Canine distemper virus isolated from a monkey efficiently replicates on Vero cells expressing non-human primate SLAM receptors but not human SLAM receptor.

PubMed

Feng, Na; Liu, Yuxiu; Wang, Jianzhong; Xu, Weiwei; Li, Tiansong; Wang, Tiecheng; Wang, Lei; Yu, Yicong; Wang, Hualei; Zhao, Yongkun; Yang, Songtao; Gao, Yuwei; Hu, Guixue; Xia, Xianzhu

2016-08-02

In 2008, an outbreak of canine distemper virus (CDV) infection in monkeys was reported in China. We isolated CDV strain (subsequently named Monkey-BJ01-DV) from lung tissue obtained from a rhesus monkey that died in this outbreak. We evaluated the ability of this virus on Vero cells expressing SLAM receptors from dog, monkey and human origin, and analyzed the H gene of Monkey-BJ01-DV with other strains. The Monkey-BJ01-DV isolate replicated to the highest titer on Vero cells expressing dog-origin SLAM (10(5.2±0.2) TCID50/ml) and monkey-origin SLAM (10(5.4±0.1) TCID50/ml), but achieved markedly lower titers on human-origin SLAM cells (10(3.3±0.3) TCID50/ml). Phylogenetic analysis of the full-length H gene showed that Monkey-BJ01-DV was highly related to other CDV strains obtained during recent CDV epidemics among species of the Canidae family in China, and these Monkey strains CDV (Monkey-BJ01-DV, CYN07-dV, Monkey-KM-01) possessed a number of amino acid specific substitutions (E276V, Q392R, D435Y and I542F) compared to the H protein of CDV epidemic in other animals at the same period. Our results suggested that the monkey origin-CDV-H protein could possess specific substitutions to adapt to the new host. Monkey-BJ01-DV can efficiently use monkey- and dog-origin SLAM to infect and replicate in host cells, but further adaptation may be required for efficient replication in host cells expressing the human SLAM receptor.

Comparison of human and monkey cells for the ability to attenuate transcripts that begin at the adenovirus major late promoter

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

Seiberg, M.; Aloni, Y.; Levine, A.J.

1989-09-01

Late transcription from the adenovirus major late promoter can terminate prematurely at a site 182 to 188 nucleotides downstream. Experiments have been designed, with run-on transcription in nuclei in vitro or riboprobe protection of RNA obtained both in vivo and in vitro, that demonstrate that the ratio of attenuator RNA to readthrough RNA is greater in monkey cells (CV-1) than in human cells (HeLa). This may explain, in part, why the human adenoviruses replicate more poorly in CV-1 cells than in HeLa cells. A mutant adenovirus that replicates better than wild-type virus in monkey cells produces less of the attenuatormore » RNA than wild-type adenovirus does in monkey cells. Monkey cell extracts have been shown to contain a factor that, when added to human cell extracts transcribing adenovirus DNA in vitro, increases the production of attenuator RNA in these reactions. These observations help to explain a portion of the block to the production of infectious adenoviruses in monkey cells.« less

Low blood cell counts in wild Japanese monkeys after the Fukushima Daiichi nuclear disaster.

PubMed

Ochiai, Kazuhiko; Hayama, Shin-ichi; Nakiri, Sachie; Nakanishi, Setsuko; Ishii, Naomi; Uno, Taiki; Kato, Takuya; Konno, Fumiharu; Kawamoto, Yoshi; Tsuchida, Shuichi; Omi, Toshinori

2014-07-24

In April 2012 we carried out a 1-year hematological study on a population of wild Japanese monkeys inhabiting the forest area of Fukushima City. This area is located 70 km from the Fukushima Daiichi Nuclear Power Plant (NPP), which released a large amount of radioactive material into the environment following the Great East Japan Earthquake of 2011. For comparison, we examined monkeys inhabiting the Shimokita Peninsula in Aomori Prefecture, located approximately 400 km from the NPP. Total muscle cesium concentration in Fukushima monkeys was in the range of 78-1778 Bq/kg, whereas the level of cesium was below the detection limit in all Shimokita monkeys. Compared with Shimokita monkeys, Fukushima monkeys had significantly low white and red blood cell counts, hemoglobin, and hematocrit, and the white blood cell count in immature monkeys showed a significant negative correlation with muscle cesium concentration. These results suggest that the exposure to some form of radioactive material contributed to hematological changes in Fukushima monkeys.

Low blood cell counts in wild Japanese monkeys after the Fukushima Daiichi nuclear disaster

PubMed Central

Ochiai, Kazuhiko; Hayama, Shin-ichi; Nakiri, Sachie; Nakanishi, Setsuko; Ishii, Naomi; Uno, Taiki; Kato, Takuya; Konno, Fumiharu; Kawamoto, Yoshi; Tsuchida, Shuichi; Omi, Toshinori

2014-01-01

In April 2012 we carried out a 1-year hematological study on a population of wild Japanese monkeys inhabiting the forest area of Fukushima City. This area is located 70 km from the Fukushima Daiichi Nuclear Power Plant (NPP), which released a large amount of radioactive material into the environment following the Great East Japan Earthquake of 2011. For comparison, we examined monkeys inhabiting the Shimokita Peninsula in Aomori Prefecture, located approximately 400 km from the NPP. Total muscle cesium concentration in Fukushima monkeys was in the range of 78–1778 Bq/kg, whereas the level of cesium was below the detection limit in all Shimokita monkeys. Compared with Shimokita monkeys, Fukushima monkeys had significantly low white and red blood cell counts, hemoglobin, and hematocrit, and the white blood cell count in immature monkeys showed a significant negative correlation with muscle cesium concentration. These results suggest that the exposure to some form of radioactive material contributed to hematological changes in Fukushima monkeys. PMID:25060710

[Hybrids of human and monkey adenoviruses (adeno-adeno hybrids) that can reproduce in monkey cells: biological and molecular genetic peculiarities].

PubMed

Grinenko, N F; Savitskaia, N V; Pashvykina, G V; Al'tshteĭn, A D

2003-06-01

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.

Cloning of the anhidrotic ectodermal dysplasia gene: Identification of cDNAs associated with CpG islands mapped near translocation breakpoint in two female patients

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

Srivastava, A.K.; Schlessinger, D.; Kere, J.

1994-09-01

The gene for the X chromosomal developmental disorder anhidrotic ectodermal dysplasia (EDA) has been mapped to Xq12-q13 by linkage analysis and is expressed in a few females with chromosomal translocations involving band Xq12-q13. A yeast artificial chromosome (YAC) contig (2.0 Mb) spanning two translocation breakpoints has been assembled by sequence-tagged site (STS)-based chromosomal walking. The two translocation breakpoints (X:autosome translocations from the affected female patients) have been mapped less than 60 kb apart within a YAC contig. Unique probes and intragenic STSs (mapped between the two translocations) have been developed and a somatic cell hybrid carrying the translocated X chromosomemore » from the AK patient has been analyzed by isolating unique probes that span the breakpoint. Several STSs made from intragenic sequences have been found to be conserved in mouse, hamster and monkey, but we have detected no mRNAs in a number of tissues tested. However, a probe and STS developed from the DNA spanning the AK breakpoint is conserved in mouse, hamster and monkey, and we have detected expressed sequences in skin cells and cDNA libraries. In addition, unique sequences have been obtained from two CpG islands in the region that maps proximal to the breakpoints. cDNAs containing these sequences are being studied as candidates for the gene affected in the etiology of EDA.« less

Optimally achieving milk bulk tank somatic cell count thresholds.

PubMed

Troendle, Jason A; Tauer, Loren W; Gröhn, Yrjo T

2017-01-01

High somatic cell count in milk leads to reduced shelf life in fluid milk and lower processed yields in manufactured dairy products. As a result, farmers are often penalized for high bulk tank somatic cell count or paid a premium for low bulk tank somatic cell count. Many countries also require all milk from a farm to be lower than a specified regulated somatic cell count. Thus, farms often cull cows that have high somatic cell count to meet somatic cell count thresholds. Rather than naïvely cull the highest somatic cell count cows, a mathematical programming model was developed that determines the cows to be culled from the herd by maximizing the net present value of the herd, subject to meeting any specified bulk tank somatic cell count level. The model was applied to test-day cows on 2 New York State dairy farms. Results showed that the net present value of the herd was increased by using the model to meet the somatic cell count restriction compared with naïvely culling the highest somatic cell count cows. Implementation of the model would be straightforward in dairy management decision software. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Androgen resistance in squirrel monkeys (Saimiri spp.).

PubMed

Gross, Katherine L; Westberry, Jenne M; Hubler, Tina R; Sadosky, Patti W; Singh, Ravinder J; Taylor, Robert L; Scammell, Jonathan G

2008-08-01

The goal of this study was to understand the basis for high androgen levels in squirrel monkeys (Saimiri spp.). Mass spectrometry was used to analyze serum testosterone, androstenedione, and dihydrotestosterone of male squirrel monkeys during the nonbreeding (n = 7) and breeding (n = 10) seasons. All hormone levels were elevated compared with those of humans, even during the nonbreeding season; the highest levels occurred during the breeding season. The ratio of testosterone to dihydrotestosterone in squirrel monkeys is high during the breeding season compared to man. Squirrel monkeys may have high testosterone to compensate for inefficient metabolism to dihydrotestosterone. We also investigated whether squirrel monkeys have high androgens to compensate for low-activity androgen receptors (AR). The response to dihydrotestosterone in squirrel monkey cells transfected with AR and AR-responsive reporter plasmids was 4-fold, compared with 28-fold in human cells. This result was not due to overexpression of cellular FKBP51, which causes glucocorticoid and progestin resistance in squirrel monkeys, because overexpression of FKBP51 had no effect on dihydrotestosterone-stimulated reporter activity in a human fibroblast cell line. To test whether the inherently low levels of FKBP52 in squirrel monkeys contribute to androgen insensitivity, squirrel monkey cells were transfected with an AR expression plasmid, an AR-responsive reporter plasmid, and a plasmid expressing FKBP52. Expression of FKBP52 decreased the EC50 or increased the maximal response to dihydrotestosterone. Therefore, the high androgen levels in squirrel monkeys likely compensate for their relatively low 5 alpha-reductase activity during the breeding season and AR insensitivity resulting from low cellular levels of FKBP52.

Evaluation of drug-induced hematotoxicity using novel in vitro monkey CFU-GM and BFU-E colony assays.

PubMed

Goto, Koichi; Goto, Mayumi; Ando-Imaoka, Masako; Kai, Kiyonori; Mori, Kazuhiko

2017-01-01

In order to evaluate drug-induced hematotoxicity in monkey cells in vitro, colony-forming unit-granulocyte, macrophage (CFU-GM), and burst-forming unit-erythroid (BFU-E) colony assays were established using mononuclear cells in the bone marrow collected from male cynomolgus monkeys. Furthermore, the effects of doxorubicin, chloramphenicol, and linezolid on CFU-GM and BFU-E colony formation were investigated using established monkey CFU-GM and BFU-E colony assays in comparison with those on human CFU-GM and BFU-E colonies acquired from human umbilical cord blood cells. Bone marrow mononuclear cells were collected from the ischial or iliac bone of male cynomolgus monkeys. The cells were subsequently processed by density gradient separation at 1.067, 1.070, or 1.077 g/mL for CFU-GM or 1.077 g/mL for BFU-E, and then cultured in methylcellulose medium for 9 or 13 days, respectively. A sufficient number of CFU-GM colonies were formed from mononuclear cells processed at a density of 1.070 g/mL. Moreover, the number of BFU-E colonies from the cells processed at a density of 1.077 g/mL was sufficient for the colony assay. The number of CFU-GM or BFU-E colonies decreased after treatment with the drugs of interest in a concentration-dependent manner. Compared with human CFU-GM, monkey CFU-GM were more sensitive to chloramphenicol and resistant to doxorubicin, whereas monkey BFU-E were more sensitive to all compounds in comparison to the sensitivity of human BFU-E. In conclusion, monkey CFU-GM and BFU-E colony assays were established and considered useful tools to evaluate the differences in drug-induced hematotoxicity between species.

Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

PubMed Central

Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

2014-01-01

Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

Application of Somatic Embryogenesis in Woody Plants.

PubMed Central

Guan, Yuan; Li, Shui-Gen; Fan, Xiao-Fen; Su, Zhen-Hong

2016-01-01

Somatic embryogenesis is a developmental process where a plant somatic cell can dedifferentiate to a totipotent embryonic stem cell that has the ability to give rise to an embryo under appropriate conditions. This new embryo can further develop into a whole plant. In woody plants, somatic embryogenesis plays a critical role in clonal propagation and is a powerful tool for synthetic seed production, germplasm conservation, and cryopreservation. A key step in somatic embryogenesis is the transition of cell fate from a somatic cell to embryo cell. Although somatic embryogenesis has already been widely used in a number of woody species, propagating adult woody plants remains difficult. In this review, we focus on molecular mechanisms of somatic embryogenesis and its practical applications in economic woody plants. Furthermore, we propose a strategy to improve the process of somatic embryogenesis using molecular means. PMID:27446166

[Comparative immunophenotypic characterization of human and monkey permanent lymphoid culture cells].

PubMed

Agrba, V Z; Lapin, B A; Medvedeva, N M; Ignatova, I E; Karal-Ogly, D D

2007-01-01

The aim of the study was to define the comparative immunophenotypic characteristics ofwidely spread lymphoid cell cultures, derived from Burkett's lymphoma named as Raji and P3HR-1 in comparison with analogous monkey cultures. It has been shown that P3HR-1 culture consists of similar type cells - activated B-lymphocytes CD23 with k phenotype, which demonstrates its monoclonality. Raji culture includes cells with markers of immature B-lymphocytes CD10 and CD24, as well as elements expressing CD10 antigens. T-cell markers were found in none of the cultures. In contrast to human cells, monkey lymphoid culture expressed both B- and T-cell markers. Moreover, in one of them, obtained from a green monkey, T-cells of suppressor type (CD8) prevailed. The immunophenotypic characteristics of primate lymphoid cell cultures, revealed by the study, are of great importance for their proper application to medical and biological studies.

Interspecies radioimmunoassay for the major structural proteins of primate type-D retroviruses

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

Colcher, D.; Teramoto, Y.A.; Schlom, J.

1977-12-01

A competition radioimmunoassay has been developed in which type-D retroviruses from three primate species compete. The assay utilizes the major structural protein (36,000 daltons) of the endogenous squirrel monkey retrovirus and antisera directed against the major structural protein (27,000 daltons) of the Mason-Pfizer monkey virus isolated from rhesus monkeys. Purified preparations of both viruses grown in heterologous cells, as well as extracts of heterologous cells infected with squirrel monkey retrovirus or Mason-Pfizer monkey virus, compete completely in the assay. Addition of an endogenous virus of the langur monkey also results in complete blocking. No blocking in the assay is observedmore » with type-C baboon viruses, woolly monkey virus, and gibbon virus. Various other type-C and type-B viruses also showed no reactivity. An interspecies assay has thus been developed that recognizes the type-D retroviruses from both Old World monkey (rhesus and langur) and New World monkey (squirrel) species.« less

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus.

PubMed

Nozu, Ryo; Horiguchi, Ryo; Murata, Ryosuke; Kobayashi, Yasuhisa; Nakamura, Masaru

2013-02-01

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.

The rhesus monkey as a model for testing the immunological effects of space flight

NASA Astrophysics Data System (ADS)

Sonnenfeld, G.; Schaffar, L.; Schmitt, D. A.; Peres, C.; Miller, E. S.

1994-08-01

The Rhesus monkey has been proposed as a model for the effects of space flight on immunity. In order to determine the feasibility of the use of the Rhesus monkey as a model, we studied the use of Rhesus monkey cells for immunological procedures that have been shown to be affected by space flight in both rodents and humans. We have shown that both lymph node cells and peripheral blood leukocytes can be stained with monoclonal antibodies to detect the following surface markers: CD4, CD-8, Ia and surface immunoglobulin. Also, the level of Ia antigen expression was increased by treatment of the cells with human interferon-gamma. In addition, cells were induced to produce interferons and interleukins. Isolated neutrophils also demonstrated increased oxidative burst. These data indicate that the Rhesus monkey will be a useful model for space flight studies of immunity.

The Rhesus monkey as a model for testing the immunological effects of space flight

NASA Technical Reports Server (NTRS)

Sonnenfeld, G.; Schaffar, L.; Schmitt, D. A.; Peres, C.; Miller, E. S.

1994-01-01

The Rhesus monkey has been proposed as a model for the effects of space flight on immunity. In order to determine the feasibility of the use of the Rhesus monkey as a model, we studied the use of Rhesus monkey cells for immunological procedures that have been shown to be affected by space flight in both rodents and humans. We have shown that both lymph node cells and peripheral blood leukocytes can be stained with monoclonal antibodies to detect the following surface markers: CD4, CD-8, Ia and surface immunoglobulin. Also, the level of Ia antigen expression was increased by treatment of the cells with human interferon-gamma. In addition, cells were induced to produce interferons and interleukins. Isolated neutrophils also demonstrated increased oxidative burst. These data indicate that the Rhesus monkey will be a useful model for space flight studies of immunity.

[Aging affects early stage direction selectivity of MT cells in rhesus monkeys].

PubMed

Liang, Zhen; Chen, Yue-Ming; Meng, Xue; Wang, Yi; Zhou, Bao-Zhuo; Xie, Ying-Ying; He, Wen-Sheng

2012-10-01

The middle temporal area (MT/V5) plays an important role in motion processing. Neurons in this area have a strongly selective response to the moving direction of objects and as such, the selectivity of MT neurons was proposed to be a neural mechanism for the perception of motion. Our previous studies have found degradation in direction selectivity of MT neurons in old monkeys, but this direction selectivity was calculated during the whole response time and the results were not able to uncover the mechanism of motion perception over a time course. Furthermore, experiments have found that direction selectivity was enhanced by attention at a later stage. Therefore, the response should be excluded in experiments with anesthesia. To further characterize the neural mechanism over a time course, we investigated the age-related changes of direction selectivity in the early stage by comparing the proportions of direction selective MT cells in old and young macaque monkeys using in vivo single-cell recording techniques. Our results show that the proportion of early-stage-direction-selective cells is lower in old monkeys than in young monkeys, and that the early stage direction bias (esDB) of old MT cells decreased relative to young MT cells. Furthermore, the proportion of MT cells having strong early stage direction selectivity in old monkeys was decreased. Accordingly, the functional degradation in the early stage of MT cells may mediate perceptual declines of old primates in visual motion tasks.

Quantification of β-cell insulin secretory function using a graded glucose infusion with C-peptide deconvolution in dysmetabolic, and diabetic cynomolgus monkeys.

PubMed

Wang, Xiaoli; Hansen, Barbara C; Shi, Da; Fang, Yupeng; Du, Fenglai; Wang, Bingdi; Chen, Yaxiong Michael; Gregoire, Francine M; Wang, Yi-Xin Jim

2013-07-25

Quantitation of β-cell function is critical in better understanding of the dynamic interactions of insulin secretion, clearance and action at different phases in the progression of diabetes. The present study aimed to quantify β-cell secretory function independently of insulin sensitivity in the context of differential metabolic clearance rates of insulin (MCRI) in nonhuman primates (NHPs). Insulin secretion rate (ISR) was derived from deconvolution of serial C-peptide concentrations measured during a 5 stage graded glucose infusion (GGI) in 12 nondiabetic (N), 8 prediabetic or dysmetabolic (DYS) and 4 overtly diabetic (DM) cynomolgus monkeys. The characterization of the monkeys was based on the fasting glucose and insulin concentrations, glucose clearance rate measured by intravenous glucose tolerance test, and insulin resistance indices measured in separate experiments. The molar ratio of C-peptide/insulin (C/I) was used as a surrogate index of hepatic MCRI. Compared to the N monkeys, the DYS with normal glycemia and hyperinsulinemia had significantly higher basal and GGI-induced elevation of insulin and C-peptide concentrations and lower C/I, however, each unit of glucose-stimulated ISR increment was not significantly different from that in the N monkeys. In contrast, the DM monkeys with β-cell failure and hyperglycemia had a depressed GGI-stimulated ISR response and elevated C/I. The present data demonstrated that in addition to β-cell hypersecretion of insulin, reduced hepatic MCRI may also contribute to the development of hyperinsulinemia in the DYS monkeys. On the other hand, hyperinsulinemia may cause the saturation of hepatic insulin extraction capacity, which in turn reduced MCRI in the DYS monkeys. The differential contribution of ISR and MCRI in causing hyperinsulinemia provides a new insight into the trajectory of β-cell dysfunction in the development of diabetes. The present study was the first to use the GGI and C-peptide deconvolution method to quantify the β-cell function in NHPs.

Reprogramming of Somatic Cells Towards Pluripotency by Cell Fusion.

PubMed

Malinowski, Andrzej R; Fisher, Amanda G

2016-01-01

Pluripotent reprogramming can be dominantly induced in a somatic nucleus upon fusion with a pluripotent cell such as embryonic stem (ES) cell. Cell fusion between ES cells and somatic cells results in the formation of heterokaryons, in which the somatic nuclei begin to acquire features of the pluripotent partner. The generation of interspecies heterokaryons between mouse ES- and human somatic cells allows an experimenter to distinguish the nuclear events occurring specifically within the reprogrammed nucleus. Therefore, cell fusion provides a simple and rapid approach to look at the early nuclear events underlying pluripotent reprogramming. Here, we describe a polyethylene glycol (PEG)-mediated cell fusion protocol to generate interspecies heterokaryons and intraspecies hybrids between ES cells and B lymphocytes or fibroblasts.

