Prediction of heparin binding sites in bone morphogenetic proteins (BMPs)

Heparin is a glycosaminoglycan known to bind bone morphogenetic proteins (BMPs) and the growth and differentiation factors (GDFs) and has strong and variable effects on BMP osteogenic activity. In this paper we report our predictions of the likely heparin binding sites for BMP-2 and 14. The N-terminal sequences upstream of TGF-β-type cysteine-knot domains in BMP-2, 7 and 14 contain the basic residues arginine and lysine, which are key components of the heparin/HS-binding sites, with these residues being highly non-conserved. Importantly, evolutionary conserved surfaces on the beta sheets are required for interactions with receptors and antagonists. Furthermore, BMP-2 has electropositive surfaces on two sides compared to BMP-7 and BMP-14. Molecular docking simulations suggest the presence of high and low affinity binding sites in dimeric BMP-2. Histidines were found to play a role in the interactions of BMP-2 with heparin; however, a pKa analysis suggests that histidines are likely not protonated. This is indicative that interactions of BMP-2 with heparin do not require acidic pH. Taken together, non-conserved amino acid residues in the N-terminus and residues protruding from the beta sheet (not overlapping with the receptor binding sites and the dimeric interface) and not C-terminal are found to be important for heparin–BMP interactions.

Role of Thiobacillus ferrooxidans and sulphur (sulphide)-dependent ferric-ion-reducing activity in the oxidation of sulphide minerals

Thiobacillus ferrooxidans oxidized the sulphide minerals e.g., pyrite, pyrrhotite and copper concentrate under anaerobic conditions in the presence of ferric ion as sole electron acceptor. Copper and iron were solubilized from sulphide ores by the sulphur (sulphide)-dependent ferric-ion oxidoreductase activity. Treatment of resting cells of T. ferrooxidans with 0.5% phenol for 30 min completely destroyed the iron- and copper-solubilizing activity. The above treatment destroyed the sulphur(sulphide)-dependent ferric-ion-reducing activity completely but did not affect the iron-oxidizing activity. The results suggest that sulphur(sulphide)-dependent ferric-ion-reducing activity actively participates in the oxidation of sulphide minerals under anaerobic conditions. The activity of sulphur(sulphide)-dependent ferric ion reduction in the solubilization of iron and copper from the sulphide ores were also observed under aerobic conditions in presence of sodium azide (0.1 μmol), which completely inhibits the iron-oxidizing activity.

FGFR1 regulated gene-expression, cell proliferation and differentiation in the developing midbrain and hindbrain

The neuroectodermal tissue close to the midbrain hindbrain boundary (MHB) is an important secondary organizer in the developing neural tube. This so-called isthmic organizer (IsO) regulates cellular survival, patterning and proliferation in the midbrain (Mb) and rhombomere 1 (R1) of the hindbrain. Signaling molecules of the IsO, such as fibroblast growth factor 8 (FGF8) and WNT1 are expressed in distinct bands of cells around the MHB. It has been previously shown that FGF-receptor 1 (FGFR1) is required for the normal development of this brain region in the mouse embryo. In the present study, we have compared the gene expression profiles of wild-type and Fgfr1 mutant embryos. We show that the loss of Fgfr1 results in the downregulation of several genes expressed close to the MHB and in the disappearance of gene expression gradients in the midbrain and R1. Our microarray screen identified several previously uncharacterized genes which may participate in the development of midbrain R1 region. Our results also show altered neurogenesis in the midbrain and R1 of the Fgfr1 mutants. Interestingly, the neuronal progenitors in midbrain and R1 show different responses to the loss of signaling through FGFR1. As Wnt1 expression at the MHB region requires the FGF signaling pathway, WNT target genes, including Drapc1, were also identified in our screen. The microarray data analysis also suggested that the cells next to the midbrain hindbrain boundary express distinct cell cycle regulators. We showed that the cells close to the border appeared to have unique features. These cells proliferate less rapidly than the surrounding cells. Unlike the cells further away from the boundary, these cells express Fgfr1 but not the other FGF receptors. The slowly proliferating boundary cells are necessary for development of the characteristic isthmic constriction. They may also contribute to compartmentalization of this brain region.

