Comparative differentiation analysis of distinct oral tissue-derived cells in response to osteogenic stimulation.

BACKGROUND: Mixed cell populations from oral tissues may be superior to pure stem cells for regenerative approaches. Therefore, the aim of the present study was to explore the osteogenic potential of mixed cells derived from oral connective tissues compared to alveolar osteoblasts. MATERIALS AND METHODS: Primary cells were isolated from the alveolar bone, periodontal ligament and gingiva. Following characterization by colony formation, growth capacity and flow cytometry, all cells were subjected to osteogenic differentiation induction and screened for a large panel of osteogenic markers using western blots, qPCR arrays, and matrix mineralization and alkaline phosphatase quantification. RESULTS: Non-induced mixed cells from gingiva showed higher colony formation efficiency but decreased proliferation compared to non-induced periodontal mixed cells, while both entities revealed similar surface markers tested in this setup. Following osteogenic induction, all cell populations individually expressed receptors with distinctively activated downstream effectors. Gene expression of induced periodontal mixed cells was similar to alveolar osteoblasts, but was differently modulated in gingival mixed cells. The latter failed to achieve osteogenic differentiation in terms of matrix mineralization and alkaline phosphatase activity, which was well observed in periodontal mixed cells and osteoblasts. CONCLUSION: Mixed cells from periodontal ligament but not from gingiva feature an inherent osteogenic capacity in vitro. From these results, it can be concluded that periodontal cells do not require further stem cell enrichment in order to qualify for bone regeneration. CLINICAL RELEVANCE: Our data contribute to the development of novel cell-based therapies using mixed cells from the periodontal ligament in regenerative periodontics.

Recurrence rate and shift in histopathological differentiation of oral squamous cell carcinoma ­ A long-term retrospective study over a period of 13.5 years.

OBJECTIVE: Little information is available as to whether recurrences of oral squamous cell carcinoma (OSCC) show different histopathological grades than the primary tumor and whether postoperative radiotherapy (PORT) influences the grade of differentiation in the case of recurrence. The objective of this study was the evaluation of recurrence rates and change in differentiation. MATERIAL AND METHODS: This retrospective, single-institution cohort study included surgically treated OSCC patients over a 13-year period (2000-2013). The relationship among tumor size, lymph node metastases, and recurrence rate of OSCC was investigated. Primary tumor differentiation was compared with differentiation of recurrence. RESULTS: A total of 429 patients (277 men and 152 women) were included in this study. Of these, 124 (28.9%) received PORT. The incidence of primary cervical metastases increased significantly with tumor size (p < 0.001). Recurrence developed in 82 patients (19.1%). Stage T1/T2 showed a significantly lower recurrence rate than stage T3/T4 (16.3% vs. 30.2%) (p < 0.01). A total of 23 (30.7%) patients with recurrence showed a change in differentiation. CONCLUSION: Increasing primary tumor size correlates with incidence of cervical metastases and recurrence rate. Initial cervical metastases show no effect on recurrence rates. Differentiation of primary tumor does not correlate with the recurrence rate. The majority of recurrences show consistent histopathological grading.

Lysyl oxidase propeptide inhibits prostate cancer cell growth by mechanisms that target FGF-2-cell binding and signaling.

Enhanced RAS signaling and decreased androgen dependence of prostate cancer cells accompany poor clinical outcomes. Elevated autocrine fibroblast growth factors 2 (FGF-2) signaling promotes prostate cancer cell growth and survival. Expression of lysyl oxidase (LOX) inhibits RAS transforming activity. LOX is secreted as 50 kDa pro-LOX protein and then undergoes extracellular proteolytic processing to form approximately 30 kDa LOX enzyme and approximately 18 kDa propeptide (LOX-PP). We have previously shown that LOX-PP inhibits breast cancer cell transformation and tumor formation, but mechanisms of action of LOX-PP have not been fully elucidated. Here we report that LOX expression is reduced in prostate cancer cell lines and that recombinant LOX-PP protein inhibits serum-stimulated DNA synthesis and MEK/ERK and PI3K/AKT pathways in DU 145 and PC-3 androgen-independent cell lines. In DU 145 cells, treatment with a pharmacologic FGF-receptor inhibitor or a neutralizing anti-FGFR1 antibody mimicked LOX-PP inhibition of serum-stimulated DNA synthesis. FGF-2-stimulated DNA synthesis, ERK1/2, AKT and FRS2alpha activation were found all to be inhibited by LOX-PP in DU 145 cells. LOX-PP reduced specific binding of FGF-2 to DU 145 cells, suggesting that LOX-PP targets FGF signaling at the receptor. Interestingly, PC-3 cells did not respond to FGF-2, consistent with previous reports. We conclude that LOX-PP inhibits proliferation of DU 145 cells by interfering with FGFR(s) binding and signaling, and that LOX-PP has other mechanisms of action in PC-3 cells.