The neonatal marmoset monkey ovary is very primitive exhibiting many oogonia

PubMed Central

Fereydouni, B; Drummer, C; Aeckerle, N; Schlatt, S; Behr, R

2014-01-01

Oogonia are characterized by diploidy and mitotic proliferation. Human and mouse oogonia express several factors such as OCT4, which are characteristic of pluripotent cells. In human, almost all oogonia enter meiosis between weeks 9 and 22 of prenatal development or undergo mitotic arrest and subsequent elimination from the ovary. As a consequence, neonatal human ovaries generally lack oogonia. The same was found in neonatal ovaries of the rhesus monkey, a representative of the old world monkeys (Catarrhini). By contrast, proliferating oogonia were found in adult prosimians (now called Strepsirrhini), which is a group of ‘lower’ primates. The common marmoset monkey (Callithrix jacchus) belongs to the new world monkeys (Platyrrhini) and is increasingly used in reproductive biology and stem cell research. However, ovarian development in the marmoset monkey has not been widely investigated. Herein, we show that the neonatal marmoset ovary has an extremely immature histological appearance compared with the human ovary. It contains numerous oogonia expressing the pluripotency factors OCT4A, SALL4, and LIN28A (LIN28). The pluripotency factor-positive germ cells also express the proliferation marker MKI67 (Ki-67), which has previously been shown in the human ovary to be restricted to premeiotic germ cells. Together, the data demonstrate the primitiveness of the neonatal marmoset ovary compared with human. This study may introduce the marmoset monkey as a non-human primate model to experimentally study the aspects of primate primitive gonad development, follicle assembly, and germ cell biology in vivo. PMID:24840529

A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

PubMed

Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

2015-09-01

A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 μI of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed.

Transplantation of iPS-Derived Tumor Cells with a Homozygous MHC Haplotype Induces GRP94 Antibody Production in MHC-Matched Macaques.

PubMed

Ishigaki, Hirohito; Maeda, Toshinaga; Inoue, Hirokazu; Akagi, Tsuyoshi; Sasamura, Takako; Ishida, Hideaki; Inubushi, Toshiro; Okahara, Junko; Shiina, Takashi; Nakayama, Misako; Itoh, Yasushi; Ogasawara, Kazumasa

2017-11-01

Immune surveillance is a critical component of the antitumor response in vivo , yet the specific components of the immune system involved in this regulatory response remain unclear. In this study, we demonstrate that autoantibodies can mitigate tumor growth in vitro and in vivo We generated two cancer cell lines, embryonal carcinoma and glioblastoma cell lines, from monkey-induced pluripotent stem cells (iPSC) carrying a homozygous haplotype of major histocompatibility complex (MHC, Mafa in Macaca fascicularis). To establish a monkey cancer model, we transplanted these cells into monkeys carrying the matched Mafa haplotype in one of the chromosomes. Neither Mafa-homozygous cancer cell line grew in monkeys carrying the matched Mafa haplotype heterozygously. We detected in the plasma of these monkeys an IgG autoantibody against GRP94, a heat shock protein. Injection of the plasma prevented growth of the tumor cells in immunodeficient mice, whereas plasma IgG depleted of GRP94 IgG exhibited reduced killing activity against cancer cells in vitro These results indicate that humoral immunity, including autoantibodies against GRP94, plays a role in cancer immune surveillance. Cancer Res; 77(21); 6001-10. ©2017 AACR . ©2017 American Association for Cancer Research.

Birth of cloned mice from vaginal smear cells after somatic cell nuclear transfer.

PubMed

Kuwayama, Hiroki; Tanabe, Yoshiaki; Wakayama, Teruhiko; Kishigami, Satoshi

2017-05-01

Less invasive methods for donor cell collection will facilitate reproduction of wild animals using somatic-cell nuclear transfer. Stages of the estrous cycle in mice have long been studies using somatic cells that can be collected from vaginal walls using cotton tipped swabs in a relatively non-invasive manner. In this study, we examined the feasibility of these cells as sources of nuclei for somatic-cell cloning using nuclear transfer. Estrous cycles generally comprise proestrus, estrus, metestrus, and diestrus stages. In the present experiments, more than 60% of cells were nucleated in vaginal smears from all but the estrus stage. However, after somatic-cell nuclear transfer of cells from proestrus, metestrus, and diestrus stages, 66%, 50%, and 72% of cloned embryos developed to the morula/blastocyst, and cloned female mouse birth rates after embryo transfer were 1.5%, 0.3%, and 1%, respectively. These results show that noninvasively collected vaginal smears contain somatic cells that can be used to clone female mice. Copyright © 2017. Published by Elsevier Inc.

Recent advancements in cloning by somatic cell nuclear transfer.

PubMed

Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

2013-01-05

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

Recent advancements in cloning by somatic cell nuclear transfer

PubMed Central

Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

2013-01-01

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model. PMID:23166393

World Conference on Pre-Erythrocytic Stage Malaria Vaccine Development: Current Status and Future Prospects Held in Bethesda, Maryland on April 12-15, 1989

DTIC Science & Technology

1989-12-01

for improving immunogenicity and efficacy using alternative delivery systems including proteosomes, liposomes, and synthetic adjuvants such as SAF...were to Identify responder T cells, clone these cells to map the T-cell epitopes, and acquire sera and lymphocytes for use as positive controls during...efficacy In Saimirl sciureus boliviensis monkeys. Monkeys were Immunized three times using either alum or muramyl tripeptide as adjuvants. Monkeys were

Hormone suppression with GnRH antagonist promotes spermatogenic recovery from transplanted spermatogonial stem cells in irradiated cynomolgus monkeys.

PubMed

Shetty, G; Uthamanthil, R K; Zhou, W; Shao, S H; Weng, C C; Tailor, R C; Hermann, B P; Orwig, K E; Meistrich, M L

2013-11-01

Hormone suppression given before or after cytotoxic treatment stimulates the recovery of spermatogenesis from endogenous and transplanted spermatogonial stem cells (SSC) and restores fertility in rodents. To test whether the combination of hormone suppression and transplantation could enhance the recovery of spermatogenesis in primates, we irradiated (7 Gy) the testes of 12 adult cynomolgus monkeys and treated six of them with gonadotropin-releasing hormone antagonist (GnRH-ant) for 8 weeks. At the end of this treatment, we transfected cryopreserved testicular cells with green fluorescent protein-lentivirus and autologously transplanted them back into one of the testes. The only significant effect of GnRH-ant treatment on endogenous spermatogenesis was an increase in the percentage of tubules containing differentiated germ cells (tubule differentiation index; TDI) in the sham-transplanted testes of GnRH-ant-treated monkeys compared with radiation-only monkeys at 24 weeks after irradiation. Although transplantation alone after irradiation did not significantly increase the TDI, detection of lentiviral DNA in the spermatozoa of one radiation-only monkey indicated that some transplanted cells colonized the testis. However, the combination of transplantation and GnRH-ant clearly stimulated spermatogenic recovery as evidenced by several observations in the GnRH-ant-treated monkeys receiving transplantation: (i) significant increases (~20%) in the volume and weight of the testes compared with the contralateral sham-transplanted testes and/or to the transplanted testes of the radiation-only monkeys; (ii) increases in TDI compared to the transplanted testes of radiation-only monkeys at 24 weeks (9.6% vs. 2.9%; p = 0.05) and 44 weeks (16.5% vs. 6.1%, p = 0.055); (iii) detection of lentiviral sequences in the spermatozoa or testes of five of the GnRH-ant-treated monkeys and (iv) significantly higher sperm counts than in the radiation-only monkeys. Thus hormone suppression enhances spermatogenic recovery from transplanted SSC in primates and may be a useful tool in conjunction with spermatogonial transplantation to restore fertility in men after cancer treatment. © 2013 American Society of Andrology and European Academy of Andrology.

Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae).

PubMed

Schelegle, E S; Gershwin, L J; Miller, L A; Fanucchi, M V; Van Winkle, L S; Gerriets, J P; Walby, W F; Omlor, A M; Buckpitt, A R; Tarkington, B K; Wong, V J; Joad, J P; Pinkerton, K B; Wu, R; Evans, M J; Hyde, D M; Plopper, C G

2001-01-01

To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4(+) lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA.

Allergic Asthma Induced in Rhesus Monkeys by House Dust Mite (Dermatophagoides farinae)

PubMed Central

Schelegle, Edward S.; Gershwin, Laurel J.; Miller, Lisa A.; Fanucchi, Michelle V.; Van Winkle, Laura S.; Gerriets, Joan P.; Walby, William F.; Omlor, Amanda M.; Buckpitt, Alan R.; Tarkington, Brian K.; Wong, Viviana J.; Joad, Jesse P.; Pinkerton, Kent B.; Wu, Reen; Evans, Michael J.; Hyde, Dallas M.; Plopper, Charles G.

2001-01-01

To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4+ lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA. PMID:11141508

Thymus development in Macaca fascicularis (Cynomolgus monkey): an approach for toxicology and embryology.

PubMed

Buse, Eberhard; Habermann, Gunnar; Vogel, Friedhelm

2006-05-01

Thymus development was studied in the cynomolgus monkey from day 35 of gestation (gd 35) to the stage of advanced involution in a 21-year-old monkey. Special emphasis was placed on thymus cell generation and cellular pattern formation. At gd 35, the epithelial bud of the thymus was visible in a sagittal position at the level of the thoracic aperture. At gd 50, first lymphocyte-like cells and few Human Leukocyte Antigen-D Region (HLA-DR) immunoreactive cells appeared. The cortico-medullary differentiation, Hassall's body precursors and faint immunoreactivity for T-lymphocytes (CD 3-positive) were detected from gd 60 onwards. First macrophages (CD 68 positive) were apparent at day 70, first CD 20 immunoreactive cells (B-lymphocyte-like cells) at gd 85, and natural killer cells (M1014 immunoreactive) at gd 100. At gd 100 all evaluated cell populations present in the adult cynomolgus monkey thymus were in place, whereas no B- and T-cell precursors or (CD 34 and CD 117, respectively) dendritic cells (CD 35 positive cells) were present. All these immunopositive cells persisted, partly with changing distribution patterns, until the advanced age of 21 years with the exception of natural killer cells, which were present only until adult ages (evaluation at 4-7 years). The rationale of this study was to analyse thymic development in the cynomolgus monkey and to evaluate the relevance of the development of thymus in non-human primate as a model for corresponding human targeted toxicological research.

Somatic embryogenesis in forestry: A practical approach to cloning the best trees

Treesearch

Alex M. Diner

1999-01-01

Trees as well as humans have two basic cell types based on genetic content: somatic cells and gametic or reproductive cells. Somatic cells, such as skin cells or the sapwood cells in a tree, have at least twice (2n) the base set of chromosomes. The reproductive cells (gametic cells) have a single (n) set of chromosomes.

Transplantation of embryonic stem cell-derived dopaminergic neurons in MPTP-treated monkeys.

PubMed

Takahashi, Jun; Takagi, Yasushi; Saiki, Hidemoto

2009-01-01

One of the target diseases of cell-replacement therapy is Parkinson's disease. Clinical experiences with fetal dopaminergic cell graft have shown that the therapy is effective, but limited and accompanied by side effects, such as dyskinesia. So, the therapy needs to be further improved and sophisticated. Embryonic stem (ES) cells are expected to be another donor cell for the treatment, because of its proliferative and differentiation capacities. For clinical application, experiments using non-human primates are important, because size, anatomy, and biological characteristics of the brain are different between rodents and primates. Here, we would like to discuss induction of dopaminergic neurons from monkey ES cells and cell transplantation into the brain of monkey Parkinson's disease model.

Somatic and neuroendocrine responses to standard and biologically salient acoustic startle stimuli in monkeys

PubMed Central

Parker, Karen J.; Hyde, Shellie A.; Buckmaster, Christine L.; Tanaka, Serena M.; Brewster, Katharine K.; Schatzberg, Alan F.; Lyons, David M.; Woodward, Steven H.

2010-01-01

SUMMARY The startle response, a simple defensive response to a sudden stimulus signaling proximal threat, has been well studied in rodents and humans, but has been rarely examined in monkeys. The first goal of the present studies was to develop a minimally immobilizing startle measurement paradigm and validate its usefulness by testing two core features of the startle response (habituation and graded responsivity) in squirrel monkey subjects. Two different types of startle stimuli were used: standard broad-band noise bursts, and species-specific alarm vocalizations (“yaps”) which are elicited in response to threat in both wild and captive animals. The second goal of the present studies was to test whether yaps produce enhanced startle responsivity due to their increased biological salience compared to simple, non-biologically relevant noise bursts. The third goal of the present studies was to evaluate the hypothalamic pituitary-adrenal (HPA) axis response to startle stimuli, as little is known about the stress-activating role of startle stimuli in any species. These experiments determined that the whole-body startle response in relatively unrestrained squirrel monkeys habituates across repeated stimulus presentations and is proportional to stimulus intensity. In addition, differential habituation was observed across biologically salient vs. standard acoustic startle stimuli. Responses to “yaps” were larger initially but attenuated more rapidly over trials. Responses to “yaps” were also larger in the early subepochs of the response window but then achieved a lower level than responses to noise bursts in the later subepochs. Finally, adrenocorticotropic hormone and cortisol concentrations were significantly elevated above baseline after startle stimuli presentation, though monkeys did not exhibit differential HPA axis responses to the two types of startle stimuli. The development of monkey startle methodology may further enhance the utility of this paradigm in translational studies of human stress-related psychiatric disorders. PMID:20869176

In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

PubMed Central

Yamauchi, Kaori; Hasegawa, Kouichi; Chuma, Shinichiro; Nakatsuji, Norio; Suemori, Hirofumi

2009-01-01

Background Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. Methods and Findings To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. Conclusion VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates. PMID:19399191

Establishment of a Somatic Cell Bank for Indian Buffalo Breeds and Assessing the Suitability of the Cryopreserved Cells for Somatic Cell Nuclear Transfer.

PubMed

Selokar, Naresh L; Sharma, Papori; Krishna, Ananth; Kumar, Deepak; Kumar, Dharmendra; Saini, Monika; Sharma, Arpna; Vijayalakshmy, Kennady; Yadav, Prem Singh

2018-06-01

Biobanks of cryopreserved gametes and embryos of domestic animals have been utilized to spread desired genotypes and to conserve the animal germplasm of endangered breeds. In principle, somatic cells can be used for the same purposes, and for reviving of animals, the somatic cells must be suitable for animal cloning techniques, such as somatic cell nuclear transfer. In the present study, we derived and cryopreserved somatic cells from three breeds of riverine and swamp-like type buffaloes and established a somatic cell bank. In total, 350 cryovials of 14 different individual animals (25 cryovials per animal) were cryopreserved and informative data such as breed value, origin, and others were documented. Immunostaining of the established cells against vimentin and cytokeratin suggested a commitment to the fibroblast lineage. In addition, microsatellite analysis was performed and documented for unambiguous parentage verification of clones in the future. Subsequently, the cryopreserved cells were tested for their suitability as nuclear donors (n = 7) using handmade cloning, and the reconstructed embryos were cultured in vitro. The cleavage rates (95.99% ± 2.17% vs. 82.18% ± 2.50%) and blastocyst rates (37.73% ± 1.54% vs. 24.31% ± 1.78%) were higher (p < 0.05) for riverine buffalo cells than that of swamp-like buffalo cells, whereas the total cell numbers of blastocysts (258.16 ± 36.25 vs. 198.16 ± 36.25, respectively) were similar. In conclusion, we demonstrated the feasibility of biobanking of buffalo somatic cells, and that the cryopreserved cells can be used to produce cloned embryos. This study encourages the development of somatic cell biobanks of domestic livestock, including endangered breeds of buffalo, to preserve valuable genotypes for future revitalization by animal cloning techniques.

Germline transmission in transgenic Huntington's disease monkeys.

PubMed

Moran, Sean; Chi, Tim; Prucha, Melinda S; Ahn, Kwang Sung; Connor-Stroud, Fawn; Jean, Sherrie; Gould, Kenneth; Chan, Anthony W S

2015-07-15

Transgenic nonhuman primate models are an increasingly popular model for neurologic and neurodegenerative disease because their brain functions and neural anatomies closely resemble those of humans. Transgenic Huntington's disease monkeys (HD monkeys) developed clinical features similar to those seen in HD patients, making the monkeys suitable for a preclinical study of HD. However, until HD monkey colonies can be readily expanded, their use in preclinical studies will be limited. In the present study, we confirmed germline transmission of the mutant huntingtin (mHTT) transgene in both embryonic stem cells generated from three male HD monkey founders (F0) and in second-generation offspring (F1) produced via artificial insemination by using intrauterine insemination technique. A total of five offspring were produced from 15 females that were inseminated by intrauterine insemination using semen collected from the three HD founders (5 of 15, 33%). Thus far, sperm collected from the HD founder (rHD8) has led to two F1 transgenic HD monkeys with germline transmission rate at 100% (2 of 2). mHTT expression was confirmed by quantitative real-time polymerase chain reaction using skin fibroblasts from the F1 HD monkeys and induced pluripotent stem cells established from one of the F1 HD monkeys (rHD8-2). Here, we report the stable germline transmission and expression of the mHTT transgene in HD monkeys, which suggest possible expansion of HD monkey colonies for preclinical and biomedical research studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Prevalence of antibody to adult T-cell leukemia virus-associated antigens (ATLA) in Japanese monkeys and other non-human primates.

PubMed

Hayami, M; Komuro, A; Nozawa, K; Shotake, T; Ishikawa, K; Yamamoto, K; Ishida, T; Honjo, S; Hinuma, Y

1984-02-15

The prevalence of adult T-cell-leukemia virus (ATLV) infection was examined in Japanese monkeys living naturally in various parts of Japan and in other species of non-human primates imported into and kept in Japan. Sera of 2,650 Japanese monkeys from 41 troops throughout Japan were tested. High incidences of anti-ATLV-associated antigen (ATLA)-positive monkeys were found in most troops, not only in the endemic area of human ATL(Southwestern Japan), but also in non-endemic areas. The incidence of sero-positive individuals increased gradually with age, reaching a maximum when the animals became adult, indicating age dependency, like that found by epidemiological studies on humans. Anti-ATLA antibodies were also detected in 90 of 815 sera of imported non-human primates of 33 species other than Japanese monkeys. All the anti-ATLA sero-positive monkeys were Catarrhines (Old World monkeys), mainly macaques of Asian origin. Some sero-positive monkeys were also found among animals of African origin, but no antibody was detected in Prosimians and Platyrrhines (New World monkeys). The clear-cut difference between the geographical distribution of sero-positive simians and that of humans indicates the improbability of direct transmission of ATLV from simians to humans.

Factors affecting the development of somatic cell nuclear transfer embryos in Cattle.

PubMed

Akagi, Satoshi; Matsukawa, Kazutsugu; Takahashi, Seiya

2014-01-01

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.

Characterization of glial-restricted precursors from rhesus monkey embryonic stem cells.

PubMed

Chen, Hongwei; Mao, Yu; Wang, Shufen; Li, Bin; Wang, Jinhuan; Li, Jian; Ma, Yuanye

2015-01-01

Glial-restricted precursor (GRP) cells, the earliest glial progenitors for both astrocytes and oligodendrocytes, have been derived from embryos and embryonic stem cells (ESC) in rodents. However, knowledge regarding the equivalent cell type in primates is limited due to restrictions imposed by ethics and resources. Here we report successful derivation and characterization of primate GRP cells from rhesus monkey ESC. The purified monkey GRP cells were A 2 B 5 -positive and FGF2-dependent for survival and proliferation. The differentiation assays indicated that they were tri-potential in vitro and bi-potential in vivo . These newly purified GRP cells will help to facilitate understanding of the molecular mechanism of glial development in primates as well as provide a source of therapeutic donor cells for use in neuroregenerative medicine.

Abnormalities in human pluripotent cells due to reprogramming mechanisms

PubMed Central

Ma, Hong; Morey, Robert; O’Neil, Ryan C.; He, Yupeng; Daughtry, Brittany; Schultz, Matthew D.; Hariharan, Manoj; Nery, Joseph R.; Castanon, Rosa; Sabatini, Karen; Thiagarajan, Rathi D.; Tachibana, Masahito; Kang, Eunju; Tippner-Hedges, Rebecca; Ahmed, Riffat; Gutierrez, Nuria Marti; Van Dyken, Crystal; Polat, Alim; Sugawara, Atsushi; Sparman, Michelle; Gokhale, Sumita; Amato, Paula; Wolf, Don P.; Ecker, Joseph R.; Laurent, Louise C.; Mitalipov, Shoukhrat

2016-01-01

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies. PMID:25008523

Somatic cell counts in bulk milk and their importance for milk processing

NASA Astrophysics Data System (ADS)

Savić, N. R.; Mikulec, D. P.; Radovanović, R. S.

2017-09-01

Bulk tank milk somatic cell counts are the indicator of the mammary gland health in the dairy herds and may be regarded as an indirect measure of milk quality. Elevated somatic cell counts are correlated with changes in milk composition The aim of this study was to assess the somatic cell counts that significantly affect the quality of milk and dairy products. We examined the somatic cell counts in bulk tank milk samples from 38 farms during the period of 6 months, from December to the May of the next year. The flow cytometry, Fossomatic was used for determination of somatic cell counts. In the same samples content of total proteins and lactose was determined by Milcoscan. Our results showed that average values for bulk tank milk samples were 273,605/ml from morning milking and 292,895/ml from evening milking. The average values for total proteins content from morning and evening milking are 3,31 and 3,34%, respectively. The average values for lactose content from morning and evening milking are 4,56 and 4,63%, respectively. The highest somatic cell count (516,000/ml) was detected in bulk tank milk sample from evening milk in the Winter and the lowest content of lactose was 4,46%. Our results showed that obtained values for bulk tank milk somatic cell counts did not significantly affected the content of total proteins and lactose.

