Published by De Gruyter

Volume 389 Issue 7

Issue of Biological Chemistry

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Contents
Journal Overview

Guest Editorial

August 5, 2008

Highlight: Self-renewal signaling in stem cells

Markus Müschen

Page range: 787-787

Commentary

August 5, 2008

Commentary: Highlight on stem cell research

Hans R. Schöler

Page range: 789-789

Highlight: Self-Renewal Signaling in Stem Cells

June 6, 2008

Prospective of Ras signaling in stem cells

Koushik Chakrabarty, Rolf Heumann

Page range: 791-798

Abstract

The Ras signaling pathway plays a predominant role during development and controls diverse biological process in all eukaryotic cells. It is a member of the large family of GTPases proteins that binds and hydrolyzes GTP. Ras is a lipid-anchored protein on the intracellular membrane compartments, and cycles between inactive GDP-bound and the signaling competent GTP-bound conformation. Studies have demonstrated Ras to be a central regulator in signal transduction pathways responding to diverse extracellular and intracellular stimuli. Much progress has been made towards delineating specific genes involved in the process of pluripotency and differentiation of stem cells. Here, we discuss recent aspects of Ras signaling pathways in mediating stem cell properties.

June 6, 2008

Prowling wolves in sheep's clothing: the search for tumor stem cells

Ibrahim Alkatout, Dieter Kabelitz, Holger Kalthoff, Sanjay Tiwari

Page range: 799-811

Abstract

The importance of a subset of cells which have ‘stem like’ characteristics and are capable of tumor initiation has been reported for a range of tumors. Isolation of these tumor-initiating cells (TICs) has largely been based on differential cell surface protein expression. However, there is still much debate on the functional significance of these markers in initiating tumors, as many properties of tumor initiation are modified by cell-cell interactions. In particular, the relationship between TICs and their microenvironment is poorly understood but has therapeutic implications, as the microenvironment can maintain tumor cells in a prolonged period of quiescence. However, a major limitation in advancing our understanding of the crosstalk between TICs and their microenvironment is the lack of sensitive techniques which allow the in vivo tracking and monitoring of TICs. Application of new in vivo cellular and molecular imaging technologies holds much promise in uncovering the mysteries of TIC behavior at the three-dimensional level. This review will describe recent advances in our understanding of the TIC concept and how the application of in vivo imaging techniques can advance our understanding of the biological fate of TICs. A supplementary resource guide describing TICs from different malignancies is also presented.

June 6, 2008

Lineage development of hematopoietic stem and progenitor cells

Bernd Giebel, Michael Punzel

Page range: 813-824

Abstract

Hematopoietic stem cells have the potential to develop into multipotent and different lineage-restricted progenitor cells that subsequently generate all mature blood cell types. The classical model of hematopoietic lineage commitment proposes a first restriction point at which all multipotent hematopoietic progenitor cells become committed either to the lymphoid or to the myeloid development, respectively. Recently, this model has been challenged by the identification of murine as well as human hematopoietic progenitor cells with lymphoid differentiation capabilities that give rise to a restricted subset of the myeloid lineages. As the classical model does not include cells with such capacities, these findings suggest the existence of alternative developmental pathways that demand the existence of additional branches in the classical hematopoietic tree. Together with some phenotypic criteria that characterize different subsets of multipotent and lineage-restricted progenitor cells, we summarize these recent findings here.

June 6, 2008

Plasticity after allogeneic hematopoietic stem cell transplantation

Alicia Rovó, Alois Gratwohl

Page range: 825-836

Abstract

The postulated almost unlimited potential of transplanted hematopoietic stem cells (HSCs) to transdifferentiate into cell types that do not belong to the hematopoietic system denotes a complete paradigm shift of the hierarchical hemopoietic tree. In several studies during the last few years, donor cells have been identified in almost all recipient tissues after allogeneic HSC transplantation (HSCT), supporting the theory that any failing organ could be accessible to regenerative cell therapy. However, the putative potential ability of the stem cells to cross beyond lineage barriers has been questioned by other studies which suggest that hematopoietic cells might fuse with non-hematopoietic cells and mimic the appearance of transdifferentiation. Proof that HSCs have preserved the capacity to transdifferentiate into other cell types remains to be demonstrated. In this review, we focus mainly on clinical studies addressing plasticity in humans who underwent allogeneic HSCT. We summarize the published data on non-hematopoietic chimerism, donor cell contribution to tissue repair, the controversies related to the methods used to detect donor-derived non-hematopoietic cells and the functional impact of this phenomenon in diverse specific target tissues and organs.

