Kinship of conditionally immortalized cells derived from fetal bone to human bone-derived mesenchymal stroma cells.

The human fetal osteoblast cell line (hFOB 1.19) has been proposed as an accessible experimental model for study of osteoblast biology relating to drug development and biomaterial engineering. For their multilineage differentiation potential, hFOB has been compared to human mesenchymal progenitor cells and used to investigate bone-metabolism in vitro. Hereby, we studied whether and to what extent the conditionally immortalized cell line hFOB 1.19 can serve as a surrogate model for bone-marrow derived mesenchymal stromal cells (bmMSC). hFOB indeed exhibit specific characteristics reminiscent of bmMSC, as colony formation, migration capacity and the propensity to grow as multicellular aggregates. After prolonged culture, in contrast to the expected effect of immortalization, hFOB acquired a delayed growth rate. In close resemblance to bmMSC at increasing passages, also hFOB showed morphological abnormalities, enlargement and finally reduced proliferation rates together with enhanced expression of the cell cycle inhibitors p21 and p16. hFOB not only have the ability to undergo multilineage differentiation but portray several important aspects of human bone marrow mesenchymal stromal cells. Superior to primary MSC and osteoblasts, hFOB enabled the generation of continuous cell lines. These provide an advanced basis for investigating age-related dysfunctions of MSCs in an in vitro 3D-stem cell microenvironment.

Multifrequency impedance measurement technique for wireless characterization of microbiological cell cultures.

An impedance measurement system with probe signal frequencies up to 50 kHz with AC-probe voltages below 30 mV rms was integrated for wireless and battery-free monitoring of microbiological cell cultures. The here presented modular design and the use of state-of-the-art components greatly eases adoptions to a wide range of biotechnological applications without the need of bulky LCR-meters or potentiostats. The device had a power consumption of less than 2.5 mA at a 3.3 V single power supply and worked trouble-free within the humid environment of a cell culture incubator. Measurements on lumped RC-elements showed an error of less than 1% for absolute values and less than 1° regarding the phase of the complex impedance. The performance of sensor devices with interdigitated electrode structures for the measurement of adherent cell cultures was tested in the presence of phosphate-buffered saline solution in the humid atmosphere of an incubator for biological cell cultures.

Increased expression of extracellular proteins as a hallmark of human endothelial cell in vitro senescence.

A convenient way to study processes of aging in distinct human tissues consists of a molecular analysis of cells from the tissue in question, that were explanted and grown in vitro until they reach senescence. Using human umbilical vein endothelial cells (HUVEC), we have established an in vitro senescence model for human endothelial cells. A major hallmark of HUVEC in vitro senescence is the increased frequency of apoptotic cell death, which occurs as a determining feature of HUVEC senescence. Senescent endothelial cells are also found in vivo in atherosclerotic lesions, suggesting that the presence of such cells may contribute to the development of vascular pathology. To elucidate mechanisms underlying endothelial cell senescence and age-associated apoptosis, gene expression analyses were carried out. In these experiments, we observed the up-regulation of genes coding for extracellular proteins in senescent HUVEC. In particular, a significant upregulation of interleukin-8, VEGI, and the IGF-binding proteins 3 and 5 was observed. Upregulation of these genes was confirmed by both RT-PCR and Western blot. In the case of interleukin-8, a roughly 50-fold upregulation of the protein was also found in cellular supernatants. The extracellular proteins encoded by these genes are well known for their ability to modulate the apoptotic response of human cells, and in the case of interleukin-8, clear links to the establishment of atherosclerotic lesions have been defined. The results described here support a new model, where changes in the secretome of human endothelial cells contribute to vascular aging and vascular pathology.

Hyal2--less active, but more versatile?

Hyal2 is one of several hyaluronidases present in vertebrates. The human gene encoding this enzyme is present on chromosome 3p.21.3, close to two additional hyaluronidase genes. cDNAs encoding Hyal2 homologues have been characterized from mouse and Xenopus laevis. These enzymes hydrolyze high molecular mass hyaluronan to intermediates of approximately 20 kDa, a finding which implies that structural domains of this size exist in this polysaccharide which was mostly thought to be a random coil. Hyal2 enzymes have an acidic pH-optimum with an activity that is considerably lower than observed for other types of hyaluronidases. Originally considered to be a typical lysosomal enzyme, more recent evidence has shown that Hyal2 proteins can also be exposed on the cell surface bound to the plasma membrane via a GPI anchor. Hyal2 is present in many tissues, one exception being the adult brain. In this tissue, the gene is silenced after birth by methylation. Current evidence about the role of Hyal2 in tumor growth, inflammation and frog embryogenesis is discussed.