Relationship between nonadiabaticity and damping in permalloy studied by current induced spin structure transformations.

By direct imaging we determine spin structure changes in Permalloy wires and disks due to spin transfer torque as well as the critical current densities for different domain wall types. Periodic domain wall transformations from transverse to vortex walls and vice versa are observed, and the transformation mechanism occurs by vortex core displacement perpendicular to the wire. The results imply that the nonadiabaticity parameter beta does not equal the damping alpha, in agreement with recent theoretical predictions. The vortex core motion perpendicular to the current is further studied in disks revealing that the displacement in opposite directions can be attributed to different polarities of the vortex core.

Erythropoietin regulations in humans under different environmental and experimental conditions.

In the adult human, the kidney is the main organ for the production and release of erythropoietin (EPO). EPO is stimulating erythropoiesis by increasing the proliferation, differentiation and maturation of the erythroid precursors. In the last decades, enormous efforts were made in the purification, molecular encoding and description of the EPO gene. This led to an incredible increase in the understanding of the EPO-feedback-regulation loop at a molecular level, especially the oxygen-dependent EPO gene expression, a key function in the regulation loop. However, studies in humans at a systemic level are still very scanty. Therefore, it is the purpose of the present review to report on the main recent investigations on EPO production and release in humans under different environmental and experimental conditions, including: (i) studies on EPO circadian, monthly and even annual variations, (ii) studies in connection with short-, medium- and long-term exercise at sea-level which will be followed (iii) by studies performed at moderate and high altitude.

Water immersion is associated with an increase in aquaporin-2 excretion in healthy volunteers.

Here, we report the alterations in renal water handling in healthy volunteers during a 6 h thermoneutral water immersion at 34 to 36 degrees C. We found that water immersion is associated with a reversible increase in total urinary AQP2 excretion.

A hypothesis: autonomic rhythms are reflected in growth lines of teeth in humans and extinct archosaurs.

A major determinant of tooth architecture is the arrangement of lines in dentin and in the enamel following the contour of the surface. Since the original description of these lines in the 19th century, they have been attributed to recurring events during tooth development. They have also attracted the attention of dental scientists and anthropologists; however, to date, studies of these structures have been largely theoretical and microscopic. We show here that the statistical properties of the spacing between the lines are similar in teeth from both ancient and modern humans and from extinct archosaurs, reptiles that lived tens or hundreds of millions of years ago-they also resemble heart rate variability of living humans. We propose that the deposition of these recurring structures is controlled by the autonomic nervous system. This control accounts for their regularity and recurrent nature and implies that the lines are an expression of a biologic rhythm which has been conserved throughout evolution. Details of the rhythms give clues to life styles in ancient civilizations and to the physiology of extinct archosaurs.

Changes in the autonomic reactivity pattern to psychological load under long-term microgravity--twelve men during 6-month spaceflights.

A complex psychophysiological test battery was applied to twelve subjects during long-term spaceflights. This experiment was designed to assess the psychophysiological reactivity to acute psychological stressors. A set of noninvasive physiological measurements (electrocardiogram, electromyogram, blood pressure, skin conductance, peripheral skin temperature) was used to describe the reactivity of the autonomic nervous system and the cardiovascular system to an induced series of changes between mental activity load and quiet relaxation. It could be shown that under space conditions the subjects react differently than on earth. On the basis of significantly lower heart rates we concluded that an extended parasympathetic cardial influence is present during later periods of long-term space flights. The peripheral skin conductance reactivity, however, indicated a tendency to higher peripheral sympathetic tonus under microgravity. Assuming individually different pathways of sympathetic-parasympathetic traffic under psychological stress, the whole set of data could be classified into autonomic outlet types (AOT) based on clinical reference data. Most of the subjects changed their AOT during flight. After flight the subjects eventually fell back into their pre-flight patterns, but seven of twelve subjects showed a significantly different autonomic system reactivity at least once after landing that was similar to that of hypertensive patients, indicating an extended sympathetic overshoot directly post-flight. In conclusion it is assumed that sympathetic effects in one measurement do not exclude less sympathetic or even parasympathetic effects in others during adaptation to extreme environments.

Differences in the autonomic reactivity pattern to psychological load in patients with hypertension and rheumatic diseases.

