Monte Carlo Study Elucidates the Type 1/Type 2 Choice in Apoptotic Death Signaling in Healthy and Cancer Cells

PubMed Central

Raychaudhuri, Subhadip; Raychaudhuri, Somkanya C

2013-01-01

Apoptotic cell death is coordinated through two distinct (type 1 and type 2) intracellular signaling pathways. How the type 1/type 2 choice is made remains a central problem in the biology of apoptosis and has implications for apoptosis related diseases and therapy. We study the problem of type 1/type 2 choice in silico utilizing a kinetic Monte Carlo model of cell death signaling. Our results show that the type 1/type 2 choice is linked to deterministic versus stochastic cell death activation, elucidating a unique regulatory control of the apoptotic pathways. Consistent with previous findings, our results indicate that caspase 8 activation level is a key regulator of the choice between deterministic type 1 and stochastic type 2 pathways, irrespective of cell types. Expression levels of signaling molecules downstream also regulate the type 1/type 2 choice. A simplified model of DISC clustering elucidates the mechanism of increased active caspase 8 generation and type 1 activation in cancer cells having increased sensitivity to death receptor activation. We demonstrate that rapid deterministic activation of the type 1 pathway can selectively target such cancer cells, especially if XIAP is also inhibited; while inherent cell-to-cell variability would allow normal cells stay protected. PMID:24709706

Genome Wide DNA Methylation Profiles Provide Clues to the Origin and Pathogenesis of Germ Cell Tumors

PubMed Central

Rijlaarsdam, Martin A.; Tax, David M. J.; Gillis, Ad J. M.; Dorssers, Lambert C. J.; Koestler, Devin C.; de Ridder, Jeroen; Looijenga, Leendert H. J.

2015-01-01

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809). PMID:25859847

Type I intrinsically photosensitive retinal ganglion cells of early post-natal development correspond to the M4 subtype.

PubMed

Sexton, Timothy J; Bleckert, Adam; Turner, Maxwell H; Van Gelder, Russell N

2015-06-21

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate circadian light entrainment and the pupillary light response in adult mice. In early development these cells mediate different processes, including negative phototaxis and the timing of retinal vascular development. To determine if ipRGC physiologic properties also change with development, we measured ipRGC cell density and light responses in wild-type mouse retinas at post-natal days 8, 15 and 30. Melanopsin-positive cell density decreases by 17% between post-natal days 8 and 15 and by 25% between days 8 and 30. This decrease is due specifically to a decrease in cells co-labeled with a SMI-32, a marker for alpha-on ganglion cells (corresponding to adult morphologic type M4 ipRGCs). On multi-electrode array recordings, post-natal day 8 (P8) ipRGC light responses show more robust firing, reduced adaptation and more rapid recovery from short and extended light pulses than do the light responses of P15 and P30 ipRGCs. Three ipRGC subtypes - Types I-III - have been defined in early development based on sensitivity and latency on multielectrode array recordings. We find that Type I cells largely account for the unique physiologic properties of P8 ipRGCs. Type I cells have previously been shown to have relatively short latencies and high sensitivity. We now show that Type I cells show have rapid and robust recovery from long and short bright light exposures compared with Type II and III cells, suggesting differential light adaptation mechanisms between cell types. By P15, Type I ipRGCs are no longer detectable. Loose patch recordings of P8 M4 ipRGCs demonstrate Type I physiology. Type I ipRGCs are found only in early development. In addition to their previously described high sensitivity and rapid kinetics, these cells are uniquely resistant to adaptation and recover quickly and fully to short and prolonged light exposure. Type I ipRGCs correspond to the SMI-32 positive, M4 subtype and largely lose melanopsin expression in development. These cells constitute a unique morphologic and physiologic class of ipRGCs functioning early in postnatal development.

Myxococcus xanthus Developmental Cell Fate Production: Heterogeneous Accumulation of Developmental Regulatory Proteins and Reexamination of the Role of MazF in Developmental Lysis

PubMed Central

Lee, Bongsoo; Holkenbrink, Carina; Treuner-Lange, Anke

2012-01-01

Myxococcus xanthus undergoes a starvation-induced multicellular developmental program during which cells partition into three known fates: (i) aggregation into fruiting bodies followed by differentiation into spores, (ii) lysis, or (iii) differentiation into nonaggregating persister-like cells, termed peripheral rods. As a first step to characterize cell fate segregation, we enumerated total, aggregating, and nonaggregating cells throughout the developmental program. We demonstrate that both cell lysis and cell aggregation begin with similar timing at approximately 24 h after induction of development. Examination of several known regulatory proteins in the separated aggregated and nonaggregated cell fractions revealed previously unknown heterogeneity in the accumulation patterns of proteins involved in type IV pilus (T4P)-mediated motility (PilC and PilA) and regulation of development (MrpC, FruA, and C-signal). As part of our characterization of the cell lysis fate, we set out to investigate the unorthodox MazF-MrpC toxin-antitoxin system which was previously proposed to induce programmed cell death (PCD). We demonstrate that deletion of mazF in two different wild-type M. xanthus laboratory strains does not significantly reduce developmental cell lysis, suggesting that MazF's role in promoting PCD is an adaption to the mutant background strain used previously. PMID:22493014

Sensory Cells of the Fish Ear: A Hairy Enigma

NASA Technical Reports Server (NTRS)

Popper, A. N.; Saidel, W. M.

1995-01-01

Analysis of the structure of the ears in teleost fishes has led to the tentative suggestion that otolithic endorgans may function differently, in different species. Recently, evidence has demonstrated different 'types' of sensory hair cells can be found in the ears of teleost fishes, and individual hair cell types are found in discrete regions of individual sensory, epithelia. The presence of multiple hair cell types in fishes provides strong support to the hypothesis of regional differences in the responses of individual otolithic sensory epithelia. The finding of hair cell types in fishes that closely resemble those found in amniote vestibular endorgans also suggests that hair cell heterogeneity arose earlier in the evolution of the vertebrate ear than previously thought.

Adult, embryonic and fetal hemoglobin are expressed in human glioblastoma cells.

PubMed

Emara, Marwan; Turner, A Robert; Allalunis-Turner, Joan

2014-02-01

Hemoglobin is a hemoprotein, produced mainly in erythrocytes circulating in the blood. However, non-erythroid hemoglobins have been previously reported in other cell types including human and rodent neurons of embryonic and adult brain, but not astrocytes and oligodendrocytes. Human glioblastoma multiforme (GBM) is the most aggressive tumor among gliomas. However, despite extensive basic and clinical research studies on GBM cells, little is known about glial defence mechanisms that allow these cells to survive and resist various types of treatment. We have shown previously that the newest members of vertebrate globin family, neuroglobin (Ngb) and cytoglobin (Cygb), are expressed in human GBM cells. In this study, we sought to determine whether hemoglobin is also expressed in GBM cells. Conventional RT-PCR, DNA sequencing, western blot analysis, mass spectrometry and fluorescence microscopy were used to investigate globin expression in GBM cell lines (M006x, M059J, M059K, M010b, U87R and U87T) that have unique characteristics in terms of tumor invasion and response to radiotherapy and hypoxia. The data showed that α, β, γ, δ, ζ and ε globins are expressed in all tested GBM cell lines. To our knowledge, we are the first to report expression of fetal, embryonic and adult hemoglobin in GBM cells under normal physiological conditions that may suggest an undefined function of those expressed hemoglobins. Together with our previous reports on globins (Ngb and Cygb) expression in GBM cells, the expression of different hemoglobins may constitute a part of series of active defence mechanisms supporting these cells to resist various types of treatments including chemotherapy and radiotherapy.

Single cell array impedance analysis in a microfluidic device

NASA Astrophysics Data System (ADS)

Altinagac, Emre; Taskin, Selen; Kizil, Huseyin

2016-10-01

Impedance analysis of single cells is presented in this paper. Following the separation of a target cell type by dielectrophoresis in our previous work, this paper focuses on capturing the cells as a single array and performing impedance analysis to point out the signature difference between each cell type. Lab-on-a-chip devices having a titanium interdigitated electrode layer on a glass substrate and a PDMS microchannel are fabricated to capture each cell in a single form and perform impedance analysis. HCT116 (homosapiens colon colorectal carcin) and HEK293 (human embryonic kidney) cells are used in our experiments.

CHZ868, a Type II JAK2 Inhibitor, Reverses Type I JAK Inhibitor Persistence and Demonstrates Efficacy in Myeloproliferative Neoplasms

PubMed Central

Meyer, Sara C.; Keller, Matthew D.; Chiu, Sophia; Koppikar, Priya; Guryanova, Olga A.; Rapaport, Franck; Xu, Ke; Manova, Katia; Pankov, Dmitry; O’Reilly, Richard J.; Kleppe, Maria; McKenney, Anna Sophia; Shih, Alan H.; Shank, Kaitlyn; Ahn, Jihae; Papalexi, Eftymia; Spitzer, Barbara; Socci, Nick; Viale, Agnes; Mandon, Emeline; Ebel, Nicolas; Andraos, Rita; Rubert, Joëlle; Dammassa, Ernesta; Romanet, Vincent; Dölemeyer, Arno; Zender, Michael; Heinlein, Melanie; Rampal, Rajit; Weinberg, Rona Singer; Hoffman, Ron; Sellers, William R.; Hofmann, Francesco; Murakami, Masato; Baffert, Fabienne; Gaul, Christoph; Radimerski, Thomas; Levine, Ross L.

2015-01-01

Summary Although clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasms (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated if CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2- and MPL-mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients. PMID:26175413

Rapid CD4(+) T-cell loss induced by human immunodeficiency virus type 1(NC) in uninfected and previously infected chimpanzees.

PubMed

Novembre, F J; de Rosayro, J; Nidtha, S; O'Neil, S P; Gibson, T R; Evans-Strickfaden, T; Hart, C E; McClure, H M

2001-02-01

To investigate the pathogenicity of a virus originating in a chimpanzee with AIDS (C499), two chimpanzees were inoculated with a plasma-derived isolate termed human immunodeficiency virus type 1(NC) (HIV-1(NC)). A previously uninfected chimpanzee, C534, experienced rapid peripheral CD4(+) T-cell loss to fewer than 26 cells/microl by 14 weeks after infection. CD4(+) T-cell depletion was associated with high plasma HIV-1 loads but a low virus burden in the peripheral lymph node. The second chimpanzee, C459, infected 13 years previously with HIV-1(LAV), experienced a more protracted course of peripheral CD4(+) T-cell loss after HIV-1(NC) inoculation, resulting in fewer than 200 cells/microl by 96 weeks postinoculation. The quantities of viral RNA in the plasma and peripheral lymph node from C459 were below the lower limits of detection prior to inoculation with HIV-1(NC) but were significantly and persistently increased after superinfection, with HIV-1(NC) representing the predominant viral genotype. These results show that viruses derived from C499 are more pathogenic for chimpanzees than any other HIV-1 isolates described to date.

Acetyl phosphate and the phosphorylation of OmpR are involved in the regulation of the cell division rate in Escherichia coli.

PubMed

Prüss, B M

1998-09-01

Carbon sources that can be converted to acetate were added to the growth medium of Escherichia coli wild-type cells. Cells responded with an increased cell division rate. The addition of acetate also caused a decreased synthesis of flagella. Mutants in phosphotransacetylase, which are incapable of synthesizing acetyl phosphate, and mutants in the osmoregulator OmpR divided at a lower rate than did wild-type cells. The mutants did not increase their cell division rate upon the addition of serine, as observed for wild-type cells. These data are consistent with the idea that the previously described effect of serine upon the cell division rate is mediated by acetyl phosphate and phosphorylation of OmpR.

Endogenous New World primate type C viruses isolated from owl monkey (Aotus trivirgatus) kidney cell line.

PubMed Central

Todaro, G J; Sherr, C J; Sen, A; King, N; Daniel, M D; Fleckenstein, B

1978-01-01

A type C virus (OMC-1) detected in a culture of owl monkey kidney cells resembled typical type C viruses morphologically, but was slightly larger than previously characterized mammalian type C viruses. OMC-1 can be transmitted to bat lung cells and cat embryo fibroblasts. The virions band at a density of 1.16 g/ml in isopycnic sucrose density gradients and contain reverse transcriptase and a 60-65S RNA genome composed of approximately 32S subunits. The reverse transcriptase is immunologically and biochemically distinct from the polymerases of othe retroviruses. Radioimmunoassays directed to the interspecies antigenic determinants of the major structure proteins of other type C viruses do not detect a related antigen in OMC-1. Nucleic acid hybridization experiments using labeled viral genomic RNA or proviral cDNA transcripts to normal cellular DNA of different species show that OMC-1 is an endogenous virus with multiple virogene copies (20-50 per haploid genome) present in normal owl monkey cells and is distinct from previously isolated type C and D viruses. Sequences related to the OMC-1 genome can be detected in other New World monkeys. Thus, similar to the Old World primates (e.g., baboons as a prototype), the New World monkeys contain endogenous type C viral genes that appear to have been transmitted in the primate germ line. Images PMID:76312

Morphological and immunohistochemical diversity of endometrial stromal sarcoma in rats.

PubMed

Kumabe, Shino; Sato, Junko; Tomonari, Yuki; Takahashi, Miwa; Inoue, Kaoru; Yoshida, Midori; Doi, Takuya; Wako, Yumi; Tsuchitani, Minoru

2018-04-01

To clarify the histopathological characteristics of rat endometrial stromal sarcoma (ESS), we morphologically reviewed 12 malignant uterine tumors protruding into the lumen in previous rat carcinogenicity studies. The 12 cases were classified into the following 6 types based on their morphological features: spindle cell and collagen rich type, pleomorphic/spindle cell and compact type, decidual alteration type, histiocytic and multinucleated giant cell mixture type, Antoni A-type schwannoma type, and Antoni B-type schwannoma type. Immunohistochemically, tumor cells in all cases exhibited focal or diffuse positive reactions for vimentin, and 11 of the 12 cases were positive for S-100. Interestingly, 9 cases were positive for desmin or αSMA, indicating tumor cells expressing smooth muscle properties. Both Antoni A- and B-type schwannoma types showed low reactions for both muscle markers. Positive results for estrogen receptor α in the 11 cases suggested that they were derived from endometrial stromal cells. On the basis of their immunohistochemical profiles, they were considered to be derived from endometrial stromal cells while they showed morphological variation. The detection of a basement membrane surrounding tumor cells might not be a definitive indicator for differential diagnosis of ESS from malignant schwannoma. In conclusion, ESS could exhibit wide morphological and immunohistochemical variation including features of schwannoma or smooth muscle tumor.

Low-level lasers affect Escherichia coli cultures in hyperosmotic stress

NASA Astrophysics Data System (ADS)

Pinheiro, C. C.; Barboza, L. L.; Paoli, F.; Fonseca, A. S.

2015-08-01

Physical characteristics and practical properties have made lasers of interest for biomedical applications. Effects of low-level lasers on biological tissues could occur or be measurable depending on cell type, presence of a pathologic process or whether the cells are in an adverse environment. The objective of this work was to evaluate the survival, morphology and filamentation of E. coli cells proficient and deficient in the repair of oxidative DNA lesions exposed low-level red and infrared lasers submitted to hyperosmotic stress. Wild type and endonuclease VIII deficient E. coli cells in exponential and stationary growth phase were exposed to red and infrared lasers and submitted to hyperosmotic stress. Cell viability, filamentation phenotype and cell morphology were evaluated. Cell viability was not significantly altered but previous laser exposure induced filamentation and an altered area of stressed cells depending on physiologic condition and presence of the DNA repair. Results suggest that previous exposure to low-level red and infrared lasers could not affect viability but induced morphologic changes in cells submitted to hyperosmotic stress depending on physiologic conditions and repair of oxidative DNA lesions.

The Adaptor Protein SAP Regulates Type II NKT Cell Development, Cytokine Production and Cytotoxicity Against Lymphoma1

PubMed Central

Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L.; Stein, Paul L.; Wang, Chyung-Ru

2014-01-01

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule-associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT cell TCR transgenic mouse model (24αβTg), we demonstrated that CD1d-expressing hematopoietic cells but not thymic epithelial cells meditate efficient selection of type II NKT cells. Further, we showed that SAP regulates type II NKT cell development by controlling Egr2 and PLZF expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IRF4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP. PMID:25236978

The adaptor protein SAP regulates type II NKT-cell development, cytokine production, and cytotoxicity against lymphoma.

PubMed

Weng, Xiufang; Liao, Chia-Min; Bagchi, Sreya; Cardell, Susanna L; Stein, Paul L; Wang, Chyung-Ru

2014-12-01

CD1d-restricted NKT cells represent a unique lineage of immunoregulatory T cells that are divided into two groups, type I and type II, based on their TCR usage. Because there are no specific tools to identify type II NKT cells, little is known about their developmental requirements and functional regulation. In our previous study, we showed that signaling lymphocytic activation molecule associated protein (SAP) is essential for the development of type II NKT cells. Here, using a type II NKT-cell TCR transgenic mouse model, we demonstrated that CD1d-expressing hematopoietic cells, but not thymic epithelial cells, meditate efficient selection of type II NKT cells. Furthermore, we showed that SAP regulates type II NKT-cell development by controlling early growth response 2 protein and promyelocytic leukemia zinc finger expression. SAP-deficient 24αβ transgenic T cells (24αβ T cells) exhibited an immature phenotype with reduced Th2 cytokine-producing capacity and diminished cytotoxicity to CD1d-expressing lymphoma cells. The impaired IL-4 production by SAP-deficient 24αβ T cells was associated with reduced IFN regulatory factor 4 and GATA-3 induction following TCR stimulation. Collectively, these data suggest that SAP is critical for regulating type II NKT cell responses. Aberrant responses of these T cells may contribute to the immune dysregulation observed in X-linked lymphoproliferative disease caused by mutations in SAP. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

PubMed

Tsukamoto, Kentaro; Ozeki, Chikako; Kohda, Tomoko; Tsuji, Takao

2015-01-01

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

Insight into Genotype-Phenotype Associations through eQTL Mapping in Multiple Cell Types in Health and Immune-Mediated Disease

PubMed Central

Peters, James E.; Lyons, Paul A.; Lee, James C.; Richard, Arianne C.; Fortune, Mary D.; Newcombe, Paul J.; Richardson, Sylvia; Smith, Kenneth G. C.

2016-01-01

Genome-wide association studies (GWAS) have transformed our understanding of the genetics of complex traits such as autoimmune diseases, but how risk variants contribute to pathogenesis remains largely unknown. Identifying genetic variants that affect gene expression (expression quantitative trait loci, or eQTLs) is crucial to addressing this. eQTLs vary between tissues and following in vitro cellular activation, but have not been examined in the context of human inflammatory diseases. We performed eQTL mapping in five primary immune cell types from patients with active inflammatory bowel disease (n = 91), anti-neutrophil cytoplasmic antibody-associated vasculitis (n = 46) and healthy controls (n = 43), revealing eQTLs present only in the context of active inflammatory disease. Moreover, we show that following treatment a proportion of these eQTLs disappear. Through joint analysis of expression data from multiple cell types, we reveal that previous estimates of eQTL immune cell-type specificity are likely to have been exaggerated. Finally, by analysing gene expression data from multiple cell types, we find eQTLs not previously identified by database mining at 34 inflammatory bowel disease-associated loci. In summary, this parallel eQTL analysis in multiple leucocyte subsets from patients with active disease provides new insights into the genetic basis of immune-mediated diseases. PMID:27015630

Apocrine hidradenocarcinoma showing Paget's disease and mucinous metaplasia.

PubMed

Wahl, Carter E; Todd, Douglas H; Binder, Scott W; Cassarino, David S

2009-05-01

A 54-year-old man presented with a solitary, erythematous, rapidly growing 1-cm nodule on his scalp that had arisen over the previous 3 months. He had no history of skin cancer. An excisional biopsy of the lesion showed a fairly well-circumscribed but focally invasive tumor consisting of areas of typical-appearing clear cell hidradenoma as well as areas with mucinous goblet-type cells and cells with eosinophilic cytoplasm and decapitation-type secretion. There was marked cellular atypia, numerous atypical mitotic figures and focal necrosis. The tumor cells focally involved the overlying epidermis (Paget's disease). Large areas of mucin were identified throughout the lesion. The tumor cells stained with markers for cytokeratin 7 and focally for EMA and CEA, confirming ductal differentiation. The goblet cells and mucinous areas stained with mucicarmine and PASD. The patient was diagnosed with hidradenocarcinoma with mucinous differentiation. Associated Paget's disease has only rarely been reported, and mucinous metaplasia is a previously unreported feature in hidradenocarcinoma.

A Molecular Census of Arcuate Hypothalamus and Median Eminence Cell Types

PubMed Central

Campbell, John N.; Macosko, Evan Z.; Fenselau, Henning; Pers, Tune H.; Lyubetskaya, Anna; Tenen, Danielle; Goldman, Melissa; Verstegen, Anne M.J.; Resch, Jon M.; McCarroll, Steven A.; Rosen, Evan D.; Lowell, Bradford B.; Tsai, Linus

2017-01-01

The hypothalamic arcuate-median eminence complex (Arc-ME) controls energy balance, fertility, and growth through molecularly distinct cell types, many of which remain unknown. To catalog cell types in an unbiased way, we profiled gene expression in 20,921 individual cells in and around the adult mouse Arc-ME using Drop-seq. We identify 50 transcriptionally distinct Arc-ME cell populations, including a rare tanycyte population at the Arc-ME diffusion barrier, a novel leptin-sensing neuronal population, multiple AgRP and POMC subtypes, and an orexigenic somatostatin neuronal population. We extended Drop-seq to detect dynamic expression changes across relevant physiological perturbations, revealing cell type-specific responses to energy status, including distinctly responsive subtypes of AgRP and POMC neurons. Finally, integrating our data with human GWAS data implicates two previously unknown neuronal subtypes in the genetic control of obesity. This resource will accelerate biological discovery by providing insights into molecular and cell type diversity from which function can be inferred. PMID:28166221

Differential requirement for the IKKβ/NF-κB signaling module in regulating TLR versus RLR-induced type 1 IFN expression in dendritic cells1

PubMed Central

Wang, Xingyu; Wang, Junmei; Zheng, Hong; Xie, Mengyu; Hopewell, Emily L.; Albrecht, Randy A.; Nogusa, Shoko; García-Sastre, Adolfo; Balachandran, Siddharth; Beg, Amer A.

2014-01-01

Host innate-immune responses are tailored by cell-type to control and eradicate specific infectious agents. For example, an acute RNA virus infection can result in high-level expression of type 1 interferons (IFNs) by both conventional (cDCs) and plasmacytoid dendritic cells (pDCs), but while cDCs preferentially utilize RIG-I-like Receptor (RLR) signaling to produce type 1 IFNs, pDCs predominantly employ Toll-like Receptors (TLR) to induce these cytokines. We previously found that the IKKβ/NF-κB pathway regulates early IFN-β expression but not the magnitude of type 1 IFN expression following RLR engagement. In this study, we use IKKβ inhibition and mice deficient in IKKβ or canonical NF-κB subunits (p50, RelA/p65 and cRel) to demonstrate that the IKKβ/NF-κB axis is critically important for virus-induced type 1 IFN expression in pDCs, but not in cDCs. We also reveal a crucial and more general requirement for IKKβ/NF-κB in TLR - but not RLR- induced expression of type 1 IFNs and inflammatory cytokines. Together, these findings reveal a previously unappreciated specificity of the IKKβ/NF-κB signaling axis in regulation of anti-microbial responses by different classes of PRR, and therefore by individual cell-types reliant on particular PRRs for their innate-immune transcriptional responses. PMID:25057006

Identification of cyst nematode B-type CLE peptides and modulation of the vascular stem cell pathway for feeding cell formation

USDA-ARS?s Scientific Manuscript database

Stem cells are important in the continuous formation of various tissues during postembryonic organogenesis. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular procambium/cambium are regulated by CLE-receptor kinase-WOX signaling modules. Previous data showed ...

Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice.

PubMed

Li, Yafan; Wheeler, Deric L; Ananthaswamy, Honnavara N; Verma, Ajit K; Oberley, Terry D

2007-12-01

Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

Publisher Correction: Single-cell analysis of experience-dependent transcriptomic states in the mouse visual cortex.

PubMed

Hrvatin, Sinisa; Hochbaum, Daniel R; Nagy, M Aurel; Cicconet, Marcelo; Robertson, Keiramarie; Cheadle, Lucas; Zilionis, Rapolas; Ratner, Alex; Borges-Monroy, Rebeca; Klein, Allon M; Sabatini, Bernardo L; Greenberg, Michael E

2018-05-11

In the version of this article initially published, the x-axis labels in Fig. 3c read Vglut, Gad1/2, Aldh1l1 and Pecam1; they should have read Vglut + , Gad1/2 + , Aldh1l1 + and Pecam1 + . In Fig. 4, the range values were missing from the color scales; they are, from left to right, 4-15, 0-15, 4-15 and 0-15 in Fig. 4a and 4-15, 4-15 and 4-8 in Fig. 4h. In the third paragraph of the main text, the phrase reading "Previous approaches have analyzed a limited number of inhibitory cell types, thus masking the full diversity of excitatory populations" should have read "Previous approaches have analyzed a limited number of inhibitory cell types and masked the full diversity of excitatory populations." In the second paragraph of Results section "Diversity of experience-regulated ERGs," the phrase reading "thus suggesting considerable divergence within the gene expression program responding to early stimuli" should have read "thus suggesting considerable divergence within the early stimulus-responsive gene expression program." In the fourth paragraph of Results section "Excitatory neuronal LRGs," the sentence reading "The anatomical organization of these cell types into sublayers, coupled with divergent transcriptional responses to a sensory stimulus, suggested previously unappreciated functional subdivisions located within the laminae of the mouse visual cortex and resembling the cytoarchitecture in higher mammals" should have read "The anatomical organization of these cell types into sublayers, coupled with divergent transcriptional responses to a sensory stimulus, suggests previously unappreciated functional subdivisions located within the laminae of the mouse visual cortex, resembling the cytoarchitecture in higher mammals." In the last sentence of the Results, "sensory-responsive genes" should have read "sensory-stimulus-responsive genes." The errors have been corrected in the HTML and PDF versions of the article.

The candidate sour taste receptor, PKD2L1, is expressed by type III taste cells in the mouse.

PubMed

Kataoka, Shinji; Yang, Ruibiao; Ishimaru, Yoshiro; Matsunami, Hiroaki; Sévigny, Jean; Kinnamon, John C; Finger, Thomas E

2008-03-01

The transient receptor potential channel, PKD2L1, is reported to be a candidate receptor for sour taste based on molecular biological and functional studies. Here, we investigated the expression pattern of PKD2L1-immunoreactivity (IR) in taste buds of the mouse. PKD2L1-IR is present in a few elongate cells in each taste bud as reported previously. The PKD2L1-expressing cells are different from those expressing PLCbeta2, a marker of Type II cells. Likewise PKD2L1-immunoreactive taste cells do not express ecto-ATPase which marks Type I cells. The PKD2L1-positive cells are immunoreactive for neural cell adhesion molecule, serotonin, PGP-9.5 (ubiquitin carboxy-terminal transferase), and chromogranin A, all of which are present in Type III taste cells. At the ultrastructural level, PKD2L1-immunoreactive cells form synapses onto afferent nerve fibers, another feature of Type III taste cells. These results are consistent with the idea that different taste cells in each taste bud perform distinct functions. We suggest that Type III cells are necessary for transduction and/or transmission of information about "sour", but have little or no role in transmission of taste information of other taste qualities.

Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats.

PubMed

Lee, Sang-Bok; Lee, Cil-Han; Kim, Se-Nyun; Chung, Ki-Myung; Cho, Young-Kyung; Kim, Kyung-Nyun

2009-12-01

Glutamate-induced cobalt uptake reveals that non-NMDA glutamate receptors (GluRs) are present in rat taste bud cells. Previous studies involving glutamate induced cobalt staining suggest this uptake mainly occurs via kainate type GluRs. It is not known which of the 4 types of taste bud cells express subunits of kainate GluR. Circumvallate and foliate papillae of Sprague-Dawley rats (45~60 days old) were used to search for the mRNAs of subunits of non-NMDA GluRs using RT-PCR with specific primers for GluR1-7, KA1 and KA2. We also performed RT-PCR for GluR5, KA1, PLCbeta2, and NCAM/SNAP 25 in isolated single cells from taste buds. Taste epithelium, including circumvallate or foliate papilla, express mRNAs of GluR5 and KA1. However, non-taste tongue epithelium expresses no subunits of non-NMDA GluRs. Isolated single cell RT-PCR reveals that the mRNAs of GluR5 and KA1 are preferentially expressed in Type II and Type III cells over Type I cells.

Rapid CD4+ T-Cell Loss Induced by Human Immunodeficiency Virus Type 1NC in Uninfected and Previously Infected Chimpanzees

PubMed Central

Novembre, Francis J.; de Rosayro, Juliette; Nidtha, Soumya; O'Neil, Shawn P.; Gibson, Terri R.; Evans-Strickfaden, Tammy; Hart, Clyde E.; McClure, Harold M.

2001-01-01

To investigate the pathogenicity of a virus originating in a chimpanzee with AIDS (C499), two chimpanzees were inoculated with a plasma-derived isolate termed human immunodeficiency virus type 1NC (HIV-1NC). A previously uninfected chimpanzee, C534, experienced rapid peripheral CD4+ T-cell loss to fewer than 26 cells/μl by 14 weeks after infection. CD4+ T-cell depletion was associated with high plasma HIV-1 loads but a low virus burden in the peripheral lymph node. The second chimpanzee, C459, infected 13 years previously with HIV-1LAV, experienced a more protracted course of peripheral CD4+ T-cell loss after HIV-1NC inoculation, resulting in fewer than 200 cells/μl by 96 weeks postinoculation. The quantities of viral RNA in the plasma and peripheral lymph node from C459 were below the lower limits of detection prior to inoculation with HIV-1NC but were significantly and persistently increased after superinfection, with HIV-1NC representing the predominant viral genotype. These results show that viruses derived from C499 are more pathogenic for chimpanzees than any other HIV-1 isolates described to date. PMID:11152525

Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes

PubMed Central

Bolzer, Andreas; Kreth, Gregor; Solovei, Irina; Koehler, Daniela; Saracoglu, Kaan; Fauth, Christine; Müller, Stefan; Eils, Roland; Cremer, Christoph; Speicher, Michael R

2005-01-01

Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes—independently of their gene density—were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding. PMID:15839726

The candidate sour taste receptor, PKD2L1, is expressed by type III taste cells in the mouse

PubMed Central

Kataoka, Shinji; Yang, Ruibiao; Ishimaru, Yoshiro; Matsunami, Hiroaki; Kinnamon, John C.; Finger, Thomas E.

2008-01-01

The transient receptor potential (TRP) channel, PKD2L1, is reported to be a candidate receptor for sour taste based on molecular biological and functional studies. Here, we investigated the expression pattern of PKD2L1-immunoreactivity (IR) in taste buds of the mouse. PKD2L1-IR is present in a few elongate cells in each taste bud as reported previously. The PKD2L1-expressing cells are different from those expressing PLCβ2, a marker of Type II cells. Likewise PKD2L1-immunoreactive taste cells do not express ecto-ATPase which marks Type I cells. The PKD2L1 positive cells are immunoreactive for NCAM, serotonin, PGP-9.5 (ubiquitin carboxy terminal transferase) and chromogranin A, all of which are present in Type III taste cells. At the ultrastructural level, PKD2L1-immunoreactive cells form synapses onto afferent nerve fibers, another feature of Type III taste cells. These results are consistent with the idea that different taste cells in each taste bud perform distinct functions. We suggest that Type III cells are necessary for transduction and/or transmission of information about “sour”, but have little or no role in transmission of taste information of other taste qualities. PMID:18156604

Activation of ERK signaling and induction of colon cancer cell death by piperlongumine

USDA-ARS?s Scientific Manuscript database

Piperlongumine (PPLGM) is a bioactive compound isolated from long peppers that shows selective toxicity towards a variety of cancer cell types including colon cancer. The signaling pathways that lead to cancer cell death in response to PPLGM exposure have not been previously identified. Our objectiv...

Cuticular Waxes of Arabidopsis thaliana Shoots: Cell-Type-Specific Composition and Biosynthesis

PubMed Central

Hegebarth, Daniela; Jetter, Reinhard

2017-01-01

It is generally assumed that all plant epidermis cells are covered with cuticles, and the distinct surface geometries of pavement cells, guard cells, and trichomes imply functional differences and possibly different wax compositions. However, experiments probing cell-type-specific wax compositions and biosynthesis have been lacking until recently. This review summarizes new evidence showing that Arabidopsis trichomes have fewer wax compound classes than pavement cells, and higher amounts of especially long-chain hydrocarbons. The biosynthesis machinery generating this characteristic surface coating is discussed. Interestingly, wax compounds with similar, long hydrocarbon chains had been identified previously in some unrelated species, not all of them bearing trichomes. PMID:28686187

Dilute passage promotes expression of genetic and phenotypic variants of human immunodeficiency virus type 1 in cell culture.

PubMed Central

Sánchez-Palomino, S; Rojas, J M; Martínez, M A; Fenyö, E M; Nájera, R; Domingo, E; López-Galíndez, C

1993-01-01

We have studied the extent of genetic and phenotypic diversification of human immunodeficiency virus type 1 (HIV-1) upon 15 serial passages of clonal viral populations in MT-4 cell cultures. Several genetic and phenotypic modifications previously noted during evolution of HIV-1 in infected humans were also observed upon passages of the virus in cell culture. Notably, the transition from non-syncytium-inducing to syncytium-inducing phenotype (previously observed during disease progression) and fixation of amino acid substitutions at the main antigenic loop V3 of gp120 were observed in the course of replication of the virus in MT-4 cell cultures in the absence of immune selection. Interestingly, most genetic and phenotypic alterations occurred upon passage of the virus at a low multiplicity of infection (0.001 infectious particles per cell) rather than at a higher multiplicity of infection (0.1 infectious particles per cell). The degree of genetic diversification attained by HIV-1, estimated by the RNase A mismatch cleavage method and by nucleotide sequencing, is of about 0.03% of genomic sites mutated after 15 serial passages. This value is not significantly different from previous estimates for foot-and-mouth disease virus when subjected to a similar process and analysis. We conclude that several genetic and phenotypic modifications of HIV-1 previously observed in vivo occur also in the constant environment provided by a cell culture system. Dilute passage promotes in a highly significant way the expression of deviant HIV-1 genomes. Images PMID:8474182

Selective expression of muscarinic acetylcholine receptor subtype M3 by mouse type III taste bud cells.

PubMed

Mori, Yusuke; Eguchi, Kohgaku; Yoshii, Kiyonori; Ohtubo, Yoshitaka

2016-11-01

Each taste bud cell (TBC) type responds to a different taste. Previously, we showed that an unidentified cell type(s) functionally expresses a muscarinic acetylcholine (ACh) receptor subtype, M3, and we suggested the ACh-dependent modification of its taste responsiveness. In this study, we found that M3 is expressed by type III TBCs, which is the only cell type that possesses synaptic contacts with taste nerve fibers in taste buds. The application of ACh to the basolateral membrane of mouse fungiform TBCs in situ increased the intracellular Ca 2+ concentration in 2.4 ± 1.4 cells per taste bud (mean ± SD, n = 14). After Ca 2+ imaging, we supravitally labeled type II cells (phospholipase C β2 [PLCβ2]-immunoreactive cells) with Lucifer yellow CH (LY), a fluorescent dye and investigated the positional relationship between ACh-responding cells and LY-labeled cells. After fixation, the TBCs were immunohistostained to investigate the positional relationships between immunohistochemically classified cells and LY-labeled cells. The overlay of the two positional relationships obtained by superimposing the LY-labeled cells showed that all of the ACh-responding cells were type III cells (synaptosomal-associated protein 25 [SNAP-25]-immunoreactive cells). The ACh responses required no added Ca 2+ in the bathing solution. The addition of 1 μM U73122, a phospholipase C inhibitor, decreased the magnitude of the ACh response, whereas that of 1 μM U73343, a negative control, had no effect. These results suggest that type III cells respond to ACh and release Ca 2+ from intracellular stores. We also discuss the underlying mechanism of the Ca 2+ response and the role of M3 in type III cells.

An International Intercomparison of Argon Triple Point Calibration Facilities, Accommodating Long-stem Thermometers

NASA Astrophysics Data System (ADS)

Bloembergen, P.; Bonnier, G.; Ronsin, H.

1990-01-01

Argon triple point calibration facilities have been compared among eight laboratories with one transfer system, employing local long-stem standard platinum resistance thermometers. The apparatus intercompared, included a sealed cell and its associated cryostat. As is evidenced by the results of long-term investigations, previously performed at the INM, cells of the type employed may show a triple-point temperature, which is stable within the reproducibility of the measurements (simeq0,1 mK) over a period of about 10 years. At each laboratory the mean difference between the Argon triple-point temperature of the transfer cell (t) and the local cell (i) has been determined, using a standard resistance thermometer previously calibrated at the fixed points, according to the IPTS-68; associated repeatabilities are typically of the order of 0,1 mK. The reproducibility attained by measuring the mean difference in different laboratories, using cells of the same type and origin (INM), amounts to 0,4 mK.

Immunocytochemical analysis of syntaxin-1 in rat circumvallate taste buds.

PubMed

Yang, Ruibiao; Ma, Huazhi; Thomas, Stacey M; Kinnamon, John C

2007-06-20

Mammalian buds contain a variety of morphological taste cell types, but the type III taste cell is the only cell type that has synapses onto nerve processes. We hypothesize that taste cell synapses utilize the SNARE protein machinery syntaxin, SNAP-25, and synaptobrevin, as is used by synapses in the central nervous system (CNS) for Ca2+-dependent exocytosis. Previous studies have shown that taste cells with synapses display SNAP-25- and synaptobrevin-2-like immunoreactivity (LIR) (Yang et al. [2000a] J Comp Neurol 424:205-215, [2004] J Comp Neurol 471:59-71). In the present study we investigated the presynaptic membrane protein, syntaxin-1, in circumvallate taste buds of the rat. Our results indicate that diffuse cytoplasmic and punctate syntaxin-1-LIR are present in different subsets of taste cells. Diffuse, cytoplasmic syntaxin-1-LIR is present in type III cells while punctate syntaxin-1-LIR is present in type II cells. The punctate syntaxin-1-LIR is believed to be associated with Golgi bodies. All of the synapses associated with syntaxin-1-LIR taste cells are from type III cells onto nerve processes. These results support the proposition that taste cell synapses use classical SNARE machinery such as syntaxin-1 for neurotransmitter release in rat circumvallate taste buds. (c) 2007 Wiley-Liss, Inc.

Morphology of retinal ganglion cells in the ferret (Mustela putorius furo).

PubMed

Isayama, Tomoki; O'Brien, Brendan J; Ugalde, Irma; Muller, Jay F; Frenz, Aaron; Aurora, Vikas; Tsiaras, William; Berson, David M

2009-12-01

The ferret is the premiere mammalian model of retinal and visual system development, but the spectrum and properties of its retinal ganglion cells are less well understood than in another member of the Carnivora, the domestic cat. Here, we have extensively surveyed the dendritic architecture of ferret ganglion cells and report that the classification scheme previously developed for cat ganglion cells can be applied with few modifications to the ferret retina. We confirm the presence of alpha and beta cells in ferret retina, which are very similar to those in cat retina. Both cell types exhibited an increase in dendritic field size with distance from the area centralis (eccentricity) and with distance from the visual streak. Both alpha and beta cell populations existed as two subtypes whose dendrites stratified mainly in sublamina a or b of the inner plexiform layer. Six additional morphological types of ganglion cells were identified: four monostratified cell types (delta, epsilon, zeta, and eta) and two bistratified types (theta and iota). These types closely resembled their counterparts in the cat in terms of form, relative field size, and stratification. Our data indicate that, among carnivore species, the retinal ganglion cells resemble one another closely and that the ferret is a useful model for studies of the ontogenetic differentiation of ganglion cell types.

Adenovirus Vector Pseudotyping in Fiber-Expressing Cell Lines: Improved Transduction of Epstein-Barr Virus-Transformed B Cells

PubMed Central

Von Seggern, Dan J.; Huang, Shuang; Fleck, Shonna Kaye; Stevenson, Susan C.; Nemerow, Glen R.

2000-01-01

While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.βgal.ΔF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.βgal.ΔF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types. PMID:10590124

Desert hedgehog (Dhh) gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules.

PubMed

Clark, A M; Garland, K K; Russell, L D

2000-12-01

Testes from adult and prepubertal mice lacking the Desert hedgehog (DHH:) gene were examined in order to describe further the role of Dhh in spermatogenesis because, in a previous report, DHH:-null male mice were shown to be sterile. Dhh is a signaling molecule expressed by Sertoli cells. Its receptor, patched (Ptc), has been previously localized to Leydig cells and is herein described as being localized also to peritubular cells. Two phenotypes of the mice were observed: masculinized (7.5% of DHH:-null males) and feminized (92.5%), both of which displayed abnormal peritubular tissue and severely restricted spermatogenesis. Testes from adult feminized animals lacked adult-type Leydig cells and displayed numerous undifferentiated fibroblastic cells in the interstitium that produced abundant collagen. The basal lamina, normally present between the myoid cells and Sertoli cells, was focally absent. We speculate that the abnormal basal lamina contributed to other characteristics, such as extracordal gonocytes, apolar Sertoli cells, and anastomotic seminiferous tubules. The two DHH:-null phenotypes described have common peritubular cell defects that may be indicative of the essential role of peritubular cells in development of tubular morphology, the differentiation of Leydig cells, and the ultimate support of spermatogenesis.

Genetic identification of brain cell types underlying schizophrenia.

PubMed

Skene, Nathan G; Bryois, Julien; Bakken, Trygve E; Breen, Gerome; Crowley, James J; Gaspar, Héléna A; Giusti-Rodriguez, Paola; Hodge, Rebecca D; Miller, Jeremy A; Muñoz-Manchado, Ana B; O'Donovan, Michael C; Owen, Michael J; Pardiñas, Antonio F; Ryge, Jesper; Walters, James T R; Linnarsson, Sten; Lein, Ed S; Sullivan, Patrick F; Hjerling-Leffler, Jens

2018-06-01

With few exceptions, the marked advances in knowledge about the genetic basis of schizophrenia have not converged on findings that can be confidently used for precise experimental modeling. By applying knowledge of the cellular taxonomy of the brain from single-cell RNA sequencing, we evaluated whether the genomic loci implicated in schizophrenia map onto specific brain cell types. We found that the common-variant genomic results consistently mapped to pyramidal cells, medium spiny neurons (MSNs) and certain interneurons, but far less consistently to embryonic, progenitor or glial cells. These enrichments were due to sets of genes that were specifically expressed in each of these cell types. We also found that many of the diverse gene sets previously associated with schizophrenia (genes involved in synaptic function, those encoding mRNAs that interact with FMRP, antipsychotic targets, etc.) generally implicated the same brain cell types. Our results suggest a parsimonious explanation: the common-variant genetic results for schizophrenia point at a limited set of neurons, and the gene sets point to the same cells. The genetic risk associated with MSNs did not overlap with that of glutamatergic pyramidal cells and interneurons, suggesting that different cell types have biologically distinct roles in schizophrenia.

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

PubMed

Huang, Y; Ohtani, K; Iwanaga, R; Matsumura, Y; Nakamura, M

2001-03-01

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells

PubMed Central

Masujin, Kentaro; Okada, Hiroyuki; Ushiki-Kaku, Yuko; Matsuura, Yuichi; Yokoyama, Takashi

2017-01-01

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population. PMID:28636656

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells.

PubMed

Miyazawa, Kohtaro; Masujin, Kentaro; Okada, Hiroyuki; Ushiki-Kaku, Yuko; Matsuura, Yuichi; Yokoyama, Takashi

2017-01-01

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population.

Electrical filtering in gerbil isolated type I semicircular canal hair cells

NASA Technical Reports Server (NTRS)

Rennie, K. J.; Ricci, A. J.; Correia, M. J.

1996-01-01

1. Membrane potential responses of dissociated gerbil type I semicircular canal hair cells to current injections in whole cell current-clamp have been measured. The input resistance of type I cells was 21.4 +/- 14.3 (SD) M omega, (n = 25). Around the zero-current potential (Vz = -66.6 +/- 9.3 mV, n = 25), pulsed current injections (from approximately -200 to 750 pA) produced only small-amplitude, pulse-like changes in membrane potential. 2. Injecting constant current to hyperpolarize the membrane to around -100 mV resulted in a approximately 10-fold increase in membrane resistance. Current pulses superimposed on this constant hyperpolarization produced larger and more complex membrane potential changes. Depolarizing currents > or = 200 pA caused a rapid transient peak voltage before a plateau. 3. Membrane voltage was able to faithfully follow sine-wave current injections around Vz over the range 1-1,000 Hz with < 25% attenuation at 1 kHz. A previously described K conductance, IKI, which is active at Vz, produces the low input resistance and frequency response. This was confirmed by pharmacologically blocking IKI. This conductance, present in type I cells but not type II hair cells, would appear to confer on type I cells a lower gain, but a much broader bandwidth at Vz, than seen in type II cells.

The study of integrated coal-gasifier molten carbonate fuel cell systems

NASA Technical Reports Server (NTRS)

1983-01-01

A novel integration concept for a coal-fueled coal gasifier-molten carbonate fuel cell power plant was studied. Effort focused on determining the efficiency potential of the concept, design, and development requirements of the processes in order to achieve the efficiency. The concept incorporates a methane producing catalytic gasifier of the type previously under development by Exxon Research and Development Corp., a reforming molten carbonate fuel cell power section of the type currently under development by United Technologies Corp., and a gasifier-fuel cell recycle loop. The concept utilizes the fuel cell waste heat, in the form of hydrogen and carbon monoxide, to generate additional fuel in the coal gasifier, thereby eliminating the use of both an O2 plant and a stream bottoming cycle from the power plant. The concept has the potential for achieving coal-pile-to-busbar efficiencies of 50-59%, depending on the process configuration and degree of process configuration and degree of process development requirements. This is significantly higher than any previously reported gasifier-molten carbonate fuel cell system.

Schistosoma Infection and Schistosoma-Derived Products Modulate the Immune Responses Associated with Protection against Type 2 Diabetes

PubMed Central

Tang, Chun-Lian; Liu, Zhi-Ming; Gao, Yan Ru; Xiong, Fei

2018-01-01

Studies on parasite-induced immunoregulatory mechanisms could contribute to the development of new therapies for inflammatory diseases such as type 2 diabetes (T2D), which is a chronic inflammatory disease characterized by persistent elevated glucose levels due to insulin resistance. The association between previous Schistosoma infection and T2D has been confirmed—Schistosoma infection and Schistosoma-derived products modulate the immune system, including innate and acquired immune responses, contributing to T2D disease control. Schistosoma infections and Schistosoma-derived molecules affect the immune cell composition in adipose tissue, dampening inflammation and improving glucose tolerance. This protective role includes the polarization of immune cells to alternatively activated macrophages, dendritic cells, eosinophils, and group 2 innate lymphoid cells. Furthermore, Schistosoma infection and Schistosoma products are effective for the treatment of T2D, as they increase the number of type 2 helper T cells (Th2) and regulatory T cells (Tregs) and decrease type 1 helper T cells (Th1) and type 17 helper T cells (Th17) cells. Thus, our aim was to comprehensively review the mechanism through which Schistosoma infection and Schistosoma products modulate the immune response against T2D. PMID:29387059

Schistosoma Infection and Schistosoma-Derived Products Modulate the Immune Responses Associated with Protection against Type 2 Diabetes.

PubMed

Tang, Chun-Lian; Liu, Zhi-Ming; Gao, Yan Ru; Xiong, Fei

2017-01-01

Studies on parasite-induced immunoregulatory mechanisms could contribute to the development of new therapies for inflammatory diseases such as type 2 diabetes (T2D), which is a chronic inflammatory disease characterized by persistent elevated glucose levels due to insulin resistance. The association between previous Schistosoma infection and T2D has been confirmed- Schistosoma infection and Schistosoma -derived products modulate the immune system, including innate and acquired immune responses, contributing to T2D disease control. Schistosoma infections and Schistosoma -derived molecules affect the immune cell composition in adipose tissue, dampening inflammation and improving glucose tolerance. This protective role includes the polarization of immune cells to alternatively activated macrophages, dendritic cells, eosinophils, and group 2 innate lymphoid cells. Furthermore, Schistosoma infection and Schistosoma products are effective for the treatment of T2D, as they increase the number of type 2 helper T cells (Th2) and regulatory T cells (Tregs) and decrease type 1 helper T cells (Th1) and type 17 helper T cells (Th17) cells. Thus, our aim was to comprehensively review the mechanism through which Schistosoma infection and Schistosoma products modulate the immune response against T2D.

Blood types of the native Americans of Oklahoma.

PubMed

Kasprisin, D O; Crow, M; McClintock, C; Lawson, J

1987-05-01

Large numbers of Indians from Oklahoma were screened for a variety of red cell antigens. Sufficient numbers of Cherokees, Creeks, and Choctaws were studied to calculate gene frequencies. These tribes originated in the Southeastern United States and were forcibly moved to Oklahoma. The Creeks and Choctaws have not been studied previously. A small number of Cherokees remained in North Carolina, and their blood types have been reported. The blood types of the Oklahoma Cherokees are quite similar to those observed there but one important difference was discovered. The data previously reported concerning the Eastern Cherokees revealed the absence of the Dia antigen. The present study found that the Oklahoma Cherokees do have the Dia antigen, although in a lower percentage than the other southeastern tribes. The Creeks and Choctaws share a linguistic heritage as well as having similar red cell phenotypes.

Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

PubMed

Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E

2011-01-01

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

Proton and Fe Ion-Induced Early and Late Chromosome Aberrations in Different Cell Types

NASA Technical Reports Server (NTRS)

Wu, Honglu; Lu, Tao; Yeshitla, Samrawit; Zhang, Ye; Kadhim, Munira

2016-01-01

An early stage of cancer development is believed to be genomic instability (GI) which accelerates the mutation rate in the descendants of the cells surviving radiation exposure. To investigate GI induced by charged particles, we exposed human lymphocytes, human fibroblast cells, and human mammary epithelial cells to high energy protons and Fe ions. In addition, we also investigated GI in bone marrow cells isolated from CBA/CaH (CBA) and C57BL/6 (C57) mice, by analyzing cell survival and chromosome aberrations in the cells after multiple cell divisions. Results analyzed so far from the experiments indicated different sensitivities to charged particles between CBA/CaH (CBA) and C57BL/6 (C57) mouse strains, suggesting that there are two main types of response to irradiation: 1) responses associated with survival of damaged cells and 2) responses associated with the induction of non-clonal chromosomal instability in the surviving progeny of stem cells. Previously, we reported that the RBE for initial chromosome damages was high in human lymphocytes exposed to Fe ions. Our results with different cell types demonstrated different RBE values between different cell types and between early and late chromosomal damages. This study also attempts to offer an explanation for the varying RBE values for different cancer types.

Mycoplasma-induced BALB/c 3T3 collagenase is a mammalian enzyme.

PubMed Central

Kluve, B; Merrick, W C; Gershman, H

1983-01-01

A collagenase previously reported to accumulate in the medium of cultures of BALB/c 3T3 cells on infection with Mycoplasma orale [Kluve, Merrick, Stanbridge & Gershman (1981) Nature (London) 292, 855-857] was partially purified and characterized. With regard to purification properties, activation, sensitivity to inhibitors and relative molecular mass the enzyme was similar to previously reported vertebrate collagenases, but could not be unequivocally distinguished from bacterial collagenases. With regard to substrate-specificity and reaction products, however, the collagenase was typical of vertebrate collagenases and distinct from bacterial collagenases. Specifically, the enzyme displayed a preference for type III collagen and type I collagen, a somewhat decreased ability to degrade type II collagen, and a very limited ability to degrade type IV collagen. The initial products of the action of the collagenase on type I collagen were characterized as fragments one-quarter and three-quarters of the length of the intact collagen molecule. Because the properties of the collagenase produced by cultures of mycoplasma-infected BALB/c 3T3 cells are those of a mammalian-type (vertebrate-type) enzyme, we have concluded that the collagenase is a product of the mouse (BALB/c 3T3) genome, and is not produced by the mycoplasma. Therefore it appears that infection of BALB/c 3T3 mouse fibroblasts with Mycoplasma orale induces the mouse cells to produce and secrete collagenase. PMID:6309150

Recent advances in the ITO/InP solar cell

NASA Technical Reports Server (NTRS)

Gessert, T. A.; Li, X.; Wanlass, M. W.; Coutts, T. J.

1991-01-01

It was demonstrated that Indium Tin Oxide (ITO)/InP solar cells can now be made on as-received p(-) bulk substrates which are of nearly equal quality to those which could previously only be made on epitaxially grown p(-) InP base layers. Although this advancement is due in part to both increases in substrate quality and a better understanding of back contact formation, it appears that the passivation/compensation effects resulting from having H2 in the sputtering gas tends to reduce significantly the performance differences previously observed between these two substrates. It is shown that since high efficiency ITO/InP cells can be made from as-received substrates, and since the type conversion process is not highly spatially dependent, large area ITO/InP cells (4 sq cm) with efficiencies approaching 17 percent (Global) can be made. Furthermore, the measured open circuit voltages (V sub OC) and quantum efficiencies (QEs) from these large cells suggest that, when they are processed using optimum grid designs, the efficiencies will be nearly equal to that of the smaller cells thus far produced. It has been shown, through comparative experiments involving ITO/InP and IO/InP cells, that Sn may not be the major cause of type conversion of the InP surface and thus further implies that the ITO may not be an essential element in this type of device. Specifically, very efficient photovoltaic solar cells were made by sputtering (Sn free) In2O3 showing that type conversion and subsequent junction formation will occur even in the absence of the sputtered SN species. The result suggests that sputter damage may indeed be the important mechanism(s) of type conversion. Finally, an initial study of the stability of the ITO/InP cell done over the course of about one year has indicated that the J(sub SC) (short circuit current) and the fill factor (FF) are measurably stable within experimental certainty.

Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.

PubMed

Sviderskaya, Elena V; Easty, David J; Lawrence, Mark A; Sánchez, Daniel P; Negulyaev, Yuri A; Patel, Ricken H; Anand, Praveen; Korchev, Yuri E; Bennett, Dorothy C

2009-09-01

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.

Accelerated retinal ganglion cell death in mice deficient in the Sigma-1 receptor.

PubMed

Mavlyutov, Timur A; Nickells, Robert W; Guo, Lian-Wang

2011-04-26

The sigma-1 receptor (σR1), a ligand-operated chaperone, has been inferred to be neuroprotective in previous studies using σR1 ligands. The σR1 specificity of the protective function, however, has yet to be firmly established, due to the existence of non-σR1 targets of the ligands. Here, we used the σR1-knockout mouse (Sigmar1(-/-)) to demonstrate unambiguously the role of the σR1 in protecting the retinal ganglion cells against degeneration after acute damage to the optic nerve. Retinal σR binding sites were labeled with radioiodinated σR ligands and analyzed by autoradiography. Localization of the σR1 was performed by indirect immunofluorescence on frozen retinal sections. Retinal ganglion cell death was induced by acute optic nerve crush in wild-type and Sigmar1(-/-) mice. Surviving cells in the ganglion cell layer were counted on Nissl-stained retinal whole mounts 7 days after the crush surgery. Photoaffinity labeling indicated the presence of the σR1 in the retina, in concentrations equivalent to those in liver tissue. Immunolabeling detected this receptor in cells of both the ganglion cell layer and the photoreceptor cell layer in wild-type retinas. Quantification of cells remaining after optic nerve crush showed that 86.8±7.9% cells remained in the wild-type ganglion cell layer, but only 68.3±3.4% survived in the Sigmar1(-/-), demonstrating a significant difference between the wild-type and the Sigmar1(-/-) in crush-induced ganglion cell loss. Our data indicated faster retinal ganglion cell death in Sigmar1(-/-) than in wild-type mice under the stresses caused by optic nerve crush, providing direct evidence for a role of the σR1 in alleviating retinal degeneration. This conclusion is consistent with the previous pharmacological studies using σR1 agonists. Thus, our study supports the idea that the σR1 is a promising therapeutic target for neurodegenerative retinal diseases, such as glaucoma.

Accelerated retinal ganglion cell death in mice deficient in the Sigma-1 receptor

PubMed Central

Mavlyutov, Timur A.; Nickells, Robert W.

2011-01-01

Purpose The sigma-1 receptor (σR1), a ligand-operated chaperone, has been inferred to be neuroprotective in previous studies using σR1 ligands. The σR1 specificity of the protective function, however, has yet to be firmly established, due to the existence of non-σR1 targets of the ligands. Here, we used the σR1-knockout mouse (Sigmar1−/−) to demonstrate unambiguously the role of the σR1 in protecting the retinal ganglion cells against degeneration after acute damage to the optic nerve. Methods Retinal σR binding sites were labeled with radioiodinated σR ligands and analyzed by autoradiography. Localization of the σR1 was performed by indirect immunofluorescence on frozen retinal sections. Retinal ganglion cell death was induced by acute optic nerve crush in wild-type and Sigmar1−/− mice. Surviving cells in the ganglion cell layer were counted on Nissl-stained retinal whole mounts 7 days after the crush surgery. Results Photoaffinity labeling indicated the presence of the σR1 in the retina, in concentrations equivalent to those in liver tissue. Immunolabeling detected this receptor in cells of both the ganglion cell layer and the photoreceptor cell layer in wild-type retinas. Quantification of cells remaining after optic nerve crush showed that 86.8±7.9% cells remained in the wild-type ganglion cell layer, but only 68.3±3.4% survived in the Sigmar1−/−, demonstrating a significant difference between the wild-type and the Sigmar1−/− in crush-induced ganglion cell loss. Conclusions Our data indicated faster retinal ganglion cell death in Sigmar1−/− than in wild-type mice under the stresses caused by optic nerve crush, providing direct evidence for a role of the σR1 in alleviating retinal degeneration. This conclusion is consistent with the previous pharmacological studies using σR1 agonists. Thus, our study supports the idea that the σR1 is a promising therapeutic target for neurodegenerative retinal diseases, such as glaucoma. PMID:21541278

Laminin and collagen modulate expression of the small leucine-rich proteoglycan fibromodulin in rat anterior pituitary gland.

PubMed

Syaidah, Rahimi; Horiguchi, Kotaro; Fujiwara, Ken; Tsukada, Takehiro; Kikuchi, Motoshi; Yashiro, Takashi

2013-11-01

The anterior pituitary is a complex organ consisting of five types of hormone-producing cells, non–hormone-producing cells such as folliculostellate (FS) cells and vascular cells (endothelial cells and pericytes). We have previously shown that FS cells and pericytes produce fibromodulin, a small leucine-rich proteoglycan (SLRP). SLRPs are major proteoglycans of the extracellular matrix (ECM) and are important in regulating cell signaling pathways and ECM assembly. However, the mechanism regulating fibromodulin expression in the anterior pituitary has not been elucidated. Here, we investigate whether fibromodulin expression is modulated by major anterior pituitary ECM components such as laminin and type I collagen. Using transgenic rats expressing green fluorescent protein (GFP) specifically in FS cells, we examine fibromodulin expression in GFP-positive (FS cells) and GFP-negative cells (e.g., pericytes, endocrine cells and endothelial cells). Immunostaining and Western blot analysis were used to assess protein expression in the presence and absence of laminin or type I collagen. We confirmed fibromodulin expression in the pituitary and observed the up-regulation of fibromodulin in FS cells in the presence of ECM components. However, neither laminin nor type I collagen affected expression in GFP-negative cells. This suggests that laminin and type I collagen support the function of FS cells by increasing fibromodulin protein expression in the anterior pituitary.

TGFbeta type II receptor signaling controls Schwann cell death and proliferation in developing nerves.

PubMed

D'Antonio, Maurizio; Droggiti, Anna; Feltri, M Laura; Roes, Jürgen; Wrabetz, Lawrence; Mirsky, Rhona; Jessen, Kristján R

2006-08-16

During development, Schwann cell numbers are precisely adjusted to match the number of axons. It is essentially unknown which growth factors or receptors carry out this important control in vivo. Here, we tested whether the type II transforming growth factor (TGF) beta receptor has a role in this process. We generated a conditional knock-out mouse in which the type II TGFbeta receptor is specifically ablated only in Schwann cells. Inactivation of the receptor, evident at least from embryonic day 18, resulted in suppressed Schwann cell death in normally developing and injured nerves. Notably, the mutants also showed a strong reduction in Schwann cell proliferation. Consequently, Schwann cell numbers in wild-type and mutant nerves remained similar. Lack of TGFbeta signaling did not appear to affect other processes in which TGFbeta had been implicated previously, including myelination and response of adult nerves to injury. This is the first in vivo evidence for a growth factor receptor involved in promoting Schwann cell division during development and the first genetic evidence for a receptor that controls normal developmental Schwann cell death.

Formation and regeneration of the urothelium.

PubMed

Yamany, Tammer; Van Batavia, Jason; Mendelsohn, Cathy

2014-06-01

This review addresses significant changes in our understanding of urothelial development and regeneration. Understanding urothelial differentiation will be important in the push to find new methods of bladder reconstruction and augmentation, as well as identification of bladder cancer stem cells. This review will cover recent findings including the identification of novel progenitor cells in the embryo and adult urothelium, function of the urothelium, and regeneration of the urothelium. Using Cre-lox recombination with cell-type-specific Cre lines, lineage studies from our laboratory have revealed novel urothelial cell types and progenitors that are critical for formation and regeneration of the urothelium. Interestingly, our studies indicate that Keratin-5-expressing basal cells, which have previously been proposed to be urothelial stem cells, are a self-renewing unipotent population, whereas P-cells, a novel urothelial cell type, are progenitors in the embryo, and intermediate cells serve as a progenitor pool in the adult. These findings could have important implications for our understanding of cancer tumorigenesis and could move the fields of regeneration and reconstruction forward.

A POX on Renal Cancer Cells | Center for Cancer Research

Cancer.gov

Proline oxidase, or POX, is an enzyme responsible for metabolizing the amino acid proline. POX contributes to the regulation of cell death that occurs when cellular systems malfunction, a process called apoptosis. Previous studies have determined that levels of POX are reduced in several types of human cancer. Likewise, many cancer cells become resistant to apoptosis,

76 FR 45838 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-01

...-effects, reducing their pain burden during treatment. IL13-receptor-alpha-2 (IL13-R[alpha]2) is a cell... kill only those cancer cells which express IL13-R[alpha]2. Our inventors previously constructed fusion... overexpressed IL13-R[alpha]2, as well as other types of diseased cells (asthma, pulmonary fibrosis) which...

Stearidonic and eicosapentaenoic acids inhibit interleukin-6 expression in ob/ob mouse adipose stem cells via toll-like receptor-2-mediated pathways

USDA-ARS?s Scientific Manuscript database

Increases in adipose tissue weight positively correlates with increased circulating inflammatory cytokines such as interleukin-6 (IL-6). We previously have shown that adipose stem cell produce significantly higher levels of IL-6 when compared to other cell types in the adipose tissue in genetically ...

Poorly Differentiated Squamous Cell Carcinoma Arising in Tattooed Skin

PubMed Central

Sarma, Deba P.; Dentlinger, Renee B.; Forystek, Amanda M.; Stevens, Todd; Huerter, Christopher

2010-01-01

Introduction. Tattoos have increasingly become accepted by mainstream Western society. As a result, the incidence of tattoo-associated dermatoses is on the rise. The presence of a poorly differentiated squamous cell carcinoma in an old tattooed skin is of interest as it has not been previously documented. Case Presentation. A 79-year-old white homeless man of European descent presented to the dermatology clinic with a painless raised nodule on his left forearm arising in a tattooed area. A biopsy of the lesion revealed a poorly differentiated squamous cell carcinoma infiltrating into a tattoo. The lesion was completely excised and the patient remains disease-free one year later. Conclusion. All previous reports of squamous cell carcinomas arising in tattoos have been well-differentiated low-grade type or keratoacanthoma-type and are considered to be coincidental rather than related to any carcinogenic effect of the tattoo pigments. Tattoo-associated poorly differentiated invasive carcinoma appears to be extremely rare. PMID:21274289

A POX on Renal Cancer Cells | Center for Cancer Research

Cancer.gov

Proline oxidase, or POX, is an enzyme responsible for metabolizing the amino acid proline. POX contributes to the regulation of cell death that occurs when cellular systems malfunction, a process called apoptosis. Previous studies have determined that levels of POX are reduced in several types of human cancer. Likewise, many cancer cells become resistant to apoptosis, suggesting a link between POX and cancer cell survival.

Discovering cell types in flow cytometry data with random matrix theory

NASA Astrophysics Data System (ADS)

Shen, Yang; Nussenblatt, Robert; Losert, Wolfgang

Flow cytometry is a widely used experimental technique in immunology research. During the experiments, peripheral blood mononuclear cells (PBMC) from a single patient, labeled with multiple fluorescent stains that bind to different proteins, are illuminated by a laser. The intensity of each stain on a single cell is recorded and reflects the amount of protein expressed by that cell. The data analysis focuses on identifying specific cell types related to a disease. Different cell types can be identified by the type and amount of protein they express. To date, this has most often been done manually by labelling a protein as expressed or not while ignoring the amount of expression. Using a cross correlation matrix of stain intensities, which contains both information on the proteins expressed and their amount, has been largely ignored by researchers as it suffers from measurement noise. Here we present an algorithm to identify cell types in flow cytometry data which uses random matrix theory (RMT) to reduce noise in a cross correlation matrix. We demonstrate our method using a published flow cytometry data set. Compared with previous analysis techniques, we were able to rediscover relevant cell types in an automatic way. Department of Physics, University of Maryland, College Park, MD 20742.

Morphology-based optical separation of subpopulations from a heterogeneous murine breast cancer cell line.

PubMed

Tamura, Masato; Sugiura, Shinji; Takagi, Toshiyuki; Satoh, Taku; Sumaru, Kimio; Kanamori, Toshiyuki; Okada, Tomoko; Matsui, Hirofumi

2017-01-01

Understanding tumor heterogeneity is an urgent and unmet need in cancer research. In this study, we used a morphology-based optical cell separation process to classify a heterogeneous cancer cell population into characteristic subpopulations. To classify the cell subpopulations, we assessed their morphology in hydrogel, a three-dimensional culture environment that induces morphological changes according to the characteristics of the cells (i.e., growth, migration, and invasion). We encapsulated the murine breast cancer cell line 4T1E, as a heterogeneous population that includes highly metastatic cells, in click-crosslinkable and photodegradable gelatin hydrogels, which we developed previously. We observed morphological changes within 3 days of encapsulating the cells in the hydrogel. We separated the 4T1E cell population into colony- and granular-type cells by optical separation, in which local UV-induced degradation of the photodegradable hydrogel around the target cells enabled us to collect those cells. The obtained colony- and granular-type cells were evaluated in vitro by using a spheroid assay and in vivo by means of a tumor growth and metastasis assay. The spheroid assay showed that the colony-type cells formed compact spheroids in 2 days, whereas the granular-type cells did not form spheroids. The tumor growth assay in mice revealed that the granular-type cells exhibited lower tumor growth and a different metastasis behavior compared with the colony-type cells. These results suggest that morphology-based optical cell separation is a useful technique to classify a heterogeneous cancer cell population according to its cellular characteristics.

Evolution of resistance and progression to disease during clonal expansion of cancer.