Cell lineage analysis in human brain using endogenous retroelements

PubMed Central

Evrony, Gilad D.; Lee, Eunjung; Mehta, Bhaven K.; Benjamini, Yuval; Johnson, Robert M.; Cai, Xuyu; Yang, Lixing; Haseley, Psalm; Lehmann, Hillel S.; Park, Peter J.; Walsh, Christopher A.

2015-01-01

Summary Somatic mutations occur during brain development and are increasingly implicated as a cause of neurogenetic disease. However, the patterns in which somatic mutations distribute in the human brain are unknown. We used high-coverage whole-genome sequencing of single neurons from a normal individual to identify spontaneous somatic mutations as clonal marks to track cell lineages in human brain. Somatic mutation analyses in >30 locations throughout the nervous system identified multiple lineages and sub-lineages of cells marked by different LINE-1 (L1) retrotransposition events and subsequent mutation of poly-A microsatellites within L1. One clone contained thousands of cells limited to the left middle frontal gyrus, whereas a second distinct clone contained millions of cells distributed over the entire left hemisphere. These patterns mirror known somatic mutation disorders of brain development, and suggest that focally distributed mutations are also prevalent in normal brains. Single-cell analysis of somatic mutation enables tracing of cell lineage clones in human brain. PMID:25569347

The large Maf factor Traffic Jam controls gonad morphogenesis in Drosophila.

PubMed

Li, Michelle A; Alls, Jeffrey D; Avancini, Rita M; Koo, Karen; Godt, Dorothea

2003-11-01

Interactions between somatic and germline cells are critical for the normal development of egg and sperm. Here we show that the gene traffic jam (tj) produces a soma-specific factor that controls gonad morphogenesis and is required for female and male fertility. tj encodes the only large Maf factor in Drosophila melanogaster, an orthologue of the atypical basic Leu zipper transcription factors c-Maf and MafB/Kreisler in vertebrates. Expression of tj occurs in somatic gonadal cells that are in direct contact with germline cells throughout development. In tj mutant gonads, somatic cells fail to inter-mingle and properly envelop germline cells, causing an early block in germ cell differentiation. In addition, tj mutant somatic cells show an increase in the level of expression for several adhesion molecules. We propose that tj is a critical modulator of the adhesive properties of somatic cells, facilitating germline-soma interactions that are essential for germ cell differentiation.

Cloning of pigs from somatic cells and its prospects.

PubMed

Onishi, Akira

2002-01-01

The technology of somatic cell cloning in pigs is valuable for agricultural and therapeutic purposes. This paper will focus on the current methods of cloning pigs, including our successful microinjection#10; of somatic cell nuclei and its application. #10;

Insights from Proteomic Studies into Plant Somatic Embryogenesis.

PubMed

Heringer, Angelo Schuabb; Santa-Catarina, Claudete; Silveira, Vanildo

2018-03-01

Somatic embryogenesis is a biotechnological approach mainly used for the clonal propagation of different plants worldwide. In somatic embryogenesis, embryos arise from somatic cells under appropriate culture conditions. This plasticity in plants is a demonstration of true cellular totipotency and is the best approach among the genetic transformation protocols used for plant regeneration. Despite the importance of somatic embryogenesis, knowledge regarding the control of the somatic embryogenesis process is limited. Therefore, the elucidation of both the biochemical and molecular processes is important for understanding the mechanisms by which a single somatic cell becomes a whole plant. Modern proteomic techniques rely on an alternative method for the identification and quantification of proteins with different abundances in embryogenic cell cultures or somatic embryos and enable the identification of specific proteins related to somatic embryogenesis development. This review focuses on somatic embryogenesis studies that use gel-free shotgun proteomic analyses to categorize proteins that could enhance our understanding of particular aspects of the somatic embryogenesis process and identify possible targets for future studies. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The protons of space and brain tumors II. Cellular and molecular considerations

NASA Astrophysics Data System (ADS)

Nagle, W. A.; Moss, A. J.; Dalrymple, G. V.; Cox, A. B.; Wigle, J. F.; Mitchell, J. C.

1989-05-01

An increased incidence of highly malignant gliomas, termed glioblastoma multiforme has been observed in Rhesus monkeys irradiated with 55 MeV protons, and in humans treated with therapeutic irradiation to the head. The results suggest a radiation etiology for these tumors. In this paper, we review briefly some characteristics of glioma tumors, and summarize the genetic changes associated with malignant gliomas in experimental animals and in humans. The genetic abnormalities include cytogenetic alterations, and changes in the structure and expression of specific oncogenes. We discuss the potential for these genetic changes to contribute to several putative mechanism leading to aberrant growth stimulation and, ultimately, to tumorigenesis. In addition, we review briefly some recent data concerning the molecular nature of radiation-induced somatic cell mutation and oncogene activation, and discuss the significance of these results for the radiation etiology of malignant gliomas. Finally, some implications of these results are discussed in relation to human radiation exposure in space.

A polymorphic DNA marker that represents a conserved expressed sequence in the region of the Huntington disease gene.

PubMed Central

Hayden, M R; Hewitt, J; Wasmuth, J J; Kastelein, J J; Langlois, S; Conneally, M; Haines, J; Smith, B; Hilbert, C; Allard, D

1988-01-01

A polymorphic marker (D4S62) that is genetically closely linked to D4S10 and is in the region of the gene for Huntington disease is described. A four-allele polymorphism is detected when HincII-digested DNA is hybridized with D4S62. D4S62 maps, by Southern blot analysis using somatic-cell hybrids, to 4p16.1 closer to the centromere than does D4S10. The use of the polymorphisms detected by D4S62 increases the informativeness of markers close to the gene for Huntington disease and will be useful for preclinical diagnosis. D4S62 detects transcripts of approximately 6,000 nucleotides in rat, mouse, and monkey liver and brain. This represents the first demonstration of conserved expressed sequences close to the gene for Huntington disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 PMID:2892395

Attenuation of microglial RANTES by NEMO-binding domain peptide inhibits the infiltration of CD8(+) T cells in the nigra of hemiparkinsonian monkey.

PubMed

Roy, A; Mondal, S; Kordower, J H; Pahan, K

2015-08-27

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Despite intense investigations, little is known about its pathological mediators. Here, we report the marked upregulation of RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin, chemokines that are involved in T cell trafficking, in the serum of hemiparkinsonian monkeys. Interestingly, 1-methyl-4-phenylpyridinium (MPP(+)), a Parkinsonian toxin, increased the expression of RANTES and eotaxin in mouse microglial cells. The presence of NF-κB binding sites in promoters of RANTES and eotaxin and down-regulation of these genes by NEMO-binding domain (NBD) peptide, selective inhibitor of induced NF-κB activation, in MPP(+)-stimulated microglial cells suggest that the activation of NF-κB plays an important role in the upregulation of these two chemokines. Consistently, serum enzyme-linked immuno assay (ELISA) and nigral immunohistochemistry further confirmed that these chemokines were strongly upregulated in MPTP-induced hemiparkinsonian monkeys and that treatment with NBD peptides effectively inhibited the level of these chemokines. Furthermore, the microglial upregulation of RANTES in the nigra of hemiparkinsonian monkeys could be involved in the altered adaptive immune response in the brain as we observed greater infiltration of CD8(+) T cells around the perivascular niche and deep brain parenchyma of hemiparkinsonian monkeys as compared to control. The treatment of hemiparkinsonian monkeys with NBD peptides decreased the microglial expression of RANTES and attenuated the infiltration of CD8(+) T cells in nigra. These results indicate the possible involvement of chemokine-dependent adaptive immune response in Parkinsonism. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle

PubMed Central

AKAGI, Satoshi; MATSUKAWA, Kazutsugu; TAKAHASHI, Seiya

2014-01-01

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle. PMID:25341701

Tissue-specific expression of squirrel monkey chorionic gonadotropin

PubMed Central

Vasauskas, Audrey A.; Hubler, Tina R.; Boston, Lori; Scammell, Jonathan G.

2010-01-01

Pituitary gonadotropins LH and FSH play central roles in reproductive function. In Old World primates, LH stimulates ovulation in females and testosterone production in males. Recent studies have found that squirrel monkeys and other New World primates lack expression of LH in the pituitary. Instead, chorionic gonadotropin (CG), which is normally only expressed in the placenta of Old World primates, is the active luteotropic pituitary hormone in these animals. The goal of this study was to investigate the tissue-specific regulation of squirrel monkey CG. We isolated the squirrel monkey CGβ gene and promoter from genomic DNA from squirrel monkey B-lymphoblasts and compared the promoter sequence to that of the common marmoset, another New World primate, and human CGβ and LHβ. Using reporter gene assays, we found that a squirrel monkey CGβ promoter fragment (−1898/+9) is active in both mouse pituitary LβT2 and human placenta JEG3 cells, but not in rat adrenal PC12 cells. Furthermore, within this construct separate cis-elements are responsible for pituitary- and placenta-specific expression. Pituitary-specific expression is governed by Egr-1 binding sites in the proximal 250 bp of the promoter, whereas placenta-specific expression is controlled by AP-2 sites further upstream. Thus, selective expression of the squirrel monkey CGβ promoter in pituitary and placental cells is governed by distinct cis-elements that exhibit homology with human LHβ and marmoset CGβ promoters, respectively. PMID:21130091

Effect of space flight on cytokine production and other immunologic parameters of rhesus monkeys

NASA Technical Reports Server (NTRS)

Sonnenfeld, G.; Davis, S.; Taylor, G. R.; Mandel, A. D.; Konstantinova, I. V.; Lesnyak, A.; Fuchs, B. B.; Peres, C.; Tkackzuk, J.; Schmitt, D. A.

1996-01-01

During a recent flight of a Russian satellite (Cosmos #2229), initial experiments examining the effects of space flight on immunologic responses of rhesus monkeys were performed to gain insight into the effect of space flight on resistance to infection. Experiments were performed on tissue samples taken from the monkeys before and immediately after flight. Additional samples were obtained approximately 1 month after flight for a postflight restraint study. Two types of experiments were carried out throughout this study. The first experiment determined the ability of leukocytes to produce interleukin-1 and to express interleukin-2 receptors. The second experiment examined the responsiveness of rhesus bone marrow cells to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Human reagents that cross-reacted with monkey tissue were utilized for the bulk of the studies. Results from both studies indicated that there were changes in immunologic function attributable to space flight. Interleukin-1 production and the expression of interleukin-2 receptors was decreased after space flight. Bone marrow cells from flight monkeys showed a significant decrease in their response to GM-CSF compared with the response of bone marrow cells from nonflight control monkeys. These results suggest that the rhesus monkey may be a useful surrogate for humans in future studies that examine the effect of space flight on immune response, particularly when conditions do not readily permit human study.

Cancer treatment in childhood and testicular function: the importance of the somatic environment.

PubMed

Stukenborg, Jan-Bernd; Jahnukainen, Kirsi; Hutka, Marsida; Mitchell, Rod T

2018-02-01

Testicular function and future fertility may be affected by cancer treatment during childhood. Whilst survival of the germ (stem) cells is critical for ensuring the potential for fertility in these patients, the somatic cell populations also play a crucial role in providing a suitable environment to support germ cell maintenance and subsequent development. Regulation of the spermatogonial germ-stem cell niche involves many signalling pathways with hormonal influence from the hypothalamo-pituitary-gonadal axis. In this review, we describe the somatic cell populations that comprise the testicular germ-stem cell niche in humans and how they may be affected by cancer treatment during childhood. We also discuss the experimental models that may be utilized to manipulate the somatic environment and report the results of studies that investigate the potential role of somatic cells in the protection of the germ cells in the testis from cancer treatment. © 2018 The authors.

Cancer treatment in childhood and testicular function: the importance of the somatic environment

PubMed Central

Stukenborg, Jan-Bernd; Jahnukainen, Kirsi; Hutka, Marsida

2018-01-01

Testicular function and future fertility may be affected by cancer treatment during childhood. Whilst survival of the germ (stem) cells is critical for ensuring the potential for fertility in these patients, the somatic cell populations also play a crucial role in providing a suitable environment to support germ cell maintenance and subsequent development. Regulation of the spermatogonial germ-stem cell niche involves many signalling pathways with hormonal influence from the hypothalamo-pituitary-gonadal axis. In this review, we describe the somatic cell populations that comprise the testicular germ-stem cell niche in humans and how they may be affected by cancer treatment during childhood. We also discuss the experimental models that may be utilized to manipulate the somatic environment and report the results of studies that investigate the potential role of somatic cells in the protection of the germ cells in the testis from cancer treatment. PMID:29351905

Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae).

PubMed

Correia, Sandra M; Canhoto, Jorge M

2010-06-01

The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.

Age-related increase of sIAHP in prefrontal pyramidal cells of monkeys: relationship to cognition

PubMed Central

Luebke, Jennifer I.; Amatrudo, Joseph M.

2010-01-01

Reduced excitability, due to an increase in the slow afterhyperpolarization (and its underlying current sIAHP), occurs in CA1 pyramidal cells in aged cognitively-impaired, but not cognitively-unimpaired, rodents. We sought to determine whether similar age-related changes in the sIAHP occur in pyramidal cells in the rhesus monkey dorsolateral prefrontal cortex (dlPFC). Whole-cell patch-clamp recordings were obtained from layer 3 (L3) and layer 5 (L5) pyramidal cells in dlPFC slices prepared from young (9.6 ± 0.7 years old) and aged (22.3 ± 0.7 years old) behaviorally characterized subjects. The amplitude of the sIAHP was significantly greater in L3 (but not L5) cells from aged-impaired compared to both aged-unimpaired and young monkeys, which did not differ. Aged L3, but not L5, cells exhibited significantly increased action potential firing rates, but there was no relationship between sIAHP and firing rate. Thus, in monkey dlPFC L3 cells, an increase in sIAHP is associated with age-related cognitive decline; however, this increase is not associated with a reduction in excitability. PMID:20727620

Immunophenotypic characterization of the cutaneous exanthem of SIV-infected rhesus monkeys. Apposition of degenerative Langerhans cells and cytotoxic lymphocytes during the development of acquired immunodeficiency syndrome.

PubMed Central

Ringler, D. J.; Hancock, W. W.; King, N. W.; Letvin, N. L.; Daniel, M. D.; Desrosiers, R. C.; Murphy, G. F.

1987-01-01

A T-cell tropic retrovirus, simian immunodeficiency virus (SIV), has recently been isolated from immunodeficient rhesus monkeys. This virus has remarkable similarities to human immunodeficiency virus (HIV), the etiologic agent of acquired immunodeficiency syndrome. Subsequent studies of simian infection with SIV have shown it to be a relevant animal model for studying the pathogenesis of AIDS in man. In both HIV-infected humans and SIV-infected monkeys, a cutaneous maculopapular eruption has been described. To date, the pathogenesis and possible relationship of these exanthema to the evolution of systemic immunosuppression have remained obscure. In this study, the mononuclear cell infiltrates that characterize skin rashes of SIV-infected rhesus monkeys were found to be composed predominantly of cells with phenotypic characteristics of cytotoxic/suppressor (T8+) lymphocytes and natural killer cells. Many of these cells expressed membrane-bound interleukin-2 receptor molecules. Double labeling and immunoelectron microscopy revealed these cells in direct contact with degenerative Langerhans cells within the epidermis and dermis. These observations suggest that the cutaneous rash associated with SIV infection may be the consequence of target cell injury of Langerhans cells by effector cells with cytotoxic potential. Images Figure 1 Figure 2 Figure 3 PMID:3030113

Five classic articles in somatic cell reprogramming.

PubMed

Park, In-Hyun

2010-09-01

Research on somatic cell reprogramming has progressed significantly over the past few decades, from nuclear transfer into frogs' eggs in 1952 to the derivation of human-induced pluripotent stem (iPS) cells in the present day. In this article, I review five landmark papers that have laid the foundation for current efforts to apply somatic cell reprogramming in the clinic.

Sporadic Premature Aging in a Japanese Monkey: A Primate Model for Progeria

PubMed Central

Oishi, Takao; Imai, Hiroo; Go, Yasuhiro; Imamura, Masanori; Hirai, Hirohisa; Takada, Masahiko

2014-01-01

In our institute, we have recently found a child Japanese monkey who is characterized by deep wrinkles of the skin and cataract of bilateral eyes. Numbers of analyses were performed to identify symptoms representing different aspects of aging. In this monkey, the cell cycle of fibroblasts at early passage was significantly extended as compared to a normal control. Moreover, both the appearance of senescent cells and the deficiency in DNA repair were observed. Also, pathological examination showed that this monkey has poikiloderma with superficial telangiectasia, and biochemical assay confirmed that levels of HbA1c and urinary hyaluronan were higher than those of other (child, adult, and aged) monkey groups. Of particular interest was that our MRI analysis revealed expansion of the cerebral sulci and lateral ventricles probably due to shrinkage of the cerebral cortex and the hippocampus. In addition, the conduction velocity of a peripheral sensory but not motor nerve was lower than in adult and child monkeys, and as low as in aged monkeys. However, we could not detect any individual-unique mutations of known genes responsible for major progeroid syndromes. The present results indicate that the monkey suffers from a kind of progeria that is not necessarily typical to human progeroid syndromes. PMID:25365557

Chronic Nicotine Treatment Increases nAChRs and Microglial Expression in Monkey Substantia Nigra after Nigrostriatal Damage

PubMed Central

Campos, Carla; Parameswaran, Neeraja; William Langston, J.; Michael McIntosh, J.; Yeluashvili, Michael

2010-01-01

Our previous work had shown that long-term nicotine administration improved dopaminergic markers and nicotinic receptors (nAChRs) in the striatum of monkeys with nigrostriatal damage. The present experiments were done to determine whether nicotine treatment also led to changes in the substantia nigra, the region containing dopaminergic cell bodies. Monkeys were chronically treated with nicotine in the drinking water for 6 months after which they were injected with low dose MPTP for a further 6-month period. Nicotine was administered until the monkeys were euthanized 2 months after the last MPTP injection. Nicotine treatment did not affect the dopamine transporter or the number of tyrosine hydroxylase positive cells in the substantia nigra of lesioned monkeys. However, nicotine administration did lead to a greater increase in α3/α6β2* and α4β2* nAChRs in lesioned monkeys compared to controls. Nicotine also significantly elevated microglia and reduced the number of extracellular neuromelanin deposits in the substantia nigra of MPTP-lesioned monkeys. These findings indicate that long-term nicotine treatment modulates expression of several molecular measures in monkey substantia nigra that may result in an improvement in nigral integrity and/or function. These observations may have therapeutic implications for Parkinson’s disease. PMID:19685015

Molecular Mechanisms of Induced Pluripotency

PubMed Central

Muchkaeva, I.A.; Dashinimaev, E.B.; Terskikh, V.V.; Sukhanov, Y.V.; Vasiliev, A.V.

2012-01-01

In this review the distinct aspects of somatic cell reprogramming are discussed. The molecular mechanisms of generation of induced pluripotent stem (iPS) cells from somatic cells via the introduction of transcription factors into adult somatic cells are considered. Particular attention is focused on the generation of iPS cells without genome modifications via the introduction of the mRNA of transcription factors or the use of small molecules. Furthermore, the strategy of direct reprogramming of somatic cells omitting the generation of iPS cells is considered. The data concerning the differences between ES and iPS cells and the problem of epigenetic memory are also discussed. In conclusion, the possibility of using iPS cells in regenerative medicine is considered. PMID:22708059

Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

PubMed

Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

2011-05-01

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

Cell-Type-Specific Optogenetics in Monkeys.

PubMed

Namboodiri, Vijay Mohan K; Stuber, Garret D

2016-09-08

The recent advent of technologies enabling cell-type-specific recording and manipulation of neuronal activity spurred tremendous progress in neuroscience. However, they have been largely limited to mice, which lack the richness in behavior of primates. Stauffer et al. now present a generalizable method for achieving cell-type specificity in monkeys. Copyright © 2016 Elsevier Inc. All rights reserved.

Infection and persistence of rhesus monkey rhadinovirus in immortalized B-cell lines.

PubMed

Bilello, John P; Lang, Sabine M; Wang, Fred; Aster, Jon C; Desrosiers, Ronald C

2006-04-01

Similar to its close relative human herpesvirus 8, rhesus monkey rhadinovirus (RRV) persists predominantly in B cells of its natural host. Rhesus monkey B-cell lines immortalized by the Epstein-Barr-related virus from rhesus monkeys (rhEBV) were used as targets for infection by RRV. These cultured B cells were susceptible to infection by RRV and continued to produce low titers of RRV for months of continuous culture. Infection by RRV did not detectably alter the growth rates of these B-cell lines when it was measured at standard or reduced serum concentrations. Depending on the cell line, 5 to 40% of the B cells stained positive for the RRV genome by fluorescence in situ hybridization (FISH). Most RRV-positive cells showed a fine punctate nuclear staining pattern consistent with latent infection, while a small minority of cells (0.2 to 1%) contained large, intensely staining nuclear foci consistent with productive, replicative infection. Greater than 90% of the cells were rhEBV genome positive in a pattern consistent with latent infection, and again only a small minority of cells showed a productive, replicative staining pattern. Dual, two-color FISH staining revealed coinfection of numerous cells with both RRV and rhEBV, but productive replication of RRV and rhEBV was always observed in separate cells, never in the same cell. Thus, productive replication of RRV is unlinked to that of rhEBV; factors that influence activation to productive replication act separately on RRV and rhEBV, even within the same cell. The percentage of B cells expressing green fluorescent protein (GFP) early after infection with a recombinant RRV containing a GFP reporter gene was dose dependent and at a low multiplicity of infection increased progressively over time until 14 to 17 days after infection. These results establish a naturalistic cell culture system for the study of infection and persistence by RRV in rhesus monkey B cells.

Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

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

Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer

2005-07-01

The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requiresmore » permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.« less

Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing

PubMed Central

Li, Zhoufang; Liu, Guangjie; Tong, Yin; Zhang, Meng; Xu, Ying; Qin, Li; Wang, Zhanhui; Chen, Xiaoping; He, Jiankui

2015-01-01

Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys. PMID:25961410

L1-Associated Genomic Regions are Deleted in Somatic Cells of the Healthy Human Brain

PubMed Central

Erwin, Jennifer A.; Paquola, Apuã C.M.; Singer, Tatjana; Gallina, Iryna; Novotny, Mark; Quayle, Carolina; Bedrosian, Tracy; Ivanio, Francisco; Butcher, Cheyenne R.; Herdy, Joseph R.; Sarkar, Anindita; Lasken, Roger S.; Muotri, Alysson R.; Gage, Fred H.

2016-01-01

The healthy human brain is a mosaic of varied genomes. L1 retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that Somatic L1-Associated Variants (SLAVs) are actually composed of two classes: L1 retrotransposition insertions and retrotransposition-independent L1-associated variants. We demonstrate that a subset of SLAVs are, in fact, somatic deletions generated by L1 endonuclease cutting activity. Retrotransposition- independent rearrangements within inherited L1s resulted in the deletion of proximal genomic regions. These rearrangements were resolved by microhomology-mediated repair, which suggests that L1-associated genomic regions are hotspots for somatic copy number variants in the brain and therefore a heritable genetic contributor to somatic mosaicism. We demonstrate that SLAVs are present in crucial neural genes, such as DLG2/PSD93, and affect between 44–63% of cells of the cells in the healthy brain. PMID:27618310

Tissue-specific expression of squirrel monkey chorionic gonadotropin.

PubMed

Vasauskas, Audrey A; Hubler, Tina R; Boston, Lori; Scammell, Jonathan G

2011-02-01

Pituitary gonadotropins LH and FSH play central roles in reproductive function. In Old World primates, LH stimulates ovulation in females and testosterone production in males. Recent studies have found that squirrel monkeys and other New World primates lack expression of LH in the pituitary. Instead, chorionic gonadotropin (CG), which is normally only expressed in the placenta of Old World primates, is the active luteotropic pituitary hormone in these animals. The goal of this study was to investigate the tissue-specific regulation of squirrel monkey CG. We isolated the squirrel monkey CGβ gene and promoter from genomic DNA from squirrel monkey B-lymphoblasts and compared the promoter sequence to that of the common marmoset, another New World primate, and human and rhesus macaque CGβ and LHβ. Using reporter gene assays, we found that a squirrel monkey CGβ promoter fragment (-1898/+9) is active in both mouse pituitary LβT2 and human placenta JEG3 cells, but not in rat adrenal PC12 cells. Furthermore, within this construct separate cis-elements are responsible for pituitary- and placenta-specific expression. Pituitary-specific expression is governed by Egr-1 binding sites in the proximal 250 bp of the promoter, whereas placenta-specific expression is controlled by AP-2 sites further upstream. Thus, selective expression of the squirrel monkey CGβ promoter in pituitary and placental cells is governed by distinct cis-elements that exhibit homology with human LHβ and marmoset CGβ promoters, respectively. Copyright © 2010 Elsevier Inc. All rights reserved.

Fibroblast growth factor signaling is required for early somatic gonad development in zebrafish.

PubMed

Leerberg, Dena M; Sano, Kaori; Draper, Bruce W

2017-09-01

The vertebrate ovary and testis develop from a sexually indifferent gonad. During early development of the organism, primordial germ cells (the gamete lineage) and somatic gonad cells coalesce and begin to undergo growth and morphogenesis to form this bipotential gonad. Although this aspect of development is requisite for a fertile adult, little is known about the genetic regulation of early gonadogenesis in any vertebrate. Here, we provide evidence that fibroblast growth factor (Fgf) signaling is required for the early growth phase of a vertebrate bipotential gonad. Based on mutational analysis in zebrafish, we show that the Fgf ligand 24 (Fgf24) is required for proliferation, differentiation, and morphogenesis of the early somatic gonad, and as a result, most fgf24 mutants are sterile as adults. Additionally, we describe the ultrastructural elements of the early zebrafish gonad and show that distinct somatic cell populations can be identified soon after the gonad forms. Specifically, we show that fgf24 is expressed in an epithelial population of early somatic gonad cells that surrounds an inner population of mesenchymal somatic gonad cells that are in direct contact with the germ cells, and that fgf24 is required for stratification of the somatic tissue. Furthermore, based on gene expression analysis, we find that differentiation of the inner mesenchymal somatic gonad cells into functional cell types in the larval and early juvenile-stage gonad is dependent on Fgf24 signaling. Finally, we argue that the role of Fgf24 in zebrafish is functionally analogous to the role of tetrapod FGF9 in early gonad development.

Consequence of changing standards for somatic cell count on US Dairy Herd Improvement herds

USDA-ARS?s Scientific Manuscript database

Consequence of noncompliance with European Union (EU) and current US standards for somatic cell count (SCC) as well as SCC standards proposed by the National Milk Producers Federation was examined for US herds. Somatic cell scores (SCS) from 14,854 Dairy Herd Improvement (DHI) herds were analyzed. H...

Phenotypic and Functional Characterization of Peripheral Blood Lymphocytes from Various Age- and Sex-Specific Groups of Owl Monkeys (Aotus nancymaae).

PubMed

Nehete, Pramod N; Nehete, Bharti P; Chitta, Sriram; Williams, Lawrence E; Abee, Christian R

2017-02-01

Owl monkeys (Aotus nancymaae) are New World NHP that serve an important role in vaccine development and as a model for human disease conditions such as malaria. Despite the past contributions of this animal model, limited information is available about the phenotype and functional properties of peripheral blood lymphocytes in reference to sex and age. Using a panel of human antibodies and a set of standardized human immune assays, we identified and characterized various peripheral blood lymphocyte subsets, evaluated the immune functions of T cells, and analyzed cytokines relative to sex and age in healthy owl monkeys. We noted age- and sex-dependent changes in CD28+ (an essential T cell costimulatory molecule) and CD95+ (an apoptotic surface marker) T cells and various levels of cytokines in the plasma. In immune assays of freshly isolated peripheral blood mononuclear cells, IFNγ and perforin responses were significantly higher in female than in male monkeys and in young adults than in juvenile and geriatric groups, despite similar lymphocyte (particularly T cell) populations in these groups. Our current findings may be useful in exploring Aotus monkeys as a model system for the study of aging, susceptibility to infectious diseases, and age-associated differences in vaccine efficacy, and other challenges particular to pediatric and geriatric patients.

Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana.

PubMed

Portillo, L; Olmedilla, A; Santacruz-Ruvalcaba, F

2012-10-01

In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.

The contribution of mammary infections by coagulase-negative staphylococci to the herd bulk milk somatic cell count.

PubMed

Rainard, P; Ducelliez, M; Poutrel, B

1990-01-01

Quarter foremilk samples were taken at 2-3 weekly intervals for several years in an experimental herd comprising about 45 cows. The samples were submitted to bacteriological analysis and somatic cell counting. The most prevalent quarter infections from 1982 to 1988 were by coagulase-negative staphylococci (15-20% of all the quarters sampled). Most of these (75.6%) persisted until drying-off. Dry cow therapy eliminated 86.5% of these infections. Comparison of udder quarters within cows, involving 775 samples from pairs of non-infected quarters and quarters infected by coagulase-negative staphylococci, yielded geometric means of somatic cell counts of 210,000 and 420,000 cells/ml, respectively. The correlation (r = 0.87) between the herd bulk milk somatic cell count (SCC) and its estimation from the quarter milk somatic cell count performed on the same day allowed us to evaluate the contribution of the different categories of quarters, according to their infection status, to the herd bulk milk SCC. Quarters infected by a major pathogen (8.5% of samples) gave rise to 46.6% of the total number of cells, while quarters infected by coagulase-negative staphylococci (17.8% of samples) gave rise to 18.1%. Although coagulase-negative staphylococci represented only a secondary source of somatic cells as compared to major pathogens, they were not a negligible source considering the threshold of 300,000 somatic cells advocated for herd milk of good quality.

Problems and potentialities of cultured plant cells in retrospect and prospect

NASA Technical Reports Server (NTRS)

Steward, F. C.; Krikorian, A. D.

1979-01-01

The past, present and expected future accomplishments and limitations of plant cell and tissue culture are reviewed. Consideration is given to the pioneering insights of Haberlandt in 1902, the development of culture techniques, and past work on cell division, cell and tissue growth and development, somatic embryogenesis, and metabolism and respiration. Current activity in culture media and technique development for plant regions, organs, tissues, cells, protoplasts, organelles and embryos, totipotency, somatic embryogenesis and clonal propagation under normal and space conditions, biochemical potentialities, and genetic engineering is surveyed. Prospects for the investigation of the induced control of somatic cell division, the division of isolated protoplasts, the improvement of haploid cell cultures, liquid cultures for somatic embryogenesis, and the genetic control of development are outlined.

Chromatin remodeling in somatic cells injected into mature pig oocytes.

PubMed

Bui, Hong-Thuy; Van Thuan, Nguyen; Wakayama, Teruhiko; Miyano, Takashi

2006-06-01

We examined the involvement of histone H3 modifications in the chromosome condensation and decondensation of somatic cell nuclei injected into mature pig oocytes. Nuclei of pig granulosa cells were transferred into in vitro matured intact pig oocytes, and histone H3 phosphorylation, acetylation, and methylation were examined by immunostaining with specific antibodies in relation to changes in chromosome morphology. In the condensed chromosomes of pig oocytes at metaphase II, histone H3 was phosphorylated at serine 10 (H3-S10) and serine 28 (H3-S28), and methylated at lysine 9 (H3-K9), but was not acetylated at lysine 9, 14 and 18 (H3-K9, H3-K14 and H3-K18). During the first 2 h after nuclear transfer, a series of events were observed in the somatic nuclei: nuclear membrane disassembly; chromosome condensation to form a metaphase-like configuration; an increase in histone H3 phosphorylation levels (H3-S10 and H3-S28). Next, pig oocytes injected with nuclei of somatic cells were electroactivated and the chromosome morphology of oocytes and somatic cells was examined along with histone modifications. Generally, chromosomes of the somatic cells showed a similar progression of cell cycle stage to that of oocytes, through anaphase II- and telophase II-like stages then formed pronucleus-like structures, although the morphology of the spindles differed from that of oocyte spindles. The chromosomes of somatic cells also showed changes in histone H3 dephosphorylation and reacetylation, similar to oocytes. In contrast, histone H3 methylation (H3-K9) of somatic cell nuclei did not show any significant change after injection and electroactivation of the oocytes. These results suggest that nuclear remodeling including histone H3 phosphorylation and acetylation of injected somatic nuclei took place in the oocytes under regulation by the oocyte cytoplasm.

Retinoic acid signaling is dispensable for somatic development and function in the mammalian ovary.

PubMed

Minkina, Anna; Lindeman, Robin E; Gearhart, Micah D; Chassot, Anne-Amandine; Chaboissier, Marie-Christine; Ghyselinck, Norbert B; Bardwell, Vivian J; Zarkower, David

2017-04-15

Retinoic acid (RA) is a potent inducer of cell differentiation and plays an essential role in sex-specific germ cell development in the mammalian gonad. RA is essential for male gametogenesis and hence fertility. However, RA can also disrupt sexual cell fate in somatic cells of the testis, promoting transdifferentiation of male Sertoli cells to female granulosa-like cells when the male sexual regulator Dmrt1 is absent. The feminizing ability of RA in the Dmrt1 mutant somatic testis suggests that RA might normally play a role in somatic cell differentiation or cell fate maintenance in the ovary. To test for this possibility we disrupted RA signaling in somatic cells of the early fetal ovary using three genetic strategies and one pharmaceutical approach. We found that deleting all three RA receptors (RARs) in the XX somatic gonad at the time of sex determination did not significantly affect ovarian differentiation, follicle development, or female fertility. Transcriptome analysis of adult triple mutant ovaries revealed remarkably little effect on gene expression in the absence of somatic RAR function. Likewise, deletion of three RA synthesis enzymes (Aldh1a1-3) at the time of sex determination did not masculinize the ovary. A dominant-negative RAR transgene altered granulosa cell proliferation, likely due to interference with a non-RA signaling pathway, but did not prevent granulosa cell specification and oogenesis or abolish fertility. Finally, culture of fetal XX gonads with an RAR antagonist blocked germ cell meiotic initiation but did not disrupt sex-biased gene expression. We conclude that RA signaling, although crucial in the ovary for meiotic initiation, is not required for granulosa cell specification, differentiation, or reproductive function. Copyright © 2017 Elsevier Inc. All rights reserved.

[Direct and indirect somatic embryogenesis in Freesia refracta].

PubMed

Wang, L; Duan, X G; Hao, S

1999-06-01

Somatic embryogenesis can be induced in tissue cultures of Freesia refracta either directly from the epidermal cells of explant, or indirectly via intervening callus. In direct pathway, somatic embryos were in contact with maternal tissue in a suspensor-like structure. In indirect pathway, the explants first proliferacted to give rise to calluses before embryoids were induced. The two sorts of calluses were defined to embryogenic callus and non-embryogenic callus according to producing of somatic embryos. An indirect somatic embryo is developed from a pre-embryogenically determined cell. This kind of somatic embryo has no suspensor structure instead of a complex with maternal tissue. Somatic embryos have their own vascular tissues, and can develop new plantlets independently.

Innexin2 gap junctions in somatic support cells are required for cyst formation and for egg chamber formation in Drosophila.

PubMed

Mukai, Masanori; Kato, Hirotaka; Hira, Seiji; Nakamura, Katsuhiro; Kita, Hiroaki; Kobayashi, Satoru

2011-01-01

Germ cells require intimate associations with surrounding somatic cells during gametogenesis. During oogenesis, gap junctions mediate communication between germ cells and somatic support cells. However, the molecular mechanisms by which gap junctions regulate the developmental processes during oogenesis are poorly understood. We have identified a female sterile allele of innexin2 (inx2), which encodes a gap junction protein in Drosophila. In females bearing this inx2 allele, cyst formation and egg chamber formation are impaired. In wild-type germaria, Inx2 is strongly expressed in escort cells and follicle cells, both of which make close contact with germline cells. We show that inx2 function in germarial somatic cells is required for the survival of early germ cells and promotes cyst formation, probably downstream of EGFR pathway, and that inx2 function in follicle cells promotes egg chamber formation through the regulation of DE-cadherin and Bazooka (Baz) at the boundary between germ cells and follicle cells. Furthermore, genetic experiments demonstrate that inx2 interacts with the zero population growth (zpg) gene, which encodes a germline-specific gap junction protein. These results indicate a multifunctional role for Inx2 gap junctions in somatic support cells in the regulation of early germ cell survival, cyst formation and egg chamber formation. Inx2 gap junctions may mediate the transfer of nutrients and signal molecules between germ cells and somatic support cells, as well as play a role in the regulation of cell adhesion. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Lentivirus-mediated RNA interference of vascular endothelial growth factor in monkey eyes with iris neovascularization.

PubMed

Yuan, Meng-Ke; Tao, Yong; Yu, Wen-Zhen; Kai, Wang; Jiang, Yan-Rong

2010-08-25

To explore the in vivo anti-angiogenesis effects resulting from lentivirus-mediated RNAi of vascular endothelial growth factor (VEGF) in monkeys with iris neovascularization (INV). Five specific recombinant lentiviral vectors for RNA interference, targeting Macaca mulatta VEGFA, were designed and the one with best knock down efficacy (LV-GFP-VEGFi1) in H1299 cells and RF/6A cells was selected by real-time PCR for in vivo use. A laser-induced retinal vein occlusion model was established in one eye of seven cynomolgus monkeys. In monkeys number 1, 3, and 5 (Group 1), the virus (1x10(8) particles) was intravitreally injected into the preretinal space of the animal's eye immediately after laser coagulation; and in monkeys number 2, 4, and 6 (Group 2), the virus (1x10(8) particles) was injected at 10 days after laser coagulation. In monkey number 7, a blank control injection was performed. In monkeys number 1 and 2, virus without RNAi sequence was used; in monkeys number 3 and 4, virus with nonspecific RNAi sequence was used; and in monkeys 5 and 6, LV-GFP-VEGFi1 was used. In monkey number 5, at 23 days after laser treatment, no obvious INV was observed, while fluorescein angiography of the iris revealed high fluorescence at the margin of pupil and point posterior synechiae. At 50 days after laser treatment, only a slight ectropion uvea was found. However, in the other eyes, obvious INV or hyphema was observed. The densities of new iridic vessels all significantly varied: between monkey number 5 and number 3 (36.01+/-4.49/mm(2) versus 48.68+/-9.30/mm(2), p=0.025), between monkey number 3 and monkey number 7 (48.68+/-9.30/mm(2) versus 74.38+/-9.23/mm(2), p=0.002), and between monkey number 5 and number 7 (36.01+/-4.49/mm(2) versus 74.38+/-9.23/mm(2), p<0.001). Lentivirus-mediated RNAi of VEGF may be a new strategy to treat iris neovascularization, while further studies are needed to investigate the long-term effect.

BMP signalling in human fetal ovary somatic cells is modulated in a gene-specific fashion by GREM1 and GREM2

PubMed Central

Bayne, Rosemary A.; Donnachie, Douglas J.; Kinnell, Hazel L.; Childs, Andrew J.; Anderson, Richard A.

2016-01-01

STUDY QUESTION Do changes in the expression of bone morphogenetic proteins (BMPs) 2 and 4, and their antagonists Gremlin1 (GREM1) and Gremlin2 (GREM2) during human fetal ovarian development impact on BMP pathway activity and lead to changes in gene expression that may influence the fate and/or function of ovarian somatic cells? STUDY FINDING BMPs 2 and 4 differentially regulate gene expression in cultured human fetal ovarian somatic cells. Expression of some, but not all BMP target genes is antagonised by GREM1 and GREM2, indicating the existence of a mechanism to fine-tune BMP signal intensity in the ovary. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker of immature ovarian somatic cells, is identified as a novel transcriptional target of BMP4. WHAT IS KNOWN ALREADY Extensive re-organisation of the germ and somatic cell populations in the feto-neonatal ovary culminates in the formation of primordial follicles, which provide the basis for a female's future fertility. BMP growth factors play important roles at many stages of ovarian development and function. GREM1, an extracellular antagonist of BMP signalling, regulates the timing of primordial follicle formation in the mouse ovary, and mRNA levels of BMP4 decrease while those of BMP2 increase prior to follicle formation in the human fetal ovary. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Expression of genes encoding BMP pathway components, BMP antagonists and markers of ovarian somatic cells were determined by quantitative (q)RT-PCR in human fetal ovaries (from 8 to 21 weeks gestation) and fetal ovary-derived somatic cell cultures. Ovarian expression of GREM1 protein was confirmed by immunoblotting. Primary human fetal ovarian somatic cell cultures were derived from disaggregated ovaries by differential adhesion and cultured in the presence of recombinant human BMP2 or BMP4, with or without the addition of GREM1 or GREM2. MAIN RESULTS AND THE ROLE OF CHANCE We demonstrate that the expression of BMP antagonists GREM1, GREM2 and CHRD  increases in the lead-up to primordial follicle formation in the human fetal ovary, and that the BMP pathway is active in cultured ovarian somatic cells. This leads to differential changes in the expression of a number of genes, some of which are further modulated by GREM1 and/or GREM2. The positive transcriptional regulation of LGR5 (a marker of less differentiated somatic cells) by BMP4 in vitro suggests that increasing levels of GREM1 and reduced levels of BMP4 as the ovary develops in vivo may act to reduce LGR5 levels and allow pre-granulosa cell differentiation. LIMITATIONS, REASONS FOR CAUTION While we have demonstrated that markers of different somatic cell types are expressed in the cultured ovarian somatic cells, their proportions may not represent the same cells in the intact ovary which also contains germ cells. WIDER IMPLICATIONS OF THE FINDINGS This study extends previous work identifying germ cells as targets of ovarian BMP signalling, and suggests BMPs may regulate the development of both germ and somatic cells in the developing ovary around the time of follicle formation. LARGE SCALE DATA Not applicable. STUDY FUNDING/COMPETING INTERESTS This work was supported by The UK Medical Research Council (Grant No.: G1100357 to RAA), and Medical Research Scotland (Grant No. 345FRG to AJC). The authors have no competing interests to declare. PMID:27385727

BMP signalling in human fetal ovary somatic cells is modulated in a gene-specific fashion by GREM1 and GREM2.

