Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions.

Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.

Impact of a primary care physician workshop on osteoporosis medical practices.

SUMMARY: Attendance at a fragility-fractures-prevention workshop by primary care physicians was associated with higher rates of osteoporosis screening and treatment initiation in elderly female patients and higher rates of treatment initiation in high-risk male and female patients. However, osteoporosis management remained sub-optimal, particularly in men. INTRODUCTION: Rates of osteoporosis-related medical practices of primary care physicians exposed to a fragility-fractures-prevention workshop were compared with those of unexposed physicians. METHODS: In a cluster cohort study, 26 physicians exposed to a workshop were matched with 260 unexposed physicians by sex and year of graduation. For each physician, rates of bone mineral density (BMD) testing and osteoporosis treatment initiation among his/her elderly patients 1 year following the workshop were computed. Rates were compared using multilevel logistic regression models controlling for potential patient- and physician-level confounders. RESULTS: Twenty-five exposed physicians (1,124 patients) and 209 unexposed physicians (9,663 patients) followed at least one eligible patient. In women, followed by exposed physicians, higher rates of BMD testing [8.5% versus 4.2%, adjusted OR (aOR) = 2.81, 95% CI 1.60-4.94] and treatment initiation with bone-specific drugs (BSDs; 4.8% vs. 2.4%, aOR = 1.95, 1.06-3.60) were observed. In men, no differences were detected. In patients on long-term glucocorticoid therapy or with a previous osteoporotic fracture, higher rates of treatment initiation with BSDs were observed in women (12.0% vs. 1.9%, aOR = 7.38, 1.55-35.26), and men were more likely to initiate calcium/vitamin D (5.3% vs. 0.8%, aOR = 7.14, 1.16-44.06). CONCLUSIONS: Attendance at a primary care physician workshop was associated with higher rates of osteoporosis medical practices for elderly women and high-risk men and women. However, osteoporosis detection and treatment remained sub-optimal, particularly in men.

Nomenclature of prominin-1 (CD133) splice variants - an update.

Prominin-1 (CD133), a pentaspan membrane glycoprotein that constitutes an important cell surface marker of various, either normal or cancerous, stem cell populations is widely used to isolate or characterize such cells in different systems. Occurring throughout the metazoan evolution with a remarkably conserved genomic organization, it may be expressed as different splice variants with distinctive characteristics. A rational nomenclature has been proposed earlier for their consistent designation across species. Although generally accepted, it seems to be misunderstood in view of the recent report of novel prominin-1 complementary DNAs in rhesus monkey and humans with improper naming. As this may lead to confusion, we have reexamined the genomic organization of prominin-1 in various primates to provide an update that should further clarify the rationale of the nomenclature for prominin-1 gene products. This report comprises (i) the determination of the genomic organization of prominin-1 gene in two non-human primates, i.e. Macaca mulatta and Pan troglodytes, commonly used in research, (ii) the mapping of a new exon that creates an alternative cytoplasmic C-terminal end of prominin-1, (iii) the identification of various potential PDZ-binding domains generated by alternative cytoplasmic C-terminal tails, suggesting that different prominin-1 splice variants might interact with distinct protein partners, and (iv) a summing up of the different prominin-1 splice variants.

Immunohistochemical in situ characterization of orthopedic implants on polymethyl metacrylate embedded cutting and grinding sections.

When investigating the tissue reaction on orthopedic implants, the cellular activity at the bone-implant interface is of special interest. Preparation of undecalcified bone sections with methylmetacrylate (MMA)-based resins allows evaluation of the host tissue reactions with the implant in situ. However, the technical workup is demanding and few reports exist on the immunohistochemical characterization of these sections. Rat (R), sheep (S), and human (H) samples were investigated. R specimens contained intramedullary rods in the rat tibia. S specimens were sheep tibiae with an external fixator. H specimens were obtained from deceased patients. Specimens were embedded in MMA-based Technovit 9100N using cold polymerization. Sections of 10-15 microm thickness were obtained and prepared for immunohistochemical staining. Good morphological detail was preserved in all specimens providing information about mineralization, recent bone formation, and bone-implant contact. The following antibodies could reproducibly be detected specifically: Osteopontin (R, S, H), Osteonectin, Cathepsin D (R, S), von Willebrand factor (R, H), Osteocalcin, ED 1 (R), CD 3, CD 68, Keratin (H). Control procedures without adding primary antibodies showed no unspecific staining. Reliable detection of immunohistochemical markers of bone resorption, bone formation, inflammation, and angiogenesis at undecalcified sections with the implant in situ appears promising in enhancing our understanding of the cellular activity and cell-matrix interactions at the bone-implant interface.