Fine-tuning of the signalling network controlling morphogenesis and stem cell development in teeth

Tooth development is regulated by sequential and reciprocal interactions between epithelium and mesenchyme. The molecular mechanisms underlying this regulation are conserved and most of the participating molecules belong to several signalling families. Research focusing on mouse teeth has uncovered many aspects of tooth development, including molecular and evolutionary specifi cs, and in addition offered a valuable system to analyse the regulation of epithelial stem cells. In mice the spatial and temporal regulation of cell differentiation and the mechanisms of patterning during development can be analysed both in vivo and in vitro. Follistatin (Fst), a negative regulator of TGFβ superfamily signalling, is an important inhibitor during embryonic development. We showed the necessity of modulation of TGFβ signalling by Fst in three different regulatory steps during tooth development. First we showed that tinkering with the level of TGFβ signalling by Fst may cause variation in the molar cusp patterning and crown morphogenesis. Second, our results indicated that in the continuously growing mouse incisors asymmetric expression of Fst is responsible for the labial-lingual patterning of ameloblast differentiation and enamel formation. Two TGFβ superfamily signals, BMP and Activin, are required for proper ameloblast differentiation and Fst modulates their effects. Third, we identifi ed a complex signalling network regulating the maintenance and proliferation of epithelial stem cells in the incisor, and showed that Fst is an essential modulator of this regulation. FGF3 in cooperation with FGF10 stimulates proliferation of epithelial stem cells and transit amplifying cells in the labial cervical loop. BMP4 represses Fgf3 expression whereas Activin inhibits the repressive effect of BMP4 on the labial side. Thus, Fst inhibits Activin rather than BMP4 in the cervical loop area and limits the proliferation of lingual epithelium, thereby causing the asymmetric maintenance and proliferation of epithelial stem cells. In addition, we detected Lgr5, a Wnt target gene and an epithelial stem cell marker in the intestine, in the putative epithelial stem cells of the incisor, suggesting that Lgr5 is a marker of incisor stem cells but is not regulated by Wnt/β-catenin signalling in the incisor. Thus the epithelial stem cells in the incisor may not be directly regulated by Wnt/β-catenin signalling. In conclusion, we showed in the mouse incisors that modulating the balance between inductive and inhibitory signals constitutes a key mechanism regulating the epithelial stem cells and ameloblast differentiation. Furthermore, we found additional support for the location of the putative epithelial stem cells and for the stemness of these cells. In the mouse molar we showed the necessity of fi ne-tuning the signalling in the regulation of the crown morphogenesis, and that altering the levels of an inhibitor can cause variation in the crown patterning.

Regulation of Osteoblast Differentiation and Gene Expression by Phosphodiesterases and Bioactive Peptides