Xenopus kidney hyaluronidase-1 (XKH1), a novel type of membrane-bound hyaluronidase solely degrades hyaluronan at neutral pH.

In search for Xenopus laevis hyaluronidase genes, a cDNA encoding a putative PH-20-like enzyme was isolated. In the adult frog, this mRNA was only found to be expressed in the kidney and therefore named XKH1. When expressed by means of cRNA injection into frog oocytes, XKH1 solely exhibited at physiologic ionic strength hyaluronidase activity at neutral pH and in weakly acidic solutions. The enzyme was inactive below pH 5.4. In addition to hyaluronic acid hydrolysis, chondroitin sulfate also was degraded at low yield as assessed by fluorophore-assisted carbohydrate electrophoresis analysis of the degradation products. The enzyme is sorted to the outer surface of the cell membrane of XKH1 expressing oocytes. From there, it could not be removed by phospholipase C nor was secreted hyaluronidase activity detectable. We conclude that XKH1 represents a membrane-bound hyaluronan-degrading enzyme exclusively expressed in cells of the adult frog kidney where it either may be involved in the reorganization of the extracellular architecture or in supporting physiological demands for proper renal functions.

The mammalian homologue of the novel peptide Bv8 is expressed in the central nervous system and supports neuronal survival by activating the MAP kinase/PI-3-kinase pathways.

Previous studies have identified the mammalian homologue of Bv8 (mBv8), a small protein originally isolated from skin secretions of the frog, Bombina variegata. In situ hybridization showed that mBv8 RNA was widely expressed in the rodent CNS, with high levels being detected in layer II of the cerebral cortex, limbic regions, cerebellar Purkinje cells, and dorsal and ventral horns of the spinal cord. A similar pattern of distribution was found by examining the presence of mBv8 protein by immunocytochemistry. Addition of frog Bv8 to cultured cerebellar granule cells reduced the extent of apoptotic death induced by switching the growing medium from 25 to 5 mM K+. Bv8 could also protect cultured cortical neurons against excitotoxic death. Both effects were prevented by PD98059 and LY294002, which inhibit the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI-3-K) pathways, respectively. In cultured cerebellar granule cells, Bv8 stimulated both the MAPK and the PI-3-K pathways, as revealed by Western blot analysis of phosphorylated p44/p42 MAPKs and phosphorylated Akt, respectively. We conclude that mBv8 acts as an endogenous neurotrophic factor and supports neuronal survival through the activation of the MAPK/PI-3-K pathways.

Xenopus brevican is expressed in the notochord and the brain during early embryogenesis.

A complete cDNA encoding the Xenopus laevis homologue of the aggrecan/versican family member, brevican (Xbcan) was cloned from an embryonic stage 42 cDNA library. In the deduced amino acid sequence, 1152 in length, similarity to the hyaluronan-binding (link) domains of brevicans from other species were present in the N-terminal region as well as EGF-, lectin- and complement regulatory protein-like domains in the C-terminal part, the latter three being characteristic for brevican found within the extracellular matrix (J. Biol. Chem. 269 (1994) 10119). Indeed, Xbcan was secreted into the extracellular space as a soluble protein when expressed in oocytes. No cDNAs encoding a GPI-anchored bcan variant could be isolated from that cDNA library. During embryonic development, the expression of this gene was first observed in the notochord of neurula stage embryos. In addition to this, in tailbuds, Xbcan was also found to be expressed within the fifth and sixth rhombomere of the hindbrain. In tadpole stage embryos, expression was furthermore observed in periventricular regions of the developing brain and the rostral part of the spinal cord.

Brix from xenopus laevis and brx1p from yeast define a new family of proteins involved in the biogenesis of large ribosomal subunits.