Comparative analysis of proliferative potential and clonogenicity of MACS-immunomagnetic isolated CD34+ and CD133+ blood stem cells derived from a single donor.

A novel stem cell marker prominin-1 (CD133) has been shown to be expressed on a subpopulation of CD34(+) haematopoietic stem and progenitor cells. The aim of this study was to compare in parallel commercially available CD34(+) and CD133(+) isolation methods based on paramagnetic bead-coupled antibodies using clinical-grade samples of mobilized peripheral blood from 10 individual healthy donors under identical conditions. The CD133 negative fraction from the first selection was used for CD34(+) enrichment to obtain an additional CD34(+)/CD133(-) population. Although no significant difference in total cell expansion between cells isolated from the three procedures was observed in a 7-day cytokine-driven suspension culture, the long-term culture-initiating cell assay demonstrated that cells derived by CD34(+) isolation contain less primitive progenitors than those isolated based on CD133(+) selection. Interestingly, CD34(+)-enriched progenitors, especially the CD34(+)/CD133(-) fraction, contained a significantly higher proportion of erythroid colony-forming cells, whereas the highest content of myeloid colony-forming cells was concentrated in the CD133(+) selected cells. These subtle differences between CD34(+) and CD133(+) immunomagnetic selection will have to be explored for their potential clinical relevance.

Rat prominin, like its mouse and human orthologues, is a pentaspan membrane glycoprotein.

Mouse prominin is the first characterized member of a novel family of membrane glycoproteins. It displays a characteristic membrane topology with five transmembrane segments and two large glycosylated extracellular loops. Prominin orthologues and paralogues have been identified in human, fish, fly, and worm. Recently, a cDNA sequence encoding the rat homologue of mouse prominin has been reported [Zhu et al. (2001) Biochem. Biophys. Res. Commun. 281, 951-956]. Surprisingly, due to a single nucleotide deletion that shifts the reading frame and introduces a premature stop codon, the protein predicted from this cDNA would correspond to a C-terminally truncated form of prominin with only four transmembrane segments. Here we report evidence that is in contrast to the report of Zhu et al. (2001). We isolated a rat prominin cDNA devoid of any frameshift mutation, demonstrate that rat prominin, like the other mammalian prominins, is a full-length 120-kDa pentaspan membrane glycoprotein, and have not been able to detect any C-terminally truncated form of rat prominin.

Prominin: a story of cholesterol, plasma membrane protrusions and human pathology.

Prominin is the first identified member of a novel family of polytopic membrane proteins conserved throughout the animal kingdom. It has an unusual membrane topology, containing five transmembrane domains and two large glycosylated extracellular loops. In mammals, prominin is expressed in various embryonic and adult epithelial cells, as well as in nonepithelial cells, such as hematopoietic stem cells. At the subcellular level, prominin is selectively localized in microvilli and other plasma membrane protrusions, irrespective of cell type. At the molecular level, prominin specifically interacts with membrane cholesterol and is a marker of a novel type of cholesterol-based lipid 'raft'. A frameshift mutation in the human prominin gene, which results in a truncated protein that is no longer transported to the cell surface, is associated with retinal degeneration. Given that prominin is concentrated in the plasma membrane evaginations at the base of the outer segment of rod photoreceptor cells, which are essential precursor structures in the biogenesis of photoreceptive disks, it is proposed that prominin has a role in the generation of plasma membrane protrusions, their lipid composition and organization and their membrane-to-membrane interactions.

Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane.

Membrane cholesterol-sphingolipid 'rafts', which are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold, have been implicated in the sorting of certain membrane proteins, such as placental alkaline phosphatase (PLAP), to the apical plasma membrane domain of epithelial cells. Here we show that prominin, an apically sorted pentaspan membrane protein, becomes associated in the trans-Golgi network with a lipid raft that is soluble in Triton X-100 but insoluble in another non-ionic detergent, Lubrol WX. At the cell surface, prominin remains insoluble in Lubrol WX and is selectively associated with microvilli, being largely segregated from the membrane subdomains containing PLAP. Cholesterol depletion results in the loss of prominin's microvillus-specific localization but does not lead to its complete intermixing with PLAP. We propose the coexistence within a membrane domain, such as the apical plasma membrane, of different cholesterol-based lipid rafts, which underlie the generation and maintenance of membrane subdomains.

The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions.

