Wolfgang Voelter

Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR.

Histone deacetylases (HDACs) play a key role in regulating eukaryotic gene expression. The HDAC domain, homologous to the yeast repressors RPD3 and HDA1, is considered necessary and sufficient for enzymatic activity. Here, we show that the catalytic domain of HDAC4 interacts with HDAC3 via the transcriptional corepressor N-CoR/SMRT. All experimental conditions leading to the suppression of HDAC4 binding to SMRT/N-CoR and to HDAC3 result in the loss of enzymatic activity associated with HDAC4. In vitro reconstitution experiments indicate that HDAC4 and other class II HDACs are inactive in the context of the SMRT/N-CoR-HDAC3 complex and do not contribute to its enzymatic activity. These observations indicate that class II HDACs regulate transcription by bridging the enzymatically active SMRT/N-CoR-HDAC3 complex and select transcription factors independently of any intrinsic HDAC activity.

Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo

Angiogenesis is an ordered process requiring the inter‐play of numerous cellular and humoral factors. Studies over the past 20 years have identified several growth factors, cytokines, and enzymes that promote blood vessel formation. Most have revealed how individual factors promote an angiogenic phenotype in endothelial cells in vitro or contribute to blood vessel formation in vivo. However, the fundamental question that remains unanswered is how the cellular microenvironment contributes to angiogenesis. Fibrocytes are a recently characterized mesenchymal cell type isolated from peripheral blood that rapidly enter subcutaneously implanted wound chambers and sites of tissue injury. Here we describe the induction of an angiogenic phenotype in microvascular endothelial cells in vitro and promotion of angiogenesis in vivo by cultured fibrocytes. Fibrocytes constitutively secrete extracellular matrix‐degrading enzymes, primarily matrix metalloproteinase 9, which promotes endothelial cell invasion. In addition, fibrocytes secrete several proangiogenic factors including VEGF, bFGF, IL‐8, PDGF, and hematopoietic growth factors that promote endothelial cell migration, proliferation, and/or tube formation. By contrast, they do not produce representative antiangiogenic factors. Finally, both autologous fibrocytes and fibrocyte‐conditioned media were found to induce blood vessel formation in vivo using the Matrigel angiogenesis model.—Hartlapp, I., Abe, R., Saeed, R. W., Peng, T., Voelter, W., Bucala, R., Metz, C. N. Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo. FASEB J. 15, 2215–2224 (2001)

A new family of human histone deacetylases related to Saccharomyces cerevisiae HDA1p.

Histone deacetylases are the catalytic subunits of multiprotein complexes that are targeted to specific promoters through their interaction with sequence-specific DNA-binding factors. We have cloned and characterized a new human cDNA, HDAC-A, with homology to the yeast HDA1 family of histone deacetylases. Analysis of the predicted amino acid sequence of HDAC-A revealed an open reading frame of 967 amino acids containing two domains: a NH2-terminal domain with no homology to known proteins and a COOH-terminal domain with homology to known histone deacetylases (42% similarity to RPD3, 60% similarity to HDA1). Three additional human cDNAs with high homology to HDAC-A were identified in sequence data bases, indicating that HDAC-A itself is a member of a new family of human histone deacetylases. The mRNA encoding HDAC-A was differentially expressed in a variety of human tissues. The expressed protein, HDAC-Ap, exhibited histone deacetylase activity and this activity mapped to the COOH-terminal region (amino acids 495–967) with homology to HDA1p. In immunoprecipitation experiments, HDAC-A interacted specifically with several cellular proteins, indicating that it might be part of a larger multiprotein complex.

Human HDAC7 Histone Deacetylase Activity Is Associated with HDAC3 in Vivo

Histone deacetylases (HDACs) are part of transcriptional corepressor complexes and play key roles in regulating chromatin structure. Three different classes of human HDACs have been defined based on their homology to HDACs found in Saccharomyces cerevisiae: RPD3 (class I), HDA1 (class II), and SIR2 (class III). Here we describe the identification and functional characterization of HDAC7, a new member of the human class II HDAC family. Although HDAC7 is localized mostly to the cell nucleus, it is also found in the cytoplasm, suggesting nucleocytoplasmic shuttling. The HDAC activity of HDAC7 maps to a carboxyl-terminal domain and is dependent on the interaction with the class I HDAC, HDAC3, in the cell nucleus. Cytoplasmic HDAC7 that is not bound to HDAC3 is enzymatically inactive. We provide evidence that the transcriptional corepressors SMRT and N-CoR could serve as critical mediators of HDAC7 activity by binding class II HDACs and HDAC3 by two distinct repressor domains. Different class II HDACs reside in the cell nucleus in stable and autonomous complexes with enzymatic activity, but the enzymatic activities associated with HDAC7 and HDAC4 rely on shared cofactors, including HDAC3 and SMRT/N-CoR.

Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes

The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non‐specific actin polymerization during the assay, fluorescent G‐actin was mixed with thymosin β4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.

Pheromone Cross-Inhibition between Staphylococcus aureus and Staphylococcus epidermidis

ABSTRACT Cross-inhibition by quorum-sensing pheromones betweenStaphylococcus aureus and Staphylococcus epidermidis was investigated using all known S. aureus agr pheromone subgroups. All S. aureus subgroups were sensitive towards the S. epidermidis pheromone, with the exception of the recently identified subgroup 4. The subgroup 4 pheromone was also the only S. aureus pheromone able to inhibit the S. epidermidis agr response. The close relation of subgroup 4 to subgroup 1 suggests that subgroup 4 might have evolved from subgroup 1 by mutation under the selective pressure of competition with S. epidermidis. The competition between S. aureus and S. epidermidis by means of quorum-sensing cross talk seems to be generally in favor of S. epidermidis, which might explain the predominance of S. epidermidis on the skin and in infections on indwelling medical devices.

Determination of selected pyrimidines, purines and their metabolites in serum and urine by reversed-phase ion-pair chromatography.

The qualitative and quantitative determination of selected pyrimidines, purines and azapurines and their metabolites by reversed-phase ion-pair high-performance liquid chromatography, using tetrabutylammonium hydroxide as pairing ion and isocratic chromatrographic conditions, is described. The method provides a selective and sensitive assay for these classes of compounds examined in complex biological fluids.

The Extracellular Human Melanoma Inhibitory Activity (Mia) Protein Adopts an SH3 Domain-Like Fold.

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain‐like fold in solution, with two perpendicular, antiparallel, three‐ and five‐stranded β‐sheets. In contrast to known structures with the SH3 domain fold, MIA is a single‐domain protein, and contains an additional antiparallel β‐sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain‐like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin α4β1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.

A novel method for the syntheses of symmetrical disulfides using CsF–Celite as a solid base

Abstract The oxidative coupling of aliphatic, aromatic and heteroaromatic thiols to disulfides using cesium fluoride–Celite is described. CsF–Celite provides an efficient, convenient and practical method for the syntheses of symmetrical disulfides.

A study on purified bovine erythrocuprein

Abstract Erythrocuprein was isolated from bovine blood by precipitation with organic solvents and chromatography (Sephadex G-75 and DEAE-Sephadex A-50). The preparation was electrophoretically pure, had an absorption ratio A 259 nm A 680 nm = 31.5 and contained 2 atoms of copper and 2 atoms of zinc per mole. Preincubation at 45° in the presence of 1% sodium dodecyl sulphate resulted in a striking change of the electrophoretic properties of the protein: The molecular weights of the two bands observed were calculated to be 16 000 (monomeric species) and 64 000 (tetrameric species). Surprisingly, no dimeric form was obtained. However, the monomeric unit was obtained exclusively by preincubation with sodium dodecyl sulphate at 100° for 1 min. Upon cooling of erythrocuprein solutions to 77°K a marked increase of the absorption in the visible region was observed and the shoulder at 430 nm was more distinct. The EPR signal of Cu 2+ in the protein showed axial symmetry. Superhyperfine splittings were observed in the g ⊥ region, indicating possible coordination of the copper to one or more nitrogen atoms. Reductive titration with dithionite gave evidence of a transfer of two electrons per molecule or of one electron per Cu 2+ . The optical rotatory dispersion and magnetic optical rotatory dispersion spectra, recorded from 220 to 600 nm, show only one trough (225 nm) or peak (around 225 nm), respectively. In the spectral range from 200 to 250 nm the circular dichroism (CD) spectrum shows only one negative Cotton effect at λ = 208 nm which is modified only slightly in sodium dodecyl sulphate or urea. From these spectral characteristics it is suggested that the helical content of erythrocuprein is low. It is demonstrated that magnetic circular dichroism is a tool for resolving overlapping CD and absorption bands in the region of the copper d → d transition.