Cell Biology

Interrelationships between nuclear architecture and gene expression were examined by comparing the representation of nuclear matrix proteins in ROS 17/2.8 rat and MG-63 human osteosarcoma cells with those in normal diploid osteoblasts. The tumor-derived cells coexpress genes which are expressed in a sequential and mutually exclusive manner during the progressive stages of osteoblast differentiation. In osteosarcoma cells two-dimensional electrophoretic analysis indicates a composite representation of nuclear matrix proteins characteristic of both the proliferative and postproliferative periods of osteoblast phenotype development. In addition, nuclear matrix proteins unique to the tumor cells and the absence of nuclear matrix proteins found only in normal diploid osteoblasts are observed. Tumor-specific nuclear matrix proteins include those expressed in a proliferation-dependent and independent manner. There is a parallel relationship between nuclear matrix proteins and the expressio...

We have identified a set of retinal basal glia, designated RBC cells, in the axon layer of thedeveloping Drosophila eye disc. In vivo pulse labeling with bromodeoxyuridine shows that these cells originate in the optic stalk and migrate into the disc. In mutants lacking photoreceptor axons, RBC cells accumulate in the optic stalk, but do not invade the disc. The association of RBG cells with photoreceptor axons, their origin in the optic stalk, and their migration into the retina are in common with the behavior of astrocytes in the developing mammalian retina.

The formation and maintenance of cell polarity is essential for epithelial morphogenesis. Dpatj (Drosophila homolog of mammalian Patj) is a multi-PDZ domain protein that localizes to the apical cell membrane and forms a protein complex with cell polarity proteins, Crumbs (Crb) and Stardust (Sdt). Whereas Crb and Sdt are known to be required for the organization of adherens junctions (AJs) and rhabdomeres in differentiating photoreceptors, the in vivo function of Dpatj as a member of the Crb complex in developing eye has been unclear due to the lack of loss-of-function mutations specifically affecting the dpatj gene. Our genetic analysis of hypomorph, null, and RNA interference reveals distinct dual functions of Dpatj in developing and mature photoreceptors. The C-terminal region (PDZ domains 2-4) of Dpatj is not essential for development of the animal but is required to prevent late-onset photoreceptor degeneration. In contrast, the N-terminal region of Dpatj is essential for animal viability and photoreceptor morphogenesis during development. The localization and maintenance of Crb and Sdt in the apical photoreceptor membrane are strongly affected by reduced levels of Dpatj. Dpatj is necessary for proper positioning of AJs and the integrity of photoreceptors in the developing retina as well as for the maintenance of adult photoreceptors. Our study provides evidence that Dpatj has domain-specific early and late functions in regulating the localization and stability of the Crb-Sdt complex in photoreceptor cells.

Organogenesis involves an initial surge of cell proliferation, leading to differentiation. This is followed by cell death in order to remove extra cells. During early development, there is little or no cell death. However,there is a lack of information concerning the genes required for survival during the early cell-proliferation phase. Here, we show that Lobe(L) and the Notch (N) ligand Serrate (Ser), which are both involved in ventral eye growth, are required for cell survival in the early eye disc. We observed that the loss-of-ventral-eye phenotype in L or Ser mutants is due to the induction of cell death and the upregulation of secreted Wingless (Wg). This loss-of-ventral-eye phenotype can be rescued by (i) increasing the levels of cell death inhibitors, (ii)reducing the levels of Hid-Reaper-Grim complex, or (iii) reducing canonical Wg signaling components. Blocking Jun-N-terminal kinase (JNK) signaling, which can induce caspase-independent cell death, significantly rescued vent...

AJs, are larger in size than the vertebrate TJs, and only * Howard Hughes Medical Institute appear relatively late in embryogenesis. However, SJs † Department of Molecular and Human Genetics show similarities to TJs, suggesting that they play an ‡ Department of Cell Biology equivalent role (Woods and Bryant, 1993; Marfatia et al., § Program in Developmental Biology 1994; Baumgartner et al., 1996). Baylor College of Medicine Formation of polarized epithelia in most species is Houston, Texas 77030 dependent on and controlled by specific transmembrane proteins (TPs) (Tepass, 1997). The only known apically localized TP required for the maintenance of Summary cell polarity is the CRB protein of Drosophila (Wodarz et al., 1993, 1995; Tepass, 1996). Overexpression and Polarization of epithelial cells depends on a hierarchiloss-of-function studies show that much of the function cal process whereby specific membrane-associated of CRB seems to be confined to its short 37 amino acid proteins become targeted to specialized membrane cytoplasmic domain (Wodarz et al., 1993, 1995). The domains. Here, we describe a novel Drosophila prolaterally localized TPs required for the maintenance of tein, Discs Lost (DLT), that plays a crucial role in the epithelial polarity are Cadherins, enriched at AJs (Teppolarization of embryonic epithelia during cellular ass et al., 1996; Uemura et al., 1996; Yap et al., 1997), blastoderm formation. At subsequent stages of develand NRX IV, localized to SJs (Baumgartner et al., 1996; opment, DLT interacts with the apical determinant M. A. B. and H. J. B., unpublished data). Finally, basally Crumbs (CRB) and the laterally localized protein Neulocated TPs like integrins play a major role in the interrexin IV (NRX IV). Mutations in dlt or double-stranded action with basal substratum and are required for adhe-RNA interference lead to aberrant localization of CRB sion of epithelial cells to their extracellular environment and NRX IV and cause a concomitant loss of epithelial (Brown, 1993; Hynes, 1996). The activity of each of these cell polarity. Hence, DLT is required to establish and proteins causes only a partial polarization, and similarly, maintain cell polarity and participates in different moloss of any of these proteins leads only to a partial loss lecular complexes that define apical and lateral memof polarity (Tepass, 1997). brane domains. In vertebrates, several nontransmembrane proteins have been shown to be associated with AJs and TJs.