PubMed

Durrett, Richard; Moseley, Stephen

2010-02-01

Inspired by previous work of Iwasa et al. (2006) and Haeno et al. (2007), we consider an exponentially growing population of cancerous cells that will evolve resistance to treatment after one mutation or display a disease phenotype after two or more mutations. We prove results about the distribution of the first time when k mutations have accumulated in some cell, and about the growth of the number of type-k cells. We show that our results can be used to derive the previous results about a tumor grown to a fixed size. Copyright 2009 Elsevier Inc. All rights reserved.

Collagen type I and III synthesis by Tenon's capsule fibroblasts in culture: individual patient characteristics and response to mitomycin C, 5-fluorouracil, and ascorbic acid.

PubMed Central

Gross, R L

1999-01-01

PURPOSE: This study was performed to better understand the differences between patients in specific components of wound healing as it may pertain to glaucoma filtration surgery, including the use of antimetabolites. METHODS: Human Tenon's capsule fibroblasts were obtained at the time of glaucoma filtering surgery and established in individual cell cultures from 35 glaucoma patients. The dose-response to 5-fluorouracil (5FU) and mitomycin C (MMC) was determined. The individual cell lines were exposed to the antimetabolites and ascorbic acid with measurement of collagen type I and III production by an ELISA-type dot blot assay. These results were then statistically compared to the individual patient characteristics including age, race, previous surgery and medications, and type of glaucoma. RESULTS: 5-FU had little effect on collagen type I and III production or protein synthesis. MMC had an inhibitory effect on collagen secretion and total protein synthesis with increasing concentration. Photomicrographs of the cells after each treatment condition revealed characteristic morphologic changes when compared to controls. There was a large range of collagen type I and III production with correlation between the amounts of each collagen type secreted in response to the antimetabolites. However, there was no correlation with accepted risk factors for filtration failure. CONCLUSION: These antimetabolites act similarly on different cell lines in a nonspecific manner. The results suggest that the increased risk of filtration failure due to age, race, diagnosis, and previous conjunctival surgery is not due to differences in secretion of collagen types I and III by Tenon's capsule fibroblasts. Images FIGURE 3 PMID:10703140

First siRNA library screening in hard-to-transfect HUVEC cells

PubMed Central

Zumbansen, Markus; Altrogge, Ludger M; Spottke, Nicole UE; Spicker, Sonja; Offizier, Sheila M; Domzalski, Sandra BS; St Amand, Allison L; Toell, Andrea; Leake, Devin; Mueller-Hartmann, Herbert A

2010-01-01

Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa® Nucleofector® 96-well Shuttle® System for siRNA screening in primary cells. Lonza's Clonetics® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME® siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector® 96-well Shuttle® System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection. PMID:20628494

ERE environment- and cell type-specific transcriptional effects of estrogen in normal endometrial cells.

PubMed

Lascombe, I; Sallot, M; Vuillermoz, C; Weisz, A; Adessi, G L; Jouvenot, M

1998-04-30

Our previous results have suggested a repression of E2 (17beta-estradiol) effect on the c-fos gene of cultured guinea-pig endometrial cells. To investigate this repression, the expression of three human c-fos gene recombinants, pFC1-BL (-2250/+41), pFC2-BL (-1400/+41) and pFC2E (-1300/-1050 and -230/+41), known to be E2-responsive in Hela cells, was studied in stromal (SC) and glandular epithelial cells (GEC). In both cellular types, pFC1-BL was not induced by E2, even in the presence of growth factors or co-transfected estrogen receptor. The pattern of pFC2-BL and pFC2E expression was strikingly different and depended on the cellular type: pFC2-BL and pFC2E induction was restricted to the glandular epithelial cells and did not occur in the SCs. We argue for a repression of E2 action which is dependent on the estrogen-responsive cis-acting element (ERE) environment and also cell type-specific involving DNA/protein and/or protein/protein interactions with cellular type-specific factors.

Inhibition of membrane type-1 matrix metalloproteinase by cancer drugs interferes with the homing of diabetogenic T cells into the pancreas.

PubMed

Savinov, Alexei Y; Rozanov, Dmitri V; Golubkov, Vladislav S; Wong, F Susan; Strongin, Alex Y

2005-07-29

We have discovered that clinically tested inhibitors of matrix metalloproteinases can control the functional activity of T cell membrane type-1 matrix metalloproteinase (MT1-MMP) and the onset of disease in a rodent model of type 1 diabetes in non-obese diabetic mice. We determined that MT1-MMP proteolysis of the T cell surface CD44 adhesion receptor affects the homing of T cells into the pancreas. We also determined that both the induction of the intrinsic T cell MT1-MMP activity and the shedding of cellular CD44 follow the adhesion of insulin-specific, CD8-positive, Kd-restricted T cells to the matrix. Conversely, inhibition of these events by AG3340 (a potent hydroxamate inhibitor that was widely used in clinical trials in cancer patents) impedes the transmigration of diabetogenic T cells into the pancreas and protects non-obese diabetic mice from diabetes onset. Overall, our studies have divulged a previously unknown function of MT1-MMP and identified a promising novel drug target in type I diabetes.

Fast globally optimal segmentation of cells in fluorescence microscopy images.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2011-01-01

Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

No evidence of persisting unrepaired nuclear DNA single strand breaks in distinct types of cells in the brain, kidney, and liver of adult mice after continuous eight-week 50 Hz magnetic field exposure with flux density of 0.1 mT or 1.0 mT.

PubMed

Korr, Hubert; Angstman, Nicholas B; Born, Tatjana B; Bosse, Kerstin; Brauns, Birka; Demmler, Martin; Fueller, Katja; Kántor, Orsolya; Kever, Barbara M; Rahimyar, Navida; Salimi, Sepideh; Silny, Jiri; Schmitz, Christoph

2014-01-01

It has been hypothesized in the literature that exposure to extremely low frequency electromagnetic fields (50 or 60 Hz) may lead to human health effects such as childhood leukemia or brain tumors. In a previous study investigating multiple types of cells from brain and kidney of the mouse (Acta Neuropathologica 2004; 107: 257-264), we found increased unrepaired nuclear DNA single strand breaks (nDNA SSB) only in epithelial cells of the choroid plexus in the brain using autoradiographic methods after a continuous eight-week 50 Hz magnetic field (MF) exposure of adult mice with flux density of 1.5 mT. In the present study we tested the hypothesis that MF exposure with lower flux densities (0.1 mT, i.e., the actual exposure limit for the population in most European countries, and 1.0 mT) shows similar results to those in the previous study. Experiments and data analysis were carried out in a similar way as in our previous study. Continuous eight-week 50 Hz MF exposure with 0.1 mT or 1.0 mT did not result in increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice. MF exposure with 1.0 mT led to reduced unscheduled DNA synthesis (UDS) in epithelial cells in the choroid plexus of the fourth ventricle in the brain (EC-CP) and epithelial cells of the cortical collecting duct in the kidney, as well as to reduced mtDNA synthesis in neurons of the caudate nucleus in the brain and in EC-CP. No evidence was found for increased persisting unrepaired nDNA SSB in distinct types of cells in the brain, kidney, and liver of adult mice after continuous eight-week 50 Hz magnetic field exposure with flux density of 0.1 mT or 1.0 mT.

Changes in fine structure of pericytes and novel desmin-immunopositive perivascular cells during postnatal development in rat anterior pituitary gland.

PubMed

Jindatip, Depicha; Fujiwara, Ken; Horiguchi, Kotaro; Tsukada, Takehiro; Kouki, Tom; Yashiro, Takashi

2013-09-01

Pericytes are perivascular cells associated with capillaries. We previously demonstrated that pericytes, identified by desmin immunohistochemistry, produce type I and III collagens in the anterior pituitary gland of adult rats. In addition, we recently used desmin immunoelectron microscopy to characterize a novel type of perivascular cell, dubbed a desmin-immunopositive perivascular cell, in the anterior pituitary. These two types of perivascular cells differ in fine structure. The present study attempted to characterize the morphological features of pituitary pericytes and novel desmin-immunopositive perivascular cells during postnatal development, in particular their role in collagen synthesis. Desmin immunostaining revealed numerous perivascular cells at postnatal day 5 (P5) and P10. Transmission electron microscopy showed differences in the fine structure of the two cell types, starting at P5. Pericytes had well-developed rough endoplasmic reticulum and Golgi apparatus at P5 and P10. The novel desmin-immunopositive perivascular cells exhibited dilated cisternae of rough endoplasmic reticulum at P5-P30. In addition, during early postnatal development in the gland, a number of type I and III collagen-expressing cells were observed, as were high expression levels of these collagen mRNAs. We conclude that pituitary pericytes and novel desmin-immunopositive perivascular cells contain well-developed cell organelles and that they actively synthesize collagens during the early postnatal period.

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

PubMed Central

Halder, Sujata; Cotmore, Susan; Heimburg-Molinaro, Jamie; Smith, David F.; Cummings, Richard D.; Chen, Xi; Trollope, Alana J.; North, Simon J.; Haslam, Stuart M.; Dell, Anne; Tattersall, Peter; McKenna, Robert; Agbandje-McKenna, Mavis

2014-01-01

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-LeX identified in a previous glycan microarray screen. PMID:24475195

Cellular mechanisms of cyclophosphamide-induced taste loss in mice

PubMed Central

Mukherjee, Nabanita; Pal Choudhuri, Shreoshi; Delay, Rona J.

2017-01-01

Many commonly prescribed chemotherapy drugs such as cyclophosphamide (CYP) have adverse side effects including disruptions in taste which can result in loss of appetite, malnutrition, poorer recovery and reduced quality of life. Previous studies in mice found evidence that CYP has a two-phase disturbance in taste behavior: a disturbance immediately following drug administration and a second which emerges several days later. In this study, we examined the processes by which CYP disturbs the taste system by examining the effects of the drug on taste buds and cells responsible for taste cell renewal using immunohistochemical assays. Data reported here suggest CYP has direct cytotoxic effects on lingual epithelium immediately following administration, causing an early loss of taste sensory cells. Types II and III cells in fungiform taste buds appear to be more susceptible to this effect than circumvallate cells. In addition, CYP disrupts the population of rapidly dividing cells in the basal layer of taste epithelium responsible for taste cell renewal, manifesting a disturbance days later. The loss of these cells temporarily retards the system’s capacity to replace Type II and Type III taste sensory cells that survived the cytotoxic effects of CYP and died at the end of their natural lifespan. The timing of an immediate, direct loss of taste cells and a delayed, indirect loss without replacement of taste sensory cells are broadly congruent with previously published behavioral data reporting two periods of elevated detection thresholds for umami and sucrose stimuli. These findings suggest that chemotherapeutic disturbances in the peripheral mechanisms of the taste system may cause dietary challenges at a time when the cancer patient has significant need for well balanced, high energy nutritional intake. PMID:28950008

Cellular mechanisms of cyclophosphamide-induced taste loss in mice.

PubMed

Mukherjee, Nabanita; Pal Choudhuri, Shreoshi; Delay, Rona J; Delay, Eugene R

2017-01-01

Many commonly prescribed chemotherapy drugs such as cyclophosphamide (CYP) have adverse side effects including disruptions in taste which can result in loss of appetite, malnutrition, poorer recovery and reduced quality of life. Previous studies in mice found evidence that CYP has a two-phase disturbance in taste behavior: a disturbance immediately following drug administration and a second which emerges several days later. In this study, we examined the processes by which CYP disturbs the taste system by examining the effects of the drug on taste buds and cells responsible for taste cell renewal using immunohistochemical assays. Data reported here suggest CYP has direct cytotoxic effects on lingual epithelium immediately following administration, causing an early loss of taste sensory cells. Types II and III cells in fungiform taste buds appear to be more susceptible to this effect than circumvallate cells. In addition, CYP disrupts the population of rapidly dividing cells in the basal layer of taste epithelium responsible for taste cell renewal, manifesting a disturbance days later. The loss of these cells temporarily retards the system's capacity to replace Type II and Type III taste sensory cells that survived the cytotoxic effects of CYP and died at the end of their natural lifespan. The timing of an immediate, direct loss of taste cells and a delayed, indirect loss without replacement of taste sensory cells are broadly congruent with previously published behavioral data reporting two periods of elevated detection thresholds for umami and sucrose stimuli. These findings suggest that chemotherapeutic disturbances in the peripheral mechanisms of the taste system may cause dietary challenges at a time when the cancer patient has significant need for well balanced, high energy nutritional intake.

Revisiting Cardiac Cellular Composition

PubMed Central

Pinto, Alexander R.; Ilinykh, Alexei; Ivey, Malina J.; Kuwabara, Jill T.; D'Antoni, Michelle L.; Debuque, Ryan; Chandran, Anjana; Wang, Lina; Arora, Komal; Rosenthal, Nadia; Tallquist, Michelle D.

2015-01-01

Rationale Accurate knowledge of the cellular composition of the heart is essential to fully understand the changes that occur during pathogenesis and to devise strategies for tissue engineering and regeneration. Objective To examine the relative frequency of cardiac endothelial cells, hematopoietic-derived cells and fibroblasts in the mouse and human heart. Methods and Results Using a combination of genetic tools and cellular markers, we examined the occurrence of the most prominent cell types in the adult mouse heart. Immunohistochemistry revealed that endothelial cells constitute over 60%, hematopoietic-derived cells 5–10%, and fibroblasts under 20% of the non-myocytes in the heart. A refined cell isolation protocol and an improved flow cytometry approach provided an independent means of determining the relative abundance of non-myocytes. High dimensional analysis and unsupervised clustering of cell populations confirmed that endothelial cells are the most abundant cell population. Interestingly, fibroblast numbers are smaller than previously estimated, and two commonly assigned fibroblast markers, Sca-1 and CD90, underrepresent fibroblast numbers. We also describe an alternative fibroblast surface marker that more accurately identifies the resident cardiac fibroblast population. Conclusions This new perspective on the abundance of different cell types in the heart demonstrates that fibroblasts comprise a relatively minor population. By contrast, endothelial cells constitute the majority of non-cardiomyocytes and are likely to play a greater role in physiologic function and response to injury than previously appreciated. PMID:26635390

Purification and cultivation of human pituitary growth hormone secreting cells

NASA Technical Reports Server (NTRS)

Hymer, W. C.

1984-01-01

A multiphase study was conducted to examine the properties of growth hormone cells. Topics investigated included: (1) to determine if growth hormone (GH) cells contained within the rat pituitary gland can be separated from the other hormone producing cell types by continuous flow electrophoresis (CFE); (2) to determine what role, if any, gravity plays in the electrophoretic separation of GH cells; (3) to compare in vitro GH release from rat pituitary cells previously exposed to microgravity conditions vs release from cells not exposed to microgravity; (4) to determine if the frequency of different hormone producing pituitary cell types contained in cell suspensions can be quantitated by flow cytometry; and (5) to determine if GH contained within the human post mortem pituitary gland can be purified by CFE. Specific experimental procedures and results are included.

Conceptual Difficulties Experienced by Prospective Teachers in Electrochemistry: Half-Cell Potential, Cell Potential, and Chemical and Electrochemical Equilibrium in Galvanic Cells.

ERIC Educational Resources Information Center

Ozkaya, Ali Riza

2002-01-01

A previous study of prospective teachers found that students from different countries and different levels of electrochemistry hold common misconceptions, indicating that concepts were presented to them poorly. Reports on how prospective teachers' scientifically incorrect ideas were used to form assertion-reason-type questions and how these…

Classical macroautophagy in Lobivia rauschii (Cactaceae) and possible plastidial autophagy in Tillandsia albida (Bromeliaceae) tapetum cells.

PubMed

Papini, Alessio; Mosti, Stefano; van Doorn, Wouter G

2014-05-01

The tapetum in anthers is a tissue that undergoes programmed cell death (PCD) during the production of pollen. We observed two types of autophagy prior to cell death. In Lobivia rauschii (Cactaceae), tapetum cells showed plant-type autophagosomes-autolysosomes, which have been found previously exclusively in root meristem cells. The autophagic structures were formed by a network of tubules which apparently merged laterally, thereby sequestering a portion of the cytoplasm. The organelles observed in the sequestered material included multilamellar bodies, which have not been reported earlier in these organelles. By contrast, Tillandsia albida (Bromeliaceae) tapetum cells contained no such organelles but showed plastids that might possibly carry out autophagy, as they contained portions of the cytoplasm similar to the phenomenon reported earlier in Phaseolus and Dendrobium. However, the ultrastructure of the T. albida plastids was different from that in the previous reports. It is concluded that in L. rauschii classical plant macroautophagy was involved in degradation of the cytoplasm, while in T. albida such classical macroautophagy was not observed. Instead, the data in T. albida suggested the hypothesis that plastids are able to carry out degradation of the cytoplasm.

Epigenetic Reprogramming of the Type III Interferon Response Potentiates Antiviral Activity and Suppresses Tumor Growth

PubMed Central

Ding, Siyuan; Khoury-Hanold, William; Iwasaki, Akiko; Robek, Michael D.

2014-01-01

Type III interferon (IFN-λ) exhibits potent antiviral activity similar to IFN-α/β, but in contrast to the ubiquitous expression of the IFN-α/β receptor, the IFN-λ receptor is restricted to cells of epithelial origin. Despite the importance of IFN-λ in tissue-specific antiviral immunity, the molecular mechanisms responsible for this confined receptor expression remain elusive. Here, we demonstrate that the histone deacetylase (HDAC) repression machinery mediates transcriptional silencing of the unique IFN-λ receptor subunit (IFNLR1) in a cell-type-specific manner. Importantly, HDAC inhibitors elevate receptor expression and restore sensitivity to IFN-λ in previously nonresponsive cells, thereby enhancing protection against viral pathogens. In addition, blocking HDAC activity renders nonresponsive cell types susceptible to the pro-apoptotic activity of IFN-λ, revealing the combination of HDAC inhibitors and IFN-λ to be a potential antitumor strategy. These results demonstrate that the type III IFN response may be therapeutically harnessed by epigenetic rewiring of the IFN-λ receptor expression program. PMID:24409098

Various heterologous cells exhibit interferon induced transfer of viral resistance.

PubMed

Hughes, T K; Blalock, J E; Baron, S

1978-01-01

Previously it was shown that cocultivation of mouse L and human WISH or baby hamster kidney cells in the presence of mouse interferon resulted in decreased viral yield from both cell species. We now show that this phenomenon also occurs when rabbit kidney and human WISH cells, with their corresponding interferons, are cocultivated with human WISH and baby hamster kidney cells, respectively. This finding increases the number of donor cell types to three. The related finding that monkey VERO and chick embryo cells can be recipients of transferred resistance expands the number of heterologous recipient cell species capable of receiving transferred resistence to five. Not all cell types tested have been shown to function in this transfer system. The fact that VERO cells, which do not produce interferon, are capable of receiving transferred resistence is significant because it indicates that the mechanism of transfer does not involve production or interferon by the recipient cells.

Interleukins 12 and 15 induce cytotoxicity and early NK-cell differentiation in type 3 innate lymphoid cells.

PubMed

Raykova, Ana; Carrega, Paolo; Lehmann, Frank M; Ivanek, Robert; Landtwing, Vanessa; Quast, Isaak; Lünemann, Jan D; Finke, Daniela; Ferlazzo, Guido; Chijioke, Obinna; Münz, Christian

2017-12-26

Type 3 innate lymphoid cells (ILC3s) fulfill protective functions at mucosal surfaces via cytokine production. Although their plasticity to become ILC1s, the innate counterparts of type 1 helper T cells, has been described previously, we report that they can differentiate into cytotoxic lymphocytes with many characteristics of early differentiated natural killer (NK) cells. This transition is promoted by the proinflammatory cytokines interleukin 12 (IL-12) and IL-15, and correlates with expression of the master transcription factor of cytotoxicity, eomesodermin (Eomes). As revealed by transcriptome analysis and flow cytometric profiling, differentiated ILC3s express CD94, NKG2A, NKG2C, CD56, and CD16 among other NK-cell receptors, and possess all components of the cytotoxic machinery. These characteristics allow them to recognize and kill leukemic cells with perforin and granzymes. Therefore, ILC3s can be harnessed for cytotoxic responses via differentiation under the influence of proinflammatory cytokines.

Carbonic anhydrase enzymes regulate mast cell–mediated inflammation

PubMed Central

Soteropoulos, Patricia

2016-01-01

Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine–mediated inflammation through their enhanced ability to develop into mast cells. In this study, we show that carbonic anhydrase (Car) enzymes are up-regulated in type 2–associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent mouse mast cell responses and inflammation after Trichinella spiralis infection or the induction of food allergy–like disease. Further, we used CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively, these experiments identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be used to treat mast cell–mediated inflammation. PMID:27526715

STL-based Analysis of TRAIL-induced Apoptosis Challenges the Notion of Type I/Type II Cell Line Classification

PubMed Central

Bertaux, François; Maler, Oded; Batt, Gregory

2013-01-01

Extrinsic apoptosis is a programmed cell death triggered by external ligands, such as the TNF-related apoptosis inducing ligand (TRAIL). Depending on the cell line, the specific molecular mechanisms leading to cell death may significantly differ. Precise characterization of these differences is crucial for understanding and exploiting extrinsic apoptosis. Cells show distinct behaviors on several aspects of apoptosis, including (i) the relative order of caspases activation, (ii) the necessity of mitochondria outer membrane permeabilization (MOMP) for effector caspase activation, and (iii) the survival of cell lines overexpressing Bcl2. These differences are attributed to the activation of one of two pathways, leading to classification of cell lines into two groups: type I and type II. In this work we challenge this type I/type II cell line classification. We encode the three aforementioned distinguishing behaviors in a formal language, called signal temporal logic (STL), and use it to extensively test the validity of a previously-proposed model of TRAIL-induced apoptosis with respect to experimental observations made on different cell lines. After having solved a few inconsistencies using STL-guided parameter search, we show that these three criteria do not define consistent cell line classifications in type I or type II, and suggest mutants that are predicted to exhibit ambivalent behaviors. In particular, this finding sheds light on the role of a feedback loop between caspases, and reconciliates two apparently-conflicting views regarding the importance of either upstream or downstream processes for cell-type determination. More generally, our work suggests that these three distinguishing behaviors should be merely considered as type I/II features rather than cell-type defining criteria. On the methodological side, this work illustrates the biological relevance of STL-diagrams, STL population data, and STL-guided parameter search implemented in the tool Breach. Such tools are well-adapted to the ever-increasing availability of heterogeneous knowledge on complex signal transduction pathways. PMID:23675292

Identification of an Extracellular Polysaccharide Network Essential for Cytochrome Anchoring and Biofilm Formation in Geobacter sulfurreducens▿ †

PubMed Central

Rollefson, Janet B.; Stephen, Camille S.; Tien, Ming; Bond, Daniel R.

2011-01-01

Transposon insertions in Geobacter sulfurreducens GSU1501, part of an ATP-dependent exporter within an operon of polysaccharide biosynthesis genes, were previously shown to eliminate insoluble Fe(III) reduction and use of an electrode as an electron acceptor. Replacement of GSU1501 with a kanamycin resistance cassette produced a similarly defective mutant, which could be partially complemented by expression of GSU1500 to GSU1505 in trans. The Δ1501 mutant demonstrated limited cell-cell agglutination, enhanced attachment to negatively charged surfaces, and poor attachment to positively charged poly-d-lysine- or Fe(III)-coated surfaces. Wild-type and mutant cells attached to graphite electrodes, but when electrodes were poised at an oxidizing potential inducing a positive surface charge (+0.24 V versus the standard hydrogen electrode [SHE]), Δ1501 mutant cells detached. Scanning electron microscopy revealed fibrils surrounding wild-type G. sulfurreducens which were absent from the Δ1501 mutant. Similar amounts of type IV pili and pilus-associated cytochromes were detected on both cell types, but shearing released a stable matrix of c-type cytochromes and other proteins bound to polysaccharides. The matrix from the mutant contained 60% less sugar and was nearly devoid of c-type cytochromes such as OmcZ. The addition of wild-type extracellular matrix to Δ1501 cultures restored agglutination and Fe(III) reduction. The polysaccharide binding dye Congo red preferentially bound wild-type cells and extracellular matrix material over mutant cells, and Congo red inhibited agglutination and Fe(III) reduction by wild-type cells. These results demonstrate a crucial role for the xap (extracellular anchoring polysaccharide) locus in metal oxide attachment, cell-cell agglutination, and localization of essential cytochromes beyond the Geobacter outer membrane. PMID:21169487

Type I and type II interferons upregulate functional type I interleukin-1 receptor in a human fibroblast cell line TIG-1.

PubMed

Takii, T; Niki, N; Yang, D; Kimura, H; Ito, A; Hayashi, H; Onozaki, K

1995-12-01

The regulation of type I interleukin-1 receptor (IL-1R) expression by type I, interferon (IFN)-alpha A/D, and type II IFN, IFN-gamma, in a human fibroblast cell line TIG-1 was investigated. After 2 h stimulation with human IFN-alpha A/D or IFN-gamma, the levels of type I IL-1R mRNA increased. We previously reported that IL-1 upregulates transcription and cell surface molecules of type I IL-1R in TIG-1 cells through induction of prostaglandin (PG) E2 and cAMP accumulation. However, indomethacin was unable to inhibit the effect of IFNs, indicating that IFNs augment IL-1R expression through a pathway distinct from that of IL-1. The augmentation was also observed in other fibroblast cell lines. Nuclear run-on assays and studies of the stability of mRNA suggested that the increase in IL-1R mRNA was a result of the enhanced transcription of IL-1R gene. Binding studies using 125I-IL-1 alpha revealed that the number of cell surface IL-1R increased with no change in binding affinity by treatment with these IFNs. Pretreatment of the cells with IFNs enhanced IL-1-induced IL-6 production, indicating that IFNs upregulate functional IL-1R. IL-1 and IFNs are produced by the same cell types, as well as by the adjacent different cell types, and are concomitantly present in lesions of immune and inflammatory reactions. These results therefore suggest that IFNs exhibit synergistic effects with IL-1 through upregulation of IL-1R. Augmented production of IL-6 may also contribute to the reactions.

Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma.

PubMed

Laouar, Yasmina; Sutterwala, Fayyaz S; Gorelik, Leonid; Flavell, Richard A

2005-06-01

Interferon-gamma and interleukin 12 produced by the innate arm of the immune system are important regulators of T helper type 1 (T(H)1) cell development, but signals that negatively regulate their expression remain controversial. Here we show that transforming growth factor-beta (TGF-beta) controlled T(H)1 differentiation through the regulation of interferon-gamma produced by natural killer (NK) cells. Blockade of TGF-beta signaling in NK cells caused the accumulation of a large pool of NK cells secreting copious interferon-gamma, responsible for T(H)1 differentiation and protection from leishmania infection. In contrast, blockade of TGF-beta signaling in dendritic cells did not affect dendritic cell homeostasis or interleukin 12 production, thus indicating a previously undescribed demarcation of the function of TGF-beta in NK cells versus dendritic cells.

Susceptibility of thermally injured mice to cytomegalovirus infection.

PubMed

Kobayashi, H; Kobayashi, M; Herndon, D N; Pollard, R B; Suzuki, F

2001-11-01

Thermally injured patients are very susceptible to infection with cytomegaloviruses. In this study a role of burn-associated type 2 T cell responses on the cytomegalovirus infection was examined in a mouse model of thermal injury. A predominance of type 2 T cell responses in splenic lymphocytes of thermally injured mice has been previously demonstrated. SCID mice inoculated with splenic T cells from thermally injured mice were susceptible to infection with a small amount (5 PFU/mouse) of murine cytomegalovirus (MCMV). Conversely, SCID mice inoculated with splenic T cells from normal mice were resistant to the same infection. High levels of IL-4 and IL-10, but not IFN-gamma and IL-2, were detected in sera of thermally injured mice (TI-mice) infected with MCMV when those were compared with sera of normal mice infected with MCMV. IL-4 and IL-10 (type 2 cytokines) were produced by splenic T cells from MCMV-infected TI-mice, when they were stimulated in vitro with anti-CD3 mAb. Type 1 cytokines (IFN-gamma and IL-2), however, were not produced by these T cells after the same stimulation. In contrast, splenic T cells from MCMV-infected normal mice produced type 1 cytokines by the stimulation with anti-CD3 mAb. These results suggest that the susceptibility of mice to MCMV infection is markedly influenced by burn-associated type 2 T cell responses.

Cell metal interactions: A comparison of natural uranium to other common metals in renal cells and bone osteoblasts

NASA Astrophysics Data System (ADS)

Milgram, S.; Carrière, M.; Thiebault, C.; Berger, P.; Khodja, H.; Gouget, B.

2007-07-01

Uranium acute intoxication has been documented to induce nephrotoxicity. Kidneys are the main target organs after short term exposures to high concentrations of the toxic, while chronic exposures lead to its accumulation in the skeleton. In this paper, chemical toxicity of uranium is investigated for rat osteoblastic bone cells and compared to results previously obtained on renal cells. We show that bone cells are less sensitive to uranium than renal cells. The influence of the chemical form on U cytotoxicity is demonstrated. For both cell types, a comparison of uranium toxicity with other metals or metalloids toxicities (Mn, Ni, Co, Cu, Zn, Se and Cd) permits classification of Cd, Zn, Se IV and Cu as the most toxic and Ni, Se VI, Mn and U as the least toxic. Chemical toxicity of natural uranium proves to be far less than that of cadmium. To try to explain the differences in sensitivities observed between metals and different cell types, cellular accumulations in cell monolayers are quantified by inductively coupled plasma-mass spectroscopy (ICP-MS), function of time or function of dose: lethal doses which simulate acute intoxications and sub-lethal doses which are more realistic with regard to environmentally metals concentrations. In addition to being more resistant, bone cells accumulated much more uranium than did renal cells. Moreover, for both cell models, Mn, U-citrate and U-bicarbonate are strongly accumulated whereas Cu, Zn and Ni are weakly accumulated. On the other hand, a strong difference in Cd behaviour between the two cell types is shown: whereas Cd is very weakly accumulated in bone cells, it is very strongly accumulated in renal cells. Finally, elemental distribution of the toxics is determined on a cellular scale using nuclear microprobe analysis. For both renal and osteoblastic cells, uranium was accumulated in as intracellular precipitates similar to those observed previously by SEM/EDS.

Characteristics of allelic gene expression in human brain cells from single-cell RNA-seq data analysis.

PubMed

Zhao, Dejian; Lin, Mingyan; Pedrosa, Erika; Lachman, Herbert M; Zheng, Deyou

2017-11-10

Monoallelic expression of autosomal genes has been implicated in human psychiatric disorders. However, there is a paucity of allelic expression studies in human brain cells at the single cell and genome wide levels. In this report, we reanalyzed a previously published single-cell RNA-seq dataset from several postmortem human brains and observed pervasive monoallelic expression in individual cells, largely in a random manner. Examining single nucleotide variants with a predicted functional disruption, we found that the "damaged" alleles were overall expressed in fewer brain cells than their counterparts, and at a lower level in cells where their expression was detected. We also identified many brain cell type-specific monoallelically expressed genes. Interestingly, many of these cell type-specific monoallelically expressed genes were enriched for functions important for those brain cell types. In addition, function analysis showed that genes displaying monoallelic expression and correlated expression across neuronal cells from different individual brains were implicated in the regulation of synaptic function. Our findings suggest that monoallelic gene expression is prevalent in human brain cells, which may play a role in generating cellular identity and neuronal diversity and thus increasing the complexity and diversity of brain cell functions.

Discrimination of taste qualities among mouse fungiform taste bud cells.

PubMed

Yoshida, Ryusuke; Miyauchi, Aya; Yasuo, Toshiaki; Jyotaki, Masafumi; Murata, Yoshihiro; Yasumatsu, Keiko; Shigemura, Noriatsu; Yanagawa, Yuchio; Obata, Kunihiko; Ueno, Hiroshi; Margolskee, Robert F; Ninomiya, Yuzo

2009-09-15

Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin-expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter-best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour-best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl-best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.

Innervation of the anterior byssal retractor muscle in Mytilus edulis L. II. Ultrastructure of the glio-interstitial cells.

PubMed

Gilloteaux, J

1975-08-27

Studies on the intrinsic innervation of the anterior byssal retractor muscle (ABRM) in Mytilus edulis L. were continued at the ultrastructural level. Electron micrographs show nerve processes ensheathed by glio-interstitial cells running between muscle fibers. The glio-interstitial cells may represent all the types of osmiophilic cells previously described by the light microscopic ZIO technique in the anterior byssal retractor muscle.

Cell separation by immunoaffinity partitioning with polyethylene glycol-modified Protein A in aqueous polymer two-phase systems

NASA Technical Reports Server (NTRS)

Karr, Laurel J.; Van Alstine, James M.; Snyder, Robert S.; Shafer, Steven G.; Harris, J. Milton

1988-01-01

Previous work has shown that polyethylene glycol (PEG)-bound antibodies can be used as affinity ligands in PEG-dextran two-phase systems to provide selective partitioning of cells to the PEG-rich phase. In the present work it is shown that immunoaffinity partitioning can be simplified by use of PEG-modified Protein A which complexes with unmodified antibody and cells and shifts their partitioning into the PEG-rich phase, thus eliminating the need to prepare a PEG-modified antibody for each cell type. In addition, the paper provides a more rigorous test of the original technique with PEG-bound antibodies by showing that it is effective at shifting the partitioning of either cell type of a mixture of two cell populations.

Role of innate lymphoid cells in obesity and metabolic disease

PubMed Central

Saetang, Jirakrit; Sangkhathat, Surasak

2018-01-01

The immune system has previously been demonstrated to be associated with the pathophysiological development of metabolic abnormalities. However, the mechanisms linking immunity to metabolic disease remain to be fully elucidated. It has previously been suggested that innate lymphoid cells (ILCs) may be involved in the progression of numerous types of metabolic diseases as these cells act as suppressors and promoters for obesity and associated conditions, and are particularly involved in adipose tissue inflammation, which is a major feature of metabolic imbalance. Group 2 ILCs (ILC2s) have been revealed as anti-obese immune regulators by secreting anti-inflammatory cytokines and promoting the polarization of M2 macrophages, whereas group 1 ILCs (ILC1s), including natural killer cells, may promote adipose tissue inflammation via production of interferon-γ, which in turn polarizes macrophages toward the M1 type. The majority of studies to date have demonstrated the pathological association between ILCs and obesity in the context of adipose tissue inflammation, whereas the roles of ILCs in other organs which participate in obesity development have not been fully characterized. Therefore, identifying the roles of all types of ILCs as central components mediating obesity-associated inflammation, is of primary concern, and may lead to the discovery of novel preventative and therapeutic interventions. PMID:29138853

Forward-biased current annealing of radiation degraded indium phosphide and gallium arsenide solar cells

NASA Technical Reports Server (NTRS)

Michael, Sherif; Cypranowski, Corinne; Anspaugh, Bruce

1990-01-01

The preliminary results of a novel approach to low-temperature annealing of previously irradiated indium phosphide and gallium arsenide solar cells are reported. The technique is based on forward-biased current annealing. The two types of III-V solar cells were irradiated with 1-MeV electrons to a fluence level of (1-10) x 10 to the 14th electrons/sq cm. Several annealing attempts were made, varying all conditions. Optimum annealing was achieved when cells were injected with minority currents at a constant 90 C. The current density for each type of cell was also determined. Significant recovery of degraded parameters was achieved in both cases. However, the InP cell recovery notably exceeded the recovery in GaAs cells. The recovery is thought to be caused by current-stimulated reordering of the radiator-induced displacement damage. Both types of cell were then subjected to several cycles of irradiation and annealing. The results were also very promising. The significant recovery of degraded cell parameters at low temperature might play a major role in considerably extending the end of life of future spacecraft.

Documentation of angiotensin II receptors in glomerular epithelial cells

NASA Technical Reports Server (NTRS)

Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

1998-01-01

Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial cells participate in angiotensin II-mediated control of the glomerular filtration barrier.

Tim18, a component of the mitochondrial translocator, mediates yeast cell death induced by arsenic.

PubMed

Du, Li; Yu, Yong; Li, Zidong; Chen, Jingsi; Liu, Yan; Xia, Yongjing; Liu, Xiangjun

2007-08-01

Evidence is presented that Tim18, a mitochondria translocase, plays a role in the previously described apoptosis induced by arsenite in Saccharomyces cerevisiae. Tim18 deletion mutant exhibited resistance to arsenite. After arsenite treatment, both the wild type and Tim18-deficient cells showed reactive oxygen species (ROS) production. Arsenite induced the higher expression of tim18 in wild type yeast cells. We found that the tim18 deletion mutant also exhibited resistance to other apoptotic stresses such as acetic acid, H2O2, and hyperosmotic stress. These results suggest that Tim18 is important for yeast cell death induced by arsenic, and it may act downstream of ROS production.

Biomolecular Profiling of Jet Fuel Toxicity Using Proteomics

DTIC Science & Technology

2006-02-28

pulmonary alveolar type II cells and macrophages, and human epidermal keratinocytes in various exposure models. Results strongly suggest an injurious effect ...of exposure on all cells studied. In both pulmonary and skin cells, the protein profiles of JP-8 effect corroborates previous histological findings...potential intervention by Substance P (SP) in the pulmonary effects of JP-8 exposure , studies incorporating SP treatment along with JP-8 exposure

Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers.

PubMed

Dorrell, Craig; Abraham, Stephanie L; Lanxon-Cookson, Kelsea M; Canaday, Pamela S; Streeter, Philip R; Grompe, Markus

2008-09-01

We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or alpha-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of alpha, beta, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.

Longevity of U cells of differentiated yeast colonies grown on respiratory medium depends on active glycolysis.

PubMed

Čáp, Michal; Váchová, Libuše; Palková, Zdena

2015-01-01

Colonies of Saccharomyces cerevisiae laboratory strains pass through specific developmental phases when growing on solid respiratory medium. During entry into the so-called alkali phase, in which ammonia signaling is initiated, 2 prominent cell types are formed within the colonies: U cells in upper colony regions, which have a longevity phenotype and activate the expression of a large number of metabolic genes, and L cells in lower regions, which die more quickly and exhibit a starvation phenotype. Here, we performed a detailed analysis of the activities of enzymes of central carbon metabolism in lysates of both cell types and determined several fermentation end products, showing that previously reported expression differences are reflected in the different enzymatic capabilities of each cell type. Hence, U cells, despite being grown on respiratory medium, behave as fermenting cells, whereas L cells rely on respiratory metabolism and possess active gluconeogenesis. Using a spectrum of different inhibitors, we showed that glycolysis is essential for the formation, and particularly, the survival of U cells. We also showed that β-1,3-glucans that are released from the cell walls of L cells are the most likely source of carbohydrates for U cells.

Perturbation of auxin homeostasis by overexpression of wild-type IAA15 results in impaired stem cell differentiation and gravitropism in roots.

PubMed

Yan, Da-Wei; Wang, Jing; Yuan, Ting-Ting; Hong, Li-Wei; Gao, Xiang; Lu, Ying-Tang

2013-01-01

Aux/IAAs interact with auxin response factors (ARFs) to repress their transcriptional activity in the auxin signaling pathway. Previous studies have focused on gain-of-function mutations of domain II and little is known about whether the expression level of wild-type Aux/IAAs can modulate auxin homeostasis. Here we examined the perturbation of auxin homeostasis by ectopic expression of wild-type IAA15. Root gravitropism and stem cell differentiation were also analyzed. The transgenic lines were less sensitive to exogenous auxin and exhibited low-auxin phenotypes including failures in gravity response and defects in stem cell differentiation. Overexpression lines also showed an increase in auxin concentration and reduced polar auxin transport. These results demonstrate that an alteration in the expression of wild-type IAA15 can disrupt auxin homeostasis.

Cell type-selective disease-association of genes under high regulatory load

PubMed Central

Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

2015-01-01

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3′ UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. PMID:26338775

Large Scale Production of Stem Cells and Their Derivatives

NASA Astrophysics Data System (ADS)

Zweigerdt, Robert

Stem cells have been envisioned to become an unlimited cell source for regenerative medicine. Notably, the interest in stem cells lies beyond direct therapeutic applications. They might also provide a previously unavailable source of valuable human cell types for screening platforms, which might facilitate the development of more efficient and safer drugs. The heterogeneity of stem cell types as well as the numerous areas of application suggests that differential processes are mandatory for their in vitro culture. Many of the envisioned applications would require the production of a high number of stem cells and their derivatives in scalable, well-defined and potentially clinical compliant manner under current good manufacturing practice (cGMP). In this review we provide an overview on recent strategies to develop bioprocesses for the expansion, differentiation and enrichment of stem cells and their progenies, presenting examples for adult and embryonic stem cells alike.

Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

PubMed

Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

Novel Afferent Terminal Structure in the Crista Ampullaris of the Goldfish, Carassius auratus

NASA Technical Reports Server (NTRS)

Lanford, Pamela J.; Popper, Arthur N.

1996-01-01

Using transmission electron microscopy, we have identified a new type of afferent terminal structure in the crista ampullaris of the goldfish Carassius auratus. In addition to the bouton-type afferent terminals previously described in the ear of this species, the crista also contained enlarged afferent terminals that enveloped a portion of the basolateral hair cell membrane. The hair cell membrane was evaginated and protruded into the afferent terminal in a glove-and-finger configuration. The membranes of the two cells were regularly aligned in the protruded region of the contact and had a distinct symmetrical electron density. The electron-dense profiles of these contacts were easily identified and were present in every crista sampled. In some cases, efferent terminals synapsed onto the afferents at a point where the hair cell protruded into the terminal. The ultrastructural similarities of the goldfish crista afferents to calyx afferents found in amniotes (birds, reptiles, and mammals) are discussed. The results of the study support the hypothesis that structural variation in the vertebrate inner ear may have evolved much earlier in evolution than previously supposed.

Treatment and survival among 1594 patients with ATL.

PubMed

Katsuya, Hiroo; Ishitsuka, Kenji; Utsunomiya, Atae; Hanada, Shuichi; Eto, Tetsuya; Moriuchi, Yukiyoshi; Saburi, Yoshio; Miyahara, Masaharu; Sueoka, Eisaburo; Uike, Naokuni; Yoshida, Shinichiro; Yamashita, Kiyoshi; Tsukasaki, Kunihiro; Suzushima, Hitoshi; Ohno, Yuju; Matsuoka, Hitoshi; Jo, Tatsuro; Amano, Masahiro; Hino, Ryosuke; Shimokawa, Mototsugu; Kawai, Kazuhiro; Suzumiya, Junji; Tamura, Kazuo

2015-12-10

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature T lymphocytes caused by human T-lymphotropic virus type I. Intensive combination chemotherapy and allogeneic hematopoietic stem cell transplantation have been introduced since the previous Japanese nationwide survey was performed in the late 1980s. In this study, we delineated the current features and management of ATL in Japan. The clinical data were collected retrospectively from the medical records of patients diagnosed with ATL between 2000 and 2009, and a total of 1665 patients' records were submitted to the central office from 84 institutions in Japan. Seventy-one patients were excluded; 895, 355, 187, and 157 patients with acute, lymphoma, chronic, and smoldering types, respectively, remained. The median survival times were 8.3, 10.6, 31.5, and 55.0 months, and 4-year overall survival (OS) rates were 11%, 16%, 36%, and 52%, respectively, for acute, lymphoma, chronic, and smoldering types. The number of patients with allogeneic hematopoietic stem cell transplantation was 227, and their median survival time and OS at 4 years after allogeneic hematopoietic stem cell transplantation was 5.9 months and 26%, respectively. This study revealed that the prognoses of the patients with acute and lymphoma types were still unsatisfactory, despite the recent progress in treatment modalities, but an improvement of 4-year OS was observed in comparison with the previous survey. Of note, one-quarter of patients who could undergo transplantation experienced long survival. It is also noted that the prognosis of the smoldering type was worse than expected. © 2015 by The American Society of Hematology.

Joint annotation of chromatin state and chromatin conformation reveals relationships among domain types and identifies domains of cell-type-specific expression

PubMed Central

Libbrecht, Maxwell W.; Ay, Ferhat; Hoffman, Michael M.; Gilbert, David M.; Bilmes, Jeffrey A.; Noble, William Stafford

2015-01-01

The genomic neighborhood of a gene influences its activity, a behavior that is attributable in part to domain-scale regulation. Previous genomic studies have identified many types of regulatory domains. However, due to the difficulty of integrating genomics data sets, the relationships among these domain types are poorly understood. Semi-automated genome annotation (SAGA) algorithms facilitate human interpretation of heterogeneous collections of genomics data by simultaneously partitioning the human genome and assigning labels to the resulting genomic segments. However, existing SAGA methods cannot integrate inherently pairwise chromatin conformation data. We developed a new computational method, called graph-based regularization (GBR), for expressing a pairwise prior that encourages certain pairs of genomic loci to receive the same label in a genome annotation. We used GBR to exploit chromatin conformation information during genome annotation by encouraging positions that are close in 3D to occupy the same type of domain. Using this approach, we produced a model of chromatin domains in eight human cell types, thereby revealing the relationships among known domain types. Through this model, we identified clusters of tightly regulated genes expressed in only a small number of cell types, which we term “specific expression domains.” We found that domain boundaries marked by promoters and CTCF motifs are consistent between cell types even when domain activity changes. Finally, we showed that GBR can be used to transfer information from well-studied cell types to less well-characterized cell types during genome annotation, making it possible to produce high-quality annotations of the hundreds of cell types with limited available data. PMID:25677182

Joint annotation of chromatin state and chromatin conformation reveals relationships among domain types and identifies domains of cell-type-specific expression.

PubMed

Libbrecht, Maxwell W; Ay, Ferhat; Hoffman, Michael M; Gilbert, David M; Bilmes, Jeffrey A; Noble, William Stafford

2015-04-01

The genomic neighborhood of a gene influences its activity, a behavior that is attributable in part to domain-scale regulation. Previous genomic studies have identified many types of regulatory domains. However, due to the difficulty of integrating genomics data sets, the relationships among these domain types are poorly understood. Semi-automated genome annotation (SAGA) algorithms facilitate human interpretation of heterogeneous collections of genomics data by simultaneously partitioning the human genome and assigning labels to the resulting genomic segments. However, existing SAGA methods cannot integrate inherently pairwise chromatin conformation data. We developed a new computational method, called graph-based regularization (GBR), for expressing a pairwise prior that encourages certain pairs of genomic loci to receive the same label in a genome annotation. We used GBR to exploit chromatin conformation information during genome annotation by encouraging positions that are close in 3D to occupy the same type of domain. Using this approach, we produced a model of chromatin domains in eight human cell types, thereby revealing the relationships among known domain types. Through this model, we identified clusters of tightly regulated genes expressed in only a small number of cell types, which we term "specific expression domains." We found that domain boundaries marked by promoters and CTCF motifs are consistent between cell types even when domain activity changes. Finally, we showed that GBR can be used to transfer information from well-studied cell types to less well-characterized cell types during genome annotation, making it possible to produce high-quality annotations of the hundreds of cell types with limited available data. © 2015 Libbrecht et al.; Published by Cold Spring Harbor Laboratory Press.

High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

2016-02-01

Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

LocExpress: a web server for efficiently estimating expression of novel transcripts.

PubMed

Hou, Mei; Tian, Feng; Jiang, Shuai; Kong, Lei; Yang, Dechang; Gao, Ge

2016-12-22

The temporal and spatial-specific expression pattern of a transcript in multiple tissues and cell types can indicate key clues about its function. While several gene atlas available online as pre-computed databases for known gene models, it's still challenging to get expression profile for previously uncharacterized (i.e. novel) transcripts efficiently. Here we developed LocExpress, a web server for efficiently estimating expression of novel transcripts across multiple tissues and cell types in human (20 normal tissues/cells types and 14 cell lines) as well as in mouse (24 normal tissues/cell types and nine cell lines). As a wrapper to RNA-Seq quantification algorithm, LocExpress efficiently reduces the time cost by making abundance estimation calls increasingly within the minimum spanning bundle region of input transcripts. For a given novel gene model, such local context-oriented strategy allows LocExpress to estimate its FPKMs in hundreds of samples within minutes on a standard Linux box, making an online web server possible. To the best of our knowledge, LocExpress is the only web server to provide nearly real-time expression estimation for novel transcripts in common tissues and cell types. The server is publicly available at http://loc-express.cbi.pku.edu.cn .

Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex.

PubMed

Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A

2014-10-01

Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.

Mechanisms for the epigenetic inheritance of stress response in single cells.

PubMed

Xue, Yuan; Acar, Murat

2018-05-30

Cells have evolved to dynamically respond to different types of environmental and physiological stress conditions. The information about a previous stress stimulus experience by a mother cell can be passed to its descendants, allowing them to better adapt to and survive in new environments. In recent years, live-cell imaging combined with cell-lineage tracking approaches has elucidated many important principles that guide stress inheritance at the single-cell and population level. In this review, we summarize different strategies that cells can employ to pass the 'memory' of previous stress responses to their descendants. Among these strategies, we focus on a recent discovery of how specific features of Msn2 nucleo-cytoplasmic shuttling dynamics could be inherited across cell lineages. We also discuss how stress response can be transmitted to progenies through changes in chromatin and through partitioning of anti-stress factors and/or damaged macromolecules between mother and daughter cells during cell division. Finally, we highlight how emergent technologies will help address open questions in the field.

Effects of Elevated Pax6 Expression and Genetic Background on Mouse Eye Development

PubMed Central

Chanas, Simon A.; Collinson, J. Martin; Ramaesh, Thaya; Dorà, Natalie; Kleinjan, Dirk A.; Hill, Robert E.; West, John D.

2009-01-01

Purpose To analyze the effects of Pax6 overexpression and its interaction with genetic background on eye development. Methods Histologic features of eyes from hemizygous PAX77+/− transgenic (high Pax6 gene dose) and wild-type mice were compared on different genetic backgrounds. Experimental PAX77+/−↔wild-type and control wild-type↔wild-type chimeras were analyzed to investigate the causes of abnormal eye development in PAX77+/− mice. Results PAX77+/− mice showed an overlapping but distinct spectrum of eye abnormalities to Pax6+/− heterozygotes (low Pax6 dose). Some previously reported PAX77+/− eye abnormalities did not occur on all three genetic backgrounds examined. Several types of eye abnormalities occurred in the experimental PAX77+/−↔wild-type chimeras, and they occurred more frequently in chimeras with higher contributions of PAX77+/− cells. Groups of RPE cells intruded into the optic nerve sheath, indicating that the boundary between the retina and optic nerve may be displaced. Both PAX77+/− and wild-type cells were involved in this ingression and in retinal folds, suggesting that neither effect was cell-autonomous. Cell-autonomous effects included failure of PAX77+/− and wild-type cells to mix normally and overrepresentation of PAX77+/− in the lens epithelium and RPE. Conclusions The extent of PAX77+/− eye abnormalities depended on PAX77+/− genotype, genetic background, and stochastic variation. Chimera analysis identified two types of cell-autonomous effects of the PAX77+/− genotype. Abnormal cell mixing between PAX77+/− and wild-type cells suggests altered expression of cell surface adhesion molecules. Some phenotypic differences between PAX77+/−↔wild-type and Pax6+/−↔wild-type chimeras may reflect differences in the levels of PAX77+/− and Pax6+/− contributions to chimeric lenses. PMID:19387074

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH*

PubMed Central

Daidoji, Tomo; Watanabe, Yohei; Ibrahim, Madiha S.; Yasugi, Mayo; Maruyama, Hisataka; Masuda, Taisuke; Arai, Fumihito; Ohba, Tomoyuki; Honda, Ayae; Ikuta, Kazuyoshi; Nakaya, Takaaki

2015-01-01

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells. PMID:25673693

A solitary polypoid gastric metastasis 20 years after renal cell carcinoma: an event to be considered, and a brief review of the literature.

PubMed

Onorati, M; Petracco, G; Uboldi, P; Redaelli, D G; Romagnoli, S; Albertoni, M; Di Nuovo, F

2013-08-01

The incidence of gastric metastasis is 2.6%. Although all primary neoplasms can metastasize to the stomach, most originate from melanoma or breast and lung cancer. Their most common endoscopic appearance is a "volcano-like" polypoid mass covered by normal mucosa that may show a central ulceration. Renal cell carcinoma, clear cell type, is known to spread hematogenously, and isolated metastasis to the stomach is a rare event. In this report, we describe a gastric recurrence of RCC, clear-cell type, in a 80-year-old patient who had undergone nephrectomy 20 years before. We also performed a brief review of the literature to update the number of cases described to date. Metastatic involvement of the stomach should be suspected in any patient with a previous history of renal cell carcinoma, clear cell type, presenting with gastrointestinal symptoms, even if many years after nephrectomy. The peculiarity of our case is due to the very late presentation of the gastric metastasis. Only two cases of very late gastric metastases from RCC, clear cell type, have been described in the literature, to date.

Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells

PubMed Central

Sviderskaya, Elena V.; Easty, David J.; Lawrence, Mark A.; Sánchez, Daniel P.; Negulyaev, Yuri A.; Patel, Ricken H.; Anand, Praveen; Korchev, Yuri E.; Bennett, Dorothy C.

2009-01-01

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.—Sviderskaya, E. V., Easty, D. J., Lawrence, M. A., Sánchez, D. P., Negulyaev, Y. A., Patel, R. H., Anand, P., Korchev, Y. E., Bennett, D. C. Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells. PMID:19447881

Two alternate strategies for innate immunity to Epstein-Barr virus: One using NK cells and the other NK cells and γδ T cells

PubMed Central

Horowitz, Amir; Nemat-Gorgani, Neda; Münz, Christian

2017-01-01

Most humans become infected with Epstein–Barr virus (EBV), which then persists for life. Infrequently, EBV infection causes infectious mononucleosis (IM) or Burkitt lymphoma (BL). Type I EBV infection, particularly type I BL, stimulates strong responses of innate immune cells. Humans respond to EBV in two alternative ways. Of 24 individuals studied, 13 made strong NK and γδ T cell responses, whereas 11 made feeble γδ T cell responses but stronger NK cell responses. The difference does not correlate with sex, HLA type, or previous exposure to EBV or cytomegalovirus. Cohorts of EBV+ children and pediatric IM patients include both group 1 individuals, with high numbers of γδ T cells, and group 2 individuals, with low numbers. The even balance of groups 1 and 2 in the human population points to both forms of innate immune response to EBV having benefit for human survival. Correlating these distinctive responses with the progress of EBV infection might facilitate the management of EBV-mediated disease. PMID:28468758

Total-Body Irradiation and Fludarabine Phosphate Followed by Donor Peripheral Blood Stem Cell Transplant in Treating Patients With Hematologic Malignancies or Kidney Cancer

ClinicalTrials.gov

2017-12-11

Adult Acute Myeloid Leukemia in Remission; Childhood Acute Lymphoblastic Leukemia in Remission; Childhood Acute Myeloid Leukemia in Remission; Childhood Myelodysplastic Syndrome; Childhood Renal Cell Carcinoma; Chronic Myelomonocytic Leukemia; Clear Cell Renal Cell Carcinoma; de Novo Myelodysplastic Syndrome; Metastatic Renal Cell Cancer; Previously Treated Myelodysplastic Syndrome; Progression of Multiple Myeloma or Plasma Cell Leukemia; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Lymphoblastic Lymphoma; Recurrent Adult Non-Hodgkin Lymphoma; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Acute Myeloid Leukemia; Recurrent Childhood Lymphoblastic Lymphoma; Recurrent Childhood Non-Hodgkin Lymphoma; Refractory Anemia; Refractory Anemia With Ringed Sideroblasts; Refractory Childhood Hodgkin Lymphoma; Refractory Chronic Lymphocytic Leukemia; Renal Medullary Carcinoma; Type 1 Papillary Renal Cell Carcinoma; Type 2 Papillary Renal Cell Carcinoma; Untreated Adult Acute Lymphoblastic Leukemia; Untreated Adult Acute Myeloid Leukemia; Untreated Childhood Acute Lymphoblastic Leukemia

Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.

PubMed

Spaethling, Jennifer M; Na, Young-Ji; Lee, Jaehee; Ulyanova, Alexandra V; Baltuch, Gordon H; Bell, Thomas J; Brem, Steven; Chen, H Isaac; Dueck, Hannah; Fisher, Stephen A; Garcia, Marcela P; Khaladkar, Mugdha; Kung, David K; Lucas, Timothy H; O'Rourke, Donald M; Stefanik, Derek; Wang, Jinhui; Wolf, John A; Bartfai, Tamas; Grady, M Sean; Sul, Jai-Yoon; Kim, Junhyong; Eberwine, James H

2017-01-17

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Resistance to human immunodeficiency virus type 1 (HIV-1) generated by lentivirus vector-mediated delivery of the CCR5Δ32 gene despite detectable expression of the HIV-1 co-receptors

PubMed Central

Jin, Qingwen; Marsh, Jon; Cornetta, Kenneth; Alkhatib, Ghalib

2009-01-01

It has previously been demonstrated that there are two distinct mechanisms for genetic resistance to human immunodeficiency virus type 1 (HIV-1) conferred by the CCR5Δ32 gene: the loss of wild-type CCR5 surface expression and the generation of CCR5Δ32 protein, which interacts with CXCR4. To analyse the protective effects of long-term expression of the CCR5Δ32 protein, recombinant lentiviral vectors were used to deliver the CCR5Δ32 gene into human cell lines and primary peripheral blood mononuclear cells that had been immortalized by human T-cell leukemia virus type 1. Blasticidin S-resistant cell lines expressing the lentivirus-encoded CCR5Δ32 showed a significant reduction in HIV-1 Env-mediated fusion assays. It was shown that CD4+ T lymphocytes expressing the lentivirus-encoded CCR5Δ32 gene were highly resistant to infection by a primary but not by a laboratory-adapted X4 strain, suggesting different infectivity requirements. In contrast to previous studies that analysed the CCR5Δ32 protective effects in a transient expression system, this study showed that long-term expression of CCR5Δ32 conferred resistance to HIV-1 despite cell-surface expression of the HIV co-receptors. The results suggest an additional unknown mechanism for generating the CCR5Δ32 resistance phenotype and support the hypothesis that the CCR5Δ32 protein acts as an HIV-suppressive factor by altering the stoichiometry of the molecules involved in HIV-1 entry. The lentiviral-CCR5Δ32 vectors offer a method of generating HIV-resistant cells by delivery of the CCR5Δ32 gene that may be useful for stem cell- or T-cell-based gene therapy for HIV-1 infection. PMID:18796731

Recombinant Marburg viruses containing mutations in the IID region of VP35 prevent inhibition of Host immune responses.

PubMed

Albariño, César G; Wiggleton Guerrero, Lisa; Spengler, Jessica R; Uebelhoer, Luke S; Chakrabarti, Ayan K; Nichol, Stuart T; Towner, Jonathan S

2015-02-01

Previous in vitro studies have demonstrated that Ebola and Marburg virus (EBOV and MARV) VP35 antagonize the host cell immune response. Moreover, specific mutations in the IFN inhibitory domain (IID) of EBOV and MARV VP35 that abrogate their interaction with virus-derived dsRNA, lack the ability to inhibit the host immune response. To investigate the role of MARV VP35 in the context of infectious virus, we used our reverse genetics system to generate two recombinant MARVs carrying specific mutations in the IID region of VP35. Our data show that wild-type and mutant viruses grow to similar titers in interferon deficient cells, but exhibit attenuated growth in interferon-competent cells. Furthermore, in contrast to wild-type virus, both MARV mutants were unable to inhibit expression of various antiviral genes. The MARV VP35 mutants exhibit similar phenotypes to those previously described for EBOV, suggesting the existence of a shared immune-modulatory strategy between filoviruses. Published by Elsevier Inc.

A case of fulminant Type 1 diabetes following anti-PD1 immunotherapy in a genetically susceptible patient.

PubMed

Araújo, Manuel; Ligeiro, Dário; Costa, Luís; Marques, Filipa; Trindade, Helder; Correia, José Manuel; Fonseca, Candida

2017-06-01

Programmed cell death-1 protein (PD-1) is an immune checkpoint that has gained popularity in the treatment of several advanced cancers. Inhibiting this checkpoint is known to enhance immune response, but is also known to diminish immune tolerance and to increase autoimmune toxicity. We discuss a case of rapid onset fulminant Type 1 diabetes induced by treatment with anti-programmed cell death-1 monoclonal antibody, nivolumab, in a patient with late-stage non-small-cell lung adenocarcinoma. The patient had no history of previous diabetes but did reveal a high-risk genotype for Type 1 diabetes development (DR3-DQ2; DR4-DQ8). This finding supports that acute Type 1 diabetes can be an important adverse effect of immunotherapies targeting T-cell activation regulation. Because of the severity of this adverse effect, physicians should be aware of it, and studies directed to the detection of new biomarkers for early risk stratification (e.g., HLA) should be sought.

[The role of chromogranin-A and its derived peptide, WE-14 in the development of type 1 diabetes mellitus].

PubMed

Herold, Zoltán; Nagy, Péter; Patócs, Attila; Somogyi, Anikó

2015-02-01

Chromogranin-A is a member of the granine protein family. It is produced in neuroendocrine cells via secretory granules. Many cleavage proteins are formed from chromogranin-A, from which some have well known biological activity, while the function of others is not yet fully known. Serum chromogranin-A levels are used in neuroendocrine tumour diagnostics. Recent studies showed that one of its cleavage protein, WE-14 may also play a role in the development of type 1 diabetes. WE-14 may function as an autoantigen for T-cells involved in the destruction of β-cells. This mechanism was previously observed only in non-obese diabetic mice. Novel results show that WE-14 also serves as a target for autoreactive cells in newly diagnosed type 1 diabetic patients as well, which reaction can be increased with transglutaminase. In this paper the authors summarize the recent knowledge about chromogranin-A and its potential role in the pathomechanism of type 1 diabetes mellitus.

Characterization of C-type lectins reveals an unexpectedly limited interaction between Cryptococcus neoformans spores and Dectin-1

PubMed Central

Walsh, Naomi M.; Wuthrich, Marcel; Wang, Huafeng; Klein, Bruce; Hull, Christina M.

2017-01-01

Phagocytosis by innate immune cells is an important process for protection against multiple pathologies and is particularly important for resistance to infection. However, phagocytosis has also been implicated in the progression of some diseases, including the dissemination of the human fungal pathogen, Cryptococcus neoformans. Previously, we identified Dectin-1 as a likely phagocytic receptor for C. neoformans spores through the use of soluble components in receptor-ligand blocking experiments. In this study, we used gain-of-function and loss-of-function assays with intact cells to evaluate the in vivo role of Dectin-1 and other C-type lectins in interactions with C. neoformans spores and discovered stark differences in outcome when compared with previous assays. First, we found that non-phagocytic cells expressing Dectin-1 were unable to bind spores and that highly sensitive reporter cells expressing Dectin-1 were not stimulated by spores. Second, we determined that some phagocytes from Dectin-1-/- mice interacted with spores differently than wild type (WT) cells, but the effects varied among assays and were modest overall. Third, while we detected small but statistically significant reductions in phagocytosis by primary alveolar macrophages from Dectin-1-/- mice compared to WT, we found no differences in survival between WT and Dectin-1-/- mice challenged with spores. Further analyses to assess the roles of other C-type lectins and their adapters revealed very weak stimulation of Dectin-2 reporter cells by spores and modest differences in binding and phagocytosis by Dectin-2-/- bone marrow-derived phagocytes. There were no discernable defects in binding or phagocytosis by phagocytes lacking Mannose Receptor, Mincle, Card-9, or FcRγ. Taken together, these results lead to the conclusion that Dectin-1 and other C-type lectins do not individually play a major roles in phagocytosis and innate defense by phagocytes against C. neoformans spores and highlight challenges in using soluble receptor/ligand blocking experiments to recapitulate biologically relevant interactions. PMID:28282442

Structure of kaurane-type diterpenes from Parinari sprucei and their potential anticancer activity.

PubMed

Braca, Alessandra; Armenise, Annahil; Morelli, Ivano; Mendez, Jeannette; Mi, Qiuwen; Chai, Hee-Byung; Swanson, Steven M; Kinghorn, A Douglas; De Tommasi, Nunziatina

2004-06-01

Twenty-three kaurane-type diterpenes 1 - 23, including twenty new natural products 1 - 20, have been isolated from the leaves of Parinari sprucei and their structures elucidated by spectroscopic and chemical analysis. The isolated compounds were tested for their cytotoxic activity towards a panel of cancer cell lines. Compounds 9 and 10 showed activity against all cell lines with ED (50) values in the range of 10 - 20 microg/mL. The previously known 13-hydroxy-15-oxozoapatlin 21 was evaluated in an in vivo hollow fiber test, and found to be active with KB and LNCaP cells at the concentrations used.

Assessment of Cell Line Models of Primary Human Cells by Raman Spectral Phenotyping

PubMed Central

Swain, Robin J.; Kemp, Sarah J.; Goldstraw, Peter; Tetley, Teresa D.; Stevens, Molly M.

2010-01-01

Abstract Researchers have previously questioned the suitability of cell lines as models for primary cells. In this study, we used Raman microspectroscopy to characterize live A549 cells from a unique molecular biochemical perspective to shed light on their suitability as a model for primary human pulmonary alveolar type II (ATII) cells. We also investigated a recently developed transduced type I (TT1) cell line as a model for alveolar type I (ATI) cells. Single-cell Raman spectra provide unique biomolecular fingerprints that can be used to characterize cellular phenotypes. A multivariate statistical analysis of Raman spectra indicated that the spectra of A549 and TT1 cells are characterized by significantly lower phospholipid content compared to ATII and ATI spectra because their cytoplasm contains fewer surfactant lamellar bodies. Furthermore, we found that A549 spectra are statistically more similar to ATI spectra than to ATII spectra. The spectral variation permitted phenotypic classification of cells based on Raman spectral signatures with >99% accuracy. These results suggest that A549 cells are not a good model for ATII cells, but TT1 cells do provide a reasonable model for ATI cells. The findings have far-reaching implications for the assessment of cell lines as suitable primary cellular models in live cultures. PMID:20409492

An N-terminal Retention Module Anchors the Giant Adhesin LapA of Pseudomonas fluorescens at the Cell Surface: A Novel Sub-family of Type I Secretion Systems.

PubMed

Smith, T Jarrod; Font, Maria E; Kelly, Carolyn M; Sondermann, Holger; O'Toole, George A

2018-02-05

LapA of Pseudomonas fluorescens Pf0-1 belongs to a diverse family of cell surface associated bacterial adhesins that are secreted via the type-1 secretion system (T1SS). We previously reported that the periplasmic protease LapG cleaves the N-terminus of LapA at a canonical dialanine motif to release the adhesin from the cell surface under conditions unfavorable to biofilm formation, thus decreasing biofilm formation. Here, we characterize LapA as the first type 1 secreted substrate that does not follow the "one-step" rule of T1SS. Rather, a novel N-terminal element, called the retention module (RM), localizes LapA at the cell surface as a secretion intermediate. Our genetic, biochemical, and molecular modeling analysis support a model wherein LapA is tethered to the cell surface through its T1SS outer membrane TolC-like pore, LapE, until LapG cleaves LapA in the periplasm. We further demonstrate this unusual retention strategy is likely conserved among LapA-like proteins, and reveals a new subclass of T1SS ABC transporters involved in transporting this group of surface-associated, LapA-like adhesins. These studies demonstrate a novel cell surface retention strategy used throughout the Proteobacteria and highlight a previously unappreciated flexibility of function for T1SS. Importance. Bacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface associated adhesins is key for initiating contact with a preferred substratum to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal Proteobacteria and provide phylogenetic evidence that these adhesins are secreted by a new subclass of T1SS ABC transporters. Copyright © 2018 American Society for Microbiology.