June 6, 2008

Adult progenitor cells in vascular remodeling during atherosclerosis

Mihail Hristov, Alma Zernecke, Andreas Schober, Christian Weber

Page range: 837-844

Abstract

The mobilization and recruitment of bone marrow-derived, circulating or tissue resident progenitor cells giving rise to smooth muscle-like cells have been implicated in neointima hyperplasia after arterial injury and in accelerated forms of arterial lesion formation, e.g., transplant arteriopathy or graft vasculopathy. By contrast, convincing evidence has emerged that the vascular homing of endothelial progenitor cells (EPCs) contributes to endothelial recovery, thus limiting neointima formation after arterial injury. In the chronic context of primary atherosclerosis, plaque progression and destabilization, a more complex picture has become apparent. In patients with coronary artery disease, the number and function of EPCs have been linked with an improved endothelial function or regeneration, but have been inversely correlated with cardiovascular risk. In animal models, however, the injection of bone marrow cells or EPCs, or the application of stem-cell mobilizing factors, have been associated with an exacerbation of atherosclerosis and unstable plaque phenotypes, whereas the contribution of bone marrow-derived smooth muscle progenitors to primary atherosclerosis appears to be rather confined. Here, we discuss crucial biochemical cues, namelychemokines, adhesion molecules, growth factors and pharmacological means that guide and control the context-specific mobilization, recruitment and fate of vascular progenitor cells in arterial remodeling during atherosclerosis.

June 6, 2008

Oct4 expression revisited: potential pitfalls for data misinterpretation in stem cell research

Stefanie Liedtke, Milaid Stephan, Gesine Kögler

Page range: 845-850

Abstract

The octamer-binding transcription factor 4 gene encodes a nuclear protein (Oct4, also known as Pou5F1 and Oct3/4) that belongs to a family of transcription factors containing the POU DNA-binding domain. Expression can be detected in embryonic stem cells as well as in adult stem cells, such as bone marrow-derived mesenchymal stem cells. Expression of Oct4 is downregulated coincident with stem cell differentiation and loss of expression leading to differentiation. A role for maintaining pluripotency and self-renewal of embryonic stem cells is ascribed to Oct4 as a pluripotency marker. Results describing Oct4 expression in differentiated cells, including peripheral blood mononuclear cells (PBMCs), neonatal and adult stem cells, as well as cancer cells, must be interpreted with caution. In several publications, Oct4 has been ascribed a function in maintaining self-renewal of adult stem cells. In contrast, other publications reported Oct4 expression in human tumor cells. Here, we summarize the recent findings on Oct4 expression and present possibilities and reasons why several false positive results on Oct4 expression still occur in the recent literature. Also, simple solutions are provided to avoid these positive signals.

August 5, 2008

Generation of transducible versions of transcription factors Oct4 and Sox2

Manal Bosnali, Frank Edenhofer

Page range: 851-861

Abstract

The transcription factors Oct4 and Sox2 are two of the main regulators of pluripotency in embryonic stem cells. Since the importance of non-genetic modification is continually increasing, particularly for therapeutic application of manipulated cells, the aim of the present study was to generate cell-permeant Oct4 and Sox2 proteins for the direct cellular delivery of active proteins. Protein transduction allowing cellular manipulation to circumvent genetic modification of target cells has recently been developed. We present a new expression vector system, pSESAME, that facilitates the generation of transducible proteins. Using pSESAME, both Oct4 and Sox2 were genetically fused with a TAT protein transduction domain that promotes cellular penetration. The recombinant purified Oct4 and Sox2 fusion proteins display DNA-binding properties comparable to their endogenous counterparts, and exhibit cellular entry and the ability to modulate the transcriptional machinery maintaining pluripotency of mouse embryonic stem cells. In a rescue assay we demonstrate that transducible Oct4 and Sox2 fusion proteins can compensate knockdown of Pou5f1 and Sox2 , respectively. This study provides powerful tools for the modulation of stem cell properties without genetic interference.

