1[alpha],25-dihydroxyvitamin D(3) enhances annexin II dependent proliferation of osteoblasts.

Cells experience a variety of physiological and non-physiological stresses and consequently have appropriate mechanisms to deal with such deviations from homeostasis. Particularly subject to mechanical stress and shear forces are the cells that make up the bones. Osteoblastic cells can interpret this stress as a stimulus for proliferation; however, the molecular mechanisms underlying this phenomenon are poorly understood. We have identified annexin II as being specifically upregulated in mechanically stressed osteoblasts and found that increased levels of this protein are necessary for 1[alpha],25-dihydroxyvitamin D(3) mediated augmentation of the proliferative response of osteoblasts after mechanical stress. Our data demonstrate a novel interaction between 1[alpha],25-dihydroxyvitamin D(3) and annexin II in the proliferative response of osteoblasts as well as a novel function for annexin II in the stress response. These findings may offer new therapeutic opportunities for conditions that require regenerative osteoblastic activity such as osteoporosis.

Polyploidization and centrosome hyperamplification in inflammatory bronchi.

OBJECTIVE AND DESIGN: Inflammatory and tumorous bronchi were screened in order to obtain new tumor relevant cytogenetic parameters. MATERIAL OR SUBJECTS: Bronchial cells of 32 patients were cultivated by standard cell culture procedures. METHODS: Tetraploidy and aneuploidy was determined by enumeration of chromosome 7 and 8 versus the number of centrosomes. The resulting data were correlated with histopathological data. RESULTS: Tetra- and aneuploidy of epithelial cells were detectable in 76% of tumor cell cultures, 75% of high grade inflammatory tissues and 40% of non- and low grade-inflammatory tissues. Additionally, we observed centrosome hyper-amplification and multipolar mitoses not only in the tumor but also in the early stages of inflammation. CONCLUSION: Inflammatory bronchi already show tumor-specific features and may consequently represent the preliminary genetic stage of cancer development in bronchi.

Total synthesis of rutamycin B, a macrolide antibiotic from Streptomyces aureofaciens.

Rutamycin B (2) was synthesized from three principal subunits, spiroketal 75, keto aldehyde 83, and aldehyde 108. First, triol 62 was assembled by Julia coupling of sulfone 56 with aldehyde 58 followed by an acid-catalyzed spiroketalization. The three hydroxyl functions of 62 were successfully differentiated, leading to phosphonate 75. The latter was condensed in a Wadsworth-Emmons reaction with 83, prepared in six steps from (R)-aldehyde 76, to give 92. Coupling of the titanium enolate of 92 with 108 afforded Felkin product 109 with high stereoselectivity in a process that is critically dependent on the presence of the p-methoxybenzyl ether in the aldehyde. Transformation of 109 via aldehyde 116 to vinylboronate 122 was followed by macrocyclization under Suzuki conditions to yield 123. Exhaustive desilylation of the latter yielded rutamycin B.

The scanning near-field optical microscope as a tool for proteomics.

The identification of the entire genetic code of human DNA is more or less completed. With this knowledge, research in identifying the real information lying in the genes, will begin. This information is contained in the proteins, which are the main biological actors in the cell. For this reason proteins will be targeted in biological investigations in the future. The structure, affinity and reactivity of each identified protein has to be determined, which is a primary goal in the field of proteomics. This will require new and better strategies to identify protein-protein interaction. Our approach, based on the detection and visualization of single proteins by scanning near-field optical microscopy (SNOM), has allowed us to visualize various fixed and fluorochrome-labelled proteins at the nanometer scale. Subsequently SNOM may then be developed to efficiently detect the specific behavior of a certain protein in response to other biomolecules.

Polyploidization: a Janus-faced mechanism.

Normal human somatic cells are diploid. But sometimes certain tissues of the human body contain elevated numbers of tetraploid cells. This tetraploid cell population seems to represent the first step of an ongoing process of polyploidization. All tissues containing tetraploid cells have in common the fact that they are subjected to stress, which is caused by a variety of circumstances like inflammation, elevated metabolism, ageing, repair processes or selection pressure. Tetraploid cells are supposed to play a beneficial role in these stress situations, because they are known to be more resistant in general and because they are characterized by an elevated biosynthetic activity. In contrast to their beneficial character, they have a big potential concerning the malignant development of a tissue: they play a crucial role in early morphological stages of the pathway hyperplasia-metaplasia-dysplasia-carcinoma. This report links several intracellular mechanisms with each other, which potentially determine the real fate of tetraploid cells.

p53 and c-Jun functionally synergize in the regulation of the DNA repair gene hMSH2 in response to UV.