PubMed

Bayne, Rosemary A; Donnachie, Douglas J; Kinnell, Hazel L; Childs, Andrew J; Anderson, Richard A

2016-09-01

Do changes in the expression of bone morphogenetic proteins (BMPs) 2 and 4, and their antagonists Gremlin1 (GREM1) and Gremlin2 (GREM2) during human fetal ovarian development impact on BMP pathway activity and lead to changes in gene expression that may influence the fate and/or function of ovarian somatic cells? BMPs 2 and 4 differentially regulate gene expression in cultured human fetal ovarian somatic cells. Expression of some, but not all BMP target genes is antagonised by GREM1 and GREM2, indicating the existence of a mechanism to fine-tune BMP signal intensity in the ovary. Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker of immature ovarian somatic cells, is identified as a novel transcriptional target of BMP4. Extensive re-organisation of the germ and somatic cell populations in the feto-neonatal ovary culminates in the formation of primordial follicles, which provide the basis for a female's future fertility. BMP growth factors play important roles at many stages of ovarian development and function. GREM1, an extracellular antagonist of BMP signalling, regulates the timing of primordial follicle formation in the mouse ovary, and mRNA levels of BMP4 decrease while those of BMP2 increase prior to follicle formation in the human fetal ovary. Expression of genes encoding BMP pathway components, BMP antagonists and markers of ovarian somatic cells were determined by quantitative (q)RT-PCR in human fetal ovaries (from 8 to 21 weeks gestation) and fetal ovary-derived somatic cell cultures. Ovarian expression of GREM1 protein was confirmed by immunoblotting. Primary human fetal ovarian somatic cell cultures were derived from disaggregated ovaries by differential adhesion and cultured in the presence of recombinant human BMP2 or BMP4, with or without the addition of GREM1 or GREM2. We demonstrate that the expression of BMP antagonists GREM1, GREM2 and CHRD  increases in the lead-up to primordial follicle formation in the human fetal ovary, and that the BMP pathway is active in cultured ovarian somatic cells. This leads to differential changes in the expression of a number of genes, some of which are further modulated by GREM1 and/or GREM2. The positive transcriptional regulation of LGR5 (a marker of less differentiated somatic cells) by BMP4 in vitro suggests that increasing levels of GREM1 and reduced levels of BMP4 as the ovary develops in vivo may act to reduce LGR5 levels and allow pre-granulosa cell differentiation. While we have demonstrated that markers of different somatic cell types are expressed in the cultured ovarian somatic cells, their proportions may not represent the same cells in the intact ovary which also contains germ cells. This study extends previous work identifying germ cells as targets of ovarian BMP signalling, and suggests BMPs may regulate the development of both germ and somatic cells in the developing ovary around the time of follicle formation. Not applicable. This work was supported by The UK Medical Research Council (Grant No.: G1100357 to RAA), and Medical Research Scotland (Grant No. 345FRG to AJC). The authors have no competing interests to declare. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Multicellularity makes somatic differentiation evolutionarily stable

PubMed Central

Wahl, Mary E.; Murray, Andrew W.

2016-01-01

Many multicellular organisms produce two cell lineages: germ cells, whose descendants produce the next generation, and somatic cells, which support, protect, and disperse the germ cells. This germ-soma demarcation has evolved independently in dozens of multicellular taxa but is absent in unicellular species. A common explanation holds that in these organisms, inefficient intercellular nutrient exchange compels the fitness cost of producing nonreproductive somatic cells to outweigh any potential benefits. We propose instead that the absence of unicellular, soma-producing populations reflects their susceptibility to invasion by nondifferentiating mutants that ultimately eradicate the soma-producing lineage. We argue that multicellularity can prevent the victory of such mutants by giving germ cells preferential access to the benefits conferred by somatic cells. The absence of natural unicellular, soma-producing species previously prevented these hypotheses from being directly tested in vivo: to overcome this obstacle, we engineered strains of the budding yeast Saccharomyces cerevisiae that differ only in the presence or absence of multicellularity and somatic differentiation, permitting direct comparisons between organisms with different lifestyles. Our strains implement the essential features of irreversible conversion from germ line to soma, reproductive division of labor, and clonal multicellularity while maintaining sufficient generality to permit broad extension of our conclusions. Our somatic cells can provide fitness benefits that exceed the reproductive costs of their production, even in unicellular strains. We find that nondifferentiating mutants overtake unicellular populations but are outcompeted by multicellular, soma-producing strains, suggesting that multicellularity confers evolutionary stability to somatic differentiation. PMID:27402737

Nuclear microscopic investigations into the elemental changes in the substantia nigra of unilaterally MPTP-lesioned Parkinsonian monkeys

NASA Astrophysics Data System (ADS)

Thong, P. S. P.; He, Y.; Lee, T.; Watt, F.

1997-07-01

Various transition metals, particularly iron, have been implicated in the aetiology of the neurodegenerative disease, Parkinson's disease, in which there is a characteristic loss of cells in the substantia nigra (SN) region of the brain. In this study, monkeys were unilaterally lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) to obtain primate models of parkinsonism, with the non-lesioned side of the brain serving as controls. The monkeys were sacrificed at one day, one week, two weeks, one month and one year after lesioning to investigate the time dependent elemental changes in the parkinsonian SN. Sections of the brain encompassing both the lesioned and non-lesioned SNs were analysed using the National University of Singapore nuclear microscope. Adjacent sections were tyrosine hydroxylase (TH) immunohistochemically stained to provide complementary information on dopaminergic cell loss and to facilitate definition of the SN boundaries during data analysis. In one-day and one-week monkeys (representing early stages of the disease), there were no changes in elemental concentrations within experimental errors and the adjacent TH-stained sections did not show apparent cell loss in the SN. At two weeks, cell loss was seen in the lesioned SN compared to the control SN. Although there was no bulk increase in SN iron, localised accumulation of iron in granules containing up to 15% by weight iron was observed in the lesioned SN of one of the two-week monkeys. An average 15% increase in nigral iron, significant at the 90% confidence level ( p < 0.1), was seen in the one-month monkeys. TH-stained sections for the one-month monkeys showed cell loss in the lesioned SN. In one-year samples (representing the advanced stage of the disease) there was a significant ( p < 0.05) 56% increase in iron, 14% increase in phosphorous and a 20% decrease in copper. Here an almost complete loss of cells in the lesioned SN was apparent from the adjacent TH-stained sections. These preliminary results suggest that while bulk increase in iron may seem to follow cell death, localised accumulation of SN iron in the early stages of the disease may play an important role in initiating and/or accelerating nigral cell death.

Germ cell regeneration-mediated, enhanced mutagenesis in the ascidian Ciona intestinalis reveals flexible germ cell formation from different somatic cells.

PubMed

Yoshida, Keita; Hozumi, Akiko; Treen, Nicholas; Sakuma, Tetsushi; Yamamoto, Takashi; Shirae-Kurabayashi, Maki; Sasakura, Yasunori

2017-03-15

The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona. Copyright © 2017 Elsevier Inc. All rights reserved.

High stability of nuclear microsatellite loci during the early stages of somatic embryogenesis in Norway spruce.

PubMed

Helmersson, Andreas; von Arnold, Sara; Burg, Kornel; Bozhkov, Peter V

2004-10-01

Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death. In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes. We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis. Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid. We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines. There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin. The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants. We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.

Real-time bioluminescence imaging of macroencapsulated fibroblasts reveals allograft protection in rhesus monkeys (Macaca mulatta).

PubMed

Tarantal, Alice F; Lee, C Chang I; Itkin-Ansari, Pamela

2009-07-15

Encapsulation of cells has the potential to eliminate the need for immunosuppression for cellular transplantation. Recently, the TheraCyte device was shown to provide long-term immunoprotection of murine islets in a mouse model of diabetes. In this report, translational studies were undertaken using skin fibroblasts from an unrelated rhesus monkey donor that were transduced with an HIV-1-derived lentiviral vector expressing firefly luciferase permitting the use of bioluminescence imaging (BLI) to monitor cell survival over time and in a noninvasive manner. Encapsulated cells were transplanted subcutaneously (n=2), or cells were injected without encapsulation (n=1) and outcomes compared. BLI was performed to monitor cell survival. The BLI signal from the encapsulated cells remained robust postinsertion and in one animal persisted for up to 1 year. In contrast, the control animal that received unencapsulated cells exhibited a complete loss of cell signal within 14 days. These data demonstrate that TheraCyte encapsulation of allogeneic cells provides robust immune protection in transplanted rhesus monkeys.

Impact of Somatic Mutations in the D-Loop of Mitochondrial DNA on the Survival of Oral Squamous Cell Carcinoma Patients

PubMed Central

Lin, Jin-Ching; Wang, Chen-Chi; Jiang, Rong-San; Wang, Wen-Yi; Liu, Shih-An

2015-01-01

Objectives The aim of this study was to investigate somatic mutations in the D-loop of mitochondrial DNA (mtDNA) and their impact on survival in oral squamous cell carcinoma patients. Materials and Methods Surgical specimen confirmed by pathological examination and corresponding non-cancerous tissues were collected from 120 oral squamous cell carcinoma patients. The sequence in the D-loop of mtDNA from non-cancerous tissues was compared with that from paired cancer samples and any sequence differences were recognized as somatic mutations. Results Somatic mutations in the D-loop of mtDNA were identified in 75 (62.5%) oral squamous cell carcinoma patients and most of them occurred in the poly-C tract. Although there were no significant differences in demographic and tumor-related features between participants with and without somatic mutation, the mutation group had a better survival rate (5 year disease-specific survival rate: 64.0% vs. 43.0%, P = 0.0266). Conclusion Somatic mutation in D-loop of mtDNA was associated with a better survival in oral squamous cell carcinoma patients. PMID:25906372

L1-associated genomic regions are deleted in somatic cells of the healthy human brain.

PubMed

Erwin, Jennifer A; Paquola, Apuã C M; Singer, Tatjana; Gallina, Iryna; Novotny, Mark; Quayle, Carolina; Bedrosian, Tracy A; Alves, Francisco I A; Butcher, Cheyenne R; Herdy, Joseph R; Sarkar, Anindita; Lasken, Roger S; Muotri, Alysson R; Gage, Fred H

2016-12-01

The healthy human brain is a mosaic of varied genomes. Long interspersed element-1 (LINE-1 or L1) retrotransposition is known to create mosaicism by inserting L1 sequences into new locations of somatic cell genomes. Using a machine learning-based, single-cell sequencing approach, we discovered that somatic L1-associated variants (SLAVs) are composed of two classes: L1 retrotransposition insertions and retrotransposition-independent L1-associated variants. We demonstrate that a subset of SLAVs comprises somatic deletions generated by L1 endonuclease cutting activity. Retrotransposition-independent rearrangements in inherited L1s resulted in the deletion of proximal genomic regions. These rearrangements were resolved by microhomology-mediated repair, which suggests that L1-associated genomic regions are hotspots for somatic copy number variants in the brain and therefore a heritable genetic contributor to somatic mosaicism. We demonstrate that SLAVs are present in crucial neural genes, such as DLG2 (also called PSD93), and affect 44-63% of cells of the cells in the healthy brain.

Simian virus 40 inhibits differentiation and maturation of rhesus macaque DC-SIGN(+) dendritic cells.

PubMed

Changyong, C; Sun, M; Li, H; Brockmeyer, N; Wu, Nan Ping

2010-09-24

Dendritic cells (DC) are the initiators and modulators of the immune responses. Some species of pathogenic microorganisms have developed immune evasion strategies by controlling antigen presentation function of DC. Simian virus 40 (SV40) is a DNA tumor virus of rhesus monkey origin. It can induce cell transformation and tumorigenesis in many vertebrate species, but often causes no visible effects and persists as a latent infection in rhesus monkeys under natural conditions. To investigate the interaction between SV40 and rhesus monkey DC, rhesus monkey peripheral blood monocyte-derived DC were induced using recombinant human Interleukin-4 (rhIL-4) and infective SV40, the phenotype and function of DC-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN)(+) DC were analyzed by flow cytometry (FCM) and mixed lymphocyte reaction (MLR). Results showed that SV40 can down-regulate the expression of CD83 and CD86 on DC and impair DC-induced activation of T cell proliferation. These findings suggest that SV40 might also cause immune suppression by influencing differentiation and maturation of DC.

Establishment and Characterization of a Testicular Cell Line from the Half-Smooth Tongue Sole, Cynoglossus semilaevis

PubMed Central

Zhang, Bo; Wang, Xianli; Sha, Zhenxia; Yang, Changgeng; Liu, Shanshan; Wang, Na; Chen, Song-Lin

2011-01-01

Spermatogenesis within the adult testis is an excellent system for studying stem cell renewal and differentiation, which is under the control of testicular somatic cells. In order to understanding spermatogenesis in the half-smooth tongue sole (Cynoglossus semilaevis) as a marine fish model of aquaculture importance, we established a cell line called CSGC from a juvenile gonad of this organism. CSGC is composed of fibroblast-like cells, retains a diploid karyotype of 42 chromosomes, lacks the heterogametic W chromosome, lacks a female specific marker and expresses the dmrt, a marker for testicular somatic cells. Therefore, CSGC appears to consist of testicular somatic cell cells. We show that this cell line is effective for infection by the turbot reddish body iridovirus and flounder lymphocystis disease virus as evidenced by the appearance of cytopathic effect and virus propagation in the virus-infected cells, and most convincingly, the observation of viral particles by electon microscopy, demonstrateing that CSGC is suitable to study interactions between virus and host cells. As a first fish testicular somatic cell line of the ZZ-ZW genetic sex determination system, CSGC will be a useful tool to study sex-related events and interactions between somatic cells and germ cells during spermatogenesis. PMID:21547062

A B cell follicle sanctuary permits persistent productive SIV infection in elite controllers

PubMed Central

Fukazawa, Yoshinori; Lum, Richard; Okoye, Afam A.; Park, Haesun; Matsuda, Kenta; Bae, Jin Young; Hagen, Shoko I.; Shoemaker, Rebecca; Deleage, Claire; Lucero, Carissa; Morcock, David; Swanson, Tonya; Legasse, Alfred W.; Axthelm, Michael K.; Hesselgesser, Joseph; Geleziunas, Romas; Hirsch, Vanessa M.; Edlefsen, Paul T.; Piatak, Michael; Estes, Jacob D.; Lifson, Jeffrey D.; Picker, Louis J.

2014-01-01

Chronic phase HIV/SIV replication is reduced by as much as 10,000-fold in elite controllers (EC) compared to typical progressors, but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here, we show that productive SIV infection in rhesus monkey EC is strikingly restricted to follicular helper CD4+ T cells (TFH), suggesting that while the potent SIV-specific CD8+ T cells of these monkeys can effectively clear productive infection from extra-follicular sites, their relative exclusion from B cell follicles limits elimination of infected TFH. Indeed, CD8+ lymphocyte depletion of EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH, with TFH restriction resuming upon CD8+ T cell recovery. Thus, B cell follicles constitute sanctuaries for persistent SIV replication in the presence of potent anti-viral CD8+ T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy. PMID:25599132

Cellular Mechanisms of Somatic Stem Cell Aging

PubMed Central

Jung, Yunjoon

2014-01-01

Tissue homeostasis and regenerative capacity rely on rare populations of somatic stem cells endowed with the potential to self-renew and differentiate. During aging, many tissues show a decline in regenerative potential coupled with a loss of stem cell function. Cells including somatic stem cells have evolved a series of checks and balances to sense and repair cellular damage to maximize tissue function. However, during aging the mechanisms that protect normal cell function begin to fail. In this review, we will discuss how common cellular mechanisms that maintain tissue fidelity and organismal lifespan impact somatic stem cell function. We will highlight context-dependent changes and commonalities that define aging, by focusing on three age-sensitive stem cell compartments: blood, neural, and muscle. Understanding the interaction between extrinsic regulators and intrinsic effectors that operate within different stem cell compartments is likely to have important implications for identifying strategies to improve health span and treat age-related degenerative diseases. PMID:24439814

A single-cell and feeder-free culture system for monkey embryonic stem cells.

PubMed

Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

2014-01-01

Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

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

Ren, Zhenhua; Key Laboratory of Neurodegeneration, Ministry of Education, Beijing; Department of Anatomy, Anhui Medical University, Hefei, 230032

2011-12-10

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristicsmore » of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy. -- Highlights: Black-Right-Pointing-Pointer Spontaneous transformation of cynomolgus monkey MSCs in vitro. Black-Right-Pointing-Pointer Transformed mesenchymal cells lack multipotency. Black-Right-Pointing-Pointer Transformed mesenchymal cells are highly tumorigenic. Black-Right-Pointing-Pointer Transformed mesenchymal cells do not have the characteristics of cancer stem cells.« less

Cisplatin-induced cytotoxicity in BSO-exposed renal proximal tubular epithelial cells: sex, age, and species.

PubMed

Lu, Yongke; Kawashima, Akira; Horii, Ikuo; Zhong, Laifu

2005-01-01

Cisplatin (CP)-induced kidney damage and effects of DL-buthionine-(S,R)-sulfoximine (BSO) on it are species- and age-different. It remains unclear whether CP-induced cytotoxicity in renal proximal tubular epithelial cells (RTEC), the main target cells of CP, is also species- and age-different; and whether CP-induced cytotoxicity varies with the difference in age and species, if any, is one of the questions. In the present study, the effects of BSO on CP-induced cytotoxicity in primary cultures of RTEC isolated from monkeys and different age and sex rats were studied. The RTEC were isolated from 3-week-old, 2-month-old, or 5-month-old rats, and 6-8 year-old monkeys. After subculturing, RTEC was inoculated into type I collagen-coated 96-well culture plates; after preincubation, 40 microM BSO was added, 16 hours later, varying concentrations of CP were added. At that time, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were performed to test cell viability. The concentrations of CP that inhibited 50% cell growth (IC50) of RTEC from rats and monkeys were 1.11 and 3.03 mM at 8 hours, and 0.51 and 1.24 mM at 24 hours, respectively. The BSO made the IC50s of RTEC from rats and monkeys lower, down to 0.07 and 0.48 mM at 8 hours, and 0.02 and 0.11 mM at 24 hours, respectively. The IC50s of RTEC from different sex and age rats were almost same. These results suggested that CP-induced cytotoxicity was concentration- and time-dependent, with species-dependent differences, rat RTEC were more susceptible to CP than monkey RTEC, rat RTEC were more dependent on glutathione (GSH) during the stress state were than monkey cells; CP-induced cytotoxicity was without sex- and age-dependent differences in rat RTEC.

Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

PubMed

Wang, Zhongde

2011-01-01

Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

PubMed

Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

2006-11-01

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Producing primate embryonic stem cells by somatic cell nuclear transfer.

PubMed

Byrne, J A; Pedersen, D A; Clepper, L L; Nelson, M; Sanger, W G; Gokhale, S; Wolf, D P; Mitalipov, S M

2007-11-22

Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.

Axonal properties determine somatic firing in a model of in vitro CA1 hippocampal sharp wave/ripples and persistent gamma oscillations

PubMed Central

Traub, Roger D.; Schmitz, Dietmar; Maier, Nikolaus; Whittington, Miles A.; Draguhn, Andreas

2012-01-01

Evidence has been presented that CA1 pyramidal cells, during spontaneous in vitro sharp wave/ripple (SPW-R) complexes, generate somatic action potentials that originate in axons. ‘Participating’ (somatically firing) pyramidal cells fire (almost always) at most once during a particular SPW-R whereas non-participating cells virtually never fire during an SPW-R. Somatic spikelets were small or absent, while ripple-frequency EPSCs and IPSCs occurred during the SPW-R in pyramidal neurons. These experimental findings could be replicated with a network model in which electrical coupling was present between small pyramidal cell axonal branches. Here, we explore this model in more depth. Factors that influence somatic participation include: (i) the diameter of axonal branches that contain coupling sites to other axons, because firing in larger branches injects more current into the main axon, increasing antidromic firing probability; (ii) axonal K+ currents; and (iii) somatic hyperpolarization and shunting. We predict that portions of axons fire at high frequency during SPW-R, while somata fire much less. In the model, somatic firing can occur by occasional generation of full action potentials in proximal axonal branches, which are excited by high-frequency spikelets. When the network contains phasic synaptic inhibition, at the axonal gap junction site, gamma oscillations result, again with more frequent axonal firing than somatic firing. Combining the models, so as to generate gamma followed by sharp waves, leads to strong overlap between the population of cells firing during gamma the population of cells firing during a subsequent sharp wave, as observed in vivo. PMID:22697272

FoxP2 is a Parvocellular-Specific Transcription Factor in the Visual Thalamus of Monkeys and Ferrets

PubMed Central

Iwai, Lena; Ohashi, Yohei; van der List, Deborah; Usrey, William Martin; Miyashita, Yasushi; Kawasaki, Hiroshi

2013-01-01

Although the parallel visual pathways are a fundamental basis of visual processing, our knowledge of their molecular properties is still limited. Here, we uncovered a parvocellular-specific molecule in the dorsal lateral geniculate nucleus (dLGN) of higher mammals. We found that FoxP2 transcription factor was specifically expressed in X cells of the adult ferret dLGN. Interestingly, FoxP2 was also specifically expressed in parvocellular layers 3–6 of the dLGN of adult old world monkeys, providing new evidence for a homology between X cells in the ferret dLGN and parvocellular cells in the monkey dLGN. Furthermore, this expression pattern was established as early as gestation day 140 in the embryonic monkey dLGN, suggesting that parvocellular specification has already occurred when the cytoarchitectonic dLGN layers are formed. Our results should help in gaining a fundamental understanding of the development, evolution, and function of the parallel visual pathways, which are especially prominent in higher mammals. PMID:22791804

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

B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers.