As a ground-based reference study to a space experiment, a complex psychophysiological test battery (heart rate, blood pressure, skin conductance, finger temperature, forearm electromyogram during psychological loading task solving) was developed and first applied on two cohorts of subjects with different blood pressure levels at rest (53 hypertensive patients, and 30 normal controls). The data describing autonomic reactivity could be differentiated by cluster analysis into four Autonomic Outlet Types (AOT). The method was subsequently applied to 20 patients with systemic Lupus erythematosus and 13 subjects with rheumatoid arthritis to work out a discriminant function for classifying and testing it's validity. The AOT classification of all subjects showed a significant correlation with the different types of diseases.

Markers of coagulation, fibrinolysis and angiogenesis after strenuous short-term exercise (Wingate-test) in male subjects of varying fitness levels.

It was the aim of the study to analyse the haemostatic system during a high standardized intensive short-term (30 s) exercise (anaerobic Wingate test). Blood samples were taken from 15 male subjects before (t0 ), and within 2 (t1 ), 9 (t2 ) and 30 min (t3 ) after the test. We found that the partial thromboplastin time was markedly shortened, whereas the prothrombin time increased slightly from t0 to t1 (p < 0.002) and remained elevated (t3, p < 0.046). Factor VIII increased from t0 to t1 (p < 0.001) and remained elevated as well (t3, p < 0.001). Fibrin monomers were approximately 15 times higher immediately post-exercise (t1, p < 0.001) and continued to be elevated (t3, p < 0.004). The tissue plasminogen activator increased by 4 times after exercise (t1, p < 0.001) and remained elevated (t3, p < 0.002). The d-dimers increased from t0 to t1 (p < 0.001) as well and remained elevated (t3, p < 0.005). Thrombopoietin concentrations were unchanged, whereas the vascular endothelial growth factor increased immediately post-exercise (t0 to t1, p < 0.011 resp. at t2 p < 0.019) and returned to the control level at t3 (p < 0.878). In conclusion, it was found that prothrombotic markers and, even more pronounced, those of the fibrinolytic system were increased. The study provides evidence that due to intensive short-term exercise the balance of the haemostatic system is shifted to a higher equilibrium. Theoretically, the data show that in the case of a subject with risk factors such as impaired fibrinolysis, unfavourable conditions cannot be excluded.

Oxygen uptake in whole-body vibration exercise: influence of vibration frequency, amplitude, and external load.

Vibration exercise (VbX) is a new type of physical training to increase muscle power. The present study was designed to assess the influence of whole-body VbX on metabolic power. Specific oxygen uptake (sVO(2)) was assessed, testing the hypotheses that sVO(2) increases with the frequency of vibration (tested in 10 males) and with the amplitude (tested in 8 males), and that the VbX-related increase in sVO(2) is enhanced by increased muscle force (tested in 8 males). With a vibration amplitude of 5 mm, a linear increase in sVO(2) was found from frequencies 18 to 34 Hz (p < 0.01). Each vibration cycle evoked an oxygen consumption of approximately 2.5 micro l x kg(-1). At a vibration frequency of 26 Hz, sVO(2) increased more than proportionally with amplitudes from 2.5 to 7.5 mm. With an additional load of 40 % of the lean body mass attached to the waist, sVO(2) likewise increased significantly. A further increase was observed when the load was applied to the shoulders. The present findings indicate that metabolic power in whole-body VbX can be parametrically controlled by frequency and amplitude, and by application of additional loads. These results further substantiate the view that VbX enhances muscular metabolic power, and thus muscle activity.

Serum matrix metalloproteinase-2 in patients with malignant melanoma.

PURPOSE: Matrix metalloproteinases (MMPs) are a family of structurally related zinc-dependent endopeptidases that are able to degrade extracellular matrix components. MMPs play a role in tumor invasion and tumor metastasis. MMP-2 (also known as gelatinase A) is expressed in human melanoma cells. METHODS: In this study, we measured MMP-2 in 337 serum probes of 166 melanoma patients with a recently developed enzyme immunoassay and compared these data with the tumor stage, presence of metastases, and the levels of S100beta and soluble intracellular adhesion molecule-1 (sICAM-1) in serum. RESULTS: The mean levels were (189.2 +/- 50.8) ng/ml for MMP-2, (263.2 +/- 74.1) ng/ml for sICAM-1, and (0.424 +/- 1.568) U/ml for S100beta. There was a statistical significant correlation of MMP-2 with sICAM-1 (P=0.05) and Sl00beta (P=0.01). The mean MMP-2 levels (in ng/ml) in patients with metastatic melanoma were 196.4 +/- 54.0 versus 182.6 +/- 46.9 in non-metastasizing melanoma (P=0.037). However, there was no significant difference in MMP-2 levels between the different tumor stages. CONCLUSION: Determination of MMP-2 serum levels is of limited value as a tumor marker in melanoma, though there are higher levels in the more advanced disease.