June 6, 2008

Cytokine combinations differentially influence the SDF-1α-dependent migratory activity of cultivated murine hematopoietic stem and progenitor cells

Susannah H. Kassmer, Bernd Niggemann, Michael Punzel, Christine Mieck, Kurt S. Zänker, Thomas Dittmar

Page range: 863-872

Abstract

Stromal cell-derived factor-1α (SDF-1α) is a strong migratory stimulant for hematopoietic stem and progenitor cells (HSPCs). The hematopoietic cytokines thrombopoietin (TPO), Flt3-ligand (FL), stem cell factor (SCF) and interleukin 11 (IL-11) are able to stimulate amplification of primitive murine hematopoietic stem cells (HSCs) in vitro . The effects of these cytokines on SDF-1α-induced migratory activity of murine Lin - c-kit + HSPC were analyzed by cultivation of these cells in the presence of 12 combinations of FL, TPO, SCF and IL-11. Migratory activity was measured in a three-dimensional collagen matrix using time-lapse video microscopy. Each cytokine combination had a distinct effect on SDF-1α-stimulated migratory activity. For instance, FL- and SCF-cultivated cells showed a high migratory SDF-1α response, while cells cultivated with SCF, TPO and IL-11 did not react to SDF-1α stimulation with an elevated migration rate. Our data indicate that the differences in the migratory SDF-1α response are not related to different CXCR4 expression levels, but rather to the differential engagement of the CXCR4-dependent MAPK p42/44 and PI3K signal transduction pathways. This indicates that hematopoietic cytokines can have a significant impact on SDF-1α-stimulated migratory activity and the underlying intracellular signaling processes in cultivated HSPCs.

June 6, 2008

Bone marrow-derived mesenchymal stem cell transplantation does not improve quality of muscle reinnervation or recovery of motor function after facial nerve transection in rats

Maria Grosheva, Orlando Guntinas-Lichius, Stephan Arnhold, Emmanouil Skouras, Stefanie Kuerten, Michael Streppel, Srebrina K. Angelova, Konstantin Wewetzer, Christine Radtke, Sarah A. Dunlop, Doychin N. Angelov

Page range: 873-888

Abstract

Recently, we devised and validated a novel strategy in rats to improve the outcome of facial nerve reconstruction by daily manual stimulation of the target muscles. The treatment resulted in full recovery of facial movements (whisking), which was achieved by reducing the proportion of pathologically polyinnervated motor endplates. Here, we posed whether manual stimulation could also be beneficial after a surgical procedure potentially useful for treatment of large peripheral nerve defects, i.e., entubulation of the transected facial nerve in a conduit filled with suspension of isogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) in collagen. Compared to control treatment with collagen only, entubulation with BM-MSCs failed to decrease the extent of collateral axonal branching at the lesion site and did not improve functional recovery. Post-operative manual stimulation of vibrissal muscles also failed to promote a better recovery following entubulation with BM-MSCs. We suggest that BM-MSCs promote excessive trophic support for regenerating axons which, in turn, results in excessive collateral branching at the lesion site and extensive polyinnervation of the motor endplates. Furthermore, such deleterious effects cannot be overridden by manual stimulation. We conclude that entubulation with BM-MSCs is not beneficial for facial nerve repair.