In vitro chondrogenic commitment of human Wharton's jelly stem cells by co-culture with human articular chondrocytes.

PubMed

Pereira, R C; Costa-Pinto, A R; Frias, A M; Neves, N M; Azevedo, H S; Reis, R L

2017-06-01

Wharton's jelly stem cells (WJSCs) are a potential source of transplantable stem cells in cartilage-regenerative strategies, due to their highly proliferative and multilineage differentiation capacity. We hypothesized that a non-direct co-culture system with human articular chondrocytes (hACs) could enhance the potential chondrogenic phenotype of hWJSCs during the expansion phase compared to those expanded in monoculture conditions. Primary hWJSCs were cultured in the bottom of a multiwell plate separated by a porous transwell membrane insert seeded with hACs. No statistically significant differences in hWJSCs duplication number were observed under either of the culture conditions during the expansion phase. hWJSCs under co-culture conditions show upregulations of collagen type I and II, COMP, TGFβ1 and aggrecan, as well as of the main cartilage transcription factor, SOX9, when compared to those cultured in the absence of chondrocytes. Chondrogenic differentiation of hWJSCs, previously expanded in co-culture and monoculture conditions, was evaluated for each cellular passage using the micromass culture model. Cells expanded in co-culture showed higher accumulation of glycosaminoglycans (GAGs) compared to cells in monoculture, and immunohistochemistry for localization of collagen type I revealed a strong detection signal when hWJSCs were expanded under monoculture conditions. In contrast, type II collagen was detected when cells were expanded under co-culture conditions, where numerous round-shaped cell clusters were observed. Using a micromass differentiation model, hWJSCs, previously exposed to soluble factors secreted by hACs, were able to express higher levels of chondrogenic genes with deposition of cartilage extracellular matrix components, suggesting their use as an alternative cell source for treating degenerated cartilage. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Optofluidic Single-Cell Genome Amplification of Sub-micron Bacteria in the Ocean Subsurface

PubMed Central

Landry, Zachary C.; Vergin, Kevin; Mannenbach, Christopher; Block, Stephen; Yang, Qiao; Blainey, Paul; Carlson, Craig; Giovannoni, Stephen

2018-01-01

Optofluidic single-cell genome amplification was used to obtain genome sequences from sub-micron cells collected from the euphotic and mesopelagic zones of the northwestern Sargasso Sea. Plankton cells were visually selected and manually sorted with an optical trap, yielding 20 partial genome sequences representing seven bacterial phyla. Two organisms, E01-9C-26 (Gammaproteobacteria), represented by four single cell genomes, and Opi.OSU.00C, an uncharacterized Verrucomicrobia, were the first of their types retrieved by single cell genome sequencing and were studied in detail. Metagenomic data showed that E01-9C-26 is found throughout the dark ocean, while Opi.OSU.00C was observed to bloom transiently in the nutrient-depleted euphotic zone of the late spring and early summer. The E01-9C-26 genomes had an estimated size of 4.76–5.05 Mbps, and contained “O” and “W”-type monooxygenase genes related to methane and ammonium monooxygenases that were previously reported from ocean metagenomes. Metabolic reconstruction indicated E01-9C-26 are likely versatile methylotrophs capable of scavenging C1 compounds, methylated compounds, reduced sulfur compounds, and a wide range of amines, including D-amino acids. The genome sequences identified E01-9C-26 as a source of “O” and “W”-type monooxygenase genes related to methane and ammonium monooxygenases that were previously reported from ocean metagenomes, but are of unknown function. In contrast, Opi.OSU.00C genomes encode genes for catabolizing carbohydrate compounds normally associated with eukaryotic phytoplankton. This exploration of optofluidics showed that it was effective for retrieving diverse single-cell bacterioplankton genomes and has potential advantages in microbiology applications that require working with small sample volumes or targeting cells by their morphology.

Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

PubMed

Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

2008-06-01

Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

Role of Outer Membrane C-Type Cytochromes MtrC and OmcA in Shewanella Oneidensis MR-1 Cell Production, Accumulation, and Detachment During Respiration on Hematite

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Andrew C.; Peterson, L.; Reardon, Catherine L.

2012-07-01

Solid phase iron oxides are considered to be important terminal electron acceptors for microbial respiration in many anoxic environments. Besides the knowledge that cells attach to and reduce these substrates, other aspects of surface-associated cell behavior and the related cell surface components that influence cell-mineral interactions are not well understood. In the present study, wild-type cells of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 formed thin biofilms one-to-two cell layers in thickness when respiring on natural specular hematite under flow conditions similar to those which exist in aquatic sediments and subsurface environments. The distribution of cells within the biofilm indicatedmore » that direct contact was not required for electron transfer from cells to the mineral surface. Detached biomass in the form of single cells represented >99% of the surface-associated wild-type cell production from respiration on hematite over the biofilm life cycle. A mutant deficient in the outer membrane c35 type cytochrome OmcA, while still able to respire and replicate on hematite, established a lower steady-state cell density on the mineral surface than that of the wild-type strain. A mutant deficient in MtrC, another outer membrane c-type cytochrome, and a mutant deficient in both cytochromes were unable to reduce sufficient amounts of hematite to support detectable growth on the mineral surface. When considered in the context of previous work, the results support a growing body of evidence that the relative importance of OmcA and MtrC to cell respiration and replication depends on the form of iron oxide available as terminal electron acceptor.« less

The meaning of PIWI proteins in cancer development.

PubMed

Litwin, Monika; Szczepańska-Buda, Anna; Piotrowska, Aleksandra; Dzięgiel, Piotr; Witkiewicz, Wojciech

2017-05-01

Cancer is a histologically and genetically heterogeneous population of tumor cells that exhibits distinct molecular profiles determined by epigenetic alterations. P-element-induced wimpy testis (PIWI) proteins in complex with PIWI-interacting RNA (piRNA) have been previously demonstrated to be involved in epigenetic regulation in germline cells. Recently, reactivation of PIWI expression, primarily PIWI-like protein 1 and 2, through aberrant DNA methylation resulting in genomic silencing has been identified in various types of tumors. It has been suggested that the PIWI-piRNA complex contributes to cancer development and progression by promoting a stem-like state of cancer cells, or cancer stem cells (CSCs). It has been identified that CSCs represent the cells that have undergone epithelial-mesenchymal transition (EMT) and acquired metastatic capacities. However, the molecular association between the EMT process and the stem-cell state remains unclear. Further extensive characterization of CSCs in individual types of tumors is required to identify specific markers for the heterogeneous population of CSCs and therefore selectively target CSCs. Previous studies indicate a reciprocal regulation between PIWI proteins and a complex signaling network linking markers characterized for CSCs and transcription factors involved in EMT. In the present review, studies of PIWI function are summarized, and the potential involvement of PIWI proteins in cancer development and progression is discussed.

The meaning of PIWI proteins in cancer development

PubMed Central

Litwin, Monika; Szczepańska-Buda, Anna; Piotrowska, Aleksandra; Dzięgiel, Piotr; Witkiewicz, Wojciech

2017-01-01

Cancer is a histologically and genetically heterogeneous population of tumor cells that exhibits distinct molecular profiles determined by epigenetic alterations. P-element-induced wimpy testis (PIWI) proteins in complex with PIWI-interacting RNA (piRNA) have been previously demonstrated to be involved in epigenetic regulation in germline cells. Recently, reactivation of PIWI expression, primarily PIWI-like protein 1 and 2, through aberrant DNA methylation resulting in genomic silencing has been identified in various types of tumors. It has been suggested that the PIWI-piRNA complex contributes to cancer development and progression by promoting a stem-like state of cancer cells, or cancer stem cells (CSCs). It has been identified that CSCs represent the cells that have undergone epithelial-mesenchymal transition (EMT) and acquired metastatic capacities. However, the molecular association between the EMT process and the stem-cell state remains unclear. Further extensive characterization of CSCs in individual types of tumors is required to identify specific markers for the heterogeneous population of CSCs and therefore selectively target CSCs. Previous studies indicate a reciprocal regulation between PIWI proteins and a complex signaling network linking markers characterized for CSCs and transcription factors involved in EMT. In the present review, studies of PIWI function are summarized, and the potential involvement of PIWI proteins in cancer development and progression is discussed. PMID:28529570

Defects in a New Class of Sulfate/Anion Transporter Link Sulfur Acclimation Responses to Intracellular Glutathione Levels and Cell Cycle Control1[W][OPEN

PubMed Central

Fang, Su-Chiung; Chung, Chin-Lin; Chen, Chun-Han; Lopez-Paz, Cristina; Umen, James G.

2014-01-01

We previously identified a mutation, suppressor of mating type locus3 15-1 (smt15-1), that partially suppresses the cell cycle defects caused by loss of the retinoblastoma tumor suppressor-related protein encoded by the MAT3 gene in Chlamydomonas reinhardtii. smt15-1 single mutants were also found to have a cell cycle defect leading to a small-cell phenotype. SMT15 belongs to a previously uncharacterized subfamily of putative membrane-localized sulfate/anion transporters that contain a sulfate transporter domain and are found in a widely distributed subset of eukaryotes and bacteria. Although we observed that smt15-1 has a defect in acclimation to sulfur-limited growth conditions, sulfur acclimation (sac) mutants, which are more severely defective for acclimation to sulfur limitation, do not have cell cycle defects and cannot suppress mat3. Moreover, we found that smt15-1, but not sac mutants, overaccumulates glutathione. In wild-type cells, glutathione fluctuated during the cell cycle, with highest levels in mid G1 phase and lower levels during S and M phases, while in smt15-1, glutathione levels remained elevated during S and M. In addition to increased total glutathione levels, smt15-1 cells had an increased reduced-to-oxidized glutathione redox ratio throughout the cell cycle. These data suggest a role for SMT15 in maintaining glutathione homeostasis that impacts the cell cycle and sulfur acclimation responses. PMID:25361960

Specific Accumulation of Tumor-Derived Adhesion Factor in Tumor Blood Vessels and in Capillary Tube-Like Structures of Cultured Vascular Endothelial Cells

NASA Astrophysics Data System (ADS)

Akaogi, Kotaro; Okabe, Yukie; Sato, Junji; Nagashima, Yoji; Yasumitsu, Hidetaro; Sugahara, Kazuyuki; Miyazaki, Kaoru

1996-08-01

Tumor-derived adhesion factor (TAF) was previously identified as a cell adhesion molecule secreted by human bladder carcinoma cell line EJ-1. To elucidate the physiological function of TAF, we examined its distribution in human normal and tumor tissues. Immunochemical staining with an anti-TAF monoclonal antibody showed that TAF was specifically accumulated in small blood vessels and capillaries within and adjacent to tumor nests, but not in those in normal tissues. Tumor blood vessel-specific staining of TAF was observed in various human cancers, such as esophagus, brain, lung, and stomach cancers. Double immunofluorescent staining showed apparent colocalization of TAF and type IV collagen in the vascular basement membrane. In vitro experiments demonstrated that TAF preferentially bound to type IV collagen among various extracellular matrix components tested. In cell culture experiments, TAF promoted adhesion of human umbilical vein endothelial cells to type IV collagen substrate and induced their morphological change. Furthermore, when the endothelial cells were induced to form capillary tube-like structures by type I collagen, TAF and type IV collagen were exclusively detected on the tubular structures. The capillary tube formation in vitro was prevented by heparin, which inhibited the binding of TAF to the endothelial cells. These results strongly suggest that TAF contributes to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen.

Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes.

PubMed

Speiseder, Thomas; Hofmann-Sieber, Helga; Rodríguez, Estefanía; Schellenberg, Anna; Akyüz, Nuray; Dierlamm, Judith; Spruss, Thilo; Lange, Claudia; Dobner, Thomas

2017-01-01

Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells. Copyright © 2016 American Society for Microbiology.

Short-wavelength cone-opponent retinal ganglion cells in mammals.

PubMed

Marshak, David W; Mills, Stephen L

2014-03-01

In all of the mammalian species studied to date, the short-wavelength-sensitive (S) cones and the S-cone bipolar cells that receive their input are very similar, but the retinal ganglion cells that receive synapses from the S-cone bipolar cells appear to be quite different. Here, we review the literature on mammalian retinal ganglion cells that respond selectively to stimulation of S-cones and respond with opposite polarity to longer wavelength stimuli. There are at least three basic mechanisms to generate these color-opponent responses, including: (1) opponency is generated in the outer plexiform layer by horizontal cells and is conveyed to the ganglion cells via S-cone bipolar cells, (2) inputs from bipolar cells with different cone inputs and opposite response polarity converge directly on the ganglion cells, and (3) inputs from S-cone bipolar cells are inverted by S-cone amacrine cells. These are not mutually exclusive; some mammalian ganglion cells that respond selectively to S-cone stimulation seem to utilize at least two of them. Based on these findings, we suggest that the small bistratified ganglion cells described in primates are not the ancestral type, as proposed previously. Instead, the known types of ganglion cells in this pathway evolved from monostratified ancestral types and became bistratified in some mammalian lineages.

InAlAs photovoltaic cell design for high device efficiency

DOE PAGES

Smith, Brittany L.; Bittner, Zachary S.; Hellstroem, Staffan D.; ...

2017-04-17

This study presents a new design for a single-junction InAlAs solar cell, which reduces parasitic absorption losses from the low band-gap contact layer while maintaining a functional window layer by integrating a selective etch stop. The etch stop is then removed prior to depositing an anti-reflective coating. The final cell had a 17.9% efficiency under 1-sun AM1.5 with an anti-reflective coating. Minority carrier diffusion lengths were extracted from external quantum efficiency data using physics-based device simulation software yielding 170 nm in the n-type emitter and 4.6 um in the p-type base, which is more than four times the diffusion lengthmore » previously reported for a p-type InAlAs base. In conclusion, this report represents significant progress towards a high-performance InAlAs top cell for a triple-junction design lattice-matched to InP.« less

Epitope Mapping of Monoclonal Antibody PMab-48 Against Dog Podoplanin.

PubMed

Yamada, Shinji; Kaneko, Mika K; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Ogasawara, Satoshi; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Harada, Hiroyuki; Kato, Yukinari

2018-04-02

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on normal renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells. Increased expression of PDPN in cancers is associated with poor prognosis and hematogenous metastasis through interactions with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously reported a novel PMab-48 antibody, which is an anti-dog PDPN (dPDPN) monoclonal antibody (mAb) recognizing PDPN expressed in lymphatic endothelial cells. However, the binding epitope of PMab-48 is yet to be clarified. In this study, an enzyme-linked immunosorbent assay and flow cytometry were used to investigate epitopes of PMab-48. The results revealed that the critical epitope of PMab-48 comprises Asp29, Asp30, Ile31, Ile32, and Pro33 of dPDPN.

Anti-proliferation activity of terpenoids isolated from Euphorbia kansui in human cancer cells and their structure-activity relationship.

PubMed

Hou, Jin-Jun; Shen, Yao; Yang, Zhou; Fang, Lin; Cai, Lu-Ying; Yao, Shuai; Long, Hua-Li; Wu, Wan-Ying; Guo, De-An

2017-10-01

Euphorbia kansui is a commonly used traditional Chinese medicine for the treatment of edema, pleural effusion, and asthma, etc. According to the previous researches, terpenoids in E. kansui possess various biological activities, e.g., anti-virus, anti-allergy, antitumor effects. In this work, twenty five terpenoids were isolated from E. kansui, including thirteen ingenane- and eight jatrophane-type diterpenoids (with two new compounds, kansuinin P and Q) and four triterpenoids. Eighteen of them were analyzed by MTS assay for in vitro anticancer activity in five human cancer cell lines. Structure-activity relationship for 12 ingenane-type diterpenoids in colorectal cancer Colo205 cells were preliminary studied. Significant anti-proliferation activities were observed in human melanoma cells breast cancer MDA-MB-435 cells and Colo205 cells. More than half of the isolated ingenane-type diterpenoids showed inhibitory activities in MDA-MB-435 cells. Eight ingenane- and one jatrophane-type diterpenoids possessed much lower IC 50 values in MDA-MB-435 cells than positive control staurosporine. Preliminary structure-activity relationship analysis showed that substituent on position 20 was important for the activity of ingenane-type diterpenoids in Colo205 cells and substituent on position 3 contributed more significant biological activity of the compounds than that on position 5 in both MDA-MB-435 and Colo205 cells. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Germinal centre frequency is decreased in pancreatic lymph nodes from individuals with recent-onset type 1 diabetes.

PubMed

Willcox, Abby; Richardson, Sarah J; Walker, Lucy S K; Kent, Sally C; Morgan, Noel G; Gillespie, Kathleen M

2017-07-01

Pancreatic lymph nodes (PLNs) are critical sites for the initial interaction between islet autoantigens and autoreactive lymphocytes, but the histology of PLNs in tissue from individuals with type 1 diabetes has not been analysed in detail. The aim of this study was to examine PLN tissue sections from healthy donors compared with those at risk of, or with recent-onset and longer-duration type 1 diabetes. Immunofluorescence staining was used to examine PLN sections from the following donor groups: non-diabetic (n=15), non-diabetic islet autoantibody-positive (n=5), recent-onset (≤1.5 years duration) type 1 diabetes (n=13), and longer-duration type 1 diabetes (n=15). Staining for CD3, CD20 and Ki67 was used to detect primary and secondary (germinal centre-containing) follicles and CD21 and CD35 to detect follicular dendritic cell networks. The frequency of secondary follicles was lower in the recent-onset type 1 diabetes group compared with the non-diabetic control group. The presence of insulitis (as evidence of ongoing beta cell destruction) and diagnosis of type 1 diabetes at a younger age, however, did not appear to be associated with a lower frequency of secondary follicles. A higher proportion of primary B cell follicles were observed to lack follicular dendritic cell networks in the recent-onset type 1 diabetes group. Histological analysis of rare PLNs from individuals with type 1 diabetes suggests a previously unrecognised phenotype comprising decreased primary B cell follicle frequency and fewer follicular dendritic cell networks in recent-onset type 1 diabetes.

Cells differentiated from mouse embryonic stem cells via embryoid bodies express renal marker molecules.

PubMed

Kramer, Jan; Steinhoff, Jürgen; Klinger, Matthias; Fricke, Lutz; Rohwedel, Jürgen

2006-03-01

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.

E-Cadherin-Dependent Stimulation of Traction Force at Focal Adhesions via the Src and PI3K Signaling Pathways

PubMed Central

Jasaitis, Audrius; Estevez, Maruxa; Heysch, Julie; Ladoux, Benoit; Dufour, Sylvie

2012-01-01

The interplay between cadherin- and integrin-dependent signals controls cell behavior, but the precise mechanisms that regulate the strength of adhesion to the extracellular matrix remains poorly understood. We deposited cells expressing a defined repertoire of cadherins and integrins on fibronectin (FN)-coated polyacrylamide gels (FN-PAG) and on FN-coated pillars used as a micro-force sensor array (μFSA), and analyzed the functional relationship between these adhesion receptors to determine how it regulates cell traction force. We found that cadherin-mediated adhesion stimulated cell spreading on FN-PAG, and this was modulated by the substrate stiffness. We compared S180 cells with cells stably expressing different cadherins on μFSA and found that traction forces were stronger in cells expressing cadherins than in parental cells. E-cadherin-mediated contact and mechanical coupling between cells are required for this increase in cell-FN traction force, which was not observed in isolated cells, and required Src and PI3K activities. Traction forces were stronger in cells expressing type I cadherins than in cells expressing type II cadherins, which correlates with our previous observation of a higher intercellular adhesion strength developed by type I compared with type II cadherins. Our results reveal one of the mechanisms whereby molecular cross talk between cadherins and integrins upregulates traction forces at cell-FN adhesion sites, and thus provide additional insight into the molecular control of cell behavior. PMID:22853894

Stem cells distribution, cellular proliferation and migration in the adult Austrolebias charrua brain.

PubMed

Torres-Pérez, Maximiliano; Rosillo, Juan Carlos; Berrosteguieta, Ines; Olivera-Bravo, Silvia; Casanova, Gabriela; García-Verdugo, José Manuel; Fernández, Anabel Sonia

2017-10-15

Our previous studies demonstrated that Austrolebias charrua annual fish is an excellent model to study adult brain cell proliferation and neurogenesis due to the presence of active and fast neurogenesis in several regions during its short lifespan. Our main goal was to identify and localize the cells that compose the neurogenic areas throughout the Austrolebias brain. To do this, we used two thymidine halogenated analogs to detect cell proliferation at different survival times: 5-chloro-2'-deoxyuridine (CldU) at 1day and 5-iodo-2'-deoxyuridine (IdU) at 30days. Three types of proliferating cells were identified: I - transient amplifying or fast cycling cells that uptake CldU; II - stem cells or slow cycling cells, that were labeled with both CldU and IdU and did not migrate; and III - migrant cells that uptake IdU. Mapping and 3D-reconstruction of labeled nuclei showed that type I and type II cells were preferentially found close to ventricle walls. Type III cells appeared widespread and migrating in tangential and radial routes. Use of proliferation markers together with Vimentin or Nestin evidenced that type II cells are the putative stem cells that are located at the ventricular lumen. Double label cells with IdU+ and NeuN or HuC/D allowed us identify migrant neurons. Quantitation of labeled nuclei indicates that the proportion of putative stem cells is around 10% in all regions of the brain. This percentage of stem cells suggests the existence of a constant brain cell population in Austrolebias charrua that seems functional to the maintainance of adult neurogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

INFLUENZA-INDUCED UP-REGULATION OF TLR3 IN RESPIRATORY EPITHELIAL CELLS MAY OCCUR THROUGH A POSITIVE FEEDBACK LOOP INVOLVING TYPE I INTERFERON

EPA Science Inventory

Toll-like receptor 3 (TLR3) plays an important role in the host defense responses against viral infections, including Influenza virus infections. Based on our previous observations showing that Influenza infection of respiratory epithelial cells results in an up-regulation of Tol...

Plasticity within stem cell hierarchies in mammalian epithelia.

PubMed

Tetteh, Paul W; Farin, Henner F; Clevers, Hans

2015-02-01

Tissue homeostasis and regeneration are fueled by resident stem cells that have the capacity to self-renew, and to generate all the differentiated cell types that characterize a particular tissue. Classical models of such cellular hierarchies propose that commitment and differentiation occur unidirectionally, with the arrows 'pointing away' from the stem cell. Recent studies, all based on genetic lineage tracing, describe various strategies employed by epithelial stem cell hierarchies to replace damaged or lost cells. While transdifferentiation from one tissue type into another ('metaplasia') appears to be generally forbidden in nonpathological contexts, plasticity within an individual tissue stem cell hierarchy may be much more common than previously appreciated. In this review, we discuss recent examples of such plasticity in selected mammalian epithelia, highlighting the different modes of regeneration and their implications for our understanding of cellular hierarchy and tissue self-renewal. Copyright © 2014 Elsevier Ltd. All rights reserved.

A subset of high Gleason grade prostate carcinomas contain a large burden of prostate cancer syndecan-1 positive stromal cells.

PubMed

Sharpe, Benjamin; Alghezi, Dhafer A; Cattermole, Claire; Beresford, Mark; Bowen, Rebecca; Mitchard, John; Chalmers, Andrew D

2017-05-01

There is a pressing need to identify prognostic and predictive biomarkers for prostate cancer to aid treatment decisions in both early and advanced disease settings. Syndecan-1, a heparan sulfate proteoglycan, has been previously identified as a potential prognostic biomarker by multiple studies at the tissue and serum level. However, other studies have questioned its utility. Anti-Syndecan-1 immunohistochemistry was carried out on 157 prostate tissue samples (including cancerous, adjacent normal tissue, and non-diseased prostate) from three independent cohorts of patients. A population of Syndecan-1 positive stromal cells was identified and the number and morphological parameters of these cells quantified. The identity of the Syndecan-1-positive stromal cells was assessed by multiplex immunofluorescence using a range of common cell lineage markers. Finally, the burden of Syndecan-1 positive stromal cells was tested for association with clinical parameters. We identified a previously unreported cell type which is marked by Syndecan-1 expression and is found in the stroma of prostate tumors and adjacent normal tissue but not in non-diseased prostate. We call these cells Prostate Cancer Syndecan-1 Positive (PCSP) cells. Immunofluorescence analysis revealed that the PCSP cell population did not co-stain with markers of common prostate epithelial, stromal, or immune cell populations. However, morphological analysis revealed that PCSP cells are often elongated and displayed prominent lamellipodia, suggesting they are an unidentified migratory cell population. Analysis of clinical parameters showed that PCSP cells were found with a frequency of 20-35% of all tumors evaluated, but were not present in non-diseased normal tissue. Interestingly, a subset of primary Gleason 5 prostate tumors had a high burden of PCSP cells. The current study identifies PCSP cells as a novel, potentially migratory cell type, which is marked by Syndecan-1 expression and is found in the stroma of prostate carcinomas, adjacent normal tissue, but not in non-diseased prostate. A subset of poor prognosis high Gleason grade 5 tumors had a particularly high PCSP cell burden, suggesting an association between this unidentified cell type and tumor aggressiveness. © 2017 Wiley Periodicals, Inc.

c-Jun activation in Schwann cells protects against loss of sensory axons in inherited neuropathy

PubMed Central

Hantke, Janina; Carty, Lucy; Wagstaff, Laura J.; Turmaine, Mark; Wilton, Daniel K.; Quintes, Susanne; Koltzenburg, Martin; Baas, Frank; Mirsky, Rhona

2014-01-01

Charcot–Marie–Tooth disease type 1A is the most frequent inherited peripheral neuropathy. It is generally due to heterozygous inheritance of a partial chromosomal duplication resulting in over-expression of PMP22. A key feature of Charcot–Marie–Tooth disease type 1A is secondary death of axons. Prevention of axonal loss is therefore an important target of clinical intervention. We have previously identified a signalling mechanism that promotes axon survival and prevents neuron death in mechanically injured peripheral nerves. This work suggested that Schwann cells respond to injury by activating/enhancing trophic support for axons through a mechanism that depends on upregulation of the transcription factor c-Jun in Schwann cells, resulting in the sparing of axons that would otherwise die. As c-Jun orchestrates Schwann cell support for distressed neurons after mechanical injury, we have now asked: do Schwann cells also activate a c-Jun dependent neuron-supportive programme in inherited demyelinating disease? We tested this by using the C3 mouse model of Charcot–Marie–Tooth disease type 1A. In line with our previous findings in humans with Charcot–Marie–Tooth disease type 1A, we found that Schwann cell c-Jun was elevated in (uninjured) nerves of C3 mice. We determined the impact of this c-Jun activation by comparing C3 mice with double mutant mice, namely C3 mice in which c-Jun had been conditionally inactivated in Schwann cells (C3/Schwann cell-c-Jun−/− mice), using sensory-motor tests and electrophysiological measurements, and by counting axons in proximal and distal nerves. The results indicate that c-Jun elevation in the Schwann cells of C3 nerves serves to prevent loss of myelinated sensory axons, particularly in distal nerves, improve behavioural symptoms, and preserve F-wave persistence. This suggests that Schwann cells have two contrasting functions in Charcot–Marie–Tooth disease type 1A: on the one hand they are the genetic source of the disease, on the other, they respond to it by mounting a c-Jun-dependent response that significantly reduces its impact. Because axonal death is a central feature of much nerve pathology it will be important to establish whether an axon-supportive Schwann cell response also takes place in other conditions. Amplification of this axon-supportive mechanism constitutes a novel target for clinical intervention that might be useful in Charcot–Marie–Tooth disease type 1A and other neuropathies that involve axon loss. PMID:25216747

Romidepsin and Lenalidomide in Treating Patients With Previously Untreated Peripheral T-Cell Lymphoma

ClinicalTrials.gov

2018-01-05

Adult Nasal Type Extranodal NK/T-cell Lymphoma; Anaplastic Large Cell Lymphoma; Angioimmunoblastic T-cell Lymphoma; Hepatosplenic T-cell Lymphoma; Peripheral T-cell Lymphoma; Stage I Cutaneous T-cell Non-Hodgkin Lymphoma; Stage IA Mycosis Fungoides/Sezary Syndrome; Stage IB Mycosis Fungoides/Sezary Syndrome; Stage II Cutaneous T-cell Non-Hodgkin Lymphoma; Stage IIA Mycosis Fungoides/Sezary Syndrome; Stage IIB Mycosis Fungoides/Sezary Syndrome; Stage III Cutaneous T-cell Non-Hodgkin Lymphoma; Stage IIIA Mycosis Fungoides/Sezary Syndrome; Stage IIIB Mycosis Fungoides/Sezary Syndrome; Stage IV Cutaneous T-cell Non-Hodgkin Lymphoma; Stage IVA Mycosis Fungoides/Sezary Syndrome; Stage IVB Mycosis Fungoides/Sezary Syndrome

Giant cell lung carcinoma in a man with acquired immunodeficiency syndrome.

PubMed

Kodama, Takahide; Miyazaki, Kunihiko; Satoh, Hiroaki; Hitomi, Shigemi; Ohtsuka, Morio

2009-01-01

A 66-year-old man, who was discovered to have human immunodeficiency virus (HIV) infection 22 months previously and was treated with highly active antiretroviral (HAART) therapy, developed giant cell carcinoma of the lung. In English literature, this is the first case of such cell type of lung cancer during HAART therapy. Since giant cell carcinoma of the lung occurs mainly in elderly men who smoke heavily, there may not be a possibility that the HIV or HAART was causative in our patient.

Interactions between genetic background, insulin resistance and β-cell function.

PubMed

Kahn, S E; Suvag, S; Wright, L A; Utzschneider, K M

2012-10-01

An interaction between genes and the environment is a critical component underlying the pathogenesis of the hyperglycaemia of type 2 diabetes. The development of more sophisticated techniques for studying gene variants and for analysing genetic data has led to the discovery of some 40 genes associated with type 2 diabetes. Most of these genes are related to changes in β-cell function, with a few associated with decreased insulin sensitivity and obesity. Interestingly, using quantitative traits based on continuous measures rather than dichotomous ones, it has become evident that not all genes associated with changes in fasting or post-prandial glucose are also associated with a diagnosis of type 2 diabetes. Identification of these gene variants has provided novel insights into the physiology and pathophysiology of the β-cell, including the identification of molecules involved in β-cell function that were not previously recognized as playing a role in this critical cell. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.

PubMed

Boeva, Valentina; Louis-Brennetot, Caroline; Peltier, Agathe; Durand, Simon; Pierre-Eugène, Cécile; Raynal, Virginie; Etchevers, Heather C; Thomas, Sophie; Lermine, Alban; Daudigeos-Dubus, Estelle; Geoerger, Birgit; Orth, Martin F; Grünewald, Thomas G P; Diaz, Elise; Ducos, Bertrand; Surdez, Didier; Carcaboso, Angel M; Medvedeva, Irina; Deller, Thomas; Combaret, Valérie; Lapouble, Eve; Pierron, Gaelle; Grossetête-Lalami, Sandrine; Baulande, Sylvain; Schleiermacher, Gudrun; Barillot, Emmanuel; Rohrer, Hermann; Delattre, Olivier; Janoueix-Lerosey, Isabelle

2017-09-01

Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.

Oral administration of a Spirulina extract enriched for Braun-type lipoproteins protects mice against influenza A(H1N1) virus infection

USDA-ARS?s Scientific Manuscript database

Previous studies indicate that Immulina, a commercial extract of Arthrospira (Spirulina) platensis, is a potent activator of innate immune cells and that Braun-type lipoproteins (a principal toll-like receptor (TLR) 2 ligand) are the main active components within this product. In the present study, ...

Immune Cells from SR/CR Mice Induce the Regression of Established Tumors in BALB/c and C57BL/6 Mice

PubMed Central

Koch, Janne; Hau, Jann; Pravsgaard Christensen, Jan; Elvang Jensen, Henrik; Bagge Hansen, Morten; Rieneck, Klaus

2013-01-01

Few experimental models are available for the study of natural resistance to cancer. One of them is the SR/CR (spontaneous regression/complete resistance) mouse model in which natural resistance to a variety of cancer types appeared to be inherited in SR/CR strains of BALB/c and C57BL/6 mice. The genetic, cellular, and molecular effector mechanisms in this model are largely unknown, but cells from the innate immune system may play a significant role. In contrast to previous observations, the cancer resistance was limited to S180 sarcoma cancer cells. We were unable to confirm previous observations of resistance to EL-4 lymphoma cells and J774A.1 monocyte-macrophage cancer cells. The cancer resistance against S180 sarcoma cells could be transferred to susceptible non-resistant BALB/c mice as well as C57BL/6 mice after depletion of both CD4+/CD8+ leukocytes and B-cells from SR/CR mice. In the responding recipient mice, the cancer disappeared gradually following infiltration of a large number of polymorphonuclear granulocytes and remarkably few lymphocytes in the remaining tumor tissues. This study confirmed that the in vivo growth and spread of cancer cells depend on a complex interplay between the cancer cells and the host organism. Here, hereditary components of the immune system, most likely the innate part, played a crucial role in this interplay and lead to resistance to a single experimental cancer type. The fact that leukocytes depleted of both CD4+/CD8+ and B cells from the cancer resistant donor mice could be transferred to inhibit S180 cancer cell growth in susceptible recipient mice support the vision of an efficient and adverse event free immunotherapy in future selected cancer types. PMID:23555858

Cell type-selective disease-association of genes under high regulatory load.

PubMed

Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

2015-10-15

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3' UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Optimizing Call Patterns for Landline and Cell Phone Surveys.

PubMed

Reimer, Becky; Roth, Veronica; Montgomery, Robert

2012-01-01

Cell phone surveys have become increasingly popular and researchers have noted major challenges in conducting cost-effective surveys while achieving high response rates. Previous work has shown that calling strategies that maximize both respondent contact and completed interviews for landline surveys may not be the most cost-effective for cell phone surveys. For example, Montgomery, et al. (2011) found important differences between landline and cell samples for best times to call and declines in contact rates after repeated dialing. Using paradata from the 2010 and 2011 National Flu Surveys (sponsored by the Centers for Disease Control and Prevention), we investigate differences in calling outcomes between landline and cell surveys. Specifically, we predict respondent contact and interview completion using logistic regression models that examine the impact of calling on particular days of the week, certain times of the day, number of previous calls, outcomes of previous calls and length of time between calls. We discuss how these differences can be used to increase the likelihood of contacting cooperative respondents and completing interviews for both sample types.

Integrin Expression Regulates Neuroblastoma Attachment and Migration1

PubMed Central

Meyer, Amy; van Golen, Cynthia M.; Kim, Bhumsoo; van Golen, Kenneth L.; Feldman, Eva L.

2004-01-01

Abstract Neuroblastoma (NBL) is the most common malignant disease of infancy, and children with bone metastasis have a mortality rate greater than 90%. Two major classes of proteins, integrins and growth factors, regulate the metastatic process. We have previously shown that tumorigenic NBL cells express higher levels of the type I insulin-like growth factor receptor (IGF-IR) and that β1 integrin expression is inversely proportional to tumorigenic potential in NBL. In the current study, we analyze the effect of β1 integrin and IGF-IR on NBL cell attachment and migration. Nontumorigenic S-cells express high levels of β1 integrin, whereas tumorigenic N-cells express little β1 integrin. Alterations in β1 integrin are due to regulation at the protein level, as translation is decreased in N-type cells. Moreover, inhibition of protein synthesis shows that β1 integrin is degraded more slowly in S-type cells (SHEP) than in N-type cells (SH-SY5Y and IMR32). Inhibition of α5β1 integrin prevents SHEP (but not SH-SY5Y or IMR32) cell attachment to fibronectin and increases SHEP cell migration. Increases in IGF-IR decrease β1 integrin expression, and enhance SHEP cell migration, potentially through increased expression of αvβ3. These data suggest that specific classes of integrins in concert with IGF-IR regulate NBL attachment and migration. PMID:15256055

Cation depletion by the sodium pump in red cells with pathologic cation leaks. Sickle cells and xerocytes.

PubMed Central

Joiner, C H; Platt, O S; Lux, S E

1986-01-01

The mechanism by which sickle cells and xerocytic red cells become depleted of cations in vivo has not been identified previously. Both types of cells exhibit elevated permeabilities to sodium and potassium, in the case of sickle cells, when deoxygenated. The ouabain-insensitive fluxes of sodium and potassium were equivalent, however, in both cell types under these conditions. When incubated 18 hours in vitro, sickle cells lost cations but only when deoxygenated. This cation depletion was blocked by ouabain, removal of external potassium, or pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonate, which blocks the increase in cation permeability induced by deoxygenation. The loss of cation exhibited by oxygenated xerocytes similarly incubated was also blocked by ouabain. These data support the hypothesis that the elevated "passive" cation fluxes of xerocytes and deoxygenated sickle cells are not directly responsible for cation depletion of these cells; rather, these pathologic leaks interact with the sodium pump to produce a net loss of cellular cation. PMID:2430999

Cation depletion by the sodium pump in red cells with pathologic cation leaks. Sickle cells and xerocytes.

PubMed

Joiner, C H; Platt, O S; Lux, S E

1986-12-01

The mechanism by which sickle cells and xerocytic red cells become depleted of cations in vivo has not been identified previously. Both types of cells exhibit elevated permeabilities to sodium and potassium, in the case of sickle cells, when deoxygenated. The ouabain-insensitive fluxes of sodium and potassium were equivalent, however, in both cell types under these conditions. When incubated 18 hours in vitro, sickle cells lost cations but only when deoxygenated. This cation depletion was blocked by ouabain, removal of external potassium, or pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonate, which blocks the increase in cation permeability induced by deoxygenation. The loss of cation exhibited by oxygenated xerocytes similarly incubated was also blocked by ouabain. These data support the hypothesis that the elevated "passive" cation fluxes of xerocytes and deoxygenated sickle cells are not directly responsible for cation depletion of these cells; rather, these pathologic leaks interact with the sodium pump to produce a net loss of cellular cation.

MHCII-Mediated Dialog between Group 2 Innate Lymphoid Cells and CD4+ T Cells Potentiates Type 2 Immunity and Promotes Parasitic Helminth Expulsion

PubMed Central

Oliphant, Christopher J.; Hwang, You Yi; Walker, Jennifer A.; Salimi, Maryam; Wong, See Heng; Brewer, James M.; Englezakis, Alexandros; Barlow, Jillian L.; Hams, Emily; Scanlon, Seth T.; Ogg, Graham S.; Fallon, Padraic G.; McKenzie, Andrew N.J.

2014-01-01

Summary Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity. PMID:25088770

Formation and regression of the corpus luteum of the American alligator

USGS Publications Warehouse

Guillette, L.J.; Woodward, A.R.; You-Xiang, Q.; Cox, M.C.; Matter, J.H.; Gross, T.S.

1995-01-01

Luteal morphology of the American alligator is unique when compared to other reptiles but is similar to that of its phylogenetic relatives, the birds. The theca is extensively hypertrophied, but the granulosa never fills the cavity formed following the ovulation of the ovum. The formation of the corpus luteum (CL) is correlated with elevated plasma progesterone concentrations, which decline dramatically after oviposition with the onset of luteolysis. Unlike those of most other reptiles, the central luteal cell mass is composed of two cell types; one presumably is derived from the granulosa, whereas the other is from the theca interna. Both cell types are present throughout gravidity but only one cell type is seen during mid to late luteolysis. A significant decline in luteal volume occurs following oviposition and continues throughout the post-oviposition period. The fastest decline in luteal volume occurs in the month immediately after oviposition; this rate then slows. Luteolysis appears to continue for a year or more following oviposition, as distinct structures of luteal origin can still be identified in animals 9 months after oviposition. The size of persistent CL can be used to determine whether a given female oviposited during the previous nesting season. Females with CL having volumes greater than 0.2 cm2 or CL diameters greater than 0.4 cm were active the previous season. 

Detection of alkaline phosphatase in canine cells previously stained with Wright-Giemsa and its utility in differentiating osteosarcoma from other mesenchymal tumors.

PubMed

Ryseff, Julia K; Bohn, Andrea A

2012-09-01

Osteosarcoma (OSA) is a common primary bone tumor in dogs. Demonstration of alkaline phosphatase (ALP) reactivity by tumor cells on unstained slides is useful in differentiating osteosarcoma from other types of sarcoma. However, unstained slides are not always available. The objectives of this study were to evaluate the diagnostic utility of detecting ALP expression in differentiating osteosarcoma from other sarcomas in dogs using cytologic material previously stained with Wright-Giemsa stain and to assess the sensitivity and specificity of ALP expression for diagnosing osteosarcoma using a specific protocol. Archived aspirates of histologically confirmed sarcomas in dogs that had been previously stained with Wright-Giemsa stain were treated with 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as a substrate for ALP. Cells were evaluated for expression of ALP after incubation with BCIP/NBT for 1 hour. Sensitivity and specificity of ALP expression for diagnosis of OSA were calculated. In samples from 83 dogs, cells from 15/17 OSAs and from 4/66 tumors other than OSA (amelanotic melanoma, gastrointestinal stromal tumor, collision tumor, and anaplastic sarcoma) expressed ALP. Sensitivity and specificity of ALP expression detected using BCIP/NBT substrate applied to cells previously stained with Wright-Giemsa stain for OSA were 88 and 94%, respectively. ALP expression detected using BCIP/NBT substrate applied to previously stained cells is useful in differentiating canine OSA from other mesenchymal neoplasms. © 2012 American Society for Veterinary Clinical Pathology.

Enhanced Cell-Specific Ablation in Zebrafish Using a Triple Mutant of Escherichia Coli Nitroreductase

PubMed Central

Mathias, Jonathan R.; Zhang, Zhanying; Saxena, Meera T.

2014-01-01

Abstract Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology. PMID:24428354

Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.

PubMed

Mathias, Jonathan R; Zhang, Zhanying; Saxena, Meera T; Mumm, Jeff S

2014-04-01

Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology.

Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles

PubMed Central

Coulter, Jonathan A; Jain, Suneil; Butterworth, Karl T; Taggart, Laura E; Dickson, Glenn R; McMahon, Stephen J; Hyland, Wendy B; Muir, Mark F; Trainor, Coleman; Hounsell, Alan R; O’Sullivan, Joe M; Schettino, Giuseppe; Currell, Fred J; Hirst, David G; Prise, Kevin M

2012-01-01

Background This follow-up study aims to determine the physical parameters which govern the differential radiosensitization capacity of two tumor cell lines and one immortalized normal cell line to 1.9 nm gold nanoparticles. In addition to comparing the uptake potential, localization, and cytotoxicity of 1.9 nm gold nanoparticles, the current study also draws on comparisons between nanoparticle size and total nanoparticle uptake based on previously published data. Methods We quantified gold nanoparticle uptake using atomic emission spectroscopy and imaged intracellular localization by transmission electron microscopy. Cell growth delay and clonogenic assays were used to determine cytotoxicity and radiosensitization potential, respectively. Mechanistic data were obtained by Western blot, flow cytometry, and assays for reactive oxygen species. Results Gold nanoparticle uptake was preferentially observed in tumor cells, resulting in an increased expression of cleaved caspase proteins and an accumulation of cells in sub G1 phase. Despite this, gold nanoparticle cytotoxicity remained low, with immortalized normal cells exhibiting an LD50 concentration approximately 14 times higher than tumor cells. The surviving fraction for gold nanoparticle-treated cells at 3 Gy compared with that of untreated control cells indicated a strong dependence on cell type in respect to radiosensitization potential. Conclusion Gold nanoparticles were most avidly endocytosed and localized within cytoplasmic vesicles during the first 6 hours of exposure. The lack of significant cytotoxicity in the absence of radiation, and the generation of gold nanoparticle-induced reactive oxygen species provide a potential mechanism for previously reported radiosensitization at megavoltage energies. PMID:22701316

Intercellular junctions between palisade nerve endings and outer root sheath cells of rat vellus hairs.

PubMed

Kaidoh, T; Inoué, T

2000-05-15

Hair follicles have a longitudinal set of sensory nerve endings called palisade nerve endings (PN). We examined the junctional structures between the PN and outer root sheath (ORS) cells of hair follicles in the rat external ear. Transmission electron microscopy of serial thin sections showed that the processes of the ORS cells penetrated the basal lamina of the hair follicle, forming intercellular junctions with the PN (PN-ORS junctions). Two types of junctions were found: junctions between nerve endings and ORS cells (N-ORS junctions) and those between Schwann cell processes and ORS cells (S-ORS junctions). The N-ORS junctions had two subtypes: 1) a short process or small eminence of the ORS cell was attached to the nerve ending (type I); or 2) a process of the ORS cell was invaginated into the nerve ending (type II). The S-ORS junctions also had two subtypes: 1) a short process or small eminence of the ORS cell was abutted on the Schwann cell process (type I); or 2) a process of the ORS cell was invaginated into the Schwann cell process (type II). Vesicles, coated pits, coated vesicles, and endosomes were sometimes seen in nerve endings, Schwann cells, and ORS cells near the junctions. Computer-aided reconstruction of the serial thin sections displayed the three-dimensional structure of these junctions. These results suggested that the PN-ORS junctions provided direct relationships between the PN and ORS in at least four different patterns. The discovery of these junctions shows the PN-ORS relationship to be closer than previously realized. We speculate that these junctions may have roles in attachment of the PN to the ORS, contributing to increases in the sensitivity of the PN, and in chemical signaling between the PN and ORS.

Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors.

PubMed

Galli, Uwe M; Sauter, Marlies; Lecher, Bernd; Maurer, Simone; Herbst, Hermann; Roemer, Klaus; Mueller-Lantzsch, Nikolaus

2005-04-28

Germ cell tumors (GCTs) are among the most common malignancies in young men. We have previously documented that patients with GCT frequently produce serum antibodies directed against proteins encoded by human endogenous retrovirus (HERV) type K sequences. Transcripts originating from the env gene of HERV-K, including the rec-relative of human immunodeficiency virus rev, are highly expressed in GCTs. We report here that mice that inducibly express HERV-K rec show a disturbed germ cell development and may exhibit, by 19 months of age, changes reminiscent of carcinoma in situ, the predecessor lesion of classic seminoma in humans. This provides the first direct evidence that the expression of a human endogenous retroviral gene previously established as a marker in human germ cell tumors may contribute to organ-specific tumorigenesis in a transgenic mouse model.

Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2012-10-01

In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

The Human Airway Epithelial Basal Cell Transcriptome

PubMed Central

Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

2011-01-01

Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium. PMID:21572528

Differential Sox10 Genomic Occupancy in Myelinating Glia

PubMed Central

Lopez-Anido, Camila; Sun, Guannan; Koenning, Matthias; Srinivasan, Rajini; Hung, Holly A.; Emery, Ben; Keles, Sunduz; Svaren, John

2015-01-01

Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells, and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. PMID:25974668

Physical effects of DCNQI derivatives doping as an N type organic semiconductor in organic photovoltaic cell performance.

PubMed

Lee, Joo Hyung; Oh, Se Young

2014-08-01

In the previous work, we have reported that organic photovoltaic (OPV) cells using DMDCNQI as an n-type second dopant material showed a high power conversion efficiency (PCE). In the present work, we have synthesized a novel DHDCNQI with long alkyl chains to improve the compatibility between the DHDCNQI dopant molecule and host P3HT polymer. We have fabricated OPV cells consisting of ITO/PEDOT:PSS/P3HT:PCBM:DHDCNQI/Al. We have investigated the characteristics of theses OPV cells using DCNQI derivative dopants from the measurements of the incident photon-to-current collection efficiency and photocurrent. The OPV cell using 3 wt% DHDCNQI exhibited a high PCE of 3.29% due to the high charge separation efficiency, good compatibility and low trap site effect.

An Improved Targeted cAMP Sensor to Study the Regulation of Adenylyl Cyclase 8 by Ca2+ Entry through Voltage-Gated Channels

PubMed Central

Everett, Katy L.; Cooper, Dermot M. F.

2013-01-01

Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes. PMID:24086669

Differences in beta-cell function and insulin secretion in Black vs. White obese adolescents: Do incretin hormones play a role?

USDA-ARS?s Scientific Manuscript database

Black youth are at higher risk for type 2 diabetes (T2D) than their White peers. Previously we demonstrated that for the same degree of insulin sensitivity, Black youth have an upregulated beta-cell function and insulin hypersecretion, in response to intravenous (IV) glucose, compared with Whites. T...

Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas

PubMed Central

Rovira, Meritxell; Scott, Sherri-Gae; Liss, Andrew S.; Jensen, Jan; Thayer, Sarah P.; Leach, Steven D.

2009-01-01

The question of whether dedicated progenitor cells exist in adult vertebrate pancreas remains controversial. Centroacinar cells and terminal duct (CA/TD) cells lie at the junction between peripheral acinar cells and the adjacent ductal epithelium, and are frequently included among cell types proposed as candidate pancreatic progenitors. However these cells have not previously been isolated in a manner that allows formal assessment of their progenitor capacities. We have found that a subset of adult CA/TD cells are characterized by high levels of ALDH1 enzymatic activity, related to high-level expression of both Aldh1a1 and Aldh1a7. This allows their isolation by FACS using a fluorogenic ALDH1 substrate. FACS-isolated CA/TD cells are relatively depleted of transcripts associated with differentiated pancreatic cell types. In contrast, they are markedly enriched for transcripts encoding Sca1, Sdf1, c-Met, Nestin, and Sox9, markers previously associated with progenitor populations in embryonic pancreas and other tissues. FACS-sorted CA/TD cells are uniquely able to form self-renewing “pancreatospheres” in suspension culture, even when plated at clonal density. These spheres display a capacity for spontaneous endocrine and exocrine differentiation, as well as glucose-responsive insulin secretion. In addition, when injected into cultured embryonic dorsal pancreatic buds, these adult cells display a unique capacity to contribute to both the embryonic endocrine and exocrine lineages. Finally, these cells demonstrate dramatic expansion in the setting of chronic epithelial injury. These findings suggest that CA/TD cells are indeed capable of progenitor function and may contribute to the maintenance of tissue homeostasis in adult mouse pancreas. PMID:20018761

Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation.

PubMed

Chanda, Soham; Marro, Samuele; Wernig, Marius; Südhof, Thomas C

2013-10-08

Recent studies suggest that induced neuronal (iN) cells that are directly transdifferentiated from nonneuronal cells provide a powerful opportunity to examine neuropsychiatric diseases. However, the validity of using this approach to examine disease-specific changes has not been demonstrated. Here, we analyze the phenotypes of iN cells that were derived from murine embryonic fibroblasts cultured from littermate wild-type and mutant mice carrying the autism-associated R704C substitution in neuroligin-3. We show that neuroligin-3 R704C-mutant iN cells exhibit a large and selective decrease in AMPA-type glutamate receptor-mediated synaptic transmission without changes in NMDA-type glutamate receptor- or in GABAA receptor-mediated synaptic transmission. Thus, the synaptic phenotype observed in R704C-mutant iN cells replicates the previously observed phenotype of R704C-mutant neurons. Our data show that the effect of the R704C mutation is applicable even to neurons transdifferentiated from fibroblasts and constitute a proof-of-concept demonstration that iN cells can be used for cellular disease modeling.

Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation

PubMed Central

Chanda, Soham; Marro, Samuele; Wernig, Marius; Südhof, Thomas C.

2013-01-01

Recent studies suggest that induced neuronal (iN) cells that are directly transdifferentiated from nonneuronal cells provide a powerful opportunity to examine neuropsychiatric diseases. However, the validity of using this approach to examine disease-specific changes has not been demonstrated. Here, we analyze the phenotypes of iN cells that were derived from murine embryonic fibroblasts cultured from littermate wild-type and mutant mice carrying the autism-associated R704C substitution in neuroligin-3. We show that neuroligin-3 R704C-mutant iN cells exhibit a large and selective decrease in AMPA-type glutamate receptor-mediated synaptic transmission without changes in NMDA-type glutamate receptor- or in GABAA receptor-mediated synaptic transmission. Thus, the synaptic phenotype observed in R704C-mutant iN cells replicates the previously observed phenotype of R704C-mutant neurons. Our data show that the effect of the R704C mutation is applicable even to neurons transdifferentiated from fibroblasts and constitute a proof-of-concept demonstration that iN cells can be used for cellular disease modeling. PMID:24046374

Transcriptome analysis of cattle muscle identifies potential markers for skeletal muscle growth rate and major cell types.

PubMed

Guo, Bing; Greenwood, Paul L; Cafe, Linda M; Zhou, Guanghong; Zhang, Wangang; Dalrymple, Brian P

2015-03-13

This study aimed to identify markers for muscle growth rate and the different cellular contributors to cattle muscle and to link the muscle growth rate markers to specific cell types. The expression of two groups of genes in the longissimus muscle (LM) of 48 Brahman steers of similar age, significantly enriched for "cell cycle" and "ECM (extracellular matrix) organization" Gene Ontology (GO) terms was correlated with average daily gain/kg liveweight (ADG/kg) of the animals. However, expression of the same genes was only partly related to growth rate across a time course of postnatal LM development in two cattle genotypes, Piedmontese x Hereford (high muscling) and Wagyu x Hereford (high marbling). The deposition of intramuscular fat (IMF) altered the relationship between the expression of these genes and growth rate. K-means clustering across the development time course with a large set of genes (5,596) with similar expression profiles to the ECM genes was undertaken. The locations in the clusters of published markers of different cell types in muscle were identified and used to link clusters of genes to the cell type most likely to be expressing them. Overall correspondence between published cell type expression of markers and predicted major cell types of expression in cattle LM was high. However, some exceptions were identified: expression of SOX8 previously attributed to muscle satellite cells was correlated with angiogenesis. Analysis of the clusters and cell types suggested that the "cell cycle" and "ECM" signals were from the fibro/adipogenic lineage. Significant contributions to these signals from the muscle satellite cells, angiogenic cells and adipocytes themselves were not as strongly supported. Based on the clusters and cell type markers, sets of five genes predicted to be representative of fibro/adipogenic precursors (FAPs) and endothelial cells, and/or ECM remodelling and angiogenesis were identified. Gene sets and gene markers for the analysis of many of the major processes/cell populations contributing to muscle composition and growth have been proposed, enabling a consistent interpretation of gene expression datasets from cattle LM. The same gene sets are likely to be applicable in other cattle muscles and in other species.

Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.

PubMed

Schweizer, Patrick A; Darche, Fabrice F; Ullrich, Nina D; Geschwill, Pascal; Greber, Boris; Rivinius, Rasmus; Seyler, Claudia; Müller-Decker, Karin; Draguhn, Andreas; Utikal, Jochen; Koenen, Michael; Katus, Hugo A; Thomas, Dierk

2017-10-16

Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties. hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes. hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca 2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed. We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.

Expression of alveolar type II cell markers in acinar adenocarcinomas and adenoid cystic carcinomas arising from segmental bronchi. A study in a heterotopic bronchogenic carcinoma model in dogs.

PubMed Central

TenHave-Opbroek, A. A.; Hammond, W. G.; Benfield, J. R.; Teplitz, R. L.; Dijkman, J. H.

1993-01-01

The type II alveolar epithelial cell is one of two pluripotential stem cell phenotypes in normal mammalian lung morphogenesis; cells manifesting this phenotype have been found to constitute bronchioloalveolar regions of canine adenocarcinomas. We now studied type II cell expression in canine acinar adenocarcinomas and adenoid cystic (bronchial gland) carcinomas, using the same bronchogenic carcinoma model (subcutaneous bronchial autografts treated with 3-methylcholanthrene). Distinctive features of type II cells are the approximately cuboid cell shape, large and roundish nucleus, immunofluorescent staining of the cytoplasm for the surfactant protein SP-A, and presence of multilamellar bodies or their precursory forms. Cells with these type II cell characteristics were found in the basal epithelial layer of all tumor lesions and in upper layers as far as the lumen, singly or in clusters; they were also found in early invasive carcinomatous lesions but not in bronchial glands or bronchial epithelium before carcinogen exposure. Immunoblots of tumor homogenates showed reactive proteins within size classes of SP-A (28 to 36 kd) or its dimeric form (56 to 72 kd). These findings and those previously reported are consistent with the concept that chemical carcinogenesis in the adult bronchial epithelium may lead to type II cell carcinomas of varying glandular (acinar, adenoidcystic or bronchioloalveolar) growth patterns. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 PMID:8386445

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs

PubMed Central

Jima, Dereje D.; Zhang, Jenny; Jacobs, Cassandra; Richards, Kristy L.; Dunphy, Cherie H.; Choi, William W. L.; Yan Au, Wing; Srivastava, Gopesh; Czader, Magdalena B.; Rizzieri, David A.; Lagoo, Anand S.; Lugar, Patricia L.; Mann, Karen P.; Flowers, Christopher R.; Bernal-Mizrachi, Leon; Naresh, Kikkeri N.; Evens, Andrew M.; Gordon, Leo I.; Luftig, Micah; Friedman, Daphne R.; Weinberg, J. Brice; Thompson, Michael A.; Gill, Javed I.; Liu, Qingquan; How, Tam; Grubor, Vladimir; Gao, Yuan; Patel, Amee; Wu, Han; Zhu, Jun; Blobe, Gerard C.; Lipsky, Peter E.; Chadburn, Amy

2010-01-01

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions. PMID:20733160

Receptive Field Vectors of Genetically-Identified Retinal Ganglion Cells Reveal Cell-Type-Dependent Visual Functions

PubMed Central

Katz, Matthew L.; Viney, Tim J.; Nikolic, Konstantin

2016-01-01

Sensory stimuli are encoded by diverse kinds of neurons but the identities of the recorded neurons that are studied are often unknown. We explored in detail the firing patterns of eight previously defined genetically-identified retinal ganglion cell (RGC) types from a single transgenic mouse line. We first introduce a new technique of deriving receptive field vectors (RFVs) which utilises a modified form of mutual information (“Quadratic Mutual Information”). We analysed the firing patterns of RGCs during presentation of short duration (~10 second) complex visual scenes (natural movies). We probed the high dimensional space formed by the visual input for a much smaller dimensional subspace of RFVs that give the most information about the response of each cell. The new technique is very efficient and fast and the derivation of novel types of RFVs formed by the natural scene visual input was possible even with limited numbers of spikes per cell. This approach enabled us to estimate the 'visual memory' of each cell type and the corresponding receptive field area by calculating Mutual Information as a function of the number of frames and radius. Finally, we made predictions of biologically relevant functions based on the RFVs of each cell type. RGC class analysis was complemented with results for the cells’ response to simple visual input in the form of black and white spot stimulation, and their classification on several key physiological metrics. Thus RFVs lead to predictions of biological roles based on limited data and facilitate analysis of sensory-evoked spiking data from defined cell types. PMID:26845435

D-type Cyclins are important downstream effectors of cytokine signaling that regulate the proliferation of normal and neoplastic mammary epithelial cells

PubMed Central

Zhang, Qian; Sakamoto, Kazuhito; Wagner, Kay-Uwe

2013-01-01

In response to the ligand-mediated activation of cytokine receptors, cells decide whether to proliferate or to undergo differentiation. D-type Cyclins (Cyclin D1, D2, or D3) and their associated Cyclin-dependent Kinases (CDK4, CDK6) connect signals from cytokines to the cell cycle machinery, and they propel cells through the G1 restriction point and into the S phase, after which growth factor stimulation is no longer essential to complete cell division. D-type Cyclins are upregulated in many human malignancies including breast cancer to promote an uncontrolled proliferation of cancer cells. After summarizing important aspects of the cytokine-mediated transcriptional regulation and the posttranslational modification of D-type Cyclins, this review will highlight the physiological significance of these cell cycle regulators during normal mammary gland development as well as the initiation and promotion of breast cancer. Although the vast majority of published reports focus almost exclusively on the role of Cyclin D1 in breast cancer, we summarize here previous and recent findings that demonstrate an important contribution of the remaining two members of this Cyclin family, in particular Cyclin D3, for the growth of ErbB2-associated breast cancer cells in humans and in mouse models. New data from genetically engineered models as well as the pharmacological inhibition of CDK4/6 suggest that targeting the combined functions of D-type Cyclins could be a suitable strategy for the treatment of ErbB2-positive and potentially other types of breast cancer. PMID:23562856

P-selectin mediates Ca(2+)-dependent adhesion of activated platelets to many different types of leukocytes: detection by flow cytometry.

PubMed

de Bruijne-Admiraal, L G; Modderman, P W; Von dem Borne, A E; Sonnenberg, A

1992-07-01

Previous studies have shown that thrombin-activated platelets interact through the P-selectin with neutrophils and monocytes. To identify other types of leukocytes capable of such an interaction, eosinophils, basophils, and lymphocytes were isolated from whole blood. Binding of these cells to activated platelets was examined in a double immunofluorescence assay and the results show that activated platelets not only bind to neutrophils and monocytes, but also to eosinophils, basophils, and subpopulations of T lymphocytes. Using monoclonal antibodies (MoAbs) specific for subsets of T cells, we could further demonstrate that the T cells which bind activated platelets are natural killer (NK) cells and an undefined subpopulation of CD4+ and CD8+ cells. All these interactions were dependent on divalent cations and were completely inhibited by an MoAb against P-selectin. Thus, P-selectin mediates the binding of activated platelets to many different types of leukocytes. Studies with leukocytes treated with proteases or neuraminidase have shown that the structures recognized by P-selectin are glycoproteins carrying sialic acid residues. Because the loss of binding of activated platelets to neuraminidase-treated neutrophils was almost complete, but only partial to treated eosinophils, basophils, and monocytes, the latter cell types may have different P-selectin ligands in addition to those present on neutrophils. We found that two previously identified ligands for P-selectin, the oligosaccharides Le(x) and sialyl-Le(x), had little or no inhibitory effect on adhesion of activated platelets to leukocytes and that binding was not inhibited by MoAbs against these oligosaccharides. In addition, there was no correlation between the expression of Le(x) on several cell types and their capacity to bind activated platelets. In contrast, the expression of sialyl-Le(x) on cells was almost perfectly correlated with their ability to bind activated platelets. Thus, while Le(x) cannot be a major ligand for P-selectin, a possible role for sialyl-Le(x) in P-selectin-mediated adhesion processes cannot be dismissed. Finally, activated platelets were found to bind normally to monocytes and neutrophils of patients with paroxysmal nocturnal hemoglobulinuria (PNH) and to neutrophils from which phosphatidyl inositol (PI)-linked proteins had been removed by glycosylphosphatidyl inositol-specific phospholipase C (GPI-PLC) digestion. This suggests that at least part of the P-selectin ligands on these cells are not GPI-anchored.

Neurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells

PubMed Central

Wen, Xianjie; Xu, Shiyuan; Liu, Hongzhen; Zhang, Quinguo; Liang, Hua; Yang, Chenxiang; Wang, Hanbing

2013-01-01

There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca2+ ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation. PMID:23658789

Neuroendocrine-type prostatic adenocarcinoma with microsatellite instability in a patient with lynch syndrome.

PubMed

Wagner, David G; Gatalica, Zoran; Lynch, Henry T; Kohl, Shane; Johansson, Sonny L; Lele, Subodh M

2010-12-01

Lynch syndrome is an autosomal-dominant cancer syndrome that can be identified with microsatellite instability molecular tests or immunohistochemical stains on pathologic material from patients who meet the Amsterdam Criteria II. The development of prostatic carcinoma in situ or invasive small cell carcinoma (SCC) of the prostate has not been previously reported in a patient with this syndrome. In this report, an 87-year-old White man with the Lynch syndrome had a prostate biopsy that revealed a mixed high-grade conventional adenocarcinoma and SCC of the prostate with high-grade prostatic intraepithelial neoplasia of the small cell neuroendocrine-type (HGPIN-NE), all showing MSH2 microsatellite instability and loss of MSH2 expression, a finding not previously published. These findings suggest that HGPIN-NE is a precursor of invasive SCC and also that prostatic SCC can develop in a patient with the Lynch syndrome.

The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation.

PubMed

Holmes, Amie L; Joyce, Kellie; Xie, Hong; Falank, Carolyne; Hinz, John M; Wise, John Pierce

2014-04-01

Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation. Copyright © 2014 Elsevier B.V. All rights reserved.

Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

PubMed Central

2012-01-01

Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition. PMID:22694981

Myostatin inhibition induces muscle fibre hypertrophy prior to satellite cell activation.

PubMed

Wang, Qian; McPherron, Alexandra C

2012-05-01

Muscle fibres are multinucleated post-mitotic cells that can change dramatically in size during adulthood. It has been debated whether muscle fibre hypertrophy requires activation and fusion of muscle stem cells, the satellite cells. Myostatin (MSTN) is a negative regulator of skeletal muscle growth during development and in the adult, and MSTN inhibition is therefore a potential therapy for muscle wasting diseases, some of which are associated with a depletion of satellite cells. Conflicting results have been obtained in previous analyses of the role of MSTN on satellite cell quiescence. Here, we inhibited MSTN in adult mice with a soluble activin receptor type IIB and analysed the incorporation of new nuclei using 5-bromo-2-deoxyuridine (BrdU) labelling by isolating individual myofibres. We found that satellite cells are activated by MSTN inhibition. By varying the dose and time course for MSTN inhibition, however, we found that myofibre hypertrophy precedes the incorporation of new nuclei, and that the overall number of new nuclei is relatively low compared to the number of total myonuclei. These results reconcile some of the previous work obtained by other methods. In contrast with previous reports, we also found that Mstn null mice do not have increased satellite cell numbers during adulthood and are not resistant to sarcopaenia. Our results support a previously proposed model of hypertrophy in which hypertrophy can precede satellite cell activation. Studies of the metabolic and functional effects of postnatal MSTN inhibition are needed to determine the consequences of increasing the cytoplasm/myonuclear ratio after MSTN inhibition.

Curcumin reverses T cell-mediated adaptive immune dysfunctions in tumor-bearing hosts.

PubMed

Bhattacharyya, Sankar; Md Sakib Hossain, Dewan; Mohanty, Suchismita; Sankar Sen, Gouri; Chattopadhyay, Sreya; Banerjee, Shuvomoy; Chakraborty, Juni; Das, Kaushik; Sarkar, Diptendra; Das, Tanya; Sa, Gaurisankar

2010-07-01

Immune dysfunction is well documented during tumor progression and likely contributes to tumor immune evasion. CD8(+) cytotoxic T lymphocytes (CTLs) are involved in antigen-specific tumor destruction and CD4(+) T cells are essential for helping this CD8(+) T cell-dependent tumor eradication. Tumors often target and inhibit T-cell function to escape from immune surveillance. This dysfunction includes loss of effector and memory T cells, bias towards type 2 cytokines and expansion of T regulatory (Treg) cells. Curcumin has previously been shown to have antitumor activity and some research has addressed the immunoprotective potential of this plant-derived polyphenol in tumor-bearing hosts. Here we examined the role of curcumin in the prevention of tumor-induced dysfunction of T cell-based immune responses. We observed severe loss of both effector and memory T-cell populations, downregulation of type 1 and upregulation of type 2 immune responses and decreased proliferation of effector T cells in the presence of tumors. Curcumin, in turn, prevented this loss of T cells, expanded central memory T cell (T(CM))/effector memory T cell (T(EM)) populations, reversed the type 2 immune bias and attenuated the tumor-induced inhibition of T-cell proliferation in tumor-bearing hosts. Further investigation revealed that tumor burden upregulated Treg cell populations and stimulated the production of the immunosuppressive cytokines transforming growth factor (TGF)-beta and IL-10 in these cells. Curcumin, however, inhibited the suppressive activity of Treg cells by downregulating the production of TGF-beta and IL-10 in these cells. More importantly, curcumin treatment enhanced the ability of effector T cells to kill cancer cells. Overall, our observations suggest that the unique properties of curcumin may be exploited for successful attenuation of tumor-induced suppression of cell-mediated immune responses.

Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms.

PubMed

Clark, Alex E; Sabalza, Maite; Gordts, Philip L S M; Spector, Deborah H

2018-03-15

Human cytomegalovirus (HCMV) is the top viral cause of birth defects worldwide, and current therapies have high toxicity. We previously reported that the mTOR-independent autophagy-inducing disaccharide trehalose inhibits HCMV replication in multiple cell types. Here, we examine the mechanism of inhibition and introduce the autophagy inducer SMER28 as an additional inhibitor of HCMV acting through a different mechanism. We find that trehalose induces vacuolation and acidification of vacuoles and that debris, including debris with an appearance consistent with that of abnormal virions, is present in multivesicular bodies. Trehalose treatment increased the levels of Rab7, a protein required for lysosomal biogenesis and fusion, and slightly decreased the levels of Rab11, which is associated with recycling endosomes. We also present evidence that trehalose can promote autophagy without altering cellular glucose uptake. We show that SMER28 inhibits HCMV at the level of early protein production and interferes with viral genome replication in a cell type-dependent fashion. Finally, we show that SMER28 treatment does not cause the vacuolation, acidification, or redistribution of Rab7 associated with trehalose treatment and shows only a modest and cell type-dependent effect on autophagy. We propose a model in which the reciprocal effects on Rab7 and Rab11 induced by trehalose contribute to the redirection of enveloped virions from the plasma membrane to acidified compartments and subsequent degradation, and SMER28 treatment results in decreased expression levels of early and late proteins, reducing the number of virions produced without the widespread vacuolation characteristic of trehalose treatment. IMPORTANCE There is a need for less toxic HCMV antiviral drugs, and modulation of autophagy to control viral infection is a new strategy that takes advantage of virus dependence on autophagy inhibition. The present study extends our previous work on trehalose by showing a possible mechanism of action and introduces another autophagy-inducing compound, SMER28, that is effective against HCMV in several cell types. The mechanism by which trehalose induces autophagy is currently unknown, although our data show that trehalose does not inhibit cellular glucose uptake in cells relevant for HCMV replication but instead alters virion degradation by promoting acidic vacuolization. The comparison of our cell types and those used by others highlights the cell type-dependent nature of studying autophagy. Copyright © 2018 American Society for Microbiology.

Streptococcus mutans in a Wild, Sucrose-Eating Rat Population

PubMed Central

Coykendall, Alan L.; Specht, Patricia A.; Samol, Harry H.

1974-01-01

Streptococcus mutans, an organism implicated in dental caries and not previously found outside of man and certain laboratory animals, was isolated from the mouths of wild rats which ate sugar cane. The strains isolated fermented mannitol and sorbitol, and failed to grow in 6.5% NaCl or at 45 C. They formed in vitro plaques on nichrome wires when grown in sucrose broth. They also stored intracellular polysaccharide which could be catabolized by washed, resting cells. Deoxyribonucleic acid-deoxyribonucleic acid reassociations revealed two genetic types. One type shared extensive deoxyribonucleic acid base sequences with S. mutans strains HS6 and OMZ61, two members of a genetic type found in man and laboratory hamsters. The other type seemed unrelated to any S. mutans genetic type previously encountered. It is concluded that the ecological triad of tooth-sucrose-S. mutans is not a phenomenon unique to man and experimental animals. Images PMID:4601769

Effects of acidic amino acid antagonists upon the spectral properties of carp horizontal cells: circuitry of the outer retina.

PubMed

Mangel, S C; Ariel, M; Dowling, J E

1985-11-01

The acidic amino acid receptor antagonists, alpha-methylglutamate and alpha-aminoadipate, were applied to the carp retina to study their effects upon the spectral properties of horizontal cells and to elucidate the synaptic connections between horizontal cells and cones. Application of these antagonists strongly hyperpolarized the L-type cone horizontal cells and reduced the responses of these horizontal cells to red light more than to blue light. Application of Co2+ ions to the retina, a procedure which decreases transmitter release, also hyperpolarized the L-type cone horizontal cells but reduced the response of these horizontal cells to red and blue lights equally. These results suggest that red- or long wavelength-sensitive cones release a different transmitter onto L-type cone horizontal cells than do short wavelength-sensitive cones. Application of the acidic amino acid antagonists also revealed details of the feedback pathway from L-type cone horizontal cells to cones. Previous studies have shown that feedback varies directly with stimulus size and that the effects of feedback on the responses of cones are observed as a transient waveform at response onset (a large, hyperpolarizing potential that is quickly followed by a smaller plateau potential). Application of the acidic amino acid antagonists at a dose which partially hyperpolarized the horizontal cells selectively enhanced the response of the cells to blue lights, when full field, and not spot, stimuli were used. The antagonists also eliminated the transient at response onset. These findings are consistent with the presence of a feedback pathway from L-type cone horizontal cells to short wavelength cones but not to long (red-sensitive) cones.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Hyejin; Yoon, Min-Sik; Ryu, Kwon-Yul, E-mail: kyryu@uos.ac.kr

Highlights: •Proliferation capacity of Ubc{sup −/−} FLCs was reduced during culture in vitro. •Ubc is required for proliferation of both hepatocytes and bipotent FLEPCs. •Bipotent FLEPCs exhibit highest Ubc transcription and proliferation capacity. •Cell types responsible for Ubc{sup −/−} fetal liver developmental defect were identified. -- Abstract: We have previously demonstrated that disruption of polyubiquitin gene Ubc leads to mid-gestation embryonic lethality most likely due to a defect in fetal liver development, which can be partially rescued by ectopic expression of Ub. In a previous study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoieticmore » system. We confirmed that Ubc{sup −/−} embryonic lethality could not be attributed to impaired function of hematopoietic stem cells, which raises the question of whether or not FLECs such as hepatocytes and bile duct cells, the most abundant cell types in the liver, are affected by disruption of Ubc and contribute to embryonic lethality. To answer this, we isolated FLCs from E13.5 embryos and cultured them in vitro. We found that proliferation capacity of Ubc{sup −/−} cells was significantly reduced compared to that of control cells, especially during the early culture period, however we did not observe the increased number of apoptotic cells. Furthermore, levels of Ub conjugate, but not free Ub, decreased upon disruption of Ubc expression in FLCs, and this could not be compensated for by upregulation of other poly- or mono-ubiquitin genes. Intriguingly, the highest Ubc expression levels throughout the entire culture period were observed in bipotent FLEPCs. Hepatocytes and bipotent FLEPCs were most affected by disruption of Ubc, resulting in defective proliferation as well as reduced cell numbers in vitro. These results suggest that defective proliferation of these cell types may contribute to severe reduction of fetal liver size and potentially mid-gestation lethality of Ubc{sup −/−} embryos.« less

Labelling and targeted ablation of specific bipolar cell types in the zebrafish retina

PubMed Central

2009-01-01

Background Development of a functional retina depends on regulated differentiation of several types of neurons and generation of a highly complex network between the different types of neurons. In addition, each type of retinal neuron includes several distinct morphological types. Very little is known about the mechanisms responsible for generating this diversity of retinal neurons, which may also display specific patterns of regional distribution. Results In a screen in zebrafish, using a trapping vector carrying an engineered yeast Gal4 transcription activator and a UAS:eGFP reporter cassette, we have identified two transgenic lines of zebrafish co-expressing eGFP and Gal4 in specific subsets of retinal bipolar cells. The eGFP-labelling facilitated analysis of axon terminals within the inner plexiform layer of the adult retina and showed that the fluorescent bipolar cells correspond to previously defined morphological types. Strong regional restriction of eGFP-positive bipolar cells to the central part of the retina surrounding the optic nerve was observed in adult zebrafish. Furthermore, we achieved specific ablation of the labelled bipolar cells in 5 days old larvae, using a bacterial nitroreductase gene under Gal4-UAS control in combination with the prodrug metronidazole. Following prodrug treatment, nitroreductase expressing bipolar cells were efficiently ablated without affecting surrounding retina architecture, and recovery occurred within a few days due to increased generation of new bipolar cells. Conclusion This report shows that enhancer trapping can be applied to label distinct morphological types of bipolar cells in the zebrafish retina. The genetic labelling of these cells yielded co-expression of a modified Gal4 transcription activator and the fluorescent marker eGFP. Our work also demonstrates the potential utility of the Gal4-UAS system for induction of other transgenes, including a bacterial nitroreductase fusion gene, which can facilitate analysis of bipolar cell differentiation and how the retina recovers from specific ablation of these cells. PMID:19712466

Lethality of radiation-induced chromosome aberrations in human tumour cell lines with different radiosensitivities.

PubMed

Coco-Martin, J M; Ottenheim, C P; Bartelink, H; Begg, A C

1996-03-01

In order to find an explanation for the eventual disappearance of all chromosome aberrations in two radiosensitive human tumour cell lines, the type and stability of different aberration types was investigated in more detail. To classify the aberrations into unstable and stable types, three-colour fluorescence in situ hybridization was performed, including a whole-chromosome probe, a pancentromere probe, and a stain for total DNA. This technique enables the appropriate classification of the aberrations principally by the presence (stable) or not (unstable) of a single centromere per chromosome. Unstable-type aberrations were found to disappear within 7 days (several divisions) in the two radiosensitive and the two radioresistant tumour lines investigated. Stable-type aberrations were found to remain at an approximately constant level over the duration of the experiment (14 days; 8-10 divisions) in the two radioresistant lines. In contrast, the majority of these stable-type aberrations had disappeared by 14 days in the two radiosensitive lines. The previous findings of disappearance of total aberrations in radiosensitive cells was therefore not due to a reduced induction of stable-type aberrations, but the complete disappearance of cells with this aberration type. These results could not be explained by differences in apoptosis or G1 blocks. Two possible explanations for these unexpected findings involve non-random induction of unstable-type aberrations, or lethality of stable-type aberrations. The results suggest caution in the use of stable-type aberration numbers as a predictor for radiosensitivity.

Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors.

PubMed

Lamarche, M J; Borawski, J; Bose, A; Capacci-Daniel, C; Colvin, R; Dennehy, M; Ding, J; Dobler, M; Drumm, J; Gaither, L A; Gao, J; Jiang, X; Lin, K; McKeever, U; Puyang, X; Raman, P; Thohan, S; Tommasi, R; Wagner, K; Xiong, X; Zabawa, T; Zhu, S; Wiedmann, B

2012-10-01

Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.

Anti-Hepatitis C Virus Activity and Toxicity of Type III Phosphatidylinositol-4-Kinase Beta Inhibitors

PubMed Central

LaMarche, M. J.; Borawski, J.; Bose, A.; Capacci-Daniel, C.; Colvin, R.; Dennehy, M.; Ding, J.; Dobler, M.; Drumm, J.; Gaither, L. A.; Gao, J.; Jiang, X.; Lin, K.; McKeever, U.; Puyang, X.; Raman, P.; Thohan, S.; Tommasi, R.; Wagner, K.; Xiong, X.; Zabawa, T.; Zhu, S.

2012-01-01

Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C. PMID:22825118

Experimental results with fuel cell start-up and shut-down. Impact of type of carbon for cathode catalyst support

DOE PAGES

Lottin, Olivier; Dillet, Jerome; Maranzana, Gael; ...

2015-09-14

Separate testing protocols for fuel cell startups and shutdowns were developed to distinguish between their effects on reverse currents and CO 2 evolution. The internal currents and the local potentials were measured with different membrane-electrode assemblies (MEAs): we examined the influence of the type of carbon for cathode catalyst support as well as the mitigating effect of low anode Pt loading. In conclusion, significant differences were observed and the experiments also confirmed previous results that the evolved CO 2 accounts for less than 25% of the total exchanged charge.

Experimental results with fuel cell start-up and shut-down. Impact of type of carbon for cathode catalyst support

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lottin, Olivier; Dillet, Jerome; Maranzana, Gael

Separate testing protocols for fuel cell startups and shutdowns were developed to distinguish between their effects on reverse currents and CO 2 evolution. The internal currents and the local potentials were measured with different membrane-electrode assemblies (MEAs): we examined the influence of the type of carbon for cathode catalyst support as well as the mitigating effect of low anode Pt loading. In conclusion, significant differences were observed and the experiments also confirmed previous results that the evolved CO 2 accounts for less than 25% of the total exchanged charge.

Concurrent gradients of ribbon volume and AMPA-receptor patch volume in cochlear afferent synapses on gerbil inner hair cells.

PubMed

Zhang, Lichun; Engler, Sina; Koepcke, Lena; Steenken, Friederike; Köppl, Christine

2018-07-01

The Mongolian gerbil is a classic animal model for age-related hearing loss. As a prerequisite for studying age-related changes, we characterized cochlear afferent synaptic morphology in young adult gerbils, using immunolabeling and quantitative analysis of confocal microscopic images. Cochlear wholemounts were triple-labeled with a hair-cell marker, a marker of presynaptic ribbons, and a marker of postsynaptic AMPA-type glutamate receptors. Seven cochlear positions covering an equivalent frequency range from 0.5 - 32 kHz were evaluated. The spatial positions of synapses were determined in a coordinate system with reference to their individual inner hair cell. Synapse numbers confirmed previous reports for gerbils (on average, 20-22 afferents per inner hair cell). The volumes of presynaptic ribbons and postsynaptic glutamate receptor patches were positively correlated: larger ribbons associated with larger receptor patches and smaller ribbons with smaller patches. Furthermore, the volumes of both presynaptic ribbons and postsynaptic receptor patches co-varied along the modiolar-pillar and the longitudinal axes of their hair cell. The gradients in ribbon volume are consistent with previous findings in cat, guinea pig, mouse and rat and further support a role in differentiating the physiological properties of type I afferents. However, the positive correlation between the volumes of pre- and postsynaptic elements in the gerbil is different to the opposing gradients found in the mouse, suggesting species-specific differences in the postsynaptic AMPA receptors that are unrelated to the fundamental classes of type I afferents. Copyright © 2018 Elsevier B.V. All rights reserved.

Astrocytes surviving severe stress can still protect neighboring neurons from proteotoxic injury

PubMed Central

Gleixner, Amanda M.; Posimo, Jessica M.; Pant, Deepti B.; Henderson, Matthew P.; Leak, Rehana K.

2015-01-01

Astrocytes are one of the major cell types to combat cellular stress and protect neighboring neurons from injury. In order to fulfill this important role, astrocytes must sense and respond to toxic stimuli, perhaps including stimuli that are severely stressful and kill some of the astrocytes. The present study demonstrates that primary astrocytes that managed to survive severe proteotoxic stress were protected against subsequent challenges. These findings suggest that the phenomenon of preconditioning or tolerance can be extended from mild to severe stress for this cell type. Astrocytic stress adaptation lasted at least 96 hours, the longest interval tested. Heat shock protein 70 (Hsp70) was raised in stressed astrocytes, but inhibition of neither Hsp70 nor Hsp32 activity abolished their resistance against a second proteotoxic challenge. Only inhibition of glutathione synthesis abolished astrocytic stress adaptation, consistent with our previous report. Primary neurons were plated upon previously stressed astrocytes and the co-cultures were then exposed to another proteotoxic challenge. Severely stressed astrocytes were still able to protect neighboring neurons against this injury and the protection was unexpectedly independent of glutathione synthesis. Stressed astrocytes were even able to protect neurons after simultaneous application of proteasome and Hsp70 inhibitors, which otherwise elicited synergistic, severe loss of neurons when applied together. Astrocyte-induced neuroprotection against proteotoxicity was not elicited with astrocyte-conditioned media, suggesting that physical cell-to-cell contacts may be essential. These findings suggest that astrocytes may adapt to severe stress so that they can continue to protect neighboring cell types from profound injury. PMID:26374549

Astrocytes Surviving Severe Stress Can Still Protect Neighboring Neurons from Proteotoxic Injury.

PubMed

Gleixner, Amanda M; Posimo, Jessica M; Pant, Deepti B; Henderson, Matthew P; Leak, Rehana K

2016-09-01

Astrocytes are one of the major cell types to combat cellular stress and protect neighboring neurons from injury. In order to fulfill this important role, astrocytes must sense and respond to toxic stimuli, perhaps including stimuli that are severely stressful and kill some of the astrocytes. The present study demonstrates that primary astrocytes that managed to survive severe proteotoxic stress were protected against subsequent challenges. These findings suggest that the phenomenon of preconditioning or tolerance can be extended from mild to severe stress for this cell type. Astrocytic stress adaptation lasted at least 96 h, the longest interval tested. Heat shock protein 70 (Hsp70) was raised in stressed astrocytes, but inhibition of neither Hsp70 nor Hsp32 activity abolished their resistance against a second proteotoxic challenge. Only inhibition of glutathione synthesis abolished astrocytic stress adaptation, consistent with our previous report. Primary neurons were plated upon previously stressed astrocytes, and the cocultures were then exposed to another proteotoxic challenge. Severely stressed astrocytes were still able to protect neighboring neurons against this injury, and the protection was unexpectedly independent of glutathione synthesis. Stressed astrocytes were even able to protect neurons after simultaneous application of proteasome and Hsp70 inhibitors, which otherwise elicited synergistic, severe loss of neurons when applied together. Astrocyte-induced neuroprotection against proteotoxicity was not elicited with astrocyte-conditioned media, suggesting that physical cell-to-cell contacts may be essential. These findings suggest that astrocytes may adapt to severe stress so that they can continue to protect neighboring cell types from profound injury.

Immortalization capacity of HPV types is inversely related to chromosomal instability.

PubMed

Schütze, Denise M; Krijgsman, Oscar; Snijders, Peter J F; Ylstra, Bauke; Weischenfeldt, Joachim; Mardin, Balca R; Stütz, Adrian M; Korbel, Jan O; de Winter, Johan P; Meijer, Chris J L M; Quint, Wim G V; Bosch, Leontien; Wilting, Saskia M; Steenbergen, Renske D M

2016-06-21

High-risk human papillomavirus (hrHPV) types induce immortalization of primary human epithelial cells. Previously we demonstrated that immortalization of human foreskin keratinocytes (HFKs) is HPV type dependent, as reflected by the presence or absence of a crisis period before reaching immortality. This study determined how the immortalization capacity of ten hrHPV types relates to DNA damage induction and overall genomic instability in HFKs.Twenty five cell cultures obtained by transduction of ten hrHPV types (i.e. HPV16/18/31/33/35/45/51/59/66/70 E6E7) in two or three HFK donors each were studied.All hrHPV-transduced HFKs showed an increased number of double strand DNA breaks compared to controls, without exhibiting significant differences between types. However, immortal descendants of HPV-transduced HFKs that underwent a prior crisis period (HPV45/51/59/66/70-transduced HFKs) showed significantly more chromosomal aberrations compared to those without crisis (HPV16/18/31/33/35-transduced HFKs). Notably, the hTERT locus at 5p was exclusively gained in cells with a history of crisis and coincided with increased expression. Chromothripsis was detected in one cell line in which multiple rearrangements within chromosome 8 resulted in a gain of MYC.Together we demonstrated that upon HPV-induced immortalization, the number of chromosomal aberrations is inversely related to the viral immortalization capacity. We propose that hrHPV types with reduced immortalization capacity in vitro, reflected by a crisis period, require more genetic host cell aberrations to facilitate immortalization than types that can immortalize without crisis. This may in part explain the observed differences in HPV-type prevalence in cervical cancers and emphasizes that changes in the host cell genome contribute to HPV-induced carcinogenesis.

Immortalization capacity of HPV types is inversely related to chromosomal instability

PubMed Central

Schütze, Denise M.; Krijgsman, Oscar; Snijders, Peter J.F.; Ylstra, Bauke; Weischenfeldt, Joachim; Mardin, Balca R.; Stütz, Adrian M.; Korbel, Jan O.; Meijer, Chris J.L.M.; Quint, Wim G.V.; Bosch, Leontien; Wilting, Saskia M.; Steenbergen, Renske D.M.

2016-01-01

High-risk human papillomavirus (hrHPV) types induce immortalization of primary human epithelial cells. Previously we demonstrated that immortalization of human foreskin keratinocytes (HFKs) is HPV type dependent, as reflected by the presence or absence of a crisis period before reaching immortality. This study determined how the immortalization capacity of ten hrHPV types relates to DNA damage induction and overall genomic instability in HFKs. Twenty five cell cultures obtained by transduction of ten hrHPV types (i.e. HPV16/18/31/33/35/45/51/59/66/70 E6E7) in two or three HFK donors each were studied. All hrHPV-transduced HFKs showed an increased number of double strand DNA breaks compared to controls, without exhibiting significant differences between types. However, immortal descendants of HPV-transduced HFKs that underwent a prior crisis period (HPV45/51/59/66/70-transduced HFKs) showed significantly more chromosomal aberrations compared to those without crisis (HPV16/18/31/33/35-transduced HFKs). Notably, the hTERT locus at 5p was exclusively gained in cells with a history of crisis and coincided with increased expression. Chromothripsis was detected in one cell line in which multiple rearrangements within chromosome 8 resulted in a gain of MYC. Together we demonstrated that upon HPV-induced immortalization, the number of chromosomal aberrations is inversely related to the viral immortalization capacity. We propose that hrHPV types with reduced immortalization capacity in vitro, reflected by a crisis period, require more genetic host cell aberrations to facilitate immortalization than types that can immortalize without crisis. This may in part explain the observed differences in HPV-type prevalence in cervical cancers and emphasizes that changes in the host cell genome contribute to HPV-induced carcinogenesis. PMID:26993771

Cell Kinetic and Histomorphometric Analysis of Microgravitational Osteopenia: PARE.03B

NASA Technical Reports Server (NTRS)

Roberts, W. Eugene; Garetto, Lawrence P.

1998-01-01

Previous methods of identifying cells undergoing DNA synthesis (S-phase) utilized 3H-thymidine (3HT) autoradiography. 5-Bromo-2'-deoxyuridine (BrdU) immunohistochemistry is a nonradioactive alternative method. This experiment compared the two methods using the nuclear volume model for osteoblast histogenesis in two different embedding media. Twenty Sprague-Dawley rats were used, with half receiving 3HT (1 micro-Ci/g) and the other half BrdU (50 micro-g/g). Condyles were embedded (one side in paraffin, the other in plastic) and S-phase nuclei were identified using either autoradiography or immunohistochemistry. The fractional distribution of preosteoblast cell types and the percentage of labeled cells (within each cell fraction and label index) were calculated and expressed as mean +/- standard error. Chi-Square analysis showed only a minor difference in the fractional distribution of cell types. However, there were,significant differences (p less than 0.05) by ANOVA, in the nuclear labeling of specific cell types. With the exception of the less-differentiated A+A' cells, more BrdU label was consistently detected in paraffin than in plastic-embedded sections. In general, more nuclei were labeled with 3H-thymidine than with BrdU in both types of embedding media (Fig 2.). Labeling index data (labeled cells/total cells sampled x 100) indicated that BrdU in paraffin, but not plastic gave the same results as 3HT in either embedding method. Thus, we conclude that the two labeling methods do not yield the same results.

A Historical Perspective on the Identification of Cell Types in Pancreatic Islets of Langerhans by Staining and Histochemical Techniques

PubMed Central

2015-01-01

Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes. PMID:26216133

A simple and efficient method for deriving neurospheres from bone marrow stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang Qin; Mu Jun; Li Qi

2008-08-08

Bone marrow stromal cells (MSCs) can be differentiated into neuronal and glial-like cell types under appropriate experimental conditions. However, previously reported methods are complicated and involve the use of toxic reagents. Here, we present a simplified and nontoxic method for efficient conversion of rat MSCs into neurospheres that express the neuroectodermal marker nestin. These neurospheres can proliferate and differentiate into neuron, astrocyte, and oligodendrocyte phenotypes. We thus propose that MSCs are an emerging model cell for the treatment of a variety of neurological diseases.

Comparative radiation resistance, temperature dependence and performance of diffused junction indium phosphide solar cells

NASA Technical Reports Server (NTRS)

Weinberg, I.; Swartz, C. K.; Hart, R. E., Jr.; Ghandhi, S. K.; Borrego, J. M.

1987-01-01

Indium phosphide solar cells whose p-n junctions were processed by the open tube capped diffusion and by the closed tube uncapped diffusion of sulfur into Czochralski-grown p-type substrates are compared. Differences found in radiation resistance were attributed to the effects of increased base dopant concentration. Both sets of cells showed superior radiation resistance to that of gallium arsenide cells, in agreement with previous results. No correlation was, however, found between the open-circuit voltage and the temperature dependence of the maximum power.

Engraftment of Human Pluripotent Stem Cell-derived Progenitors in the Inner Ear of Prenatal Mice.

PubMed

Takeda, Hiroki; Hosoya, Makoto; Fujioka, Masato; Saegusa, Chika; Saeki, Tsubasa; Miwa, Toru; Okano, Hideyuki; Minoda, Ryosei

2018-01-31

There is, at present, no curative treatment for genetic hearing loss. We have previously reported that transuterine gene transfer of wild type CONNEXIN30 (CX30) genes into otocysts in CX30-deleted mice could restore hearing. Cell transplantation therapy might be another therapeutic option, although it is still unknown whether stem cell-derived progenitor cells could migrate into mouse otocysts. Here, we show successful cell transplantation of progenitors of outer sulcus cell-like cells derived from human-derived induced pluripotent stem cells into mouse otocysts on embryonic day 11.5. The delivered cells engrafted more frequently in the non-sensory region in the inner ear of CX30-deleted mice than in wild type mice and survived for up to 1 week after transplantation. Some of the engrafted cells expressed CX30 proteins in the non-sensory region. This is the first report that demonstrates successful engraftment of exogenous cells in prenatal developing otocysts in mice. Future studies using this mouse otocystic injection model in vivo will provide further clues for developing treatment modalities for congenital hearing loss in humans.

Distinct interactions of Sox5 and Sox10 in fate specification of pigment cells in medaka and zebrafish

PubMed Central

Nagao, Yusuke; Takada, Hiroyuki; Miyadai, Motohiro; Adachi, Tomoko; Kamei, Yasuhiro; Hara, Ikuyo; Naruse, Kiyoshi; Hibi, Masahiko

2018-01-01

Mechanisms generating diverse cell types from multipotent progenitors are fundamental for normal development. Pigment cells are derived from multipotent neural crest cells and their diversity in teleosts provides an excellent model for studying mechanisms controlling fate specification of distinct cell types. Zebrafish have three types of pigment cells (melanocytes, iridophores and xanthophores) while medaka have four (three shared with zebrafish, plus leucophores), raising questions about how conserved mechanisms of fate specification of each pigment cell type are in these fish. We have previously shown that the Sry-related transcription factor Sox10 is crucial for fate specification of pigment cells in zebrafish, and that Sox5 promotes xanthophores and represses leucophores in a shared xanthophore/leucophore progenitor in medaka. Employing TILLING, TALEN and CRISPR/Cas9 technologies, we generated medaka and zebrafish sox5 and sox10 mutants and conducted comparative analyses of their compound mutant phenotypes. We show that specification of all pigment cells, except leucophores, is dependent on Sox10. Loss of Sox5 in Sox10-defective fish partially rescued the formation of all pigment cells in zebrafish, and melanocytes and iridophores in medaka, suggesting that Sox5 represses Sox10-dependent formation of these pigment cells, similar to their interaction in mammalian melanocyte specification. In contrast, in medaka, loss of Sox10 acts cooperatively with Sox5, enhancing both xanthophore reduction and leucophore increase in sox5 mutants. Misexpression of Sox5 in the xanthophore/leucophore progenitors increased xanthophores and reduced leucophores in medaka. Thus, the mode of Sox5 function in xanthophore specification differs between medaka (promoting) and zebrafish (repressing), which is also the case in adult fish. Our findings reveal surprising diversity in even the mode of the interactions between Sox5 and Sox10 governing specification of pigment cell types in medaka and zebrafish, and suggest that this is related to the evolution of a fourth pigment cell type. PMID:29621239

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

PubMed Central

Dasa, Siva Sai Krishna; Kelly, Kimberly A.

2016-01-01

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

Analysis of gene expression and Ig transcription in PU.1/Spi-B-deficient progenitor B cell lines.

PubMed

Schweitzer, Brock L; DeKoter, Rodney P

2004-01-01

A number of presumptive target genes for the Ets-family transcription factor PU.1 have been identified in the B cell lineage. However, the precise function of PU.1 in B cells has not been studied because targeted null mutation of the PU.1 gene results in a block to lymphomyeloid development at an early developmental stage. In this study, we take advantage of recently developed PU.1(-/-)Spi-B(-/-) IL-7 and stromal cell-dependent progenitor B (pro-B) cell lines to analyze the function of PU.1 and Spi-B in B cell development. We show that contrary to previously published expectations, PU.1 and/or Spi-B are not required for Ig H chain (IgH) gene transcription in pro-B cells. In fact, PU.1(-/-)Spi-B(-/-) pro-B cells have increased levels of IgH transcription compared with wild-type pro-B cells. In addition, high levels of Igkappa transcription are induced after IL-7 withdrawal of wild-type or PU.1(-/-)Spi-B(-/-) pro-B cells. In contrast, we found that Iglambda transcription is reduced in PU.1(-/-)Spi-B(-/-) pro-B cells relative to wild-type pro-B cells after IL-7 withdrawal. These results suggest that Iglambda, but not IgH or Igkappa, transcription, is dependent on PU.1 and/or Spi-B. The PU.1(-/-)Spi-B(-/-) pro-B cells have other phenotypic changes relative to wild-type pro-B cells including increased proliferation, increased CD25 expression, decreased c-Kit expression, and decreased RAG-1 expression. Taken together, our observations suggest that reduction of PU.1 and/or Spi-B activity in pro-B cells promotes their differentiation to a stage intermediate between late pro-B cells and large pre-B cells.

Self-calibration of a W/Re thermocouple using a miniature Ru-C (1954 °C) eutectic cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ongrai, O.; University of Surrey, Guildford, Surrey; National Institute of Metrology, Klong 5, Klong Luang, Pathumthani

2013-09-11

Previous successful investigations of miniature cobalt-carbon (Co-C, 1324 °C) and palladium-carbon (Pd-C, 1492 °C) high temperature fixed-point cells for thermocouple self-calibration have been reported [1-2]. In the present work, we describe a series of measurements of a miniature ruthenium-carbon (Ru-C) eutectic cell (melting point 1954 °C) to evaluate the repeatability and stability of a W/Re thermocouple (type C) by means of in-situ calibration. A miniature Ru-C eutectic fixed-point cell with outside diameter 14 mm and length 30 mm was fabricated to be used as a self-calibrating device. The performance of the miniature Ru-C cell and the type C thermocouple ismore » presented, including characterization of the stability, repeatability, thermal environment influence, ITS-90 temperature realization and measurement uncertainty.« less

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH.

PubMed

Daidoji, Tomo; Watanabe, Yohei; Ibrahim, Madiha S; Yasugi, Mayo; Maruyama, Hisataka; Masuda, Taisuke; Arai, Fumihito; Ohba, Tomoyuki; Honda, Ayae; Ikuta, Kazuyoshi; Nakaya, Takaaki

2015-04-24

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dopaminergic neurons write and update memories with cell-type-specific rules

PubMed Central

Aso, Yoshinori; Rubin, Gerald M

2016-01-01

Associative learning is thought to involve parallel and distributed mechanisms of memory formation and storage. In Drosophila, the mushroom body (MB) is the major site of associative odor memory formation. Previously we described the anatomy of the adult MB and defined 20 types of dopaminergic neurons (DANs) that each innervate distinct MB compartments (Aso et al., 2014a, 2014b). Here we compare the properties of memories formed by optogenetic activation of individual DAN cell types. We found extensive differences in training requirements for memory formation, decay dynamics, storage capacity and flexibility to learn new associations. Even a single DAN cell type can either write or reduce an aversive memory, or write an appetitive memory, depending on when it is activated relative to odor delivery. Our results show that different learning rules are executed in seemingly parallel memory systems, providing multiple distinct circuit-based strategies to predict future events from past experiences. DOI: http://dx.doi.org/10.7554/eLife.16135.001 PMID:27441388

Amnion as a surrogate tissue reporter of the effects of maternal preeclampsia on the fetus.

PubMed

Suzuki, Masako; Maekawa, Ryo; Patterson, Nicole E; Reynolds, David M; Calder, Brent R; Reznik, Sandra E; Heo, Hye J; Einstein, Francine Hughes; Greally, John M

2016-01-01

Preeclampsia, traditionally characterized by high blood pressure and proteinuria, is a common pregnancy complication, which affects 2-8 % of all pregnancies. Although children born to women with preeclampsia have a higher risk of hypertension in later life, the mechanism of this increased risk is unknown. DNA methylation is an epigenetic modification that has been studied as a mediator of cellular memory of adverse exposures in utero. Since each cell type in the body has a unique DNA profile, cell subtype composition is a major confounding factor in studies of tissues with heterogeneous cell types. The best way to avoid this confounding effect is by using purified cell types. However, using purified cell types in large cohort translational studies is difficult. The amnion, the inner layer of the fetal membranes of the placenta, is derived from the epiblast and consists of two cell types, which are easy to isolate from the delivered placenta. In this study, we demonstrate the value of using amnion samples for DNA methylation studies, revealing distinctive patterns between fetuses exposed to proteinuria or hypertension and fetuses from normal pregnancies. We performed a genome-wide DNA methylation analysis, HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP)-tagging, on 62 amnion samples from the placentas of uncomplicated, normal pregnancies and from those with complications of preeclampsia or hypertension. Using a regression model approach, we found 123, 85, and 99 loci with high-confidence hypertension-associated, proteinuria-associated, and hypertension- and proteinuria-associated DNA methylation changes, respectively. A gene ontology analysis showed DNA methylation changes to be selecting genes with different biological processes in exposure status. We also found that these differentially methylated regions overlap loci previously reported as differentially methylated regions in preeclampsia. Our findings support prior observations that preeclampsia is associated with changes of DNA methylation near genes that have previously been found to be dysregulated in preeclampsia. We propose that amniotic membranes represent a valuable surrogate fetal tissue on which to perform epigenome-wide association studies of adverse intrauterine conditions.

Distribution of calmodulin in corn seedlings - Immunocytochemical localization in coleoptiles and root apices

NASA Technical Reports Server (NTRS)

Dauwalder, M.; Roux, S. J.

1986-01-01

Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in etiolated corn (Zea mays, var. Bear Hybrid) seedlings. Uniform staining was seen in the background cytoplasm of most cell types. Cell walls and vacuoles were not stained. In coleoptile mesophyll cells the nucleoplasm of most nuclei was stained as was the stroma of most amyloplasts. The lumen border of mature tracheary elements in coleoptiles also stained. In the rootcap the most intensely stained regions were the cytoplasms of columella cells and of the outermost cells enmeshed in the layer of secreted slime. Nuclei in the rootcap cells did not stain distinctly, but those in all cell types of the root meristem did. Also in the root meristem, the cytoplasm of metaxylem elements stained brightly. These results are compared and contrasted with previous data on the localization of calmodulin in pea root apices and epicotyls and discussed in relation to current hypotheses on mechanisms of gravitropism.

A TALEN genome editing system to generate human stem cell-based disease models

PubMed Central

Ding, Qiurong; Lee, Youn-Kyoung; Schaefer, Esperance A. K.; Peters, Derek T.; Veres, Adrian; Kim, Kevin; Kuperwasser, Nicolas; Motola, Daniel L.; Meissner, Torsten B.; Hendriks, William T.; Trevisan, Marta; Gupta, Rajat M.; Moisan, Annie; Banks, Eric; Friesen, Max; Schinzel, Robert T.; Xia, Fang; Tang, Alexander; Xia, Yulei; Figueroa, Emmanuel; Wann, Amy; Ahfeldt, Tim; Daheron, Laurence; Zhang, Feng; Rubin, Lee L.; Peng, Lee F.; Chung, Raymond T.; Musunuru, Kiran; Cowan, Chad A.

2012-01-01

SUMMARY Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter of which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor neuron death, and hepatitis C infection. We find little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease. PMID:23246482

Okadaic acid mediates p53 hyperphosphorylation and growth arrest in cells with wild-type p53 but increases aberrant mitoses in cells with non-functional p53.

PubMed

Milczarek, G J; Chen, W; Gupta, A; Martinez, J D; Bowden, G T

1999-06-01

The protein phosphatase inhibitor and tumor promoting agent okadaic acid (OA), has been shown previously to induce hyperphosphorylation of p53 protein, which in turn correlated with increased transactivation or apoptotic function. However, how the tumor promotion effects of OA relate to p53 tumor supressor function (or dysfunction) remain unclear. Rat embryonic fibroblasts harboring a temperature-sensitive mouse p53 transgene were treated with 50 nM doses of OA. At the wild-type permissive temperature this treatment resulted in: (i) the hyperphosphorylation of sites within tryptic peptides of the transactivation domain of p53; (ii) an increase in p53 affinity for a p21(waf1) promotor oligonucleotide; (iii) an increase in cellular steady state levels of p21(waf1) message; (iv) a G2/M cell cycle blockage in addition to the G1/S arrest previously associated with p53; and (v) no increased incidence of apoptosis. On the other hand, OA treatment at the mutated p53 permissive temperature resulted in a relatively high incidence of aberrant mitosis with no upregulation of p21(waf1) message. These results suggest that while wild-type p53 blocks the proliferative effects of OA through p21(waf1)-mediated growth arrest, cells with non-functional p53 cannot arrest and suffer relatively high levels of OA-mediated aberrant mitoses.

Overexpression of ZDHHC14 promotes migration and invasion of scirrhous type gastric cancer.

PubMed

Oo, Htoo Zarni; Sentani, Kazuhiro; Sakamoto, Naoya; Anami, Katsuhiro; Naito, Yutaka; Uraoka, Naohiro; Oshima, Takashi; Yanagihara, Kazuyoshi; Oue, Naohide; Yasui, Wataru

2014-07-01

Scirrhous type gastric cancer is highly aggressive and has a poorer prognosis than many other types of gastric carcinoma, due to its characteristic rapid cancer cell infiltration and proliferation, extensive stromal fibrosis, and frequent peritoneal dissemination. The aim of the present study was to identify novel prognostic markers or therapeutic targets for scirrhous type gastric cancer. We reviewed a list of genes with upregulated expression in scirrhous type gastric cancer and compared their expression with that in normal stomach from our previous Escherichia coli (E. coli) ampicillin secretion-trap (CAST) analysis. We focused on the ZDHHC14 gene, which encodes zinc finger, DHHC-type containing 14 protein. qRT-PCR analysis of ZDHHC14 in 41 gastric cancer cases revealed that compared to mRNA levels in normal non-neoplastic gastric mucosa, ZDHHC14 mRNA was overexpressed in 27% of gastric cancer tissue samples. The overexpression of ZDHHC14 was significantly associated with depth of tumor invasion, undifferentiated histology and scirrhous pattern. The invasiveness of ZDHHC14-knockdown HSC-44PE and 44As3 gastric cancer cells was decreased in comparison with that of the negative control siRNA-transfected cells, together with downregulation of MMP-17 mRNA. Integrins α5 and β1 were also downregulated in ZDHHC14-knockdown 44As3 cells. Forced expression of ZDHHC14 activated gastric cancer cell migration and invasion in vitro. These results indicate that ZDHHC14 is involved in tumor progression in patients with scirrhous type gastric cancer.

Adult T-cell leukaemia/lymphoma can mimic other lymphomas in a non-endemic area: dilemmas in diagnosis and treatment.

PubMed

Huang, C-T; Lee, Y-H; Chow, K-C; Yang, C-F; Chen, P C-H; Hsiao, L-T; Gau, J-P; Tzeng, C-H; Liu, C-Y; Chiou, T-J

2014-04-01

The diagnosis of Adult T-cell leukaemia/lymphoma (ATL) in non-endemic regions is challenging. This study analyses the clinicopathologic features and diagnostic processes of ATL patients in Taiwan. ATL patients diagnosed and treated at Taipei Veterans General Hospital from 1998 through 2010 were retrospectively identified. The diagnosis of ATL was confirmed by in situ detection of human T-cell leukaemia virus type 1 (HTLV-1) when necessary. Patients' data were reviewed and analysed. Fourteen ATL patients were identified, among whom six (42.9%) had an antecedent diagnosis of other malignant lymphomas before the ATL diagnosis, including two diagnosed with Hodgkin disease (HD), one with peripheral T-cell lymphoma, two with chronic lymphocytic leukaemia and one with angioimmunoblastic T-cell lymphoma. Of the 14 patients, eight (57%) were subclassified as the acute type, three (21.4%) as the lymphoma type, and three (21.4%) as the chronic type ATL. Five of six (83.3%) patients with initial non-ATL misdiagnosis were diagnosed with non-acute type ATL. In particular, a patient with an antecedent diagnosis of HD presented with typical Reed-Sternberg (RS)-like cells harbouring Epstein-Barr virus genomes in affected lymph nodes. The patient progressed to acute type ATL 3 years after the initial diagnosis, and HTLV-1 genomes were identified in the previous RS-like cells. In non-endemic areas, such as Taiwan, ATL, particularly the non-acute type, may mimic other lymphomas and easily be misdiagnosed. HTLV-1 serology should be routinely screened in all malignant lymphoma patients. In situ detection of HTLV-1 is helpful in cases with diagnostic dilemmas. © 2014 The Authors; Internal Medicine Journal © 2014 Royal Australasian College of Physicians.

Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

PubMed Central

Tateno, Hiroaki; Matsushima, Asako; Hiemori, Keiko; Onuma, Yasuko; Ito, Yuzuru; Hasehira, Kayo; Nishimura, Ken; Ohtaka, Manami; Takayasu, Satoko; Nakanishi, Mahito; Ikehara, Yuzuru; Nakanishi, Mio; Ohnuma, Kiyoshi; Chan, Techuan; Toyoda, Masashi; Akutsu, Hidenori; Umezawa, Akihiro; Asashima, Makoto

2013-01-01

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. PMID:23526252

Clonal populations of amniotic cells by dilution and direct plating: evidence for hidden diversity.

PubMed

Wilson, Patricia G; Devkota, Lorna; Payne, Tiffany; Crisp, Laddie; Winter, Allison; Wang, Zhan

2012-01-01

Fetal cells are widely considered a superior cell source for regenerative medicine; fetal cells show higher proliferative capacity and have undergone fewer replicative cycles that could generate spontaneous mutations. Fetal cells in amniotic fluid were among the first normal primary cells to be cultured ex vivo, but the undefined composition of amniotic fluid has hindered advance for regenerative applications. We first developed a highly efficient method to generate clonal populations by dilution of amniocentesis samples in media and direct plating without intervening refrigeration, centrifugation, or exposure of cells to the paracrine effects in mixed cell cultures. More than 40 clonal populations were recovered from 4 amniocentesis samples and representative clones were characterized by flow cytometry, conventional assays for differentiation potential, immunofluorescence imaging, and transcript analysis. The results revealed previously unreported diversity among stromal and epithelial cell types and identified unique cell types that could be lost or undetected in mixed cell populations. The differentiation potential of amniotic cells proved to be uncoupled from expression of definitive cell surface or cytoplasmic markers for stromal and epithelial cells. Evidence for diversity among stromal and epithelial cells in amniotic fluid bears on interpretations applied to molecular and functional tests of amniotic cell populations.

Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

PubMed Central

Cheng, Kevin P.; Kiernan, Elizabeth A.; Eliceiri, Kevin W.; Williams, Justin C.; Watters, Jyoti J.

2016-01-01

Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS. PMID:26883795

Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression.

PubMed

Cheng, Kevin P; Kiernan, Elizabeth A; Eliceiri, Kevin W; Williams, Justin C; Watters, Jyoti J

2016-02-17

Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS.

Syncytial giant-cell hepatitis due to autoimmune hepatitis type II (LKM1+) presenting as subfulminant hepatitis.

PubMed

Ben-Ari, Z; Broida, E; Monselise, Y; Kazatsker, A; Baruch, J; Pappo, O; Skappa, E; Tur-Kaspa, R

2000-03-01

Giant cell hepatitis (GCH) in adults is a rare event. The diagnosis of GCH is based on findings of syncytial giant hepatocytes. It is commonly associated with either viral infection or autoimmune hepatitis type I. A patient with GCH due to autoimmune hepatitis type II (LKM1+) is described, a combination that has not been previously reported. Corticosteroid therapy was effective in decreasing serum liver enzymes; however, the patient deteriorated rapidly and developed subfulminant hepatic failure. Although an emergency orthotopic liver transplantation was performed, the patient died because of reperfusion injury. Interestingly, only a few giant hepatocytes were noted in the explanted liver. This case stresses the association of GCH with autoimmune disorders, the possible immune mechanism involved in the formation of giant cell hepatocytes, and illustrates the rapidly progressive course and unfavorable prognosis that these patients can develop.

MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion.

PubMed

Oliphant, Christopher J; Hwang, You Yi; Walker, Jennifer A; Salimi, Maryam; Wong, See Heng; Brewer, James M; Englezakis, Alexandros; Barlow, Jillian L; Hams, Emily; Scanlon, Seth T; Ogg, Graham S; Fallon, Padraic G; McKenzie, Andrew N J

2014-08-21

Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawata, Shigehisa; Suzuki, Jun; Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871

2006-11-10

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types ofmore » cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Fabao; Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071; You, Xiaona

Highlights: • Relative to wild type HBx, HBX mutant HBxΔ127 strongly enhances cell proliferation. • Relative to wild type HBx, HBxΔ127 remarkably up-regulates miR-215 in hepatoma cells. • HBxΔ127-elevated miR-215 promotes cell proliferation via targeting PTPRT mRNA. - Abstract: The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migrationmore » in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.« less

Skeletal muscle cells express ICAM-1 after muscle overload and ICAM-1 contributes to the ensuing hypertrophic response.

PubMed

Dearth, Christopher L; Goh, Qingnian; Marino, Joseph S; Cicinelli, Peter A; Torres-Palsa, Maria J; Pierre, Philippe; Worth, Randall G; Pizza, Francis X

2013-01-01

We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells.

β-Arrestin-2-Dependent Signaling Promotes CCR4-mediated Chemotaxis of Murine T-Helper Type 2 Cells.

PubMed

Lin, Rui; Choi, Yeon Ho; Zidar, David A; Walker, Julia K L

2018-06-01

Allergic asthma is a complex inflammatory disease that leads to significant healthcare costs and reduction in quality of life. Although many cell types are implicated in the pathogenesis of asthma, CD4 + T-helper cell type 2 (Th2) cells are centrally involved. We previously reported that the asthma phenotype is virtually absent in ovalbumin-sensitized and -challenged mice that lack global expression of β-arrestin (β-arr)-2 and that CD4 + T cells from these mice displayed significantly reduced CCL22-mediated chemotaxis. Because CCL22-mediated activation of CCR4 plays a role in Th2 cell regulation in asthmatic inflammation, we hypothesized that CCR4-mediated migration of CD4 + Th2 cells to the lung in asthma may use β-arr-dependent signaling. To test this hypothesis, we assessed the effect of various signaling inhibitors on CCL22-induced chemotaxis using in vitro-polarized primary CD4 + Th2 cells from β-arr2-knockout and wild-type mice. Our results show, for the first time, that CCL22-induced, CCR4-mediated Th2 cell chemotaxis is dependent, in part, on a β-arr2-dependent signaling pathway. In addition, we show that this chemotactic signaling mechanism involves activation of P-p38 and Rho-associated protein kinase. These findings point to a proinflammatory role for β-arr2-dependent signaling and support β-arr2 as a novel therapeutic target in asthma.

Skeletal Muscle Cells Express ICAM-1 after Muscle Overload and ICAM-1 Contributes to the Ensuing Hypertrophic Response

PubMed Central

Dearth, Christopher L.; Goh, Qingnian; Marino, Joseph S.; Cicinelli, Peter A.; Torres-Palsa, Maria J.; Pierre, Philippe; Worth, Randall G.; Pizza, Francis X.

2013-01-01

We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells. PMID:23505517

High Beginning-of-Life Efficiency p/n InP Solar Cells

NASA Technical Reports Server (NTRS)

Hoffman, Richard W., Jr.; Fatemi, Navid S.; Weizer, Victor G.; Jenkins, Phillip P.; Ringel, Steven A.; Scheiman, David A.; Wilt, David M.; Brinker, David J.

2004-01-01

We have achieved a new record efficiency of 17.6%, (AM0) for a p/n InP homo-epitaxy solar cell. In addition, we have eliminated a previously observed photo-degradation of cell performance, which was due to losses in J(sub sc). Cells soaked in AM0 spectrum at one-sun intensity for an hour showed no significant change in cell performance. We have discovered carrier passivation effects when using Zn as the p-type dopant in the OMVPE growth of InP and have found a method to avoid the unexpected effects which result from typical operation of OMVPE cell growth.

Basal Cells Are a Multipotent Progenitor Capable of Renewing the Bronchial Epithelium

PubMed Central

Hong, Kyung U.; Reynolds, Susan D.; Watkins, Simon; Fuchs, Elaine; Stripp, Barry R.

2004-01-01

Commitment of the pulmonary epithelium to bronchial and bronchiolar airway lineages occurs during the transition from pseudoglandular to cannalicular phases of lung development, suggesting that regional differences exist with respect to the identity of stem and progenitor cells that contribute to epithelial maintenance in adulthood. We previously defined a critical role for Clara cell secretory protein-expressing (CE) cells in renewal of bronchiolar airway epithelium following injury. Even though CE cells are also the principal progenitor for maintenance of the bronchial airway epithelium, CE cell injury is resolved through a mechanism involving recruitment of a second progenitor cell population that we now identify as a GSI-B4 reactive, cytokeratin-14-expressing basal cell. These cells exhibit multipotent differentiation capacity as assessed by analysis of cellular phenotype within clones of LacZ-tagged cells. Clones were derived from K14-expressing cells tagged in a cell-type-specific fashion by ligand-regulable Cre recombinase-mediated genomic rearrangement of the ROSA26 recombination substrate allele. We conclude that basal cells represent an alternative multipotent progenitor cell population of bronchial airways and that progenitor cell selection is dictated by the type of airway injury. PMID:14742263

Nitric oxide production by hemocytes of the ascidian Styela plicata.

PubMed

de Barros, Cintia Monteiro; de Carvalho, Danielle Ronald; Andrade, Leonardo R; Pavão, Mauro Sérgio G; Allodi, Silvana

2009-10-01

Ascidian hemolymph contains various types of blood cells (hemocytes), which are believed to be involved in defense mechanisms. We have studied nitric-oxide (NO) synthase activity in hemocytes of the ascidian Styela plicata after exposure to lipopolysaccharide (LPS). To investigate which cell types are involved in NO production, we first identified, by electron microscopy, the types of hemocytes previously described, mainly by light microscopy, by others. Five types of blood cells could be recognized in the hemolymph: granulocytes, hemoblasts, lymphocyte-like cells, morula cells, and pigment cells. The lymphocyte-like cells produced the most NO. In agreement with studies of other invertebrates, nitrite generation did not change after LPS stimulation in assays in vitro, under either different concentrations of LPS or different time periods. Therefore, we performed an in vivo assay by injecting a known quantity of Escherichia coli into the tunic of the ascidians in order to investigate possible differences in NO levels. No increase of NO occurred accompanying the inflammatory reaction suggesting that another molecule in the pathway was involved. We found that nuclear factor kappaB (NFkappaB) was activated. Since NFkappaB is involved in the production of many substances related to immune responses, additional molecules might also be generated in response to E. coli infection. These observations may improve our understanding of the reaction of animals to eutrophic conditions.

RBE of Energetic Iron Ions for the Induction of Early and Late Chromosome Aberrations in Different Cell Types

NASA Technical Reports Server (NTRS)

Zhang, Ye; Yeshitla, Samrawit; Hada, Megumi; Kadhim, Munira; Wilson, Bobby; Wu, Honglu

2014-01-01

Numerous published studies have reported the RBE values for chromosome chromosomes induced by charged particles of different LET. The RBE for chromosome aberrations in human lymphocytes exposed ex vivo showed a similar relationship as the quality factor for cancer induction. Consequently, increased chromosome aberrations in the astronauts' white blood cells post long-duration missions are used to determine the biological doses from exposures to space radiation. The RBE value is known to be very different for different types of cancer. Previously, we reported that the RBE for initial chromosome damages was high in human lymphocytes exposed to Fe ions. After multiple cell divisions post irradiation, the RBE was significantly smaller. To test the hypothesis that the RBE values for chromosome aberrations are different between early and late damages and also different between different cell types, we exposed human lymphocytes ex vivo, and human fibroblast cells and human mammary epithelial cells in vitro to 600 MeV/u Fe ions. Post irradiation, the cells were collected at first mitosis, or cultured for multiple generations for collections of remaining or late arising chromosome aberrations. The chromosome aberrations were quantified using fluorescent in situ hybridization (FISH) with whole chromosome specific probes. This study attempts to offer an explanation for the varying RBE values for different cancer types.

Comparison Between Supervised and Unsupervised Classifications of Neuronal Cell Types: A Case Study

PubMed Central

Guerra, Luis; McGarry, Laura M; Robles, Víctor; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

2011-01-01

In the study of neural circuits, it becomes essential to discern the different neuronal cell types that build the circuit. Traditionally, neuronal cell types have been classified using qualitative descriptors. More recently, several attempts have been made to classify neurons quantitatively, using unsupervised clustering methods. While useful, these algorithms do not take advantage of previous information known to the investigator, which could improve the classification task. For neocortical GABAergic interneurons, the problem to discern among different cell types is particularly difficult and better methods are needed to perform objective classifications. Here we explore the use of supervised classification algorithms to classify neurons based on their morphological features, using a database of 128 pyramidal cells and 199 interneurons from mouse neocortex. To evaluate the performance of different algorithms we used, as a “benchmark,” the test to automatically distinguish between pyramidal cells and interneurons, defining “ground truth” by the presence or absence of an apical dendrite. We compared hierarchical clustering with a battery of different supervised classification algorithms, finding that supervised classifications outperformed hierarchical clustering. In addition, the selection of subsets of distinguishing features enhanced the classification accuracy for both sets of algorithms. The analysis of selected variables indicates that dendritic features were most useful to distinguish pyramidal cells from interneurons when compared with somatic and axonal morphological variables. We conclude that supervised classification algorithms are better matched to the general problem of distinguishing neuronal cell types when some information on these cell groups, in our case being pyramidal or interneuron, is known a priori. As a spin-off of this methodological study, we provide several methods to automatically distinguish neocortical pyramidal cells from interneurons, based on their morphologies. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 71–82, 2011 PMID:21154911

Intravascular large B-cell lymphoma presenting with anasarca-type edema and acute renal failure.

PubMed

Bilgili, Serap Gunes; Yılmaz, Deniz; Soyoral, Yasemin Usul; Karadag, Ayse Serap; Bayram, Irfan

2013-09-01

Intravascular lymphoma (IVL) is a rare extra nodal subtype (usually of B-cell origin) presenting with infiltration of large neoplastic lymphocytes into lumina of blood vessels, leading to vascular occlusion. The early diagnosis is very crucial, however it is usually diagnosed postmortem investigation in most of the cases. A 56-year-old female presented with elevated creatinine level, and anasarca-type edema that superimposed with hard, indurated, erythematous plaques extending to inguinal region, abdomen, anterior aspect of chest, and face. B-cell IVL was confirmed with skin biopsy. The patient had some degree of clinical improvement following chemotherapy. B-cell IVL presenting with anasarca edema was not previously reported in the literature. Even if its rarity, IVL should be considered in the differential diagnosis of renal failure with anasarca edema.

Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Hepatocytes

PubMed Central

Conde-Vancells, Javier; Rodriguez-Suarez, Eva; Embade, Nieves; Gil, David; Matthiesen, Rune; Valle, Mikel; Elortza, Felix; Lu, Shelly C.; Mato, Jose M.; Falcon-Perez, Juan M.

2009-01-01

Synopsis Exosomes constitute a discrete population of nanometer-sized (30-150 nm) vesicles formed in endocytic compartments and released to the extracellular environment by different cell types. In this work we demonstrated by electron microscopic, western blotting and proteomic analyses that primary hepatocytes secrete exosome-like vesicles containing proteins involved in metabolizing lipoproteins, endogenous compounds as well as xenobiotics. These new findings contribute to improve our knowledge about biology's hepatocyte and may have important diagnostic, prognosis and therapeutic implications in liver diseases Exosomes represent a discrete population of vesicles that are secreted from various cell types to the extracellular media. Their protein and lipid composition are a consequence of sorting events at the level of the multivesicular body, a central organelle which integrates endocytic and secretory pathways. Characterization of exosomes from different biological samples has shown the presence of common as well as cell-type specific proteins. Remarkably, the protein content of the exosomes is modified upon pathological or stress conditions. Hepatocytes play a central role in the body response to stress metabolizing potentially harmful endogenous substances as well as xenobiotics. In the present study we described and characterized for first time exosome secretion in non-tumoral hepatocytes, and using a systematic proteomic approach, we establish the first extensive proteome of a hepatocyte-derived exosome population which should be useful in furthering our understanding of the hepatic function and in the identification of components that may serve as biomarkers for hepatic alterations. Our analysis identifies a significant number of proteins previously described among exosomes derived from others cell types as well as proteins involved in metabolizing lipoproteins, endogenous compounds and xenobiotics, not previously described in exosomes. Furthermore, we demonstrated that exosomal membrane proteins can constitute an interesting tool to express non-exosomal proteins into exosomes with therapeutic purposes. PMID:19367702

Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

PubMed Central

Hüser, Daniela; Gogol-Döring, Andreas; Chen, Wei

2014-01-01

ABSTRACT Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. IMPORTANCE Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy. PMID:25031342

H{sub 2}S does not regulate proliferation via T-type Ca{sup 2+} channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elies, Jacobo; Johnson, Emily; Boyle, John P.

T-type Ca{sup 2+} channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca{sup 2+} channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca{sup 2+} channel Cav3.2 is selectively inhibited by hydrogen sulfide (H{sub 2}S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H{sub 2}S could account for the anti-proliferative effects of this gasotransmitter. H{sub 2}S suppressed proliferation inmore » HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H{sub 2}S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H{sub 2}S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca{sup 2+} channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H{sub 2}S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca{sup 2+} channel isoform was the H{sub 2}S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca{sup 2+} channel-mediated proliferation by H{sub 2}S is independent of the channels’ sensitivity to H{sub 2}S. - Highlights: • T-type Ca{sup 2+} channels regulate proliferation and are sensitive to the gasotransmitters CO and H{sub 2}S. • H{sub 2}S reduced proliferation in HEK293 cells expressing the H{sub 2}S sensitive Cav3.2 channel. • H{sub 2}S also inhibited proliferation in non-transfected cells and HEK293 cells expressing Cav3.1. • Native smooth muscle cells primarily express Cav3.1. Their proliferation was also inhibited by H{sub 2}S. • Unlike CO, H{sub 2}S does not regulate smooth muscle proliferation via T-type Ca{sup 2+} channel inhibition.« less

Biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach.

PubMed Central

Bogdanffy, M S

1990-01-01

An increasing number of chemicals have been identified as being toxic to the nasal mucosa of rats. While many chemicals exert their effects only after inhalation exposure, others are toxic following systemic administration, suggesting that factors other than direct deposition on the nasal mucosa may be important in mechanisms of nasal toxicity. The mucosal lining of the nasal cavity consists of a heterogeneous population of ciliated and nonciliated cells, secretory cells, sensory cells, and glandular and other cell types. For chemicals that are metabolized in the nasal mucosa, the balance between metabolic activation and detoxication within a cell type may be a key factor in determining whether that cell type will be a target for toxicity. Recent research in the area of xenobiotic metabolism in nasal mucosa has demonstrated the presence of many enzymes previously described in other tissues. In particular, carboxylesterase, aldehyde dehydrogenase, cytochromes P-450, epoxide hydrolase, and glutathione S-transferases have been localized by histochemical techniques. The distribution of these enzymes appears to be cell-type-specific and the presence of the enzyme may predispose particular cell types to enhanced susceptibility or resistance to chemical-induced injury. This paper reviews the distribution of these enzymes within the nasal mucosa in the context of their contribution to xenobiotic metabolism. The localization of the enzymes by histochemical techniques has provided important information on the potential mechanism of action of esters, aldehydes, and cytochrome P-450 substrates known to injure the nasal mucosa. Images PLATE 1. PLATE 2. PLATE 3. PMID:2200661

Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform

PubMed Central

Nishihara, Kana; Huang, Ruili; Zhao, Jinghua; Shahane, Sampada A.; Witt, Kristine L.; Smith-Roe, Stephanie L.; Tice, Raymond R.; Takeda, Shunichi; Xia, Menghang

2016-01-01

DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70 −/−/RAD54 −/− and REV3 −/−) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity—2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether—were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA. PMID:26243743

C-Terminal Region of EBNA-2 Determines the Superior Transforming Ability of Type 1 Epstein-Barr Virus by Enhanced Gene Regulation of LMP-1 and CXCR7

PubMed Central

Cancian, Laila; Bosshard, Rachel; Lucchesi, Walter; Karstegl, Claudio Elgueta; Farrell, Paul J.

2011-01-01

Type 1 Epstein-Barr virus (EBV) strains immortalize B lymphocytes in vitro much more efficiently than type 2 EBV, a difference previously mapped to the EBNA-2 locus. Here we demonstrate that the greater transforming activity of type 1 EBV correlates with a stronger and more rapid induction of the viral oncogene LMP-1 and the cell gene CXCR7 (which are both required for proliferation of EBV-LCLs) during infection of primary B cells with recombinant viruses. Surprisingly, although the major sequence differences between type 1 and type 2 EBNA-2 lie in N-terminal parts of the protein, the superior ability of type 1 EBNA-2 to induce proliferation of EBV-infected lymphoblasts is mostly determined by the C-terminus of EBNA-2. Substitution of the C-terminus of type 1 EBNA-2 into the type 2 protein is sufficient to confer a type 1 growth phenotype and type 1 expression levels of LMP-1 and CXCR7 in an EREB2.5 cell growth assay. Within this region, the RG, CR7 and TAD domains are the minimum type 1 sequences required. Sequencing the C-terminus of EBNA-2 from additional EBV isolates showed high sequence identity within type 1 isolates or within type 2 isolates, indicating that the functional differences mapped are typical of EBV type sequences. The results indicate that the C-terminus of EBNA-2 accounts for the greater ability of type 1 EBV to promote B cell proliferation, through mechanisms that include higher induction of genes (LMP-1 and CXCR7) required for proliferation and survival of EBV-LCLs. PMID:21857817

Cellular tropism of human enterovirus D species serotypes EV-94, EV-70, and EV-68 in vitro: implications for pathogenesis.

PubMed

Smura, Teemu; Ylipaasto, Petri; Klemola, Päivi; Kaijalainen, Svetlana; Kyllönen, Lauri; Sordi, Valeria; Piemonti, Lorenzo; Roivainen, Merja

2010-11-01

Enterovirus 94 (EV-94) is an enterovirus serotype described recently which, together with EV-68 and EV-70, forms human enterovirus D species. This study investigates the seroprevalences of these three serotypes and their abilities to infect, replicate, and damage cell types considered to be essential for enterovirus-induced diseases. The cell types studied included human leukocyte cell lines, primary endothelial cells, and pancreatic islets. High prevalence of neutralizing antibodies against EV-68 and EV-94 was found in the Finnish population. The virus strains studied had wide leukocyte tropism. EV-94 and EV-68 were able to produce infectious progeny in leukocyte cell lines with monocytic, granulocytic, T-cell, or B-cell characteristics. EV-94 and EV-70 were capable of infecting primary human umbilical vein endothelial cells, whereas EV-68 had only marginal progeny production and did not induce cytopathic effects in these cells. Intriguingly, EV-94 was able to damage pancreatic islet β-cells, to infect, replicate, and cause necrosis in human pancreatic islets, and to induce proinflammatory and chemoattractive cytokine expression in endothelial cells. These results suggest that HEV-D viruses may be more prevalent than has been thought previously, and they provide in vitro evidence that EV-94 may be a potent pathogen and should be considered a potentially diabetogenic enterovirus type. © 2010 Wiley-Liss, Inc.

A patient with progressive myelopathy and antibodies to human T-cell leukemia virus type I and human immunodeficiency virus type 1 in serum and cerebrospinal fluid.

PubMed

Aboulafia, D M; Saxton, E H; Koga, H; Diagne, A; Rosenblatt, J D

1990-04-01

A 52-year-old human immunodeficiency virus type 1-seropositive bisexual black man was evaluated at UCLA because of the recent onset of progressive lower-extremity weakness. Initial neurologic examination showed that the patient's distal weakness was greater than his proximal weakness, with bilateral foot drop and electrophysiologic evidence of denervation in the distal lower extremities. Magnetic resonance imaging of the brain and spinal cord disclosed no abnormalities. Subsequent neurologic evaluation 8 months later showed a myelopathy, with progression of lower-extremity weakness, spasticity, and flexor spasms, and urinary incontinence, as well as the peripheral neuropathy noted previously. A second magnetic resonance imaging scan of the brain showed patchy foci of increased signal intensity in white matter and cortex, with mild generalized cerebral and cerebellar atrophy and no lesions in the spinal cord. Specimens of the patient's serum and cerebrospinal fluid contained antibodies to human immunodeficiency virus type 1. Additionally, specimens of his serum and cerebrospinal fluid were tested for antibody to human T-cell leukemia virus type I by Western blotting and radioimmunoprecipitation, and found to be positive for human T-cell leukemia virus type I gag, env, and tax antibodies. The primary cause of severe myelopathy in this patient may be infection with human T-cell leukemia virus type I rather than with human immunodeficiency virus type 1. Treatment with prednisolone resulted in improvement of the lower-extremity weakness, reduction in flexor spasms, and slower but significant improvement in urinary symptoms. Patients who are infected with human immunodeficiency virus type 1 and have unusual motor findings should be tested for concomitant human T-cell leukemia virus type I infection.

Proteomic analysis of single mammalian cells enabled by microfluidic nanodroplet sample preparation and ultrasensitive nanoLC-MS.

PubMed

Zhu, Ying; Clair, Geremy; Chrisler, William; Shen, Yufeng; Zhao, Rui; Shukla, Anil; Moore, Ronald; Misra, Ravi; Pryhuber, Gloria; Smith, Richard; Ansong, Charles; Kelly, Ryan T

2018-05-24

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analysed by ultrasensitive nanoLC-MS. An average of ~670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Are Carotid Stent Fractures Clinically Significant?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Toca, Manuel; Rodriguez, Heron E.; Naughton, Peter A.

2012-04-15

Purpose: Late stent fatigue is a known complication after carotid artery stenting (CAS) for cervical carotid occlusive disease. The purpose of this study was to determine the prevalence and clinical significance of carotid stent fractures. Materials and Methods: A single-center retrospective review of 253 carotid bifurcation lesions treated with CAS and mechanical embolic protection from April 2001 to December 2009 was performed. Stent integrity was analyzed by two independent observers using multiplanar cervical plain radiographs with fractures classified into the following types: type I = single strut fracture; type II = multiple strut fractures; type III = transverse fracture; andmore » type IV = transverse fracture with dislocation. Mean follow-up was 32 months. Results: Follow-up imaging was completed on 106 self-expanding nitinol stents (26 closed-cell and 80 open-cell stents). Eight fractures (7.5%) were detected (type I n = 1, type II n = 6, and type III n = 1). Seven fractures were found in open-cell stents (Precise n = 3, ViVEXX n = 2, and Acculink n = 2), and 1 fracture was found in a closed-cell stent (Xact n = 1) (p = 0.67). Only a previous history of external beam neck irradiation was associated with fractures (p = 0.048). No associated clinical sequelae were observed among the patients with fractures, and only 1 patient had an associated significant restenosis ({>=}80%) requiring reintervention. Conclusions: Late stent fatigue after CAS is an uncommon event and rarely clinically relevant. Although cell design does not appear to influence the occurrence of fractures, lesion characteristics may be associated risk factors.« less

Th17 polarized cells from nonobese diabetic mice following mycobacterial adjuvant immunotherapy delay type 1 diabetes.

