEp-2 cells (A.T.C.C. CCL23) by immunoaffinity chromatography. Here we demonstrate that vigilin is part of a novel large tRNA-binding ribonucleoprotein complex (tRNP), found not only in the cytoplasm, but also in the nuclei of human cells. Compositional differences in the protein pattern were detected between the nuclear and cytoplasmic tRNPs, although some properties of the purified nuclear tRNP, such as tRNA protection against nuclease attack, were identical with those of the cytoplasmic tRNP. By using either a pool of total human nuclear RNA or radioactively labelled yeast tRNAAsp in rebinding experiments, we could show that tRNA is specifically recaptured by the RNA-depleted, vigilin-containing nuclear complex. We could also show that vigilin is capable of binding tRNA in vitro. Another tRNA-binding protein is elongation factor 1 alpha, which appears to be enriched in the cytoplasmic and nuclear tRNP complexes. This suggests that the cytoplasmic tRNP may be involved in the channelled tRNA cycle in the cytoplasm of eukaryotic cells. Our results also suggest that the nuclear vigilin-containing tRNP may be related to the nuclear export of tRNA.

Biotinylation↗
PubMed1997

Chicken vigilin gene: a distinctive pattern of hypersensitive sites is characteristic for its transcriptional activity.

Vigilin, a multidomain hn-ribonucleo-K-homologous protein, is part of a ribonucleoprotein complex with cognate tRNA and is found in both the nucleus and the cytoplasm. In an approach to identify genomic regions involved in regulation of the chicken vigilin gene, we carried out transfection studies with a reporter gene in suitable chicken cells. After including a distantly positioned 5'-sequence in the construct, we observed a 10.5-fold increase in luciferase (EC 1. 13.12.7) expression compared with basal promoter activity. Accordingly, chromatin analysis of freshly isolated embryonic tendon fibroblasts with high levels of vigilin mRNA expression shows a DNase-I-hypersensitive site (DHS1) localized 2.2 kb upstream of the transcriptional start site. Similarly, phytohaemagglutinin-stimulated lymphocytes with a 4-fold elevated expression of vigilin mRNA compared with resting lymphocytes also exhibited this unique DHS, having switched from that found at 3.3 kb (DHS2) in resting lymphocytes. Furthermore, using gel-retardation experiments with DNA representing either DHS1 or DHS2, a specific interaction with chicken nuclear extracts was seen.

Animals↗
PubMed1996

Evidence for a novel cytoplasmic tRNA-protein complex containing the KH-multidomain protein vigilin.

Vigilin, a protein found predominantly in cells and tissues with a high biosynthetic capacity, was isolated in its native form from human HEp-2 cells (A.T.C.C. CCL23) by immunoaffinity chromatography. Vigilin forms part of a novel ribonucleoprotein complex that also contains additional, as yet uncharacterized, proteins. Experimental evidence suggests that the nucleic acids entrapped in this complex are protected from RNase and belong to the tRNA family. Using either a pool of total human RNA or radioactively labelled tRNA (tRNA (Asp**)) in rebinding experiments, we could show that tRNA is selectively recaptured by the RNA-depleted vigilin-containing complex.

Carrier Proteins↗