GATA Factors Regulate Inner Ear Development and Midbrain Neurogenesis

The zinc-finger transcription factors GATA2 and GATA3 in vertebrates belong to the six-member family that are essential regulators in the development of various organs. The aim of this study was to gain new information of the roles of GATA2 and GATA3 in inner ear morphogenesis and of the function of GATA2 in neuronal fate specification in the midbrain using genetically modified mouse and chicken embryos as models. A century ago the stepwise process of inner ear epithelial morphogenesis was described, but the molecular players regulating the cellular differentiation of the otic epithelium are still not fully resolved. This study provided novel data on GATA factor roles in several developmental processes during otic development. The expression analysis in chicken suggested that GATA2 and GATA3 possess redundant roles during otic cup and vesicle formation, but complementary cell-type specific functions during vestibular and cochlear morphogenesis. The comparative analysis between mouse and chicken Gata2 and Gata3 expression revealed many conserved aspects, especially during later stages of inner ear development, while the expression was more divergent at early stages. Namely, expression of both Gata genes was initiated earlier in chicken than mouse otic epithelium relative to the morphogenetic stages. Likewise, important differences concerning Gata3 expression in the otic cup epithelium were detected between mouse and chicken, suggesting that distinct molecular mechanisms regulate otic vesicle closure in different vertebrate species. Temporally distinct Gata2 and Gata3 expression was also found during otic ganglion formation in mouse and chicken. Targeted inactivation of Gata3 in mouse embryos caused aberrant morphology of the otic vesicle that in severe cases was disrupted into two parts, a dorsal and a ventral vesicle. Detailed analyses of Gata3 mutant embryos unveiled a crucial role for GATA3 in the initial inner ear morphogenetic event, the invagination of the otic placode. A large-scale comparative expression analysis suggested that GATA3 could control cell adhesion and motility in otic epithelium, which could be important for early morphogenesis. GATA3 was also identified as the first factor to directly regulate Fgf10 expression in the otic epithelium and could thus influence the development of the semicircular ducts. Despite the serious problems in the early inner ear development, the otic sensory fate establishment and some vestibular hair cell differentiation was observable in pharmacologically rescued Gata3-/- embryos. Cochlear sensory differentiation was, however, completely blocked so that no auditory hair cells were detected. In contrast to the early morphogenetic phenotype in Gata3-/- mutants, conditional inactivation of Gata2 in mouse embryos resulted in a relatively late growth defect of the three semicircular ducts. GATA2 was required for the proliferation of the vestibular nonsensory epithelium to support growing of the three ducts. Concurrently, with the role in epithelial semicircular ducts, GATA2 was also required for the mesenchymal cell clearance from the vestibular perilymphatic region between the membranous labyrinth and bony capsule. The gamma-aminobutyric acid-secreting (GABAergic) neurons in the midbrain are clinically relevant since they contribute to fear, anxiety, and addiction regulation. The molecular mechanisms regulating the GABAergic neuronal development, however, are largely unknown. Using tissue-specific mutagenesis in mice, GATA2 was characterized as a critical determinant of the GABAergic neuronal fate in the midbrain. In Gata2-deficient mouse midbrain, GABAergic neurons were not produced, instead the Gata2-mutant cells acquired a glutamatergic neuronal phenotype. Gain-of-function experiments in chicken also revealed that GATA2 was sufficient to induce GABAergic differentiation in the midbrain.

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.

Generation of Flat and Curved Membranes by Inverse-BAR Domain Proteins

Biological membranes are tightly linked to the evolution of life, because they provide a way to concentrate molecules into partially closed compartments. The dynamic shaping of cellular membranes is essential for many physiological processes, including cell morphogenesis, motility, cytokinesis, endocytosis, and secretion. It is therefore essential to understand the structure of the membrane and recognize the players that directly sculpt the membrane and enable it to adopt different shapes. The actin cytoskeleton provides the force to push eukaryotic plasma membrane in order to form different protrusions or/and invaginations. It has now became evident that actin directly co-operates with many membrane sculptors, including BAR domain proteins, in these important events. However, the molecular mechanisms behind BAR domain function and the differences between the members of this large protein family remain largely unresolved. In this thesis, the structure and functions of the I-BAR domain family members IRSp53 and MIM were thoroughly analyzed. By using several methods such as electron microscopy and systematic mutagenesis, we showed that these I-BAR domain proteins bind to PI(4,5)P2-rich membranes, generate negative membrane curvature and are involved in the formation of plasma membrane protrusions in cells e.g. filopodia. Importantly, we characterized a novel member of the BAR-domain superfamily which we named Pinkbar. We revealed that Pinkbar is specifically expressed in kidney and epithelial cells, and it localizes to Rab13-positive vesicles in intestinal epithelial cells. Remarkably, we learned that the I-BAR domain of Pinkbar does not generate membrane curvature but instead stabilizes planar membranes. Based on structural, mutagenesis and biochemical work we present a model for the mechanism of the novel membrane deforming activity of Pinkbar. Collectively, this work describes the mechanism by which I-BAR domain proteins deform membranes and provides new information about the biological roles of these proteins. Intriguingly, this work also gives evidence that significant functional plasticity exists within the I-BAR domain family. I-BAR proteins can either generate negative membrane curvature or stabilize planar membrane sheets, depending on the specific structural properties of their I-BAR domains. The results presented in this thesis expand our knowledge on membrane sculpting mechanisms and shows for the first time how flat membranes can be generated in cells.