PubMed

Fukazawa, Yoshinori; Lum, Richard; Okoye, Afam A; Park, Haesun; Matsuda, Kenta; Bae, Jin Young; Hagen, Shoko I; Shoemaker, Rebecca; Deleage, Claire; Lucero, Carissa; Morcock, David; Swanson, Tonya; Legasse, Alfred W; Axthelm, Michael K; Hesselgesser, Joseph; Geleziunas, Romas; Hirsch, Vanessa M; Edlefsen, Paul T; Piatak, Michael; Estes, Jacob D; Lifson, Jeffrey D; Picker, Louis J

2015-02-01

Chronic-phase HIV and simian immunodeficiency virus (SIV) replication is reduced by as much as 10,000-fold in elite controllers (ECs) compared with typical progressors (TPs), but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here we show that productive SIV infection in rhesus monkey ECs, but not TPs, is markedly restricted to CD4(+) follicular helper T (TFH) cells, suggesting that these EC monkeys' highly effective SIV-specific CD8(+) T cells can effectively clear productive SIV infection from extrafollicular sites, but their relative exclusion from B cell follicles prevents their elimination of productively infected TFH cells. CD8(+) lymphocyte depletion in EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH cells, with restriction of productive infection to TFH cells resuming upon CD8(+) T cell recovery. Thus, B cell follicles constitute 'sanctuaries' for persistent SIV replication in the presence of potent anti-viral CD8(+) T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy.

Differentiation and characterization of rhesus monkey atrial and ventricular cardiomyocytes from induced pluripotent stem cells.

PubMed

Zhang, Xiaoqian; Cao, Henghua; Bai, Shuyun; Huo, Weibang; Ma, Yue

2017-04-01

The combination of non-human primate animals and their induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) provides not only transplantation models for cell-based therapy of heart diseases, but also opportunities for heart-related drug research on both cellular and animal levels. However, the subtypes and electrophysiology properties of non-human primate iPSC-CMs hadn't been detailed characterized. In this study, we generated rhesus monkey induced pluripotent stem cells (riPSCs), and efficiently differentiated them into ventricular or atrial cardiomyocytes by modulating retinoic acid (RA) pathways. Our results revealed that the electrophysiological characteristics and response to canonical drugs of riPSC-CMs were similar with those of human pluripotent stem cell derived CMs. Therefore, rhesus monkeys and their iPSC-CMs provide a powerful and practicable system for heart related biomedical research. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Lactobacillus and Pediococcus species richness and relative abundance in the vagina of rhesus monkeys (Macaca mulatta)

PubMed Central

Gravett, Michael G.; Jin, Ling; Pavlova, Sylvia I.; Tao, Lin

2012-01-01

Background The rhesus monkey is an important animal model to study human vaginal health to which lactic acid bacteria play a significant role. However, the vaginal lactic acid bacterial species richness and relative abundance in rhesus monkeys is largely unknown. Methods Vaginal swab samples were aseptically obtained from 200 reproductive aged female rhesus monkeys. Following Rogosa agar plating, single bacterial colonies representing different morphotypes were isolated and analyzed for whole-cell protein profile, species-specifc PCR, and 16S rRNA gene sequence. Results A total of 510 Lactobacillus strains of 17 species and one Pediococcus acidilactici were identified. The most abundant species was L. reuteri, which colonized the vaginas of 86% monkeys. L. johnsonii was the second most abundant species, which colonized 36% of monkeys. The majority of monkeys were colonized by multiple Lactobacillus species. Conclusions The vaginas of rhesus monkeys are frequently colonized by multiple Lactobacillus species, dominated by L. reuteri. PMID:22429090

A crypto-lymphatic unit at the uvula of the monkey Macaca fascicularis. A light- and electron-microscopic study.

PubMed

Nair, P N

1983-01-01

A crypto-lymphatic unit was observed at the left lateral aspect of the uvula of a mature female monkey, Macaca fascicularis. A light- and transmission electron-microscopic investigation revealed that the lumen of the crypt was filled with bacteria, desquamated epithelial cells, lymphocytes and neutrophils. The non-keratinized stratified squamous epithelium of the crypt was fragmented and showed heavy mononuclear cell infiltration and surface discontinuities, exposing lymphoid cells to foreign material. The lymphatic parenchyma consisted of organized lymphatic tissue including germinal centres. The resident cell population included lymphocytes of varying size, blastforming B- and T-lymphocytes and two types of reticular cells resembling the fibroblastic reticulum cell and the follicular dendritic cell, respectively. Occasionally granulocytes were encountered. At its base and laterally the crypto-lymphatic unit was ensheathed by a thin connective tissue capsule. Three other monkeys of the same species failed to reveal similar structures at the same site.

Persistence and immunogenicity of chemically attenuated blood stage Plasmodium falciparum in Aotus monkeys.

PubMed

De, Sai Lata; Stanisic, Danielle I; van Breda, Karin; Bellete, Bernadette; Harris, Ivor; McCallum, Fiona; Edstein, Michael D; Good, Michael F

2016-08-01

Malaria is a disease caused by a protozoan of the Plasmodium genus and results in 0.5-0.7million deaths per year. Increasing drug resistance of the parasite and insecticide resistance of mosquitoes necessitate alternative control measures. Numerous vaccine candidates have been identified but none have been able to induce robust, long-lived protection when evaluated in malaria endemic regions. Rodent studies have demonstrated that chemically attenuated blood stage parasites can persist at sub-patent levels and induce homologous and heterologous protection against malaria. Parasite-specific cellular responses were detected, with protection dependent on CD4+ T cells. To investigate this vaccine approach for Plasmodium falciparum, we characterised the persistence and immunogenicity of chemically attenuated P. falciparum FVO strain parasites (CAPs) in non-splenectomised Aotus nancymaae monkeys following administration of a single dose. Control monkeys received either normal red blood cells or wild-type parasites followed by drug treatment. Chemical attenuation was performed using tafuramycin A, which irreversibly binds to DNA. CAPs were detected in the peripheral blood for up to 2days following inoculation as determined by thick blood smears, and for up to 8days as determined by quantitative PCR. Parasite-specific IgG was not detected in monkeys that received CAPs; however, in vitro parasite-specific T cell proliferation was observed. Following challenge, the CAP monkeys developed an infection; however, one CAP monkey and the infection and drug-cure monkeys showed partial or complete resistance. These experiments lay the groundwork for further assessment of CAPs as a potential vaccine against malaria. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Effects of a higher dose of near-infrared light on clinical signs and neuroprotection in a monkey model of Parkinson's disease.

PubMed

Moro, Cécile; El Massri, Nabil; Darlot, Fannie; Torres, Napoleon; Chabrol, Claude; Agay, Diane; Auboiroux, Vincent; Johnstone, Daniel M; Stone, Jonathan; Mitrofanis, John; Benabid, Alim-Louis

2016-10-01

We have reported previously that intracranial application of near-infrared light (NIr) - when delivered at the lower doses of 25J and 35J - reduces clinical signs and offers neuroprotection in a subacute MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) monkey model of Parkinson's disease. In this study, we explored whether a higher NIr dose (125J) generated beneficial effects in the same MPTP monkey model (n=15). We implanted an NIr (670nm) optical fibre device within a midline region of the midbrain in macaque monkeys, close to the substantia nigra of both sides. MPTP injections (1.8-2.1mg/kg) were made over a five day period, during which time the NIr device was turned on and left on continuously throughout the ensuing three week survival period. Monkeys were evaluated clinically and their brains processed for immunohistochemistry and stereology. Our results showed that the higher NIr dose did not have any toxic impact on cells at the midbrain implant site. Further, this NIr dose resulted in a higher number of nigral tyrosine hydroxylase immunoreactive cells when compared to the MPTP group. However, the higher NIr dose monkeys showed little evidence for an increase in mean clinical score, number of nigral Nissl-stained cells and density of striatal tyrosine hydroxylase terminations. In summary, the higher NIr dose of 125J was not as beneficial to MPTP-treated monkeys as compared to the lower doses of 25J and 35J, boding well for strategies of NIr dose delivery and device energy consumption in a future clinical trial. Copyright © 2016 Elsevier B.V. All rights reserved.

Distinct mobilization of leukocytes and hematopoietic stem cells by CXCR4 peptide antagonist LY2510924 and monoclonal antibody LY2624587

PubMed Central

Peng, Sheng-Bin; Van Horn, Robert D.; Yin, Tinggui; Brown, Robin M.; Roell, William C.; Obungu, Victor H.; Ruegg, Charles; Wroblewski, Victor J.; Raddad, Eyas; Stille, John R.

2017-01-01

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a critical role in mobilization and redistribution of immune cells and hematopoietic stem cells (HSCs). We evaluated effects of two CXCR4-targeting agents, peptide antagonist LY2510924 and monoclonal antibody LY2624587, on mobilizing HSCs and white blood cells (WBCs) in humans, monkeys, and mice. Biochemical analysis showed LY2510924 peptide blocked SDF-1/CXCR4 binding in all three species; LY2624587 antibody blocked binding in human and monkey, with minimal activity in mouse. Cellular analysis showed LY2624587 antibody, but not LY2510924 peptide, down-regulated cell surface CXCR4 and induced hematological tumor cell death; both agents have been shown to inhibit SDF-1/CXCR4 interaction and downstream signaling. In animal models, LY2510924 peptide induced robust, prolonged, dose- and time-dependent WBC and HSC increases in mice and monkeys, whereas LY2624587 antibody induced only moderate, transient increases in monkeys. In clinical trials, similar pharmacodynamic effects were observed in patients with advanced cancer: LY2510924 peptide induced sustained WBC and HSC increases, while LY2624587 antibody induced only minimal, transient WBC changes. These distinct pharmacodynamic effects in two different classes of CXCR4 inhibitors are clinically important and should be carefully considered when designing combination studies with immune checkpoint inhibitors or other agents for cancer therapy. PMID:29212254

Regulation and distribution of squirrel monkey chorionic gonadotropin and secretogranin II in the pituitary.

PubMed

Vasauskas, Audrey A; Hubler, Tina R; Mahanic, Christina; Gibson, Susan; Kahn, Andrea G; Scammell, Jonathan G

2011-02-01

Secretogranin II (SgII) is a member of the granin family of proteins found in neuroendocrine and endocrine cells. The expression and storage of SgII in the pituitary gland of Old World primates and rodents have been linked with those of luteinizing hormone (LH). However, New World primates including squirrel monkeys do not express LH in the pituitary gland, but rather CG is expressed. If CG takes on the luteotropic role of LH in New World primates, SgII may be associated with the expression and storage of CG in the pituitary gland. The goal of this study was to evaluate the regulation and distribution of CG and SgII in the squirrel monkey. A DNA fragment containing approximately 750 bp of squirrel monkey SgII promoter was isolated from genomic DNA and found to contain a cyclic-AMP response element that is also present in the human SgII promoter and important for GnRH responsiveness. The squirrel monkey and human SgII promoters were similarly activated by GnRH in luciferase reporter gene assays in LβT2 cells. Double immunofluorescence microscopy demonstrated close association of SgII and CG in gonadotrophs of squirrel monkey pituitary gland. These results suggest that CG and SgII have a similar intercellular distribution and are coregulated in squirrel monkey pituitary gland. Copyright © 2010 Elsevier Inc. All rights reserved.

Regulation and distribution of squirrel monkey chorionic gonadotropin and secretogranin II in the pituitary

PubMed Central

Vasauskas, Audrey A.; Hubler, Tina R.; Mahanic, Christina; Gibson, Susan; Kahn, Andrea G.; Scammell, Jonathan G.

2011-01-01

Secretogranin II (SgII) is a member of the granin family of proteins found in neuroendocrine and endocrine cells. The expression and storage of SgII in the pituitary gland of Old World primates and rodents have been linked with those of luteinizing hormone (LH). However, New World primates including squirrel monkeys do not express LH in the pituitary gland, but rather CG is expressed. If CG takes on the luteotropic role of LH in New World primates, SgII may be associated with the expression and storage of CG in the pituitary gland. The goal of this study was to evaluate the regulation and distribution of CG and SgII in the squirrel monkey. A DNA fragment containing approximately 750 bp of squirrel monkey SgII promoter was isolated from genomic DNA and found to contain a cyclic AMP response element that is also present in the human SgII promoter and important for GnRH responsiveness. The squirrel monkey and human SgII promoters were similarly activated by GnRH in luciferase reporter gene assays in LβT2 cells. Double immunofluorescence microscopy demonstrated close association of SgII and CG in gonadotrophs of squirrel monkey pituitary gland. These results suggest that CG and SgII have a similar intercellular distribution and are coregulated in squirrel monkey pituitary gland. PMID:21095191

Establishment of an appropriate animal model for lacritin studies: cloning and characterization of lacritin in monkey eyes.

PubMed

Nakajima, T; Walkup, R D; Tochigi, A; Shearer, T R; Azuma, M

2007-11-01

Lacritin is a mitogen of human salivary gland cells as well as a stimulator of human corneal epithelial cells. It is expected to be an important factor in maintaining the surrounding ocular surface. The monkey would be a relevant animal model in which to study the role of lacritin in ophthalmic physiology and pathology. However, to our knowledge, no cDNA cloning or functional analysis of monkey lacritin has been performed. Thus, the purposes of this study were: (1) to clone the monkey ortholog of lacritin; (2) to characterize lacritin in tears from several species; and (3) to determine the tissues where lacritin is produced and secreted. cDNA for lacritin from rhesus macaque contained 547 bp, with 411 bp in an open reading frame (ORF) encoding a protein of 137 amino acids. Monkey lacritin showed 89% amino acid homology with human lacritin; one amino acid was deleted in all three monkey strains. The predicted MW of mature lacritin was 12.2 kDa, and the isoelectric point was 4.99. Lacritin showed anomalous migration at approximately 21.0 kDa on SDS-PAGE, as confirmed by immunoblotting and amino acid sequencing. Similar to native lacritin in monkey tears, a 21 kDa band was also detected in human tears. In contrast, no lacritin was observed at a similar position on SDS-PAGE in rat, rabbit and dog tears. In the monkey, lacritin mRNA was expressed highly in the lacrimal gland, moderately in the conjunctiva and the meibomian gland, and weakly in corneal epithelium. In primates, lacritin was produced in the lacrimal gland and secreted into tear fluid. These results suggest that lacritin might be important for the maintenance of the ocular surface in higher animals, such as monkeys and humans.

Hexavalent chromium induces apoptosis in male somatic and spermatogonial stem cells via redox imbalance

PubMed Central

Das, Joydeep; Kang, Min-Hee; Kim, Eunsu; Kwon, Deug-Nam; Choi, Yun-Jung; Kim, Jin-Hoi

2015-01-01

Hexavalent chromium [Cr(VI)], an environmental toxicant, causes severe male reproductive abnormalities. However, the actual mechanisms of toxicity are not clearly understood and have not been studied in detail. The present in vitro study aimed to investigate the mechanism of reproductive toxicity of Cr(VI) in male somatic cells (mouse TM3 Leydig cells and TM4 Sertoli cells) and spermatogonial stem cells (SSCs) because damage to or dysfunction of these cells can directly affect spermatogenesis, resulting in male infertility. Cr(VI) by inducing oxidative stress was cytotoxic to both male somatic cells and SSCs in a dose-dependent manner, and induced mitochondria-dependent apoptosis. Although the mechanism of Cr(VI)-induced cytotoxicity was similar in both somatic cells, the differences in sensitivity of TM3 and TM4 cells to Cr(VI) could be attributed, at least in part, to cell-specific regulation of P-AKT1, P-ERK1/2, and P-P53 proteins. Cr(VI) affected the differentiation and self-renewal mechanisms of SSCs, disrupted steroidogenesis in TM3 cells, while in TM4 cells, the expression of tight junction signaling and cell receptor molecules was affected as well as the secretory functions were impaired. In conclusion, our results show that Cr(VI) is cytotoxic and impairs the physiological functions of male somatic cells and SSCs. PMID:26355036

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

PubMed Central

Chung, H.J.; Hassan, M.M.; Park, J.O.; Kim, H.J.; Hong, S.T.

2015-01-01

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery. PMID:25742639

From fibroblasts and stem cells: implications for cell therapies and somatic cloning.

PubMed

Kues, Wilfried A; Carnwath, Joseph W; Niemann, Heiner

2005-01-01

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.

A temporary immersion system improves in vitro regeneration of peach palm through secondary somatic embryogenesis

PubMed Central

Steinmacher, D. A.; Guerra, M. P.; Saare-Surminski, K.; Lieberei, R.

2011-01-01

Background and Aims Secondary somatic embryogenesis has been postulated to occur during induction of peach palm somatic embryogenesis. In the present study this morphogenetic pathway is described and a protocol for the establishment of cycling cultures using a temporary immersion system (TIS) is presented. Methods Zygotic embryos were used as explants, and induction of somatic embryogenesis and plantlet growth were compared in TIS and solid culture medium. Light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to describe in vitro morphogenesis and accompany morpho-histological alterations during culture. Key Results The development of secondary somatic embryos occurs early during the induction of primary somatic embryos. Secondary somatic embryos were observed to develop continually in culture, resulting in non-synchronized development of these somatic embryos. Using these somatic embryos as explants allowed development of cycling cultures. Somatic embryos had high embryogenic potential (65·8 ± 3·0 to 86·2 ± 5·0 %) over the period tested. The use of a TIS greatly improved the number of somatic embryos obtained, as well as subsequent plantlet growth. Histological analyses showed that starch accumulation precedes the development of somatic embryos, and that these cells presented high nucleus/cytoplasm ratios and high mitotic indices, as evidenced by DAPI staining. Morphological and SEM observations revealed clusters of somatic embryos on one part of the explants, while other parts grew further, resulting in callus tissue. A multicellular origin of the secondary somatic embryos is hypothesized. Cells in the vicinity of callus accumulated large amounts of phenolic substances in their vacuoles. TEM revealed that these cells are metabolically very active, with the presence of numerous mitochondria and Golgi apparatuses. Light microscopy and TEM of the embryogenic sector revealed cells with numerous amyloplasts, large nuclei and nucleoli, and numerous plasmodesmata. Plantlets were obtained and after 3 months in culture their growth was significantly better in TIS than on solid culture medium. However, during acclimatization the survival rate of TIS-grown plantlets was lower. Conclusions The present study confirms the occurrence of secondary somatic embryos in peach palm and describes a feasible protocol for regeneration of peach palm in vitro. Further optimizations include the use of explants obtained from adult palms and improvement of somatic embryo conversion rates. PMID:21355009

Induced pluripotent stem cell-derived neural cells survive and mature in the nonhuman primate brain.

PubMed

Emborg, Marina E; Liu, Yan; Xi, Jiajie; Zhang, Xiaoqing; Yin, Yingnan; Lu, Jianfeng; Joers, Valerie; Swanson, Christine; Holden, James E; Zhang, Su-Chun

2013-03-28

The generation of induced pluripotent stem cells (iPSCs) opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

PubMed Central

Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

2014-01-01

SUMMARY Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency. PMID:25417163

Germ Cells Are Not Required to Establish the Female Pathway in Mouse Fetal Gonads

PubMed Central

Maatouk, Danielle M.; Mork, Lindsey; Hinson, Ashley; Kobayashi, Akio; McMahon, Andrew P.; Capel, Blanche

2012-01-01

The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells. PMID:23091613

Real-time Bioluminescence Imaging of Macroencapsulated Fibroblasts Reveals Allograft Protection in Rhesus Monkeys (Macaca mulatta)

PubMed Central

Tarantal, Alice F.; Lee, C. Chang I.; Itkin-Ansari, Pamela

2009-01-01

Background Encapsulation of cells has the potential to eliminate the need for immunosuppression for cellular transplantation. Recently, the TheraCyte® device was shown to provide long-term immunoprotection of murine islets in the NOD/SCID mouse model of diabetes. In this report, translational studies were undertaken using skin fibroblasts from an unrelated rhesus monkey donor that were transduced with an HIV-1-derived lentiviral vector expressing firefly luciferase permitting the use of bioluminescence imaging (BLI) to monitor cell survival over time and in a noninvasive manner. Methods Encapsulated cells were transplanted subcutaneously (N=2) or cells were injected without encapsulation (N=1) and outcomes compared. BLI was performed to monitor cell survival. Results The BLI signal from the encapsulated cells remained robust post-insertion, and in one animal persisted for up to 1 year. In contrast, the control animal that received unencapsulated cells exhibited a complete loss of cell signal within 14 days. Conclusions These data demonstrate that TheraCyte® encapsulation of allogeneic cells provides robust immune protection in transplanted rhesus monkeys. PMID:19584678

Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria, Anthozoa).

PubMed

Barfield, Sarah; Aglyamova, Galina V; Matz, Mikhail V

2016-01-13

The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions. © 2016 The Author(s).

Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria, Anthozoa)

PubMed Central

Barfield, Sarah; Aglyamova, Galina V.; Matz, Mikhail V.

2016-01-01

The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions. PMID:26763699

Cloning animals by somatic cell nuclear transfer – biological factors

PubMed Central

Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

2003-01-01

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle. PMID:14614770

Cloning animals by somatic cell nuclear transfer--biological factors.

PubMed

Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

2003-11-13

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other species, this review will be focused on somatic cell cloning of cattle.

Cell-poor septa separate representations of digits in the ventroposterior nucleus of the thalamus in monkeys and prosimian galagos.

