Biomedical subjects

P Neth

Publications and source records attributed to P Neth.

8 recordsLinked to original sources

PubMed2004

[Telomerase activity and telomere length of human mesenchymal stem cells. Changes during osteogenic differentiation].

Human mesenchymal stem cells (hMSC) exhibit properties of self-renewal and differentiation. Assuming that telomerase activity is associated with self-renewal, it might be useful to identify and define hMSC on the basis of their telomerase status. However, telomerase activity in hMSC remains a controversial issue. Therefore, the aim of our study was to investigate telomerase activity in proliferating and highly proliferating hMSC and to measure telomerase activity and changes in telomere restriction fragment (TRF) length of confluent hMSC and of osteogenically differentiated hMSC. For tissue engineering applications scaffolds should be seeded with cells that have not lost their ability to self-replicate and differentiate during in vitro cell culture. Telomerase activity could be used to characterise and isolate these cells.

Cell Differentiation↗

PubMed2003

Initial RNA expression in human monocytes after multiple injury: a screening pilot study on potentially trauma-sensitive factors by using the microarray-technique.

BACKGROUND: Pathological affection of the immune system is one of the initiating mechanisms for the induction of multiple organ failure (MOF) in patients suffering from multiple injuries. Potential responsible intracellular mechanisms such as initial monocyte mRNA expression of specific mediators remain poorly studied, so far. Hence, we applied the microarray technique for screening of a wide variety of genes in circulating monocytes of multiple injured patients and compare the molecular results to the clinical course of the patients (MOF-score). METHODS: In our prospective pilot study 6 patients were enclosed presenting with blunt multiple injuries (Injury Severity Score 16 to 57 points). Monocytes were isolated out of sequentially drawn samples (6, 12, 24 and 48 hours after trauma) using magnetic cell sorting (CD14) and a human microarray system was used (Atlas stress 1.2, Clontech, 1176 genes). Alterations in the sequential samples were identified by calculating ratios to baseline levels on admission and cluster analysis was performed (Spotfire Decision). RESULTS: Only 86 (ca.5%) genes displayed an obvious signal. The house-keeping genes clustered well together in all patients in contrast to a substantial inter-individual variability of the other signal giving genes. No mediator burst of the classical pro- or anti-inflammatory cascade were detected. CONCLUSION: We demonstrate for the first time a screening analysis of mRNA expression patterns in circulating monocytes of multiple injured patients indicating that only very few genes appeared to be influenced by the traumatic event. So far, no correlation to the severity of trauma or MOF could be detected.

Adult↗

PubMed2001

The mRNAs of prekallikrein, factors XI and XII, and kininogen, components of the contact phase cascade are differentially expressed in multiple non-hepatic human tissues.

Recently RT-PCR studies had demonstrated the expression of plasma prekallikrein (PPK) mRNA in extrahepatic tissues. The questions arose whether that is illegitimate or regular expression, and whether the mRNAs of blood coagulation factors XI and XII, and high molecular weight kininogen, components of the contact activation cascade of blood coagulation are also expressed in non-hepatic tissues. These questions were addressed in the present study by employing quantitative RT-PCR. The relative mRNA levels of the respective proteins determined in 16 human tissues indicate legitimate extrahepatic transcription of at least three of the genes. Transcription of all genes was highest in the liver, but only PPK mRNA was detected in all 16 tissues, especially high levels in pancreas, kidney, testis, spleen and prostate. We conclude from these results that PPK is synthesized in significant amounts in non-hepatic tissues and that this locally synthesized PPK may have special local functions.

Blood Coagulation Factors↗

PubMed1999

Expression of components of the kallikrein-kinin system in human cell lines.

Components of the kallikrein-kinin system (KKS) have been shown to be synthesized in many tissues and cells, however, a systematic investigation on which of the KKS components are expressed in the various human tissues and cells and how their expression is regulated is not yet available. As a first step towards such a study we developed highly sensitive and specific reverse transcription polymerase chain reaction (RT-PCR) procedures for detecting mRNA expression of tissue kallikrein, high and low molecular weight kininogens, and kinin receptors B1 and B2. Analyses of a variety of human fibroblast and epithelial cell lines showed that they differ significantly in their individual expression profiles of KKS components indicating that the KKS participates in specific and diverse ways in the regulation of cellular functions. The RT-PCR procedures described here permit differentiation of cell lines and tissues according to their expression profiles of mRNAs of KKS components and thus provide a valuable means for selecting appropriate cells for studies on the functional significance of the KKS and its single components.

