Biomedical subjects

Günter Lepperdinger

Publications and source records attributed to Günter Lepperdinger.

10 recordsLinked to original sources

PubMed2007

Mouse testicular hyaluronidase-like proteins SPAM1 and HYAL5 but not HYALP1 degrade hyaluronan.

Besides SPAM1 (sperm adhesion molecule 1; formerly named PH-20), further hyaluronidase-like proteins, HYAL5 (hyaluronoglucosaminidase 5) and HYALP1 (hyaluronoglucosaminidase pseudogene 1) are also expressed in murine testicular tissue. As they share a high degree of sequence similarity with known hyaluronidases, all three polypeptides could potentially exhibit hyaluronidase activity, a function that is beneficial for spermatozoa in order to penetrate the hyaluronan-rich cumulus, which surrounds the oocyte. Recently, it was reported that SPAM1-deficient mice are fertile and spermatozoa derived from mutant mice still exhibit hyaluronidase activity [Baba, Kashiwabara, Honda, Yamagata, Wu, Ikawa, Okabe and Baba (2002) J. Biol. Chem. 277, 30310-30314]. We have now recombinantly expressed mouse SPAM1, HYAL5 and HYALP1 in Xenopus laevis oocytes and determined their respective expression pattern in testis. Transcripts of all three genes are expressed in seminiferous tubules in regions where maturing spermatogenic cells reside. SPAM1 and HYAL5 but not HYALP1 proteins exhibit hyaluronidase activity at neutral pH. The two active hyaluronidases are both bound to the cell surface via a glycosylphosphatidylinositol anchor. Furthermore, structural characteristics are discussed that are necessary for hyaluronidases in order to exhibit hyaluronan cleavage.

Animals↗

PubMed2006

Strong binding of bioactive BMP-2 to nanocrystalline diamond by physisorption.

Nano-crystalline diamond (NCD)-coated surfaces were efficiently functionalized with bone morphogenetic protein-2 (BMP-2) by means of physisorption. Due to their randomly oriented texture, NCD-coated surfaces appear to bind complex molecules firmly. Applying various highly sensitive analytical methods, the interaction was found extremely stable. The strength of the experimentally measured adherence between BMP-2 and NCD was further corroborated by theoretical calculations. Oxygen treatment rendered NCD hydrophilic by the appearance of surface oxygen containing groups. This particular NCD surface exhibited even higher binding energies towards BMP-2 than the hydrophobic surface, and this surface was also favoured by cultured cells. Most importantly in this context, bound BMP-2 was found fully active. When cultured on BMP-2-treated NCD, osteosarcoma cells strongly up-regulated alkaline phosphatase, a specific marker for osteogenic differentiation. Hence, this simple method will allow generating highly versatile surfaces with complex biomimetic coatings, essentials for novel medical devices and implants as well as for innovative scaffolds in tissue engineering.

Absorption↗

PubMed2006

Techniques in gerontology: cell lines as standards for telomere length and telomerase activity assessment.

The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging.

Cell Line↗

PubMed2006

Aging of murine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are able to differentiate into distinct lineages such as adipo-, osteo-, and chondrocytes. MSCs were isolated from three mouse strains, which are short- (SAMP6, 9.7 months), medium- (SAMR1, 16.3 months), or long-lived (C57BL/6, 28 months). We investigated primary colony-forming units with regard to bone marrow stroma and found differences that correlate with mean life expectancies of the particular genetic backgrounds. However, MSC derived from the various mouse strains behaved equivalently in vitro with respect to growth rate. By genomic means, we analyzed the cellular milieu in vivo and found considerable differences among the various mouse strains. This implies that, although individual MSCs show an equivalent differentiation potential in vitro, the primary stem cells are greatly influenced by their molecular environment.

Animals↗

PubMed2005

Mesenchymal stem cell aging.

Stem cells are located throughout the adult body of higher organisms, supporting a continuous renewal and repair of tissues. Unique abilities of stem cells are self-renewal and multipotential differentiation. It is, therefore, of critical importance for an organism to maintain and control quantity and quality of stem cells within a given pool. Otherwise, when something goes awry within a stem cell, it is likely to have far-reaching effects. Mesenchymal stem cells (MSC) derived from various sources such as bone marrow or fat have been expanded in culture and differentiated in vitro into several lineages such as adipocytes, osteocytes or chondrocytes. In particular, aged human MSC show a decline in differentiation potential as well as in proliferation rate. The latter most likely reflects the fact that aged MSC suffer from eroded telomeres. Besides the individual age of the cell, stem and progenitor cell functions are influenced by the cellular environment, i.e. the niche and the architecture of the tissue, they reside in. This contribution reviews current knowledge about MSC aging (in vitro or in vivo), and respective difficulties for tissue engineering and stem cell therapy.

Cell Differentiation↗

PubMed2005

CD25-expressing CD8+ T cells are potent memory cells in old age.

We have recently described an IL-2/IL-4-producing CD8+CD25+ non-regulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 alphabeta molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25- memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25- memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25- memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25- memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.

Aged↗

PubMed2005

Profiling molecular targets of TGF-beta1 in prostate fibroblast-to-myofibroblast transdifferentiation.