The tumor suppresser protein p53 is critical for guarding the genome from incorporation of damaged DNA (Lane, D. P. (1992) Nature 358, 15-16). A relevant stress that activates p53 function is UV light (Noda, A., Toma-Aiba, Y., and Fujiwara, Y. (2000) Oncogene 19, 21-31). Another well known component of the mammalian UV response is the transcription factor c-Jun (Angel, P., and Karin, M. (1991) Biochim. Biophys. Acta 1072, 129-157). We show here that upon UV irradiation p53 activates transcription of the human mismatch repair gene MSH2. Interestingly, this up-regulation critically depends on functional interaction with c-Jun. Hence, the synergistic interaction of a proto-oncogene with a tumor suppresser gene is required for the regulation of the mammalian stress response through activation of expression of MSH2.

Molecular Nanocapsules Based on Amphiphilic Hyperbranched Polyglycerols.

Polar dyes can be solubilized in apolar media-molecular nanocapsules with hydrophilic interiors have been prepared (see schematic representation) using polyglycerols with narrow polydispersity and simple esterification with fatty acids. These unimolecular micelles offer attractive potential for a variety of applications ranging from controlled drug release to the design of microreactors and catalysts.

[Relevance of life sciences for traumatologic and orthopedic surgery]. / Die Bedeutung der Biowissenschaften für die Unfallchirurgie.

During the last 15 years biology and life science underwent a dramatic development. Daily we hear about new discoveries in this area of research. There is for example the identification of new genes, which are responsible for different phenomena: cancer genes, adipositas genes and suicide genes. Furthermore there are reports of human ears growing on the back of a mice or cloning of complex organisms (i.e. Dolly). This kind of research is mostly summarized under terms like: genetic engineering, life science, genetics, molecular biology, biochemistry, bioanalytics, tissue engineering and reproduction medicine. Because of the indiscriminate use of these terms and the complexity of the single discipline only specialists are able to understand and to assess the relevance of results. Aim of this article is to explain some of these terms and to clarify the principles of some important techniques. By this we want to show the relevance of life science for traumatology which possibly leads to new diagnostic and therapeutic concepts.

Centrosome multiplication accompanies a transient clustering of polyploid cells during tissue repair.

Cells from different human wounds were analyzed concerning their degree of ploidy. The experiments showed an increased tetraploidization rate in well-healing wounds especially during inflammation and proliferation. Recent data described a polyploidization in different tissues, which is accompanied and maybe caused by the multiplication of the centrosome. We show here for the first time that cells from nonmalignant tissue, namely human wound cells, are characterized by an extensive centrosome multiplication. In an effort to identify a certain mechanism, by which the centrosome may act as a modulator of the cells' ploidy, we focused our interest on p53, whose interaction with the centrosome was recently described. Applying a wound model onto p53-wildtype (wt) and p53-knockout (ko) mice, we could show that polyploidization was not reversible in p53-ko mice during wound healing. The lack of p53, the centrosome multiplication, and the polyploidization therefore may contribute to the physiological process of tissue repair in physiologically "normal" tissue.

Tetraploidization is a physiological enhancer of wound healing.

To investigate the role of chromosomal alterations in the process of wound healing on the cellular level, we analyzed biopsies from well-healing (10) and chronic defect (8) wounds. Classical chromosome preparation and fluorescence in situ hybridization were performed with cultured cells and smear preparations. Results from both techniques showed an unusual high rate of tetraploid cells (4 n) in granulation tissue of well-healing wounds (6.5-60%), whereas we found only a low amount of tetraploid cells (from 0 to 5.5%) in chronic wounds. In fibroblast control cultures, there was a percentage of 2-5.5%. In chromosome preparations, we noticed an increased number of nonclonal structural and numerical chromosome aberrations in both well-healing and chronic wounds. Our data show clearly that especially tetraploidization is a typical phenomenon in the well-healing wound, where it apparently supports the healing process.

Primary leiomyosarcoma of bone: clinicopathologic, immunohistochemical, and molecular biologic aspects.