June 6, 2008

In vitro differentiation of reprogrammed murine somatic cells into hepatic precursor cells

Tobias Cantz, Martina Bleidißel, Martin Stehling, Hans R. Schöler

Page range: 889-896

Abstract

Recently, a new approach to reprogram somatic cells into pluripotent stem cells was shown by fusion of somatic cells with embryonic stem (ES) cells, which results in a tetraploid karyotype. Normal hepatocytes are often polyploid, so we decided to investigate the differentiation potential of fusion hybrids into hepatic cells. We chose toxic milk mice (a model of Wilson's disease) and performed initial transplantation experiments using this potential cell therapy approach. Mononuclear bone marrow cells from Rosa26 mice were fused with OG2 (Oct4-GFP transgenic) ES cells. Unfused ES cells were eliminated by selection with G418 for OG2-Rosa26 hybrids and fusion-derived colonies could be subcloned. Using an endodermal differentiation protocol, hepatic precursor cells could be generated. After FACS depletion of contaminating Oct4-GFP-positive cells, the hepatic precursor cells were transplanted into immunosuppressed toxic milk mice by intrasplenic injection. However, five out of eight mice showed teratoma formation within 3–6 weeks after transplantation in the spleen and liver. In conclusion, a hepatic precursor cell type was achieved from mononuclear bone marrow cell-ES cell hybrids and preliminary transplantation experiments confirmed engraftment, but also showed teratoma formation, which needs to be excluded by using more stringent purification strategies.

August 5, 2008

The WNT receptor FZD7 contributes to self-renewal signaling of human embryonic stem cells

Kai Melchior, Jonathan Weiß, Holm Zaehres, Yong-mi Kim, Carolyn Lutzko, Neda Roosta, Jürgen Hescheler, Markus Müschen

Page range: 897-903

Abstract

A number of recent studies identified nuclear factors that together have the unique ability to induce pluripotency in differentiated cell types. However, little is known about the factors that are needed to maintain human embryonic stem (ES) cells in an undifferentiated state. In a search for such requirements, we performed a comprehensive meta-analysis of publicly available SAGE and microarray data. The rationale for this analysis was to identify genes that are exclusively expressed in human ES cell lines compared to 30 differentiated tissue types. The WNT receptor FZD7 was found among the genes with an ES cell-specific expression profile in both SAGE and microarray analyses. Subsequent validation by quantitative RT-PCR and flow cytometry confirmed that FZD7 mRNA levels in human ES cells are up to 200-fold higher compared to differentiated cell types. ShRNA-mediated knockdown of FZD7 in human ES cells induced dramatic changes in the morphology of ES cell colonies, perturbation of expression levels of germ layer-specific marker genes, and a rapid loss of expression of the ES cell-specific transcription factor OCT4. These findings identify the WNT receptor FZD7 as a novel ES cell-specific surface antigen with a likely important role in the maintenance of ES cell self-renewal capacity.

Genes and Nucleic Acids

June 6, 2008

Elimination of hop latent viroid upon developmental activation of pollen nucleases

Jaroslav Matoušek, Lidmila Orctová, Josef Škopek, Karel Pešina, Gerhard Steger

Page range: 905-918

Abstract

Hop latent viroid (HLVd) is not transmissible through hop generative tissues and seeds. Here we describe the process of HLVd elimination during development of hop pollen. HLVd propagates in uninucleate hop pollen, but is eliminated at stages following first pollen mitosis during pollen vacuolization and maturation. Only traces of HLVd were detected by RT-PCR in mature pollen after anthesis and no viroid was detectable in in vitro germinating pollen, suggesting complete degradation of circular and linear HLVd forms. The majority of the degraded HLVd RNA in immature pollen included discrete products in the range of 230–100 nucleotides and therefore did not correspond to siRNAs. HLVd eradication from pollen correlated with developmental expression of a pollen nuclease and specific RNAses. Activity of the pollen nuclease HBN1 was maximal during the vacuolization step and decreased in mature pollen. Total RNAse activity increased continuously up to the final steps of pollen maturation. HBN1 mRNA, which is abundant at the uninucleate microspore stage, encodes a protein of 300 amino acids (34.1 kDa, isoeletric point 5.1). Sequence comparisons revealed that HBN1 is a homolog of S1-like bifunctional plant endonucleases. The developmentally activated HBN1 and pollen ribonucleases could participate in the mechanism of HLVd recognition and degradation.