PubMed

Nikoopour, Enayat; Schwartz, Jordan A; Huszarik, Katrina; Sandrock, Christian; Krougly, Olga; Lee-Chan, Edwin; Singh, Bhagirath

2010-05-01

IL-17-producing T cells are regarded as potential pathogenic T cells in the induction of autoimmune diseases. Previously, we have shown that injection of adjuvants containing Mycobacterium, such as CFA or bacillus Calmette-Guérin, can prevent type 1 diabetes in NOD mice. We injected NOD mice with mycobacterial products s.c. and analyzed the IL-17-producing cells from the draining lymph nodes and spleen by restimulating whole-cell populations or CD4(+) T cells in vitro with or without IL-17-polarizing cytokines. Mice receiving CFA had a concomitant rise in the level of IL-17, IL-22, IL-10, and IFN-gamma in the draining lymph node and spleen. Adoptive transfer of splenocytes from CFA-injected NOD mice polarized with TGF-beta plus IL-6 or IL-23 delayed the development of diabetes in recipient mice. IL-17-producing cells induced by CFA maintained their IL-17-producing ability in the recipient mice. Injection of CFA also changed the cytokine profile of cells in pancreatic tissue by increasing IL-17, IL-10, and IFN-gamma cytokine gene expression. We suggest that the rise in the level of IL-17 after adjuvant therapy in NOD mice has a protective effect on type 1 diabetes development.

Espin cytoskeletal proteins in the sensory cells of rodent taste buds

PubMed Central

Sekerková, Gabriella; Freeman, David; Mugnaini, Enrico; Bartles, James R.

2010-01-01

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste bud cells contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of rat circumvallate taste buds. In confocal images, we counted 21.5±0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7±1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP3R3),α-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP3R3, PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP3R3, α-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of ~3 μm-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10-12 μm into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells. PMID:16841162

KSP inhibitor ARRY-520 as a substitute for Paclitaxel in Type I ovarian cancer cells

PubMed Central

Kim, Ki Hyung; Xie, Yanhua; Tytler, Ewan M; Woessner, Richard; Mor, Gil; Alvero, Ayesha B

2009-01-01

Background We previously described a sub-population of epithelial ovarian cancer (EOC) cells with a functional TLR-4/MyD88/NF-κB pathway (Type I EOC cells), which confers the capacity to respond to Paclitaxel, a known TLR-4 ligand, by enhancing NF-κB activity and upregulating cytokine secretion – events that are known to promote tumor progression. It is therefore important to distinguish those patients that should not receive Paclitaxel; it is also important to identify alternative chemotherapy options that would benefit this sub-group of patients. The objective of this study is to determine if the KSP inhibitor, ARRY-520, can be a substitute for Paclitaxel in patients with Type I EOC. Methods EOC cells isolated from either ascites or tumor tissue were treated with increasing concentrations of ARRY-520 or Paclitaxel and cell viability determined. Activation of the apoptotic pathway was determined using Western blot analysis. Mitochondrial integrity was quantified using JC1 dye. Cytokine profiling was performed from supernatants using xMAP technology. NF-κB activity was measured using a Luciferase reporter system. In vivo activity was determined using a subcutaneous xenograft mouse model. Results ARRY-520 and Paclitaxel exhibited the same cytotoxic effect on Type I and II cells. The GI50 at 48 h for Type II EOC cells was 0.0015 μM and 0.2 μM for ARRY-520 and Paclitaxel, respectively. For Type I EOC cells, the GI50 at 48 h was > 3 μM and >20 μM for ARRY-520 and Paclitaxel, respectively. Decrease in the number of viable cells was accompanied by mitochondrial depolarization and caspase activation. Unlike Paclitaxel, ARRY-520 did not induce NF-κB activation, did not enhance cytokine secretion, nor induce ERK phosphorylation in Type I EOC cells. Conclusion Administration of Paclitaxel to patients with high percentage Type I cancer cells could have detrimental effects due to Paclitaxel-induced enhancement of NF-κB and ERK activities, and cytokine production (e.g. IL-6), which promote chemoresistance and tumor progression. ARRY-520 has similar anti-tumor activity in EOC cells as that of Paclitaxel. However, unlike Paclitaxel, it does not induce these pro-tumor effects in Type I cells. Therefore, the KSP inhibitor ARRY-520 may represent an alternative to Paclitaxel in this subgroup of EOC patients. PMID:19619321

Indole-3-Acetic Acid Is Produced by Emiliania huxleyi Coccolith-Bearing Cells and Triggers a Physiological Response in Bald Cells.

PubMed

Labeeuw, Leen; Khey, Joleen; Bramucci, Anna R; Atwal, Harjot; de la Mata, A Paulina; Harynuk, James; Case, Rebecca J

2016-01-01

Indole-3-acetic acid (IAA) is an auxin produced by terrestrial plants which influences development through a variety of cellular mechanisms, such as altering cell orientation, organ development, fertility, and cell elongation. IAA is also produced by bacterial pathogens and symbionts of plants and algae, allowing them to manipulate growth and development of their host. They do so by either producing excess exogenous IAA or hijacking the IAA biosynthesis pathway of their host. The endogenous production of IAA by algae remains contentious. Using Emiliania huxleyi, a globally abundant marine haptophyte, we investigated the presence and potential role of IAA in algae. Homologs of genes involved in several tryptophan-dependent IAA biosynthesis pathways were identified in E. huxleyi. This suggests that this haptophyte can synthesize IAA using various precursors derived from tryptophan. Addition of L-tryptophan to E. huxleyi stimulated IAA production, which could be detected using Salkowski's reagent and GC × GC-TOFMS in the C cell type (coccolith bearing), but not in the N cell type (bald). Various concentrations of IAA were exogenously added to these two cell types to identify a physiological response in E. huxleyi. The N cell type, which did not produce IAA, was more sensitive to it, showing an increased variation in cell size, membrane permeability, and a corresponding increase in the photosynthetic potential quantum yield of Photosystem II (PSII). A roseobacter (bacteria commonly associated with E. huxleyi) Ruegeria sp. R11, previously shown to produce IAA, was co-cultured with E. huxleyi C and N cells. IAA could not be detected from these co-cultures, and even when stimulated by addition of L-tryptophan, they produced less IAA than axenic C type culture similarly induced. This suggests that IAA plays a novel role signaling between different E. huxleyi cell types, rather than between a bacteria and its algal host.

Putrescine importer PlaP contributes to swarming motility and urothelial cell invasion in Proteus mirabilis.

PubMed

Kurihara, Shin; Sakai, Yumi; Suzuki, Hideyuki; Muth, Aaron; Phanstiel, Otto; Rather, Philip N

2013-05-31

Previously, we reported that the speA gene, encoding arginine decarboxylase, is required for swarming in the urinary tract pathogen Proteus mirabilis. In addition, this previous study suggested that putrescine may act as a cell-to-cell signaling molecule (Sturgill, G., and Rather, P. N. (2004) Mol. Microbiol. 51, 437-446). In this new study, PlaP, a putative putrescine importer, was characterized in P. mirabilis. In a wild-type background, a plaP null mutation resulted in a modest swarming defect and slightly decreased levels of intracellular putrescine. In a P. mirabilis speA mutant with greatly reduced levels of intracellular putrescine, plaP was required for the putrescine-dependent rescue of swarming motility. When a speA/plaP double mutant was grown in the presence of extracellular putrescine, the intracellular levels of putrescine were greatly reduced compared with the speA mutant alone, indicating that PlaP functioned as the primary putrescine importer. In urothelial cell invasion assays, a speA mutant exhibited a 50% reduction in invasion when compared with wild type, and this defect could be restored by putrescine in a PlaP-dependent manner. The putrescine analog Triamide-44 partially inhibited the uptake of putrescine by PlaP and decreased both putrescine stimulated swarming and urothelial cell invasion in a speA mutant.

Projection specificity in heterogeneous locus coeruleus cell populations: implications for learning and memory

PubMed Central

Uematsu, Akira; Tan, Bao Zhen

2015-01-01

Noradrenergic neurons in the locus coeruleus (LC) play a critical role in many functions including learning and memory. This relatively small population of cells sends widespread projections throughout the brain including to a number of regions such as the amygdala which is involved in emotional associative learning and the medial prefrontal cortex which is important for facilitating flexibility when learning rules change. LC noradrenergic cells participate in both of these functions, but it is not clear how this small population of neurons modulates these partially distinct processes. Here we review anatomical, behavioral, and electrophysiological studies to assess how LC noradrenergic neurons regulate these different aspects of learning and memory. Previous work has demonstrated that subpopulations of LC noradrenergic cells innervate specific brain regions suggesting heterogeneity of function in LC neurons. Furthermore, noradrenaline in mPFC and amygdala has distinct effects on emotional learning and cognitive flexibility. Finally, neural recording data show that LC neurons respond during associative learning and when previously learned task contingencies change. Together, these studies suggest a working model in which distinct and potentially opposing subsets of LC neurons modulate particular learning functions through restricted efferent connectivity with amygdala or mPFC. This type of model may provide a general framework for understanding other neuromodulatory systems, which also exhibit cell type heterogeneity and projection specificity. PMID:26330494

Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer.

PubMed

Chung, Suyoun; Suzuki, Hanae; Miyamoto, Takashi; Takamatsu, Naofumi; Tatsuguchi, Ayako; Ueda, Koji; Kijima, Kyoko; Nakamura, Yusuke; Matsuo, Yo

2012-12-01

We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also reported to be highly upregulated in multiple types of human cancer. It was implied to play indispensable roles in cancer cell survival and indicated its involvement in the maintenance of tumor-initiating cells. We conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC₅₀ of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin-like), which we identified as novel MELK substrates and are important for stem-cell characteristics and invasiveness. The compound suppressed mammosphere formation of breast cancer cells and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. This MELK inhibitor should be a promising compound possibly to suppress the growth of tumor-initiating cells and be applied for treatment of a wide range of human cancer.

Nanosilica induced dose-dependent cytotoxicity and cell type-dependent multinucleation in HepG2 and L-02 cells

NASA Astrophysics Data System (ADS)

Yu, Yongbo; Duan, Junchao; Li, Yang; Yu, Yang; Hu, Hejing; Wu, Jing; Zhang, Yannan; Li, Yanbo; CaixiaGuo; Zhou, Xianqing; Sun, Zhiwei

2016-11-01

The prevalent exposure to nanosilica gained concerns about health effects of these particles on human beings. Although nanosilica-induced multinucleation has been confirmed previously, the underlying mechanism was still not clear; this study was to investigate the origination of multinucleated cells caused by nanosilica (62 nm) in both HepG2 and L-02 cells. Cell viability and cellular uptake was determined by MTT assay and transmission electron microscope (TEM), respectively. Giemsa staining was applied to detect multinucleation. To clarify the origination of multinucleated cells, fluorescent probes, PKH26 and PKH67, time-lapse observation were further conducted by confocal microscopy. Results indicated that nanosilica particles were internalized into cells and induced cytotoxicity in a dose-dependent manner. Quantification analysis showed that nanosilica significantly increased the rates of binucleated and multinucleated cells, which suggested mitotic catastrophe induction. Moreover, dynamic visualization verified that multinucleation resulted from cell fusion in HepG2 cells not in L-02 cells after nanosilica exposure, suggesting cell type-dependent multinucleation formation. Both multinucleation and cell fusion were involved in genetic instability, which emphasized the significance to explore the multinucleation induced by nanosilica via environmental, occupational and consumer product exposure.

Type II cGMP‑dependent protein kinase inhibits the migration, invasion and proliferation of several types of human cancer cells.

PubMed

Wu, Min; Wu, Yan; Qian, Hai; Tao, Yan; Pang, Ji; Wang, Ying; Chen, Yongchang

2017-10-01

Previous studies have indicated that type II cyclic guanosine monophosphate (cGMP)‑dependent protein kinase (PKG II) could inhibit the proliferation and migration of gastric cancer cells. However, the effects of PKG II on the biological functions of other types of cancer cells remain to be elucidated. Therefore, the aim of the present study was to investigate the effects of PKG II on cancer cells derived from various types of human tissues, including A549 lung, HepG2 hepatic, OS‑RC‑2 renal, SW480 colon cancer cells and U251 glioma cells. Cancer cells were infected with adenoviral constructs coding PKG II (Ad‑PKG II) to up‑regulate PKG II expression, and treated with 8‑(4‑chlorophenylthio) (8‑pCPT)‑cGMP to activate the kinase. A Cell Counting kit 8 assay was used to detect cell proliferation. Cell migration was measured using a Transwell assay, whereas a terminal deoxynucleotidyl transferase 2'‑deoxyuridine, 5'‑triphosphate nick‑end labeling assay was used to detect cell apoptosis. A pull‑down assay was used to investigate the activation of Ras‑related C3 botulinum toxin substrate (Rac) 1 and western blotting was used to detect the expression of proteins of interest. The present results demonstrated that EGF (100 ng/ml, 24 h) promoted the proliferation and migration of cancer cells, and it suppressed their apoptosis. In addition, treatment with EGF enhanced the activation of Rac1, and up‑regulated the protein expression of proliferating cell nuclear antigen, matrix metalloproteinase (MMP)2, MMP7 and B‑cell lymphoma (Bcl)‑2, whereas it down‑regulated the expression of Bcl‑2‑associated X protein. Transfection of cancer cells with Ad‑PKG II, and PKG II activation with 8‑pCPT‑cGMP, was identified to counteract the effects triggered by EGF. The present results suggested that PKG II may exert inhibitory effects on the proliferation and migration of various types of cancer cells.

Adenocarcinoma arising from intracranial recurrent mature teratoma and featuring mutated KRAS and wild-type BRAF genes.

PubMed

Kim, Eun Soo; Kwon, Mi Jung; Song, Joon Ho; Kim, Dong Hoon; Park, Hye-Rim

2015-02-01

Malignant transformation or recurrence of intracranial mature teratoma is an extremely rare occurrence, compared to the usual ovarian counterpart. Previously, yolk sac tumor elements have been considered to be selective progenitors of enteric-type adenocarcinoma arising from intracranial germ cell tumors. However, the present case demonstrates the occurrence of enteric-type adenocarcinoma in recurrent intracranial mature cystic teratoma 12 years after gross total removal, a case of which has not previously been documented in the literature. The 11.5-cm long, dura mater-based tumor on the right fronto-temporal lobe displaced the brain; however, the patient had no neurologic symptoms or discomfort other than pus-like discharge on the scalp. Microscopic examinations revealed a small focus of adenocarcinoma and dysplastic colonic mucosa in the mature cystic teratoma. No immature elements were seen. The cystic wall was almost denuded and showed an exuberant xanthogranulomatous reaction with foreign-body type giant cells engulfing keratin materials and cholesterol clefts, suggesting that chronic inflammation due to repeated cyst wall rupture and the previous resection may contribute to malignant transformation. The adenocarcinoma showed strong immunohistochemical expression of CK20 and p53, but CK7 in patches. The molecular profile of the adenocarcinoma showed a mutation in KRAS and wild-type BRAF, which might be associated with malignant transformation of intracranial mature teratomas. In conclusion, the intracranial mature teratomas should require long-term follow-up, and clinicians, radiologists and pathologists should be aware of the potential for malignant progression of recurrent intracranial mature cystic teratoma despite gross total resection and no neurologic symptoms. © 2014 Japanese Society of Neuropathology.

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells.

PubMed

Li, Bing; Su, Trent; Ferrari, Roberto; Li, Jing-Yu; Kurdistani, Siavash K

2014-02-01

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.

The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host-Cell Interaction.

PubMed

Gil-Bona, Ana; Reales-Calderon, Jose A; Parra-Giraldo, Claudia M; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

2016-01-01

Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a "veil growth," never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain.

The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host–Cell Interaction

PubMed Central

Gil-Bona, Ana; Reales-Calderon, Jose A.; Parra-Giraldo, Claudia M.; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

2016-01-01

Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a “veil growth,” never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain. PMID:26870022

Visual analysis of mass cytometry data by hierarchical stochastic neighbour embedding reveals rare cell types.

PubMed

van Unen, Vincent; Höllt, Thomas; Pezzotti, Nicola; Li, Na; Reinders, Marcel J T; Eisemann, Elmar; Koning, Frits; Vilanova, Anna; Lelieveldt, Boudewijn P F

2017-11-23

Mass cytometry allows high-resolution dissection of the cellular composition of the immune system. However, the high-dimensionality, large size, and non-linear structure of the data poses considerable challenges for the data analysis. In particular, dimensionality reduction-based techniques like t-SNE offer single-cell resolution but are limited in the number of cells that can be analyzed. Here we introduce Hierarchical Stochastic Neighbor Embedding (HSNE) for the analysis of mass cytometry data sets. HSNE constructs a hierarchy of non-linear similarities that can be interactively explored with a stepwise increase in detail up to the single-cell level. We apply HSNE to a study on gastrointestinal disorders and three other available mass cytometry data sets. We find that HSNE efficiently replicates previous observations and identifies rare cell populations that were previously missed due to downsampling. Thus, HSNE removes the scalability limit of conventional t-SNE analysis, a feature that makes it highly suitable for the analysis of massive high-dimensional data sets.

Immune biosensors based on the SPR and TIRE: efficiency of their application for bacteria determination

NASA Astrophysics Data System (ADS)

Starodub, N. F.; Ogorodniichuk, J.; Lebedeva, T.; Shpylovyy, P.

2013-11-01

In this work we have designed high-specific biosensors for Salmonella typhimurium detection based on the surface plasmon resonance (SPR) and total internal reflection ellipsometry (TIRE). It has been demonstrated high selectivity and sensitivity of analysis. As a registering part for our experiments the Spreeta (USA) and "Plasmonotest" (Ukraine) with flowing cell have been applied among of SPR device. Previous researches confirmed an efficiency of SPR biosensors using for detecting of specific antigen-antibody interactions therefore this type of reactions with some previous preparations of surface binding layer was used as reactive part. It has been defined that in case with Spreeta sensitivity was on the level 103 - 107 cells/ml. Another biosensor based on the SPR has shown the sensitivity within 101 - 106 cells/ml. Maximal sensitivity was on the level of several cells in 10 ml (up to the fact that less than 5 cells) which has been obtained using the biosensor based on TIRE.

A common haplotype lowers PU.1 expression in myeloid cells and delays onset of Alzheimer's disease.

PubMed

Huang, Kuan-Lin; Marcora, Edoardo; Pimenova, Anna A; Di Narzo, Antonio F; Kapoor, Manav; Jin, Sheng Chih; Harari, Oscar; Bertelsen, Sarah; Fairfax, Benjamin P; Czajkowski, Jake; Chouraki, Vincent; Grenier-Boley, Benjamin; Bellenguez, Céline; Deming, Yuetiva; McKenzie, Andrew; Raj, Towfique; Renton, Alan E; Budde, John; Smith, Albert; Fitzpatrick, Annette; Bis, Joshua C; DeStefano, Anita; Adams, Hieab H H; Ikram, M Arfan; van der Lee, Sven; Del-Aguila, Jorge L; Fernandez, Maria Victoria; Ibañez, Laura; Sims, Rebecca; Escott-Price, Valentina; Mayeux, Richard; Haines, Jonathan L; Farrer, Lindsay A; Pericak-Vance, Margaret A; Lambert, Jean Charles; van Duijn, Cornelia; Launer, Lenore; Seshadri, Sudha; Williams, Julie; Amouyel, Philippe; Schellenberg, Gerard D; Zhang, Bin; Borecki, Ingrid; Kauwe, John S K; Cruchaga, Carlos; Hao, Ke; Goate, Alison M

2017-08-01

A genome-wide survival analysis of 14,406 Alzheimer's disease (AD) cases and 25,849 controls identified eight previously reported AD risk loci and 14 novel loci associated with age at onset. Linkage disequilibrium score regression of 220 cell types implicated the regulation of myeloid gene expression in AD risk. The minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, showed association with delayed AD onset and lower expression of SPI1 in monocytes and macrophages. SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability was enriched within the PU.1 cistrome, implicating a myeloid PU.1 target gene network in AD. Finally, experimentally altered PU.1 levels affected the expression of mouse orthologs of many AD risk genes and the phagocytic activity of mouse microglial cells. Our results suggest that lower SPI1 expression reduces AD risk by regulating myeloid gene expression and cell function.

Embryonic hematopoiesis in vertebrate somites gives rise to definitive hematopoietic stem cells

PubMed Central

Qiu, Juhui; Fan, Xiaoying; Wang, Yixia; Jin, Hongbin; Song, Yixiao; Han, Yang; Huang, Shenghong; Meng, Yaping; Tang, Fuchou; Meng, Anming

2016-01-01

Hematopoietic stem cells (HSCs) replenish all types of blood cells. It is debating whether HSCs in adults solely originate from the aorta-gonad-mesonephros (AGM) region, more specifically, the dorsal aorta, during embryogenesis. Here, we report that somite hematopoiesis, a previously unwitnessed hematopoiesis, can generate definitive HSCs (dHSCs) in zebrafish. By transgenic lineage tracing, we found that a subset of cells within the forming somites emigrate ventromedially and mix with lateral plate mesoderm-derived primitive hematopoietic cells before the blood circulation starts. These somite-derived hematopoietic precursors and stem cells (sHPSCs) subsequently enter the circulation and colonize the kidney of larvae and adults. RNA-seq analysis reveals that sHPSCs express hematopoietic genes with sustained expression of many muscle/skeletal genes. Embryonic sHPSCs transplanted into wild-type embryos expand during growth and survive for life time with differentiation into various hematopoietic lineages, indicating self-renewal and multipotency features. Therefore, the embryonic origin of dHSCs in adults is not restricted to the AGM. PMID:27252540

Systematic analysis of protein turnover in primary cells.

PubMed

Mathieson, Toby; Franken, Holger; Kosinski, Jan; Kurzawa, Nils; Zinn, Nico; Sweetman, Gavain; Poeckel, Daniel; Ratnu, Vikram S; Schramm, Maike; Becher, Isabelle; Steidel, Michael; Noh, Kyung-Min; Bergamini, Giovanna; Beck, Martin; Bantscheff, Marcus; Savitski, Mikhail M

2018-02-15

A better understanding of proteostasis in health and disease requires robust methods to determine protein half-lives. Here we improve the precision and accuracy of peptide ion intensity-based quantification, enabling more accurate protein turnover determination in non-dividing cells by dynamic SILAC-based proteomics. This approach allows exact determination of protein half-lives ranging from 10 to >1000 h. We identified 4000-6000 proteins in several non-dividing cell types, corresponding to 9699 unique protein identifications over the entire data set. We observed similar protein half-lives in B-cells, natural killer cells and monocytes, whereas hepatocytes and mouse embryonic neurons show substantial differences. Our data set extends and statistically validates the previous observation that subunits of protein complexes tend to have coherent turnover. Moreover, analysis of different proteasome and nuclear pore complex assemblies suggests that their turnover rate is architecture dependent. These results illustrate that our approach allows investigating protein turnover and its implications in various cell types.

Conditioned medium as a strategy for human stem cells chondrogenic differentiation.

PubMed

Alves da Silva, M L; Costa-Pinto, A R; Martins, A; Correlo, V M; Sol, P; Bhattacharya, M; Faria, S; Reis, R L; Neves, Nuno M

2015-06-01

Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow-derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly-derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three-dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage-related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.

Islet-Derived CD4 T Cells Targeting Proinsulin in Human Autoimmune Diabetes

PubMed Central

Michels, Aaron W.; Landry, Laurie G.; McDaniel, Kristen A.; Yu, Liping; Campbell-Thompson, Martha; Kwok, William W.; Jones, Kenneth L.; Gottlieb, Peter A.; Kappler, John W.; Tang, Qizhi; Roep, Bart O.; Atkinson, Mark A.; Mathews, Clayton E.

2017-01-01

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing β-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19–35, and two clones from separate donors responded to insulin B-chain amino acids 9–23 (B:9–23), which are known to be a critical self-antigen–driving disease progress in animal models of autoimmune diabetes. These B:9–23–specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9–23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment. PMID:27920090

Immunohistochemical analysis of cytochrome P4501A induction in organs and cell types of Rivulus marmoratus exposed to waterborne 2,3,7,8-tetrachlorodibenzo-p-dioxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stegeman, J.; Smolowitz, R.; Burnett, K.

1994-12-31

Identifying target cells and organs is critical to establishing the sites and mechanisms of toxicity of Ah-receptor agonists. Previous studies have described the localization of CYPLA induced in multiple organs of fish exposed to Ah-receptor agonists. Here the authors compare the responses in multiple cell types and organs of small fish (Rivulus) exposed to waterborne TCDD. Adult fish were exposed to TCDD at concentrations from 0.01 to 10 ng/liter for 48 hours, then prepared and analyzed by immunohistochemistry with monoclonal antibody to teleost CYPIAI. At the highest dose profound induction was detected in virtually every organ. Structures staining intensely were:more » nasal and cephalic chemoreceptors, including sensory and basal cells; superficial cells in skin and pharynx; cartilage cells (chondrocytes) in the head, gills, growth plates and fins; epithelial and endothelial cells of liver, gut, kidney, and gill; pseudobranch vessels and glandular cells; eye lens epithelium; endothelium in vessels of eye, brain, skin, muscle, thymus and gonad. Lesser concentrations of TCDD elicited less strong responses, and control fish showed mild staining only in cartilage structures. The dose-dependent patterns of induction differed between different cell types. Responsive cells identified is these fish indicate sites where toxicity associated with Ah-receptor agonists or with CYPLA function may be expressed.« less

Checkpoint independence of most DNA replication origins in fission yeast

PubMed Central

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells. Conclusion The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks. PMID:18093330

The genetic locus NRC-1 within chromosome 3p12 mediates tumor suppression in renal cell carcinoma independently of histological type, tumor microenvironment, and VHL mutation.

PubMed

Lovell, M; Lott, S T; Wong, P; El-Naggar, A; Tucker, S; Killary, A M

1999-05-01

Human chromosome 3p cytogenetic abnormalities and loss of heterozygosity have been observed at high frequency in the nonpapillary form of sporadic renal cell carcinoma (RCC). The von Hippel-Lindau (VHL) gene has been identified as a tumor suppressor gene for RCC at 3p25, and functional studies as well as molecular genetic and cytogenetic analyses have suggested as many as two or three additional regions of 3p that could harbor tumor suppressor genes for sporadic RCC. We have previously functionally defined a novel genetic locus nonpapillary renal carcinoma-1 (NRC-1) within chromosome 3p12, distinct from the VHL gene, that mediates tumor suppression and rapid cell death of RCC cells in vivo. We now report the suppression of tumorigenicity of RCC cells in vivo after the transfer of a defined centric 3p fragment into different histological types of RCC. Results document the functional involvement of NRC-1 in not only different cell types of RCC (i.e., clear cell, mixed granular cell/clear cell, and sarcomatoid types) but also in papillary RCC, a less frequent histological type of RCC for which chromosome 3p LOH and genetic aberrations have only rarely been observed. We also report that the tumor suppression observed in functional genetic screens was independent of the microenvironment of the tumor, further supporting a role for NRC-1 as a more general mediator of in vivo growth control. Furthermore, this report demonstrates the first functional evidence for a VHL-independent pathway to tumorigenesis in the kidney via the genetic locus NRC-1.

Systemic immunological tolerance to ocular antigens is mediated by TRAIL-expressing CD8+ T cells.

PubMed

Griffith, Thomas S; Brincks, Erik L; Gurung, Prajwal; Kucaba, Tamara A; Ferguson, Thomas A

2011-01-15

Systemic immunological tolerance to Ag encountered in the eye restricts the formation of potentially damaging immune responses that would otherwise be initiated at other anatomical locations. We previously demonstrated that tolerance to Ag administered via the anterior chamber (AC) of the eye required Fas ligand-mediated apoptotic death of inflammatory cells that enter the eye in response to the antigenic challenge. Moreover, the systemic tolerance induced after AC injection of Ag was mediated by CD8(+) regulatory T cells. This study examined the mechanism by which these CD8(+) regulatory T cells mediate tolerance after AC injection of Ag. AC injection of Ag did not prime CD4(+) T cells and led to increased TRAIL expression by splenic CD8(+) T cells. Unlike wild-type mice, Trail(-/-) or Dr5(-/-) mice did not develop tolerance to Ag injected into the eye, even though responding lymphocytes underwent apoptosis in the AC of the eyes of these mice. CD8(+) T cells from Trail(-/-) mice that were first injected via the AC with Ag were unable to transfer tolerance to naive recipient wild-type mice, but CD8(+) T cells from AC-injected wild-type or Dr5(-/-) mice could transfer tolerance. Importantly, the transferred wild-type (Trail(+/+)) CD8(+) T cells were also able to decrease the number of infiltrating inflammatory cells into the eye; however, Trail(-/-) CD8(+) T cells were unable to limit the inflammatory cell ingress. Together, our data suggest that "helpless" CD8(+) regulatory T cells generated after AC injection of Ag enforce systemic tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye.

MANF silencing, immunity induction or autophagy trigger an unusual cell type in metamorphosing Drosophila brain.

PubMed

Stratoulias, Vassilis; Heino, Tapio I

2015-05-01

Glia are abundant cells in the brain of animals ranging from flies to humans. They perform conserved functions not only in neural development and wiring, but also in brain homeostasis. Here we show that by manipulating gene expression in glia, a previously unidentified cell type appears in the Drosophila brain during metamorphosis. More specifically, this cell type appears in three contexts: (1) after the induction of either immunity, or (2) autophagy, or (3) by silencing of neurotrophic factor DmMANF in glial cells. We call these cells MANF immunoreactive Cells (MiCs). MiCs are migratory based on their shape, appearance in brain areas where no cell bodies exist and the nuclear localization of dSTAT. They are labeled with a unique set of molecular markers including the conserved neurotrophic factor DmMANF and the transcription factor Zfh1. They possess the nuclearly localized protein Relish, which is the hallmark of immune response activation. They also express the conserved engulfment receptor Draper, therefore indicating that they are potentially phagocytic. Surprisingly, they do not express any of the common glial and neuronal markers. In addition, ultrastructural studies show that MiCs are extremely rich in lysosomes. Our findings reveal critical molecular and functional components of an unusual cell type in the Drosophila brain. We suggest that MiCs resemble macrophages/hemocytes and vertebrate microglia based on their appearance in the brain upon genetically challenged conditions and the expression of molecular markers. Interestingly, macrophages/hemocytes or microglia-like cells have not been reported in the fly nervous system before.

Generalized time-dependent model of radiation-induced chromosomal aberrations in normal and repair-deficient human cells.

PubMed

Ponomarev, Artem L; George, Kerry; Cucinotta, Francis A

2014-03-01

We have developed a model that can simulate the yield of radiation-induced chromosomal aberrations (CAs) and unrejoined chromosome breaks in normal and repair-deficient cells. The model predicts the kinetics of chromosomal aberration formation after exposure in the G₀/G₁ phase of the cell cycle to either low- or high-LET radiation. A previously formulated model based on a stochastic Monte Carlo approach was updated to consider the time dependence of DNA double-strand break (DSB) repair (proper or improper), and different cell types were assigned different kinetics of DSB repair. The distribution of the DSB free ends was derived from a mechanistic model that takes into account the structure of chromatin and DSB clustering from high-LET radiation. The kinetics of chromosomal aberration formation were derived from experimental data on DSB repair kinetics in normal and repair-deficient cell lines. We assessed different types of chromosomal aberrations with the focus on simple and complex exchanges, and predicted the DSB rejoining kinetics and misrepair probabilities for different cell types. The results identify major cell-dependent factors, such as a greater yield of chromosome misrepair in ataxia telangiectasia (AT) cells and slower rejoining in Nijmegen (NBS) cells relative to the wild-type. The model's predictions suggest that two mechanisms could exist for the inefficiency of DSB repair in AT and NBS cells, one that depends on the overall speed of joining (either proper or improper) of DNA broken ends, and another that depends on geometric factors, such as the Euclidian distance between DNA broken ends, which influences the relative frequency of misrepair.

Inhibition of l-type amino acid transporter 1 activity as a new therapeutic target for cholangiocarcinoma treatment.

PubMed

Yothaisong, Supak; Dokduang, Hasaya; Anzai, Naohiko; Hayashi, Keitaro; Namwat, Nisana; Yongvanit, Puangrat; Sangkhamanon, Sakkarn; Jutabha, Promsuk; Endou, Hitoshi; Loilome, Watcharin

2017-03-01

Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na + -independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [ 14 C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue-stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal's body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that suppression of l-type amino acid transporter-1 activity using JPH203 might be used as a new therapeutic strategy for cholangiocarcinoma treatment.

Characterization of developmental immature fiber (im) mutant and Texas Marker-1 (TM-1) cotton fibers by Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) spectroscopy

USDA-ARS?s Scientific Manuscript database

The immature fiber (im) mutant is one type of cotton fiber mutants with unique characteristics of non-fluffy cotton bolls. Compared to its near-isogenic wild type Texas Marker-1 (TM-1), im fiber has thin secondary cell wall and is less mature. In this work, we applied the previously proposed princip...

Fluorescent-Magnetic-Biotargeting Multifunctional Nanobioprobes for Detecting and Isolating Multiple Types of Tumor Cells

PubMed Central

Song, Er-Qun; Hu, Jun; Wen, Cong-Ying; Tian, Zhi-Quan; Yu, Xu; Zhang, Zhi-Ling; Shi, Yun-Bo; Pang, Dai-Wen

2011-01-01

Fluorescent-magnetic-biotargeting multifunctional nanobioprobes (FMBMNs) have attracted great attention in recent years due to their increasing, important applications in biomedical research, clinical diagnosis, and biomedicine. We have previously developed such nanobioprobes for the detection and isolation of a single kind of tumor cells. Detection and isolation of multiple tumor markers or tumor cells from complex samples sensitively and with high efficiency is critical for the early diagnosis of tumors, especially malignant tumors or cancers, which will improve clinical diagnosis outcomes and help to select effective treatment approaches. Here, we expanded the application of the monoclonal antibody (mAb)-coupled FMBMNs for multiplexed assays. Multiple types of cancer cells, such as leukemia cells and prostate cancer cells, were detected and collected from mixed samples within 25 minutes by using a magnet and an ordinary fluorescence microscope. The capture efficiencies of mAb-coupled FMBMNs for the above mentioned two types of cells were 96% and 97% respectively. Furthermore, by using the mAb-coupled FMBMNs, specific and sensitive detection and rapid separation of a small number of spiked leukemia cells and prostate cancer cells in a large population of cultured normal cells (about 0.01% were tumor cells) were achieved simply and inexpensively without any sample pretreatment before cell analysis. Therefore, mAb-coupled multicolour FMBMNs may be used for very sensitive detection and rapid isolation of multiple cancer cells in biomedical research and medical diagnostics. PMID:21250650

Epstein-Barr Virus Type 2 Infects T Cells in Healthy Kenyan Children.

PubMed

Coleman, Carrie B; Daud, Ibrahim I; Ogolla, Sidney O; Ritchie, Julie A; Smith, Nicholas A; Sumba, Peter O; Dent, Arlene E; Rochford, Rosemary

2017-09-15

The 2 strains of Epstein-Barr virus (EBV), EBV type 1 (EBV-1) and EBV-2, differ in latency genes, suggesting that they use distinct mechanisms to establish latency. We previously reported that EBV-2 infects T cells in vitro. In this study, we tested the possibility that EBV-2 infects T cells in vivo. Purified T-cell fractions isolated from children positive for EBV-1 or EBV-2 and their mothers were examined for the presence of EBV and for EBV type. We detected EBV-2 in all T-cell samples obtained from EBV-2-infected children at 12 months of age, with some children retaining EBV-2-positive T cells through 24 months of age, suggesting that EBV-2 persists in T cells. We were unable to detect EBV-2 in T-cell samples from mothers but could detect EBV-2 in samples of their breast milk and saliva. These data suggest that EBV-2 uses T cells as an additional latency reservoir but that, over time, the frequency of infected T cells may drop below detectable levels. Alternatively, EBV-2 may establish a prolonged transient infection in the T-cell compartment. Collectively, these novel findings demonstrate that EBV-2 infects T cells in vivo and suggest EBV-2 may use the T-cell compartment to establish latency. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Hematopoietic stem cells with controllable tEpoR transgenes have a competitive advantage in bone marrow transplantation.

PubMed

Kirby, S; Walton, W; Smithies, O

2000-06-15

In a previous study, it was found that a truncated erythropoietin receptor transgene (tEpoR tg) enables multilineage hematopoietic progenitor amplification after treatment with erythropoietin (epo) in vitro and in vivo. This study used competitive bone marrow (BM) repopulation to show that tEpoR tg facilitates transplantation by hematopoietic stem cells (HSC). Individual multilineage colonies, committed myeloid progenitor colonies, and lymphoid colonies (pre-B colony-forming units) were grown from the marrow of animals 6 months after they received a 50/50 mixture of transgene and wild-type BM cells. In epo-treated recipients, the transgene-bearing cells significantly outcompeted the wild-type cells (84%-100% versus 16%-0%, respectively). In recipients treated with phosphate-buffered saline, the repopulation was minimally different from the donor mixture (49%-64% transgene versus 51%-36% wild-type). The epo-induced repopulation advantage is maintained in secondary transplants. In addition, neither accelerated HSC depletion nor uncontrollable proliferation occurred during epo-stimulated serial transplants of transgene-containing BM. Thus, the tEpoR tg functions in a benign fashion in HSC and allows for a significant and controllable repopulation advantage in vivo without excessive HSC depletion relative to wild-type BM. (Blood. 2000;95:3710-3715)

Single-Cell Detection of Secreted Aβ and sAPPα from Human IPSC-Derived Neurons and Astrocytes.

PubMed

Liao, Mei-Chen; Muratore, Christina R; Gierahn, Todd M; Sullivan, Sarah E; Srikanth, Priya; De Jager, Philip L; Love, J Christopher; Young-Pearse, Tracy L

2016-02-03

Secreted factors play a central role in normal and pathological processes in every tissue in the body. The brain is composed of a highly complex milieu of different cell types and few methods exist that can identify which individual cells in a complex mixture are secreting specific analytes. By identifying which cells are responsible, we can better understand neural physiology and pathophysiology, more readily identify the underlying pathways responsible for analyte production, and ultimately use this information to guide the development of novel therapeutic strategies that target the cell types of relevance. We present here a method for detecting analytes secreted from single human induced pluripotent stem cell (iPSC)-derived neural cells and have applied the method to measure amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα), analytes central to Alzheimer's disease pathogenesis. Through these studies, we have uncovered the dynamic range of secretion profiles of these analytes from single iPSC-derived neuronal and glial cells and have molecularly characterized subpopulations of these cells through immunostaining and gene expression analyses. In examining Aβ and sAPPα secretion from single cells, we were able to identify previously unappreciated complexities in the biology of APP cleavage that could not otherwise have been found by studying averaged responses over pools of cells. This technique can be readily adapted to the detection of other analytes secreted by neural cells, which would have the potential to open new perspectives into human CNS development and dysfunction. We have established a technology that, for the first time, detects secreted analytes from single human neurons and astrocytes. We examine secretion of the Alzheimer's disease-relevant factors amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα) and present novel findings that could not have been observed without a single-cell analytical platform. First, we identify a previously unappreciated subpopulation that secretes high levels of Aβ in the absence of detectable sAPPα. Further, we show that multiple cell types secrete high levels of Aβ and sAPPα, but cells expressing GABAergic neuronal markers are overrepresented. Finally, we show that astrocytes are competent to secrete high levels of Aβ and therefore may be a significant contributor to Aβ accumulation in the brain. Copyright © 2016 the authors 0270-6474/16/361730-17$15.00/0.

Overexpression of the ADP (E3-11.6K) protein increases cell lysis and spread of adenovirus.

PubMed

Doronin, Konstantin; Toth, Karoly; Kuppuswamy, Mohan; Krajcsi, Peter; Tollefson, Ann E; Wold, William S M

2003-01-20

Adenoviruses replicate in the nucleus and induce lytic cell death. We have shown previously that efficient cell lysis and release of adenovirus from infected cells requires an 11.6-kDa protein named Adenovirus Death Protein (ADP). The adp gene is located in the early E3 transcription unit, but the gene is expressed primarily at very late stages of infection. The putative function of ADP was discerned previously from the use of virus mutants that lack functional ADP. Here we describe two adenovirus mutants, named VRX-006 and VRX-007, that overexpress ADP. VRX-006 lacks all other genes in the E3 region, and VRX-007 lacks all other E3 genes except 12.5K. VRX-006 and VRX-007 display the phenotype predicted by the proposed function for ADP: they produce early cytopathic effect, early cell lysis, large plaques, and increased cell-to-cell spread. They grow as well in cultured cells as does adenovirus type 5. These results are consistent with the conclusion that ADP functions in adenovirus infections to promote virus release from cells at the culmination of infection.

The Herbal Medicine Cordyceps sinensis Protects Pancreatic Beta Cells from Streptozotocin-Induced Endoplasmic Reticulum Stress.

PubMed

Liu, Hong; Cao, Diyong; Liu, Hua; Liu, Xinghai; Mai, Wenli; Lan, Haitao; Huo, Wen; Zheng, Qian

2016-08-01

Our previous work found that Cordyceps sinensis (CS) improves the activity and secretory function of pancreatic islet beta cells. The objective was to observe a further possible role of CS in the protection of insulin-secreting cells. A rat model of type 2 diabetes mellitus was developed with streptozotocin (STZ) and a high-energy fat diet (HFD). CS was administered in the successful model of rats with type 2 diabetes. After 4 weeks, the biochemistry index of blood samples was measured, and pathologic observation was performed by immunohistochemistry. In the rats with type 2 diabetes induced by a HFD and STZ, the levels of fasting blood glucose and fasting insulin were elevated, and the insulin sensitivity index was decreased. Pathologic examination found an increased number of apoptotic cells, an elevated protein expression of pro-apoptotic C/EBP homologous protein (CHOP) and an increased c-Jun level by means of JNK phosphorylation, responsive to the endoplasmic reticulum stress of islet beta cells. With treatment by CS for 4 weeks, the elevated levels of both fasting blood glucose and fasting insulin in the rats with type 2 diabetes were significantly lower, and the decreased insulin sensitivity index was reversed. Compared to the control rats with type 2 diabetes, CS application significantly reduced the number of apoptotic cells and decreased protein expression of both CHOP and c-Jun. The herbal compound CS could protect pancreatic beta cells from the pro-apoptotic endoplasmic reticulum stress induced by HFD-STZ. This suggests an alternative approach to treating type 2 diabetes. Copyright © 2016 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

Versatility and nuances of the architecture of haematopoiesis - Implications for the nature of leukaemia.

PubMed

Brown, Geoffrey; Hughes, Philip J; Ceredig, Rhodri; Michell, Robert H

2012-01-01

For many years there was a widely accepted picture of how a haematopoietic stem cell (HSC) gives rise to the multiple types of blood and immune cells. This described the general nature of stem and progenitor cells and the pathways of cell development. Recent years have seen many attempts to re-draw the map of haematopoiesis. These have become increasingly complex, and they often envisage multiples routes to some cell types. The 'established' view that self-renewal in haematopoiesis only occurs in HSCs has been challenged by the recognition of self-renewing HSC-derived progenitor cells that display at least some fate restriction. This evolution of how normal haematopoiesis is viewed has inevitable implications for understanding the origins, disease progression and classification of the leukaemias. In essence, some progenitor cells are now seen as possessing a larger repertoire of routes to end-fates than was previously thought. This leads one to ask whether leukaemia stem cells are equally or less versatile than their normal counterparts? Copyright © 2011 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujimura, Hiroaki

Mating pheromones, a- and {alpha}-factors, arrest the division of cells of opposite mating types, {alpha} and a cells, respectively. The author has isolated a sterile mutant of Saccharomyces cerevisiae using EMS that is defective in division arrest in response to {alpha}-factor but not defective in morphological changes and agglutinin induction. The mutation was designated dac2 for division arrest control by mating pheromones. The dac2 mutation was closely linked to gal1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11, ste12, ste18, and dac1). Although dac2 cells had no phenotype in the absence ofmore » pheromones, they showed morphological alterations and divided continuously in the presence of pheromones. As a result, dac2 cells had a mating defect. The dac2 mutation could suppress the lethality caused by the disruption of the GPA1 gene. These results suggest that the DAC2 product may control the signal for G-protein-mediated cell-cycle arrest and indicate that the synchronization of haploid yeast cell cycles by mating pheromones is essential for cell fusion during conjugation.« less

MicroRNA-139 suppresses proliferation in luminal type breast cancer cells by targeting Topoisomerase II alpha

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Wei; State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi'an; Sa, Ke-Di

The classification of molecular subtypes of breast cancer improves the prognostic accuracy and therapeutic benefits in clinic. However, because of the complexity of breast cancer, more biomarkers and functional molecules need to be explored. Here, analyzing the data in a huge cohort of breast cancer patients, we found that Topoisomerase II alpha (TOP2a), an important target of chemotherapy is a biomarker for prognosis in luminal type breast cancer patients, but not in basal like or HER2 positive breast cancer patients. We identified that miR-139, a previous reported anti-metastatic microRNA targets 3’-untranslated region (3′UTR) of TOP2a mRNA. Further more, we revealedmore » that the forced expression of miR-139 reduces the TOP2a expression at both mRNA and protein levels. And our functional experiments showed that the ectopic expression of miR-139 remarkably inhibits proliferation in luminal type breast cancer cells, while exogenous TOP2a expression could rescue inhibition of cell proliferation mediated by miR-139. Collectively, our present study demonstrates the miR-139-TOP2a regulatory axis is important for proliferation in luminal type breast cancer cells. This functional link may help us to further understand the specificity of subtypes of breast cancer and optimize the strategy of cancer treatment. - Highlights: • High levels of TOP2a expression are closely associated with poor prognosis in luminal type breast cancer patients. • TOP2a is a novel target of miR-139. • Overexpression of miR-139 inhibits proliferation in luminal type breast cancer cells. • TOP2a is essential for miR-139-induced growth arrest in luminal type breast cancer cells.« less

Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase.

PubMed

Miyazawa, Ken; Yoshimi, Akira; Zhang, Silai; Sano, Motoaki; Nakayama, Mayumi; Gomi, Katsuya; Abe, Keietsu

2016-09-01

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.

Overexpression of NGF ameliorates ethanol neurotoxicity in the developing cerebellum.

PubMed

Heaton, M B; Mitchell, J J; Paiva, M

2000-11-05

Transgenic mice overexpressing NGF in the central nervous system under the control of the glial fibrillary acidic protein (GFAP) promoter were exposed to ethanol via vapor inhalation on postnatal days 4 and 5 (P4-5), the period of maximal cerebellar Purkinje cell sensitivity to ethanol. Wild-type controls were exposed in a similar manner. There were no differences in body weight or size following these procedures, but the transgenic brain weights at this age were significantly greater than wild-type controls. In the wild-type animals, a significant 33.3% ethanol-mediated loss of Purkinje cells in lobule I was detected via unbiased three-dimensional stereological counting on P5. In the GFAP-NGF transgenic animals, however, the 17.6% difference in Purkinje cell number in control and ethanol-exposed animals was not significant. There was a similar difference in Purkinje cell density in both groups, which did reach statistical significance (-32.7% in wild-type ethanol-treated animals, -17% in transgenic ethanol-exposed animals). These results suggest that endogenous overexpression of neurotrophic factors, which have previously been shown to protect against ethanol neurotoxicity in culture, can serve a similar protective function in the intact animal. Copyright 2000 John Wiley & Sons, Inc.

The Ron Receptor Regulates Kupffer Cell-Dependent Cytokine Production and Hepatocyte Survival Following Endotoxin Exposure in Mice

PubMed Central

Stuart, William D.; Kulkarni, Rishikesh M.; Gray, Jerilyn K.; Vasiliauskas, Juozas; Leonis, Mike A.; Waltz, Susan E.

2011-01-01

Previous studies demonstrated that targeted deletion of the Ron receptor tyrosine kinase (TK) domain in mice leads to marked hepatocyte protection in a well-characterized model of lipopolysaccharide (LPS)-induced acute liver failure in D-galactosamine (GalN)-sensitized mice. Hepatocyte protection in TK−/− mice was observed despite paradoxically elevated serum levels of tumor necrosis factor alpha (TNFα). To understand the role of Ron in the liver, purified populations of Kupffer cells and hepatocytes from wild-type (TK+/+) and TK−/− mice were studied. Utilizing quantitative RT-PCR, we demonstrated that Ron is expressed in these cell-types. Moreover, we also recapitulated the protected hepatocyte phenotype and exaggerated cytokine production observed in the TK−/− mice in vivo through the use of purified cultured cells ex vivo. We show that isolated TK−/− Kupffer cells produce increased levels of TNFα and select cytokines compared to TK+/+ cells following LPS stimulation. We also show that conditioned media from LPS-treated TK−/− Kupffer cells was more toxic to hepatocytes than control media, suggesting the exaggerated levels of cytokines produced from the TK−/− Kupffer cells are detrimental to wild type hepatocytes. In addition, we observed that TK−/− hepatocytes were more resistant to cell death compared to TK+/+ hepatocytes, suggesting that Ron functions in both the epithelial and inflammatory cell compartments to regulate acute liver injury. These findings were confirmed in vivo in mice with hepatocyte and macrophage cell-type-specific conditional Ron deletions. Mice with Ron loss selectively in hepatocytes exhibited less liver damage and increased survival compared to mice with Ron loss in macrophages. In conclusion, we have dissected cell-type-specific roles for Ron such that this receptor modulates cytokine production from Kupffer cells and inhibits hepatocyte survival in response to injury. PMID:21520175

Role of STAT1 in Chlamydia-Induced Type-1 Interferon Production in Oviduct Epithelial Cells

PubMed Central

Hosey, Kristen Lynette; Hu, Sishun

2015-01-01

We previously reported that Chlamydia muridarum-infected murine oviduct epithelial cells (OE cells) secrete interferon β (IFN-β) in a mostly TLR3-dependent manner. However, C. muridarum-infected TLR3-deficient OE cells were still able to secrete detectable levels of IFN-β into the supernatants, suggesting that other signaling pathways contribute to Chlamydia-induced IFN-β synthesis in these cells. We investigated the role of STAT1 as a possible contributor in the Chlamydia-induced type-1 IFN production in wild-type (WT) and TLR3-deficient OE cells to ascertain its putative role at early- and late-times during Chlamydia infection. Our data show that C. muridarum infection significantly increased STAT1 gene expression and protein activation in WT OE cells; however, TLR3-deficient OE cells showed diminished STAT1 protein activation and gene expression. There was significantly less IFN-β detected in the supernatants of C. muridarum-infected OE cells derived from mice deficient in STAT1 when compared with WT OE cells, which suggest that STAT1 is required for the optimal synthesis of IFN-β during infection. Real-time quantitative polymerase chain reaction analyses of signaling components of the type-1 IFN signaling pathway demonstrated equal upregulation in the expression of STAT2 and IRF7 genes in the WT and TLR3-deficient OE cells, but no upregulation in these genes in the STAT1-deficient OE cells. Finally, experiments in which INFAR1 was blocked with neutralizing antibody revealed that IFNAR1-mediated signaling was critical to the Chlamydia-induced upregulation in IFN-α gene transcription, but had no role in the Chlamydia-induced upregulation in IFN-β gene transcription. PMID:26262558

Variable promoter methylation contributes to differential expression of key genes in human placenta-derived venous and arterial endothelial cells.

PubMed

Joo, Jihoon E; Hiden, Ursula; Lassance, Luciana; Gordon, Lavinia; Martino, David J; Desoye, Gernot; Saffery, Richard

2013-07-15

The endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. The molecular mechanisms underpinning this process in utero have yet to be fully elucidated. The aim of this study was to investigate the potential for DNA methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells. Placenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. DNA methylation was measured at >450,000 CpG sites in parallel with expression measurements taken from 25,000 annotated genes. A consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. This included many loci previously not investigated in relation to endothelial function. An inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including NOS3, encoding endothelial Nitric Oxide Synthase. Endothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes. These are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life.

Lack of HXK2 induces localization of active Ras in mitochondria and triggers apoptosis in the yeast Saccharomyces cerevisiae.

PubMed

Amigoni, Loredana; Martegani, Enzo; Colombo, Sonia

2013-01-01

We recently showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type cells growing exponentially on glucose, while in the hxk2Δ strain they accumulated mainly in mitochondria. An aberrant accumulation of activated Ras in these organelles was previously reported and correlated to mitochondrial dysfunction, accumulation of ROS, and cell death. Here we show that addition of acetic acid to wild-type cells results in a rapid recruitment of Ras-GTP from the nucleus and the plasma membrane to the mitochondria, providing a further proof that Ras proteins might be involved in programmed cell death. Moreover, we show that Hxk2 protects against apoptosis in S. cerevisiae. In particular, cells lacking HXK2 and showing a constitutive accumulation of activated Ras at the mitochondria are more sensitive to acetic-acid-induced programmed cell death compared to the wild type strain. Indeed, deletion of HXK2 causes an increase of apoptotic cells with several morphological and biochemical changes that are typical of apoptosis, including DNA fragmentation, externalization of phosphatidylserine, and ROS production. Finally, our results suggest that apoptosis induced by lack of Hxk2 may not require the activation of Yca1, the metacaspase homologue identified in yeast.

RBE of Energetic Iron Ions for the Induction of Early and Late Chromosome Aberrations in Different Cell Types

NASA Technical Reports Server (NTRS)

Zhang, Ye; Yeshitla, Samrawit; Hada, Megumi; Kadhim, Munira; Wilson, Bobby; Wu, Honglu

2015-01-01

Numerous published studies have reported the Relative Biological Effectiveness (RBE) values for chromosome aberrations induced by charged particles of different LET. The RBE for chromosome aberrations in human lymphocytes exposed ex vivo has been suggested to show a similar relationship as the quality factor for cancer induction. Therefore, increased chromosome aberrations in the astronauts' white blood cells post long-duration missions are used to determine the biological doses from exposures to space radiation. However, the RBE value is known to be very different for different types of cancer. Previously, we reported that, even though the RBE for initial chromosome damages was high in human lymphocytes exposed to Fe ions, the RBE was significantly reduced after multiple cell divisions post irradiation. To test the hypothesis that RBE values for chromosome aberrations are cell type dependent, and different between early and late damages, we exposed human lymphocytes ex vivo, and human mammary epithelial cells in vitro to various charged particles. Chromosome aberrations were quantified using the samples collected at first mitosis post irradiation for initial damages, and the samples collected after multiple generations for the remaining or late arising aberrations. Results of the study suggested that the effectiveness of high-LET charged particles for late chromosome aberrations may be cell type dependent, even though the RBE values are similar for early damages.

Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity

USGS Publications Warehouse

Park, J.-W.; Moon, C.H.; Harmache, A.; Wargo, A.R.; Purcell, M.K.; Bremont, M.; Kurath, G.

2011-01-01

Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 μm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 μm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

Aseptic cure of pulmonary paracoccidioidomycosis can be achieved after a previous subcutaneous immunization of susceptible but not resistant mice.

PubMed

Arruda, Celina; Vaz, Celidéia A C; Calich, Vera L G

2007-05-01

The murine model of paracoccidioidomycosis, the most important South American endemic mycosis, mimics the human disease: resistance is associated with preserved cellular immunity while T-cell anergy is related with susceptibility. In the present study we asked whether a previous s.c. infection which induces strong cellular immunity would protect mice against a lethal pulmonary challenge. It was found that susceptible but not resistant mice developed immunoprotection and aseptic cure of infection. Immunoprotection led to reversal of DTH anergy, increased levels of antibodies and pulmonary IL-12, IL-2 and IL-4 indicating a balanced type 1/type 2 response. On the contrary, no marked differences in A/Sn infection and immunity were observed. Depletion experiments showed that immunoprotection required the cooperative action of CD4(+) and CD8(+) T cells in association with IFN-gamma and IL-12. Altogether, these observations demonstrated that susceptible hosts can develop sterilizing immunity and defined the main immunological requirements to control secondary paracoccidioidomycosis.

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

PubMed Central

Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F.; Mistretta, Charlotte M.; Mishina, Yuji; Liu, Hong-Xiang

2016-01-01

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC. PMID:26741369

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

PubMed

Boggs, Kristin; Venkatesan, Nandakumar; Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F; Mistretta, Charlotte M; Mishina, Yuji; Liu, Hong-Xiang

2016-01-01

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

Characteristics of chromosome instability in the human lymphoblast cell line WTK1

NASA Technical Reports Server (NTRS)

Schwartz, J. L.; Jordan, R.; Evans, H. H.

2001-01-01

The characteristics of spontaneous and radiation-induced chromosome instability were determined in each of 50 individual clones isolated from control populations of human lymphoblasts (WTK1), as well as from populations of these cells previously exposed to two different types of ionizing radiation, Fe-56 and Cs-137. The types of chromosome instability did not appear to change in clones surviving radiation exposure. Aneuploidy, polyploidy, chromosome dicentrics and translocations, and chromatid breaks and gaps were found in both control and irradiated clones. The primary effect of radiation exposure was to increase the number of cells within any one clone that had chromosome alterations. Chromosome instability was associated with telomere shortening and elevated levels of apoptosis. The results suggest that the proximal cause of chromosome instability is telomere shortening.

Defining the Diverse Cell Populations Contributing to Lignification in Arabidopsis Stems.

PubMed

Smith, Rebecca A; Schuetz, Mathias; Karlen, Steven D; Bird, David; Tokunaga, Naohito; Sato, Yasushi; Mansfield, Shawn D; Ralph, John; Samuels, A Lacey

2017-06-01

Many land plants evolved tall and sturdy growth habits due to specialized cells with thick lignified cell walls: tracheary elements that function in water transport and fibers that function in structural support. The objective of this study was to define how and when diverse cell populations contribute lignin precursors, monolignols, to secondary cell walls during lignification of the Arabidopsis ( Arabidopsis thaliana ) inflorescence stem. Previous work demonstrated that, when lignin biosynthesis is suppressed in fiber and tracheary element cells with thickened walls, fibers become lignin-depleted while vascular bundles still lignify, suggesting that nonlignifying neighboring xylem cells are contributing to lignification. In this work, we dissect the contributions of different cell types, specifically xylary parenchyma and fiber cells, to lignification of the stem using cell-type-specific promoters to either knock down an essential monolignol biosynthetic gene or to introduce novel monolignol conjugates. Analysis of either reductions in lignin in knockdown lines, or the addition of novel monolignol conjugates, directly identifies the xylary parenchyma and fiber cell populations that contribute to the stem lignification and the developmental timing at which each contribution is most important. © 2017 American Society of Plant Biologists. All Rights Reserved.

Cyclic phosphatidic acid induces G0/G1 arrest, inhibits AKT phosphorylation, and downregulates cyclin D1 expression in colorectal cancer cells.

PubMed

Tsukahara, Tamotsu; Haniu, Hisao; Matsuda, Yoshikazu

2015-03-01

Lysophosphatidic acid (LPA) and its analogs are well-known mitogens for various cell types. Many reports have confirmed that several types of cancer cell produce LPA to promote survival, growth and tumorigenesis. This indicates that the interface between the LPA signaling pathway and the cell cycle signaling system is critical to the control of cancer cell proliferation. However, our previous study indicated that cyclic phosphatidic acid (cPA), which is structurally similar to LPA, inhibits the proliferation and migration of colon cancer cells. It has been reported that cPA shows several biological activities not shown by LPA. However, understanding of the detailed molecular and cellular mechanism underlying the regulation of the cell cycle by cPA is still in its infancy. In this study, we investigated the effect of cPA treatment on human DLD-1 colon cancer cells by analyzing cell cycle dynamics, gene expression, and AKT phosphorylation. Our findings indicate that cPA inhibits cell cycle progression in DLD-1 colon cancer cells via the downregulation of cyclin D1 and the inhibition of AKT phosphorylation.

Measuring cell viscoelastic properties using a force-spectrometer: influence of protein-cytoplasm interactions

PubMed Central

Canetta, Elisabetta; Duperray, Alain; Leyrat, Anne; Verdier, Claude

2005-01-01

Cell adhesive and rheological properties play a very important role in cell transmigration through the endothelial barrier, in particular in the case of inflammation (leukocytes) or cancer metastasis (cancer cells). In order to characterize cell viscoelastic properties, we have designed a force spectrometer (AFM) which can stretch cells thereby allowing measurement of their rheological properties. This custom-made force spectrometer allows two different visualizations, one lateral and one from below. It allows investigation of the effects of rheology involved during cell stretching. To test the ability of our system to characterize such viscoelastic properties, ICAM-1 transfected CHO cells were analyzed. Two forms of ICAM-1 were tested; wild type ICAM-1, which can interact with the cytoskeleton, and a mutant form which lacks the cytoplasmic domain, and is unable to associate with the cytoskeleton. Stretching experiments carried out on these cells show the formation of long filaments. Using a previous model of filament elongation, we could determine the viscoelastic properties of a single cell. As expected, different viscoelastic components were found between the wild type and the mutant, which reveal that the presence of interactions between ICAM-1 and the cytoskeleton increases the stiffness of the cell. PMID:16308464

Measuring cell viscoelastic properties using a force-spectrometer: influence of protein-cytoplasm interactions.

PubMed

Canetta, Elisabetta; Duperray, Alain; Leyrat, Anne; Verdier, Claude

2005-01-01

Cell adhesive and rheological properties play a very important role in cell transmigration through the endothelial barrier, in particular in the case of inflammation (leukocytes) or cancer metastasis (cancer cells). In order to characterize cell viscoelastic properties, we have designed a force spectrometer (AFM) which can stretch cells thereby allowing measurement of their rheological properties. This custom-made force spectrometer allows two different visualizations, one lateral and one from below. It allows investigation of the effects of rheology involved during cell stretching. To test the ability of our system to characterize such viscoelastic properties, ICAM-1 transfected CHO cells were analyzed. Two forms of ICAM-1 were tested; wild type ICAM-1, which can interact with the cytoskeleton, and a mutant form which lacks the cytoplasmic domain, and is unable to associate with the cytoskeleton. Stretching experiments carried out on these cells show the formation of long filaments. Using a previous model of filament elongation, we could determine the viscoelastic properties of a single cell. As expected, different viscoelastic components were found between the wild type and the mutant, which reveal that the presence of interactions between ICAM-1 and the cytoskeleton increases the stiffness of the cell.

Comparison of hematological alterations and markers of B-cell activation in workers exposed to benzene, formaldehyde and trichloroethylene

PubMed Central

Bassig, Bryan A.; Zhang, Luoping; Vermeulen, Roel; Tang, Xiaojiang; Li, Guilan; Hu, Wei; Guo, Weihong; Purdue, Mark P.; Yin, Songnian; Rappaport, Stephen M.; Shen, Min; Ji, Zhiying; Qiu, Chuangyi; Ge, Yichen; Hosgood, H.Dean; Reiss, Boris; Wu, Banghua; Xie, Yuxuan; Li, Laiyu; Yue, Fei; Freeman, Laura E.Beane; Blair, Aaron; Hayes, Richard B.; Huang, Hanlin; Smith, Martyn T.; Rothman, Nathaniel; Lan, Qing

2016-01-01

Benzene, formaldehyde (FA) and trichloroethylene (TCE) are ubiquitous chemicals in workplaces and the general environment. Benzene is an established myeloid leukemogen and probable lymphomagen. FA is classified as a myeloid leukemogen but has not been associated with non-Hodgkin lymphoma (NHL), whereas TCE has been associated with NHL but not myeloid leukemia. Epidemiologic associations between FA and myeloid leukemia, and between benzene, TCE and NHL are, however, still debated. Previously, we showed that these chemicals are associated with hematotoxicity in cross-sectional studies of factory workers in China, which included extensive personal monitoring and biological sample collection. Here, we compare and contrast patterns of hematotoxicity, monosomy 7 in myeloid progenitor cells (MPCs), and B-cell activation biomarkers across these studies to further evaluate possible mechanisms of action and consistency of effects with observed hematologic cancer risks. Workers exposed to benzene or FA, but not TCE, showed declines in cell types derived from MPCs, including granulocytes and platelets. Alterations in lymphoid cell types, including B cells and CD4+ T cells, and B-cell activation markers were apparent in workers exposed to benzene or TCE. Given that alterations in myeloid and lymphoid cell types are associated with hematological malignancies, our data provide biologic insight into the epidemiological evidence linking benzene and FA exposure with myeloid leukemia risk, and TCE and benzene exposure with NHL risk. PMID:27207665

Inflammatory pseudo-tumours of the abdomen: plasma cell granulomas

PubMed Central

Wu, Jane P.; Yunis, Eduardo J.; Fetterman, George; Jaeschke, Walter F.; Gilbert, Enid F.

1973-01-01

Pseudo-tumours of the plasma cell granuloma type are reported in two patients. One was retroperitoneal and the other intraabdominal. Most of the cases of plasma cell granulomas described in the literature have been in the lung (Brunn, 1939; Childress and Adie, 1950; Cotton, 1952; Umiker and Iverson, 1954; Lane, Krohn, Kolozai, and Whitehead, 1955; Liebow and Hubbell, 1956; Titus, Harrison, Clagett, Anderson, and Knaff, 1962; Mason, Keats, and Baker, 1963; Wentworth, Lynch, Fallis, Turner, Lowden, and Conen, 1968; Bahadori and Liebow, 1973). A retroperitoneal site has not to our knowledge been reported previously. The postinflammatory nature of such lesions and the significance of a previous history of abdominal surgery are emphasized. It is important to be aware of such benign lesions which may simulate malignant tumours so that unnecessary radical treatment can be avoided. Images PMID:4784503

Fabric-based alkaline direct formate microfluidic fuel cells.

PubMed

Domalaon, Kryls; Tang, Catherine; Mendez, Alex; Bernal, Franky; Purohit, Krutarth; Pham, Linda; Haan, John; Gomez, Frank A

2017-04-01

Fabric-based microfluidic fuel cells (MFCs) serve as a novel, cost-efficient alternative to traditional FCs and batteries, since fluids naturally travel across fabric via capillary action, eliminating the need for an external pump and lowering production and operation costs. Building on previous research with Y-shaped paper-based MFCs, fabric-based MFCs mitigate fragility and durability issues caused by long periods of fuel immersion. In this study, we describe a microfluidic fabric-based direct formate fuel cell, with 5 M potassium formate and 30% hydrogen peroxide as the anode fuel and cathode oxidant, respectively. Using a two-strip, stacked design, the optimized parameters include the type of encasement, the barrier, and the fabric type. Surface contact of the fabric and laminate sheet expedited flow and respective chemical reactions. The maximum current (22.83 mA/cm 2 ) and power (4.40 mW/cm 2 ) densities achieved with a 65% cotton/35% polyester blend material are a respective 8.7% and 32% higher than previous studies with Y-shaped paper-based MFCs. In series configuration, the MFCs generate sufficient energy to power a handheld calculator, a thermometer, and a spectrum of light-emitting diodes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Back wall solar cell

NASA Technical Reports Server (NTRS)

Brandhorst, H. W., Jr. (Inventor)

1978-01-01

A solar cell is disclosed which comprises a first semiconductor material of one conductivity type with one face having the same conductivity type but more heavily doped to form a field region arranged to receive the radiant energy to be converted to electrical energy, and a layer of a second semiconductor material, preferably highly doped, of opposite conductivity type on the first semiconductor material adjacent the first semiconductor material at an interface remote from the heavily doped field region. Instead of the opposite conductivity layer, a metallic Schottky diode layer may be used, in which case no additional back contact is needed. A contact such as a gridded contact, previous to the radiant energy may be applied to the heavily doped field region of the more heavily doped, same conductivity material for its contact.