A fundamental feature of developing tissues and organs is generation of planar polarity of cells in an epithelium with respect to the body axis. The Drosophila compound eye shows two-tier dorsoventral (DV) planar polarity. At the individual ommatidium level, the eight photoreceptors in each unit eye form a dorsoventrally asymmetric cluster. At the level of eye field, hundreds of ommatidia in the upper and lower halves of an eye are uniformly polarized dorsally or ventrally, respectively. This results in DV mirror symmetries about the equator. The uniform orientations of photoreceptor clusters over long distance in the eye field provide an excellent model for studying the genetic basis of long-range planar polarity. Ommatidial DV polarity can be detected in third instar eye imaginal disc during the early stage of retinal diVerentiation. Recent studies have strongly suggested that the foundation for this DV polarity pattern is laid much earlier in undiVerentiated eye disc. The eye disc primordium is partitioned into the DV compartments of independent cell lineages. The Iroquois-Complex genes specify the dorsal fate whereas their absence in the ventral compartment results in the ventral fate with expression of the fringe gene. Genetic evidence suggests that the interaction of dorsal and ventral cells at the DV boundary in early eye disc generates an organizing center for growth and patterning of the eye field. In this chapter, we outline key genetic events involved in early DV patterning and growth of eye disc, and its potential role in organizing long-range signaling for DV planar polarity during later diVerentiation of the eye.

The POU homeodomain protein Oct-4 and the Forkhead Box protein FoxD3 (previously Genesis) are transcriptional regulators expressed in embryonic stem cells. Down-regulation of Oct-4 during gastrulation is essential for proper endoderm development. After gastrulation, FoxD3 is generally down-regulated during early endoderm formation, although it specifically remains expressed in the embryonic neural crest. In these studies, we have found that Oct-4 and FoxD3 can bind to identical regulatory DNA sequences. In addition, Oct-4 physically interacted with the FoxD3 DNA-binding domain. Cotransfection of Oct-4 and FoxD3 expression vectors activated the osteopontin enhancer, which is expressed in totipotent embryonic stem cells. FoxA1 and FoxA2 (previously HNF-3α and HNF-3β) are Forkhead Box transcription factors that participate in liver and lung formation from foregut endoderm. Although FoxD3 activated the FoxA1 and FoxA2 promoters, Oct-4 inhibited FoxD3 activation of the FoxA1 and FoxA2 en...

The complex and diverse biological effects of retinoic acid (RA) are mediated through specific receptors that are members of the steroid hormone family of nuclear transcription factors. The RA receptor family consists of multiple structurally distinct RA receptors, which diverge primarily at the NH2-terminal domain. The evolutionary conservation of this divergent region in individual RA receptors among different species together with their tissue-specific patterns of expression suggest that the biological function and activity of the individual RA receptors may be confined to specific tissues. To test this hypothesis in hematopoietic cells, we used retrovirus-mediated gene transduction to introduce the RA receptors RAR-a, RAR-13, and RAR-y as well as RXR-a into a mutant subclone of the HL-60 promyelocytic leukemia cell line (designated HL-60R) that is relatively resistant to RA-induced granulocytic differentiation. We found that each of these structurally distinct RA receptors could restore sensitivity of the HL-60R cells to RA. A critical threshold number of transduced receptors per cell appears to be necessary to restore this functional activity. Thus, the capability to mediate granulocytic differentiation of HL-60 cells is shared among distinctly different RA receptors. The retinoids and in particular retinoic acid (RA) exert a wide range of biological effects related to cell proliferation and differentiation. RA functions as a natural morphogen in regulating fetal development during vertebrate embryogenesis (2, 34). RA also influences adult epithelial cell growth and differentiation (27) and suppresses malignant transformation both in vitro and in vivo (5, 20, 29). In addition, RA induces the terminal differentiation of human promyelocytic leuke- mia cells . RA is thought to mediate these diverse and complex biological effects through specific re- ceptors that are members of the steroid/thyroid hormone receptor superfamily of nuclear transcription factors (15). These receptors exhibit a modular structure that includes discrete DNA-binding and hormone (ligand)-binding do- mains. The RA receptors presumably bind to specific cis- acting DNA sequences that are important for regulating transcription of specific target genes, and this target gene expression may be modified in the presence of ligand. The RA receptors, in contrast with the glucocorticoid receptors, exhibit a relatively large number of structurally distinct isoforms. The RAR-ot, RAR-,B, and RAR-,y receptors share marked structural similarity in their DNA-binding and ligand-binding domains but markedly diverge in their NH2terminal regions. In contrast, the RA receptors designated RXR exhibit marked divergence in both the NH2-terminal and ligand-binding domains compared with the RAR-a, RAR-,B, and RAR--y receptors and appear to define a distinct and separate family of RA receptors (28). Why are there so many different types of RA receptors? What specific physiological role does each distinct receptor play in regulating cell growth and differentiation? Several lines of indirect evidence suggest that the activity of each of * Corresponding author.