Protein Structure and Function

June 6, 2008

Solution structure of Phl p 3, a major allergen from timothy grass pollen

Kristian Schweimer, Arnd Petersen, Roland Suck, Wolf-Meinhard Becker, Paul Rösch, Irena Matecko

Page range: 919-923

Abstract

The major 97-aa timothy grass ( Phleum pratense ) allergen Phl p 3 was recently isolated from an extract of timothy grass pollen. Sequence comparison classifies this protein as a group 3 allergen. The solution structure of Phl p 3 as determined by nuclear magnetic resonance spectroscopy reveals that the protein consists of a core of hydrophobic amino-acid side chains from two β-sheets of five and four anti-parallel β-strands, respectively. This conformation is very similar to the crystal structure published for Phl p 2 and strongly resembles the known conformation of the carboxy-terminal domain of Phl p 1, the major difference being the loop orientations. Phl p 2 and Phl p 3 show virtually identical immunoreactivity, and comparison of the charged surface amino acids of the two proteins gives initial clues as to the IgE recognition epitopes of these proteins.

June 6, 2008

A hypoallergenic hybrid molecule with increased immunogenicity consisting of derivatives of the major grass pollen allergens, Phl p 2 and Phl p 6

Birgit Linhart, Nadine Mothes-Luksch, Susanne Vrtala, Michael Kneidinger, Peter Valent, Rudolf Valenta

Page range: 925-933

Abstract

Allergen-specific immunotherapy is currently based on the administration of allergen extracts containing natural allergens. However, its broad application is limited by the poor quality of these extracts. Based on recombinant allergens, well-defined allergy vaccines for allergen-specific immunotherapy can be produced. Furthermore, they can be modified to reduce their allergenic activity and to avoid IgE-mediated side effects. Here, we demonstrate that the immunogenicity of two grass pollen-derived hypoallergenic allergen derivatives could be increased by engineering them as a single hybrid molecule. We used a hypoallergenic Phl p 2 mosaic, generated by fragmentation of the Phl p 2 sequence and reassembly of the resulting peptides in an altered order, and a truncated Phl p 6 allergen, to produce a hybrid protein. The hybrid retained the reduction of IgE reactivity and allergenic activity of its components as shown by ELISA and basophil activation assays. Immunization with the hybrid molecule demonstrated the increased immunogenicity of this molecule, leading to higher levels of allergen-specific IgG antibodies compared to the single components. These antibodies could inhibit patients' IgE binding to the wild-type allergens. Thus, the described strategy allows the development of safer and more efficacious vaccines for the treatment of grass pollen allergy.

Cell Biology and Signaling

June 6, 2008

Epigallocatechin gallate inhibits endothelial exocytosis

Munekazu Yamakuchi, Clare Bao, Marcella Ferlito, Charles J. Lowenstein

Page range: 935-941

Abstract

Consumption of green tea is associated with a decrease in cardiovascular mortality. The beneficial health effects of green tea are attributed in part to polyphenols, organic compounds found in tea that lower blood pressure, reduce body fat, decrease LDL cholesterol, and inhibit inflammation. We hypothesized that epigallocatechin gallate (EGCG), the most abundant polyphenol in tea, inhibits endothelial exocytosis, the initial step in leukocyte trafficking and vascular inflammation. To test this hypothesis, we treated human umbilical-vein endothelial cells with EGCG and other polyphenols, and then measured endothelial exocytosis. We found that EGCG decreases endothelial exocytosis in a concentration-dependent manner, with the effects most prominent after 4 h of treatment. Other catechin polyphenols had no effect on endothelial cells. By inhibiting endothelial exocytosis, EGCG decreases leukocyte adherence to endothelial cells. In searching for the mechanism by which EGCG affects endothelial cells, we found that EGCG increases Akt phosphorylation, eNOS phosphorylation, and nitric oxide (NO) production. NOS inhibition revealed that NO mediates the anti-inflammatory effects of EGCG. Our data suggest that polyphenols can decrease vascular inflammation by increasing the synthesis of NO, which blocks endothelial exocytosis.