A clone was isolated from a cDNA library from early embryos of Xenopus laevis that codes for a highly charged protein containing 339 amino acids. Two putative nuclear localization signals could be identified in its sequence, but no other known motifs or domains. Closely related ORFs are present in the genomes of man, C. elegans, yeast and Arabidopsis. A fusion protein with GFP expressed in HeLa cells or Xenopus oocytes was found to be localized in the nucleolus and coiled (Cajal) bodies. Moreover, immunoprecipitation experiments demonstrated that the new Xenopus protein interacts with 5S, 5.8S and 28S RNAs of large ribosomal subunits. The name Brix (biogenesis of ribosomes in Xenopus) is proposed for this protein and the corresponding gene. In Saccharomyces cerevisiae, the essential gene YOL077c, now named BRX1, codes for the Brix homolog, which is also localized in the nucleolus. Depletion of Brx1 p in a conditional yeast mutant leads to defects in rRNA processing, and a block in the assembly of large ribosomal subunits.

Amphibian choroid plexus lipocalin, Cpl1.

Choroid plexus lipocalin 1 (Cpl1) has been isolated from the African clawed toad (Xenopus laevis) and the cane toad (Bufo marinus). Xcpl1 has been used as a marker for studying early neural development. Due to its retinoid binding properties and the fact that it causes dysmorphogenesis when overexpressed in the early embryo, the protein product is considered to be part of the retinoic acid signalling pathway. Later in development and during adulthood, the epithelial cell sheet of the choroid plexus which forms the blood-cerebrospinal fluid barrier expresses cpl1 as the predominant secretory protein. These data, the similarity of Cpl1 to prostaglandin D(2) synthase and its functional homology to transthyretin will be discussed.

Murine Bv8 gene maps near a synteny breakpoint of mouse chromosome 6 and human 3p21.

The genomic structure of the murine Bv8 gene was determined in 129/SvJ mouse, and the chromosomal localization was identified. Bv8 has first been characterized from skin secretion of the yellow-bellied toad, Bombina variegata. When injected into rat brain, this polypetide causes hyperalgesia. The murine Bv8 gene was shown to consist of four exons and was localized on chromosome 6 between the microsatellite markers D6Mit66 and D6Mit36 near the gene mem1, whereas the human counterpart was assigned to the non-syntenic region 3p21.1. Furthermore, the primary Bv8 transcript appeared to be alternatively spliced. The first variant contained all four exons yielding a product with a stretch highly enriched in basic amino acids in its central part. This domain is absent in the peptides from frog as well as in a splice variant expressed in mouse testis. A third variant gives rise to a truncated polypeptide.

Irradiation-induced expression of hyaluronan (HA) synthase 2 and hyaluronidase 2 genes in rat lung tissue accompanies active turnover of HA and induction of types I and III collagen gene expression.

Hyaluronan (HA) is a linear glycosaminoglycan that accumulates in the interstitium of injured lung and inhibits gas exchange between air and blood. In the present study we investigated the molecular mechanisms behind the local turnover of HA during the early phase of irradiation-evoked lung fibrosis in rats. Irradiation with a single dose of 30 Gy to the lower part of the right lung of rats induced an accumulation of HA in bronchoalveolar lavage fluid 6 wk after irradiation, followed by return to almost normal levels at 10 wk after irradiation. This was parallelled with a transient downregulation of HA receptors on alveolar macrophages (AMs); 4 and 6 wk after irradiation the binding of [(3)H]HA to AMs was decreased to about 50% of that of AMs from nonirradiated control rats, returning to almost normal level at 10 wk after irradiation. Analysis of the expression of rat HA synthase (HAS) isoforms (rHAS1, rHAS2, and rHAS3) and rat hyaluronidases (rHYAL1 and rHYAL2) by Northern blotting revealed an upregulation of rHAS2 messenger RNA at 4, 6, and 10 wk after irradiation, but a progressive decrease in the constitutive expression of rHYAL2 at 6 and 10 wk after irradiation; rHAS1 was undetectable, whereas rHAS3 and rHYAL1 were faintly detectable. Although transforming growth factor-beta1 stimulated HA production by normal lung fibroblasts, it inhibited HYAL activity in lysosomes and HYAL activity released into the culture media. Another interesting observation was that HA fragments, which likely result from the action of HYAL, induced expression of types I and III collagen genes. Our results indicate that rHAS2 and rHYAL2 are involved in the turnover of HA during the early phase of lung injury and that rHAS2 and rHYAL2 as well as HA fragments may play important roles in the pathogenesis of lung fibrosis.