Bone is a mineralized tissue that enables multiple mechanical and metabolic functions to be carried out in the skeleton. Bone contains distinct cell types: osteoblasts (bone-forming cells), osteocytes (mature osteoblast that embedded in mineralized bone matrix) and the osteoclasts (bone-resorbing cells). Remodelling of bone begins early in foetal life, and once the skeleton is fully formed in young adults, almost all of the metabolic activity is in this form. Bone is constantly destroyed or resorbed by osteoclasts and then replaced by osteoblasts. Many bone diseases, i.e. osteoporosis, also known as bone loss, typically reflect an imbalance in skeletal turnover. The cyclic adenosine monophosphate (cAMP) and the cyclic guanosine monophosphate (cGMP) are second messengers involved in a variety of cellular responses to such extracellular agents as hormones and neurotransmitters. In the hormonal regulation of bone metabolism, i.e. via parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrp) and prostaglandin E2 signal via cAMP. cAMP and cGMP are formed by adenylate and guanylate cyclases and are degraded by phosphodiesterases (PDEs). PDEs determine the amplitudes of cyclic nucleotide-mediated hormonal responses and modulate the duration of the signal. The activities of the PDEs are regulated by multiple inputs from other signalling systems and are crucial points of cross-talk between the pathways. Food-derived bioactive peptides are reported to express a variety of functions in vivo. The angiotensin-converting enzymes (ACEs) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. The bioactive peptides offer also a nutriceutical and a nutrigenomic aspect to bone cell biology. The aim of this study was to investigate the influence of PDEs and bioactive peptides on the activation and the differentiation of human osteoblast cells. The profile of PDEs in human osteoblast-like cells and the effect of glucocorticoids on the function of cAMP PDEs, were investigated at the mRNA and enzyme levels. The effects of PDEs on bone formation and osteoblast gene expression were determined with chemical inhibitors and siRNAs (short interfering RNAs). The influence of bioactive peptides on osteoblast gene expression and proliferation was studied at the mRNA and cellular levels. This work provides information on how PDEs are involved in the function and the differentiation of osteoblasts. The findings illustrate that gene-specific silencing with an RNA interference (RNAi) method is useful in inhibiting, the gene expression of specific PDEs and further, PDE7 inhibition upregulates several osteogenic genes and increases bALP activity and mineralization in human mesenchymal stem cells-derived osteoblasts. PDEs appear to be involved in a mechanism by which glucocorticoids affect cAMP signaling. This may provide a potential route in the formation of glucocorticoid-induced bone loss, involving the down-regulation of cAMP-PDE. PDEs may play an important role in the regulation of osteoblastic differentiation. Isoleucine-proline-proline (IPP), a bioactive peptide, possesses the potential to increase osteoblast proliferation, differentiation and signalling.

Identification of alpha- and beta-Hydroxy Acid Containing Cyclodepsipeptides in Natural Peptide Mixtures Using Negative Ion Mass Spectrometry

Natural peptide libraries often contain cyclodepsipeptides containing alpha or beta hydroxy residues. Extracts of fungal hyphae of Isaria yield a microheterogenous cyclodepsipeptide mixture in which two classes of molecules can be identified by mass spectral fragmentation of negative ions. In the case of isaridins, which contain an alpha-hydroxy residue and a beta-amino acid residue, a characteristic product ion corresponding to a neutral loss of 72 Da is obtained. hi addition, neutral loss of water followed by a 72 Da loss is also observed. Two distinct modes of fragmentation rationalize the observed product ion distribution. The neutral loss of 72 Da has also been obtained for a roseotoxin component, which is also an alpha-hydroxy residue containing cyclodepsipeptide. In the case of isariins, which contain a beta-hydroxy acid residue, ring opening and subsequent loss of the terminal residue as an unsaturated ketene fragment, rationalizes the observed product ion formation. Fragmentation of negative ions provide characteristic neutral losses, which are diagnostic of the presence of alpha-hydroxy or beta-hydroxy residues.

A CD8(+) T cell clone specific for antigen also recognizes peptidomimics present in anti-idiotypic antibody: Implications for T cell memory

The relay hypothesis [R. Nayak, S. Mitra-Kaushik, M.S. Shaila, Perpetuation of immunological memory: a relay hypothesis, Immunology 102 (2001) 387-395] was earlier proposed to explain perpetuation of immunological memory without requiring long lived memory cells or persisting antigen. This hypothesis envisaged cycles of interaction and proliferation of complementary idiotypic B cells (Burnet cells) and anti-idiotypic B cells (Jerne cells) as the primary reason for perpetuation of immunological memory. The presence of pepti-domimics of antigen in anti-idiotypic antibody and their presentation to antigen specific T cells was postulated to be primary reason for perpetuation of T cell memory. Using a viral hemagglutinin as a model, in this work, we demonstrate the presence of peptidomimics in the variable region of ail anti-idiotypic antibody capable of functionally mimicking the antigen derived peptides. A CD8(+) CTL clone was generated against the hemagglutinin protein which specifically responds to either peptidomimic synthesizing cells or peptidomimic pulsed antigen presenting cells. Thus, it appears reasonable that a population of activated antigen specific T cells is maintained in the body by presentation of peptidomimic through Jerne cells and other antigen presenting cells long after immunization. (C) 2007 Elsevier Inc. All rights reserved.