Proteolysis

June 6, 2008

Dimerization of endogenous MT1-MMP is a regulatory step in the activation of the 72-kDa gelatinase MMP-2 on fibroblasts and fibrosarcoma cells

Signe Ingvarsen, Daniel H. Madsen, Thore Hillig, Leif R. Lund, Kenn Holmbeck, Niels Behrendt, Lars H. Engelholm

Page range: 943-953

Abstract

The secreted gelatinase matrix metalloprotease-2 (MMP-2) and the membrane-anchored matrix metalloprotease MT1-MMP (MMP-14), are central players in pericellular proteolysis in extracellular matrix degradation. In addition to possessing a direct collagenolytic and gelatinolytic activity, these enzymes take part in a cascade pathway in which MT1-MMP activates the MMP-2 proenzyme. This reaction occurs in an interplay with the matrix metalloprotease inhibitor, TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex with one TIMP-2 molecule. We provide positive evidence that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of fibroblasts and fibrosarcoma cells. Even in the absence of transfection and overexpression, dimerization of MT1-MMP markedly stimulated the formation of active MMP-2 products. The effect demonstrated here was brought about by a monoclonal antibody that binds specifically to MT1-MMP as shown by immunofluorescence experiments. The antibody has no effect on the catalytic activity. The effect on proMMP-2 activation involves MT1-MMP dimerization because it requires the divalent monoclonal antibody, with no effect obtained with monovalent Fab fragments. Since only a negligible level of proMMP-2 activation was obtained with MT1-MMP-expressing cells in the absence of dimerization, our results identify the dimerization event as a critical level of proteolytic cascade regulation.

June 6, 2008

Reconstitution of human azurocidin catalytic triad and proteolytic activity by site-directed mutagenesis

Mariusz Olczak, Katarzyna Indyk, Teresa Olczak

Page range: 955-962

Abstract

Azurocidin belongs to the serprocidin family, but it is devoid of proteolytic activity due to a substitution of His and Ser residues in the catalytic triad. The aim of this study was to reconstitute the active site of azurocidin by site-directed mutagenesis, analyze its processing and restored proteolytic activity. Azurocidin expressed in Sf 9 insect cells possessing the reconstituted His41-Asp89-Ser175 triad exhibited significant proteolytic activity toward casein with a pH optimum of approximately 8–9, but a reconstitution of only one active site amino acid did not result in proteolytically active protein. Enzymatically active recombinant azurocidin caused cleavage of the C-terminal fusion tag with the primary cleavage site after lysine at Lys-Leu and after alanine at Ala-Ala, and the secondary cleavage site after arginine at Arg-Gln, as well as with low efficiency caused cleavage of insulin chain B after leucine at Leu-Tyr and Leu-Cys, and after alanine at Ala-Leu. We demonstrate that cleavage of the azurocidin C-terminal tripeptide is not necessary for its enzymatic activity. The first isoleucine present in mature azurocidin can be replaced by similar amino acids, such as leucine or valine, but its substitution by histidine or arginine decreases proteolytic activity.

About this journal

Objective
Biological Chemistry keeps you up-to-date with all new developments in the molecular life sciences. In addition to original research reports, authoritative reviews written by leading researchers in the field keep you informed about the latest advances in the molecular life sciences. Rapid, yet rigorous reviewing ensures fast access to recent research results of exceptional significance in the biological sciences.

Topics

general biochemistry
pathobiochemistry
structural biology
molecular and cellular biology
genetics and epigenetics
molecular medicine
cancer research
virology
immunology
plant molecular biology and biochemistry
novel experimental methodologies

Article formats
Research Articles, Short Communications, Reviews and Minireviews
Reviews are published by invitation only, but suggestions to the Editor-in-Chief are welcome.

The journal is associated with the German Society for Biochemistry and Molecular Biology (GBM).