BACKGROUND: Primary leiomyosarcoma of bone is a very rare malignant tumor with uncertain pathogenicity. METHODS: The authors studied five cases of surgically treated primary leiomyosarcoma of bone. Clinical histories and radiographic findings were recorded. Regular clinical and radiographic controls were obtained postoperatively. In all cases, immunohistochemical studies were used to confirm the diagnosis. Molecular biologic examinations, using the polymerase chain reaction technique with microsatellite DNA markers from regions of tumor-relevant genes, were performed to determine the stability of the genome or to detect some typical genomic changes. RESULTS: The study included three women and two men, with an average age of 42 years. The tumor was located in the pelvis in two patients, in the femur in two patients, and in the proximal tibia in one patient. All tumors were classified as high-grade tumors (four stage IIB, one stage IIA). Radiographically, all tumors appear as purely osteolytic lesions, with a geographic or moth-eaten appearance and without any sclerotic margin. Three patients underwent limb salvage surgery followed by endoprosthetic replacement. The other two patients required amputation. The mean follow-up was 19 months (range, 8-29 months). Three patients died of disease, with a mean postoperative survival period of 18 months (range, 6-27 months). Four patients developed diffuse pulmonary metastases after an average of 10.5 months. One of those patients responded well to chemotherapy. In all cases, immunohistochemistry showed strong reactivity of the tumor cells for (alpha-SMA and vimentin. Molecular biologic investigations revealed a high rate of genomic instabilities in all of the stage IIB tumors. CONCLUSION: Clinical follow-up suggests that primary osseous leiomyosarcoma has an aggressive biologic behavior. The immunohistochemical studies are useful tools and suggest that osseous leiomyosarcoma arise from the vascular smooth muscle cells within the bone. The molecular biologic findings of a high rate of genomic instability confirm the hypothesis that this rare entity is of an aggressive nature.

Invasion of human cartilage by cultured multicellular spheroids of rheumatoid synovial cells--a novel in vitro model system for rheumatoid arthritis.

OBJECTIVE: A new 3-dimensional cell culture system was established to examine the invasion of rheumatoid synovial cells into cartilage. METHODS: Synovial cells from 20 patients with rheumatoid arthritis (RA) and 15 patients with osteoarthritis (OA) were co-cultured as multicellular spheroids with cartilage fragments. RESULTS: After 4 weeks the rheumatoid spheroids eroded the cartilage. The destruction of cartilage was supported by a high expression level of cathepsin D and matrix metalloproteinases. In contrast, the OA tissue showed attachment to the cartilage only. CONCLUSION: Our results indicate that spheroid/cartilage co-cultures seem to be a suitable in vitro model for the study of destructive cellular mechanisms that occur in RA.

[Significance of molecular biology research for trauma surgery exemplified by wound and bone healing]. / Die Bedeutung der molekularbiologischen Forschung für die Unfallchirurgie am Beispiel der Wund- und Knochenheilung.

During the past years molecular biology has become increasing interesting for medical research. These techniques allow the identification of intra-, extra- and intercellular mechanisms, which are important for physiological and pathophysiological processes. Because healing takes place at the cellular level, molecular biology is also relevant for traumatological research. As of yet, there are only a few papers which deal with molecular biological and traumatological problems. For this reason, we only have little knowledge of the function of genes and proteins during wound and fracture healing, for example. To demonstrate the possibilities of molecular biology, we present an experimental strategy by which these techniques can help to answer special traumatological questions.

[Quantitative determination of cell composition of human granulation tissue by fluorescence activated cell sorting (FAC)]. / Quantitative Bestimmung der Zellzusammensetzung von humanem Granulationsgewebe durch Fluoreszenz Aktiviertes Cell Sorting (FACS).

Granulation tissue of normal and non-healing human wounds showed a similar distribution pattern of the cell populations investigated by FACS analysis, whereas only non-healing wounds revealed a reduced density of cells and intercellular matrix. Thus, supporting the thesis that impaired wound healing is not caused by changes of the cellular distribution pattern of granulation tissue, but possibly by lessened cell function.

Heat-preconditioning confers protection from Ca(2+)-mediated cell toxicity in renal tubular epithelial cells (BSC-1).

A rise in intracellular calcium may mediate ischemic damage by phospholipid hydrolysis and proteolysis. Heat shock proteins have been shown to provide protection from various forms of cell stress, but not from models of Ca(2+)-mediated injury. The effect of heat preconditioning in a model of ionomycin-induced injury in cultured renal tubular epithelial cells (BSC-1) was examined. Hsp70-mRNA expression was induced by hyperthermia (HT) (42 degrees C, 60 min). Hsp70 protein accumulation was maximal after 12-18 h and returned to baseline levels by 96 h. Treatment of BSC-1 cells with ionomycin (7.0 microM) produced lethal cell injury characterized by LDH release. Cells examined at 18 h after HT were significantly less damaged than cells studied at 96 h after HT. Our data are the first to demonstrate that heat preconditioning confers protection from Ca(2+)-mediated cell injury. The state of increased tolerance is transient and closely parallels kinetics of Hsp70 expression.