Growth factor induction in intervertebral disc tissue: Observations in basic mechanisms of disc degeneration and rearrangement

The intervertebral disc is composed of concentrically arranged components: annulus fibrosus, the transition zone, and central nucleus pulposus. The major disc cell type differs in various parts of the intervertebral disc. In annulus fibrosus a spindle shaped fibroblast-like cell mainly dominates, whereas in central nucleus pulposus the more rounded chondrocyte-like disc cell is the major cell type. At birth the intervertebral disc is well vascularized, but during childhood and adolescence blood vessels become smaller and less numerous. The adult intervertebral disc is avascular and is nourished via the cartilage endplates. On the other hand, degenerated and prolapsed intervertebral discs are again vascularized, and show many changes compared to normal discs, including: nerve ingrowth, change in collagen turnover, and change in water content. Furthermore, the prolapsed intervertebral disc tissue has a tendency to decrease in size over time. Growth factors are polypeptides which regulate cell growth, extracellular matrix protease activity, and vascularization. Oncoproteins c-Fos and c-Jun heterodimerize, forming the AP-1 transcription factor which is expressed in activated cells. In this thesis the differences of growth factor expression in normal intervertebral disc, the degenerated intervertebral disc and herniated intervertebral disc were analyzed. Growth factors of particular interest were basic fibroblast growth factor (bFGF or FGF-2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGFβ). Cell activation was visualized by the expression of the AP-1 transcription promoters c-Fos and c-Jun. The expression was shown with either mono- or polyclonal antibodies by indirect avidin-biotin-peroxidase immunohistochemical staining method. The normal control material was collected from a tissue bank of five organ donors. The degenerated disc material was from twelve patients operated on for painful degenerative disc disease, and herniated disc tissue material was obtained from 115 patients operated on for sciatica. Normal control discs showed only TGFβ immunopositivity. All other factors studied were immunonegative in the control material. Prolapsed disc material was immunopositive for all factors studied, and this positivity was located either in the disc cells or in blood vessels. Furthermore, neovascularization was noted. Disc cell immunoreaction was shown in chondrocyte-like disc cells or in fibroblast-like disc cells, the former being expressed especially in conglomerates (clusters of disc cells). TGFβ receptor induction was prominent in prolapsed intervertebral disc tissue. In degenerated disc material, the expression of growth factors was analyzed in greater detail in various parts of the disc: nucleus pulposus, anterior annulus fibrosus and posterior annulus fibrosus. PDGF did not show any immunoreactivity, whereas all other studied growth factors were localized either in chondrocyte-like disc cells, often forming clusters, in fibroblast-like disc cells, or in small capillaries. Many of the studied degenerated discs showed tears in the posterior region of annulus fibrosus, but expression of immunopositive growth factors was detected throughout the entire disc. Furthermore, there was a difference in immunopositive cell types for different growth factors. The main conclusion of the thesis, supported by all substudies, is the occurrence of growth factors in disc cells. They may be actively participating in a network regulating disc cell growth, proliferation, extracellular matrix turnover, and neovascularization. Chondrocyte-like disc cells, in particular, expressed growth factors and oncoproteins, highlighting the importance of this cell type in the basic pathophysiologic events involved in disc degeneration and disc rearrangement. The thesis proposes a hypothesis for cellular remodelling in intervertebral disc tissue. In summary, the model presents an activation pattern of different growth factors at different intervertebral disc stages, mechanisms leading to neovascularization of the intervertebral disc in pathological conditions, and alteration of disc cell shape, especially in annulus fibrosus. Chondrocyte-like disc cells become more numerous, and these cells are capable of forming clusters, which appear to be regionally active within the disc. The alteration of the phenotype of disc cells expressing growth factors from fibroblast-like disc cells to chondrocyte-like cells in annulus fibrosus, and the numerous expression of growth factor expressing disc cells in nucleus pulposus, may be a key element both during pathological degeneration of the intervertebral disc, and during the healing process after trauma.