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-γ-activated C57BL/6 (H-2b) vascular endothelial cells as stimulators and CD8+ T lymphocytes isolated from CBA/J (H-2k) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-γ. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8+ T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44low naive CD8+ T lymphocytes. Activated CD8+ T lymphocytes had the ability to produce IFN-γ and IL-2, acq...

Vascular endothelium is an important site for a wide array of immunological processes such as inflammation, atherosclerosis and allograft rejection. Culture methods of mouse vascular endothelium would provide an important in vitro correlate to immunological murine in vivo models. We describe a simple method to culture mouse vascular endothelium Ž . from thoracic aorta. Our cultured cells express typical phenotypic CD105, CD31, CD106 , morphological and ultrastructural Ž . intercellular junctions, Weibel-Palade bodies markers of vascular endothelium. They also possess functional receptors for uptake and processing of acetylated low-density lipoproteins. The mouse vascular endothelium within our system expresses high levels of MHC class I and MHC class II after activation with IFN-g. In addition, these cells express the accessory molecules CD80 and CD54, while they lack constitutive expression of CD86 and CD40, providing them the means to function as antigen presenting cells. Alloreactive CD4 q and CD8 q T lymphocytes demonstrate evidence of DNA synthesis after co-culture with activated vascular endothelium indicating their commitment to proliferation. In conclusion, we describe a simple culture system to isolate and grow mouse vascular endothelium, which provides a powerful tool to study biological interactions in vitro.

Highlights d NAD + induces transcription of stress and metabolic genes through the circadian clock d BMAL1 is required for chromatin remodeling and HSF1 recruitment in response to NAD + d NAD + regulates PER2 acetylation and localization to control feedback repression

The adenoviral E1A proteins are able to promote proliferation and transformation, inhibit differentiation, induce apoptosis, and suppress tumor growth. The extreme N terminus and conserved region one of E1A, which are indispensable for transcriptional regulation and for binding to p300/CBP, TBP, and pCAF, play essential roles in these abilities. These observations strongly suggest an intrinsic link between E1A-mediated transcriptional regulation and other effects. In this report, we show that E1A inhibits the normal growth of Saccharomyces cerevisiae HF7c, and this inhibition also depends on the domains required for transcriptional regulation. We demonstrate that E1A associates with histone acetyltransferase activity and represses the transactivation activity of transcription factor in S. cerevisiae, suggesting that E1A may suppress the expression of genes required for normal growth. Based on yeast growth rescue, we present a genetic screening strategy that identified RACK1 as an E1A antagonizing factor. Expression of human RACK1 efficiently relieves E1A-mediated growth inhibition in HF7c and protects human tumor cells from E1A-induced apoptosis. Finally, we show that RACK1 decreases E1A-associated histone acetyltransferase activity in yeast and mammalian cells, and physically interacts with E1A. Our data demonstrate that RACK1 is a repressor of E1A, possibly by antagonizing the effects of E1A on host gene transcription.

The kinesin family is greatly expanded in plants compared with animals and, with more than a third up-regulated in expression during cell division, it has been suggested that this expansion facilitated complex plant-specific cytoskeletal rearrangements. The cell cycle-regulated kinesins include two with an N-terminal malectin domain, a protein domain that has been shown to bind polysaccharides and peptides when found extracellularly in receptor-like kinases. Although malectin domain kinesins are evolutionarily deep rooted, their function in plants remains unclear. Here we show that loss of MALECTIN DOMAIN KINESIN 2 (MDKIN2) results in stochastic developmental defects in pollen, embryo, and endosperm. High rates of seed abnormalities and abortion occur in mdkin2 mutants through a partial maternal effect. No additive effect or additional developmental defects were noted in mdkin1 mdkin2 double mutants. MDKIN2 is expressed in regions of cell division throughout the plant. Subcellular l...