Pentoxifylline improves sperm capacitation and in vitro fertilization of oocytes in the golden hamster

Pentoxifylline (PF) is used to improve motility of spermatozoa from subfertile or nonfertile males to accomplish in vitro fertilization in humans. The possible adverse effect of PF on pre- and peri- implantation stage embryo development in a suitable rodent model, such as the golden hamster, is yet to be determined. In this study, hamster cauda epididymal spermatozoa were exposed to different concentrations (0.23 to 3.6 mM) of PF, and their quantitative [percentage of motility] and qualitative [Score 0 to 5] motility were assessed and values expressed as the sperm motility index. Upon addition of spermatozoa to dishes containing PF, an immediate increase in sperm motility and sperm motility index was evident, which increased up to 4 to 6 h and then declined. The sperm motility index increase by PF was dose-dependant, and greater than or equal to 1.8 mM PF was detrimental after 4 h. The optimum dose of PF was found to be 0.45 mM. To assess the fertilizing ability of PF-treated spermatozoa, in vitro fertilization was carried out. Fertilization rates for spermatozoa treated with 3.6 mM PF were lower (53.8 +/- 7.8) than for the controls (69.5 +/- 10.2), whereas, treatment with 0.45 mM PF increased the rates (91.6 +/- 4.3) compared with that of the controls (80.2 +/- 5.9). In conclusion, low concentrations (0.23 to 0.45 mM) of PF improve sperm capacitation and fertilization of oocytes in vitro in the golden hamster.

Effect of chronic administration of Tamoxifen on fertility in male bonnet monkeys (Macaca radiata)

Administration of Tamoxifen via the Alzet pump at a rate of 50 mu g hr(-1) for 90 days in the adult male bonnet monkeys Macaca radiata had no effect on the serum testosterone concentration determined at 10 AM and 10 PM as well as total sperm count determined at 15-day intervals over a period of 260 days. However, a significant reduction in sperm motility was observed beyond 90 days up until the 225th day. Breeding studies conducted from day 90 to 260 revealed that these males were infertile.

Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+](i)) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline [0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 +/- 9.2 versus 141 +/- 6.0 fmoles/10(6) spermatozoa; mean +/- SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10(6) spermatozoal, cGMP values (similar to 27 fmoles/10(6) spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl-/HCO(3)(-)antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 mu M) A protein kinase G inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR, A phospholipase A(2) inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2+](i) was measured using fura-2-AM, there was an early rise 271 nM at 0.5 hi in pentoxifylline(-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR(20.5 +/- 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2+/-](i) and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A;A(2) and protein kinase C activity.

Synthesis, biological studies of linear and branched arabinofuranoside-containing glycolipids and their interaction with surfactant protein A

Oligoarabinofuranoside-containing glycolipids relevant to mycobacterial cell wall components were synthesized in order to understand the functional roles of such glycolipids. A series of linear tetra-, hexa-, octa-and a branched heptasaccharide oligoarabinofuranosides, with 1 -> 2 and 1 -> 5 a-linkages between the furanoside residues, were synthesized by chemical methods from readily available monomer building blocks. Upon the synthesis of glycolipids, constituted with a double alkyl chain-substituted sn-glycerol core and oligosaccharide fragments, biological studies were performed to identify the effect of synthetic glycolipids on the biofilm formation and sliding motilities of Mycobacterium smegmatis. Synthetic glycolipids and arabinofuranosides displayed an inhibitory effect on the growth profile, but mostly on the biofilm formation and maturation. Similarly, synthetic compounds also influenced the sliding motility of the bacteria. Further, biophysical studies were undertaken, so as to identify the interactions of the glycolipids with a pulmonary surfactant protein, namely surfactant protein A (SP-A), with the aid of the surface plasmon resonance technique. Specificities of each glycolipid interacting with SP-A were thus evaluated. From this study, glycolipids were found to exhibit higher apparent association constants than the corresponding oligosaccharide portion alone, without the double alkyl group-substituted glycerol core.

