Novel benzimidazole phosphonates as potential inhibitors of protein prenylation

Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.     

Soft tissue fibroblasts from well healing and chronic human wounds show different rates of myofibroblasts in vitro

Due to an increasing life expectancy in western countries, chronic wound treatment will be an emerging challenge in the next decades. Because therapies are improving slowly appropriate diagnostic tools enabling the early prediction of the healing success remain to be developed. We used a well-established in vitro assay in combination with the analysis of 27 cytokines to discriminate between fibroblasts from chronic (n = 6) and well healing (n = 8) human wounds. Proliferation and migration of the cells as well as their response to hypoxia and their behaviour in co-culture with microvascular endothelial cells were analyzed. Myofibroblast differentiation, a time-limited essential process of regular wound healing, was also quantified. Besides weaker proliferation and migration significantly higher rates of myofibroblasts were detected in chronic wounds. With respect to the cytokine release, there was a clear trend within the group of chronic wound fibroblasts, which were releasing interferon-γ, monocyte chemotactic protein-1, granulocyte–macrophage colony stimulating factor and basic fibroblast growth factor in higher amounts than fibroblasts from healing wounds. Although the overall response of both groups of fibroblasts to hypoxia and to the contact with endothelial cells was similar, especially chronic wound fibroblasts seemed to benefit from the endothelial interaction during hypoxia and displayed better migration characteristics. The study shows (1) that the assay can identify specific features of fibroblasts derived from different human wounds and (2) that wound fibroblasts are varying in their response to the chosen parameters. Thus, current therapeutic approaches and individual healing prediction might benefit from this assay.     

A quantitative method for separation of livingHydra cells

Summary We describe a rapid method for the isolation of large numbers of livingHydra cells of defined cell type in an isotonic cell medium (Gierer et al. 1972). Intact animals are enzymatically dissociated into a single cell suspension and the various cell types separated in less than one hour by counterflow centrifugation elutriation. Cell loss is minimal. RNA isolated from various fractions can be probed with cell type specific cDNA-clones.     

Cell sorting during the regeneration of Hydra from reaggregated cells.

The role of cell sorting in the reorganization of Hydra cell reaggregates was studied. We quantitatively labeled ectodermal and endodermal cells by incubating whole animals in fluorescent beads or by injecting the beads into the gastric cavity. Beads were stably incorporated into the cells by phagocytosis. Our data show that dramatic cell sorting processes drive the formation of ectoderm and endoderm within the first 12 hr of reaggregation. After the ectoderm is established, no further rearrangement could be observed. We also tested the ability of cells to sort out with respect to their original position in Hydra by dissociating labeled apical and basal pieces of Hydra and measuring the clumping of labeled cells during reorganization. There was no increase in the clumping of cells during reorganization indicating that cell sorting is not involved in the formation of early activation centers. There was also no preferential incorporation of apically derived (presumptive head) tissue into tentacles that subsequently formed, indicating that after dissociation into single cells there is no predisposition of erstwhile presumptive head tissue to form heads.     

Method of non-destructive mechanical testing of new surface coatings for prostheses]

Research into new surface coatings and surface processing methods for prostheses is subject to numerous studies. The aim of this study was to test an innovative biomechanical measuring method for the examination of the ingrowth of bone implants. Using a transcortical model, coated (n=14) or uncoated (n=14) titanic cylinders were implanted into the lateral condyle of 28 New Zealand White Rabbits. After 6 weeks or 6 months the animals were sacrificed and the osseointegration of the implants was evaluated biomechanically and histologically. Up to traction of 50 N the load dependent movement between bone and testing cylinder did not lead to a destruction of the bone-implant-interface. Therefore, biomechanical and histological investigations could be performed in the same specimen. The results of both evaluations showed a significant correlation (correlation coefficient -0.79; p < 0.01) and were absolutely reproducible. With the method of non-destructive mechanical testing, it is possible to halve the number of required animals. Additionally, the results of the biomechanical and histological analysis can be compared and thus serve as an internal control. In summary, the method of non-destructive mechanical testing represents an ideal tool to study new surface coatings and surface processing methods for prostheses.     

The Leydig cell of the human testis —A new member of the diffuse neuroendocrine system

A number of marker substances for neuronal and neuroendocrine cells have been demonstrated in the cytoplasm of the interstitial Leydig cells of human testes using basic immunocytochemical methods and some of their modifications. We were able to reveal immunoreactivity for enzymes involved in the synthesis of the catecholamines dopamine and noradrenaline (tryosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine-β-hydroxylase), for the indolamine 5-hydroxytryptamine (serotonin), as well as for a number of wellknown neuronal markers such as the neurofilament protein 200, synaptophysin, chromogranin A+B, the neural cell-adhesion molecule (N-CAM), the microtubule-associated protein (MAP-2), and the calcium-binding proteins: S-100, calbindin and parvalbumin. Immunoreactivity for these substances was found in the majority of the interstitial cells although differences in the staining intensity among the individual Leydig cells and among Leydig cells from different patients were observed. At the electron-microscopic level the Leydig cell cytoplasm was seen to contain microtubules, intermediate- and microfilaments as well as clear (40–60 nm) and dense-core (100–300 nm) vesicles, providing a morphological correlate for some of the immunocytochemical results. Although individual marker substances are not absolutely specific for nerve and neuroendocrine cells, the results obtained, together with the already established neuronspecific enolase-, substance P-, methionine-enkephalinand proopiomelanocortin (POMC)-derived peptide-like immunoreactivity, provide strong evidence for the neuroendocrine (paraneuronal, APUD-like) nature of the Leydig cells of the human testis. Dedicated to Professor Dr. Werner Hilscher on the occasion of this 65th birthday     

Intraepithelial lymphocytes and macrophages in the human epididymis

The epididymides from 7 young adults and 5 older patients with prostatic cancer were investigated by means of light and electron microscopy. The existence of lymphocytes and macrophages between epithelial cells of various segments of the ductuli efferentes and the ductus epididymidis is proven, and the distribution and morphology of these cells are described. A possible relationship between these two intraepithelial cell types, as well as their presumptive role with regard to an immunological barrier-function are discussed.     

Mobile phase modifier effects in multimodal cation exchange chromatography

This study examines protein adsorption behavior and the effects of mobile phase modifiers in multimodal chromatographic systems. Chromatography results with a diverse protein library indicate that multimodal and ion exchange resins have markedly different protein binding behavior and selectivity. NMR results corroborate the stronger binding observed for the multimodal system and provide insight into the structural basis for the observed binding behavior. Protein-binding affinity and selectivity in multimodal and ion exchange systems are then examined using a variety of mobile phase modifiers. Arginine and guanidine are found to have dramatic effects on protein adsorption, yielding changes in selectivity in both chromatographic systems. While sodium caprylate leads to slightly weaker chromatographic retention for most proteins, certain proteins exhibit significant losses in retention in both systems. The presence of a competitive binding mechanism between the multimodal ligand and sodium caprylate for binding to ubiquitin is confirmed using STD NMR. Polyol mobile phase modifiers are shown to result in increased retention for weakly bound proteins and decreased retention for strongly bound proteins, indicating that the overall retention behavior is determined by a balance between changes in electrostatic and hydrophobic interactions. This work provides an improved understanding of protein adsorption and mobile phase modifier effects in multimodal chromatographic systems and sets the stage for future work to develop more selective protein separation systems.     

Proteomic analysis of the androgen receptor via MS-compatible purification of biotinylated protein on streptavidin resin

The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.