Cell Line↗

PubMed1999

Expression of plasma prekallikrein mRNA in human nonhepatic tissues and cell lineages suggests special local functions of the enzyme.

At present it is generally accepted that plasma prekallikrein (PPK) is synthesized in the liver and secreted into the bloodstream. Surprisingly, it has recently been shown that PPK mRNA is present also in RNA from the kidney, adrenal gland and placenta. In spite of its novelty and possible important physiological implications this finding has been neglected. Here we report that PPK mRNA is expressed also in the human brain, heart, lung, trachea, endothelial cells and leukocytes as well as in a variety of fibroblast and epithelial cell lines. Expression of PPK mRNA in fibroblasts, endothelial cells and leukocytes suggests that PPK mRNA detected in RNA preparations from whole tissue may originate solely from these ubiquitously occurring cells. However, PPK mRNA expression in various epithelial cell lines demonstrates that tissue-specific cells also transcribe the PPK gene. The presence of PPK mRNA in nonhepatic tissues and cells indicates that they have the capacity to synthesize the protein. The physiological role of PPK synthesized in extrahepatic tissue is unknown. It may participate in local actions within tissues as well as contributing to the PPK pool in blood plasma. Cultured cells will provide a valuable model for exploring the physiological significance of extrahepatic PPK expression.

Base Sequence↗

PubMed1997

A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase.

CFTR-NBF-2 expressed and purified in fusion with the maltose-binding protein was shown to catalyse the reaction ATP-->ADP+Pi by three different assays, monitoring ATP turnover, formation of ADP and release of Pi (Km 86 microM, rate constant 0.37 min(-1)). The reaction product ADP inhibits this ATPase activity. In a similar manner the hydrolysis of GTP to GDP and Pi was demonstrated (Km 40 microM, rate constant 0.29 min(-1)). In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP. As typical for adenylate kinases a distinct AMP-binding site could be verified for CFTR-NBF-2 by the inability of TNP-ATP and AMP to compete for binding. All three enzymatic activities were inhibited by the symmetric double-substrate-mimicking inhibitor Ap5A. As NBF-2 plays a central role in CFTR channel opening and closing the results reported here are fundamental in understanding mechanisms of CFTR channel activity regulation.

Adenosine Diphosphate↗

PubMed1996

A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein.

Association reactions of a recombinant CFTR-NBF-2 polypeptide fused to glutathione S-transferase with guanine nucleotides were monitored quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled GTP after binding to NBF-2. Binding of TNP-GTP to the recombinant NBF-2 polypeptide was characterized by a Kd value of 3.9 microM. The corrected Kd values for unlabelled guanine nucleotides were determined to be 33 microM for GTP, 92 microM for GDP and 217 microM for GMP. TNP-ATP bound to NBF-2 was competitively displaced by GTP indicating a common binding site for both nucleotides. The recombinant NBF-2 did not show an intrinsic GTPase activity above a detection limit of 0.007 min(-1). Our findings provide the first experimental evidence that NBF-2 can act as a GTP-binding subunit that would favor the release of GDP after GTP hydrolysis.

Adenosine Triphosphate↗

PubMedDate not supplied

[3D-Culturing of human osteoblastic cells with vessel like nutrient supply].

AIM: The treatment of large, critical-size bone defects is a major therapeutic problem in orthopaedic and reconstructive surgery. The engineering of bone tissue could be used to replace lost bone mass. However, scaffolds seeded with vital cells and cultured in vitro suffer from poor oxygen and nutrient supply centrally, when the constructs exceed a critical volume. Therefore, we have established an osteoblastic cell culture in a new 3D-culture chamber with an artificial, vessel-like central membrane, allowing continuous nutrient supply. METHOD: Human osteoblasts were cultured in a 3D-like manner using a perfusion chamber for one week. In this system, the nutrient supply is guaranteed by a vessel-like, semipermeable polysulfone membrane with a continuous flow of medium. After fixation and cryosectioning, histological and immunohistological staining and scanning electron microscopy was carried out. RESULTS: Examinations reveal 3D cell growth around the central vessel. Formation of an extracellular matrix, rich in collagen type I and fibronectin, was detected immunohistochemically. Furthermore, we demonstrated cell adherence to the membrane and examined the surface morphology by scanning electron microscopy. CONCLUSION: The innovative approach for 3D-culturing of human osteoblasts in a system with a central nutrient supply opens up new possibilities for the in vitro cultivation for tissue engineering.

Cell Culture Techniques↗