A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) plays an important role in a variety of cellular functions, including biofilm formation, alterations in the cell surface, host colonization and regulation of bacterial flagellar motility, which enable bacteria to survive changing environmental conditions. The cellular level of c-di-GMP is regulated by a balance between opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDE-As). Here, we report the presence and importance of a protein, MSDGC-1 (an orthologue of Rv1354c in Mycobacterium tuberculosis), involved in c-di-GMP turnover in Mycobacterium smegmatis. MSDGC-1 is a multidomain protein, having GAF, GGDEF and EAL domains arranged in tandem, and exhibits both c-di-GMP synthesis and degradation activities. Most other proteins containing GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. Unlike other bacteria, which harbour several copies of the protein involved in c-di-GMP turnover, M. smegmatis has a single genomic copy, deletion of which severely affects long-term survival under conditions of nutrient starvation. Overexpression of MSDGC-1 alters the colony morphology and growth profile of M. smegmatis. In order to gain insights into the regulation of the c-di-GMP level, we cloned individual domains and tested their activities. We observed a loss of activity in the separated domains, indicating the importance of full-length MSDGC-1 for controlling bifunctionality.

Comparative Transcriptomics of H. pylori Strains AM5, SS1 and Their hpyAVIBM Deletion Mutants: Possible Roles of Cytosine Methylation

Helicobacter pylori is an important human pathogen and one of the most successful chronic colonizers of the human body. H. pylori uses diverse mechanisms to modulate its interaction with the host in order to promote chronic infection and overcome host immune response. Restriction-modification genes are a major part of strain-specific genes present in H. pylori. The role of N-6 -adenine methylation in bacterial gene regulation and virulence is well established but not much is known about the effect of C-5 -cytosine methylation on gene expression in prokaryotes. In this study, it was observed by microarray analysis and RT-PCR, that deletion of an orphan C-5 -cytosine methyltransferase, hpyAVIBM in H. pylori strains AM5and SS1 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. AM Delta DhpyAVIBM mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. hpyAVIBM from strain 26695 is able to complement mutant SS1 and AM5 strains. This study highlights a possible significance of cytosine methylation in the physiology of H. pylori.

Role of outer membrane exopolymers of Acidithiobacillus ferrooxidans in adsorption of cells onto pyrite and chalcopyrite

Bacterial surface polymers play a major role in the adhesion of bacterial cells to solid surfaces. Lipopolysaccharides (LPS) are essential constituents of the cell walls of almost all Gram-negative bacteria. This paper reports the results of the investigations on the role of outer membrane exopolymers (LPS) of the chemolithotroph, Acidithiobacillus ferrooxidans, in adsorption of the cells onto pyrite and chalcopyrite. Optimization of EDTA treatment for removal of LPS from cell surface and the surface characterization of EDTA-treated cells are outlined. There was no change in cell morphology or loss in cell motility upon treatment with upto 0.04 mM EDTA for 1 h. Partial removal of LPS by EDTA treatment resulted in reduced adsorption of the cells on both pyrite and chalcopyrite. The protein profile of the EDTA-extractable fraction showed presence of certain outer membrane proteins indicating that EDTA treatment results in temporary gaps in the outer membrane. Also, specificity towards pyrite compared to chalcopyrite that was exhibited by untreated cells was lost when their exopolymer layers were stripped off, which could be attributed to the role of outer membrane proteins in the mineral-specificity exhibited by the bacteria. (C) 2013 Elsevier B.V. All rights reserved.

Potential ultrastructural changes in rat epididymal cell types induced by Boswellia papyrifera and Boswellia carterii incense

Boswellia papyrifera and Boswellia carterii, known as Arabian incense, diffuses smoke, contaminating the air, which adversely affects human health. Therefore, this study was designed to ascertain the effect of these plants on histopathological and ultrastructure changes in cauda epididymis of Albino rats. Animals were exposed to 4 g/kg body weight of B. papyrifera and B. carterii daily for 120 days along with suitable controls. Our study indicates a significant reduction in epithelial heights. Cells showed signs of degeneration. The ultrastructural study revealed that the cauda epididymis was affected, including its cell types. Furthermore, a decrease in the size of mitochondria, Golgi complex, and both ERs was observed. In all treated groups, plasma fructose decreased considerably, indicating the sign of reduced energy, vital for motility and other sperm functions. The results of this study suggest that these plants systematically affect cauda epididymal cell types and its lumen through its potential toxicity. (C) 2013 Published by Elsevier Masson SAS on behalf of Academie des sciences.