Limitations of Commercializing Fuel Cell Technologies

NASA Astrophysics Data System (ADS)

Nordin, Normayati

2010-06-01

Fuel cell is the technology that, nowadays, is deemed having a great potential to be used in supplying energy. Basically, fuel cells can be categorized particularly by the kind of employed electrolyte. Several fuel cells types which are currently identified having huge potential to be utilized, namely, Solid Oxide Fuel Cells (SOFC), Molten Carbonate Fuel Cells (MCFC), Alkaline Fuel Cells (AFC), Phosphoric Acid Fuel Cells (PAFC), Polymer Electron Membrane Fuel Cell (PEMFC), Direct Methanol Fuel Cells (DMFC) and Regenerative Fuel Cells (RFC). In general, each of these fuel cells types has their own characteristics and specifications which assign the capability and suitability of them to be utilized for any particular applications. Stationary power generations and transport applications are the two most significant applications currently aimed for the fuel cell market. It is generally accepted that there are lots of advantages if fuel cells can be excessively commercialized primarily in context of environmental concerns and energy security. Nevertheless, this is a demanding task to be accomplished, as there is some gap in fuel cells technology itself which needs a major enhancement. It can be concluded, from the previous study, cost, durability and performance are identified as the main limitations to be firstly overcome in enabling fuel cells technology become viable for the market.

Clonal Populations of Amniotic Cells by Dilution and Direct Plating: Evidence for Hidden Diversity

PubMed Central

Wilson, Patricia G.; Devkota, Lorna; Payne, Tiffany; Crisp, Laddie; Winter, Allison; Wang, Zhan

2012-01-01

Fetal cells are widely considered a superior cell source for regenerative medicine; fetal cells show higher proliferative capacity and have undergone fewer replicative cycles that could generate spontaneous mutations. Fetal cells in amniotic fluid were among the first normal primary cells to be cultured ex vivo, but the undefined composition of amniotic fluid has hindered advance for regenerative applications. We first developed a highly efficient method to generate clonal populations by dilution of amniocentesis samples in media and direct plating without intervening refrigeration, centrifugation, or exposure of cells to the paracrine effects in mixed cell cultures. More than 40 clonal populations were recovered from 4 amniocentesis samples and representative clones were characterized by flow cytometry, conventional assays for differentiation potential, immunofluorescence imaging, and transcript analysis. The results revealed previously unreported diversity among stromal and epithelial cell types and identified unique cell types that could be lost or undetected in mixed cell populations. The differentiation potential of amniotic cells proved to be uncoupled from expression of definitive cell surface or cytoplasmic markers for stromal and epithelial cells. Evidence for diversity among stromal and epithelial cells in amniotic fluid bears on interpretations applied to molecular and functional tests of amniotic cell populations. PMID:23024659

Radiation-induced genomic instability and bystander effects: related inflammatory-type responses to radiation-induced stress and injury? A review.

PubMed

Lorimore, S A; Wright, E G

2003-01-01

To review studies of radiation responses in the haemopoietic system in the context of radiation-induced genomic instability, bystander effects and inflammatory-type processes. There is considerable evidence that cells that themselves are not exposed to ionizing radiation but are the progeny of cells irradiated many cell divisions previously may express a high frequency of gene mutations, chromosomal aberrations and cell death. These effects are collectively known as radiation-induced genomic instability. A second untargeted effect results in non-irradiated cells exhibiting responses typically associated with direct radiation exposure but occurs as a consequence of contact with irradiated cells or by receiving soluble signals from irradiated cells. These effects are collectively known as radiation-induced bystander effects. Reported effects include increases or decreases in damage-inducible and stress-related proteins; increases or decreases in reactive oxygen species, cell death or cell proliferation, and induction of mutations and chromosome aberrations. This array of responses is reminiscent of effects mediated by cytokines and other similar regulatory factors that may involve, but do not necessarily require, gap junction-mediated transfer, have multiple inducers and a variety of context-dependent consequences in different cell systems. That chromosomal instability in haemopoietic cells can be induced by an indirect bystander-type mechanism both in vitro and in vivo provides a potential link between these two untargeted effects and there are radiation responses in vivo consistent with the microenvironment contributing secondary cell damage as a consequence of an inflammatory-type response to radiation-induced injury. Intercellular signalling, production of cytokines and free radicals are features of inflammatory responses that have the potential for both bystander-mediated and persisting damage as well as for conferring a predisposition to malignancy. The induction of bystander effects and instabilities may reflect interrelated aspects of a non-specific inflammatory-type response to radiation-induced stress and injury and be involved in a variety of the pathological consequences of radiation exposures.

NIFLUMIC ACID BLOCKS NATIVE AND RECOMBINANT T-TYPE CHANNELS

PubMed Central

Balderas, E; Arteaga-Tlecuitl, R; Rivera, M; Gomora, JC; Darszon, A

2012-01-01

Voltage-dependent calcium channels are widely distributed in animal cells, including spermatozoa. Calcium is fundamental in many sperm functions such as: motility, capacitation and the acrosome reaction, all essential for fertilization. Pharmacological evidence has suggested T-type calcium channels participate in the acrosome reaction. Niflumic acid (NA), a non-steroidal anti-inflammatory drug commonly used as chloride channel blocker, blocks T-currents in mouse spermatogenic cells and Cl− channels in testicular sperm. Here we examine the mechanism of NA blockade and explore if it can be used to separate the contribution of different CaV3 members previously detected in these cells. Electrophysiological patch-clamp recordings were performed in isolated mouse spermatogenic cells and in HEK cells heterologously expressing CaV3 channels. NA blocks mouse spermatogenic cell T-type currents with an IC50 of 73.5 µM, without major voltage-dependent effects. The NA blockade is more potent in the open and in the inactivated state than in the closed state of the T-type channels. Interestingly, we found that heterologously expressed CaV3.1 and CaV3.3 channels were more sensitive to NA than CaV3.2 channels, and this drug substantially slowed the recovery from inactivation of the three isoforms. Molecular docking modeling of drug-channel binding predicts that NA binds preferentially to the extracellular face of CaV3.1 channels. The biophysical characteristics of mouse spermatogenic cell T-type currents more closely resemble those from heterologously expressed CaV3.1 channels, including their sensitivity to NA. As CaV3.1 null mice maintain their spermatogenic cell T-currents, it is likely that a novel CaV3.2 isoform is responsible for them. PMID:21898399

Triterpenoid saponins from the roots of Clematis argentilucida.

PubMed

Zhao, Mei; Ma, Ning; Qiu, Feng; Tian, Xiangrong; Zhang, Yan; Tang, Haifeng; Liu, Xinyou

2014-09-01

Reinvestigation of the n-BuOH extract of the roots of Clematis argentilucida led to the isolation of a new ursane-type triterpenoid saponin 1 and a new taraxerane-type saponin 2, four known saponins 3-6 first isolated from the species, together with seven saponins 7-13 reported in the previous papers. The structures of saponins 1-6 were elucidated by extensive spectroscopic analysis and chemical evidences. The ursane-type and taraxerane-type triterpenoid saponins were obtained from genus Clematis for the first time, and the aglycone of saponin 1, 3β,28-dihydroxy-18αH-ursan-20-en was first encountered. The cytotoxicity of all the saponins was evaluated against human glioblastoma U251MG cell lines. The monodesmosidic saponins 1, 2 and 4-8 exhibited cytotoxic activity against the cells with IC50 values ranging from 6.95 to 38.51 μM. Copyright © 2014 Elsevier B.V. All rights reserved.

Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes.

PubMed

Deckmann, Klaus; Filipski, Katharina; Krasteva-Christ, Gabriela; Fronius, Martin; Althaus, Mike; Rafiq, Amir; Papadakis, Tamara; Renno, Liane; Jurastow, Innokentij; Wessels, Lars; Wolff, Miriam; Schütz, Burkhard; Weihe, Eberhard; Chubanov, Vladimir; Gudermann, Thomas; Klein, Jochen; Bschleipfer, Thomas; Kummer, Wolfgang

2014-06-03

Chemosensory cells in the mucosal surface of the respiratory tract ("brush cells") use the canonical taste transduction cascade to detect potentially hazardous content and trigger local protective and aversive respiratory reflexes on stimulation. So far, the urogenital tract has been considered to lack this cell type. Here we report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system, but not in further centrally located parts of the urinary tract, such as the bladder, ureter, and renal pelvis. Urethral brush cells express bitter and umami taste receptors and downstream components of the taste transduction cascade; respond to stimulation with bitter (denatonium), umami (monosodium glutamate), and uropathogenic Escherichia coli; and release acetylcholine to communicate with other cells. They are approached by sensory nerve fibers expressing nicotinic acetylcholine receptors, and intraurethral application of denatonium reflexively increases activity of the bladder detrusor muscle in anesthetized rats. We propose a concept of urinary bladder control involving a previously unidentified cholinergic chemosensory cell monitoring the chemical composition of the urethral luminal microenvironment for potential hazardous content.

Onecut1 and Onecut2 redundantly regulate early retinal cell fates during development

PubMed Central

Sapkota, Darshan; Chintala, Hemabindu; Wu, Fuguo; Fliesler, Steven J.; Hu, Zihua; Mu, Xiuqian

2014-01-01

Previously, we have shown that Onecut1 (Oc1) and Onecut2 (Oc2) are expressed in retinal progenitor cells, developing retinal ganglion cells (RGCs), and horizontal cells (HCs). However, in Oc1-null mice, we only observed an 80% reduction in HCs, but no defects in other cell types. We postulated that the lack of defects in other cell types in Oc1-null retinas was a result of redundancy with Oc2. To test this theory, we have generated Oc2-null mice and now show that their retinas also only have defects in HCs, with a 50% reduction in their numbers. However, when both Oc1 and Oc2 are knocked out, the retinas exhibit more profound defects in the development of all early retinal cell types, including completely failed genesis of HCs, compromised generation of cones, reduced production (by 30%) of RGCs, and absence of starburst amacrine cells. Cone subtype diversification and RGC subtype composition also were affected in the double-null retina. Using RNA-Seq expression profiling, we have identified downstream genes of Oc1 and Oc2, which not only confirms the redundancy between the two factors and renders a molecular explanation for the defects in the double-null retinas, but also shows that the onecut factors suppress the production of the late cell type, rods, indicating that the two factors contribute to the competence of retinal progenitor cells for the early retinal cell fates. Our results provide insight into how onecut factors regulate the creation of cellular diversity in the retina and, by extension, in the central nervous system in general. PMID:25228773

Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb.

PubMed

Neuveut, C; Low, K G; Maldarelli, F; Schmitt, I; Majone, F; Grassmann, R; Jeang, K T

1998-06-01

Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16(INK4a), thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16(INK4a), Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16(INK4a).

Type XVII collagen (BP180) can function as a cell-matrix adhesion molecule via binding to laminin 332

PubMed Central

Van den Bergh, F.; Eliason, S.L.; Giudice, G.J.

2010-01-01

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knockdown of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix. PMID:21034821

β-Catenin signaling regulates temporally discrete phases of anterior taste bud development

PubMed Central

Thirumangalathu, Shoba; Barlow, Linda A.

2015-01-01

The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of β-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of β-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of β-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of β-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, β-catenin activation a day later within Shh+ placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of β-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of β-catenin within Shh+ precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by β-catenin. PMID:26525674

Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments.

PubMed

Sandh, Gustaf; Ramström, Margareta; Stensjö, Karin

2014-12-04

In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts. Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme. The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.

Immunohistochemical detection of p53 protein in ameloblastoma types.

PubMed

el-Sissy, N A

1999-05-01

Overexpression of p53 protein in unicystic ameloblastoma (uAB) is denser than in the conventional ameloblastoma (cAB) type, indicating increased wild type p53--suppressing the growth potential of uAB and denoting the early event of neoplastic transformation, probably of a previous odontogenic cyst. Overexpression of p53 in borderline cAB and malignant ameloblastoma (mAB) types might reflect a mutational p53 protein playing an oncogenic role, promoting tumour growth. Overexpression of p53 protein could be a valid screening method for predicting underlying malignant genetic changes in AB types, through increased frequency of immunoreactive cells or increased staining density.

Efficiency of introns from various origins in fish cells.

PubMed

Bétancourt, O H; Attal, J; Théron, M C; Puissant, C; Houdebine, L M

1993-06-01

Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.

The efficacy of polycaprolactone/hydroxyapatite scaffold in combination with mesenchymal stem cells for bone tissue engineering.

PubMed

Chuenjitkuntaworn, Boontharika; Osathanon, Thanaphum; Nowwarote, Nunthawan; Supaphol, Pitt; Pavasant, Prasit

2016-01-01

Major drawbacks of using an autograft are the possibilities of insufficient bony source and patient's morbidity after operation. Bone tissue engineering technology, therefore, has been applied for repairing bony defects. Previous study showed that a novel fabricated 3D-Polycaprolactone/Hydroxyapatite (PCL/HAp) scaffold possessed a good biocompatibility for bone cells. This study aimed to determine the ability of PCL/HAp for supporting cell growth, gene expression, and osteogenic differentiation in three types of mesenchymal stem cells, including bone marrow-derived mesenchymal stem cells (BMSCs), dental pulp stem cells (DPSCs), and adiposed-derived mesenchymal stem cells (ADSCs). These were assessed by cell viability assay (MTT), reverse-transcription polymerase chain reaction (RT-PCR) analysis, alkaline phosphatase activity, and osteogenic differentiation by alizarin red-S staining. The results showed that PCL/HAp scaffold could support growth of all three types of mesenchymal stem cells. In addition, DPSCs with PCL/HAp showed the highest level of calcium deposition compared to other groups. In conclusion, DPSCs exhibited a better compatibility with these scaffolds compared to BMSCs and ADSCs. However, the PCL/HAp could be a good candidate scaffold for all tested mesenchymal stem cells in bone tissue engineering. © 2015 Wiley Periodicals, Inc.

Activity of human papillomavirus type 16 P97 promoter in immortal and tumorigenic human oral keratinocytes.

PubMed

Kook, J K; Kim, J H; Min, B M

1998-10-01

We previously immortalized normal human oral keratinocytes (NHOK) by transfection with cloned human papillomavirus type 16 (HPV-16) genome and converted these immortalized cells to tumorigenic cells with chemical carcinogens. Since the tumorigenic cells expressed higher level of HPV-16 E6/E7 transcripts, we predicted that enhanced E6/E7 expression was induced by mutations at the long control region (LCR) of the viral genome integrated into cellular chromosome. To test this possibility, we sequenced the entire HPV-16 LCR from immortalized and tumorigenic cells, but no difference in the sequences in all of the tested cells was observed. However, it is possible that such differences in the expression of E6/E7 could have originated from different activities of cellular transcription factors in the different cells. To examine this prospect, we subcloned entire LCR into a reporter gene and determined the promoter activity of LCR in immortalized and tumorigenic cells. We found that the LCR promoter activity was significantly higher in tumorigenic cells when comparing to immortalized cells. We also observed that at least 477 nucleotides upstream of E6 open reading frame are needed for the maximum LCR promoter activity in tumorigenic cells.

N-acyl Taurines and Acylcarnitines Cause an Imbalance in Insulin Synthesis and Secretion Provoking β Cell Dysfunction in Type 2 Diabetes.

PubMed

Aichler, Michaela; Borgmann, Daniela; Krumsiek, Jan; Buck, Achim; MacDonald, Patrick E; Fox, Jocelyn E Manning; Lyon, James; Light, Peter E; Keipert, Susanne; Jastroch, Martin; Feuchtinger, Annette; Mueller, Nikola S; Sun, Na; Palmer, Andrew; Alexandrov, Theodore; Hrabe de Angelis, Martin; Neschen, Susanne; Tschöp, Matthias H; Walch, Axel

2017-06-06

The processes contributing to β cell dysfunction in type 2 diabetes (T2D) are uncertain, largely because it is difficult to access β cells in their intact immediate environment. We examined the pathophysiology of β cells under T2D progression directly in pancreatic tissues. We used MALDI imaging of Langerhans islets (LHIs) within mouse tissues or from human tissues to generate in situ-omics data, which we supported with in vitro experiments. Molecular interaction networks provided information on functional pathways and molecules. We found that stearoylcarnitine accumulated in β cells, leading to arrest of insulin synthesis and energy deficiency via excessive β-oxidation and depletion of TCA cycle and oxidative phosphorylation metabolites. Acetylcarnitine and an accumulation of N-acyl taurines, a group not previously detected in β cells, provoked insulin secretion. Thus, β cell dysfunction results from enhanced insulin secretion combined with an arrest of insulin synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Pathways of cell-cell transmission of HTLV-1

PubMed Central

Pique, Claudine; Jones, Kathryn S.

2012-01-01

The deltaretroviruses human T cell lymphotropic virus type 1 (HTLV-1) and human T cell lymphotropic virus type 2 (HTLV-2) have long been believed to differ from retroviruses in other genera by their mode of transmission. While other retroviruses were thought to primarily spread by producing cell-free particles that diffuse through extracellular fluids prior to binding to and infecting target cells, HTLV-1 and HTLV-2 were believed to transmit the virus solely by cell–cell interactions. This difference in transmission was believed to reflect the fact that, relative to other retroviruses, the cell-free virions produced by HTLV-infected cells are very poorly infectious. Since HTLV-1 and HTLV-2 are primarily found in T cells in the peripheral blood, spread of these viruses was believed to occur between infected and uninfected, T cells, although little was known about the cellular and viral proteins involved in this interaction. Recent studies have revealed that the method of transmission of HTLV is not unique: other retroviruses including human immunodeficiency virus (HIV) are also transmitted from cell-to-cell, and this method is dramatically more efficient than cell-free transmission. Moreover, cell–cell transmission of HTLV-1, as well as HIV, can occur following interactions between dendritic cells and T cells, as well as between T cells. Conversely, other studies have shown that cell-free HTLV-1 is not as poorly infectious as previously thought, since it is capable of infecting certain cell types. Here we summarize the recent insights about the mechanisms of cell–cell transmission of HTLV-1 and other retroviruses. We also review in vitro and in vivo studies of infection and discuss how these finding may relate to the spread of HTLV-1 between individuals. PMID:23109932

Macroglial cells of the teleost central nervous system: a survey of the main types.

PubMed

Cuoghi, Barbara; Mola, Lucrezia

2009-12-01

Following our previous review of teleost microglia, we focus here on the morphological and histochemical features of the three principal macroglia types in the teleost central nervous system (ependymal cells, astrocyte-like cells/radial glia and oligodendrocytes). This review is concerned with recent literature and not only provides insights into the various individual aspects of the different types of macroglial cells plus a comparison with mammalian glia, but also indicates the several potentials that the neural tissue of teleosts exhibits in neurobiological research. Indeed, some areas of the teleost brain are particularly suitable in terms of the establishment of a "simple" but complete research model (i.e. the visual pathway complex and the supramedullary neuron cluster in puffer fish). The relationships between neurons and glial cells are considered in fish, with the aim of providing an integrated picture of the complex ways in which neurons and glia communicate and collaborate in normal and injured neural tissues. The recent setting up of successful protocols for fish glia and mixed neuron-glia cultures, together with the molecular facilities offered by the knowledge of some teleost genomes, should allow consistent input towards the achievement of this aim.

Adult T-cell leukemia-lymphoma in a pregnant woman diagnosed as a human T-cell lymphotropic virus type 1 carrier.

PubMed

Fuchi, Naoki; Miura, Kiyonori; Imaizumi, Yoshitaka; Hasegawa, Hiroo; Yanagihara, Katsunori; Miyazaki, Yasushi; Masuzaki, Hideaki

2016-03-01

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia-lymphoma (ATL), which is difficult to cure. In Japan, a nationwide HTLV-1 screening test in pregnant women has been recommended since 2011. A 30-year-old woman was diagnosed as being an HTLV-1 carrier in her previous pregnancy. During the current pregnancy, she had persistent fever and cough. Although she had treatment with antibiotics, peripheral white blood cell count remained high, with an abnormal lymphocyte count. Given that she was an HTLV-1 carrier, she was diagnosed with unfavorable chronic ATL (aggressive ATL) at 12 weeks gestation. After pregnancy termination, her ATL status became favorable chronic ATL (indolent ATL). Therefore, watchful waiting was performed until disease progression. This is the first case report of chronic ATL in early pregnancy, in a woman already diagnosed as an HTLV-1 carrier on screening test. © 2015 Japan Society of Obstetrics and Gynecology.

Cell Wall Structure of Coccoid Green Algae as an Important Trade-Off Between Biotic Interference Mechanisms and Multidimensional Cell Growth.

PubMed

Dunker, Susanne; Wilhelm, Christian

2018-01-01

Coccoid green algae can be divided in two groups based on their cell wall structure. One group has a highly chemical resistant cell wall (HR-cell wall) containing algaenan. The other group is more susceptible to chemicals (LR-cell wall - Low resistant cell wall). Algaenan is considered as important molecule to explain cell wall resistance. Interestingly, cell wall types (LR- and HR-cell wall) are not in accordance with the taxonomic classes Chlorophyceae and Trebouxiophyceae, which makes it even more interesting to consider the ecological function. It was already shown that algaenan helps to protect against virus, bacterial and fungal attack, but in this study we show for the first time that green algae with different cell wall properties show different sensitivity against interference competition with the cyanobacterium Microcystis aeruginosa . Based on previous work with co-cultures of M. aeruginosa and two green algae ( Acutodesmus obliquus and Oocystis marssonii ) differing in their cell wall structure, it was shown that M. aeruginosa could impair only the growth of the green algae if they belong to the LR-cell wall type. In this study it was shown that the sensitivity to biotic interference mechanism shows a more general pattern within coccoid green algae species depending on cell wall structure.

Titan Cell Production Enhances the Virulence of Cryptococcus neoformans

PubMed Central

Crabtree, Juliet N.; Okagaki, Laura H.; Wiesner, Darin L.; Strain, Anna K.; Nielsen, Judith N.

2012-01-01

Infection with Cryptococcus neoformans begins when desiccated yeast cells or spores are inhaled and lodge in the alveoli of the lungs. A subset of cryptococcal cells in the lungs differentiate into enlarged cells, referred to as titan cells. Titan cells can be as large as 50 to 100 μm in diameter and exhibit a number of features that may affect interactions with host immune defenses. To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously described C. neoformans mutant, the gpr4Δ gpr5Δ mutant, which has minimal titan cell production in vivo. The gpr4Δ gpr5Δ mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with the gpr4Δ gpr5Δ mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor in C. neoformans that promotes establishment of the initial pulmonary infection and plays a key role in disease progression. PMID:22890995

Titan cell production enhances the virulence of Cryptococcus neoformans.

PubMed

Crabtree, Juliet N; Okagaki, Laura H; Wiesner, Darin L; Strain, Anna K; Nielsen, Judith N; Nielsen, Kirsten

2012-11-01

Infection with Cryptococcus neoformans begins when desiccated yeast cells or spores are inhaled and lodge in the alveoli of the lungs. A subset of cryptococcal cells in the lungs differentiate into enlarged cells, referred to as titan cells. Titan cells can be as large as 50 to 100 μm in diameter and exhibit a number of features that may affect interactions with host immune defenses. To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously described C. neoformans mutant, the gpr4Δ gpr5Δ mutant, which has minimal titan cell production in vivo. The gpr4Δ gpr5Δ mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with the gpr4Δ gpr5Δ mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor in C. neoformans that promotes establishment of the initial pulmonary infection and plays a key role in disease progression.

Pathological aspects of so called "hilar cholangiocarcinoma"

PubMed Central

Castellano-Megías, Víctor M; Ibarrola-de Andrés, Carolina; Colina-Ruizdelgado, Francisco

2013-01-01

Cholangiocarcinoma (CC) arising from the large intrahepatic bile ducts and extrahepatic hilar bile ducts share clinicopathological features and have been called hilar and perihilar CC as a group. However, “hilar and perihilar CC” are also used to refer exclusively to the intrahepatic hilar type CC or, more commonly, the extrahepatic hilar CC. Grossly, a major distinction can be made between papillary and non-papillary tumors. Histologically, most hilar CCs are well to moderately differentiated conventional type (biliary) carcinomas. Immunohistochemically, CK7, CK20, CEA and MUC1 are normally expressed, being MUC2 positive in less than 50% of cases. Two main premalignant lesions are known: biliary intraepithelial neoplasia (BilIN) and intraductal papillary neoplasm of the biliary tract (IPNB). IPNB includes the lesions previously named biliary papillomatosis and papillary carcinoma. A series of 29 resected hilar CC from our archives is reviewed. Most (82.8%) were conventional type adenocarcinomas, mostly well to moderately differentiated, although with a broad morphological spectrum; three cases exhibited a poorly differentiated cell component resembling signet ring cells. IPNB was observed in 5 (17.2%), four of them with an associated invasive carcinoma. A clear cell type carcinoma, an adenosquamous carcinoma and two gastric foveolar type carcinomas were observed. PMID:23919110

Pathological aspects of so called "hilar cholangiocarcinoma".

PubMed

Castellano-Megías, Víctor M; Ibarrola-de Andrés, Carolina; Colina-Ruizdelgado, Francisco

2013-07-15

Cholangiocarcinoma (CC) arising from the large intrahepatic bile ducts and extrahepatic hilar bile ducts share clinicopathological features and have been called hilar and perihilar CC as a group. However, "hilar and perihilar CC" are also used to refer exclusively to the intrahepatic hilar type CC or, more commonly, the extrahepatic hilar CC. Grossly, a major distinction can be made between papillary and non-papillary tumors. Histologically, most hilar CCs are well to moderately differentiated conventional type (biliary) carcinomas. Immunohistochemically, CK7, CK20, CEA and MUC1 are normally expressed, being MUC2 positive in less than 50% of cases. Two main premalignant lesions are known: biliary intraepithelial neoplasia (BilIN) and intraductal papillary neoplasm of the biliary tract (IPNB). IPNB includes the lesions previously named biliary papillomatosis and papillary carcinoma. A series of 29 resected hilar CC from our archives is reviewed. Most (82.8%) were conventional type adenocarcinomas, mostly well to moderately differentiated, although with a broad morphological spectrum; three cases exhibited a poorly differentiated cell component resembling signet ring cells. IPNB was observed in 5 (17.2%), four of them with an associated invasive carcinoma. A clear cell type carcinoma, an adenosquamous carcinoma and two gastric foveolar type carcinomas were observed.

A Membrane-Type-1 Matrix Metalloproteinase (MT1-MMP) – Discoidin Domain Receptor 1 Axis Regulates Collagen-Induced Apoptosis in Breast Cancer Cells

PubMed Central

Assent, Delphine; Bourgot, Isabelle; Hennuy, Benoît; Geurts, Pierre; Noël, Agnès; Foidart, Jean-Michel; Maquoi, Erik

2015-01-01

During tumour dissemination, invading breast carcinoma cells become confronted with a reactive stroma, a type I collagen-rich environment endowed with anti-proliferative and pro-apoptotic properties. To develop metastatic capabilities, tumour cells must acquire the capacity to cope with this novel microenvironment. How cells interact with and respond to their microenvironment during cancer dissemination remains poorly understood. To address the impact of type I collagen on the fate of tumour cells, human breast carcinoma MCF-7 cells were cultured within three-dimensional type I collagen gels (3D COL1). Using this experimental model, we have previously demonstrated that membrane type-1 matrix metalloproteinase (MT1-MMP), a proteinase overexpressed in many aggressive tumours, promotes tumour progression by circumventing the collagen-induced up-regulation of BIK, a pro-apoptotic tumour suppressor, and hence apoptosis. Here we performed a transcriptomic analysis to decipher the molecular mechanisms regulating 3D COL1-induced apoptosis in human breast cancer cells. Control and MT1-MMP expressing MCF-7 cells were cultured on two-dimensional plastic plates or within 3D COL1 and a global transcriptional time-course analysis was performed. Shifting the cells from plastic plates to 3D COL1 activated a complex reprogramming of genes implicated in various biological processes. Bioinformatic analysis revealed a 3D COL1-mediated alteration of key cellular functions including apoptosis, cell proliferation, RNA processing and cytoskeleton remodelling. By using a panel of pharmacological inhibitors, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase specifically activated by collagen, as the initiator of 3D COL1-induced apoptosis. Our data support the concept that MT1-MMP contributes to the inactivation of the DDR1-BIK signalling axis through the cleavage of collagen fibres and/or the alteration of DDR1 receptor signalling unit, without triggering a drastic remodelling of the transcriptome of MCF-7 cells. PMID:25774665

Granulocyte-macrophage colony-stimulating factor induces the differentiation of murine erythroleukaemia cells into dendritic cells.

PubMed Central

Cao, X; Zhao, Y; Yu, Y; Wang, Y; Zhang, M; Zhang, W; Wang, J

1998-01-01

Dendritic cells (DC) are professional antigen-presenting cells (APC) within the immune system and antigen-pulsed DC can be used as an effective vaccine for active immunotherapy of cancer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the generation of DC. We previously showed that GM-CSF can induce murine erythroleukaemia cells (FBL-3) to differentiate into monocyte-like cells. To develop a new vaccinating method to stimulate the host immune response to leukaemia, we further investigate whether FBL-3 cells induced by GM-CSF can differentiate into DC in the present study. After being treated with GM-CSF, FBL-3 cells expressed high levels of 33D1 and NLDC-145, which are the specific markers of DC. The expression of MHC-II, B7-1, B7-2 and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated markedly; the typical morphology of DC were also observed by electron microscopy. Functionally, the GM-CSF-induced FBL-3 cells could apparently stimulate the proliferation of naive allogeneic and autologous T lymphocytes and induce the generation of specific CTL more efficiently than the wild-type FBL-3 cells. Mice immunized with GM-CSF-induced FBL-3 cells could resist the subsequent challenge with the wild-type FBL-3 cells. Collectively, these data indicate that GM-CSF differentiates murine erythroleukaemia cells into DC phenotypically, morphologically and functionally. FBL-3-derived DC can be used as a new type of vaccine. Our results may have important implications for the immunotherapy of leukaemia. Images Figure 3 Figure 4 PMID:9767469

Adaptation of Rhodopseudomonas acidophila strain 7050 to growth at different light intensities: what are the benefits to changing the type of LH2?

PubMed

Gardiner, A T; Niedzwiedzki, D M; Cogdell, R J

2018-04-01

Typical purple bacterial photosynthetic units consist of light harvesting one/reaction centre 'core' complexes surrounded by light harvesting two complexes. Factors such as the number and size of photosynthetic units per cell, as well as the type of light harvesting two complex that is produced, are controlled by environmental factors. In this paper, the change in the type of LH2 present in the Rhodopsuedomonas acidophila strain 7050 is described when cells are grown at a range of different light intensities. This species contains multiple pucBA genes that encode the apoproteins that form light-harvesting complex two, and a more complex mixture of spectroscopic forms of this complex has been found than was previously thought to be the case. Femto-second time resolved absorption has been used to investigate how the energy transfer properties in the membranes of high-light and low-light adapted cells change as the composition of the LH2 complexes varies.

De Novo Chromosome Structure Prediction

NASA Astrophysics Data System (ADS)

di Pierro, Michele; Cheng, Ryan R.; Lieberman-Aiden, Erez; Wolynes, Peter G.; Onuchic, Jose'n.

Chromatin consists of DNA and hundreds of proteins that interact with the genetic material. In vivo, chromatin folds into nonrandom structures. The physical mechanism leading to these characteristic conformations, however, remains poorly understood. We recently introduced MiChroM, a model that generates chromosome conformations by using the idea that chromatin can be subdivided into types based on its biochemical interactions. Here we extend and complete our previous finding by showing that structural chromatin types can be inferred from ChIP-Seq data. Chromatin types, which are distinct from DNA sequence, are partially epigenetically controlled and change during cell differentiation, thus constituting a link between epigenetics, chromosomal organization, and cell development. We show that, for GM12878 lymphoblastoid cells we are able to predict accurate chromosome structures with the only input of genomic data. The degree of accuracy achieved by our prediction supports the viability of the proposed physical mechanism of chromatin folding and makes the computational model a powerful tool for future investigations.

Human leukocyte antigen-G expression and polymorphisms promote cancer development and guide cancer diagnosis/treatment.

PubMed

Zhang, Yanwen; Yu, Shuwen; Han, Yali; Wang, Yunshan; Sun, Yuping

2018-01-01

Human leukocyte antigen-G (HLA-G) is a non-classical HLA molecule, predominantly expressed in cytotrophoblast cells to protect the fetus during pregnancy. Notably, a high frequency of HLA-G expression has been observed in a wide variety of cancer types in previous studies. Furthermore, HLA-G expression in cancer has been considered to be detrimental, since it can protect cancer cells from natural killer cell cytotoxic T lymphocyte-mediated destruction, promote tumor spreading and shorten the survival time of patients by facilitating tumor immune evasion. In addition, HLA-G polymorphisms have been investigated in numerous types of cancer and are considered as risk factors and predictive markers of cancer. This review focuses on HLA-G expression and its polymorphisms in cancer, analyzing the mechanisms of HLA-G in promoting cancer development, and evaluating the potential and value of its clinical application as a diagnostic and prognostic biomarker, or even as a prospective therapeutic target in certain types of tumors.

Molecular Dissection of Mesenchymal–Epithelial Interactions in the Hair Follicle

PubMed Central

Rendl, Michael; Lewis, Lisa

2005-01-01

De novo hair follicle formation in embryonic skin and new hair growth in adult skin are initiated when specialized mesenchymal dermal papilla (DP) cells send cues to multipotent epithelial stem cells. Subsequently, DP cells are enveloped by epithelial stem cell progeny and other cell types to form a niche orchestrating hair growth. Understanding the general biological principles that govern the mesenchymal–epithelial interactions within the DP niche, however, has been hampered so far by the lack of systematic approaches to dissect the complete molecular make-up of this complex tissue. Here, we take a novel multicolor labeling approach, using cell type–specific transgenic expression of red and green fluorescent proteins in combination with immunolabeling of specific antigens, to isolate pure populations of DP and four of its surrounding cell types: dermal fibroblasts, melanocytes, and two different populations of epithelial progenitors (matrix and outer root sheath cells). By defining their transcriptional profiles, we develop molecular signatures characteristic for the DP and its niche. Validating the functional importance of these signatures is a group of genes linked to hair disorders that have been largely unexplored. Additionally, the DP signature reveals novel signaling and transcription regulators that distinguish them from other cell types. The mesenchymal–epithelial signatures include key factors previously implicated in ectodermal-neural fate determination, as well as a myriad of regulators of bone morphogenetic protein signaling. These findings establish a foundation for future functional analyses of the roles of these genes in hair development. Overall, our strategy illustrates how knowledge of the genes uniquely expressed by each cell type residing in a complex niche can reveal important new insights into the biology of the tissue and its associated disease states. PMID:16162033

Cell Kinetic and Histomorphometric Analysis of Microgravitational Osteopenia: PARE.03B

NASA Technical Reports Server (NTRS)

Roberts, W. Eugene; Garetto, Lawrence P.

1998-01-01

Previous methods of identifying cells undergoing DNA synthesis (S-phase) utilized H-3 thymidine (3HT) autoradiography. 5-Bromo-2'-deoxyuridine (BrdU) immunohistochemistry is a nonradioactive alternative method. This experiment compared the two methods using the nuclear volume model for osteoblast histogenesis in two different embedding media. Twenty Sprague-Dawley rats were used, with half receiving 3HT (1 micro Ci/g) and the other half BrdU (50 microgram/g). Condyies were embedded (one side in paraffin, the other in plastic) and S-phase nuclei were identified using either autoradiography or immunohistochemistry. The fractional distribution of preosteoblast cell types and the percentage of labeled cells (within each cell fraction and label index) were calculated and expressed as mean q standard error. Chi-Square analysis showed only a minor difference in the fractional distribution of cell types. However, there were significant differences (p less than 0.05) by ANOVA, in the nuclear labeling of specific cell types. With the exception of the less-differentiated A+A'cells, more BrdU label was consistently detected in paraffin than in plastic-embedded sections. In general, more nuclei were labeled with 3H-thymidine than with BrdU in both types of embedding media. Labeling index data (labeled cells/total cells sampled x 100) indicated that BrdU in paraffin, but not plastic gave the same results as 3HT in either embedding method. Thus, we conclude that the two labeling methods do not yield the same results for the nuclear volume model and that embedding media is an important factor whenusing BrdU. As a result of this work, 3HT was chosen for used in the PARE.03 flight experiments.

Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B

PubMed Central

2012-01-01

Background Although the peptidyl-prolyl isomerase, cyclophilin-A (peptidyl-prolyl isomerase, PPIA), has been studied for decades in the context of its intracellular functions, its extracellular roles as a major contributor to both inflammation and multiple cancers have more recently emerged. A wide range of activities have been ascribed to extracellular PPIA that include induction of cytokine and matrix metalloproteinase (MMP) secretion, which potentially underlie its roles in inflammation and tumorigenesis. However, there have been conflicting reports as to which particular signaling events are under extracellular PPIA regulation, which may be due to either cell-dependent responses and/or the use of commercial preparations recently shown to be highly impure. Methods We have produced and validated the purity of recombinant PPIA in order to subject it to a comparative analysis between different cell types. Specifically, we have used a combination of multiple methods such as luciferase reporter screens, translocation assays, phosphorylation assays, and nuclear magnetic resonance to compare extracellular PPIA activities in several different cell lines that included epithelial and monocytic cells. Results Our findings have revealed that extracellular PPIA activity is cell type-dependent and that PPIA signals via multiple cellular receptors beyond the single transmembrane receptor previously identified, Extracellular Matrix MetalloPRoteinase Inducer (EMMPRIN). Finally, while our studies provide important insight into the cell-specific responses, they also indicate that there are consistent responses such as nuclear factor kappa B (NFκB) signaling induced in all cell lines tested. Conclusions We conclude that although extracellular PPIA activates several common pathways, it also targets different receptors in different cell types, resulting in a complex, integrated signaling network that is cell type-specific. PMID:22631225

Molecules mediating adhesion of T and B cells, monocytes and granulocytes to vascular endothelial cells.

PubMed Central

Prieto, J; Beatty, P G; Clark, E A; Patarroyo, M

1988-01-01

Leucocytes interact with vascular endothelial cells (EC), and adhesion between these two cell types in vitro is modulated by phorbol ester. Monocytes were found to display the highest basal adhesion to EC, followed by Epstein-Barr virus-immortalized normal B cells (EBV-B), T cells and granulocytes. Phorbol ester treatment increased the adhesion of all types of leucocytes, except monocytes. In the presence of this compound, monoclonal antibody 60.3 to GP90 (CD18, a leucocyte-adhesion protein which is non-covalently associated to either GP160, GP155, or GP130) was found to inhibit the adhesion of the four types of leucocytes to a considerable extent, while anti-lymphocyte function-associated antigen-1 (LFA-1) antibody to GP160 (CD11a) inhibited the adhesion of T and B cells only. Antibody 60.1 to GP155 (CD11b) had a major inhibitory activity exclusively on granulocytes, while antibody LB-2, which recognizes a distinct adhesion molecule (GP84) and, in contrast to the previous antibodies, reacts with EC, mainly inhibited adhesion of EBV-B and did not increase the inhibition obtained with antibody 60.3 alone. Fab fragments of antibody 60.3 inhibited leucocyte adhesion more efficiently, in either the absence or presence of phorbol ester, than the intact antibody molecule. It is concluded the GP90, either alone or associated to the larger glycoproteins, mediates the adhesion in all types of leucocytes, while GP84 mediates the adhesion of the activated B cells. Images Figure 2 PMID:3259203

Mutation in the peb1A Locus of Campylobacter jejuni Reduces Interactions with Epithelial Cells and Intestinal Colonization of Mice

PubMed Central

Pei, Zhiheng; Burucoa, Christophe; Grignon, Bernadette; Baqar, Shahida; Huang, Xiao-Zhe; Kopecko, Dennis J.; Bourgeois, A. L.; Fauchere, Jean-Louis; Blaser, Martin J.

1998-01-01

Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays in C. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model. PMID:9488379

Morphological evidence for local microcircuits in rat vestibular maculae

NASA Technical Reports Server (NTRS)

Ross, M. D.

1997-01-01

Previous studies suggested that intramacular, unmyelinated segments of vestibular afferent nerve fibers and their large afferent endings (calyces) on type I hair cells branch. Many of the branches (processes) contain vesicles and are presynaptic to type II hair cells, other processes, intramacular nerve fibers, and calyces. This study used serial section transmission electron microscopy and three-dimensional reconstruction methods to document the origins and distributions of presynaptic processes of afferents in the medial part of the adult rat utricular macula. The ultrastructural research focused on presynaptic processes whose origin and termination could be observed in a single micrograph. Results showed that calyces had 1) vesiculated, spine-like processes that invaginated type I cells and 2) other, elongate processes that ended on type II cells pre- as well as postsynaptically. Intramacular, unmyelinated segments of afferent nerve fibers gave origin to branches that were presynaptic to type II cells, calyces, calyceal processes, and other nerve fibers in the macula. Synapses with type II cells occurred opposite subsynaptic cisternae (C synapses); all other synapses were asymmetric. Vesicles were pleomorphic but were differentially distributed according to process origin. Small, clear-centered vesicles, approximately 40-60 nm in diameter, predominated in processes originating from afferent nerve fibers and basal parts of calyces. Larger vesicles approximately 70-120 nm in diameter having approximately 40-80 nm electron-opaque cores were dominant in processes originating from the necks of calyces. Results are interpreted to indicate the existence of a complex system of intrinsic feedforward (postsynaptic)-feedback (presynaptic) connections in a network of direct and local microcircuits. The morphological findings support the concept that maculae dynamically preprocess linear acceleratory information before its transmission to the central nervous system.

Regulation of Neuronal Cav3.1 Channels by Cyclin-Dependent Kinase 5 (Cdk5)

PubMed Central

González-Ramírez, Ricardo; González-Billault, Christian; Felix, Ricardo

2015-01-01

Low voltage-activated (LVA) T-type Ca2+ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca2+ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Cav3.1 to Cav3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca2+ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Cav3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Cav3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Cav3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca2+ channels. PMID:25760945

Regulation of neuronal cav3.1 channels by cyclin-dependent kinase 5 (Cdk5).

PubMed

Calderón-Rivera, Aida; Sandoval, Alejandro; González-Ramírez, Ricardo; González-Billault, Christian; Felix, Ricardo

2015-01-01

Low voltage-activated (LVA) T-type Ca2+ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca2+ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Cav3.1 to Cav3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca2+ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Cav3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Cav3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Cav3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca2+ channels.

Adrenoceptors in Brain: Cellular Gene Expression and Effects on Astrocytic Metabolism and [Ca2+]i

PubMed Central

Hertz, Leif; Lovatt, Ditte; Goldman, Steven A.; Nedergaard, Maiken

2010-01-01

Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., Cereb. Cortex 18, 2789–2795), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of α1-, α2- and β-adrenergic receptors and their subtypes was determined in freshly-isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10–12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. α1A-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and α1B-receptors were not expressed in any cell population. Among α2-receptors only α2A-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. β1-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas β2-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of α2-adrenoceptors increases glycogen formation and oxidative metabolism, the latter by a mechanism depending on intramitochondrial Ca2+, whereas α1-adrenoceptor stimulation enhances glutamate uptake, and β-adrenoceptor activation causes glycogenolysis and increased Na+,K+-ATPase activity. The Ca2+- and cAMP-mediated association between energy-consuming and energy-yielding processes is emphasized. PMID:20380860

Targeting of phage particles towards endothelial cells by antibodies selected through a multi-parameter selection strategy.

PubMed

Mandrup, Ole A; Lykkemark, Simon; Kristensen, Peter

2017-02-10

One of the hallmarks of cancer is sustained angiogenesis. Here, normal endothelial cells are activated, and their formation of new blood vessels leads to continued tumour growth. An improved patient condition is often observed when angiogenesis is prevented or normalized through targeting of these genomically stable endothelial cells. However, intracellular targets constitute a challenge in therapy, as the agents modulating these targets have to be delivered and internalized specifically to the endothelial cells. Selection of antibodies binding specifically to certain cell types is well established. It is nonetheless a challenge to ensure that the binding of antibodies to the target cell will mediate internalization. Previously selection of such antibodies has been performed targeting cancer cell lines; most often using either monovalent display or polyvalent display. In this article, we describe selections that isolate internalizing antibodies by sequential combining monovalent and polyvalent display using two types of helper phages, one which increases display valence and one which reduces background. One of the selected antibodies was found to mediate internalization into human endothelial cells, although our results confirms that the single stranded nature of the DNA packaged into phage particles may limit applications aimed at targeting nucleic acids in mammalian cells.

Adiponectin modulates inflammatory reactions via calreticulin receptor–dependent clearance of early apoptotic bodies

PubMed Central

Takemura, Yukihiro; Ouchi, Noriyuki; Shibata, Rei; Aprahamian, Tamar; Kirber, Michael T.; Summer, Ross S.; Kihara, Shinji; Walsh, Kenneth

2007-01-01

Obesity and type 2 diabetes are associated with chronic inflammation. Adiponectin is an adipocyte-derived hormone with antidiabetic and antiinflammatory actions. Here, we demonstrate what we believe to be a previously undocumented activity of adiponectin, facilitating the uptake of early apoptotic cells by macrophages, an essential feature of immune system function. Adiponectin-deficient (APN-KO) mice were impaired in their ability to clear apoptotic thymocytes in response to dexamethasone treatment, and these animals displayed a reduced ability to clear early apoptotic cells that were injected into their intraperitoneal cavities. Conversely, adiponectin administration promoted the clearance of apoptotic cells by macrophages in both APN-KO and wild-type mice. Adiponectin overexpression also promoted apoptotic cell clearance and reduced features of autoimmunity in lpr mice whereas adiponectin deficiency in lpr mice led to a further reduction in apoptotic cell clearance, which was accompanied by exacerbated systemic inflammation. Adiponectin was capable of opsonizing apoptotic cells, and phagocytosis of cell corpses was mediated by the binding of adiponectin to calreticulin on the macrophage cell surface. We propose that adiponectin protects the organism from systemic inflammation by promoting the clearance of early apoptotic cells by macrophages through a receptor-dependent pathway involving calreticulin. PMID:17256056

Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells.

PubMed

Gledhill, Karl; Guo, Zongyou; Umegaki-Arao, Noriko; Higgins, Claire A; Itoh, Munenari; Christiano, Angela M

2015-01-01

The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs) present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes.

JACKDAW controls epidermal patterning in the Arabidopsis root meristem through a non-cell-autonomous mechanism.

PubMed

Hassan, Hala; Scheres, Ben; Blilou, Ikram

2010-05-01

In Arabidopsis, specification of the hair and non-hair epidermal cell types is position dependent, in that hair cells arise over clefts in the underlying cortical cell layer. Epidermal patterning is determined by a network of transcriptional regulators that respond to an as yet unknown cue from underlying tissues. Previously, we showed that JACKDAW (JKD), a zinc finger protein, localizes in the quiescent centre and the ground tissue, and regulates tissue boundaries and asymmetric cell division by delimiting SHORT-ROOT movement. Here, we provide evidence that JKD controls position-dependent signals that regulate epidermal-cell-type patterning. JKD is required for appropriately patterned expression of the epidermal cell fate regulators GLABRA2, CAPRICE and WEREWOLF. Genetic interaction studies indicate that JKD operates upstream of the epidermal patterning network in a SCRAMBLED (SCM)-dependent fashion after embryogenesis, but acts independent of SCM in embryogenesis. Tissue-specific induction experiments indicate non-cell-autonomous action of JKD from the underlying cortex cell layer to specify epidermal cell fate. Our findings are consistent with a model where JKD induces a signal in every cortex cell that is more abundant in the hair cell position owing to the larger surface contact of cells located over a cleft.

Dietary polyphenols influence antimetabolite agents: methotrexate, 6-mercaptopurine and 5-fluorouracil in leukemia cell lines

PubMed Central

Mahbub, Amani; Le Maitre, Christine; Haywood-Small, Sarah; Cross, Neil; Jordan-Mahy, Nicola

2017-01-01

Polyphenols have been previously shown to sensitize leukemia cell lines to topoisomerase inhibitors. Here, we assess the effects of five polyphenols when used alone and in combination with antimetabolites: methotrexate, 6-mercaptopurine and 5-fluorouracil; in lymphoid and myeloid leukemia cells lines, and non-tumor control cells. The effects of combined treatments were investigated on ATP and glutathione levels, cell-cycle progression, DNA damage and apoptosis. Polyphenols antagonized methotrexate and 6-mercaptopurine induced cell-cycle arrest and apoptosis in most leukemia cell lines. This was associated with reduced DNA damage and increased glutathione levels, greater than that seen following individual treatments alone. In contrast, 5-fluorouracil when combined with quercetin, apigenin and rhein caused synergistic decrease in ATP levels, induction of cell-cycle arrest and apoptosis in some leukemia cell lines. However, antagonistic effects were observed when 5-fluorouracil was combined with rhein and cis-stilbene in myeloid cell lines. The effects were dependant on polyphenol type and chemotherapy agent investigated, and cell type treated. Interestingly treatment of non-tumor control cells with polyphenols protected cells from antimetabolite treatments. This suggests that polyphenols modulate the action of antimetabolite agents; more importantly they antagonized methotrexate and 6-mercaptopurine actions, thus suggesting the requirement of polyphenol-exclusion during their use. PMID:29285220

Breast cancer instructs dendritic cells to prime interleukin 13–secreting CD4+ T cells that facilitate tumor development

PubMed Central

Aspord, Caroline; Pedroza-Gonzalez, Alexander; Gallegos, Mike; Tindle, Sasha; Burton, Elizabeth C.; Su, Dan; Marches, Florentina; Banchereau, Jacques; Palucka, A. Karolina

2007-01-01

We previously reported (Bell, D., P. Chomarat, D. Broyles, G. Netto, G.M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K.A. Palucka, and J. Banchereau. 1999. J. Exp. Med. 190: 1417–1426) that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which cluster with CD4+ T cells. We now show that CD4+ T cells infiltrating breast cancer tumors secrete type 1 (interferon γ) as well as high levels of type 2 (interleukin [IL] 4 and IL-13) cytokines. Immunofluorescence staining of tissue sections revealed intense IL-13 staining on breast cancer cells. The expression of phosphorylated signal transducer and activator of transcription 6 in breast cancer cells suggests that IL-13 actually delivers signals to cancer cells. To determine the link between breast cancer, DCs, and CD4+ T cells, we implanted human breast cancer cell lines in nonobese diabetic/LtSz-scid/scid β2 microglobulin–deficient mice engrafted with human CD34+ hematopoietic progenitor cells and autologous T cells. There, CD4+ T cells promote early tumor development. This is dependent on DCs and can be partially prevented by administration of IL-13 antagonists. Thus, breast cancer targets DCs to facilitate its development. PMID:17438063

Ribbon Synaptic Plasticity in Gravity Sensors of Rats Flown on Neurolab

NASA Technical Reports Server (NTRS)

Ross, Muriel D.; Varelas, Joseph

2003-01-01

Previous spaceflight experiments (Space Life Sciences-1 and -2 (SLS-1 and SLS-2)) first demonstrated the extraordinary ability of gravity sensor hair cells to change the number, kind, and distribution of connections (synapses) they make to other cells while in weightlessness. The number of synapses in hair cells in one part of the inner ear (the utricle) was markedly elevated on flight day 13 (FD13) of SLS-2. Unanswered questions, however, were whether these increases in synapses occur rapidly and whether they remain stable in weightlessness. The answers have implications for long-duration human space travel. If gravity sensors can adapt quickly, crews may be able to move easily between different gravity levels, since the sensors will adapt rapidly to weightlessness on the spacecraft and then back to Earth's gravity when the mission ends. This ability to adapt is also important for recovery from balance disorders. To further our understanding of this adaptive potential (a property called neuronal synaptic plasticity), the present Neurolab research was undertaken. Our experiment examined whether: (a) increases in synapses would remain stable throughout the flight, (b) changes in the number of synapses were uniform across different portions of the gravity sensors (the utricle and saccule), and (c) synaptic changes were similar for the different types of hair cells (Type I and Type II). Utricular and saccular maculae (the gravity-sensing portions of the inner ear) were collected in flight from rats on FD2 and FD14. Samples were also collected from control rats on the ground. Tissues were prepared for ultrastructural study. Hair cells and their ribbon synapses were examined in a transmission electron microscope. Synapses were counted in all hair cells in 50 consecutive sections that crossed the striolar zone. Results indicate that utricular hair cell synapses initially increased significantly in number in both types of hair cells by FD2. Counts declined by FD14, but the mean number of synapses in utricular Type II cells remained significantly higher than in the ground control rats. For saccular samples, synaptic number in Type I and Type II cells declined on FD2, but returned to near-baseline values by FD14. These findings indicate that: (a) synaptic plasticity occurs rapidly in weightlessness, and (b) synaptic changes are not identical for the two types of hair cells or for the two maculae.

Topologically associating domains are stable units of replication-timing regulation.

PubMed

Pope, Benjamin D; Ryba, Tyrone; Dileep, Vishnu; Yue, Feng; Wu, Weisheng; Denas, Olgert; Vera, Daniel L; Wang, Yanli; Hansen, R Scott; Canfield, Theresa K; Thurman, Robert E; Cheng, Yong; Gülsoy, Günhan; Dennis, Jonathan H; Snyder, Michael P; Stamatoyannopoulos, John A; Taylor, James; Hardison, Ross C; Kahveci, Tamer; Ren, Bing; Gilbert, David M

2014-11-20

Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program. In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400-800 kilobases ('replication domains'), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements. Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs). Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure. Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell-type-specific sub-nuclear compartmentalization and replication timing with developmentally stable structural domains and offer a unified model for large-scale chromosome structure and function.

CsSCL1 is differentially regulated upon maturation in chestnut microshoots and is specifically expressed in rooting-competent cells.

PubMed

Vielba, Jesús M; Díaz-Sala, Carmen; Ferro, Enrique; Rico, Saleta; Lamprecht, María; Abarca, Dolores; Ballester, Antonio; Sánchez, Conchi

2011-10-01

The Castanea sativa SCL1 gene (CsSCL1) has previously been shown to be induced by auxin during adventitious root (AR) formation in rooting-competent microshoots. However, its expression has not previously been analyzed in rooting-incompetent shoots. This study focuses on the regulation of CsSCL1 during maturation and the role of the gene in the formation of AR. The expression of CsSCL1 in rooting-incompetent microshoots and other tissues was investigated by quantitative reverse transcriptase--polymerase chain reaction. The analysis was complemented by in situ hybridization of the basal segments of rooting-competent and --incompetent microshoots during AR induction, as well as in AR and lateral roots. It was found that CsSCL1 is upregulated by auxin in a cell-type- and phase-dependent manner during the induction of AR. In root-forming shoots, CsSCL1 mRNA was specifically located in the cambial zone and derivative cells, which are rooting-competent cells, whereas in rooting-incompetent shoots the hybridization signal was more diffuse and evenly distributed through the phloem and parenchyma. CsSCL1 expression was also detected in lateral roots and axillary buds. The different CsSCL1 expression patterns in rooting-competent and -incompetent microshoots, together with the specific location of transcripts in cell types involved in root meristem initiation and in the root primordia of AR and lateral roots, indicate an important role for the gene in determining whether certain cells will enter the root differentiation pathway and its involvement in meristem maintenance.

T-cell-dependent control of acute Giardia lamblia infections in mice.

PubMed

Singer, S M; Nash, T E

2000-01-01

We have studied immune mechanisms responsible for control of acute Giardia lamblia and Giardia muris infections in adult mice. Association of chronic G. lamblia infection with hypogammaglobulinemia and experimental infections of mice with G. muris have led to the hypothesis that antibodies are required to control these infections. We directly tested this hypothesis by infecting B-cell-deficient mice with either G. lamblia or G. muris. Both wild-type mice and B-cell-deficient mice eliminated the vast majority of parasites between 1 and 2 weeks postinfection with G. lamblia. G. muris was also eliminated in both wild-type and B-cell-deficient mice. In contrast, T-cell-deficient and scid mice failed to control G. lamblia infections, as has been shown previously for G. muris. Treatment of wild-type or B-cell-deficient mice with antibodies to CD4 also prevented elimination of G. lamblia, confirming a role for T cells in controlling infections. By infecting mice deficient in either alphabeta- or gammadelta-T-cell receptor (TCR)-expressing T cells, we show that the alphabeta-TCR-expressing T cells are required to control parasites but that the gammadelta-TCR-expressing T cells are not. Finally, infections in mice deficient in production of gamma interferon or interleukin 4 (IL-4) and mice deficient in responding to IL-4 and IL-13 revealed that neither the Th1 nor the Th2 subset is absolutely required for protection from G. lamblia. We conclude that a T-cell-dependent mechanism is essential for controlling acute Giardia infections and that this mechanism is independent of antibody and B cells.

Gemcitabine Hydrochloride, Carboplatin, Dexamethasone, and Rituximab in Treating Patients With Previously Treated Lymphoid Malignancies

ClinicalTrials.gov

2017-05-28

Adult Nasal Type Extranodal NK/T-cell Lymphoma; Anaplastic Large Cell Lymphoma; Angioimmunoblastic T-cell Lymphoma; Cutaneous B-cell Non-Hodgkin Lymphoma; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Hepatosplenic T-cell Lymphoma; Intraocular Lymphoma; Nodal Marginal Zone B-cell Lymphoma; Noncutaneous Extranodal Lymphoma; Peripheral T-cell Lymphoma; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Grade III Lymphomatoid Granulomatosis; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Adult Lymphoblastic Lymphoma; Recurrent Adult T-cell Leukemia/Lymphoma; Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma; Small Intestine Lymphoma; Splenic Marginal Zone Lymphoma; Testicular Lymphoma; Waldenstrom Macroglobulinemia

Primary focal T-cell lymphoma of the liver: a case report and review of the literature.

PubMed

Cerban, Razvan; Gheorghe, Liana; Becheanu, Gabriel; Serban, Valentin; Gheorghe, Cristian

2012-06-01

We present the case of a previously healthy 62 year old man who developed primary non-Hodgkin lymphoma of the liver. Biopsy confirmed that it was a diffuse large anaplastic T-cell lymphoma of an extremely rare type. The diagnosis of this type of lesions is suggested by the presence of a hepatic mass without lymphadenopathy, splenomegaly or bone marrow involvement associated with normal tumor markers (carcinoembryonic antigen, alpha-fetoprotein and CA 19-9 levels). Histological examination of tissue is essential to confirm the diagnosis. Treatment options are surgical resection and/or chemotherapy but the rate of response to treatment varies widely. Some patients can achieve prolonged remission.

Immunological Prevention of Spontaneous Mammary Carcinoma in Transgenic Mice

DTIC Science & Technology

2001-08-01

respectively, and a sera Mouse Typer Iso- TUBO cell inhibition was found in BALB/c mice injected with typing kit (Bio-Rad, Richmond , CA) as previously described...Curr. Opin. Immunol., 6: 41. Lapp6, M. A., and Prehn . R. T. Immunologic surveillance at the macroscopic level: 414-419. 1994. nonselective

Dragon (repulsive guidance molecule b) inhibits IL-6 expression in macrophages.

PubMed

Xia, Yin; Cortez-Retamozo, Virna; Niederkofler, Vera; Salie, Rishard; Chen, Shanzhuo; Samad, Tarek A; Hong, Charles C; Arber, Silvia; Vyas, Jatin M; Weissleder, Ralph; Pittet, Mikael J; Lin, Herbert Y

2011-02-01

Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon, and RGMc/hemojuvelin were found recently to act as bone morphogenetic protein (BMP) coreceptors that enhance BMP signaling activity. Although our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have shown previously that Dragon is expressed in neural cells, germ cells, and renal epithelial cells. In this study, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand-dependent manner via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared with that in respective cells derived from wild-type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.

Epstein-Barr virus recombinants from overlapping cosmid fragments.

PubMed

Tomkinson, B; Robertson, E; Yalamanchili, R; Longnecker, R; Kieff, E

1993-12-01

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)

Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture

PubMed Central

Takeuchi, Hiroki; Nakatsuji, Norio; Suemori, Hirofumi

2014-01-01

Insulin-producing cells (IPCs) derived from human pluripotent stem cells (hPSCs) may be useful in cell therapy and drug discovery for diabetes. Here, we examined various growth factors and small molecules including those previously reported to develop a robust differentiation method for induction of mature IPCs from hPSCs. We established a protocol that induced PDX1-positive pancreatic progenitor cells at high efficiency, and further induced mature IPCs by treatment with forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide in 3D culture. The cells that differentiated into INSULIN-positive and C-PEPTIDE-positive cells secreted insulin in response to glucose stimulation, indicating a functional IPC phenotype. We also found that this method was applicable to different types of hPSCs. PMID:24671046

High-frequency microrheology reveals cytoskeleton dynamics in living cells

NASA Astrophysics Data System (ADS)

Rigato, Annafrancesca; Miyagi, Atsushi; Scheuring, Simon; Rico, Felix

2017-08-01

Living cells are viscoelastic materials, dominated by an elastic response on timescales longer than a millisecond. On shorter timescales, the dynamics of individual cytoskeleton filaments are expected to emerge, but active microrheology measurements on cells accessing this regime are scarce. Here, we develop high-frequency microrheology experiments to probe the viscoelastic response of living cells from 1 Hz to 100 kHz. We report the viscoelasticity of different cell types under cytoskeletal drug treatments. On previously inaccessible short timescales, cells exhibit rich viscoelastic responses that depend on the state of the cytoskeleton. Benign and malignant cancer cells revealed remarkably different scaling laws at high frequencies, providing a unique mechanical fingerprint. Microrheology over a wide dynamic range--up to the frequency characterizing the molecular components--provides a mechanistic understanding of cell mechanics.

Dragon (repulsive guidance molecule RGMb) inhibits E-cadherin expression and induces apoptosis in renal tubular epithelial cells.

PubMed

Liu, Wenjing; Li, Xiaoling; Zhao, Yueshui; Meng, Xiao-Ming; Wan, Chao; Yang, Baoxue; Lan, Hui-Yao; Lin, Herbert Y; Xia, Yin

2013-11-01

Dragon is one of the three members of the repulsive guidance molecule (RGM) family, i.e. RGMa, RGMb (Dragon), and RGMc (hemojuvelin). We previously identified the RGM members as bone morphogenetic protein (BMP) co-receptors that enhance BMP signaling. Our previous studies found that Dragon is highly expressed in the tubular epithelial cells of mouse kidneys. However, the roles of Dragon in renal epithelial cells are yet to be defined. We now show that overexpression of Dragon increased cell death induced by hypoxia in association with increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 levels in mouse inner medullary collecting duct (IMCD3) cells. Dragon also inhibited E-cadherin expression but did not affect epithelial-to-mesenchymal transition induced by TGF-β in IMCD3 cells. Previous studies suggest that the three RGM members can function as ligands for the receptor neogenin. Interestingly, our present study demonstrates that the Dragon actions on apoptosis and E-cadherin expression in IMCD3 cells were mediated by the neogenin receptor but not through the BMP pathway. Dragon expression in the kidney was up-regulated by unilateral ureteral obstruction in mice. Compared with wild-type mice, heterozygous Dragon knock-out mice exhibited 45-66% reduction in Dragon mRNA expression, decreased epithelial apoptosis, and increased tubular E-cadherin expression and had attenuated tubular injury after unilateral ureteral obstruction. Our results suggest that Dragon may impair tubular epithelial integrity and induce epithelial apoptosis both in vitro and in vivo.

Evaluation of telomere length in human cardiac tissues using cardiac quantitative FISH.

PubMed

Sharifi-Sanjani, Maryam; Meeker, Alan K; Mourkioti, Foteini

2017-09-01

Telomere length has been correlated with various diseases, including cardiovascular disease and cancer. The use of currently available telomere-length measurement techniques is often restricted by the requirement of a large amount of cells (Southern-based techniques) or the lack of information on individual cells or telomeres (PCR-based methods). Although several methods have been used to measure telomere length in tissues as a whole, the assessment of cell-type-specific telomere length provides valuable information on individual cell types. The development of fluorescence in situ hybridization (FISH) technologies enables the quantification of telomeres in individual chromosomes, but the use of these methods is dependent on the availability of isolated cells, which prevents their use with fixed archival samples. Here we describe an optimized quantitative FISH (Q-FISH) protocol for measuring telomere length that bypasses the previous limitations by avoiding contributions from undesired cell types. We have used this protocol on small paraffin-embedded cardiac-tissue samples. This protocol describes step-by-step procedures for tissue preparation, permeabilization, cardiac-tissue pretreatment and hybridization with a Cy3-labeled telomeric repeat complementing (CCCTAA) 3 peptide nucleic acid (PNA) probe coupled with cardiac-specific antibody staining. We also describe how to quantify telomere length by means of the fluorescence intensity and area of each telomere within individual nuclei. This protocol provides comparative cell-type-specific telomere-length measurements in relatively small human cardiac samples and offers an attractive technique to test hypotheses implicating telomere length in various cardiac pathologies. The current protocol (from tissue collection to image procurement) takes ∼28 h along with three overnight incubations. We anticipate that the protocol could be easily adapted for use on different tissue types.

Association of ORMDL3 with rhinovirus-induced endoplasmic reticulum stress and type I Interferon responses in human leukocytes

PubMed Central

Liu, Yi-Ping; Rajamanikham, Victoria; Baron, Marissa; Patel, Sagar; Mathur, Sameer K.; Schwantes, Elizabeth A.; Ober, Carole; Jackson, Daniel J.; Gern, James E.; Lemanske, Robert F.; Smith, Judith A

2017-01-01

Background Children with risk alleles at the 17q21 genetic locus who wheeze during rhinovirus illnesses have a greatly increased likelihood of developing childhood asthma. In mice, overexpression of the 17q21 gene ORMDL3 leads to airway remodeling and hyper-responsiveness. However, the mechanisms by which ORMDL3 predisposes to asthma are unclear. Previous studies have suggested that ORMDL3 induces endoplasmic reticulum (ER) stress and production of the type I interferon (IFN) regulated chemokine CXCL10. Objective The purpose of this study was to determine the relationship between ORMDL3 and rhinovirus-induced ER stress and type I IFN in human leukocytes. Methods ER stress was monitored by measuring HSPA5, CHOP and spliced XBP1 gene expression, and type I IFN by measuring IFNB1 (IFN-β) and CXCL10 expression in human cell lines and primary leukocytes following treatment with rhinovirus. Requirements for cell contact and specific cell type in ORMDL3 induction were examined by transwell assay and depletion experiments, respectively. Finally, the effects of 17q21 genotype on the expression of ORMDL3, IFNB1, and ER stress genes were assessed. Results THP-1 monocytes overexpressing ORMDL3 responded to rhinovirus with increased IFNB1 and HSPA5. Rhinovirus-induced ORMDL3 expression in primary leukocytes required cell-cell contact, and induction was abrogated by plasmacytoid dendritic cell depletion. The degree of rhinovirus induced ORMDL3, HSPA5, and IFNB1 expression varied by leukocyte type and 17q21 genotype, with the highest expression of these genes in the asthma-associated genotype. Conclusions & Clinical Relevance Multiple lines of evidence support an association between higher ORMDL3 and increased rhinovirus-induced HSPA5 and type I IFN gene expression. These associations with ORMDL3 are cell-type specific, with the most significant 17q21 genotype effects on ORMDL3 expression and HSPA5 induction evident in B cells. Together, these findings have implications for how the interaction of increased ORMDL3 and rhinovirus may predispose to asthma. PMID:28192616

Single-cell RNA-sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine.