Sticking and erosion at carbon-containing plasma-facing materials in fusion reactors

Controlled nuclear fusion is one of the most promising sources of energy for the future. Before this goal can be achieved, one must be able to control the enormous energy densities which are present in the core plasma in a fusion reactor. In order to be able to predict the evolution and thereby the lifetime of different plasma facing materials under reactor-relevant conditions, the interaction of atoms and molecules with plasma first wall surfaces have to be studied in detail. In this thesis, the fundamental sticking and erosion processes of carbon-based materials, the nature of hydrocarbon species released from plasma-facing surfaces, and the evolution of the components under cumulative bombardment by atoms and molecules have been investigated by means of molecular dynamics simulations using both analytic potentials and a semi-empirical tight-binding method. The sticking cross-section of CH3 radicals at unsaturated carbon sites at diamond (111) surfaces is observed to decrease with increasing angle of incidence, a dependence which can be described by a simple geometrical model. The simulations furthermore show the sticking cross-section of CH3 radicals to be strongly dependent on the local neighborhood of the unsaturated carbon site. The erosion of amorphous hydrogenated carbon surfaces by helium, neon, and argon ions in combination with hydrogen at energies ranging from 2 to 10 eV is studied using both non-cumulative and cumulative bombardment simulations. The results show no significant differences between sputtering yields obtained from bombardment simulations with different noble gas ions. The final simulation cells from the 5 and 10 eV ion bombardment simulations, however, show marked differences in surface morphology. In further simulations the behavior of amorphous hydrogenated carbon surfaces under bombardment with D^+, D^+2, and D^+3 ions in the energy range from 2 to 30 eV has been investigated. The total chemical sputtering yields indicate that molecular projectiles lead to larger sputtering yields than atomic projectiles. Finally, the effect of hydrogen ion bombardment of both crystalline and amorphous tungsten carbide surfaces is studied. Prolonged bombardment is found to lead to the formation of an amorphous tungsten carbide layer, regardless of the initial structure of the sample. In agreement with experiment, preferential sputtering of carbon is observed in both the cumulative and non-cumulative simulations

Infra-red absorption bands of co-ordinated water in titanium complexes

ORANGE red and amorphous peroxy-titanium complexes of oxalic, malonic and maleic acids1-3, when vacuum-dried, have co-ordinated water molecules firmly bonded to the central titanium atom as shown in formula (I). The peroxy-oxygen from these compounds is slowly lost even at room temperature because of the strained peroxy-group3,4. The compounds, when kept at 95°-100°C. for about three days, give deperoxygenated compounds of the type (II). However, a sample of peroxy-titanium oxalate sealed in a glass tube lost all its peroxy-oxygen in about four years and gave a white crystalline basic oxalate (II). The amorphous nature of the compounds may be due to random hydrogen bonding in the complexes. The crystallinity observed in one of the deperoxygenated titanyl oxalates may be due to the rearrangement of the molecules during ageing for more than four years. The infra-red absorption of these compounds was studied to find out the effect of co-ordination and hydrogen bonding on the infra-red bands of the free water.

Relative Contribution of Cell Proliferation and Microvascularity to Metastasis in Malignant Uveal Melanoma