Quorum sensing and pathogenesis: role of small signalling molecules in bacterial persistence

The pathogenesis of Mycobacterium tuberculosis is associated with its ability to survive inside the human host and the bacteria use a variety of mechanism to evade the host's defence. A clearer understanding of the host pathogen interaction is needed to follow the pathogenicity and virulence. Recent advances in the study of inter and intra-cellular communication in bacteria had prompted us to study the role of quorum sensing in bacterial survival and pathogenicity. The cell cell communication in bacteria (quorum sensing) is mediated through the exchange of small molecules called as autoinducers that allow bacteria to modulate their gene expression in response to change in cell-population density. It is a coordinated response that confers multicellularity to a bacterial population in response to stress from external environment. Quorum sensing molecules are the global regulators and regulate a wide range of physiological processes including biofilm formation, motility, cell differentiation, long-term survival and many others. Many bacterial pathogens require quorum sensing to produce the virulence factors in response to host pathogen interaction. Here, we summarize our current understanding on small molecule signalling and their role in the bacterial persistence. New discoveries in these areas have enriched our knowledge on intracellular signalling and their role in the long-term survival of mycobacteria under nutrient starvation.

Kinematic and Mechanical Profile of the Self-Actuation of Thermosalient Crystal Twins of 1,2,4,5-Tetrabromobenzene: A Molecular Crystalline Analogue of a Bimetallic Strip

A paradigm shift from hard to flexible, organic-based optoelectronics requires fast and reversible mechanical response from actuating materials that are used for conversion of heat or light into mechanical motion. As the limits in the response times of polymer-based actuating materials are reached, which are inherent to the less-than-optimal coupling between the light/heat and mechanical energy in them, 1 a conceptually new approach to mechanical actuation is required to leapfrog the performance of organic actuators. Herein, we explore single crystals of 1,2,4,5-tetrabromobenzene (TBB) as actuating elements and establish relations between their kinematic profile and mechanical properties. Centimeter-size acicular crystals of TBB are the only naturally twinned crystals out of about a dozen known materials that exhibit the thermosalient effect-an extremely rare and visually impressive crystal locomotion. When taken over a phase transition, crystals of this material store mechanical strain and are rapidly self-actuated to sudden jumps to release the internal strain, leaping up to several centimeters. To establish the structural basis for this colossal crystal motility, we investigated the mechanical profile of the crystals from macroscale, in response to externally induced deformation under microscope, to nanoscale, by using nanoindentation. Kinematic analysis based on high-speed recordings of over 200 twinned TBB crystals exposed to directional or nondirectional heating unraveled that the crystal locomotion is a kinematically complex phenomenon that includes at least six kinematic effects. The nanoscale tests confirm the highly elastic nature, with an elastic deformation recovery (60%) that is far superior to those of molecular crystals reported earlier. This property appears to be critical for accumulation of stress required for crystal jumping. Twinned crystals of TBB exposed to moderate directional heating behave as all-organic analogue of a bimetallic `strip, where the lattice misfit between the two crystal components drives reveriible deformation of the crystal.

Ultrastructural and hormonal changes in rat cauda epididymal spermatozoa induced by Boswellia papyrifera and Boswellia carterii

Boswellia papyrifera and Boswellia carterii diffuses smoke polluting air that adversely affects indoor environment that certainly harm human health. Therefore, this study aims at ascertaining the effect of these plants on gonadal hormones and molecular changes in rat spermatozoa. The animals were exposed to 4 g/kg body weight of B. papyrifera and B. carterii daily for 120 days along with suitable controls. Significant decreases in FSH, LH and testosterone levels were evidenced, along with a reduction of protein, sialic acid, and carnitine levels. In sperm physiology, sperm count, motility, speed decrease, whereas sperm anomalies increase. TEM observation indicates morphological changes in plasma and acrosomal membranes, cytoplasmic droplet in the tail region, vacuolated, and disorganization of the mitochondrial sheath. These findings demonstrate that B. papyrifera and B. carterii smoke affects the process of sperm formation and maturation, which indicates the detrimental effects of these plants on the reproductive system. (c) 2014 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.d

Clusters, asters, and collective oscillations in chemotactic colloids

The creation of synthetic systems that emulate the defining properties of living matter, such as motility, gradient-sensing, signaling, and replication, is a grand challenge of biomimetics. Such imitations of life crucially contain active components that transform chemical energy into directed motion. These artificial realizations of motility point in the direction of a new paradigm in engineering, through the design of emergent behavior by manipulating properties at the scale of the individual components. Catalytic colloidal swimmers are a particularly promising example of such systems. Here we present a comprehensive theoretical description of gradient-sensing of an individual swimmer, leading controllably to chemotactic or anti-chemotactic behavior, and use it to construct a framework for studying their collective behavior. We find that both the positional and the orientational degrees of freedom of the active colloids can exhibit condensation, signaling formation of clusters and asters. The kinetics of catalysis introduces a natural control parameter for the range of the interaction mediated by the diffusing chemical species. For various regimes in parameter space in the long-ranged limit our system displays precise analogs to gravitational collapse, plasma oscillations, and electrostatic screening. We present prescriptions for how to tune the surface properties of the colloids during fabrication to achieve each type of behavior.