PubMed

Glass, Leslie L; Calero-Nieto, Fernando J; Jawaid, Wajid; Larraufie, Pierre; Kay, Richard G; Göttgens, Berthold; Reimann, Frank; Gribble, Fiona M

2017-10-01

To identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity. We performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression. PPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated and would be differentially targetable. Our findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Mechanical strain induces involution-associated events in mammary epithelial cells

PubMed Central

Quaglino, Ana; Salierno, Marcelo; Pellegrotti, Jesica; Rubinstein, Natalia; Kordon, Edith C

2009-01-01

Background Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture. Results We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition. Conclusion Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device specifically designed for such a purpose. We believe that our results indicate the relevance of mechanical stress among the early post-lactation events that lead to mammary gland involution. PMID:19615079

The Cell Cycle Regulator CCDC6 Is a Key Target of RNA-Binding Protein EWS

PubMed Central

Duggimpudi, Sujitha; Larsson, Erik; Nabhani, Schafiq; Borkhardt, Arndt; Hoell, Jessica I

2015-01-01

Genetic translocation of EWSR1 to ETS transcription factor coding region is considered as primary cause for Ewing sarcoma. Previous studies focused on the biology of chimeric transcription factors formed due to this translocation. However, the physiological consequences of heterozygous EWSR1 loss in these tumors have largely remained elusive. Previously, we have identified various mRNAs bound to EWS using PAR-CLIP. In this study, we demonstrate CCDC6, a known cell cycle regulator protein, as a novel target regulated by EWS. siRNA mediated down regulation of EWS caused an elevated apoptosis in cells in a CCDC6-dependant manner. This effect was rescued upon re-expression of CCDC6. This study provides evidence for a novel functional link through which wild-type EWS operates in a target-dependant manner in Ewing sarcoma. PMID:25751255

The Transcriptomes of Two Heritable Cell Types Illuminate the Circuit Governing Their Differentiation

PubMed Central

Homann, Oliver R.; Hernday, Aaron D.; Monighetti, Cinna K.; De La Vega, Francisco M.; Johnson, Alexander D.

2010-01-01

The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate their differences, we performed strand-specific, massively-parallel sequencing of RNA from C. albicans white and opaque cells. With these data we first annotated the C. albicans transcriptome, finding hundreds of novel differentially-expressed transcripts. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by a master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of a master regulator and the differential expression of adjacent genes, a discrepancy observed in several other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also find that 5′ and 3′ UTRs of mRNAs in the circuit are unusually long and that 5′ UTRs often differ in length between cell-types, suggesting UTRs encode important regulatory information and that use of alternative promoters is widespread. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes. PMID:20808890

Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction.

PubMed

Broaddus, W C; Liu, Y; Steele, L L; Gillies, G T; Lin, P S; Loudon, W G; Valerie, K; Schmidt-Ullrich, R K; Fillmore, H L

1999-12-01

The goal of this study was to determine whether adenoviral vector-mediated expression of human wildtype p53 can enhance the radiosensitivity of malignant glioma cells that express native wild-type p53. The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth. Recent studies suggest that reconstitution of wild-type p53 can render cells more susceptible to radiation-mediated death, primarily by p53-mediated apoptosis. Rat RT2 glioma cells were analyzed for native p53 status by reverse transcriptase-polymerase chain reaction and sequence analysis and for p53 expression by Western blot analysis. Clonogenic survival and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were used to characterize RT2 cell radiosensitivity and apoptosis, respectively, with and without prior transduction with p53-containing and control adenoviral vectors. Animal survival length was monitored after intracerebral implantation with transduced and nontransduced RT2 cells, with and without cranial radiation. The RT2 cells were demonstrated to express native rat wild-type p53 and to markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation resulted in marked decreases in RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. Animals receiving cranial radiation after intracerebral implantation with RT2 cells previously transduced with p53 survived significantly longer than control animals (p<0.01). The ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 offers hope for this approach as a therapeutic strategy, not only in human gliomas that express mutant p53, but also in those that express wild-type p53.

Encoding of Tactile Stimuli by Mechanoreceptors and Interneurons of the Medicinal Leech

PubMed Central

Kretzberg, Jutta; Pirschel, Friederice; Fathiazar, Elham; Hilgen, Gerrit

2016-01-01

For many animals processing of tactile information is a crucial task in behavioral contexts like exploration, foraging, and stimulus avoidance. The leech, having infrequent access to food, developed an energy efficient reaction to tactile stimuli, avoiding unnecessary muscle movements: The local bend behavior moves only a small part of the body wall away from an object touching the skin, while the rest of the animal remains stationary. Amazingly, the precision of this localized behavioral response is similar to the spatial discrimination threshold of the human fingertip, although the leech skin is innervated by an order of magnitude fewer mechanoreceptors and each midbody ganglion contains only 400 individually identified neurons in total. Prior studies suggested that this behavior is controlled by a three-layered feed-forward network, consisting of four mechanoreceptors (P cells), approximately 20 interneurons and 10 individually characterized motor neurons, all of which encode tactile stimulus location by overlapping, symmetrical tuning curves. Additionally, encoding of mechanical force was attributed to three types of mechanoreceptors reacting to distinct intensity ranges: T cells for touch, P cells for pressure, and N cells for strong, noxious skin stimulation. In this study, we provide evidences that tactile stimulus encoding in the leech is more complex than previously thought. Combined electrophysiological, anatomical, and voltage sensitive dye approaches indicate that P and T cells both play a major role in tactile information processing resulting in local bending. Our results indicate that tactile encoding neither relies on distinct force intensity ranges of different cell types, nor location encoding is restricted to spike count tuning. Instead, we propose that P and T cells form a mixed type population, which simultaneously employs temporal response features and spike counts for multiplexed encoding of touch location and force intensity. This hypothesis is supported by our finding that previously identified local bend interneurons receive input from both P and T cells. Some of these interneurons seem to integrate mechanoreceptor inputs, while others appear to use temporal response cues, presumably acting as coincidence detectors. Further voltage sensitive dye studies can test these hypotheses how a tiny nervous system performs highly precise stimulus processing. PMID:27840612

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

PubMed Central

Hickmott, Jack W; Chen, Chih-yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

2016-01-01

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

PubMed

Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

2016-01-01

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

Advances in the theory and application of BSF cells. [including electrical resistivity and photovoltaic cells

NASA Technical Reports Server (NTRS)

Mandelkorn, J.; Lamneck, J. H.

1975-01-01

The characteristics and behavior of p(+), p solar cells were investigated. The p(+), p cells were made by the removal of the n(+) surface layers from n(+), p p(+), BSF cells followed by application of a suitable contact to the resultant p(+), p structures. The open circuit voltage of p(+), p cells was found to increase with increasing 'p' bulk resistivity. The measured open circuit velocity-temperature coefficients were positive and increased with increasing resistivity. An outline of prior limitations in solar cell design is presented, and the removal of these limitations through use of BSF effects is pointed out. The study of BSF effects made feasible production of very thin high efficiency silicon cells as well as high resistivity-high efficiency cells, two desirable types of silicon cells which were previously impossible to make.

Normal Untreated Jurkat Cells

NASA Technical Reports Server (NTRS)

2004-01-01

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. The objective of the research was to define a way to differentiate between effects due to microgravity and those due to possible stress from non-optimal spaceflight conditions. These Jurkat cells, a human acute T-cell leukemia was obtained to evaluate three types of potential experimental stressors: a) Temperature elevation; b) Serum starvation; and c) Centrifugal force. The data from previous spaceflight experiments showed that actin filaments and cell shape are significantly different for the control. These normal cells serve as the baseline for future spaceflight experiments.

Cytoplasmic vacuolization in cell death and survival

PubMed Central

Komissarov, Alexey A.; Rafieva, Lola M.; Kostrov, Sergey V.

2016-01-01

Cytoplasmic vacuolization (also called cytoplasmic vacuolation) is a well-known morphological phenomenon observed in mammalian cells after exposure to bacterial or viral pathogens as well as to various natural and artificial low-molecular-weight compounds. Vacuolization often accompanies cell death; however, its role in cell death processes remains unclear. This can be attributed to studying vacuolization at the level of morphology for many years. At the same time, new data on the molecular mechanisms of the vacuole formation and structure have become available. In addition, numerous examples of the association between vacuolization and previously unknown cell death types have been reported. Here, we review these data to make a deeper insight into the role of cytoplasmic vacuolization in cell death and survival. PMID:27331412

Human Embryonic Stem Cells Undergo Osteogenic Differentiation in Human Bone Marrow Stromal Cell Microenvironments

PubMed Central

Tong, Wilbur; Brown, Shelley E.; Krebsbach, Paul H.

2009-01-01

Human embryonic stem cells (hESCs) may offer an unlimited supply of cells that can be directed to differentiate into all cell types within the body and used in regenerative medicine for tissue and cell replacement therapies. Previous work has shown that exposing hESCs to exogenous factors such as dexamethasone, ascorbic acid and β-glycerophosphate can induce osteogenesis. The specific factors that induce osteogenic differentiation of hESCs have not been identified yet, however, it is possible that differentiated human bone marrow stromal cells (hMBSCs) may secrete factors within the local microenvironment that promote osteogenesis. Here we report that the lineage progression of hESCs to osteoblasts is achieved in the presence of soluble signaling factors derived from differentiated hBMSCs. For 28 days, hESCs were grown in a transwell co-culture system with hBMSCs that had been previously differentiated in growth medium containing defined osteogenic supplements for 7-24 days. As a control. hESCs were co-cultured with undifferentiated hBMSCs and alone. Von Kossa and Alizarin Red staining as well as immunohistochemistry confirmed that the hESCs co-cultured with differentiated hBMSCs formed mineralized bone nodules and secreted extracellular matrix protein osteocalcin (OCN). Quantitative Alizarin Red assays showed increased mineralization as compared to the control with undifferentiated hBMSCs. RT-PCR revealed the loss of pluripotent hESC markers with the concomitant gain of osteoblastic markers such as collagen type I, runx2, and osterix. We demonstrate that osteogenic growth factors derived from differentiated hBMSCs within the local microenvironment may help to promote hESC osteogenic differentiation. PMID:20671800

Developmental expression and function analysis of protein tyrosine phosphatase receptor type D in oligodendrocyte myelination

PubMed Central

Zhu, Qiang; Tan, Zhou; Zhao, Shufang; Huang, Hao; Zhao, Xiaofeng; Hu, Xuemei; Zhang, Yiping; Shields, Christopher B; Uetani, Noriko; Qiu, Mengsheng

2015-01-01

Receptor protein tyrosine phosphatases (RPTPs) are extensively expressed in the central nervous system (CNS), and have distinct spatial and temporal patterns in different cell types during development. Previous studies have demonstrated possible roles for RPTPs in axon outgrowth, guidance, and synaptogenesis. In the present study, our results revealed that protein tyrosine phosphatase, receptor type D (PTPRD) was initially expressed in mature neurons in embryonic CNS, and later in oligodendroglial cells at postnatal stages when oligodendrocyte undergo active axonal myelination process. In PTPRD mutants, oligodendrocyte differentiation was normal and a transient myelination delay occurred at early postnatal stages, indicating the contribution of PTPRD to the initiation of axonal myelination. Our results also showed that the remyelination process was not affected in the absence of PTPRD function after a cuprizone-induced demyelination in adult animals. PMID:26341907

RGMb protects against acute kidney injury by inhibiting tubular cell necroptosis via an MLKL-dependent mechanism.

PubMed

Liu, Wenjing; Chen, Binbin; Wang, Yang; Meng, Chenling; Huang, Huihui; Huang, Xiao-Ru; Qin, Jinzhong; Mulay, Shrikant R; Anders, Hans-Joachim; Qiu, Andong; Yang, Baoxue; Freeman, Gordon J; Lu, Hua Jenny; Lin, Herbert Y; Zheng, Zhi-Hua; Lan, Hui-Yao; Huang, Yu; Xia, Yin

2018-02-13

Tubular cell necrosis is a key histological feature of acute kidney injury (AKI). Necroptosis is a type of programed necrosis, which is executed by mixed lineage kinase domain-like protein (MLKL) upon its binding to the plasma membrane. Emerging evidence indicates that necroptosis plays a critical role in the development of AKI. However, it is unclear whether renal tubular cells undergo necroptosis in vivo and how the necroptotic pathway is regulated during AKI. Repulsive guidance molecule (RGM)-b is a member of the RGM family. Our previous study demonstrated that RGMb is highly expressed in kidney tubular epithelial cells, but its biological role in the kidney has not been well characterized. In the present study, we found that RGMb reduced membrane-associated MLKL levels and inhibited necroptosis in cultured cells. During ischemia/reperfusion injury (IRI) or oxalate nephropathy, MLKL was induced to express on the apical membrane of proximal tubular (PT) cells. Specific knockout of Rgmb in tubular cells (Rgmb cKO) increased MLKL expression at the apical membrane of PT cells and induced more tubular cell death and more severe renal dysfunction compared with wild-type mice. Treatment with the necroptosis inhibitor Necrostatin-1 or GSK'963 reduced MLKL expression on the apical membrane of PT cells and ameliorated renal function impairment after IRI in both wild-type and Rgmb cKO mice. Taken together, our results suggest that proximal tubular cell necroptosis plays an important role in AKI, and that RGMb protects against AKI by inhibiting MLKL membrane association and necroptosis in proximal tubular cells.

Intracellular survival and vascular cell-to-cell transmission of Porphyromonas gingivalis

PubMed Central

Li, Ling; Michel, Raynald; Cohen, Joshua; DeCarlo, Arthur; Kozarov, Emil

2008-01-01

Background Porphyromonas gingivalis is associated with periodontal disease and invades different cell types including epithelial, endothelial and smooth muscle cells. In addition to P. gingivalis DNA, we have previously identified live invasive bacteria in atheromatous tissue. However, the mechanism of persistence of this organism in vascular tissues remains unclear. Therefore, the objective of this study was to analyze the ability of intracellular P. gingivalis to persist for extended periods of time, transmit to and possibly replicate in different cell types. Results Using antibiotic protection assays, immunofluorescent and laser confocal microscopy, we found that after a prolonged intracellular phase, while P. gingivalis can still be detected by immunostaining, the intracellular organisms lose their ability to be recovered in vitro. Surprisingly however, intracellular P. gingivalis could be recovered in vitro upon co incubation with fresh vascular host cells. We then demonstrated that the organism was able to exit the initially infected host cells, then enter and multiply in new host cells. Further, we found that cell-to-cell contact increased the transmission rate but was not required for transmission. Finally, we found that the invasion of new host cells allowed P. gingivalis to increase its numbers. Conclusion Our results suggest that the persistence of vascular tissue-embedded P. gingivalis is due to its ability to transmit among different cell types. This is the first communication demonstrating the intercellular transmission as a likely mechanism converting latent intracellular bacteria from state of dormancy to a viable state allowing for persistence of an inflammatory pathogen in vascular tissue. PMID:18254977

Lectin histochemistry of the rat lymph node: visualisation of stroma, blood vessels, sinuses, and macrophages. A contribution to the concept of an immune accessory role of sinus-lining endothelia.

PubMed

Düllmann, Jochen; Van Damme, Els J M; Peumans, Willy J; Ziesenitz, Maike; Schumacher, Udo

2002-01-01

The lectin Chelidonium majus agglutinin (CMA) was previously shown to visualise endothelia of all blood vessels and those lining sinuses of red pulp, stromal reticular meshwok (RM) and dendritic cells of lymphatic follicles in white pulp of the spleen in rats. The aim of the present study was the analysis of CMA and some other lectins in labelling RM, vascular structures and macrophages in lymph nodes of rats. It appeared that CMA stained the entire RM, dendritic cells, lining cells of sinuses and all types of blood vessels. Sinus-lining cells of lymph nodes were labelled with CMA and mannose-, GalNac-, and sialic acid-specific lectins. Moreover, lymph node macrophages were labelled above all by mannose specific lectins. The broad lectin-binding pattern of sinuses--not observed in rat spleen- and CMA-reactivity of both sinus-lining and dendritic cells corroborates the hypothesis that lymph node sinus-lining endothelia are precursors or a special type of immune accessory cells.

Hyper-dependence of breast cancer cell types on the nuclear transporter Importin β1.

PubMed

Kuusisto, Henna V; Jans, David A

2015-08-01

We previously reported that overexpression of members of the Importin (Imp) superfamily of nuclear transporters results in increased nuclear trafficking through conventional transport pathways in tumour cells. Here we show for the first time that the extent of overexpression of Impβ1 correlates with disease state in the MCF10 human breast tumour progression system. Excitingly, we find that targeting Impβ1 activity through siRNA is >30 times more efficient in decreasing the viability of malignant ductal carcinoma cells compared to isogenic non-transformed counterparts, and is highly potent and tumour selective at subnanomolar concentrations. Tumour cell selectivity of the siRNA effects was unique to Impβ1 and not other Imps, with flow cytometric analysis showing >60% increased cell death compared to controls concomitant with reduced nuclear import efficiency as indicated by confocal microscopic analysis. This hypersensitivity of malignant cell types to Impβ1 knockdown raises the exciting possibility of anti-cancer therapies targeted at Impβ1. Copyright © 2015 Elsevier B.V. All rights reserved.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy.

PubMed

Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

2015-01-08

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

An immunohistochemical study of endocrine cells in the alimentary tract of the grass lizard, Takydromus wolteri Fischer (Laceridae).

PubMed

Lee, Hyeung Sik; Ku, Sae Kwang

2004-01-01

Distribution patterns and the relative frequency of different types of endocrine cells were demonstrated in the alimentary tract of the grass lizard, Takydromus wolteri, using nine specific antibodies raised against mammalian regulatory peptides. The alimentary tract of the lizard was divided into six portions from the esophagus to the rectum. Most endocrine cells were found in the epithelial lining and were generally spindle shaped with long cytoplasmic processes ending in the lumen (open cell type), whereas cells that were spherical in shape (closed cell type) were occasionally found in gastric, esophageal and intestinal glands. Endocrine cells were stained for the following regulatory peptides: bovine Sp-1/chromogranin (BCG), serotonin, somatostatin, gastrin, cholecystokinin (CCK)-8, glucagon, insulin, human pancreatic polypeptide (HPP) and secretin. Cells stained for BCG and serotonin were present throughout the entire gastrointestinal tract and they occurred with the highest frequency in stomach and pylorus, respectively. Somatostatin-positive cells were detected throughout the entire gastrointestinal tract except for the esophagus and large intestine, and were most predominant in pylorus and duodenum. Cells stained for gastrin were restricted to the pylorus and duodenum and occurred with a relatively low frequency. CCK-8-positive cells were observed from pylorus to small intestine and showed the highest frequency in the pylorus. Glucagon- and insulin-containing cells were located in duodenum and small intestine but were found only rarely. HPP-stained cells were detected in duodenum and small intestine with the highest frequency in duodenum. Cells stained for secretin were restricted to duodenum and were found only rarely. In conclusion, distribution patterns and the relative frequency of these endocrine cells correspond well with previous reports on distribution patterns of endocrine cells in reptile species but some deviating patterns were also observed.

Toward Stable Genetic Engineering of Human O-Glycosylation in Plants1[C][W][OA

PubMed Central

Yang, Zhang; Bennett, Eric P.; Jørgensen, Bodil; Drew, Damian P.; Arigi, Emma; Mandel, Ulla; Ulvskov, Peter; Levery, Steven B.; Clausen, Henrik; Petersen, Bent L.

2012-01-01

Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating GalNAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O-glycoproteins was obtained, but a high degree of proline hydroxylation and hydroxyproline-linked arabinosides, on a mucin (MUC1)-derived substrate, was also observed. Addition of the prolyl 4-hydroxylase inhibitor 2,2-dipyridyl, however, effectively suppressed proline hydroxylation and arabinosylation of MUC1 in Bright Yellow-2 cells. In summary, stably engineered mammalian type O-glycosylation was established in transgenic plants, demonstrating that plants may serve as host cells for the production of recombinant O-glycoproteins. However, the present stable implementation further strengthens the notion that elimination of endogenous posttranslational modifications may be needed for the production of protein therapeutics. PMID:22791304

Reduced IL-10 Production in Fetal Type II Epithelial Cells Exposed to Mechanical Stretch Is Mediated via Activation of IL-6-SOCS3 Signaling Pathway

PubMed Central

Hawwa, Renda L.; Huang, Zheping; Sharma, Surendra; Sanchez-Esteban, Juan

2013-01-01

An imbalance between pro-inflammatory and anti-inflammatory cytokines is a key factor in the lung injury of premature infants exposed to mechanical ventilation. Previous studies have shown that lung cells exposed to stretch produces reduced amounts of the anti-inflammatory cytokine IL-10. The objective of these studies was to analyze the signaling mechanisms responsible for the decreased IL-10 production in fetal type II cells exposed to mechanical stretch. Fetal mouse type II epithelial cells isolated at embryonic day 18 were exposed to 20% stretch to simulate lung injury. We show that IL-10 receptor gene expression increased with gestational age. Mechanical stretch decreased not only IL-10 receptor gene expression but also IL-10 secretion. In contrast, mechanical stretch increased release of IL-6. We then investigated IL-10 signaling pathway-associated proteins and found that in wild-type cells, mechanical stretch decreased activation of JAK1 and TYK2 and increased STAT3 and SOCS3 activation. However, opposite effects were found in cells isolated from IL-10 knockout mice. Reduction in IL-6 secretion by stretch was observed in cells isolated from IL-10 null mice. To support the idea that stretch-induced SOCS3 expression via IL-6 leads to reduced IL-10 expression, siRNA-mediated inhibition of SOCS3 restored IL-10 secretion in cells exposed to stretch and decreased IL-6 secretion. Taken together, these studies suggest that the inhibitory effect of mechanical stretch on IL-10 secretion is mediated via activation of IL-6-STAT3-SOCS3 signaling pathway. SOCS3 could be a therapeutic target to increase IL-10 production in lung cells exposed to mechanical injury. PMID:23527226

Reduced IL-10 production in fetal type II epithelial cells exposed to mechanical stretch is mediated via activation of IL-6-SOCS3 signaling pathway.

PubMed

Hokenson, Michael A; Wang, Yulian; Hawwa, Renda L; Huang, Zheping; Sharma, Surendra; Sanchez-Esteban, Juan

2013-01-01

An imbalance between pro-inflammatory and anti-inflammatory cytokines is a key factor in the lung injury of premature infants exposed to mechanical ventilation. Previous studies have shown that lung cells exposed to stretch produces reduced amounts of the anti-inflammatory cytokine IL-10. The objective of these studies was to analyze the signaling mechanisms responsible for the decreased IL-10 production in fetal type II cells exposed to mechanical stretch. Fetal mouse type II epithelial cells isolated at embryonic day 18 were exposed to 20% stretch to simulate lung injury. We show that IL-10 receptor gene expression increased with gestational age. Mechanical stretch decreased not only IL-10 receptor gene expression but also IL-10 secretion. In contrast, mechanical stretch increased release of IL-6. We then investigated IL-10 signaling pathway-associated proteins and found that in wild-type cells, mechanical stretch decreased activation of JAK1 and TYK2 and increased STAT3 and SOCS3 activation. However, opposite effects were found in cells isolated from IL-10 knockout mice. Reduction in IL-6 secretion by stretch was observed in cells isolated from IL-10 null mice. To support the idea that stretch-induced SOCS3 expression via IL-6 leads to reduced IL-10 expression, siRNA-mediated inhibition of SOCS3 restored IL-10 secretion in cells exposed to stretch and decreased IL-6 secretion. Taken together, these studies suggest that the inhibitory effect of mechanical stretch on IL-10 secretion is mediated via activation of IL-6-STAT3-SOCS3 signaling pathway. SOCS3 could be a therapeutic target to increase IL-10 production in lung cells exposed to mechanical injury.

Hypoxic augmentation of Ca2+ channel currents requires a functional electron transport chain.

PubMed

Brown, Stephen T; Scragg, Jason L; Boyle, John P; Hudasek, Kristin; Peers, Chris; Fearon, Ian M

2005-06-10

The incidence of Alzheimer disease is increased following ischemic episodes, and we previously demonstrated that following chronic hypoxia (CH), amyloid beta (Abeta) peptide-mediated increases in voltage-gated L-type Ca(2+) channel activity contribute to the Ca(2+) dyshomeostasis seen in Alzheimer disease. Because in certain cell types mitochondria are responsible for detecting altered O(2) levels we examined the role of mitochondrial oxidant production in the regulation of recombinant Ca(2+) channel alpha(1C) subunits during CH and exposure to Abeta-(1-40). In wild-type (rho(+)) HEK 293 cells expressing recombinant L-type alpha(1C) subunits, Ca(2+) currents were enhanced by prolonged (24 h) exposure to either CH (6% O(2)) or Abeta-(1-40) (50 nm). By contrast the response to CH was absent in rho(0) cells in which the mitochondrial electron transport chain (ETC) was depleted following long term treatment with ethidium bromide or in rho(+) cells cultured in the presence of 1 microm rotenone. CH was mimicked in rho(0) cells by the exogenous production of O2(-.). by xanthine/xanthine oxidase. Furthermore Abeta-(1-40) enhanced currents in rho(0) cells to a degree similar to that seen in cells with an intact ETC. The antioxidants ascorbate (200 microm) and Trolox (500 microm) ablated the effect of CH in rho(+) cells but were without effect on Abeta-(1-40)-mediated augmentation of Ca(2+) current in rho(0) cells. Thus oxidant production in the mitochondrial ETC is a critical factor, acting upstream of amyloid beta peptide production in the up-regulation of Ca(2+) channels in response to CH.

Comparison of hematological alterations and markers of B-cell activation in workers exposed to benzene, formaldehyde and trichloroethylene.

PubMed

Bassig, Bryan A; Zhang, Luoping; Vermeulen, Roel; Tang, Xiaojiang; Li, Guilan; Hu, Wei; Guo, Weihong; Purdue, Mark P; Yin, Songnian; Rappaport, Stephen M; Shen, Min; Ji, Zhiying; Qiu, Chuangyi; Ge, Yichen; Hosgood, H Dean; Reiss, Boris; Wu, Banghua; Xie, Yuxuan; Li, Laiyu; Yue, Fei; Freeman, Laura E Beane; Blair, Aaron; Hayes, Richard B; Huang, Hanlin; Smith, Martyn T; Rothman, Nathaniel; Lan, Qing

2016-07-01

Benzene, formaldehyde (FA) and trichloroethylene (TCE) are ubiquitous chemicals in workplaces and the general environment. Benzene is an established myeloid leukemogen and probable lymphomagen. FA is classified as a myeloid leukemogen but has not been associated with non-Hodgkin lymphoma (NHL), whereas TCE has been associated with NHL but not myeloid leukemia. Epidemiologic associations between FA and myeloid leukemia, and between benzene, TCE and NHL are, however, still debated. Previously, we showed that these chemicals are associated with hematotoxicity in cross-sectional studies of factory workers in China, which included extensive personal monitoring and biological sample collection. Here, we compare and contrast patterns of hematotoxicity, monosomy 7 in myeloid progenitor cells (MPCs), and B-cell activation biomarkers across these studies to further evaluate possible mechanisms of action and consistency of effects with observed hematologic cancer risks. Workers exposed to benzene or FA, but not TCE, showed declines in cell types derived from MPCs, including granulocytes and platelets. Alterations in lymphoid cell types, including B cells and CD4+ T cells, and B-cell activation markers were apparent in workers exposed to benzene or TCE. Given that alterations in myeloid and lymphoid cell types are associated with hematological malignancies, our data provide biologic insight into the epidemiological evidence linking benzene and FA exposure with myeloid leukemia risk, and TCE and benzene exposure with NHL risk. Published by Oxford University Press 2016.

Interfacial stress affects rat alveolar type II cell signaling and gene expression.

PubMed

Hobi, Nina; Ravasio, Andrea; Haller, Thomas

2012-07-01

Previous work from our group (Ravasio A, Hobi N, Bertocchi C, Jesacher A, Dietl P, Haller T. Am J Physiol Cell Physiol 300: C1456-C1465, 2011.) showed that contact of alveolar epithelial type II cells with an air-liquid interface (I(AL)) leads to a paradoxical situation. It is a potential threat that can cause cell injury, but also a Ca(2+)-dependent stimulus for surfactant secretion. Both events can be explained by the impact of interfacial tensile forces on cellular structures. Here, the strength of this mechanical stimulus became also apparent in microarray studies by a rapid and significant change on the transcriptional level. Cells challenged with an I(AL) in two different ways showed activation/inactivation of cellular pathways involved in stress response and defense, and a detailed Pubmatrix search identified genes associated with several lung diseases and injuries. Altogether, they suggest a close relationship of interfacial stress sensation with current models in alveolar micromechanics. Further similarities between I(AL) and cell stretch were found with respect to the underlying signaling events. The source of Ca(2+) was extracellular, and the transmembrane Ca(2+) entry pathway suggests the involvement of a mechanosensitive channel. We conclude that alveolar type II cells, due to their location and morphology, are specific sensors of the I(AL), but largely protected from interfacial stress by surfactant release.

Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

1986-07-15

The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. Asmore » reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.« less

The interaction of the carbon nanoparticles with human cell plasma membrane

NASA Astrophysics Data System (ADS)

Overchuk, M.; Prylutska, S.; Bilyy, Rostyslav; Prylutsky, Yu.; Ritter, U.

2013-09-01

The study of carbon nanostructures is a highly topical branch of bionanotechnology because of their potential application in biomedicine. Carbon nanotubes (CNTs) are known for their ability to kill tumor cells causing hyperthermia shock and can be used in photothermal therapy respectively. Also chemically modified CNTs can be used for drug delivery. The needle-like shape of CNTs allows them to penetrate into the cell plasma membrane without killing the cell. C60 fullerenes are regarded as valuable nanocarriers for different hydrophobic molecules as well as potential antiviral agents or photosensitizers. In our previous studies we have demonstrated that all types of carbon nanoparticles cause externalization of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane in the small local patches (points of contact), leaving the other parts of plasma membrane PS-negative. In the current work there were studied the interactions of pristine C60 fullerenes and different types of CNTs with human blood cells (erythrocytes and Jurkat T-cells). We have shown, that carbon nanoparticles do not have any hemolytic effects, if judged by the dynamics of acidic hemolysis, although they are capable of permeabilizating the cells and facilitating the internalization of propidium iodide into the nuclei.

Inheritance of gene density–related higher order chromatin arrangements in normal and tumor cell nuclei

PubMed Central

Cremer, Marion; Küpper, Katrin; Wagler, Babett; Wizelman, Leah; Hase, Johann v.; Weiland, Yanina; Kreja, Ludwika; Diebold, Joachim; Speicher, Michael R.; Cremer, Thomas

2003-01-01

A gene density–related difference in the radial arrangement of chromosome territories (CTs) was previously described for human lymphocyte nuclei with gene-poor CT #18 located toward the nuclear periphery and gene-dense CT #19 in the nuclear interior (Croft, J.A., J.M. Bridger, S. Boyle, P. Perry, P. Teague, and W.A. Bickmore. 1999. J. Cell Biol. 145:1119–1131). Here, we analyzed the radial distribution of chromosome 18 and 19 chromatin in six normal cell types and in eight tumor cell lines, some of them with imbalances and rearrangements of the two chromosomes. Our findings demonstrate that a significant difference in the radial distribution of #18 and #19 chromatin is a common feature of higher order chromatin architecture in both normal and malignant cell types. However, in seven of eight tumor cell lines, the difference was less pronounced compared with normal cell nuclei due to a higher fraction of nuclei showing an inverted CT position, i.e., a CT #18 located more internally than a CT #19. This observation emphasizes a partial loss of radial chromatin order in tumor cell nuclei. PMID:12952935

Donor interleukin-22 and host type I interferon signaling pathway participate in intestinal graft-versus-host disease via STAT1 activation and CXCL10.

PubMed

Lamarthée, B; Malard, F; Gamonet, C; Bossard, C; Couturier, M; Renauld, J-C; Mohty, M; Saas, P; Gaugler, B

2016-03-01

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation, limiting the success of this therapy. We previously reported that interleukin-22 (IL-22) participates to aGVHD development, but the underlying mechanisms of its contribution remain poorly understood. In this study, we analyzed the mechanism of the pathological function of IL-22 in intestinal aGVHD. Ex-vivo colon culture experiments indicated that IL-22 was able to induce Th1-like inflammation via signal transducer and activator of transcription factor-1 (STAT1) and CXCL10 induction in the presence of type I interferon (IFN). To evaluate a potential synergy between IL-22 and type I IFN in aGVHD, we transplanted recipient mice, either wild-type (WT) or type I IFN receptor deficient (IFNAR(-/-)), with bone marrow cells and WT or IL-22 deficient (IL-22(-/-)) T cells. We observed a decreased GVHD severity in IFNAR(-/-) recipient of IL-22(-/-) T cells, which was associated with a lower level of STAT1 activation and reduced CXCL10 expression in the large intestine. Finally, immunohistochemistry staining of STAT1 performed on gastrointestinal biopsies of 20 transplanted patients showed exacerbated STAT1 activation in gastrointestinal tissues of patients with aGVHD as compared with those without aGVHD. Thus, interfering with both IL-22 and type I IFN signaling may provide a novel approach to limit aGVHD.

Characterization of Actinomyces Isolates from Infected Root Canals of Teeth: Description of Actinomyces radicidentis sp. nov.

PubMed Central

Collins, Matthew D.; Hoyles, Lesley; Kalfas, Sotos; Sundquist, Goran; Monsen, Tor; Nikolaitchouk, Natalia; Falsen, Enevold

2000-01-01

Two strains of a previously undescribed Actinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain of Actinomyces radicidentis is CCUG 36733. PMID:10970390

Oxygen consumption rate of cells in 3D culture: the use of experiment and simulation to measure kinetic parameters and optimise culture conditions.

PubMed

Streeter, Ian; Cheema, Umber

2011-10-07

Understanding the basal O(2) and nutrient requirements of cells is paramount when culturing cells in 3D tissue models. Any scaffold design will need to take such parameters into consideration, especially as the addition of cells introduces gradients of consumption of such molecules from the surface to the core of scaffolds. We have cultured two cell types in 3D native collagen type I scaffolds, and measured the O(2) tension at specific locations within the scaffold. By changing the density of cells, we have established O(2) consumption gradients within these scaffolds and using mathematical modeling have derived rates of consumption for O(2). For human dermal fibroblasts the average rate constant was 1.19 × 10(-17) mol cell(-1) s(-1), and for human bone marrow derived stromal cells the average rate constant was 7.91 × 10(-18) mol cell(-1) s(-1). These values are lower than previously published rates for similar cells cultured in 2D, but the values established in this current study are more representative of rates of consumption measured in vivo. These values will dictate 3D culture parameters, including maximum cell-seeding density and maximum size of the constructs, for long-term viability of tissue models.

Aggregation and lack of secretion of most newly synthesized proinsulin in non-beta-cell lines.

PubMed

Zhu, Yong Lian; Abdo, Alexander; Gesmonde, Joan F; Zawalich, Kathleen C; Zawalich, Walter; Dannies, Priscilla S

2004-08-01

Myoblasts transfected with HB10D insulin secrete more hormone than those transfected with wild-type insulin, as published previously, indicating that production of wild-type insulin is not efficient in these cells. The ability of non-beta-cells to produce insulin was examined in several cell lines. In clones of neuroendocrine GH(4)C(1) cells stably transfected with proinsulin, two thirds of (35)S-proinsulin was degraded within 3 h of synthesis, whereas (35)S-prolactin was stable. In transiently transfected neuroendocrine AtT20 cells, half of (35)S-proinsulin was degraded within 3 h after synthesis, whereas (35)S-GH was stable. In transiently transfected fibroblast COS cells, (35)S-proinsulin was stable for longer, but less than 10% was secreted 8 h after synthesis. Proinsulin formed a concentrated patch detected by immunofluorescence in transfected cells that did not colocalize with calreticulin or BiP, markers for the endoplasmic reticulum, but did colocalize with membrin, a marker for the cis-medial Golgi complex. Proinsulin formed a Lubrol-insoluble aggregate within 30 min after synthesis in non-beta-cells but not in INS-1E cells, a beta-cell line that normally produces insulin. More than 45% of (35)S-HB10D proinsulin was secreted from COS cells 3 h after synthesis, and this mutant formed less Lubrol-insoluble aggregate in the cells than did wild-type hormone. These results indicate that proinsulin production from these non-beta-cells is not efficient and that proinsulin aggregates in their secretory pathways. Factors in the environment of the secretory pathway of beta-cells may prevent aggregation of proinsulin to allow efficient production.

Investigation of anodic and chemical oxides grown on p-type InP with applications to surface passivation for n(+)-p solar cell fabrication

NASA Technical Reports Server (NTRS)

Faur, Maria; Faur, Mircea; Goradia, Manju; Goradia, Chandra; Jenkins, Phillip; Jayne, Douglas; Weinberg, Irving

1991-01-01

Most of the previously reported InP anodic oxides were grown on a n-type InP with applications to fabrication of MISFET structures and were described as a mixture of In2O3 and P2O5 stoichiometric compounds or nonstoichiometric phases which have properties similar to crystalline compounds In(OH)3, InPO4, and In(PO3)3. Details of the compositional change of the anodic oxides grown under different anodization conditions were previously reported. The use of P-rich oxides grown either by anodic or chemical oxidation are investigated for surface passivation of p-type InP and as a protective cap during junction formation by closed-ampoule sulfur diffusion. The investigation is based on but not limited to correlations between PL intensity and X-ray photoelectron spectroscopy (XPS) chemical composition data.

Phosphorylation of rat kidney Na-K pump at Ser938 is required for rapid angiotensin II-dependent stimulation of activity and trafficking in proximal tubule cells

PubMed Central

Massey, Katherine J.; Li, Quanwen; Rossi, Noreen F.; Keezer, Susan M.; Mattingly, Raymond R.

2015-01-01

How angiotensin (ANG) II acutely stimulates the Na-K pump in proximal tubules is only partially understood, limiting insight into how ANG II increases blood pressure. First, we tested whether ANG II increases the number of pumps in plasma membranes of native rat proximal tubules under conditions of rapid activation. We found that exposure to 100 pM ANG II for 2 min, which was previously shown to increase affinity of the Na-K pump for Na and stimulate activity threefold, increased the amount of the Na-K pump in plasma membranes of native tubules by 33%. Second, we tested whether previously observed increases in phosphorylation of the Na-K pump at Ser938 were part of the stimulatory mechanism. These experiments were carried out in opossum kidney cells, cultured proximal tubules stably coexpressing the ANG type 1 (AT1) receptor, and either wild-type or a S938A mutant of rat kidney Na-K pump under conditions found by others to stimulate activity. We found that 10 min of incubation in 10 pM ANG II stimulated activity of wild-type pumps from 2.3 to 3.5 nmol K·mg protein−1·min−1 and increased the amount of the pump in the plasma membrane by 80% but had no effect on cells expressing the S938A mutant. We conclude that acute stimulation of Na-K pump activity in native rat proximal tubules includes increased trafficking to the plasma membrane and that phosphorylation at Ser938 is part of the mechanism by which ANG II directly stimulates activity and trafficking of the rat kidney Na-K pump in opossum kidney cells. PMID:26582472

Cytology of the minor-vein phloem in 320 species from the subclass Asteridae suggests a high diversity of phloem-loading modes†

PubMed Central

Batashev, Denis R.; Pakhomova, Marina V.; Razumovskaya, Anna V.; Voitsekhovskaja, Olga V.; Gamalei, Yuri V.

2013-01-01

The discovery of abundant plasmodesmata at the bundle sheath/phloem interface in Oleaceae (Gamalei, 1974) and Cucurbitaceae (Turgeon et al., 1975) raised the questions as to whether these plasmodesmata are functional in phloem loading and how widespread symplasmic loading would be. Analysis of over 800 dicot species allowed the definition of “open” and “closed” types of the minor vein phloem depending on the abundance of plasmodesmata between companion cells and bundle sheath (Gamalei, 1989, 1990). These types corresponded to potential symplasmic and apoplasmic phloem loaders, respectively; however, this definition covered a spectrum of diverse structures of phloem endings. Here, a review of detailed cytological analyses of minor veins in 320 species from the subclass Asteridae is presented, including data on companion cell types and their combinations which have not been reported previously. The percentage of Asteridae species with “open” minor vein cytology which also contain sieve-element-companion cell complexes with “closed” cytology, i.e., that show specialization for both symplasmic and apoplasmic phloem loading, was determined. Along with recent data confirming the dissimilar functional specialization of structurally different parts of minor vein phloem in the stachyose-translocating species Alonsoa meridionalis (Voitsekhovskaja et al., 2009), these findings suggest that apoplasmic loading is indispensable in a large group of species previously classified as putative symplasmic loaders. Altogether, this study provides formal classifications of companion cells and of minor veins, respectively, in 24 families of the Asteridae based on their structural features, opening the way to a close investigation of the relationship between structure and function in phloem loading. PMID:23970890

Muscle layer histopathology and manometry pattern of primary esophageal motility disorders including achalasia.

PubMed

Nakajima, N; Sato, H; Takahashi, K; Hasegawa, G; Mizuno, K; Hashimoto, S; Sato, Y; Terai, S

2017-03-01

Histopathology of muscularis externa in primary esophageal motility disorders has been characterized previously. We aimed to correlate the results of high-resolution manometry with those of histopathology. During peroral endoscopic myotomy, peroral esophageal muscle biopsy was performed in patients with primary esophageal motility disorders. Immunohistochemical staining for c-kit was performed to assess the interstitial cells of Cajal (ICCs). Hematoxylin Eosin and Azan-Mallory staining were used to detect muscle atrophy, inflammation, and fibrosis, respectively. Slides from 30 patients with the following motility disorders were analyzed: achalasia (type I: 14, type II: 5, type III: 3), one diffuse esophageal spasm (DES), two outflow obstruction (OO), four jackhammer esophagus (JE), and one nutcracker esophagus (NE). ICCs were preserved in high numbers in type III achalasia (n=9.4±1.2 cells/high power field [HPF]), compared to types I (n=3.7±0.3 cells/HPF) and II (n=3.5±1.0 cells/HPF). Moreover, severe fibrosis was only observed in type I achalasia and not in other types of achalasia, OO, or DES. Four of five patients with JE and NE had severe inflammation with eosinophilic infiltration of the esophageal muscle layer (73.8±50.3 eosinophils/HPF) with no epithelial eosinophils. One patient with JE showed a visceral myopathy pattern. Compared to types I and II, type III achalasia showed preserved ICCs, with variable data regarding DES and OO. In disorders considered as primary esophageal motility disorders, a disease category exists, which shows eosinophilic infiltration in the esophageal muscle layer with no eosinophils in the epithelium. © 2016 John Wiley & Sons Ltd.

A mechanistic model for bromodeoxyuridine dilution naturally explains labelling data of self-renewing T cell populations

PubMed Central

Ganusov, Vitaly V.; De Boer, Rob J.

2013-01-01

Bromodeoxyuridine (BrdU) is widely used in immunology to detect cell division, and several mathematical models have been proposed to estimate proliferation and death rates of lymphocytes from BrdU labelling and de-labelling curves. One problem in interpreting BrdU data is explaining the de-labelling curves. Because shortly after label withdrawal, BrdU+ cells are expected to divide into BrdU+ daughter cells, one would expect a flat down-slope. As for many cell types, the fraction of BrdU+ cells decreases during de-labelling, previous mathematical models had to make debatable assumptions to be able to account for the data. We develop a mechanistic model tracking the number of divisions that each cell has undergone in the presence and absence of BrdU, and allow cells to accumulate and dilute their BrdU content. From the same mechanistic model, one can naturally derive expressions for the mean BrdU content (MBC) of all cells, or the MBC of the BrdU+ subset, which is related to the mean fluorescence intensity of BrdU that can be measured in experiments. The model is extended to include subpopulations with different rates of division and death (i.e. kinetic heterogeneity). We fit the extended model to previously published BrdU data from memory T lymphocytes in simian immunodeficiency virus-infected and uninfected macaques, and find that the model describes the data with at least the same quality as previous models. Because the same model predicts a modest decline in the MBC of BrdU+ cells, which is consistent with experimental observations, BrdU dilution seems a natural explanation for the observed down-slopes in self-renewing populations. PMID:23034350

β-Catenin signaling regulates temporally discrete phases of anterior taste bud development.

PubMed

Thirumangalathu, Shoba; Barlow, Linda A

2015-12-15

The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of β-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of β-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of β-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of β-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, β-catenin activation a day later within Shh(+) placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of β-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of β-catenin within Shh(+) precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by β-catenin. © 2015. Published by The Company of Biologists Ltd.

Inhibition of Notch signaling alters the phenotype of orthotopic tumors formed from glioblastoma multiforme neurosphere cells but does not hamper intracranial tumor growth regardless of endogene Notch pathway signature.

PubMed

Kristoffersen, Karina; Nedergaard, Mette Kjølhede; Villingshøj, Mette; Borup, Rehannah; Broholm, Helle; Kjær, Andreas; Poulsen, Hans Skovgaard; Stockhausen, Marie-Thérése

2014-07-01

Brain cancer stem-like cells (bCSC) are cancer cells with neural stem cell (NSC)-like properties found in the devastating brain tumor glioblastoma multiforme (GBM). bCSC are proposed a central role in tumor initiation, progression, treatment resistance and relapse and as such present a promising target in GBM research. The Notch signaling pathway is often deregulated in GBM and we have previously characterized GBM-derived bCSC cultures based on their expression of the Notch-1 receptor and found that it could be used as predictive marker for the effect of Notch inhibition. The aim of the present project was therefore to further elucidate the significance of Notch pathway activity for the tumorigenic properties of GBM-derived bCSC. Human-derived GBM xenograft cells previously established as NSC-like neurosphere cultures were used. Notch inhibition was accomplished by exposing the cells to the gamma-secretase inhibitor DAPT prior to gene expression analysis and intracranial injection into immunocompromised mice. By analyzing the expression of several Notch pathway components, we found that the cultures indeed displayed different Notch pathway signatures. However, when DAPT-treated neurosphere cells were injected into the brain of immunocompromised mice, no increase in survival was obtained regardless of Notch pathway signature and Notch inhibition. We did however observe a decrease in the expression of the stem cell marker Nestin, an increase in the proliferative marker Ki-67 and an increased number of abnormal vessels in tumors formed from DAPT-treated, high Notch-1 expressing cultures, when compared with the control. Based on the presented results we propose that Notch inhibition partly induces differentiation of bCSC, and selects for a cell type that more strongly induces angiogenesis if the treatment is not sustained. However, this more differentiated cell type might prove to be more sensitive to conventional therapies.

Modelling and simulation of thermal behaviour of vanadium redox flow battery

NASA Astrophysics Data System (ADS)

Yan, Yitao; Li, Yifeng; Skyllas-Kazacos, Maria; Bao, Jie

2016-08-01

This paper extends previous thermal models of the vanadium redox flow battery to predict temperature profiles within multi-cell stacks. This involves modelling the thermal characteristics of the stack as a whole to modelling each individual cell. The study investigates the thermal behaviour for two different scenarios: during standby periods when the pumps are turned off, and in a residential power arbitrage scenario for two types of membranes. It was found that the temperature gradient across the cells is most significant during the standby case, with the simulation results showing completely different thermal behaviours between the two systems.

Left-right asymmetry is formed in individual cells by intrinsic cell chirality.

PubMed

Hatori, Ryo; Ando, Tadashi; Sasamura, Takeshi; Nakazawa, Naotaka; Nakamura, Mitsutoshi; Taniguchi, Kiichiro; Hozumi, Shunya; Kikuta, Junichi; Ishii, Masaru; Matsuno, Kenji

2014-08-01

Many animals show left-right (LR) asymmetric morphology. The mechanisms of LR asymmetric development are evolutionarily divergent, and they remain elusive in invertebrates. Various organs in Drosophila melanogaster show stereotypic LR asymmetry, including the embryonic gut. The Drosophila embryonic hindgut twists 90° left-handedly, thereby generating directional LR asymmetry. We recently revealed that the hindgut epithelial cell is chiral in shape and other properties; this is termed planar cell chirality (PCC). We previously showed by computer modeling that PCC is sufficient to induce the hindgut rotation. In addition, both the PCC and the direction of hindgut twisting are reversed in Myosin31DF (Myo31DF) mutants. Myo31DF encodes Drosophila MyosinID, an actin-based motor protein, whose molecular functions in LR asymmetric development are largely unknown. Here, to understand how PCC directs the asymmetric cell-shape, we analyzed PCC in genetic mosaics composed of cells homozygous for mutant Myo31DF, some of which also overexpressed wild-type Myo31DF. Wild-type cell-shape chirality only formed in the Myo31DF-overexpressing cells, suggesting that cell-shape chirality was established in each cell and reflects intrinsic PCC. A computer model recapitulating the development of this genetic mosaic suggested that mechanical interactions between cells are required for the cell-shape behavior seen in vivo. Our mosaic analysis also suggested that during hindgut rotation in vivo, wild-type Myo31DF suppresses the elongation of cell boundaries, supporting the idea that cell-shape chirality is an intrinsic property determined in each cell. However, the amount and distribution of F-actin and Myosin II, which are known to help generate the contraction force on cell boundaries, did not show differences between Myo31DF mutant cells and wild-type cells, suggesting that the static amount and distribution of these proteins are not involved in the suppression of cell-boundary elongation. Taken together, our results suggest that cell-shape chirality is intrinsically formed in each cell, and that mechanical force from intercellular interactions contributes to its formation and/or maintenance. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

How MMPs Impact Bone Responses to Metastatic Prostate Cancer

DTIC Science & Technology

2011-04-30

various tumor cell types including myeloma ( Barille et al., 1997; Vacca et al., 1998; Barille et al., 1999; Vacca et al., 1999). Previous studies have...useful in vivo mouse model of human multiple myeloma. Hematol. J. 1, 351-356. Barille , S., Akhoundi, C., Collette, M., Mellerin, M. P., Rapp, M. J...activation of proMMP-2, and induction of MMP-1 by myeloma cells. Blood 90, 1649-1655. Barille , S., Bataille, R., Rapp, M. J., Harousseau, J. L. and Amiot, M

Cloning of an ets-Related Transcription Factor Involved in a Novel Epigenetic Mechanism of Mammary Carcinogenesis

DTIC Science & Technology

2001-05-01

dystrophin gene promoter is regulated by YY1 and DPBF (33). Other studies showed that serum response factor (SRF) is required for muscle-specific...transcriptional activation through CArG boxes (68) and that SRF competes with YY1 for binding to wild-type CArG elements (51). CBF-A has significant...between SRF and YY1 proteins at CArG elements has been described in chicken skeletal muscle cells(36). Our previous study in a rat mammary carcinoma cell

Lanostane-type triterpenoids from the fruiting body of Ganoderma calidophilum.

PubMed

Huang, Sheng-Zhuo; Ma, Qing-Yun; Kong, Fan-Dong; Guo, Zhi-Kai; Cai, Cai-Hong; Hu, Li-Li; Zhou, Li-Man; Wang, Qi; Dai, Hao-Fu; Mei, Wen-Li; Zhao, You-Xing

2017-11-01

To search for active anti-cancer constituents in the fruiting body of Ganoderma calidophilum, we have successfully isolated four previously undescribed spiro-lactone lanostane triterpenoids (spiroganocalitones A-D), two previously undescribed lanostanoids (ganodecalones A and B) together with twenty-three known ones. The structures of the six previously undescribed compounds were elucidated based on 1D, 2D-NMR, and HRMS analyses. Ganoderone A showed moderate cytotoxic activity against K562, BEL7402, and SGC790 cell lines with IC 50 values of 7.62, 6.28, and 3.55 μM, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chitinase and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae.

PubMed

Cabib, E; Silverman, S J; Shaw, J A

1992-01-01

Previous results [E. Cabib, A. Sburlati, B. Bowers & S. J. Silverman (1989) Journal of Cell Biology 108, 1665-1672] strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation. Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis. The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis. Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis. The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene. Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor. These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.

In vitro study of cell differentiation by two type mouse embryo stem cells on mono- and multilayer nanocarbon tubes

NASA Astrophysics Data System (ADS)

Imai, Koichi; Akasaka, Tsukasa; Watari, Fumio; Tanoue, Akito; Nakamura, Kazuaki; Suese, Kazuhiko; Takashima, Hiromasa; Nishikawa, Tetsunari; Tanaka, Akio; Takeda, Shoji

2012-09-01

The effects of nanomaterials on human reproduction and development remain unknown. The risks of nanomaterials for future generations should be elucidated. Thus, it is important to establish an experimental method to accurately examine embryotoxicity. We previously investigated the myocardial cell differentiation of ES-D3 cells using monolayer (SWCNTs) and multilayer (MWCNTs) nanocarbon tubes. As a result, in spite of having the same carbon composition, the effects on the cell differentiation levels differed between the tubes. We investigated their cell differentiation and cytotoxic effects on EL M3 and ES-R1-EGFP B2/EGFP cells, which require feeder cells. As a result, myocardial pulse rates differed between the presence of SWCNTs and MWCNTs even when feeder cells existed between the samples and cells. The different surface structures of SWCNTs and MWCNTs may have influenced ES cell differentiation.

A single-cell survey of the small intestinal epithelium

PubMed Central

Haber, Adam L.; Biton, Moshe; Rogel, Noga; Herbst, Rebecca H.; Shekhar, Karthik; Smillie, Christopher; Burgin, Grace; Delorey, Toni M.; Howitt, Michael R.; Katz, Yarden; Tirosh, Itay; Beyaz, Semir; Dionne, Danielle; Zhang, Mei; Raychowdhury, Raktima; Garrett, Wendy S.; Rozenblatt-Rosen, Orit; Shi, Hai Ning; Yilmaz, Omer; Xavier, Ramnik J.; Regev, Aviv

2018-01-01

Intestinal epithelial cells (IECs) absorb nutrients, respond to microbes, provide barrier function and help coordinate immune responses. We profiled 53,193 individual epithelial cells from mouse small intestine and organoids, and characterized novel subtypes and their gene signatures. We showed unexpected diversity of hormone-secreting enteroendocrine cells and constructed their novel taxonomy. We distinguished between two tuft cell subtypes, one of which expresses the epithelial cytokine TSLP and CD45 (Ptprc), the pan-immune marker not previously associated with non-hematopoietic cells. We also characterized how cell-intrinsic states and cell proportions respond to bacterial and helminth infections. Salmonella infection caused an increase in Paneth cells and enterocytes abundance, and broad activation of an antimicrobial program. In contrast, Heligmosomoides polygyrus caused an expansion of goblet and tuft cell populations. Our survey highlights new markers and programs, associates sensory molecules to cell types, and uncovers principles of gut homeostasis and response to pathogens. PMID:29144463

A new human male diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain.

PubMed

Yamamoto, K; Kaji, K; Kondo, H; Matsuo, M; Shibata, Y; Tasaki, Y; Utakoji, T; Ooka, H

1991-01-01

A new human diploid cell strain, TIG-7, which has the male karyotype, was established and characterized. Isozyme and histocompatibility typing of the cell strain was performed. The average in vitro life span of the cells is 73 population doublings. Changes in cell volume, doubling time, saturation density, the efficiency of cell attachment, plating efficiency, and relative DNA content were examined during in vitro cellular aging. Hydrocortisone slightly prolongs the life span of the cell strain when the hormone is administered to the cultures during middle passages. The age-related changes in the parameters of TIG-7 are not appreciably different from those of the previously established TIG-1 cell strain. These results show that this cell strain is useful for research on cellular aging; further profit is anticipated from research using a combination of these two sexually different cell strains.

Destiny of autologous bone marrow-derived stromal cells implanted in the vocal fold.

PubMed

Kanemaru, Shin-ichi; Nakamura, Tatsuo; Yamashita, Masaru; Magrufov, Akhmar; Kita, Tomoko; Tamaki, Hisanobu; Tamura, Yoshihiro; Iguchi, Fuku-ichiro; Kim, Tae Soo; Kishimoto, Masanao; Omori, Koichi; Ito, Juichi

2005-12-01

The aim of this study was to investigate the destiny of implanted autologous bone marrow-derived stromal cells (BSCs) containing mesenchymal stem cells. We previously reported the successful regeneration of an injured vocal fold through implantation of BSCs in a canine model. However, the fate of the implanted BSCs was not examined. In this study, implanted BSCs were traced in order to determine the type of tissues resulting at the injected site of the vocal fold. After harvest of bone marrow from the femurs of green fluorescent transgenic mice, adherent cells were cultured and selectively amplified. By means of a fluorescence-activated cell sorter, it was confirmed that some cells were strongly positive for mesenchymal stem cell markers, including CD29, CD44, CD49e, and Sca-1. These cells were then injected into the injured vocal fold of a nude rat. Immunohistologic examination of the resected vocal folds was performed 8 weeks after treatment. The implanted cells were alive in the host tissues and showed positive expression for keratin and desmin, markers for epithelial tissue and muscle, respectively. The implanted BSCs differentiated into more than one tissue type in vivo. Cell-based tissue engineering using BSCs may improve the quality of the healing process in vocal fold injuries.

A systems biology approach to Down syndrome: identification of Notch/Wnt dysregulation in a model of stem cells aging.

PubMed

Cairney, C J; Sanguinetti, G; Ranghini, E; Chantry, A D; Nostro, M C; Bhattacharyya, A; Svendsen, C N; Keith, W N; Bellantuono, I

2009-04-01

Stem cells are central to the development and maintenance of many tissues. This is due to their capacity for extensive proliferation and differentiation into effector cells. More recently it has been shown that the proliferative and differentiative ability of stem cells decreases with age, suggesting that this may play a role in tissue aging. Down syndrome (DS), is associated with many of the signs of premature tissue aging including T-cell deficiency, increased incidence of early Alzheimer-type, Myelodysplastic-type disease and leukaemia. Previously we have shown that both hematopoietic (HSC) and neural stem cells (NSC) in patients affected by DS showed signs of accelerated aging. In this study we tested the hypothesis that changes in gene expression in HSC and NSC of patients affected by DS reflect changes occurring in stem cells with age. The profiles of genes expressed in HSC and NSC from DS patients highlight pathways associated with cellular aging including a downregulation of DNA repair genes and increases in proapoptotic genes, s-phase cell cycle genes, inflammation and angiogenesis genes. Interestingly, Notch signaling was identified as a potential hub, which when deregulated may drive stem cell aging. These data suggests that DS is a valuable model to study early events in stem cell aging.

Conjugative type IVb pilus recognizes lipopolysaccharide of recipient cells to initiate PAPI-1 pathogenicity island transfer in Pseudomonas aeruginosa

USDA-ARS?s Scientific Manuscript database

Pseudomonas aeruginosa pathogenicity island 1 (PAPI-1) is one of the largest genomic islands of this important opportunistic human pathogen. Previous studies have shown that PAPI-1 encodes several putative virulence factors, a major regulator of biofilm formation, and antibiotic-resistance traits, a...

Mice deficient in carbonic anhydrase type 8 exhibit motor dysfunctions and abnormal calcium dynamics in the somatic region of cerebellar granule cells.

PubMed

Lamont, Matthew G; Weber, John T

2015-06-01

The waddles (wdl) mouse is characterized by a namesake "side-to-side" waddling gait due to a homozygous mutation of the Car8 gene. This mutation results in non-functional copies of the protein carbonic anhydrase type 8. Rota-rod testing was conducted to characterize the wdl mutations' effect on motor output. Results indicated that younger homozygotes outperformed their older cohorts, an effect not seen in previous studies. Heterozygotes, which were thought to be free of motor impairment, displayed motor learning deficiencies when compared with wild type performance. Acute cerebellar slices were then utilized for fluorescent calcium imaging experiments, which revealed significant alterations in cerebellar granule cell somatic calcium signaling when exposed to glutamate. The contribution of GABAergic signaling to these alterations was also verified using bath application of bicuculline. Changes in somatic calcium signals were found to be applicable to an in vivo scenario by comparing group responses to electrical stimulation of afferent mossy fiber projections. Finally, intracellular calcium store function was also found to be altered by the wdl mutation when slices were treated with thapsigargin. These findings, taken together with previous work on the wdl mouse, indicate a widespread disruption in cerebellar circuitry hampering proper neuronal communication. Copyright © 2015 Elsevier B.V. All rights reserved.

Abnormal Labyrinthine Zone in the Hectd1-null Placenta

PubMed Central

Sarkar, Anjali A.; Sabatino, Julia A.; Sugrue, Kelsey F.; Zohn, Irene E.

2016-01-01

Introduction The labyrinthine zone of the placenta is where exchange of nutrients and waste occurs between maternal and fetal circulations. Proper development of the placental labyrinth is essential for successful growth of the developing fetus and abnormalities in placental development are associated with intrauterine growth restriction (IUGR), preeclampsia and fetal demise. Our previous studies demonstrate that Hectd1 is essential for development of the junctional and labyrinthine zones of the placenta. Here we further characterize labyrinthine zone defects in the Hectd1 mutant placenta. Methods The structure of the mutant placenta was compared to wildtype littermates using histological methods. The expression of cell type specific markers was examined by immunohistochemistry and in situ hybridization. Results Hectd1 is expressed in the labyrinthine zone throughout development and the protein is enriched in syncytiotrophoblast layer type I cells (SynT-I) and Sinusoidal Trophoblast Giant cells (S-TGCs) in the mature placenta. Mutation of Hectd1 results in pale placentas with frequent hemorrhages along with gross abnormalities in the structure of the labyrinthine zone including a smaller overall volume and a poorly elaborated fetal vasculature that contain fewer fetal blood cells. Examination of molecular markers of labyrinthine trophoblast cell types reveals increased Dlx3 positive cells and Syna positive SynT-I cells, along with decreased Hand1 and Ctsq positive sinusoidal trophoblast giant cells (S-TGCs). Discussion Together these defects indicate that Hectd1 is required for development of the labyrinthine zone or the mouse placenta. PMID:26907377

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters.

PubMed

Kumar, Vibhor; Rayan, Nirmala Arul; Muratani, Masafumi; Lim, Stefan; Elanggovan, Bavani; Xin, Lixia; Lu, Tess; Makhija, Harshyaa; Poschmann, Jeremie; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

2016-05-01

Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation. © 2016 Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters

PubMed Central

Kumar, Vibhor; Rayan, Nirmala Arul; Muratani, Masafumi; Lim, Stefan; Elanggovan, Bavani; Xin, Lixia; Lu, Tess; Makhija, Harshyaa; Poschmann, Jeremie; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

2016-01-01

Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation. PMID:26957309

Flower-deficient mice have reduced susceptibility to skin papilloma formation

PubMed Central

Petrova, Evgeniya; López-Gay, Jesús M.; Rhiner, Christa; Moreno, Eduardo

2012-01-01

SUMMARY Skin papillomas arise as a result of clonal expansion of mutant cells. It has been proposed that the expansion of pretumoral cell clones is propelled not only by the increased proliferation capacity of mutant cells, but also by active cell selection. Previous studies in Drosophila describe a clonal selection process mediated by the Flower (Fwe) protein, whereby cells that express certain Fwe isoforms are recognized and forced to undergo apoptosis. It was further shown that knock down of fwe expression in Drosophila can prevent the clonal expansion of dMyc-overexpressing pretumoral cells. Here, we study the function of the single predicted mouse homolog of Drosophila Fwe, referred to as mFwe, by clonal overexpression of mFwe isoforms in Drosophila and by analyzing mFwe knock-out mice. We show that clonal overexpression of certain mFwe isoforms in Drosophila also triggers non-autonomous cell death, suggesting that Fwe function is evolutionarily conserved. Although mFwe-deficient mice display a normal phenotype, they develop a significantly lower number of skin papillomas upon exposure to DMBA/TPA two-stage skin carcinogenesis than do treated wild-type and mFwe heterozygous mice. Furthermore, mFwe expression is higher in papillomas and the papilloma-surrounding skin of treated wild-type mice compared with the skin of untreated wild-type mice. Thus, we propose that skin papilloma cells take advantage of mFwe activity to facilitate their clonal expansion. PMID:22362363

Dose- and time-dependent gene expression alterations in prostate and colon cancer cells after in vitro exposure to carbon ion and X-irradiation

PubMed Central

Suetens, Annelies; Moreels, Marjan; Quintens, Roel; Soors, Els; Buset, Jasmine; Chiriotti, Sabina; Tabury, Kevin; Gregoire, Vincent; Baatout, Sarah

2015-01-01

Hadrontherapy is an advanced form of radiotherapy that uses beams of charged particles (such as protons and carbon ions). Compared with conventional radiotherapy, the main advantages of carbon ion therapy are the precise absorbed dose localization, along with an increased relative biological effectiveness (RBE). This high ballistic accuracy of particle beams deposits the maximal dose to the tumor, while damage to the surrounding healthy tissue is limited. Currently, hadrontherapy is being used for the treatment of specific types of cancer. Previous in vitro studies have shown that, under certain circumstances, exposure to charged particles may inhibit cell motility and migration. In the present study, we investigated the expression of four motility-related genes in prostate (PC3) and colon (Caco-2) cancer cell lines after exposure to different radiation types. Cells were irradiated with various absorbed doses (0, 0.5 and 2 Gy) of accelerated 13C-ions at the GANIL facility (Caen, France) or with X-rays. Clonogenic assays were performed to determine the RBE. RT-qPCR analysis showed dose- and time-dependent changes in the expression of CCDC88A, FN1, MYH9 and ROCK1 in both cell lines. However, whereas in PC3 cells the response to carbon ion irradiation was enhanced compared with X-irradiation, the effect was the opposite in Caco-2 cells, indicating cell-type–specific responses to the different radiation types. PMID:25190155

Abnormal labyrinthine zone in the Hectd1-null placenta.

PubMed

Sarkar, Anjali A; Sabatino, Julia A; Sugrue, Kelsey F; Zohn, Irene E

2016-02-01

The labyrinthine zone of the placenta is where exchange of nutrients and waste occurs between maternal and fetal circulations. Proper development of the placental labyrinth is essential for successful growth of the developing fetus and abnormalities in placental development are associated with intrauterine growth restriction (IUGR), preeclampsia and fetal demise. Our previous studies demonstrate that Hectd1 is essential for development of the junctional and labyrinthine zones of the placenta. Here we further characterize labyrinthine zone defects in the Hectd1 mutant placenta. The structure of the mutant placenta was compared to wildtype littermates using histological methods. The expression of cell type specific markers was examined by immunohistochemistry and in situ hybridization. Hectd1 is expressed in the labyrinthine zone throughout development and the protein is enriched in syncytiotrophoblast layer type I cells (SynT-I) and Sinusoidal Trophoblast Giant cells (S-TGCs) in the mature placenta. Mutation of Hectd1 results in pale placentas with frequent hemorrhages along with gross abnormalities in the structure of the labyrinthine zone including a smaller overall volume and a poorly elaborated fetal vasculature that contain fewer fetal blood cells. Examination of molecular markers of labyrinthine trophoblast cell types reveals increased Dlx3 positive cells and Syna positive SynT-I cells, along with decreased Hand1 and Ctsq positive sinusoidal trophoblast giant cells (S-TGCs). Together these defects indicate that Hectd1 is required for development of the labyrinthine zonethe mouse placenta. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of Escherichia coli Type 1 Pilus Mutants with Altered Binding Specificities

PubMed Central

Harris, Sandra L.; Spears, Patricia A.; Havell, Edward A.; Hamrick, Terri S.; Horton, John R.; Orndorff, Paul E.