Activation of the focal adhesion kinase signaling pathway by structural alterations in the carboxyl-terminal region of c-Crk II.

The Crk II adaptor protein encodes an SH2/SH3-domain containing adaptor protein with an SH2-SH3-SH3 domain structure that transmits signals from tyrosine kinases. The two SH3 domains are separated by a 54 amino acid linker region, whose length is highly conserved in xenopus, chicken, and mamalian Crk II proteins. To gain a better understanding into the role of the C-terminal region of Crk, we generated a series of C-terminal SH3 domain and SH3 linker mutants and examined their role in tyrosine kinase pathways. Expression of point mutations in the C-terminal SH3 domain (W276K Crk), at the tyrosine phosphorylation site (Y222F Crk II), or truncation of the entire C-terminus (Crk I or Crk Delta242), all increased c-Abl binding to the N-terminal SH3 domain of Crk and, where relevant, increased Tyr(222) phosphorylation. Deletion analysis of c-Crk II also revealed the presence of a C-terminal segment important for trans-activation of FAK. Such mutants, Crk Delta255 or Crk Delta242 Extended Linker (Crk Delta242([EL])), characterized by a disruption in the SH3 linker/C-terminal SH3 boundary, induced robust hyperphosphorylation of focal adhesion kinase (FAK) on Tyr(397), hyperphosphorylation of focal adhesion proteins p130(cas) and paxillin and increased focal adhesion formation in NIH3T3 cells. The effects of Crk Delta242([EL]) could be abrogated by co-expression of dominant negative c-Src or the protein tyrosine phosphatase PTP-PEST, but not by dominant negative Abl. Our results suggest that the C-terminal region of Crk contains negative regulatory elements important for both Abl and FAK dependent signal pathways, and offers a paradigm for an autoinhibitory region in the SH3 linker/C-terminal SH3 domain.

The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction.

CMS/CD2AP is a cytoplasmic protein critical for the integrity of the kidney glomerular filtration and the T cell function. CMS contains domains and motifs characteristic for protein-protein interactions, and it is involved in the regulation of the actin cytoskeleton. We report here that the individual SH3 domains of CMS bind to phosphotyrosine proteins of approximately 80, 90, and 180 kDa in cell lysates stimulated with epidermal growth factor. The second SH3 domain of CMS bound specifically to a tyrosine-phosphorylated protein of 120 kDa, which we identified as the proto-oncoprotein c-Cbl. The c-Cbl-binding site for CMS mapped to the carboxyl terminus of c-Cbl and is different from the proline-rich region known to bind SH3-containing proteins. CMS binding to c-Cbl was markedly attenuated in a tyrosine phosphorylation-defective c-Cbl mutant indicating that this interaction is dependent on the tyrosine phosphorylation of CMS. It also implies that CMS interacts with c-Cbl in an inducible fashion upon stimulation of a variety of cell-surface receptors. Immunofluorescence analysis revealed that both proteins colocalize at lamellipodia and leading edges of cells, and we propose that the interaction of CMS with c-Cbl offers a mechanism by which c-Cbl associates and regulates the actin cytoskeleton.

Voice stress monitoring in space--possibilities and limits.

An overview of experimental investigations into the use of the voice pitch mode (F0 m) as a distance tool for the monitoring of emotional stress reactions is given. Seven experiments are summarized describing the relations between voice reaction and psychological and physical load in laboratory and field applications. The reactivity of F0 m to mental load depends on psychological personality traits. F0 m is less related to physical load. The evaluation of F0 m as voice stress indicator requires an individual calibration. This calibration has to include the assessment of the individual Autonomic Outlet Type (AOT) because the physiological relevance of voice reactions depends on the individual AOT. The calibration is based on statistical reference data which have to be improved in the future. The calibration has to be done under comparable environmental circumstances. A 3-yr application of the method on board Mir station documents the applicability of the procedure.

Serum protein s100beta in patients with malignant melanoma detected by an immunoluminometric assay.