A series of N,N-disubstituted piperazines were synthesized containing the structural elements of both methylenedioxybenzylpiperazine (MDBP) and trifluoromethylphenylpiperazine (TFMPP) in a single molecule. These six potential designer drug molecules having a regioisomeric relationship were compared in gas chromatography-mass spectrometry (GC-MS), gas chromatography-infrared spectroscopy and serotonin receptor affinity studies. These compounds were separated by capillary gas chromatography on an Rxi Ò -17Sil MS stationary phase film and the elution order appears to be determined by the position of aromatic ring substitution. The majority of electron ionization mass spectral fragment ions occur via processes initiated by one of the two nitrogen atoms of the piperazine ring. The major electron ionization mass spectrometry (EI-MS) fragment ions observed in all six of these regioisomeric substances occur at m/z = 364, 229, 163 and 135. The relative intensity of the various fragment ions is also equivalent in each of the six EI-MS spectra. The vapour phase infrared spectra provide a number of absorption bands to differentiate among the six individual compounds on this regioisomeric set. Thus, the mass spectra place these compounds into a single group and the vapour phase infrared spectra differentiate among the six regioisomeric possibilities. All of the TFMPP-MDBP regioisomers displayed significant binding to 5-HT 2B receptors and in contrast to 3-TFMPP, most of these TFMPP-MDBP isomers did not show significant binding at 5-HT 1 receptor subtypes. Only the 3-TFMPP-3,4-MDBP (Compound 5) isomer displayed affinity comparable to 3-TFMPP at 5-HT 1A receptors (K i = 188 nmol/L).

Astrocytes are versatile cells involved in synaptic information processing, energy metabolism, redox homeostasis, inflammatory response and structural support of the brain. Recently, we established a routine protocol of cultured astrocytes derived from adult and aged Wistar rats, which present several different responses compared to newborn astrocytes, commonly used to characterize the role of the astrocytes in the central nervous system. Previous studies hypothesized that astrocyte cultures prepared from adult animals derive from immature precursors present in the adult tissue throughout life. Since our group has already demonstrated that the glial functionality of adult astrocytes differs from newborn cultures, the aim of this study was to confirm that our in vitro astrocytes were derived from mature cells. Therefore, we evaluated cytoskeleton proteins, such as glial fibrillary acidic protein and vimentin, as well as Sox10, an essential marker of immature glial cells, in ex vivo tissue and in in vitro astrocytes from the same animals (1, 90 and 180 days old). In addition, we examined the mitochondrial functionality and the cellular redox homeostasis. Our results suggest that adult and aged astrocytes are derived from mature cells and that changes in mitochondrial parameters in ex vivo tissue were reproduced in in vitro astrocytes.

Mitochondria perform crucial roles in many biochemical processes, and mitochondrial depolarization is an early sign of platelet apoptosis. The mitochondrial membrane potential is usually evaluated through JC‐1 probe, but it can also be assessed with MitoTracker probes. Our aim was to evaluate mitochondrial viability in stored canine platelet concentrates (PCs) with the fluorescent probes JC‐1 and MitoTracker. Platelets from 22 canine PCs were stained with JC‐1 and MitoTracker probes on days 1, 3, and 5 of storage. Data on metabolic parameters were also collected for correlation studies. Results of JC‐1 and MitoTracker revealed a decrease in mitochondrial membrane potential in day 5 of storage compared to days 1 and 3, providing evidence of mitochondrial depolarization, a finding that was confirmed by the data on metabolic parameters. MitoTracker probes also added information regarding platelet swelling. In conclusion, MitoTracker probes offered a more complete mitochondrial analysis...

Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin's (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin's effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3β and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.

The mitostatic action of the commonly used fungicide methyl 2-benzimidazolecarbamate MBC was evaluated in primary cultures of human ovarian granulosa cells with respect to the organization and stability of spindle microtubules and mitotic centrosomes. MBC caused metaphase arrest and abnormal chromosome organization following a 3-15 h treatment at a concentration of 30 mM. While microtubules were retained in MBC-treated cells, alterations in spindle shape and microtubule composition were noted. Exposure to MBC resulted in an increased number of spindle poles associated with chromosomes displaced from the metaphase plate. A gradual increase from tri-to multipolar spindles was noted with Ž . prolonged treatment although a relatively constant fraction 50% of bipolar spindles was maintained. In non-dividing cells, MBC had no effect on microtubule organization. Analysis of mitotic figures by immunofluorescence microscopy showed a reduction in interpolar and astral microtubules in response to MBC treatment while acetylated kinetochore microtubules were retained and their plus-ends were attached to metaphase chromosomes. In multipolar spindles, analysis of microtubule Ž . Ž . organizing centers MTOCs with antisera to stable centrosomal markers SPJ and 5051 revealed that only poles associated Ž . with displaced chromosomes retained these markers. In contrast, transient centrosome markers NuMA and centrophilin were localized to all poles of multipolar spindles. Since MBC alters centrosome organization during mitosis, the results suggest that one mechanism of action of this agent is impairment of spindle microtubule dynamics at the centrosome.