The human AC133 antigen and mouse prominin are structurally related plasma membrane proteins. However, their tissue distribution is distinct, with the AC133 antigen being found on hematopoietic stem and progenitor cells and prominin on various epithelial cells. To determine whether the human AC133 antigen and mouse prominin are orthologues or distinct members of a protein family, we examined the human epithelial cell line Caco-2 for the possible expression of the AC133 antigen. By both immunofluorescence and immunoprecipitation, the AC133 antigen was found to be expressed on the surface of Caco-2 cells. Interestingly, immunoreactivity for the AC133 antigen, but not its mRNA level, was down-regulated upon differentiation of Caco-2 cells. The AC133 antigen was specifically located at the apical rather than basolateral plasma membrane. An apical localization of the AC133 antigen was also observed in various human embryonic epithelia including the neural tube, gut, and kidney. Electron microscopy revealed that, within the apical plasma membrane of Caco-2 cells, the AC133 antigen was confined to microvilli and absent from the planar, intermicrovillar regions. This specific subcellular localization did not depend on an epithelial phenotype, because the AC133 antigen on hematopoietic stem cells, as well as that ectopically expressed in fibroblasts, was selectively found in plasma membrane protrusions. Hence, the human AC133 antigen shows the features characteristic of mouse prominin in epithelial and transfected non-epithelial cells, i.e. a selective association with apical microvilli and plasma membrane protrusions, respectively. Conversely, flow cytometry of murine CD34(+) bone marrow progenitors revealed the cell surface expression of prominin. Taken together, the data strongly suggest that the AC133 antigen is the human orthologue of prominin.

A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration.

The disks of vertebrate photoreceptors are produced by outgrowths of the plasma membrane. Hence genes that encode retinal proteins targeted to plasma membrane protrusions represent candidates for inherited retinal degenerations. One such candidate is the gene encoding human prominin (mouse)-like 1 (PROML1, previously known as AC133 antigen) which belongs to the prominin family of 5-transmembrane domain proteins. Murine prominin (prom) shows a strong preference for plasma membrane protrusions in a variety of epithelial cells whereas PROML1 is expressed in retinoblastoma cell lines and adult retina. In the present study, molecular genetic analyses of a pedigree segregating for autosomal recessive retinal degeneration indicated that the affected individuals were homozygous for a nucleotide 1878 deletion in PROML1. This alteration is predicted to result in a frameshift at codon 614 with premature termination of translation. Expression of a similar prom deletion mutant in CHO cells indicated that the truncated protein does not reach the cell surface. Immunocytochemistry revealed that prom is concentrated in the plasma membrane evaginations at the base of the outer segments of rod photoreceptors. These findings suggest that loss of prominin causes retinal degeneration, possibly because of impaired generation of the evaginations and/or impaired conversion of the evaginations to disks.

Selective localization of the polytopic membrane protein prominin in microvilli of epithelial cells - a combination of apical sorting and retention in plasma membrane protrusions.

Prominin is a recently identified polytopic membrane protein expressed in various epithelial cells, where it is selectively associated with microvilli. When expressed in non-epithelial cells, prominin is enriched in plasma membrane protrusions. This raises the question of whether the selective association of prominin with microvilli in epithelial cells is solely due to its preference for, and stabilization in, plasma membrane protrusions, or is due to both sorting to the apical plasma membrane domain and subsequent enrichment in plasma membrane protrusions. To investigate this question, we have generated stably transfected MDCK cells expressing either full-length or C-terminally truncated forms of mouse prominin. Confocal immunofluorescence and domain-selective cell surface biotinylation experiments on transfected MDCK cells grown on permeable supports demonstrated the virtually exclusive apical localization of prominin at steady state. Pulse-chase experiments in combination with domain-selective cell surface biotinylation showed that newly synthesized prominin was directly targeted to the apical plasma membrane domain. Immunoelectron microscopy revealed that prominin was confined to microvilli rather than the planar region of the apical plasma membrane. Truncation of the cytoplasmic C-terminal tail of prominin impaired neither its apical cell surface expression nor its selective retention in microvilli. Both the apical-specific localization of prominin and its selective retention in microvilli were maintained when MDCK cells were cultured in low-calcium medium, i.e. in the absence of tight junctions. Taken together, our results show that: (i) prominin contains dual targeting information, for direct delivery to the apical plasma membrane domain and for the enrichment in the microvillar subdomain; and (ii) this dual targeting does not require the cytoplasmic C-terminal tail of prominin and still occurs in the absence of tight junctions. The latter observation suggests that entry into, and retention in, plasma membrane protrusions may play an important role in the establishment and maintenance of the apical-basal polarity of epithelial cells.

Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase-2.

Tyrosylprotein sulfotransferase (TPST) is a 54- to 50-kDa integral membrane glycoprotein of the trans-Golgi network found in essentially all tissues investigated, catalyzing the tyrosine O-sulfation of soluble and membrane proteins passing through this compartment. Here we describe (i) an approach to identify the TPST protein, referred to as MSC (modification after substrate crosslinking) labeling, which is based on the crosslinking of a substrate peptide to TPST followed by intramolecular [35S]sulfate transfer from the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS); and (ii) the molecular characterization of a human TPST, referred to as TPST-2, whose sequence is distinct from that reported [TPST-1; Ouyang, Y.-B., Lane, W. S. & Moore, K. L. (1998) Proc. Natl. Acad. Sci. USA 95, 2896-2901] while this study was in progress. Human TPST-2 is a type II transmembrane protein of 377 aa residues that is encoded by a ubiquitously expressed 1.9-kb mRNA originating from seven exons of a gene located on chromosome 22 (22q12.1). A 304-residue segment in the luminal domain of TPST-2 shows 75% amino acid identity to the corresponding segment of TPST-1, including conservation of the residues implicated in the binding of PAPS. Expression of the TPST-2 cDNA in CHO cells resulted in an approximately 13-fold increase in both TPST protein, as determined by MSC labeling, and TPST activity. A predicted 359-residue type II transmembrane protein in Caenorhabditis elegans with 45% amino acid identity to TPST-2 in a 257-residue segment of the luminal domain points to the evolutionary conservation of the TPST protein family.

Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells.

Using a new mAb raised against the mouse neuroepithelium, we have identified and cDNA-cloned prominin, an 858-amino acid-containing, 115-kDa glycoprotein. Prominin is a novel plasma membrane protein with an N-terminal extracellular domain, five transmembrane segments flanking two short cytoplasmic loops and two large glycosylated extracellular domains, and a cytoplasmic C-terminal domain. DNA sequences from Caenorhabditis elegans predict the existence of a protein with the same features, suggesting that prominin is conserved between vertebrates and invertebrates. Prominin is found not only in the neuroepithelium but also in various other epithelia of the mouse embryo. In the adult mouse, prominin has been detected in the brain ependymal layer, and in kidney tubules. In these epithelia, prominin is specific to the apical surface, where it is selectively associated with microvilli and microvilli-related structures. Remarkably, upon expression in CHO cells, prominin is preferentially localized to plasma membrane protrusions such as filopodia, lamellipodia, and microspikes. These observations imply that prominin contains information to be targeted to, and/or retained in, plasma membrane protrusions rather than the planar cell surface. Moreover, our results show that the mechanisms underlying targeting of membrane proteins to microvilli of epithelial cells and to plasma membrane protrusions of non-epithelial cells are highly related.

Proteolytic processing of the alpha-subunit of rat endopeptidase-24.18 by furin.

Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.

Expression of rat endopeptidase-24.18 in COS-1 cells: membrane topology and activity.

Endopeptidase-24.18 (E-24.18; EC 3.4.24.18) is a metallopeptidase of the astacin family and is highly expressed in kidney brush-border membranes of rodents. Rat E-24.18 consists of two disulphide-linked alpha/beta dimers [(alpha/beta)2]. In order to investigate the mechanisms of assembly and the importance of each subunit in the enzymic process, the cloned cDNAs for the rat alpha and beta subunits were transiently expressed either alone or together in COS-1 cells. Immunoblotting of cell extracts and spent culture media showed that, when expressed alone, the alpha subunit is secreted, whereas the beta subunit is membrane-bound. In alpha/beta-transfected cells, the alpha subunit remained membrane-bound, but could be released from the cell surface after papain treatment or after incubation with 10 mM dithiothreitol. Furthermore, mutants of the alpha subunit in which the putative C-terminal anchor domain was deleted could still form cell-associated alpha/beta dimers. These results are consistent with a topological model of E-24.18 in which the beta subunit is anchored in the plasma membrane and the alpha subunit is retained at the cell surface through disulphide bridge(s) with the beta subunit. Both the alpha and beta recombinant subunits expressed in COS-1 cells showed little azocasein-degrading activity. However, activity of either individual subunits of alpha/beta dimers was increased after mild trypsin digestion, suggesting that in COS-1 cells the enzymes are synthesized as zymogens. Finally, inactivation of the alpha subunit by site-directed mutagenesis of Glu-157, which is believed to play a role in catalysis, showed that both subunits participate in the enzymic activity of the heterodimer.