S100 protein is well established as a diagnostic tool in malignant melanoma immunohistology. In this study we measured S100beta in serum with a recently developed luminometric immunoassay with a detection limit of 0.02 microg/l. By measuring S-100beta in a group of apparently healthy individuals a mean value of 0.031 +/- 0.026 microg/l was found. In the reference group, serum S100beta was below 0.12 microg/l in all cases. To assess the sensitivity of the assay we investigated serum S-100beta levels in 371 serum samples of 315 patients with histological proven malignant melanoma at different disease stages. Staging was performed according to the German Society of Dermatology classification. Significant differences were observed between the control group and stages IIb (P = 0.01) and IV (P = 0.001). In tumour-bearing patients of stages IIIb and IV, the difference was highly significant (P < 0.0001). S100beta > 0.20 microg/l helps to distinguish between tumour-free and tumour-bearing patients with a specificity of 97.0% and a sensitivity of 64.6%. Our results demonstrate the serum S100beta is of limited value as a melanoma marker. However, it has clinical significance for identifying tumour-positive patients in advanced malignant melanoma stages III and IV.

Stabilization and productive positioning roles of the C2 domain of PTEN tumor suppressor.

PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancer. It acts as a phosphoinositide phosphatase and consists of an amino-terminal phosphatase domain tightly linked to a COOH-terminal C2 domain involved in lipid membrane-binding. We investigated the functions of the C2 domain and their relevance for tumor growth. To discriminate between PTEN C2 domain ability to recruit or to position the active site to the membrane, we artificially membrane-targeted PTEN by a myristoylation signal. This modification increased wild-type PTEN growth inhibition but did not rescue a C2 mutant defective in lipid-binding, suggesting a model in which PTEN C2 domain positions the active site productively with respect to the membrane-bound phosphoinositide substrate. When tumor-derived mutations in the loops that connect the C2 beta-strands were analyzed, we found that these generally destabilized the protein but had variable effects on the phosphatase activity and tumor growth. The magnitude of these effects was dependent on the presence of the COOH-terminal PEST sequences and on the cell type where the mutant proteins were expressed, suggesting the existence of fluctuating structural defects of the mutant protein. One of the C2 loop mutants induced a total loss of PTEN tumor-suppressor function, most likely by affecting both the membrane binding and the protein stability. These data support a double role for PTEN C2 domain in protein stability and in productive orientation of the catalytic site.

The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region.

PTEN is a recently identified tumor suppressor inactivated in a variety of cancers such as glioblastoma and endometrial and prostate carcinoma. It contains an amino-terminal phosphatase domain and acts as a phosphatidylinositol 3,4,5-trisphosphate phosphatase antagonizing the activity of the phosphatidylinositol 3-OH kinase. PTEN also contains a carboxyl-terminal domain, and we addressed the role of this region that, analogous to the amino-terminal phosphatase domain, is the target of many mutations identified in tumors. Expression of carboxyl-terminal mutants in PTEN-deficient glioblastoma cells permitted the anchorage-independent growth of the cells that otherwise was suppressed by wild-type PTEN. The stability of these mutants in cells was reduced because of rapid degradation. Although the carboxyl-terminal region contains regulatory PEST sequences and a PDZ-binding motif, these specific elements were dispensable for the tumor-suppressor function. The study of carboxyl-terminal point mutations affecting the stability of PTEN revealed that these were located in strongly predicted beta-strands. Surprisingly, the phosphatase activity of these mutants was affected in correlation with the degree of disruption of these structural elements. We conclude that the carboxyl-terminal region is essential for regulating PTEN stability and enzymatic activity and that mutations in this region are responsible for the reversion of the tumor-suppressor phenotype. We also propose that the molecular conformational changes induced by these mutations constitute the mechanism for PTEN inactivation.

CMS: an adapter molecule involved in cytoskeletal rearrangements.

Cas ligand with multiple Src homology (SH) 3 domains (CMS) is an ubiquitously expressed signal transduction molecule that interacts with the focal adhesion protein p130(Cas). CMS contains three SH3 in its NH2 terminus and proline-rich sequences in its center region. The latter sequences mediate the binding to the SH3 domains of p130(Cas), Src-family kinases, p85 subunit of phosphatidylinositol 3-kinase, and Grb2. The COOH-terminal region contains putative actin binding sites and a coiled-coil domain that mediates homodimerization of CMS. CMS is a cytoplasmic protein that colocalizes with F-actin and p130(Cas) to membrane ruffles and leading edges of cells. Ectopic expression of CMS in COS-7 cells resulted in alteration in arrangement of the actin cytoskeleton. We observed a diffuse distribution of actin in small dots and less actin fiber formation. Altogether, these features suggest that CMS functions as a scaffolding molecule with a specialized role in regulation of the actin cytoskeleton.