PubMed

Qi, Hui-Xin; Gharbawie, Omar A; Wong, Peiyan; Kaas, Jon H

2011-03-01

The architectonic features of the ventroposterior nucleus (VP) were visualized in coronal brain sections from two macaque monkeys, two owl monkeys, two squirrel monkeys, and three galagos that were processed for cytochrome oxidase, Nissl bodies, or the vesicular glutamate transporter 2 (vGluT2). The traditional ventroposterior medial (VPM) and ventroposterior lateral (VPL) subnuclei were easily identified, as well as the forelimb and hindlimb compartments of VPL, as they were separated by poorly staining, cell-poor septa. Septa also separated other cell groups within VPM and VPL, specifically in the medial compartment of VPL representing the hand (hand VPL). In one squirrel monkey and one galago we demonstrated that these five groups of cells represent digits 1-5 in a mediolateral sequence by injecting tracers into the cortical representation of single digits, defined by microelectrode recordings, and relating concentrations of labeled neurons to specific cell groups in hand VPL. The results establish the existence of septa that isolate the representation of the five digits in VPL of primates and demonstrate that the isolated cell groups represent digits 1-5 in a mediolateral sequence. The present results show that the septa are especially prominent in brain sections processed for vGluT2, which is expressed in the synaptic terminals of excitatory neurons in most nuclei of the brainstem and thalamus. As vGluT2 is expressed in the synaptic terminations from dorsal columns and trigeminal brainstem nuclei, the effectiveness of vGluT2 preparations in revealing septa in VP likely reflects a lack of synapses using glutamate in the septa. Copyright © 2010 Wiley-Liss, Inc.

Cell-Poor Septa Separate Representations of Digits in the Ventroposterior Nucleus of the Thalamus in Monkeys and Prosimian Galagos

PubMed Central

Qi, Hui-Xin; Gharbawie, Omar A.; Wong, Peiyan; Kaas, Jon H.

2013-01-01

The architectonic features of the ventroposterior nucleus (VP) were visualized in coronal brain sections from two macaque monkeys, two owl monkeys, two squirrel monkeys, and three galagos that were processed for cytochrome oxidase, Nissl bodies, or the vesicular glutamate transporter 2 (vGluT2). The traditional ventroposterior medial (VPM) and ventroposterior lateral (VPL) subnuclei were easily identified, as well as the forelimb and hindlimb compartments of VPL, as they were separated by poorly staining, cell-poor septa. Septa also separated other cell groups within VPM and VPL, specifically in the medial compartment of VPL representing the hand (hand VPL). In one squirrel monkey and one galago we demonstrated that these five groups of cells represent digits 1–5 in a mediolateral sequence by injecting tracers into the cortical representation of single digits, defined by microelectrode recordings, and relating concentrations of labeled neurons to specific cell groups in hand VPL. The results establish the existence of septa that isolate the representation of the five digits in VPL of primates and demonstrate that the isolated cell groups represent digits 1–5 in a mediolateral sequence. The present results show that the septa are especially prominent in brain sections processed for vGluT2, which is expressed in the synaptic terminals of excitatory neurons in most nuclei of the brainstem and thalamus. As vGluT2 is expressed in the synaptic terminations from dorsal columns and trigeminal brainstem nuclei, the effectiveness of vGluT2 preparations in revealing septa in VP likely reflects a lack of synapses using glutamate in the septa. J. Comp. Neurol. 519:738–758, 2011. PMID:21246552

The differentiation of hepatocyte-like cells from monkey embryonic stem cells.

PubMed

Ma, Xiaocui; Duan, Yuyou; Jung, Christine J; Wu, Jian; VandeVoort, Catherine A; Zern, Mark A

2008-12-01

Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.

[Quantitative analysis of the structure of neuronal dendritic spines in the striatum using the Leitz-ASM system].

PubMed

Leontovich, T A; Zvegintseva, E G

1985-10-01

Two principal classes of striatum long axonal neurons (sparsely ramified reticular cells and densely ramified dendritic cells) were analyzed quantitatively in four animal species: hedgehog, rabbit, dog and monkey. The cross section area, total dendritic length and the area of dendritic field were measured using "LEITZ-ASM" system. Classes of neurons studied were significantly different in dogs and monkeys, while no differences were noted between hedgehog and rabbit. Reticular neurons of different species varied much more than dendritic ones. Quantitative analysis has revealed the progressive increase in the complexity of dendritic tree in mammals from rabbit to monkey.

vasa is expressed in somatic cells of the embryonic gonad in a sex-specific manner in Drosophila melanogaster.

PubMed

Renault, Andrew D

2012-10-15

Vasa is a DEAD box helicase expressed in the Drosophila germline at all stages of development. vasa homologs are found widely in animals and vasa has become the gene of choice in identifying germ cells. I now show that Drosophila vasa expression is not restricted to the germline but is also expressed in a somatic lineage, the embryonic somatic gonadal precursor cells. This expression is sexually dimorphic, being maintained specifically in males, and is regulated post-transcriptionally. Although somatic Vasa expression is not required for gonad coalescence, these data support the notion that Vasa is not solely a germline factor.

Determining the Origin of Human Germinal Center B Cell-Derived Malignancies.

PubMed

Seifert, Marc; Küppers, Ralf

2017-01-01

Most human B cell lymphomas originate from germinal center (GC) B cells. This is partly caused by the high proliferative activity of GC B cells and the remodeling processes acting at the immunoglobulin (Ig) loci of these cells, i.e., somatic hypermutation and class-switching. Mistargeting of these processes can cause chromosomal translocations, and the hypermutation machinery may also target non-Ig genes. As somatic hypermutation is exclusively active in GC B cells, the presence of somatic mutations in rearranged IgV genes is a standard criterium for a GC or post-GC B cell origin of lymphomas. Beyond this, ongoing somatic hypermutation during lymphoma clone expansion indicates that the lymphoma has an active GC B cell differentiation program. The proto-oncogene BCL6 is specifically expressed in GC B cells and also acquires somatic mutations as a physiological by-product of the somatic hypermutation process, albeit at a lower level than IgV genes. Thus, detection of BCL6 mutations is a further genetic trait of a GC experience of a B cell lymphoma. Typically, B cell lymphomas retain key features of their specific cells of origin, including a differentiation stage-specific gene expression pattern. This is at least partly due to genetic lesions, which "freeze" the lymphoma cells at the differentiation stage at which the transformation occurred. Therefore, identification of the normal B cell subset with the most similar gene expression pattern to a particular type of B cell lymphoma has been instrumental to deduce the precise cell of origin of lymphomas.We present here protocols to analyze human B cell lymphomas for a potential origin from GC B cells by determining the presence of mutations in rearranged IgV genes and the BCL6 gene, and by comparing the gene expression pattern of lymphoma cells with those of normal B cell subsets by genechip or RNA-sequencing analysis.

Use of primary cell cultures to measure the late effects in the skins of rhesus monkeys irradiated with protons

NASA Astrophysics Data System (ADS)

Cox, A. B.; Wood, D. H.; Lett, J. T.

Previous pilot investigations of the uses of primary cell cultures to study late damage in stem cells of the skin of the New Zealand white (NZW) rabbit and the rhesus monkey /1-3/, have been extended to individual monkeys exposed to 55 MeV protons. Protons of this energy have a larger range in tissue of (~2.6 cm) than the 32 MeV protons (~0.9 cm) to which the animals in our earlier studies had been exposed. Although the primary emphases in the current studies were improvement and simplification in the techniques and logistics of transportation of biopsies to a central analytical facility, comparison of the quantitative measurements obtained thus far for survival of stem cells in the skins from animals irradiated 21 years ago reveals that the effects of both proton energies are similar.

Induction of Encephalitis in Rhesus Monkeys Infused with Lymphocryptovirus-Infected B-Cells Presenting MOG34–56 Peptide

PubMed Central

Haanstra, Krista G.; Wubben, Jacqueline A. M.; Jonker, Margreet; Hart, Bert A. ‘t.

2013-01-01

The overlapping epidemiology of multiple sclerosis (MS) and Epstein-Barr virus (EBV), the increased risk to develop MS after infectious mononucleosis (IM) and the localization of EBV-infected B-cells within the MS brain suggest a causal link between EBV and MS. However, the underlying mechanism is unknown. We hypothesize that EBV-infected B-cells are capable of eliciting a central nervous system (CNS) targeting autoimmune reaction. To test this hypothesis we have developed a novel experimental model in rhesus monkeys of IM-like disease induced by infusing autologous B-lymphoblastoid cells (B-LCL). Herpesvirus papio (HVP) is a lymphocryptovirus related to EBV and was used to generate rhesus monkey B-LCL. Three groups of five animals were included; each group received three intravenous infusions of B-LCL that were either pulsed with the encephalitogenic self peptide MOG34–56 (group A), a mimicry peptide (981–1003) of the major capsid protein of cytomegalovirus (CMVmcp981–1003; group B) or the citrullinated MOG34–56 (cMOG34–56; group C). Groups A and B received on day 98 a single immunization with MOG34–56 in incomplete Freund’s adjuvant (IFA). Group C monkeys were euthanized just prior to day 98 without booster immunization. We observed self-peptide-specific proliferation of T-cells, superimposed on similar strong proliferation of CD3+CD8+ T-cells against the B-LCL as observed in IM. The brains of several monkeys contained perivascular inflammatory lesions of variable size, comprising CD3+ and CD68+ cells. Moreover, clusters of CD3+ and CD20+ cells were detected in the meninges. The only evident clinical sign was substantial loss of bodyweight (>15%), a symptom observed both in early autoimmune encephalitis and IM. In conclusion, this model suggests that EBV-induced B-LCL can elicit a CNS targeting inflammatory (auto)immune reaction. PMID:23977076

Non-stochastic reprogramming from a privileged somatic cell state

PubMed Central

Guo, Shangqin; Zi, Xiaoyuan; Schulz, Vincent P.; Cheng, Jijun; Zhong, Mei; Koochaki, Sebastian H.J.; Megyola, Cynthia M.; Pan, Xinghua; Heydari, Kartoosh; Weissman, Sherman M.; Gallagher, Patrick G.; Krause, Diane S.; Fan, Rong; Lu, Jun

2014-01-01

SUMMARY Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient, and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a non-stochastic manner. Subsets of murine hematopoietic progenitors are privileged, whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after 4–5 divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of ~8 hours. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression, and is increased by p53-knockdown. This ultrafast-cycling population accounts for >99% of the bulk reprogramming activity in wildtype or p53-knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state, and suggest that cell cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PMID:24486105

Regulators of Mitochondrial Quality Control Differs in Subcutaneous Fat of Metabolically Healthy and Unhealthy Obese Monkeys

PubMed Central

Kavanagh, Kylie; Davis, Ashley T; Peters, Diane E; Le Grand, Andre; Bharadwaj, Manish S; Molina, Anthony JA

2016-01-01

Objectives Obesity exists with and without accompanying cardiometabolic disease, termed metabolically unhealthy obesity (MUO) and healthy obesity respectively (MHO). Underlying differences in the ability of subcutaneous (SQ) fat to respond to nutrient excess is emerging as a key pathway. We aimed to document the first spontaneous animal model of MHO and MUO and differences in SQ adipose tissue. Methods Vervet monkeys (Chlorocebus aethiops; n=171) were screened for Metabolic Syndrome. A subset of MHO and MUO monkeys (n=6/group) had SQ fat biopsies collected for histologic evaluations and examination of key mitochondrial proteins. Results Obesity was seen in 20% of monkeys, and within this population, 31% were healthy which mirrors human prevalence estimates. MUO monkeys had more than 60% lower adiponectin concentrations despite similar fat cell size, uncoupling protein 3, and activated macrophage abundance. However, alternatively activated/anti-inflammatory macrophages were 70% lower. Deficiencies of 50% or more in mitochondrial quality control regulators, and selected mitochondrial fission and fusion markers were observed in the SQ fat of MUO monkeys despite comparable mitochondrial content. Conclusions We characterized a novel and translatable spontaneously obese animal model of healthy and unhealthy obesity, occurring independently of dietary factors. Differences in mitochondrial quality and inflammatory cell populations of subcutaneous fat may underpin divergent metabolic health. PMID:28236433

Regulators of mitochondrial quality control differ in subcutaneous fat of metabolically healthy and unhealthy obese monkeys.

PubMed

Kavanagh, Kylie; Davis, Ashley T; Peters, Diane E; LeGrand, Andre C; Bharadwaj, Manish S; Molina, Anthony J A

2017-04-01

Obesity exists with and without accompanying cardiometabolic disease, termed metabolically unhealthy obesity (MUO) and healthy obesity (MHO), respectively. Underlying differences in the ability of subcutaneous (SQ) fat to respond to nutrient excess are emerging as a key pathway. This study aimed to document the first spontaneous animal model of MHO and MUO and differences in SQ adipose tissue. Vervet monkeys (Chlorocebus aethiops; N = 171) were screened for metabolic syndrome. A subset of MHO and MUO monkeys (n = 6/group) had SQ fat biopsies collected for histological evaluations and examination of key mitochondrial proteins. Obesity was seen in 20% of monkeys, and within this population, 31% were healthy, which mirrors human prevalence estimates. MUO monkeys had more than 60% lower adiponectin concentrations despite similar fat cell size, uncoupling protein 3, and activated macrophage abundance. However, alternatively activated/anti-inflammatory macrophages were 70% lower. Deficiencies of 50% or more in mitochondrial quality control regulators and selected mitochondrial fission and fusion markers were observed in the SQ fat of MUO monkeys despite comparable mitochondrial content. A novel and translatable spontaneously obese animal model of MHO and MUO, occurring independently of dietary factors, was characterized. Differences in mitochondrial quality and inflammatory cell populations of subcutaneous fat may underpin divergent metabolic health. © 2017 The Obesity Society.

Intersection of diverse neuronal genomes and neuropsychiatric disease: The Brain Somatic Mosaicism Network.

PubMed

McConnell, Michael J; Moran, John V; Abyzov, Alexej; Akbarian, Schahram; Bae, Taejeong; Cortes-Ciriano, Isidro; Erwin, Jennifer A; Fasching, Liana; Flasch, Diane A; Freed, Donald; Ganz, Javier; Jaffe, Andrew E; Kwan, Kenneth Y; Kwon, Minseok; Lodato, Michael A; Mills, Ryan E; Paquola, Apua C M; Rodin, Rachel E; Rosenbluh, Chaggai; Sestan, Nenad; Sherman, Maxwell A; Shin, Joo Heon; Song, Saera; Straub, Richard E; Thorpe, Jeremy; Weinberger, Daniel R; Urban, Alexander E; Zhou, Bo; Gage, Fred H; Lehner, Thomas; Senthil, Geetha; Walsh, Christopher A; Chess, Andrew; Courchesne, Eric; Gleeson, Joseph G; Kidd, Jeffrey M; Park, Peter J; Pevsner, Jonathan; Vaccarino, Flora M

2017-04-28

Neuropsychiatric disorders have a complex genetic architecture. Human genetic population-based studies have identified numerous heritable sequence and structural genomic variants associated with susceptibility to neuropsychiatric disease. However, these germline variants do not fully account for disease risk. During brain development, progenitor cells undergo billions of cell divisions to generate the ~80 billion neurons in the brain. The failure to accurately repair DNA damage arising during replication, transcription, and cellular metabolism amid this dramatic cellular expansion can lead to somatic mutations. Somatic mutations that alter subsets of neuronal transcriptomes and proteomes can, in turn, affect cell proliferation and survival and lead to neurodevelopmental disorders. The long life span of individual neurons and the direct relationship between neural circuits and behavior suggest that somatic mutations in small populations of neurons can significantly affect individual neurodevelopment. The Brain Somatic Mosaicism Network has been founded to study somatic mosaicism both in neurotypical human brains and in the context of complex neuropsychiatric disorders. Copyright © 2017, American Association for the Advancement of Science.

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes – A method for quantitative transcriptional reprogramming

PubMed Central

Halley-Stott, R.P.; Pasque, V.; Astrand, C.; Miyamoto, K.; Simeoni, I.; Jullien, J.; Gurdon, J.B.

2010-01-01

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein. PMID:20123126

Global transcriptome analysis of the C57BL/6J mouse testis by SAGE: evidence for nonrandom gene order.

PubMed

Divina, Petr; Vlcek, Cestmír; Strnad, Petr; Paces, Václav; Forejt, Jirí

2005-03-05

We generated the gene expression profile of the total testis from the adult C57BL/6J male mice using serial analysis of gene expression (SAGE). Two high-quality SAGE libraries containing a total of 76 854 tags were constructed. An extensive bioinformatic analysis and comparison of SAGE transcriptomes of the total testis, testicular somatic cells and other mouse tissues was performed and the theory of male-biased gene accumulation on the X chromosome was tested. We sorted out 829 genes predominantly expressed from the germinal part and 944 genes from the somatic part of the testis. The genes preferentially and specifically expressed in total testis and testicular somatic cells were identified by comparing the testis SAGE transcriptomes to the available transcriptomes of seven non-testis tissues. We uncovered chromosomal clusters of adjacent genes with preferential expression in total testis and testicular somatic cells by a genome-wide search and found that the clusters encompassed a significantly higher number of genes than expected by chance. We observed a significant 3.2-fold enrichment of the proportion of X-linked genes specific for testicular somatic cells, while the proportions of X-linked genes specific for total testis and for other tissues were comparable. In contrast to the tissue-specific genes, an under-representation of X-linked genes in the total testis transcriptome but not in the transcriptomes of testicular somatic cells and other tissues was detected. Our results provide new evidence in favor of the theory of male-biased genes accumulation on the X chromosome in testicular somatic cells and indicate the opposite action of the meiotic X-inactivation in testicular germ cells.

Global transcriptome analysis of the C57BL/6J mouse testis by SAGE: evidence for nonrandom gene order

PubMed Central

Divina, Petr; Vlček, Čestmír; Strnad, Petr; Pačes, Václav; Forejt, Jiří

2005-01-01

Background We generated the gene expression profile of the total testis from the adult C57BL/6J male mice using serial analysis of gene expression (SAGE). Two high-quality SAGE libraries containing a total of 76 854 tags were constructed. An extensive bioinformatic analysis and comparison of SAGE transcriptomes of the total testis, testicular somatic cells and other mouse tissues was performed and the theory of male-biased gene accumulation on the X chromosome was tested. Results We sorted out 829 genes predominantly expressed from the germinal part and 944 genes from the somatic part of the testis. The genes preferentially and specifically expressed in total testis and testicular somatic cells were identified by comparing the testis SAGE transcriptomes to the available transcriptomes of seven non-testis tissues. We uncovered chromosomal clusters of adjacent genes with preferential expression in total testis and testicular somatic cells by a genome-wide search and found that the clusters encompassed a significantly higher number of genes than expected by chance. We observed a significant 3.2-fold enrichment of the proportion of X-linked genes specific for testicular somatic cells, while the proportions of X-linked genes specific for total testis and for other tissues were comparable. In contrast to the tissue-specific genes, an under-representation of X-linked genes in the total testis transcriptome but not in the transcriptomes of testicular somatic cells and other tissues was detected. Conclusion Our results provide new evidence in favor of the theory of male-biased genes accumulation on the X chromosome in testicular somatic cells and indicate the opposite action of the meiotic X-inactivation in testicular germ cells. PMID:15748293

Differentiation as symbiosis.

PubMed

Chigira, M; Watanabe, H

1994-07-01

Preservation of the identity of DNA is the ultimate goal of multicellular organisms. An abnormal DNA sequence in cells within an individual means its parasitic nature in cell society as shown in tumors. Somatic gene arrangement and gene mutation in development may be considered as de novo formation of parasites. It is likely that the developmental process with genetic alterations means symbiosis between altered cells and germ line cells preserving genetic information without alterations, when somatic alteration of DNA sequence is a major mechanism of differentiation. According to the selfish gene theory of Dawkins, germ line cells permit symbiosis when somatic cell society derives clear profit for the replication of original DNA copies.

Totipotency, Pluripotency and Nuclear Reprogramming

NASA Astrophysics Data System (ADS)

Mitalipov, Shoukhrat; Wolf, Don

Mammalian development commences with the totipotent zygote which is capable of developing into all the specialized cells that make up the adult animal. As development unfolds, cells of the early embryo proliferate and differentiate into the first two lineages, the pluripotent inner cell mass and the trophectoderm. Pluripotent cells can be isolated, adapted and propagated indefinitely in vitro in an undifferentiated state as embryonic stem cells (ESCs). ESCs retain their ability to differentiate into cells representing the three major germ layers: endoderm, mesoderm or ectoderm or any of the 200+ cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human ESCs represent an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating conditions. Pluripotent cells resembling ESCs can also be derived experimentally by the nuclear reprogramming of somatic cells. Reprogrammed somatic cells may have an even more important role in cell replacement therapies since the patient's own somatic cells can be used for reprogramming thereby eliminating immune based rejection of transplanted cells. In this review, we summarize two major approaches to reprogramming: (1) somatic cell nuclear transfer and (2) direct reprogramming using genetic manipulations.

Cross-species transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony.