Uveal melanoma (UM) is the second most common primary intraocular cancer worldwide. It is a relatively rare cancer, but still the second most common type of primary malignant melanoma in humans. UM is a slowly growing tumor, and gives rise to distant metastasis mainly to the liver via the bloodstream. About 40% of patients with UM die of metastatic disease within 10 years of diagnosis, irrespective of the type of treatment. During the last decade, two main lines of research have aimed to achieve enhanced understanding of the metastasis process and accurate prognosis of patients with UM. One emphasizes the characteristics of tumor cells, particularly their nucleoli, and markers of proliferation, and the other the characteristics of tumor blood vessels. Of several morphometric measurements, the mean diameter of the ten largest nucleoli (MLN) has become the most widely applied. A large MLN has consistently been associated with high likelihood of dying from UM. Blood vessels are of paramount importance in metastasis of UM. Different extravascular matrix patterns can be seen in UM, like loops and networks. This presence is associated with death from metastatic melanoma. However, the density of microvessels is also of prognostic importance. This study was undertaken to help understanding some histopathological factors which might contribute to developing metastasis in UM patients. Factors which could be related to tumor progression to metastasis disease, namely nucleolar size, MLN, microvascular density (MVD), cell proliferation, and The Insulin-like Growth Factor 1 Receptor(IGF-1R), were investigated. The primary aim of this thesis was to study the relationship between prognostic factors such as tumor cell nucleolar size, proliferation, extravascular matrix patterns, and dissemination of UM, and to assess to what extent there is a relationship to metastasis. The secondary goal was to develop a multivariate model which includes MLN and cell proliferation in addition to MVD, and which would fit better with population-based, melanoma-related survival data than previous models. I studied 167 patients with UM, who developed metastasis even after a very long time following removal of the eye, metastatic disease was the main cause of death, as documented in the Finnish Cancer Registry and on death certificates. Using an independent population-based data set, it was confirmed that MLN and extravascular matrix loops and networks were unrelated, independent predictors of survival in UM. Also, it has been found that multivariate models including MVD in addition to MLN fitted significantly better with survival data than models which excluded MVD. This supports the idea that both the characteristics of the blood vessels and the cells are important, and the future direction would be to look for the gene expression profile, whether it is associated more with MVD or MLN. The former relates to the host response to the tumor and may not be as tightly associated with the gene expression profile, yet most likely involved in the process of hematogenous metastasis. Because fresh tumor material is needed for reliable genetic analysis, such analysis could not be performed Although noninvasive detection of certain extravascular matrix patterns is now technically possible,in managing patients with UM, this study and tumor genetics suggest that such noninvasive methods will not fully capture the process of clinical metastasis. Progress in resection and biopsy techniques is likely in the near future to result in fresh material for the ophthalmic pathologist to correlate angiographic data, histopathological characteristics such as MLN, and genetic data. This study supported the theory that tumors containing epithelioid cells grow faster and have poorer prognosis when studied by cell proliferation in UM based on Ki-67 immunoreactivity. Cell proliferation index fitted best with the survival data when combined with MVD, MLN, and presence of epithelioid cells. Analogous with the finding that high MVD in primary UM is associated with shorter time to metastasis than low MVD, high MVD in hepatic metastasis tends to be associated with shorter survival after diagnosis of metastasis. Because the liver is the main organ for metastasis from UM, growth factors largely produced in the liver hepatocyte growth factor, epidermal growth factor and insulin-like growth factor-1 (IGF-1) together with their receptors may have a role in the homing and survival of metastatic cells. Therefore the association between immunoreactivity for IGF-1R in primary UM and metastatic death was studied. It was found that immunoreactivity for IGF-IR did not independently predict metastasis from primary UM in my series.

Functions of human papillomavirus E5 oncogene in epithelial cells and in the onset of cervical cancer

Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.

Impurity diffusion in bismuth single-crystals

Measurements of impurity diffusion of 86Rb, 90Sr, 133Ba, and 137Cs in single crystal Bi were carried out. Diffusion samples were prepared from single crystal Bi by ion implantation. About 1012-1013 ions were implanted, resulting in surface activities approx =104 cpm. After implantation, specimens were annealed for specified times at 220-265 deg C, and tracer penetration profiles were determined by an electrolytic method. A typical penetration profile for 137Cs in Bi showed a linear relationship for log C vs x in with Fick's law for volume diffusion. Laws of grain boundary diffusion were not obeyed and the order of magnitude of the penetration distances was much less than on a grain boundary mechanism. Results were interpreted in terms of a modified Fischer analysis using a kinetic trapping term. Effective half lengths for trapping at a twin boundary were determined for each impurity.

Temperature-Sensitive Src-Transformed Mdck Cells nn 3d Cultures as a Model of Malignant Transformation of Epithelial Cells