A stem cell niche is defined as the most suitable tissue microenvironment where somatic cells (parent cells) or in some cases extracellular matrix elements and stem cells are present in an exclusive interaction. In recent years, promising data were published revealing the molecular and functional significance of cell-cell contacts in stem cell proliferation (self-renewal) and differentiation into mature cells. In this brief review, recent progress has been documented with special emphasis on the structural-functional relationship between stem cells and surrounding cells. Hematopoietic stem cells and their niches are among the most studied stem cell types and cell-cell interactions, which have specific as well as some common properties observed in other niche sites in the human body. Some of the signaling pathways, which are illustrated by original drawings herein, obviously play major roles in stem cell self-renewal capacity and differentiation status. Therefore, stem cell niches stand as the most critical biological sites in stem cell physiology and could also be considered as the new targets for the treatment of certain diseases.

A coupled analysis of the reaction kinetics and microstructural changes of Fe 2 O 3 /Al 2 O 3 oxygen carriers during redox reaction cycles for chemical looping systems was conducted. The microstructural changes in the oxygen carrier particles were investigated using an image analysis method with cross-sectional backscattered electron images, and microstructural information such as the particle size, porosity, and two-phase boundary between the iron oxide and pores was obtained. The microstructural changes and the degradation kinetics during redox cycles were investigated under various operating conditions (reaction temperatures, reduction times, oxygen partial pressures during the oxidation process, and weight ratios of the oxygen carriers). The degradation coefficient and the reaction enthalpy of the particles in the oxidation process are linearly related, implying that increasing the local temperature of the oxygen carriers causes the coarsening of iron oxide particles via sintering and microstructural changes. Our analysis contributes to the design of highly stable oxygen carrier particles and the improvement of the operating conditions for chemical looping systems.

A coupled analysis of the reaction kinetics and microstructural changes of Fe 2 O 3 /Al 2 O 3 oxygen carriers during redox reaction cycles for chemical looping systems was conducted. The microstructural changes in the oxygen carrier particles were investigated using an image analysis method with cross-sectional backscattered electron images, and microstructural information such as the particle size, porosity, and two-phase boundary between the iron oxide and pores was obtained. The microstructural changes and the degradation kinetics during redox cycles were investigated under various operating conditions (reaction temperatures, reduction times, oxygen partial pressures during the oxidation process, and weight ratios of the oxygen carriers). The degradation coefficient and the reaction enthalpy of the particles in the oxidation process are linearly related, implying that increasing the local temperature of the oxygen carriers causes the coarsening of iron oxide particles via sintering and microstructural changes. Our analysis contributes to the design of highly stable oxygen carrier particles and the improvement of the operating conditions for chemical looping systems.

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors -including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material -are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and aged cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation.

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays ...

Background: In animal cells, cytokinesis begins shortly after the sister chromatids move to the spindle poles. The inner centromere protein (Incenp) has been implicated in both chromosome segregation and cytokinesis, but it is not known exactly how it mediates these two distinct processes. We identified two Caenorhabditis elegans proteins, ICP-1 and ICP-2, with significant homology in their carboxyl termini to the corresponding region of vertebrate Incenp. Embryos depleted of ICP-1 by RNA-mediated interference had defects in both chromosome segregation and cytokinesis. Depletion of the Aurora-like kinase AIR-2 resulted in a similar phenotype. The carboxy-terminal region of Incenp is also homologous to that in Sli15p, a budding yeast protein that functions with the yeast Aurora kinase Ipl1p. ICP-1 bound C. elegans AIR-2 in vitro, and the corresponding mammalian orthologs Incenp and AIRK2 could be co-immunoprecipitated from cell extracts. A significant fraction of embryos depleted of ICP-1 and AIR-2 completed one cell division over the course of several cell cycles. ICP-1 promoted the stable localization of ZEN-4 (also known as CeMKLP1), a kinesin-like protein required for central spindle assembly. Conclusions: ICP-1 and AIR-2 are part of a complex that is essential for chromosome segregation and for efficient completion of cytokinesis. We propose that this complex acts by promoting dissolution of sister chromatid cohesion and the assembly of the central spindle.

During anaphase, spindle elongation pulls sister chromatids apart until each pair is fully separated. In turn, cytokinesis cleaves the cell between the separated chromosomes. What ensures that cytokinesis proceeds only after that all chromosome arms are pulled out of the cleavage plane was unknown. Here, we show that a signaling pathway, which we call NoCut, delays the completion of cytokinesis in cells with spindle-midzone defects. NoCut depends on the Aurora kinase Ipl1 and the anillin-related proteins Boi1 and Boi2, which localize to the site of cleavage in an Ipl1-dependent manner and act as abscission inhibitors. Inactivation of NoCut leads to premature abscission and chromosome breakage by the cytokinetic machinery and is lethal in cells with spindle-elongation defects. We propose that NoCut monitors clearance of chromatin from the midzone to ensure that cytokinesis completes only after all chromosomes have migrated to the poles.