Synthesis of hyaluronan of distinctly different chain length is regulated by differential expression of Xhas1 and 2 during early development of Xenopus laevis.

The localization of hyaluronan has been determined in tailbud stage embryos of Xenopus laevis using a neurocan-alkaline phosphatase fusion protein. This polysaccharide was located between the germ layers and enriched in mesenchyme, the lumen of the neural tube, the embryonic gut, the hepatic cavity and the heart. A full-length cDNA for a hyaluronan synthase, Xhas2 has been cloned. The expression pattern of Xhas1 and 2 is closely similar to the distribution of hyaluronan in the embryo. Xhas1 produces hyaluronan with a molecular mass of around 40-200 kDa, while the product formed by Xhas2 has a molecular mass above 1 million Da.

The mammalian homologues of frog Bv8 are mainly expressed in spermatocytes.

Bv8, a protein from skin secretions of Bombina variegata, reacts with receptors present in mammalian brain and intestine (Mollay et al. (1999) Eur. J. Pharmacol. 374, 189-196). As deduced from cloned cDNAs, the murine and human Bv8 homologues have identical amino-terminal sequences and also contain 10 cysteines. From mouse testes, two forms of Bv8 mRNA have been characterized, of which one contains an additional exon which codes for 21 mostly basic amino acids. The mouse Bv8 gene is most active in mid-late pachytene spermatocytes. In mouse testes, Bv8 mRNA can first be detected at the end of the second week post partum.

Hyaluronidase-2 overexpression accelerates intracerebral but not subcutaneous tumor formation of murine astrocytoma cells.

Gliomas are highly invasive, invariably fatal intracerebral tumors. It seems that receptors for hyaluronan are required for the invasive process. Hyaluronan is a major component of the extracellular matrix in the brain, and all of the gliomas express CD44, the principal receptor for hyaluronan. To investigate the role of lysosomal hyaluronidases on tumor invasion we overexpressed hyaluronidase-2 (HYAL2) in murine astrocytoma cells. We found that high expression of HYAL2 accelerated intracerebral tumor growth dramatically, whereas the same cells formed s.c. tumors within the same time as the parental cells. The brain tumors were highly vascularized and more invasive than the control tumors. It seems that the interactions of the HYAL2-expressing tumor cells with the hyaluronan-containing extracellular matrix in the brain mediate these effects, whereas the same cells in a s.c. environment, which lacks the high hyaluronan level, behave like the parental cells.

Structural organization and chromosomal localization of Hyal2, a gene encoding a lysosomal hyaluronidase.

The human HYAL2 gene encodes a lysosomal hyaluronidase that is related to the testicular PH-20 hyaluronidase. Regions conserved in these proteins have been used to design PCR primers suitable for the isolation of a fragment of the murine Hyal2 gene. This fragment was used to isolate the Hyal2 cDNA from a cDNA library. The cloned cDNA has an open reading frame of 473 codons and a 3'-untranslated region of 302 bases plus a poly(A) tail. Using this cDNA, the corresponding genomic DNA was characterized from 129SVJ mice. The murine Hyal2 gene is approximately 3.5 kb, contains the coding sequence for the mRNA on four exons, and is localized on chromosome 9 between the microsatellite markers D9Mit183 and D9Mit17 near the genes for dystroglycan and transferrin. The gene is expressed ubiquitously, the sole exception being adult brain.

HYAL2, a human gene expressed in many cells, encodes a lysosomal hyaluronidase with a novel type of specificity.

Using Expressed Sequence Tags (ESTs) deposited in the data banks, a cDNA has been assembled that encodes a protein related to the hyaluronidases from bee venom and mammalian sperm. Expression of this cDNA yielded a polypeptide termed HYAL2, which is located in lysosomes. The HYAL2 protein was shown to have hyaluronidase activity below pH 4. However, it only hydrolyzed hyaluronan of high molecular mass from umbilical cord, rooster comb, and a Streptococcus strain. The reaction product was a polysaccharide of about 20 kDa, which was further hydrolyzed to small oligosaccharides by the sperm hyaluronidase. Conversely, hyaluronan fragments from vitreous humor, which had a molecular mass of about 20 kDa, were not cleaved by the HYAL2 enzyme to any detectable extent. These results provide evidence for the existence of structural domains in hyaluronan, which are resistant to the action of this enzyme. The structural and functional implications of these findings are discussed.