Identification of human semaphorin E gene expression in rheumatoid synovial cells by mRNA differential display.

Rheumatoid arthritis (RA) is characterised by chronic inflammation of synovial tissue with aggressive proliferation of synovial cells causing destruction of cartilage and bone. Immunopathological mechanisms, infectious causes and genetic factors have been discussed, but the etiology of the disease has not been understood until now. Especially, the mechanisms driving tumourlike growth and invasive behaviour of fibroblastoid synovial cells have not been identified yet. Our aim is to find cellular factors which are mediators for such pathways. One possibility to approach this, is searching for disease-relevant genes. We applied the mRNA-differential display technique to compare mRNA expression patterns of normal and rheumatic synovial fibroblasts. We identified an upregulation of the human semaphorin E gene in rheumatoid synovial fibroblastoid cells. Interestingly semaphorin E is a member of a protein-family described to play an immunosuppressive role via inhibition cytokines. A relevance of this finding towards the pathogenesis of RA is discussed.

Evidence for a functional link between stress response and vascular control in hepatic portal circulation.

Heme oxygenase (HO)-derived carbon monoxide (CO) may contribute to vascular control through elevation of guanosine 3',5'-cyclic monophosphate. In the present study, we investigated the functional significance of expression of the isoenzyme HO-1 (heat-shock protein 32) in liver after hemorrhage/resuscitation (H/R) in rats anesthetized with pentobarbital sodium. An increase of mRNA levels for HO-1 was observed at 3 h after resuscitation, followed by induction of the protein at 6 h in pericentral hepatocytes and sinusoidal lining cells. Concomitantly, lower portal resistance was observed in H/R (0.33 +/- 0.060 mmHg.ml-1.min) compared with control rats (0.47 +/- 0.035 mmHg.ml-1.min). Blockade of the HO-CO pathway by tin protoporphyrin-IX (SnPP-IX) led to a transient increase in portal pressure with no effect on portal low in controls, whereas an increase in pressure and a decrease in flow contributed to the sustained increase in portal resistance after H/R. These results indicate that HO contributes to maintenance of hepatic perfusion in vivo under stressful conditions, suggesting a functional link between stress response and vascular control in portal circulation.

Constitutive expression of c-fos and c-jun, overexpression of ets-2, and reduced expression of metastasis suppressor gene nm23-H1 in rheumatoid arthritis.

OBJECTIVES: To identify genes that are involved in the development and progression of rheumatoid arthritis (RA). METHODS: We used a multiple gene analysis system and a set of available genes participating in processes such as proliferation, differentiation, tumour progression, and metastasis, to identify their RA related expression. Synovial tissues from 22 patients with RA were evaluated in comparison with those from six patients with osteoarthritis and two patients with non-inflamed joints as controls, using northern blot and reverse transcriptase polymerase chain reaction experiments. RESULTS: Our data confirm the role of c-fos and c-jun as constitutive signal transmitters in solid RA tissues, thus demonstrating the potential of the approach. Activation of both genes persisted through multiple passages of the cells in tissue cultures derived from the synovial lining of RA tissues. There was an increased expression of ets-2 in 30% of RA samples and an up to 30-fold decreased expression of the potential metastasis suppressor gene nm23-H1 in 90% of RA tissues, compared with control tissues. CONCLUSIONS: The data presented show for the first time a significant decrease of nm23-H1 expression in RA, which is possibly involved in local invasiveness, and a strong activation of the ets-2 nuclear oncogene in about one third of RA tissues, which may also be part of a pathway leading to advanced disease stages. The constitutive expression of c-fos and c-jun in RA tissue most probably results from a continuing inflammatory stimulus. These findings with cell cultures suggest an intrinsic activation mechanism of these early response genes in RA.

Differential expression of heat shock protein 70 in well healing and chronic human wound tissue.

Heat shock protein 70 (hsp 70) is an important member of the heat shock protein family, which is induced by different forms of stress. We attempted to find out if hsp 70 is also involved in wound healing, which likewise resembles a stress situation for cells too. Therefore we collected tissue samples from well healing and chronic human wound tissue. We used Northern- and Western-blot analysis to study the expression of hsp 70. At the protein level we found a strong correlation between well healing wounds and high expression of hsp 70, whereas chronic wounds showed no or weak expression. Interestingly hsp 70 mRNA did not show this significant correlation, displaying a variant expression pattern in the same kind of wound tissue, possibly due to unknown posttranscriptional regulating step, which has to be investigated in further studies. To localize hsp 70 mRNA and protein was used insitu hybridization and immunohistochemistry. Both displayed an overexpression in endothelial cells of capillary vessels.