Arsenic (As) is a carcinogen and it is ubiquitous in the environment. Its accumulation in plants can enter the food chain, thereby causing adverse impacts on human health. Based on studies using Xenopus oocytes, engineered microbes, and model plants, aquaporins play key roles in arsenite (AsIII) passage across the cell membrane. Herein, we summarized the roles of aquaporins in AsIII uptake, transport, and accumulation. The coupling action between polar-localized aquaporins and positive transporters probably explains the efficient As transport mediated by bi-directional channels. The expression of aquaporins is regulated at both transcriptional and posttranscriptional levels via microRNAs. However, in response to As, the proteins can be modified through phosphorylation and be degraded via the ubiquitin 26S proteasome system. Further, a proposed model comprising reactive oxygen species, Ca, Ca-dependent kinases, and transcriptional factors is constructed to reveal the signaling pathways and aquaporins' responses to As in model plants. We also provided strategies to identify and investigate putative aquaporins in plants. Identification and modification of aquaporins' residues critical for their substrate selectivity, and optimization of their expression pattern by employing appropriate promoters can be employed to reduce As accumulation in crops. Based on the characteristics of their functions and expression patterns, it is promising to employ aquaporins for genetic-engineering of crops to reduce As content and increase food safety.

Arsenic (As) is a carcinogen and it is ubiquitous in the environment. Its accumulation in plants can enter the food chain, thereby causing adverse impacts on human health. Based on studies using Xenopus oocytes, engineered microbes, and model plants, aquaporins play key roles in arsenite (AsIII) passage across the cell membrane. Herein, we summarized the roles of aquaporins in AsIII uptake, transport, and accumulation. The coupling action between polar-localized aquaporins and positive transporters probably explains the efficient As transport mediated by bi-directional channels. The expression of aquaporins is regulated at both transcriptional and posttranscriptional levels via microRNAs. However, in response to As, the proteins can be modified through phosphorylation and be degraded via the ubiquitin 26S proteasome system. Further, a proposed model comprising reactive oxygen species, Ca, Ca-dependent kinases, and transcriptional factors is constructed to reveal the signaling pathways and aquaporins' responses to As in model plants. We also provided strategies to identify and investigate putative aquaporins in plants. Identification and modification of aquaporins' residues critical for their substrate selectivity, and optimization of their expression pattern by employing appropriate promoters can be employed to reduce As accumulation in crops. Based on the characteristics of their functions and expression patterns, it is promising to employ aquaporins for genetic-engineering of crops to reduce As content and increase food safety.

Moringa oleifera (MO) is renowned for its remarkable medicinal uses, supported by claims across various cultures and growing scientific evidence. Preclinical experimental evidence indicated that MO may effectively reduce bone loss and promote bone remodelling through its effects on osteoclasts and osteoblasts. In vivo studies demonstrated that MO enhances critical aspects of bone health, such as bone volume, trabecular thickness and overall bone density. Furthermore, MO positively influenced bone biomarkers including alkaline phosphatase and procollagen type 1 N-terminal propeptide, reflecting improved bone formation. Additionally, in vitro and ex vivo studies revealed that MO boosted bone regeneration, stimulated osteoblast activity and reduced inflammation. In terms of mechanisms, MO may modulate signalling pathways related to bone metabolism, such as BMP2, PI3K/Akt/FOXO1, p38α/MAPK14 and RANKL/RANK//OPG pathways. This evidence provides a strong foundation for future clinical research and potential therapeutic applications in managing and preventing bone loss conditions.

OPCT-1 is a heterogeneous prostate cancer cell line derived from primary (rather than metastatic) disease which contains epithelial, mesenchymal, and CD44 high / CD24 low cancer stem cell (CSC) subpopulations and from which we have previously generated and characterized stable mesenchymal (P4B6B) and epithelial (P5B3) cell subpopulations. In this contribution, we explore the effect of tissue culture surface chemistry (standard tissue culture plastic (TCP) and a fluoroalkylsilica (FS) culture surface with inherently low surface energy) on the phenotype and adherent capacity of mesenchymal and epithelial cell populations. We demonstrate that OPCT-1 cells adherent to FS surfaces comprise both epithelial-and mesenchymal-like populations; a mesenchymal subpopulation derived from OPCT1 (P4B6B) poorly adheres to FS and formed spheroids, whereas an epithelial subpopulation derived from OPCT1 (P5B3) forms an adherent monolayer. In contrast, P4B6B cells do adhere to FS when cocultured with P5B3 cells. Taken together, these findings demonstrate that EMT/cell differentiation status dictates cell adhesive capacity and provide a novel insight into the relationship between epithelial and mesenchymal cell populations in metastasis. Importantly, the differences in adherence capacity between P4B6B and P5B3 are not apparent using standard TCP-based culture, thereby highlighting the value of using alternative culture surfaces for studying cell surface interaction/adhesion phenomena and interrogating mechanisms involved in adhesion and detachment of metastatic tumor cells.