2001-01-01

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket. PMID:11395476

Inkjet Gene Printing: A Novel Approach to Achieve Gene Modified Cells for Tissue Engineering

DTIC Science & Technology

2008-12-01

and pIRES-VEGF-GFP (BD Biosciences, Bedford, MA) encoding the cDNAs of jellyfish Aequorea victoria green fluorescent protein, driven by the...prepared from rat-tail Type I collagen gels using a previously reported protocol(Xu et al. 2005). Briefly, rat- tail Type I collagen (BD Biosciences...aliquots of the mixture were dispersed onto coverslips and cured in an incubator for 3–5 h. Once the gel set, the collagen bio-paper was ready for

Clinicopathological study of severe chronic active Epstein-Barr virus infection that developed in association with lymphoproliferative disorder and/or hemophagocytic syndrome.

PubMed

Ohshima, K; Suzumiya, J; Sugihara, M; Nagafuchi, S; Ohga, S; Kikuchi, M

1998-12-01

Chronic active Epstein-Barr virus (CAEBV) infection has been previously reported to be sometimes associated with an aggressive clinical course. However, the role of EBV in the CAEBV is not well clarified. A retrospective study was performed on nine adult and five child patients (eight males and six females). Histologically, at first admission, the presence of neoplastic lesions could not be confirmed. The lymph nodes in half of all cases revealed paracortical hyperplasia with transformed lymphocytes (hyperplastic type). Half of the cases showed non-suppurative necrosis and an increased number of histiocytes with phagocytosis (histiocytic type). Activated histiocytes with lymphokine positivity were frequently detected in the histiocytic type. In the phenotypical study, 10 of the examined 11 cases showed increased numbers of natural killer (NK) cells and/or CD8-positive T lymphocytes. In situ hybridization (ISH) showed EBV-infected lymphoid cells, but the number of EBV-infected cells varied. Double-labeling immunochemistry/ISH demonstrated EBV-infected T cells, including NK cells, but not B cells. In addition, three cases showed a monoclonal dissemination of EBV terminal repetitive sequence (TR), and two cases showed oligoclonal dissemination. From those findings, monoclonal, oligoclonal and polyclonal populations of EBV-infected T or NK cells were considered to be present in CAEBV states. During the clinical course, 12 of the 14 cases died within 5 years. Six cases died from EBV-associated hematopoietic tumors (histiocytic tumor, T cell lymphoma, B cell lymphoma, plasmacytoma, and NK cell leukemia); one from non-EBV-associated acute myelogenous leukemia, and five due to hemophagocytic syndrome. The examined EBV-associated hematopoietic tumors showed monoclonal EBV terminal repetitive sequences. There is a possibility that the monoclonal dissemination of EBV-infected cells develops from oligoclonal or polyclonal EBV-infected cells. And active histiocytes with lymphokine positivity were frequently detected in the cases with histologically histiocytic type. These findings seem to be related with the causes of death due to hemophagocytic syndrome.

Haemocytes control stem cell activity in the Drosophila intestine.

PubMed

Ayyaz, Arshad; Li, Hongjie; Jasper, Heinrich

2015-06-01

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. Here we show that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. We propose that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans.

Hemocytes control stem cell activity in the Drosophila intestine

PubMed Central

Ayyaz, Arshad; Li, Hongjie; Jasper, Heinrich

2015-01-01

SUMMARY Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. Here we show that intestinal stem cells (ISCs) are regulated by macrophage-like hemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. Upon tissue damage, hemocytes are recruited to the intestine and secrete the TGFβ/BMP homologue Dpp, inducing ISC proliferation by activating the Type I receptor Saxophone and the Smad homologue Smox. Activated ISCs then switch their response to Dpp by inducing expression of Thickveins, a second Type I receptor that has previously been shown to re-establish ISC quiescence by activating Mad. The interaction between hemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in aging flies. We propose that similar interactions influence pathologies like inflammatory bowel disease and colorectal cancer in humans. PMID:26005834

Mitotic position and morphology of committed precursor cells in the zebrafish retina adapt to architectural changes upon tissue maturation.

PubMed

Weber, Isabell P; Ramos, Ana P; Strzyz, Paulina J; Leung, Louis C; Young, Stephen; Norden, Caren

2014-04-24

The development of complex neuronal tissues like the vertebrate retina requires the tight orchestration of cell proliferation and differentiation. Although the complexity of transcription factors and signaling pathways involved in retinogenesis has been studied extensively, the influence of tissue maturation itself has not yet been systematically explored. Here, we present a quantitative analysis of mitotic events during zebrafish retinogenesis that reveals three types of committed neuronal precursors in addition to the previously known apical progenitors. The identified precursor types present at distinct developmental stages and exhibit different mitotic location (apical versus nonapical), cleavage plane orientation, and morphology. Interestingly, the emergence of nonapically dividing committed bipolar cell precursors can be linked to an increase in apical crowding caused by the developing photoreceptor cell layer. Furthermore, genetic interference with neuronal subset specification induces ectopic divisions of committed precursors, underlining the finding that progressing morphogenesis can effect precursor division position. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids.

PubMed

Hsiao, Amy Y; Tung, Yi-Chung; Qu, Xianggui; Patel, Lalit R; Pienta, Kenneth J; Takayama, Shuichi

2012-05-01

We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through Z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. Copyright © 2011 Wiley Periodicals, Inc.

384 Hanging Drop Arrays Give Excellent Z-factors and Allow Versatile Formation of Co-culture Spheroids

PubMed Central

Hsiao, Amy Y.; Tung, Yi-Chung; Qu, Xianggui; Patel, Lalit R.; Pienta, Kenneth J.; Takayama, Shuichi

2012-01-01

We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. PMID:22161651

Estrogen Receptor Alpha Binding to ERE is Required for Full Tlr7- and Tlr9-Induced Inflammation

PubMed Central

Cunningham, Melissa A; Wirth, Jena R; Naga, Osama; Eudaly, Jackie; Gilkeson, Gary S

2014-01-01

We previously found that a maximum innate inflammatory response induced by stimulation of Toll-like receptors (TLRs) 3, 7 and 9 requires ERα, but does not require estrogen in multiple cell types from both control and lupus-prone mice. Given the estrogen-independence, we hypothesized that ERα mediates TLR signaling by tethering to, and enhancing, the activity of downstream transcription factors such as NFκB, rather than acting classically by binding EREs on target genes. To investigate the mechanism of ERα impact on TLR signaling, we utilized mice with a knock-in ERα mutant that is unable to bind ERE. After stimulation with TLR ligands, both ex vivo spleen cells and bone marrow-derived dendritic cells (BM-DCs) isolated from mutant ERα (“KIKO”) mice produced significantly less IL-6 compared with cells from wild-type (WT) littermates. These results suggest that ERα modulation of TLR signaling does indeed require ERE binding for its effect on the innate immune response. PMID:25061615

Nanobiotechnological Nanocapsules Containing Polyhemoglobin-Tyrosinase: Effects on Murine B16F10 Melanoma Cell Proliferation and Attachment

PubMed Central

Wang, Yun; Chang, Thomas M. S.

2012-01-01

We have reported previously that daily intravenous infusions of a soluble nanobiotechnological complex, polyhemoglobin-tyrosinase [polyHb-Tyr], can suppress the growth of murine B16F10 melanoma in a mouse model. In order to avoid the need for daily intravenous injections, we have now extended this further as follows. We have prepared two types of biodegradable nanocapsules containing [polyHb-Tyr]. One type is to increase the circulation time and decrease the frequency of injection and is based on polyethyleneglycol-polylactic acid (PEG-PLA) nanocapsules containing [polyHb-Tyr]. The other type is to allow for intratumoural or local injection and is based on polylactic acid (PLA) nanocapsules containing [polyHb-Tyr]. Cell culture studies show that it can inhibit the proliferation of murine B16F10 melanoma cells in the “proliferation model”. It can also inhibit the attachment of murine B16F10 melanoma cells in the “attachment model.” This could be due to the action of tyrosinase on the depletion of tyrosine or the toxic effect of tyrosine metabolites. The other component, polyhemoglobin (polyHb), plays a smaller role in nanocapsules containing [polyHb-Tyr], and this is most likely by its depletion of nitric oxide needed for melanoma cell growth. PMID:23209910

A high-resolution map of the three-dimensional chromatin interactome in human cells.

PubMed

Jin, Fulai; Li, Yan; Dixon, Jesse R; Selvaraj, Siddarth; Ye, Zhen; Lee, Ah Young; Yen, Chia-An; Schmitt, Anthony D; Espinoza, Celso A; Ren, Bing

2013-11-14

A large number of cis-regulatory sequences have been annotated in the human genome, but defining their target genes remains a challenge. One strategy is to identify the long-range looping interactions at these elements with the use of chromosome conformation capture (3C)-based techniques. However, previous studies lack either the resolution or coverage to permit a whole-genome, unbiased view of chromatin interactions. Here we report a comprehensive chromatin interaction map generated in human fibroblasts using a genome-wide 3C analysis method (Hi-C). We determined over one million long-range chromatin interactions at 5-10-kb resolution, and uncovered general principles of chromatin organization at different types of genomic features. We also characterized the dynamics of promoter-enhancer contacts after TNF-α signalling in these cells. Unexpectedly, we found that TNF-α-responsive enhancers are already in contact with their target promoters before signalling. Such pre-existing chromatin looping, which also exists in other cell types with different extracellular signalling, is a strong predictor of gene induction. Our observations suggest that the three-dimensional chromatin landscape, once established in a particular cell type, is relatively stable and could influence the selection or activation of target genes by a ubiquitous transcription activator in a cell-specific manner.

Non-conventional protrusions: the diversity of cell interactions at short and long distance.

PubMed

Caviglia, Sara; Ober, Elke A

2018-06-08

Cells use different means to communicate within and between tissues and thereby coordinate their behaviours. Following the initial observations of enigmatic long filopodia unrelated to cell movement, it became clear that the roles of cellular protrusions are not restricted to sensing functions or motility and are much more diverse than previously appreciated. Advances in live-imaging and genetic tools revealed several types of non-conventional cell protrusions and their functions, ranging from tissue patterning, proliferation and differentiation control, tissue matching and cell spacing to more unexpected roles such as priming of cell adhesion as well as bidirectional coordination of tissue movements. Here, we will highlight exciting new insights into highly diverse cell behaviours elicited by protrusions and contact-dependent cell communication, essential for embryonic development across species. Copyright © 2018. Published by Elsevier Ltd.

Relationship between axenic growth of Dictyostelium discoideum strains and their track morphology on substrates coated with gold particles

PubMed Central

1983-01-01

Amoebae of Dictyostelium discoideum produce tracks with two distinct morphologies on gold-coated coverslips. The wild-type strain and other strains that feed only by phagocytosis produced indistinct, fuzzy tracks, whereas mutants capable of axenic growth produced clear, sharp tracks. The sharp track morphology was found to be a recessive phenotype that segregates with axenicity and probably requires a previously unidentified axenic mutation. Axenic and nonaxenic strains also differed in their ability to pinocytose. When the two types of cells were shifted from bacterial growth plates to nutrient media, within 24 h the axenic strain established a rapid rate of pinocytosis, approximately 100-fold higher than the low rate detectable for the nonaxenic strain. However, track formation did not appear to be directly related to endocytosis. Electron microscopic examination of cells during track formation showed that both axenic and nonaxenic strains accumulated gold particles on their surfaces, but neither strain internalized the gold to any significant degree. Observation of living cells revealed that axenic strains collected all particles that they contacted, whereas wild-type strains left many particles undisturbed. The size of the gold particle clusters discarded by the cells also contributed to track morphology. PMID:6619183

Ultrastructural and functional characterization of circulating hemocytes from Plutella xylostella larva: cell types and their role in phagocytosis.

PubMed

Huang, Fang; Yang, Yan-yan; Shi, Min; Li, Jun-ying; Chen, Zong-qi; Chen, Fu-shou; Chen, Xue-xin

2010-12-01

The hemocytes of different types encountered in the diamondback moth Plutella xylostella larvae of each instar and the development of the differential hemocytes counts were herein presented. Hemocytes classes/populations characterized based on their affinity with fluorescent dye (acridine orange) and ultrastructural differences comprised the prohemcoytes (<10-16%), plasmatocytes (22-65%), granulocytes (25-72%), oenocytoids (<1-9%), and spherulocytes (<1%). Prohemcoytes were the smallest cells with a comparatively tremendous nucleus. Plasmatocytes and granulocytes occupied the main proportion of total cell numbers. Oenocytoids were in a most stable presence, i.e. rotund in a diameter of 10 μm and with a nucleus deviated from the central location; however, sometimes with two nuclei which were adjoining with each other. Spherulocytes were rare and only could be observed occasionally. Ultrastructural investigation revealed that hemocytes in the diamondback moth larvae were of the typical model as in the Lepidoptera insect larvae. It is interesting to find that the cell which could phagocytize bacteria in vitro was granulocyte, not the other types of hemocytes, although plasmatocyte was usually declared to participate in this reaction in various previous studies. Copyright © 2010 Elsevier Ltd. All rights reserved.

The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes

PubMed Central

Burroughs, Amelia; Wise, Andrew K.; Xiao, Jianqiang; Houghton, Conor; Tang, Tianyu; Suh, Colleen Y.; Lang, Eric J.

2016-01-01

Key points Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes.Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets.The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear.We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number.This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Abstract Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake cats. In addition, complex spikes with a greater number of spikelets were associated with a subsequent reduction in simple spike firing rate. We therefore suggest that one important function of spikelets is the modulation of Purkinje cell simple spike firing frequency, which has implications for controlling cerebellar cortical output and motor learning. PMID:27265808

Development of a model system to analyze chondrogenic differentiation of mesenchymal stem cells

PubMed Central

Ruedel, Anke; Hofmeister, Simone; Bosserhoff, Anja-Katrin

2013-01-01

High-density cell culture is widely used for the analysis of cartilage development of human mesenchymal stem cells (HMSCs) in vitro. Several cell culture systems, as micromass, pellet culture and alginate culture, are applied by groups in the field to induce chondrogenic differentiation of HMSCs. A draw back of all model systems is the high amount of cells necessary for the experiments. Further, handling of large experimental approaches is difficult due to culturing e.g. in 15 ml tubes. Therefore, we aimed to develop a new model system based on “hanging drop” cultures using 10 to 100 fold less cells. Here, we demonstrate that differentiation of chondrogenic cells was induced as previously shown in other model systems. Real time RT-PCR analysis demonstrated that Collagen type II and MIA/CD-RAP were upregulated during culturing whereas for induction of hypertrophic markers like Collagen type X and AP-2 epsilon treatment with TGF beta was needed. To further test the system, siRNA against Sox9 was used and effects on chondrogenic gene expression were evaluated. In summary, the hanging drop culture system was determined to be a promising tool for in vitro chondrogenic studies. PMID:24294400

Single-Cell Tracking Reveals a Role for Pre-Existing CCR5+ Memory Th1 Cells in the Control of Rhinovirus-A39 After Experimental Challenge in Humans.

PubMed

Muehling, Lyndsey M; Turner, Ronald B; Brown, Kenneth B; Wright, Paul W; Patrie, James T; Lahtinen, Sampo J; Lehtinen, Markus J; Kwok, William W; Woodfolk, Judith A

2018-01-17

Little is known about T cells that respond to human rhinovirus in vivo, due to timing of infection, viral diversity, and complex T-cell specificities. We tracked circulating CD4+ T cells with identical epitope specificities that responded to intranasal challenge with rhinovirus (RV)-A39, and we assessed T-cell signatures in the nose. Cells were monitored using a mixture of 2 capsid-specific major histocompatibility complex II tetramers over a 7-week period, before and after RV-A39 challenge, in 16 human leukocyte antigen-DR4+ subjects who participated in a trial of Bifidobacterium lactis (Bl-04) supplementation. Pre-existing tetramer+ T cells were linked to delayed viral shedding, enriched for activated CCR5+ Th1 effectors, and included a minor interleukin-21+ T follicular helper cell subset. After RV challenge, expansion and activation of virus-specific CCR5+ Th1 effectors was restricted to subjects who had a rise in neutralizing antibodies, and tetramer-negative CCR5+ effector memory types were comodulated. In the nose, CXCR3-CCR5+ T cells present during acute infection were activated effector memory type, whereas CXCR3+ cells were central memory type, and cognate chemokine ligands were elevated over baseline. Probiotic had no T-cell effects. We conclude that virus-specific CCR5+ effector memory CD4+ T cells primed by previous exposure to related viruses contribute to the control of rhinovirus. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

PubMed

Nah, H D; Pacifici, M; Gerstenfeld, L C; Adams, S L; Kirsch, T

2000-03-01

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.

An auxin-induced β-type endo-1,4-β-glucanase in poplar is involved in cell expansion and lateral root formation.

PubMed

Yu, Liangliang; Li, Qiong; Zhu, Yingying; Afzal, Muhammad Saddique; Li, Laigeng

2018-05-01

PtrGH9A7, a poplar β-type endo-1,4-β-glucanase gene induced by auxin, promotes both plant growth and lateral root development by enhancing cell expansion. Endo-1,4-β-glucanase (EGase) family genes function in multiple aspects of plant growth and development. Our previous study found that PtrCel9A6, a poplar EGase gene of the β subfamily, is specifically expressed in xylem tissue and is involved in the cellulose biosynthesis required for secondary cell wall formation (Yu et al. in Mol Plant 6:1904-1917, 2013). To further explore the functions and regulatory mechanism of β-subfamily EGases, we cloned and characterized another poplar β-type EGase gene PtrGH9A7, a close homolog of PtrCel9A6. In contrast to PtrCel9A6, PtrGH9A7 is predominantly expressed in parenchyma tissues of the above-ground part; in roots, PtrGH9A7 expression is specifically restricted to lateral root primordia at all stages from initiation to emergence and is strongly induced by auxin application. Heterologous overexpression of PtrGH9A7 promotes plant growth by enhancing cell expansion, suggesting a conserved role for β-type EGases in 1,4-β-glucan chains remodeling, which is required for cell wall loosening. Moreover, the overexpression of PtrGH9A7 significantly increases lateral root number, which might result from improved lateral root primordium development due to enhanced cell expansion. Taken together, these results demonstrate that this β-type EGase induced by auxin signaling has a novel role in promoting lateral root formation as well as in enhancing plant growth.

Interleukin-17A Promotes CD8+ T Cell Cytotoxicity To Facilitate West Nile Virus Clearance.

PubMed

Acharya, Dhiraj; Wang, Penghua; Paul, Amber M; Dai, Jianfeng; Gate, David; Lowery, Jordan E; Stokic, Dobrivoje S; Leis, A Arturo; Flavell, Richard A; Town, Terrence; Fikrig, Erol; Bai, Fengwei

2017-01-01

CD8 + T cells are crucial components of immunity and play a vital role in recovery from West Nile virus (WNV) infection. Here, we identify a previously unrecognized function of interleukin-17A (IL-17A) in inducing cytotoxic-mediator gene expression and promoting CD8 + T cell cytotoxicity against WNV infection in mice. We find that IL-17A-deficient (Il17a -/- ) mice are more susceptible to WNV infection and develop a higher viral burden than wild-type (WT) mice. Interestingly, the CD8 + T cells isolated from Il17a -/- mice are less cytotoxic and express lower levels of cytotoxic-mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo Importantly, treatment of WNV-infected mice with recombinant IL-17A, as late as day 6 postinfection, significantly reduces the viral burden and increases survival, suggesting a therapeutic potential for IL-17A. In conclusion, we report a novel function of IL-17A in promoting CD8 + T cell cytotoxicity, which may have broad implications in other microbial infections and cancers. Interleukin-17A (IL-17A) and CD8 + T cells regulate diverse immune functions in microbial infections, malignancies, and autoimmune diseases. IL-17A is a proinflammatory cytokine produced by diverse cell types, while CD8 + T cells (known as cytotoxic T cells) are major cells that provide immunity against intracellular pathogens. Previous studies have demonstrated a crucial role of CD8 + T cells in recovery from West Nile virus (WNV) infection. However, the role of IL-17A during WNV infection remains unclear. Here, we demonstrate that IL-17A protects mice from lethal WNV infection by promoting CD8 + T cell-mediated clearance of WNV. In addition, treatment of WNV-infected mice with recombinant IL-17A reduces the viral burden and increases survival of mice, suggesting a potential therapeutic. This novel IL-17A-CD8 + T cell axis may also have broad implications for immunity to other microbial infections and cancers, where CD8 + T cell functions are crucial. Copyright © 2016 American Society for Microbiology.

Microgravity

NASA Image and Video Library

2004-04-15

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. The objective of the research was to define a way to differentiate between effects due to microgravity and those due to possible stress from non-optimal spaceflight conditions. These Jurkat cells, a human acute T-cell leukemia was obtained to evaluate three types of potential experimental stressors: a) Temperature elevation; b) Serum starvation; and c) Centrifugal force. The data from previous spaceflight experiments showed that actin filaments and cell shape are significantly different for the control. These normal cells serve as the baseline for future spaceflight experiments.

Fetal stem cell transplantation: Past, present, and future

PubMed Central

Ishii, Tetsuya; Eto, Koji

2014-01-01

Since 1928, human fetal tissues and stem cells have been used worldwide to treat various conditions. Although the transplantation of the fetal midbrain substantia nigra and dopaminergic neurons in patients suffering from Parkinson’s disease is particularly noteworthy, the history of other types of grafts, such as those of the fetal liver, thymus, and pancreas, should be addressed as there are many lessons to be learnt for future stem cell transplantation. This report describes previous practices and complications that led to current clinical trials of isolated fetal stem cells and embryonic stem (ES) cells. Moreover, strategies for transplantation are considered, with a particular focus on donor cells, cell processing, and the therapeutic cell niche, in addition to ethical issues associated with fetal origin. With the advent of autologous induced pluripotent stem cells and ES cells, clinical dependence on fetal transplantation is expected to gradually decline due to lasting ethical controversies, despite landmark achievements. PMID:25258662

Colon Stem Cell and Crypt Dynamics Exposed by Cell Lineage Reconstruction

PubMed Central

Itzkovitz, Shalev; Elbaz, Judith; Maruvka, Yosef E.; Segev, Elad; Shlush, Liran I.; Dekel, Nava; Shapiro, Ehud

2011-01-01

Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems. PMID:21829376

Comparative modelling of human β tubulin isotypes and implications for drug binding

NASA Astrophysics Data System (ADS)

Torin Huzil, J.; Ludueña, Richard F.; Tuszynski, Jack

2006-02-01

The protein tubulin is a target for several anti-mitotic drugs, which affect microtubule dynamics, ultimately leading to cell cycle arrest and apoptosis. Many of these drugs, including the taxanes and Vinca alkaloids, are currently used clinically in the treatment of several types of cancer. Another tubulin binding drug, colchicine, although too toxic to be used as a chemotherapeutic agent, is commonly used for the treatment of gout. The main disadvantage that all of these drugs share is that they bind tubulin indiscriminately, leading to the death of both cancerous and healthy cells. However, the broad cellular distribution of several tubulin isotypes provides a platform upon which to construct novel chemotherapeutic drugs that could differentiate between different cell types, reducing the undesirable side effects associated with current chemotherapeutic treatments. Here, we report an analysis of ten human β tubulin isotypes and discuss differences within each of the previously characterized paclitaxel, colchicine and vinblastine binding sites.

Identification of centromere regions in chromosomes of a unicellular red alga, Cyanidioschyzon merolae.

PubMed

Kanesaki, Yu; Imamura, Sousuke; Matsuzaki, Motomichi; Tanaka, Kan

2015-05-08

To investigate the evolution of centromere architecture in plant cells, it is important to identify centromere regions of primitive algae, such as Cyanidioschyzon merolae. In a previous genome project, in silico analysis predicted an AT-rich region in each chromosome as putative centromere regions. Here, we identified a centromere position in each chromosome by ChIP-on-chip analysis using an anti-CENP-A antibody. The identified centromeres were of the regional type, about 2-3 kb in length and contained no consensus or repeat elements. Centromeres in primitive eukaryotic plant cells may have originated from these regional type centromeres. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

3D printing of natural organic materials by photochemistry

NASA Astrophysics Data System (ADS)

Da Silva Gonçalves, Joyce Laura; Valandro, Silvano Rodrigo; Wu, Hsiu-Fen; Lee, Yi-Hsiung; Mettra, Bastien; Monnereau, Cyrille; Schmitt Cavalheiro, Carla Cristina; Pawlicka, Agnieszka; Focsan, Monica; Lin, Chih-Lang; Baldeck, Patrice L.

2016-03-01

In previous works, we have used two-photon induced photochemistry to fabricate 3D microstructures based on proteins, anti-bodies, and enzymes for different types of bio-applications. Among them, we can cite collagen lines to guide the movement of living cells, peptide modified GFP biosensing pads to detect Gram positive bacteria, anti-body pads to determine the type of red blood cells, and trypsin columns in a microfluidic channel to obtain a real time biochemical micro-reactor. In this paper, we report for the first time on two-photon 3D microfabrication of DNA material. We also present our preliminary results on using a commercial 3D printer based on a video projector to polymerize slicing layers of gelatine-objects.

Classes and continua of hippocampal CA1 inhibitory neurons revealed by single-cell transcriptomics.

PubMed

Harris, Kenneth D; Hochgerner, Hannah; Skene, Nathan G; Magno, Lorenza; Katona, Linda; Bengtsson Gonzales, Carolina; Somogyi, Peter; Kessaris, Nicoletta; Linnarsson, Sten; Hjerling-Leffler, Jens

2018-06-18

Understanding any brain circuit will require a categorization of its constituent neurons. In hippocampal area CA1, at least 23 classes of GABAergic neuron have been proposed to date. However, this list may be incomplete; additionally, it is unclear whether discrete classes are sufficient to describe the diversity of cortical inhibitory neurons or whether continuous modes of variability are also required. We studied the transcriptomes of 3,663 CA1 inhibitory cells, revealing 10 major GABAergic groups that divided into 49 fine-scale clusters. All previously described and several novel cell classes were identified, with three previously described classes unexpectedly found to be identical. A division into discrete classes, however, was not sufficient to describe the diversity of these cells, as continuous variation also occurred between and within classes. Latent factor analysis revealed that a single continuous variable could predict the expression levels of several genes, which correlated similarly with it across multiple cell types. Analysis of the genes correlating with this variable suggested it reflects a range from metabolically highly active faster-spiking cells that proximally target pyramidal cells to slower-spiking cells targeting distal dendrites or interneurons. These results elucidate the complexity of inhibitory neurons in one of the simplest cortical structures and show that characterizing these cells requires continuous modes of variation as well as discrete cell classes.

High Affinity Binding of Epibatidine to Serotonin Type 3 Receptors*

PubMed Central

Drisdel, Renaldo C.; Sharp, Douglas; Henderson, Tricia; Hales, Tim G.; Green, William N.

2008-01-01

Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of 125I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT3Rs) block the remaining 25%. 125I-Epibatidine binds with a high affinity to native 5-HT3Rs of N1E-115 cells and to receptors composed of only 5-HT3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT3R-specific antagonist MDL72222, and the 5-HT3R agonist chlorophenylbiguanide readily competed with 125I-epibatidine binding to 5-HT3Rs. Nicotine was a poor competitor for 125I-epibatidine binding to 5-HT3Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of 125I-epibatidine binding to 5-HT3Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT3Rs in neuroblastoma cell lines and 5-HT3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT3Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT3R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT3Rs. PMID:17702741

Macrophages Are the Major Reservoir of Latent Murine Gammaherpesvirus 68 in Peritoneal Cells

PubMed Central

Weck, Karen E.; Kim, Susanne S.; Virgin, Herbert W.; Speck, Samuel H.

1999-01-01

B cells have previously been identified as the major hematopoietic cell type harboring latent gammaherpesvirus 68 (γHV68) (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275–3279, 1992). However, we have shown that γHV68 efficiently establishes latency in B-cell-deficient mice (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775–6780, 1996), demonstrating that B cells are not required for γHV68 latency. To understand this dichotomy, we determined whether hematopoietic cell types, in addition to B cells, carry latent γHV68. We observed a high frequency of cells that reactivate latent γHV68 in peritoneal exudate cells (PECs) derived from both B-cell-deficient and normal C57BL/6 mice. PECs were composed primarily of macrophages in B-cell-deficient mice and of macrophages plus B cells in normal C57BL/6 mice. To determine which cells in PECs from C57BL/6 mice carry latent γHV68, we developed a limiting-dilution PCR assay to quantitate the frequency of cells carrying the γHV68 genome in fluorescence-activated cell sorter-purified cell populations. We also quantitated the contribution of individual cell populations to the total frequency of cells carrying latent γHV68. At early times after infection, the frequency of PECs that reactivated γHV68 correlated very closely with the frequency of PECs carrying the γHV68 genome, validating measurement of the frequency of viral-genome-positive cells as a measure of latency in this cell population. F4/80-positive macrophage-enriched, lymphocyte-depleted PECs harbored most of the γHV68 genome and efficiently reactivated γHV68, while CD19-positive, B-cell-enriched PECs harbored about a 10-fold lower frequency of γHV68 genome-positive cells. CD4-positive, T-cell-enriched PECs contained only a very low frequency of γHV68 genome-positive cells, consistent with previous analyses indicating that T cells are not a reservoir for γHV68 latency (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275–3279, 1992). Since macrophages are bone marrow derived, we determined whether elicitation of a large inflammatory response in the peritoneum would recruit additional latent cells into the peritoneum. Thioglycolate inoculation increased the total number of PECs by about 20-fold but did not affect the frequency of cells that reactivate γHV68, consistent with a bone marrow reservoir for latent γHV68. These experiments demonstrate γHV68 latency in two different hematopoietic cell types, F4/80-positive macrophages and CD19-positive B cells, and argue for a bone marrow reservoir for latent γHV68. PMID:10074181

Biological characterization of HIV type 1 envelope V3 regions from mothers and infants associated with perinatal transmission.

PubMed

Matala, E; Hahn, T; Yedavalli, V R; Ahmad, N

2001-12-10

Our previous study has shown that the human immunodeficiency virus type 1 (HIV-1) envelope V3 region minor genotypes of infected mothers were transmitted to their infants and predominated initially as a homogeneous virus population in the infants (Ahmad N, Baroudy BM, Baker RC, et al.: J Virol 1995;69:1001-1012). Here we have characterized the biological properties, including cellular tropism, replication efficiency, cytopathic effects, and coreceptor utilization, of these V3 region isolates from mothers and infants. Nineteen V3 region sequences from three mother-infant pairs, including the minor variants of mothers and the major variants of infants as characterized in our previous study, were reciprocally inserted into an HIV-1 infectious molecular clone, pNL4-3, and chimeric viruses were generated by DNA transfections into HeLa cells. Equal amounts of chimeric viruses were then used to infect T lymphocyte cell lines (A3.01 and MT-2), primary blood lymphocytes (PBLs), primary monocyte-derived macrophages (MDMs), and coreceptor cell lines. We found that the V3 region chimeras failed to replicate in T lymphocyte cell lines but replicated in MDMs and PBLs, albeit at reduced levels compared with R5 laboratory HIV-1 strains. In addition, the V3 region chimeras were able to infect the HOS-CD4(+)CCR5(+) cell line, suggesting CCR5 coreceptor utilization. Moreover, the V3 region chimeras were unable to induce syncytia in MT-2 cells, indicative of non-syncytium-inducing (NSI) phenotypes. In conclusion, the HIV-1 minor genotypes of infected mothers with macrophage-tropic and NSI or R5 phenotypes are transmitted to their infants and are initially maintained with the same properties.

viking: identification and characterization of a second type IV collagen in Drosophila.

PubMed

Yasothornsrikul, S; Davis, W J; Cramer, G; Kimbrell, D A; Dearolf, C R

1997-10-01

We have taken an enhancer trap approach to identify genes that are expressed in hematopoietic cells and tissues of Drosophila. We conducted a molecular analysis of two P-element insertion strains that have reporter gene expression in embryonic hemocytes, strain 197 and vikingICO. This analysis has determined that viking encodes a collagen type IV gene, alpha2(IV). The viking locus is located adjacent to the previously described DCg1, which encodes collagen alpha1(IV), and in the opposite orientation. The alpha2(IV) and alpha1(IV) collagens are structurally very similar to one another, and to vertebrate type IV collagens. In early development, viking and DCg1 are transcribed in the same tissue-specific pattern, primarily in the hemocytes and fat body cells. Our results suggest that both the alpha1 and alpha2 collagen IV chains may contribute to basement membranes in Drosophila. This work also provides the foundation for a more complete genetic dissection of collagen type IV molecules and their developmental function in Drosophila.

Pap-tests with non-hyperchromatic dyskariosis are often associated with squamous intraepithelial lesions of the cervix uteri with eosinophilic features.

PubMed

Bellisano, Giulia; Ambrosini-Spaltro, Andrea; Faa, Gavino; Ravarino, Alberto; Piccin, Andrea; Peer, Irmgard; Kasal, Armin; Vittadello, Fabio; Negri, Giovanni

2016-10-01

Squamous intraepithelial lesions of the cervix uteri with eosinophilic features (eosinophilic dysplasia, ED) are a peculiar type of dysplasia with metaplastic phenotype which was described in histological specimens. The cytological features of these lesions have not been studied yet. Histological samples from 66 women with features of ED and positive p16(INK4a) staining were included in the study. Within the previous year, all women had at least one pap-test, whose features were recorded and compared with 31 control samples with high-grade dysplasia of usual type. The previous pap-test showed high-grade dysplastic cells with non-hyperchromatic nuclei in 56/66 (84.8%) cases and metaplastic features in 60/66 (90.9%) cases. Conversely, the dysplastic cells of the usual lesions showed non-hyperchromatic nuclei in 6/31 (19.4%) and metaplastic features in 4/31 (12.9%) cases. Statistical analysis showed significant differences in distribution of the non-hyperchromatic nuclei (P < 0.001), metaplastic features (P < 0.001), presence of both non-hyperchromatic nuclei and metaplastic features (P < 0.001) and usual dysplastic features (P < 0.001) among the study and control groups. A high-grade squamous intraepithelial lesion with non-hyperchromatic nuclei or metaplastic features is often found in the pap-test previous to the histological diagnosis of ED and may represent the cytologic correlate of this particular type of dysplasia. Diagn. Cytopathol. 2016;44:783-786. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Loss of neurofibromatosis type 1 (NF1) gene expression in pheochromocytomas from patients without NF1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geist, R.T.; Gutmann, D.H.; Moley, J.F.

The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, termed neurofibromin. Loss of NF1 gene expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 as well as malignant and neuroblastomas from patients without NF1. Previously, we demonstrated the lack of neurofibromin expression in six pheochromocytomas from patients with NF1, suggesting that neurofibromin loss is associated with the progression to neoplasia in pheochromocytomas in these patients. The lack of NF1 gene expression in NF1 patient pheochromocytomas supports the notion that neurofibromin might be an essential regulator of cell growth in these cells. To determine whethermore » NF1 gene expression is similarly altered in pheochromocytomas from patients without NF1, twenty pheochromocytomas were examined for the presence of NF1 RNA by reverse-transcribed PCR (RT-PCR). Lack of NF1 gene expression was documented in four of these twenty tumors (20%) which corresponds to previously reported numbers for malignant melanomas and neuroblastomas in non-NF1 patients. Of these twenty pheochromocytomas, one of four sporadic tumors, one of ten tumors from patients with MEN2A, one of four tumors from patients with MEN2B, and one of two tumors from patients with von Hippel-Lindau syndrome demonstrated loss of NF1 gene expression. In all cases, the quality and quantity of tumor RNA was determined by RT-PCR amplification using primers which amplify cyclophilin RNA. We previously demonstrated that these tumors do not harbor activating mutations of the N-ras, K-ras or H-ras proto-oncogenes. These results suggest that loss of NF1 gene expression is frequently associated with the progression to neoplasia in tumors derived from adrenal medullary tissue in patients without clinical manifestations of neurofibromatosis and supports the notion that neurofibromin is a tumor suppressor gene product involved in the pathogenesis of a wide variety of tumor types.« less

Bergamot juice extract inhibits proliferation by inducing apoptosis in human colon cancer cells.

PubMed

Visalli, Giuseppa; Ferlazzo, Nadia; Cirmi, Santa; Campiglia, Pietro; Gangemi, Sebastiano; Di Pietro, Angela; Calapai, Gioacchino; Navarra, Michele

2014-01-01

Colorectal cancer (CRC) is a leading cause of cancer mortality in the industrialized world, second to lung cancer. A lot of evidences highlight that a diet rich in fruits and vegetables may reduce the risk of some types of cancer including CRC. In this study we demonstrate that Citrus bergamia juice extracts (BJe) reduces CRC cell growth by multiple mechanisms. Low BJe concentrations inhibit MAPKs pathway and alter apoptosis-related proteins, that in turn induce cell cycle arrest and apoptosis in HT-29 cells. Instead, high concentrations of BJe induce oxidative stress causing DNA damage. Our study highlights the role of BJe as modulator of cell apoptosis in CRC cells and strengthens our previous hypothesis that the flavonoid fraction of bergamot juice may play a role as anti-cancer drug.

Directed differentiation of embryonic stem cells using a bead-based combinatorial screening method.

PubMed

Tarunina, Marina; Hernandez, Diana; Johnson, Christopher J; Rybtsov, Stanislav; Ramathas, Vidya; Jeyakumar, Mylvaganam; Watson, Thomas; Hook, Lilian; Medvinsky, Alexander; Mason, Chris; Choo, Yen

2014-01-01

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.

Two-Stage, In Silico Deconvolution of the Lymphocyte Compartment of the Peripheral Whole Blood Transcriptome in the Context of Acute Kidney Allograft Rejection

PubMed Central

Shannon, Casey P.; Balshaw, Robert; Ng, Raymond T.; Wilson-McManus, Janet E.; Keown, Paul; McMaster, Robert; McManus, Bruce M.; Landsberg, David; Isbel, Nicole M.; Knoll, Greg; Tebbutt, Scott J.

2014-01-01

Acute rejection is a major complication of solid organ transplantation that prevents the long-term assimilation of the allograft. Various populations of lymphocytes are principal mediators of this process, infiltrating graft tissues and driving cell-mediated cytotoxicity. Understanding the lymphocyte-specific biology associated with rejection is therefore critical. Measuring genome-wide changes in transcript abundance in peripheral whole blood cells can deliver a comprehensive view of the status of the immune system. The heterogeneous nature of the tissue significantly affects the sensitivity and interpretability of traditional analyses, however. Experimental separation of cell types is an obvious solution, but is often impractical and, more worrying, may affect expression, leading to spurious results. Statistical deconvolution of the cell type-specific signal is an attractive alternative, but existing approaches still present some challenges, particularly in a clinical research setting. Obtaining time-matched sample composition to biologically interesting, phenotypically homogeneous cell sub-populations is costly and adds significant complexity to study design. We used a two-stage, in silico deconvolution approach that first predicts sample composition to biologically meaningful and homogeneous leukocyte sub-populations, and then performs cell type-specific differential expression analysis in these same sub-populations, from peripheral whole blood expression data. We applied this approach to a peripheral whole blood expression study of kidney allograft rejection. The patterns of differential composition uncovered are consistent with previous studies carried out using flow cytometry and provide a relevant biological context when interpreting cell type-specific differential expression results. We identified cell type-specific differential expression in a variety of leukocyte sub-populations at the time of rejection. The tissue-specificity of these differentially expressed probe-set lists is consistent with the originating tissue and their functional enrichment consistent with allograft rejection. Finally, we demonstrate that the strategy described here can be used to derive useful hypotheses by validating a cell type-specific ratio in an independent cohort using the nanoString nCounter assay. PMID:24733377

Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells

PubMed Central

Gledhill, Karl; Guo, Zongyou; Umegaki-Arao, Noriko; Higgins, Claire A.; Itoh, Munenari; Christiano, Angela M.

2015-01-01

The current utility of 3D skin equivalents is limited by the fact that existing models fail to recapitulate the cellular complexity of human skin. They often contain few cell types and no appendages, in part because many cells found in the skin are difficult to isolate from intact tissue and cannot be expanded in culture. Induced pluripotent stem cells (iPSCs) present an avenue by which we can overcome this issue due to their ability to be differentiated into multiple cell types in the body and their unlimited growth potential. We previously reported generation of the first human 3D skin equivalents from iPSC-derived fibroblasts and iPSC-derived keratinocytes, demonstrating that iPSCs can provide a foundation for modeling a complex human organ such as skin. Here, we have increased the complexity of this model by including additional iPSC-derived melanocytes. Epidermal melanocytes, which are largely responsible for skin pigmentation, represent the second most numerous cell type found in normal human epidermis and as such represent a logical next addition. We report efficient melanin production from iPSC-derived melanocytes and transfer within an entirely iPSC-derived epidermal-melanin unit and generation of the first functional human 3D skin equivalents made from iPSC-derived fibroblasts, keratinocytes and melanocytes. PMID:26308443

The Pacific bluefin tuna (Thunnus orientalis) dead end gene is suitable as a specific molecular marker of type A spermatogonia.

PubMed

Yazawa, Ryosuke; Takeuchi, Yutaka; Morita, Tetsuro; Ishida, Masashi; Yoshizaki, Goro

2013-10-01

We developed a spermatogonial transplantation technique to produce donor-derived gametes in surrogate fish. Our ultimate aim is to establish surrogate broodstock that can produce bluefin tuna. We previously determined that only type A spermatogonia (ASG) could colonize recipient gonads in salmonids. Therefore, it is necessary to develop a precise molecular marker that can distinguish ASG in order to develop efficient spermatogonial transplantation methods. In this study, the Pacific bluefin tuna (Thunnus orientalis) dead end (BFTdnd) gene was identified as a specific marker for ASG. In situ hybridization and RT-PCR analysis with various types of spermatogenic cell populations captured by laser microdissection revealed that localization of BFTdnd mRNA was restricted to ASG, and not detected in other differentiated spermatogenic cells. In order to determine if BFTdnd can be used as a molecular marker to identify germ cells with high transplantability, transplantation of dissociated testicular cells isolated from juvenile, immature, and mature Pacific bluefin tuna, which have different proportions of dnd-positive ASG, were performed using chub mackerel as the surrogate recipient species. Colonization of transplanted donor germ cells was only successful with testicular cells from immature Pacific Bluefin tuna, which contained higher proportions of dnd-positive ASG than juvenile and mature fish. Thus, BFTdnd is a useful tool for identifying highly transplantable ASG for spermatogonial transplantation. © 2013 Wiley Periodicals, Inc.

Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

PubMed

Salzillo, Marzia; Vastano, Valeria; Capri, Ugo; Muscariello, Lidia; Marasco, Rosangela

2017-04-01

Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acid, basic, and neutral peptidases present different profiles in chromophobe renal cell carcinoma and in oncocytoma.

PubMed

Blanco, Lorena; Larrinaga, Gorka; Pérez, Itxaro; López, José I; Gil, Javier; Agirregoitia, Ekaitz; Varona, Adolfo

2008-04-01

Renal cell carcinomas (RCCs) are neoplasias with high prevalence and mortality. We previously reported that several peptidases may be involved in the pathophysiology of clear cell renal cell carcinoma (CCRCC). Now, to gain insight into the reasons that lead the various RCC types to behave very differently with regard to aggressiveness and response to anticancer treatments, we analyzed subsets of chromophobe renal cell carcinoma (ChRCC), and renal oncocytoma (RO), a benign tumor; as well as different grades and stages of CCRCCs. Particulate APN, APB, and APA activities were decreased in both ChRCC and RO (tumor vs. nontumor tissues). Interestingly, activities were downregulated in a tumor-type specific way and the intensities of the decreases were stronger in the benign tumor than in the malignant type. Moreover, when two key histopathological parameters for tumor prognosis (high vs. low stage and grade) were analyzed, increases of activity were also observed in several of these cell surface peptidases (APN, APB). Some soluble activities (APB, Asp-AP) were also downregulated in the RCCs. With respect to genetic expression, PSA and APN were in a positive correlation related to their activities in both ChRCC and RO; but not APB, Asp-AP, APA, and PGI. These results may suggest an involvement of several peptidases in the pathophysiology of renal cancer, since they presented different patterns of activity and expression in tumors with different behaviors.

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses

DOE PAGES

Nafisi, Majse; Stranne, Maria; Fimognari, Lorenzo; ...

2015-07-22

Here we report that the epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed andmore » surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.« less

Auto-immune encephalitis as differential diagnosis of infectious encephalitis

PubMed Central

Armangue, Thaís; Leypoldt, Frank; Dalmau, Josep

2014-01-01

Purpose of review To describe the main types of autoimmune encephalitis with special emphasis on those associated with antibodies against neuronal cell surface or synaptic proteins, and the differential diagnosis with infectious encephalitis. Recent findings There is a continuous expansion of the number of cell surface or synaptic proteins that are targets of autoimmunity. The most recently identified include the mGluR5, DPPX, and the GABAAR. In these and previously known autoimmune encephalitis (NMDAR, AMPAR, GABABR, LGI1, CASPR2), the prodromal symptoms or types of presentations often suggest a viral encephalitis. We review here clues that help in the differential diagnosis with infectious encephalitis. Moreover, recent investigations indicate that viral encephalitis (e.g., herpes simplex) can trigger synaptic autoimmunity. In all these disorders immunotherapy is usually effective. Summary Autoimmune encephalitis comprises an expanding group of potentially treatable disorders that should be included in the differential diagnosis of any type of encephalitis. PMID:24792345

Dragon (Repulsive Guidance Molecule RGMb) Inhibits E-cadherin Expression and Induces Apoptosis in Renal Tubular Epithelial Cells*

PubMed Central

Liu, Wenjing; Li, Xiaoling; Zhao, Yueshui; Meng, Xiao-Ming; Wan, Chao; Yang, Baoxue; Lan, Hui-Yao; Lin, Herbert Y.; Xia, Yin

2013-01-01

Dragon is one of the three members of the repulsive guidance molecule (RGM) family, i.e. RGMa, RGMb (Dragon), and RGMc (hemojuvelin). We previously identified the RGM members as bone morphogenetic protein (BMP) co-receptors that enhance BMP signaling. Our previous studies found that Dragon is highly expressed in the tubular epithelial cells of mouse kidneys. However, the roles of Dragon in renal epithelial cells are yet to be defined. We now show that overexpression of Dragon increased cell death induced by hypoxia in association with increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-3 levels in mouse inner medullary collecting duct (IMCD3) cells. Dragon also inhibited E-cadherin expression but did not affect epithelial-to-mesenchymal transition induced by TGF-β in IMCD3 cells. Previous studies suggest that the three RGM members can function as ligands for the receptor neogenin. Interestingly, our present study demonstrates that the Dragon actions on apoptosis and E-cadherin expression in IMCD3 cells were mediated by the neogenin receptor but not through the BMP pathway. Dragon expression in the kidney was up-regulated by unilateral ureteral obstruction in mice. Compared with wild-type mice, heterozygous Dragon knock-out mice exhibited 45–66% reduction in Dragon mRNA expression, decreased epithelial apoptosis, and increased tubular E-cadherin expression and had attenuated tubular injury after unilateral ureteral obstruction. Our results suggest that Dragon may impair tubular epithelial integrity and induce epithelial apoptosis both in vitro and in vivo. PMID:24052264

Mice completely lacking immunoproteasomes display major alterations in antigen presentation

PubMed Central

Kincaid, Eleanor Z; Che, Jenny W; York, Ian; Escobar, Hernando; Reyes-Vargas, Eduardo; Delgado, Julio C.; Welsh, Raymond M; Karow, Margaret L.; Murphy, Andrew J.; Valenzuela, David M.; Yancopoulos, George D.; Rock, Kenneth L

2011-01-01

The importance of immunoproteasomes to antigen presentation has been unclear because animals totally lacking immunoproteasomes have not been previously developed. Here we show that dendritic cells from mice lacking the three immunoproteasome catalytic subunits display defects in presenting multiple major histocompatability (MHC) class I epitopes. During viral infection in vivo, the presentation of a majority of MHC class I epitopes is markedly reduced in immunoproteasome-deficient animals, while presentation of MHC class II peptides is unaffected. By mass spectrometry the repertoire of MHC class I-presented peptides is ~50% different and these differences are sufficient to stimulate robust transplant rejection of wild type cells in mutant mice. These results indicate that immunoproteasomes play a much more important role in antigen presentation than previously thought. PMID:22197977

Impact of the p53 status of tumor cells on extrinsic and intrinsic apoptosis signaling.

PubMed

Wachter, Franziska; Grunert, Michaela; Blaj, Cristina; Weinstock, David M; Jeremias, Irmela; Ehrhardt, Harald

2013-04-17

The p53 protein is the best studied target in human cancer. For decades, p53 has been believed to act mainly as a tumor suppressor and by transcriptional regulation. Only recently, the complex and diverse function of p53 has attracted more attention. Using several molecular approaches, we studied the impact of different p53 variants on extrinsic and intrinsic apoptosis signaling. We reproduced the previously published results within intrinsic apoptosis induction: while wild-type p53 promoted cell death, different p53 mutations reduced apoptosis sensitivity. The prediction of the impact of the p53 status on the extrinsic cell death induction was much more complex. The presence of p53 in tumor cell lines and primary xenograft tumor cells resulted in either augmented, unchanged or reduced cell death. The substitution of wild-type p53 by mutant p53 did not affect the extrinsic apoptosis inducing capacity. In summary, we have identified a non-expected impact of p53 on extrinsic cell death induction. We suggest that the impact of the p53 status of tumor cells on extrinsic apoptosis signaling should be studied in detail especially in the context of therapeutic approaches that aim to restore p53 function to facilitate cell death via the extrinsic apoptosis pathway.

Silencing by nuclear matrix attachment distinguishes cell-type specificity: association with increased proliferation capacity.

PubMed

Linnemann, Amelia K; Krawetz, Stephen A

2009-05-01

DNA loop organization by nuclear scaffold/matrix attachment is a key regulator of gene expression that may provide a means to modulate phenotype. We have previously shown that attachment of genes to the NaCl-isolated nuclear matrix correlates with their silencing in HeLa cells. In contrast, expressed genes were associated with the lithium 3,5-diiodosalicylate (LIS)-isolated nuclear scaffold. To define their role in determining phenotype matrix attached regions (MARs) on human chromosomes 14-18 were identified as a function of expression in a primary cell line. The locations of MARs in aortic adventitial fibroblast (AoAF) cells were very stable (r = 0.909) and 96% of genes attached at MARs are silent (P < 0.001). Approximately one-third of the genes uniquely expressed in AoAF cells were associated with the HeLa cell nuclear matrix and silenced. Comparatively, 81% were associated with the AoAF cell nuclear scaffold (P < 0.001) and expressed. This suggests that nuclear scaffold/matrix association mediates a portion of cell type-specific gene expression thereby modulating phenotype. Interestingly, nuclear matrix attachment and thus silencing of specific genes that regulate proliferation and maintain the integrity of the HeLa cell genome suggests that transformation may at least in part be achieved through aberrant nuclear matrix attachment.

Increase in Pancreatic Proinsulin and Preservation of β-Cell Mass in Autoantibody-Positive Donors Prior to Type 1 Diabetes Onset

PubMed Central

Rodriguez-Calvo, Teresa; Zapardiel-Gonzalo, Jose; Amirian, Natalie; Castillo, Ericka; Lajevardi, Yasaman; Krogvold, Lars; Dahl-Jørgensen, Knut

2017-01-01

Type 1 diabetes is characterized by the loss of insulin production caused by β-cell dysfunction and/or destruction. The hypothesis that β-cell loss occurs early during the prediabetic phase has recently been challenged. Here we show, for the first time in situ, that in pancreas sections from autoantibody-positive (Ab+) donors, insulin area and β-cell mass are maintained before disease onset and that production of proinsulin increases. This suggests that β-cell destruction occurs more precipitously than previously assumed. Indeed, the pancreatic proinsulin-to-insulin area ratio was also increased in these donors with prediabetes. Using high-resolution confocal microscopy, we found a high accumulation of vesicles containing proinsulin in β-cells from Ab+ donors, suggesting a defect in proinsulin conversion or an accumulation of immature vesicles caused by an increase in insulin demand and/or a dysfunction in vesicular trafficking. In addition, islets from Ab+ donors were larger and contained a higher number of β-cells per islet. Our data indicate that β-cell mass (and function) is maintained until shortly before diagnosis and declines rapidly at the time of clinical onset of disease. This suggests that secondary prevention before onset, when β-cell mass is still intact, could be a successful therapeutic strategy. PMID:28137793

Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

PubMed

Ramírez-Carvajal, Lisbeth; Singh, Neetu; de los Santos, Teresa; Rodríguez, Luis L; Long, Charles R

2016-01-01

Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Neurite differentiation is modulated in neuroblastoma cells engineered for altered acetylcholinesterase expression.

PubMed

Koenigsberger, C; Chiappa, S; Brimijoin, S

1997-10-01

Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level (p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.

Further Studies on the Effect of SiN x Refractive Index and Emitter Sheet Resistance on Potential-Induced Degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Jaewon; Dauksher, Bill; Bowden, Stuart

We present the impacts of silicon nitride (SiNx) antireflection coating refractive index and emitter sheet resistance on potential-induced degradation of the shunting type (PID-s). Previously, it has been shown that the cell becomes more PID-s-susceptible as the refractive index decreases or the emitter sheet resistance increases. To verify the effect of refractive index on PID-s, we fabricated cells with varying SiN x refractive index (1.87, 1.94, 2.05) on typical p-type base solar cells with ~60 Ω/sq emitters. However, none of these cells showed output power degradation, regardless of the refractive index. Further investigation of the emitter showed that the PID-smore » was suppressed at ~60 Ω/sq due to the extremely high surface phosphorus concentration (6 x 10 21 cm -3), as measured by secondary ion mass spectrometry. Furthermore, PID-s was observed on cells possessing a high emitter sheet resistance (~80 Ω/sq). In conclusion, the emitter surface phosphorus concentration plays an important role in determining PID-s susceptibility.« less

Further Studies on the Effect of SiN x Refractive Index and Emitter Sheet Resistance on Potential-Induced Degradation

DOE PAGES

Oh, Jaewon; Dauksher, Bill; Bowden, Stuart; ...

2017-01-11

We present the impacts of silicon nitride (SiNx) antireflection coating refractive index and emitter sheet resistance on potential-induced degradation of the shunting type (PID-s). Previously, it has been shown that the cell becomes more PID-s-susceptible as the refractive index decreases or the emitter sheet resistance increases. To verify the effect of refractive index on PID-s, we fabricated cells with varying SiN x refractive index (1.87, 1.94, 2.05) on typical p-type base solar cells with ~60 Ω/sq emitters. However, none of these cells showed output power degradation, regardless of the refractive index. Further investigation of the emitter showed that the PID-smore » was suppressed at ~60 Ω/sq due to the extremely high surface phosphorus concentration (6 x 10 21 cm -3), as measured by secondary ion mass spectrometry. Furthermore, PID-s was observed on cells possessing a high emitter sheet resistance (~80 Ω/sq). In conclusion, the emitter surface phosphorus concentration plays an important role in determining PID-s susceptibility.« less

Changes in ganglion cell physiology during retinal degeneration influence excitability by prosthetic electrodes

NASA Astrophysics Data System (ADS)

Cho, Alice; Ratliff, Charles; Sampath, Alapakkam; Weiland, James

2016-04-01

Objective. Here we investigate ganglion cell physiology in healthy and degenerating retina to test its influence on threshold to electrical stimulation. Approach. Age-related Macular Degeneration and Retinitis Pigmentosa cause blindness via outer retinal degeneration. Inner retinal pathways that transmit visual information to the central brain remain intact, so direct electrical stimulation from prosthetic devices offers the possibility for visual restoration. Since inner retinal physiology changes during degeneration, we characterize physiological properties and responses to electrical stimulation in retinal ganglion cells (RGCs) of both wild type mice and the rd10 mouse model of retinal degeneration. Main results. Our aggregate results support previous observations that elevated thresholds characterize diseased retinas. However, a physiology-driven classification scheme reveals distinct sub-populations of ganglion cells with thresholds either normal or strongly elevated compared to wild-type. When these populations are combined, only a weakly elevated threshold with large variance is observed. The cells with normal threshold are more depolarized at rest and exhibit periodic oscillations. Significance. During degeneration, physiological changes in RGCs affect the threshold stimulation currents required to evoke action potentials.

Tax posttranslational modifications and interaction with calreticulin in MT-2 cells and human peripheral blood mononuclear cells of human T cell lymphotropic virus type-I-associated myelopathy/tropical spastic paraparesis patients.

PubMed

Medina, Fernando; Quintremil, Sebastian; Alberti, Carolina; Barriga, Andres; Cartier, Luis; Puente, Javier; Ramírez, Eugenio; Ferreira, Arturo; Tanaka, Yuetsu; Valenzuela, Maria Antonieta

2014-04-01

The human retrovirus human T cell lymphotropic virus type-I (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Axonal degeneration in HAM/TSP patients occurs without neuron infection, with the secreted viral Tax protein proposed to be involved. We previously found that Tax secreted into the culture medium of MT-2 cells (HTLV-1-infected cell line) produced neurite retraction in neuroblastoma cells differentiated to neuronal type. To assess the relevance of Tax posttranslational modifications on this effect, we addressed the question of whether Tax secreted by MT-2 cells and peripheral blood mononuclear cells (PBMCs) of HTLV-1-infected subjects is modified. The interaction of Tax with calreticulin (CRT) that modulates intracellular Tax localization and secretion has been described. We studied Tax localization and modifications in MT-2 cells and its interaction with CRT. Intracellular Tax in MT-2 cells was assessed by flow cytometry, corresponding mainly to a 71-kDa protein followed by western blot. This protein reported as a chimera with gp21 viral protein-confirmed by mass spectrometry-showed no ubiquitination or SUMOylation. The Tax-CRT interaction was determined by confocal microscopy and coimmunoprecipitation. Extracellular Tax from HAM/TSP PBMCs is ubiquitinated according to western blot, and its interaction with CRT was shown by coimmunoprecipitation. A positive correlation between Tax and CRT secretion was observed in HAM/TSP PBMCs and asymptomatic carriers. For both proteins inhibitors and activators of secretion showed secretion through the endoplasmic reticulum-Golgi complex. Tax, present in PBMC culture medium, produced neurite retraction in differentiated neuroblastoma cells. These results suggest that Tax, whether ubiquitinated or not, is active for neurite retraction.

The Aryl Hydrocarbon Receptor Mediates Leflunomide-Induced Growth Inhibition of Melanoma Cells

PubMed Central

O’Donnell, Edmond F.; Kopparapu, Prasad Rao; Koch, Daniel C.; Jang, Hyo Sang; Phillips, Jessica Lynne; Tanguay, Robert L.; Kerkvliet, Nancy I.; Kolluri, Siva Kumar

2012-01-01

A novel role of the dihydroorotatedehydrogenase (DHODH) inhibitor leflunomide as a potential anti-melanoma therapy was recently reported (Nature 471∶518-22, 2011). We previously reported that leflunomide strongly activates the transcriptional activity of the Aryl Hydrocarbon Receptor (AhR). We therefore tested whether the AhR regulates the anti-proliferative effects of leflunomide in melanoma. We first evaluated the expression of AhR in melanoma cells and found that AhR is highly expressed in A375 melanoma as well as in several other cancer cell types. To evaluate whether AhR plays a role in regulating the growth inhibitory effects of leflunomide in A375 cells, we generated a stable cell line from parental A375 cells expressing a doxycycline (DOX) inducible AhR shRNA. Using these cells in the absence or presence of DOX (normal AhR levels or AhR-knockdown, respectively) we found that the anti-proliferative effects of leflunomide, but not its metabolite A771726, were strongly dependent upon AhR expression. It has been well established that supplementation of cells with exogenous uridine completely rescues the anti-proliferative effects due to DHODH inhibition. Thus, we performed uridine rescue experiments in A375 cells to determine whether the anti-proliferative effects of leflunomide are solely due to DHODH inhibition as previously reported. Interestingly, saturating levels of uridine only modestly rescued A375 cells from the anti-proliferative effects of both leflunomide and A771726, indicating additional mechanism(s), apart from DHODH inhibition are responsible for the anti-proliferative effects of leflunomide in melanoma cells. Uridine also did not rescue MDA-MB-435S melanoma cell proliferation after leflunomide treatment. Our results reveal that the AhR is a molecular target of leflunomide and support the feasibility of the clinical application of leflunomide for treating melanoma. Furthermore, analysis of expression data from 967 cancer cell lines revealed that AhR is expressed in multiple different cancer types supporting the intriguing possibility of targeting the AhR for therapy in a number of cancers. PMID:22815870

The role of alveolar epithelial cells in initiating and shaping pulmonary immune responses: communication between innate and adaptive immune systems.

PubMed

Chuquimia, Olga D; Petursdottir, Dagbjort H; Rahman, Muhammad J; Hartl, Katharina; Singh, Mahavir; Fernández, Carmen

2012-01-01

Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.

Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats

DOE PAGES

Botcheva, Krassimira; McCorkle, Sean R.

2014-11-21

The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We reportmore » distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). In conclusion, our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways.« less

Hsl7 is a substrate-specific type II protein arginine methyltransferase in yeast

PubMed Central

Sayegh, Joyce; Clarke, Steven G.

2008-01-01

The Saccharomyces cerevisiae protein Hsl7 is a regulator of the Swe1 protein kinase in cell cycle checkpoint control. Hsl7 has been previously described as a type III protein arginine methyltransferase, catalyzing the formation of ω-monomethylarginine residues on non-physiological substrates. However, we show here that Hsl7 can also display type II activity, generating symmetric dimethylarginine residues on calf thymus histone H2A. Symmetric dimethylation is only observed when enzyme and the methyl-accepting substrate were incubated for extended times. We confirmed the Hsl7-dependent formation of symmetric dimethylarginine by amino acid analysis and thin layer chromatography with wild type and mutant recombinant enzymes expressed from both bacteria and yeast. This result is significant because no type II activity has been previously demonstrated in S. cerevisiae. We also show that Hsl7 has little or no activity on GST-GAR, a commonly used substrate for protein arginine methyltransferases, and only minimal activity on myelin basic protein. This enzyme thus may only recognize only a small subset of potential substrate proteins in yeast, in contrast to the situation with Rmt1, the major type I methyltransferase. PMID:18515076

Satellite cell senescence underlies myopathy in a mouse model of limb-girdle muscular dystrophy 2H

PubMed Central

Kudryashova, Elena; Kramerova, Irina; Spencer, Melissa J.

2012-01-01

Mutations in the E3 ubiquitin ligase tripartite motif-containing 32 (TRIM32) are responsible for the disease limb-girdle muscular dystrophy 2H (LGMD2H). Previously, we generated Trim32 knockout mice (Trim32–/– mice) and showed that they display a myopathic phenotype accompanied by neurogenic features. Here, we used these mice to investigate the muscle-specific defects arising from the absence of TRIM32, which underlie the myopathic phenotype. Using 2 models of induced atrophy, we showed that TRIM32 is dispensable for muscle atrophy. Conversely, TRIM32 was necessary for muscle regrowth after atrophy. Furthermore, TRIM32-deficient primary myoblasts underwent premature senescence and impaired myogenesis due to accumulation of PIAS4, an E3 SUMO ligase and TRIM32 substrate that was previously shown to be associated with senescence. Premature senescence of myoblasts was also observed in vivo in an atrophy/regrowth model. Trim32–/– muscles had substantially fewer activated satellite cells, increased PIAS4 levels, and growth failure compared with wild-type muscles. Moreover, Trim32–/– muscles exhibited features of premature sarcopenia, such as selective type II fast fiber atrophy. These results imply that premature senescence of muscle satellite cells is an underlying pathogenic feature of LGMD2H and reveal what we believe to be a new mechanism of muscular dystrophy associated with reductions in available satellite cells and premature sarcopenia. PMID:22505452

The RNA-binding protein Spo5 promotes meiosis II by regulating cyclin Cdc13 in fission yeast.

PubMed

Arata, Mayumi; Sato, Masamitsu; Yamashita, Akira; Yamamoto, Masayuki

2014-03-01

Meiosis comprises two consecutive nuclear divisions, meiosis I and II. Despite this unique progression through the cell cycle, little is known about the mechanisms controlling the sequential divisions. In this study, we carried out a genetic screen to identify factors that regulate the initiation of meiosis II in the fission yeast Schizosaccharomyces pombe. We identified mutants deficient in meiosis II progression and repeatedly isolated mutants defective in spo5, which encodes an RNA-binding protein. Using fluorescence microscopy to visualize YFP-tagged protein, we found that spo5 mutant cells precociously lost Cdc13, the major B-type cyclin in fission yeast, before meiosis II. Importantly, the defect in meiosis II was rescued by increasing CDK activity. In wild-type cells, cdc13 transcripts increased during meiosis II, but this increase in cdc13 expression was weaker in spo5 mutants. Thus, Spo5 is a novel regulator of meiosis II that controls the level of cdc13 expression and promotes de novo synthesis of Cdc13. We previously reported that inhibition of Cdc13 degradation is necessary to initiate meiosis II; together with the previous information, the current findings indicate that the dual control of Cdc13 by de novo synthesis and suppression of proteolysis ensures the progression of meiosis II. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Genomic and transcriptomic comparison of allergen and silver nanoparticle-induced mast cell degranulation reveals novel non-immunoglobulin E mediated mechanisms.

PubMed

Johnson, Monica; Alsaleh, Nasser; Mendoza, Ryan P; Persaud, Indushekhar; Bauer, Alison K; Saba, Laura; Brown, Jared M

2018-01-01

Mast cells represent a crucial cell type in host defense; however, maladaptive responses are contributing factors in the pathogenesis of allergic diseases. Previous work in our laboratory has shown that exposure to silver nanoparticles (AgNPs) results in mast cell degranulation via a non-immunoglobulin E (IgE) mechanism. In this study, we utilized a systems biology approach to identify novel genetic factors playing a role in AgNP-induced mast cell degranulation compared to the classical activation by antigen-mediated FcεRI crosslinking. Mast cell degranulation was assessed in bone marrow-derived mast cells isolated from 23 strains of mice following exposure to AgNPs or FcεRI crosslinking with dinitrophenyl (DNP). Utilizing strain-dependent mast cell degranulation, an association mapping study identified 3 chromosomal regions that were significantly associated with mast cell degranulation by AgNP and one non-overlapping region associated with DNP-mediated degranulation. Two of the AgNP-associated regions correspond to genes previously reported to be associated with allergic disorders (Trac2 on chromosome 1 and Traf6 on chromosome 2) and an uncharacterized gene identified on chromosome 1 (Fam126b). In conjunction, RNA-sequencing performed on mast cells from the high and low responder strains revealed 3754 and 34 differentially expressed genes that were unique to DNP and AgNP exposures, respectively. Select candidate genes include Ptger4, a gene encoding a G-protein coupled receptor in addition to a multifunctional adaptor protein, Txnip, that may be driving mast cell degranulation by AgNP. Taken together, we identified novel genes that have not been previously shown to play a role in nanoparticle-mediated mast cell activation. With further functional evaluation in the future, these genes may be potential therapeutic targets in the treatment of non-IgE mediated mast cell-linked disorders.