The development of age-related proliferative disorders of the prostate gland is supported by transdifferentiation and cellular senescence processes in the stroma. Both processes are involved in remodeling of stromal tissue, as observed in benign prostatic hyperplasia (BPH), and in "reactive stroma" adjacent to prostate cancer (PCa). It has been assumed that TGF-beta1 plays a key role in the aging prostate by inducing premature senescence and favoring myofibroblast differentiation. Therefore, we evaluated the stromal cell phenotypes of human primary adult prostatic fibroblasts (n=3) and the molecular and cellular mechanisms of growth arrest after treatment with TGF-beta1 and of in vitro cellular senescence. Microarray analysis, quantitative PCR, immunofluorescence and western blot revealed that cellular senescence and transdifferentiation of fibroblasts have distinct underlying mechanisms, pathways and gene and protein expression profiles in human PrSCs. In clear contrast to senescent cells, TGF-beta1-treated cells morphologically transdifferentiated into myofibroblasts with dense cytoskeletal fibers and increased expression of smooth muscle cell alpha-actin, calponin and tenascin. TGF-beta1 induced neither expression of senescence-associated markers nor genes involved in terminal growth arrest, such as senescence-associated beta-galactosidase and cyclin-dependent kinase (cdk) inhibitors p16(Ink4A) and p21(Cip1) but increased p15(Ink4B) protein expression. Differentiation inhibitor (Id-1) protein level down-regulation was observed under both conditions. Genes specifically up-regulated by transdifferentiation but not by cellular senescence of PrSCs were metalloproteinase 1 tissue inhibitor (Timp1), transgelin (Tagln), gamma 2 actin (Actg2), plasminogen activator inhibitor 1 (Serpinel), insulin-like growth factor binding protein 3 (Igfbp3), parathyroid hormone-like hormone (Pthlp), Tgfb-1, four and a half LIM domains 2 (Fhl-2), hydrogen peroxide-inducible clone 5 (Hic5) and cartilage oligomeric matrix protein (Comp). Other genes, such as Cdc28 protein kinase 1 (Cks1b), v-myb myeloblastosis viral oncogene homolog (MybL2), pyruvate kinase, muscle 2 (Pkm2) and Forkhead box M1 (FoxM1), were down-regulated only upon TGF-beta1 treatment but not by cellular senescence. Pyruvate dehydrogenase kinase 3 (Pdk3) and connective tissue growth factor (Ctgf) were up-regulated and hyaluronan synthase 3 (Has3) down-regulated under both conditions. Moreover, GageC1, a prostate/testis-specific protein overexpressed in symptomatic BPH and PCa was induced in transdifferentiated stromal cells. Genes such as GageC1 could be promising targets for therapeutic inhibitors of stromal tissue remodeling and progression of BPH and PCa.

Cell Differentiation↗

PubMed2003

'Piggy-back' transport of Xenopus hyaluronan synthase (XHAS1) via the secretory pathway to the plasma membrane.

Hyaluronan is the sole glycosaminoglycan whose biosynthesis takes place directly at the plasma membrane. The mechanism by which hyaluronan synthase (HAS) becomes inserted there, as well as the question of how the enzyme discriminates between particular membrane species in polarized cells, are largely unknown. In vitro translation of HAS suggested that the nascent protein becomes stabilized in the presence of microsomal membranes, but would not insert spontaneously into membranes after being translated in the absence of those. We therefore monitored the membrane attachment of enzymatically active fusion proteins consisting of Xenopus HAS1 and green fluorescent protein shortly after de novo synthesis in Vero cells. Our data strongly suggest that HAS proteins are directly translated on the ER membrane without exhibiting an N-terminal signal sequence. From there the inactive protein is transferred to the plasma membrane via the secretory pathway. For unknown reasons, HAS inserted into membranes other than the plasma membrane remains inactive.

Animals↗

PubMed2002

Hyaluronan is an abundant constituent of the extracellular matrix of Xenopus embryos.

The spatiotemporal distribution of hyaluronan (HA), a major constituent of the vertebrate extracellular matrix, was analyzed during early embryonic development of Xenopus laevis. This polysaccharide is abundantly present in ventricular structures such as the blastocoel, the archenteron as well as later on in the hepatic cavity, the brain ventricles and the developing heart. At the blastula stage, HA was detected in the extracellular matrix of the ecto- and mesodermal primordia. Shortly before gastrulation, it becomes enriched at the basal site of the superficial cell layer of the ectoderm. During gastrulation, enhanced synthesis of HA takes place in the involuting marginal zone, shortly before invagination starts, hence, resulting in a torus-like deposition in the deep layer of the equatorial mesodermal primordium. After gastrulation, HA appears to accumulate within the extracellular matrix demarcating the primary germ layers. During tailbud stages, it is found highly enriched in many mesodermal derivatives, e.g., in mesenchyme, the heart, precordal cartilage and the lung primordia. Furthermore, extracellular matrix of the ventral mesodermal cell layer in the trunk region and the immediate proximity of blood vessels contain high amounts of HA.

Animals↗

PubMed2002

Degradation of hyaluronan by a Hyal2-type hyaluronidase affects pattern formation of vitelline vessels during embryogenesis of Xenopus laevis.

A Hyal2-type hyaluronidase of Xenopus laevis (Xhyal2) was characterized by molecular cloning, biochemical analysis and ectopic overexpression in embryos. When expressed in Xenopus oocytes, Xhyal2 exists as a soluble protein in the extracellular space and in intercellular compartments as well as being attached to the cell surface through a glycosyl-phosphatidyl-inositol anchor. This enzyme specifically degrades hyaluronan not only at acidic pH values but more slowly also under physiological conditions. Xhyal2 is differentially expressed during embryogenesis. Particularly striking is the high level of expression in the developing brain, the head mesenchyme and the pronephros. Elevated levels of mRNA were also found in endothelial cells which will later form vascular structures. Ectopic overexpression of Xhyal2 in frog embryos causes loss of hyaluronan in the cellular environment. This causes severe defects in the assembly of the highly structured plexus of the vitelline vessels from prevascular endothelial cells. Our data support the notion that the level of Xhyal2 expression determines the organization of the extracellular environment so that cells can merge and/or migrate within an originally impenetrable matrix.

Amino Acid Sequence↗