Traditional materials from new sourcesconflicts in analytical methods for calcium carbonate. Calcium carbonate, (E170) is a common food and pharmaceutical additive/ingredient. In addition to a source of calcium, the carbonate has uses including as a colour, acidity regulator and bulking agent. Globally, a range of regulatory agencies and pharmacopoeia control the analyses and specification of additives in food, supplements, pharmaceutical substances and excipients. Accordingly, a range of specifications and analyses exist for calcium carbonate depending on the application and market of the product. In this contribution, we analyse calcium carbonates from geological, synthetic and biogenic sources, focussing on acid insoluble impurities, a test required by current monographs. Analysis of calcium carbonate from different origins may require modification of existing tests to comply with regulatory bodies, due to the variation of impurities specific to the source of the material. We suggest an analytical approach involving centrifugation that improves analytical efficiency (up to 85% time reduction), especially for calcium carbonate of biological origin.

Hair follicle stem cells in the epithelial bulge are responsible for the continual regeneration of the hair follicle during cycling. The bulge cells reside in a niche composed of dermal cells. The dermal compartment of the hair follicle consists of the dermal papilla and dermal sheath. Interactions between hair follicle epithelial and dermal cells are necessary for hair follicle morphogenesis during development and in hair reconstitution assays. Dermal papilla and dermal sheath cells express specific markers and possess distinctive morphology and behavior in culture. These cells can induce hair follicle differentiation in epithelial cells and are required in hair reconstitution assays either in the form of intact tissue, dissociated freshly-prepared cells or cultured cells. This review will focus on hair follicle dermal cells since most therapeutic efforts to date have concentrated on this aspect of the hair follicle, with the idea that enriching hair-inductive dermal cell populations and expanding their number by culture while maintaining their properties, will establish an efficient hair reconstitution assay that could eventually have therapeutic implications.

Enhancers are context-specific regulators of expression that drive biological complexity and variation through the redeployment of conserved genes. An example of this is the enhancermediated control of Engrailed 1 (EN1), a pleiotropic gene whose expression is required for the formation of mammalian eccrine sweat glands. We previously identified the En1 candidate enhancer (ECE) 18 cis-regulatory element that has been highly and repeatedly derived on the human lineage to potentiate ectodermal EN1 and induce our species' uniquely high eccrine gland density. Intriguingly, ECE18 quantitative activity is negligible outside of primates and ECE18 is not required for En1 regulation and eccrine gland formation in mice, raising the possibility that distinct enhancers have evolved to modulate the same trait. Here we report the identification of the ECE20 enhancer and show it has conserved functionality in mouse and human developing skin ectoderm. Unlike ECE18, knock-out of ECE20 in mice reduces ectodermal En1 and eccrine gland number. Notably, we find ECE20, but not ECE18, is also required for En1 expression in the embryonic mouse brain, demonstrating that ECE20 is a pleiotropic En1 enhancer. Finally, that ECE18 deletion does not potentiate the eccrine phenotype of ECE20 knock-out mice supports the secondary incorporation of ECE18 into the regulation of this trait in primates. Our findings reveal that the mammalian En1 regulatory machinery diversified to incorporate both shared and lineage-restricted enhancers to regulate the same phenotype, and also have implications for understanding the forces that shape the robustness and evolvability of developmental traits.

Alopecia, or hair loss, is a condition of diverse origins, from genetic underpinnings to scarring arising from inflammation or trauma. Current treatments modulate hair growth by influencing existing precursor populations, however treatments aimed at de-novo production of hair follicles remain elusive. An in-vitro model studying de-novo human hair follicle formation from iPS cells or adult cells would open the potential for such novel treatments to be identified and tested. Due to the similarities between human and mouse hair follicles during development, we aimed to optimize current in-vitro conditions for de-novo murine hair follicle formation from neonatal cells. We used a modified mouse iPS cell organoid culture system in which a mixture of neonatal dermal and epidermal cells cultured in differentiation medium formed skin organoids that spontaneously produced de novo hair follicles in maturation medium. We then tested the activity, temporality, and length of modulation of different signaling pathways implicated in hair follicle formation. We observed through organoid culture activation of the WNT or SHH pathway with CHIR99021 or Hh-Ag1.5, respectively, in maturation medium facilitates more robust de-novo hair follicle production. We also observed that inhibition of the BMP pathway with LDN reduces hair follicle formation. Our results suggest that additional factors, regulated by concentration and temporality, are required for robust in-vitro murine hair follicle formation from mouse neonatal cells.

Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode ''budding'' is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions (AJs) is clearly required for budding. Snail-mediated downregulation of AJ component E-cadherin is important for placode budding in mice. Beta-catenin, another AJ component, has been more difficult to study owing to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes, and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent AJ dissolution.

Putative epithelial stem cells were identified in the hair follicle bulge as quiescent ''label retaining cells''. The study of these cells was hindered until the identification of bulge cell molecular markers, such as CD34 expression and K15 promoter activity. This allowed for the isolation and characterization of bulge cells from mouse follicles. Bulge cells possess stem cell characteristics, including multipotency, high proliferative potential, and their cardinal feature of quiescence. Lineage analysis demonstrated that all epithelial layers within the adult follicle and hair originated from bulge cells. Bulge cells only contribute to the epidermis during wound healing, but after isolation, when combined with neonatal dermal cells, they regenerate new hair follicles, epidermis, and sebaceous glands. Bulge cells maintain their stem cell characteristics after propagation in vitro, thus ultimately they may be useful for tissue engineering applications. Understanding the signals important for directing movement and differentiation of bulge cells into different lineages will be important for developing treatments based on stem cells as well as clarifying their role in skin disease.

During cellular senescence cells undergo permanent cell cycle arrest, and this protects from malignant transformation. Though important to control cancerous neoplasia, the same mechanisms may fundamentally drive aging and age-related disorders. Senescent cells in many instances release a battery of soluble factors, collectively termed as senescence associated secretory phenotype (SASP). Depending on histogenetic origin of cells, SASP can vary in components and composition. Of note, SASP spreads senescence within tissues and organs and promotes several age associated disorders, including skin aging and impairment of chronic wound healing. Employing already published gene interaction data, we developed a Boolean network model-based gene regulatory network of SASP. We have simulated the network with the aim to inhibit SASP during DNA damage induced senescence. This simulation allowed us to predict different in-silico gene knockouts those can prevent damaging effects of key SASP-factors. The most promising in-silico knockout candidates originating from this simulation was NF-kB Essential Modulator (NEMO) or IKK-g. Using in vitro experiments in murine dermal fibroblasts, we confirmed the importance of NEMO to successfully inhibit key SASP following DNA-damage. Therefore, we experimentally strengthened the speculated regulatory role of NF-kB signaling in the onset as well as maintenance of SASP using in-silico and in-vitro approaches. These data, thus, give access to potential therapeutic targets for SASP-associated aging diseases.

Conventionally, signaling through BCR initiates sequence of events necessary for activation and differentiation of B cells. We report an alternative approach, independent of BCR, for stimulating resting B (RB) cells, by involving TLR-2 and CD40molecules crucial for innate and adaptive immunity. CD40 triggering of TLR-2 stimulated RB cells significantly augments their activation, proliferation and differentiation. It also substantially ameliorates the calcium flux, antigen uptake capacity and ability of B cells to activate T cells. The survival of RB cells was improved and it increases the number of cells expressing activation induced deaminase (AID), signifying class switch recombination (CSR). Further, we also observed increased activation rate and decreased threshold period required for optimum stimulation of RB cells. These results corroborate well with microarray gene expression data. This study provides novel insights into coordination between the molecules of innate and adaptive immunity in activating B cells, in a BCR independent manner. This strategy can be exploited to design vaccines to bolster B cell activation and antigen presenting efficiency, leading to faster and better immune response.

B cells are an integral component in mounting humoral immune responses and they are also crucial in programming T cell mediated immunity. Usually, B cell activation is initiated by recognition of antigen through B cell receptor (BCR), followed by its processing and presentation to T cells. But very little is known about BCR independent activation of B cells. Now, there is an increasing body of evidence indicating the combinatorial effect of innate and adaptive immune components in modulating the functions of B cells. In this study, we demonstrate the activation of resting B cells (RB) by simultaneous involvement of Toll like Receptor-2 (TLR-2) and costimulatory molecule, CD86. Interestingly, these B cells exhibited significant level of activation and proliferation. Furthermore, this process of activation leads to the differentiation of RB cells, preferably into marginal zone precursors (CD19 + IgD hi IgM hi CD21/35 hi CD23 hi ) in a shorter time window and showed increased secretion of IgG isotype. These RB cells also showed enhanced antigen uptake capacity. These observations were also substantiated by microarray gene expression results, which strengthen the notion that combinatorial signaling through innate and adaptive immune components enhances B cell mediated immune response. Thus, the present study elucidates a novel BCR independent B cell activation mechanism that links TLR-2 and CD86. Hence signaling of TLRs in conjunction with costimulatory molecules will substantially help in bolstering humoral immune response, which can be extrapolated to formulate vaccination strategies for diseases involving B cell-mediated immunity.