B-50/growth-associated protein-43, a marker of neural development in Xenopus laevis.

To study the regulation and function of the growth-associated protein B-50/growth-associated protein-43 (mol. wt 43,000) in Xenopus laevis, B-50/growth-associated protein-43 complementary DNAs were isolated and characterized. The deduced amino acid sequence revealed potential functional domains of Xenopus B-50/growth-associated protein-43 that may be involved in G-protein interaction, membrane-binding, calmodulin-binding and protein kinase C phosphorylation. The expression of B-50/growth-associated protein-43 at the RNA and protein level during development was investigated using the Xenopus complementary DNA and the monoclonal B-50/growth-associated protein-43 antibody NM2. The antibody NM2 recognized the gene product on western blot and in whole-mount immunocytochemistry of Xenopus embryos. Moreover, visualization of the developmentally regulated appearance of B-50/growth-associated protein-43 immunoreactivity showed that this mode of detection may be used to monitor axonogenesis under various experimental conditions. In the adult Xenopus, XB-50/growth-associated protein-43 messenger RNA was shown to be expressed at high levels in brain, spinal cord and eye using northern blotting. The earliest expression detected on northern blot was at developmental stage 13 with poly(A) RNA. By whole-mount immunofluorescence, applying the confocal laser scanning microscope, the protein was first detected in embryos from stage 20, where it was expressed in the developing trigeminal ganglion. Also later in development the expression of the B-50/growth-associated protein-43 gene was restricted to the nervous system in Xenopus, as was previously found for the mouse. In conclusion, we find that XB-50/growth-associated protein-43 is a good marker to study the development of the nervous system in Xenopus laevis.

A retinoid-binding lipocalin, Xlcpl1, relevant for embryonic pattern formation is expressed in the nervous system of Xenopus laevis.

The importance of retinoids in early development has been increasingly recognized during the past decade. Their transport and action are mediated by extracellular, intracellular and nuclear proteins. Here we describe the isolation and characterization of a cDNA, Xlcpl1, coding for a lipocalin that is most likely involved in the transport of retinoids in the embryonic nervous system of Xenopus laevis. Lipocalins generally are carriers for small hydrophobic molecules. The expression of Xlcpl1 shows a distinct spatial and temporal pattern: transcription was observed in the anterior-most part of the neural plate at embryonic stages 13-14 whereas in adult brain, Xlcpl1 is expressed exclusively in the choroid plexus. In addition, expression was also found at the dorsal borders of the eye anlagen and in the otic vesicle of mid-neurula stage embryos. In order to gain insight into the molecular function of Xlcpl1, we caused ectopic overexpression by injecting fertilized eggs with either an excess of sense mRNA or DNA constructs under the control of the EF1α promotor. The resulting embryos developed a dysmorphogenised anterior neural system, in particular malformed heads and eyes. Phenotypic defects were therefore similar to those obtained by retinoic acid treatment at the gastrula stage or overexpression of other proteins involved in retinoic acid signalling in the early embryo. Retinoic acid treatment disrupts the endogenous gradient of retinoids that supposedly controls antero-posterior differentiation. By analogy, we propose that local overexpression of Xlcpl1 reflects precisely the situation of exogenous application of retinoic acid. In fact, we have shown biochemically that Xlcpl1 is suited to mediate the cellular retinoid signal.

The lipocalin Xlcpl1 expressed in the neural plate of Xenopus laevis embryos is a secreted retinaldehyde binding protein.

The cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate of Xenopus laevis embryos and the adult choroid plexus have been investigated. It was found that this lipocalin, termed Xlcpl1, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol. Furthermore, this protein also binds D/L thyroxine. The Xlcpl1 cDNA was expressed in cell culture using the vaccinia virus expression system. In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway. We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids. Therefore, cpl1-expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers. These retinoids may enter the cytosol by diffusion or receptor-controlled mechanisms, as has been shown for exogenously applied retinoids. Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development.