The equilibrium between cell proliferation, differentiation, and apoptosis is crucial for maintaining homeostasis in epithelial tissues. In order for the epithelium to function properly, individual cells must gain normal structural and functional polarity. The junctional proteins have an important role both in binding the cells together and in taking part in cell signaling. Cadherins form adherens junctions. Cadherins initiate the polarization process by first recognizing and binding the neighboring cells together, and then guiding the formation of tight junctions. Tight junctions form a barrier in dividing the plasma membranes to apical and basolateral membrane domains. In glandular tissues, single layered and polarized epithelium is folded into tubes or spheres, in which the basal side of the epithelial layer faces the outer basal membrane, and the apical side the lumen. In carcinogenesis, the differentiated architecture of an epithelial layer is disrupted. Filling of the luminal space is a hallmark of early epithelial tumors in tubular and glandular structures. In order for the transformed tumor cells to populate the lumen, enhanced proliferation as well as inhibition of apoptosis is required. Most advances in cancer biology have been achieved by using two-dimensional (2D) cell culture models, in which the cells are cultured on flat surfaces as monolayers. However, the 2D cultures are limited in their capacity to recapitulate the structural and functional features of tubular structures and to represent cell growth and differentiation in vivo. The development of three-dimensional (3D) cell culture methods enables the cells to grow and to be studied in a more natural environment. Despite the wide use of 2D cell culture models and the development of novel 3D culture methods, it is not clear how the change of the dimensionality of culture conditions alters the polarization and transformation process and the molecular mechanisms behind them. Src is a well-known oncogene. It is found in focal and adherens junctions of cultured cells. Active src disrupts cell-cell junctions and interferes with cell-matrix binding. It promotes cell motility and survival. Src transformation in 2D disrupts adherens junctions and the fibroblastic phenotype of the cells. In 3D, the adherens junctions are weakened, and in glandular structures, the lumen is filled with nonpolarized vital cells. Madin-Darby canine kidney (MDCK) cells are an epithelial cell type commonly used as a model for cell polarization. Its-src-transformed variants are useful model systems for analyzing the changes in cell morphology, and they play a role in src-induced malignant transformation. This study investigates src-transformed cells in 3D cell cultures as a model for malignant transformation. The following questions were posed. Firstly: What is the role of the composition and stiffness of the extracellular matrix (ECM) on the polarization and transformation of ts v-src MDCK cells in 3D cell cultures? Secondly: How do the culture conditions affect gene expression? What is the effect of v-src transformation in 2D and in 3D cell models? How does the shift from 2D to 3D affect cell polarity and gene expression? Thirdly: What is the role of survivin and its regulator phosphatase and tensin homolog protein (PTEN) in cell polarization and transformation, and in determining cell fate? How does their expression correlate with impaired mitochondrial function in transformed cells? In order to answer the above questions, novel methods of culturing and monitoring cells had to be created: novel 3D methods of culturing epithelial cells were engineered, enabling real time monitoring of a polarization and transformation process, and functional testing of 3D cell cultures. Novel 3D cell culture models and imaging techniques were created for the study. Attention was focused especially on confocal microscopy and live-cell imaging. Src-transformation disturbed the polarization of the epithelium by disrupting cell adhesion, and sensitized the cells to their environment. With active src, the morphology of the cell cluster depended on the composition and stiffness of the matrix. Gene expression studies revealed a broader impact of src transformation than mere continuous activity of src-kinase. In 2D cultures, src transformation altered the expression of immunological, actin cytoskeleton and extracellular matrix (ECM). In 3D, the genes regulating cell division, inhibition of apoptosis, cell metabolism, mitochondrial function, actin cytoskeleton and mechano-sensing proteins were altered. Surprisingly, changing the culture conditions from 2D to 3D affected also gene expression considerably. The microarray hit survivin, an inhibitor of apoptosis, played a crucial role in the survival and proliferation of src-transformed cells.

On the evaluation of stability of rare earth oxides as face coats for investment casting of titanium

Attempts have been made to evaluate the thermal stability of rare earth oxide face coats against liquid titanium. Determination of microhardness profiles and concentration profiles of oxygen and metallic constituents of oxide in investment cast titanium rods has allowed grActation of relative stability of rare earth oxides. The relative stability of evaluated oxides in the order of increasing stability follows the sequence CeO2 — ZrO2 — Gd2O3 — didymium oxide — Sm2O3 —Nd2O3 — Y2O3. The grading does not follow the free energy data of the formation of these oxides. A better correlation with the experimental observations is obtained when the solubility of the metallic species in titanium is also taken into consideration.