PubMed

Chen, Eunice C; Yagi, Shigeo; Kelly, Kristi R; Mendoza, Sally P; Tarara, Ross P; Canfield, Don R; Maninger, Nicole; Rosenthal, Ann; Spinner, Abigail; Bales, Karen L; Schnurr, David P; Lerche, Nicholas W; Chiu, Charles Y

2011-07-01

Adenoviruses are DNA viruses that naturally infect many vertebrates, including humans and monkeys, and cause a wide range of clinical illnesses in humans. Infection from individual strains has conventionally been thought to be species-specific. Here we applied the Virochip, a pan-viral microarray, to identify a novel adenovirus (TMAdV, titi monkey adenovirus) as the cause of a deadly outbreak in a closed colony of New World monkeys (titi monkeys; Callicebus cupreus) at the California National Primate Research Center (CNPRC). Among 65 titi monkeys housed in a building, 23 (34%) developed upper respiratory symptoms that progressed to fulminant pneumonia and hepatitis, and 19 of 23 monkeys, or 83% of those infected, died or were humanely euthanized. Whole-genome sequencing of TMAdV revealed that this adenovirus is a new species and highly divergent, sharing <57% pairwise nucleotide identity with other adenoviruses. Cultivation of TMAdV was successful in a human A549 lung adenocarcinoma cell line, but not in primary or established monkey kidney cells. At the onset of the outbreak, the researcher in closest contact with the monkeys developed an acute respiratory illness, with symptoms persisting for 4 weeks, and had a convalescent serum sample seropositive for TMAdV. A clinically ill family member, despite having no contact with the CNPRC, also tested positive, and screening of a set of 81 random adult blood donors from the Western United States detected TMAdV-specific neutralizing antibodies in 2 individuals (2/81, or 2.5%). These findings raise the possibility of zoonotic infection by TMAdV and human-to-human transmission of the virus in the population. Given the unusually high case fatality rate from the outbreak (83%), it is unlikely that titi monkeys are the native host species for TMAdV, and the natural reservoir of the virus is still unknown. The discovery of TMAdV, a novel adenovirus with the capacity to infect both monkeys and humans, suggests that adenoviruses should be monitored closely as potential causes of cross-species outbreaks.

Responses of Cells in the Midbrain Near-Response Area in Monkeys with Strabismus

PubMed Central

Das, Vallabh E.

2012-01-01

Purpose. To investigate whether neuronal activity within the supraoculomotor area (SOA—monosynaptically connected to medial rectus motoneurons and encode vergence angle) of strabismic monkeys was correlated with the angle of horizontal misalignment and therefore helps to define the state of strabismus. Methods. Single-cell neural activity was recorded from SOA neurons in two monkeys with exotropia as they performed eye movement tasks during monocular viewing. Results. Horizontal strabismus angle varied depending on eye of fixation (dissociated horizontal deviation) and the activity of SOA cells (n = 35) varied in correlation with the angle of strabismus. Both near-response (cells that showed larger firing rates for smaller angles of exotropia) and far-response (cells that showed lower firing rates for smaller angles of exotropia) cells were identified. SOA cells showed no modulation of activity with changes in conjugate eye position as tested during smooth-pursuit, thereby verifying that the responses were related to binocular misalignment. SOA cell activity was also not correlated with change in horizontal misalignment due to A-patterns of strabismus. Comparison of SOA population activity in strabismic animals and normal monkeys (described in the literature) show that both neural thresholds and neural sensitivities are altered in the strabismic animals compared with the normal animals. Conclusions. SOA cell activity is important in determining the state of horizontal strabismus, possibly by altering vergence tone in extraocular muscle. The lack of correlated SOA activity with changes in misalignment due to A/V patterns suggest that circuits mediating horizontal strabismus angle and those that mediate A/V patterns are different. PMID:22562519

Control of Anther Cell Differentiation by the Small Protein Ligand TPD1 and Its Receptor EMS1 in Arabidopsis

PubMed Central

Huang, Jian; Zhang, Tianyu; Linstroth, Lisa; Tillman, Zachary; Otegui, Marisa S.; Owen, Heather A.

2016-01-01

A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive microsporocytes (pollen mother cells) and surrounding somatic cells. The microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS MICROSPOROCYTES1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction. PMID:27537183

Metaphase yields from staphylococcal enterotoxin A stimulated peripheral blood lymphocytes of unirradiated and irradiated aged rhesus monkeys

NASA Technical Reports Server (NTRS)

Hill, F. S.; Cox, A. B.; Salmon, Y. L.; Cantu, A. O.; Lucas, J. N.

1994-01-01

The mitogen phytohemagglutinin (PHA) works well in both human and cynomolgus monkey (Macaca fascicularis) lymphocyte cultures to stimulate T cell proliferation. T cells from rhesus monkeys (Macaca mulatta) are less responsive than human cells, producing few metaphases when thousands are required, e.g. in biological dosimetry studies. We show that staphylococcal enterotoxin A (SEA), one of the most potent mitogens known, at a concentration of 0.5 microgram/ml stimulated peripheral lymphocytes to grow with a mitotic index (MI) averaging 0.13 metaphases/cell in old, irradiated rhesus macaques. This was significantly greater (p < 0.001) than that produced by PHA (MI < 0.01) in lymphocytes from the same animals. Whole blood was cultured for 96, 120 and 144 h for five irradiated individuals and for two controls. All cells cultured with SEA produced a high MI with a peak response at 120 h whereas the same cultures showed low MI for each PHA stimulated culture.

Role of Somatic Testicular Cells during Mouse Spermatogenesis in Three-Dimensional Collagen Gel Culture System

PubMed Central

Khajavi, Noushafarin; Akbari, Mohammad; Abolhassani, Farid; Dehpour, Ahmad Reza; Koruji, Morteza; Habibi Roudkenar, Mehryar

2014-01-01

Objective Spermatogonial stem cells (SSCs) are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional (3D) culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. Materials and Methods In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting (MACS) using GDNF family receptor alpha-1 (Gfrα-1) antibody. Two experimental designs were investigated. 1. Gfrα-1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfrα-1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 (SCP3)-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 (TTF1) as post meiotic markers. For statistical analysis student t test was performed. Results Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Conclusion Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro. PMID:24518977

vasa is expressed in somatic cells of the embryonic gonad in a sex-specific manner in Drosophila melanogaster

PubMed Central

Renault, Andrew D.

2012-01-01

Summary Vasa is a DEAD box helicase expressed in the Drosophila germline at all stages of development. vasa homologs are found widely in animals and vasa has become the gene of choice in identifying germ cells. I now show that Drosophila vasa expression is not restricted to the germline but is also expressed in a somatic lineage, the embryonic somatic gonadal precursor cells. This expression is sexually dimorphic, being maintained specifically in males, and is regulated post-transcriptionally. Although somatic Vasa expression is not required for gonad coalescence, these data support the notion that Vasa is not solely a germline factor. PMID:23213382

Immune and Genetic Correlates of Vaccine Protection Against Mucosal Infection by SIV in Monkeys

PubMed Central

Letvin, Norman L.; Rao, Srinivas S.; Montefiori, David C.; Seaman, Michael S.; Sun, Yue; Lim, So-Yon; Yeh, Wendy W.; Asmal, Mohammed; Gelman, Rebecca S.; Shen, Ling; Whitney, James B.; Seoighe, Cathal; Lacerda, Miguel; Keating, Sheila; Norris, Philip J.; Hudgens, Michael G.; Gilbert, Peter B.; Buzby, Adam P.; Mach, Linh V.; Zhang, Jinrong; Balachandran, Harikrishnan; Shaw, George M.; Schmidt, Stephen D.; Todd, John-Paul; Dodson, Alan; Mascola, John R.; Nabel, Gary J.

2013-01-01

The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was an about one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01–negative monkeys challenged with SIVsmE660, no CD8+ T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4+ T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanism of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies. PMID:21543722

Immune and Genetic Correlates of Vaccine Protection Against Mucosal Infection by SIV in Monkeys.

PubMed

Letvin, Norman L; Rao, Srinivas S; Montefiori, David C; Seaman, Michael S; Sun, Yue; Lim, So-Yon; Yeh, Wendy W; Asmal, Mohammed; Gelman, Rebecca S; Shen, Ling; Whitney, James B; Seoighe, Cathal; Lacerda, Miguel; Keating, Sheila; Norris, Philip J; Hudgens, Michael G; Gilbert, Peter B; Buzby, Adam P; Mach, Linh V; Zhang, Jinrong; Balachandran, Harikrishnan; Shaw, George M; Schmidt, Stephen D; Todd, John-Paul; Dodson, Alan; Mascola, John R; Nabel, Gary J

2011-05-04

The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was about a one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01-negative monkeys challenged with SIVsmE660, no CD8(+) T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4(+) T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanisms of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.

Human somatic cell nuclear transfer and cloning.

PubMed

2012-10-01

This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Integration of Genomic, Biologic, and Chemical Approaches to Target p53 Loss and Gain-of-Function in Triple Negative Breast Cancer

DTIC Science & Technology

2014-09-01

in this renewal: p53 triple negative breast cancer subtypes gene expression somatic cell genetics CRISPR /Cas 3. OVERALL PROJECT SUMMARY...to the efficacy of the synthetic lethality screen. In addition, we have optimized the use of CRISPR /Cas, a novel somatic cell recombination...completing this stage of the research within the upcoming Year 2 of the award period. Figure 1. CRISPR /Cas-mediated in vitro somatic cell

Microinjection of Follicle-Enclosed Mouse Oocytes

NASA Astrophysics Data System (ADS)

Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.

Gravity separation of fat, somatic cells, and bacteria in raw and pasteurized milks.

PubMed

Caplan, Z; Melilli, C; Barbano, D M

2013-04-01

The objective of experiment 1 was to determine if the extent of gravity separation of milk fat, bacteria, and somatic cells is influenced by the time and temperature of gravity separation or the level of contaminating bacteria present in the raw milk. The objective of experiment 2 was to determine if different temperatures of milk heat treatment affected the gravity separation of milk fat, bacteria, and somatic cells. In raw milk, fat, bacteria, and somatic cells rose to the top of columns during gravity separation. About 50 to 80% of the fat and bacteria were present in the top 8% of the milk after gravity separation of raw milk. Gravity separation for 7h at 12°C or for 22h at 4°C produced equivalent separation of fat, bacteria, and somatic cells. The completeness of gravity separation of fat was influenced by the level of bacteria in the milk before separation. Milk with a high bacterial count had less (about 50 to 55%) gravity separation of fat than milk with low bacteria count (about 80%) in 22h at 4°C. Gravity separation caused fat, bacteria, and somatic cells to rise to the top of columns for raw whole milk and high temperature, short-time pasteurized (72.6°C, 25s) whole milk. Pasteurization at ≥76.9°C for 25s prevented all 3 components from rising, possibly due to denaturation of native bovine immunoglobulins that normally associate with fat, bacteria, and somatic cells during gravity separation. Gravity separation can be used to produce reduced-fat milk with decreased bacterial and somatic cell counts, and may be a critical factor in the history of safe and unique traditional Italian hard cheeses produced from gravity-separated raw milk. A better understanding of the mechanism of this natural process could lead to the development of new nonthermal thermal technology (that does not involve heating the milk to high temperatures) to remove bacteria and spores from milk or other liquids. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Developmental Localization and Methylesterification of Pectin Epitopes during Somatic Embryogenesis of Banana (Musa spp. AAA)

PubMed Central

Xu, Chunxiang; Zhao, Lu; Pan, Xiao; Šamaj, Jozef

2011-01-01

Background The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development. Methodology/Principal Findings Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment. Conclusions/Significance These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana. PMID:21826225

Transcriptional Noise and Somatic Mutations in the Aging Pancreas.

PubMed

Swisa, Avital; Kaestner, Klaus H; Dor, Yuval

2017-12-05

The underlying mechanisms and functional significance of pancreatic β cell heterogeneity are an intensive area of investigation. In a recent Cell paper, Enge and colleagues (2017) performed single-cell RNA sequencing of human pancreatic cells and concluded that with age, pancreatic cells become transcriptionally noisy and accumulate somatic mutations. Copyright © 2017. Published by Elsevier Inc.

Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm

PubMed Central

Zhou, Yang; Connor, Erin E; Bickhart, Derek M; Li, Congjun; Baldwin, Ransom L; Schroeder, Steven G; Rosen, Benjamin D; Yang, Liguo; Van Tassell, Curtis P

2018-01-01

Abstract Background Although sperm DNA methylation has been studied in humans and other species, its status in cattle is largely unknown. Results Using whole-genome bisulfite sequencing (WGBS), we profiled the DNA methylome of cattle sperm through comparison with three somatic tissues (mammary gland, brain, and blood). Large differences between cattle sperm and somatic cells were observed in the methylation patterns of global CpGs, pericentromeric satellites, partially methylated domains (PMDs), hypomethylated regions (HMRs), and common repeats. As expected, we observed low methylation in the promoter regions and high methylation in the bodies of active genes. We detected selective hypomethylation of megabase domains of centromeric satellite clusters, which may be related to chromosome segregation during meiosis and their rapid transcriptional activation upon fertilization. We found more PMDs in sperm cells than in somatic cells and identified meiosis-related genes such asKIF2B and REPIN1, which are hypomethylated in sperm but hypermethylated in somatic cells. In addition to the common HMRs around gene promoters, which showed substantial differences between sperm and somatic cells, the sperm-specific HMRs also targeted to distinct spermatogenesis-related genes, including BOLL, MAEL, ASZ1, SYCP3, CTCFL, MND1, SPATA22, PLD6, DDX4, RBBP8, FKBP6, and SYCE1. Although common repeats were heavily methylated in both sperm and somatic cells, some young Bov-A2 repeats, which belong to the SINE family, were hypomethylated in sperm and could affect the promoter structures by introducing new regulatory elements. Conclusions Our study provides a comprehensive resource for bovine sperm epigenomic research and enables new discoveries about DNA methylation and its role in male fertility. PMID:29635292

Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm.

PubMed

Zhou, Yang; Connor, Erin E; Bickhart, Derek M; Li, Congjun; Baldwin, Ransom L; Schroeder, Steven G; Rosen, Benjamin D; Yang, Liguo; Van Tassell, Curtis P; Liu, George E

2018-05-01

Although sperm DNA methylation has been studied in humans and other species, its status in cattle is largely unknown. Using whole-genome bisulfite sequencing (WGBS), we profiled the DNA methylome of cattle sperm through comparison with three somatic tissues (mammary gland, brain, and blood). Large differences between cattle sperm and somatic cells were observed in the methylation patterns of global CpGs, pericentromeric satellites, partially methylated domains (PMDs), hypomethylated regions (HMRs), and common repeats. As expected, we observed low methylation in the promoter regions and high methylation in the bodies of active genes. We detected selective hypomethylation of megabase domains of centromeric satellite clusters, which may be related to chromosome segregation during meiosis and their rapid transcriptional activation upon fertilization. We found more PMDs in sperm cells than in somatic cells and identified meiosis-related genes such asKIF2B and REPIN1, which are hypomethylated in sperm but hypermethylated in somatic cells. In addition to the common HMRs around gene promoters, which showed substantial differences between sperm and somatic cells, the sperm-specific HMRs also targeted to distinct spermatogenesis-related genes, including BOLL, MAEL, ASZ1, SYCP3, CTCFL, MND1, SPATA22, PLD6, DDX4, RBBP8, FKBP6, and SYCE1. Although common repeats were heavily methylated in both sperm and somatic cells, some young Bov-A2 repeats, which belong to the SINE family, were hypomethylated in sperm and could affect the promoter structures by introducing new regulatory elements. Our study provides a comprehensive resource for bovine sperm epigenomic research and enables new discoveries about DNA methylation and its role in male fertility.

Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

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

Yue, Xiao-shan; Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501; Fujishiro, Masako

In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells weremore » tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.« less

A new nonhuman primate model of severe dry eye.

PubMed

Qin, Yi; Tan, Xiaobo; Zhang, Yingnan; Jie, Ying; Labbe, Antoine; Pan, Zhiqiang

2014-05-01

The aim of this study was to establish a new rhesus monkey model of severe dry eye. A total of 8 rhesus monkeys were used for the study. Four monkeys had their main lacrimal gland and nictitating membrane surgically removed (group 1). Another 4 monkeys had a similar surgery with further application of 50% trichloroacetic acid on the bulbar conjunctiva (group 2). The ocular surface was evaluated before and after the surgery (1, 4, 8, 12, and 24 weeks) using Schirmer-1 test, corneal fluorescein staining, and the lissamine green test. Conjunctival impression cytology was also performed before and 24 weeks after the surgery. Finally, the cornea and the conjunctiva were evaluated using light microscopy. A significant decrease in tear secretion was observed in all operated eyes. Schirmer test data measured were ≤4 mm in all the operated eyes. Slit-lamp examination also revealed abnormal staining in all the operated eyes that remained stable until the end of the experiment. In group 2, corneal fluorescein staining and lissamine green test values were always ≥5 (max 12) and ≥4 (max 9), respectively. Impression cytology specimens of both the treated groups showed conjunctival squamous metaplasia and a decreased number of goblet cells. Under light microscopy, the corneal epithelium appeared irregular with edematous basal epithelial cells. The conjunctiva showed a decreased goblet cell density with infiltration of inflammatory cells. Complete removal of the principal lacrimal gland and nictitating membrane associated with the application of 50% trichloroacetic acid on the conjunctiva could induce severe dry eye in rhesus monkeys.

Intra-Prostate Cancer Vaccine Inducer

DTIC Science & Technology

2006-02-01

analyzed by flowcytometry for Ii and MHC class II expression. The active constructs were used for the Ii suppression in the experiments planned in...care guidelines under an approved protocol. Cell lines and antibodies Green monkey kidney COS cells (#CRL-1650), cultured in RPMI-1640 medium with...AIDS vaccine protection in rhesus monkeys . J Virol 2004;78(14):7490-7. 12. Letvin NL, Montefiori DC, Yasutomi Y, et al. Potent, protective anti-HIV

Somatic Embryogenesis: Still a Relevant Technique in Citrus Improvement.

PubMed

Omar, Ahmad A; Dutt, Manjul; Gmitter, Frederick G; Grosser, Jude W

2016-01-01

The genus Citrus contains numerous fresh and processed fruit cultivars that are economically important worldwide. New cultivars are needed to battle industry threatening diseases and to create new marketing opportunities. Citrus improvement by conventional methods alone has many limitations that can be overcome by applications of emerging biotechnologies, generally requiring cell to plant regeneration. Many citrus genotypes are amenable to somatic embryogenesis, which became a key regeneration pathway in many experimental approaches to cultivar improvement. This chapter provides a brief history of plant somatic embryogenesis with focus on citrus, followed by a discussion of proven applications in biotechnology-facilitated citrus improvement techniques, such as somatic hybridization, somatic cybridization, genetic transformation, and the exploitation of somaclonal variation. Finally, two important new protocols that feature plant regeneration via somatic embryogenesis are provided: protoplast transformation and Agrobacterium-mediated transformation of embryogenic cell suspension cultures.

Inducible somatic embryogenesis in Theobroma cacao achieved using the DEX-activatable transcription factor-glucocorticoid receptor fusion.

PubMed

Shires, Morgan E; Florez, Sergio L; Lai, Tina S; Curtis, Wayne R

2017-11-01

To carry out mass propagation of superior plants to improve agricultural and silvicultural production though advancements in plant cell totipotency, or the ability of differentiated somatic plant cells to regenerate an entire plant. The first demonstration of a titratable control over somatic embryo formation in a commercially relevant plant, Theobroma cacao (Chocolate tree), was achieved using a dexamethasone activatable chimeric transcription factor. This four-fold enhancement in embryo production rate utilized a glucocorticoid receptor fused to an embryogenic transcription factor LEAFY COTYLEDON 2. Where previous T. cacao somatic embryogenesis has been restricted to dissected flower parts, this construct confers an unprecedented embryogenic potential to leaves. Activatable chimeric transcription factors provide a means for elucidating the regulatory cascade associated with plant somatic embryogenesis towards improving its use for somatic regeneration of transgenics and plant propagation.

Ganglion Cell and Displaced Amacrine Cell Density Distribution in the Retina of the Howler Monkey (Alouatta caraya)

PubMed Central

Muniz, José Augusto Pereira Carneiro; de Athaide, Luana Modesto; Gomes, Bruno Duarte; Finlay, Barbara L.; Silveira, Luiz Carlos de Lima

2014-01-01

Unlike all other New World (platyrrine) monkeys, both male and female howler monkeys (Alouatta sp.) are obligatory trichromats. In all other platyrrines, only females can be trichromats, while males are always dichromats, as determined by multiple behavioral, electrophysiological, and genetic studies. In addition to obligatory trichromacy, Alouatta has an unusual fovea, with substantially higher peak cone density in the foveal pit than every other diurnal anthropoid monkey (both platyrrhines and catarrhines) and great ape yet examined, including humans. In addition to documenting the general organization of the retinal ganglion cell layer in Alouatta, the distribution of cones is compared to retinal ganglion cells, to explore possible relationships between their atypical trichromacy and foveal specialization. The number and distribution of retinal ganglion cells and displaced amacrine cells were determined in six flat-mounted retinas from five Alouatta caraya. Ganglion cell density peaked at 0.5 mm between the fovea and optic nerve head, reaching 40,700–45,200 cells/mm2. Displaced amacrine cell density distribution peaked between 0.5–1.75 mm from the fovea, reaching mean values between 2,050–3,100 cells/mm2. The mean number of ganglion cells was 1,133,000±79,000 cells and the mean number of displaced amacrine cells was 537,000±61,800 cells, in retinas of mean area 641±62 mm2. Ganglion cell and displaced amacrine cell density distribution in the Alouatta retina was consistent with that observed among several species of diurnal Anthropoidea, both platyrrhines and catarrhines. The principal alteration in the Alouatta retina appears not to be in the number of any retinal cell class, but rather a marked gradient in cone density within the fovea, which could potentially support high chromatic acuity in a restricted central region. PMID:25546077

Eomesodermin(lo) CTLA4(hi) Alloreactive CD8+ Memory T Cells Are Associated With Prolonged Renal Transplant Survival Induced by Regulatory Dendritic Cell Infusion in CTLA4 Immunoglobulin-Treated Nonhuman Primates.

PubMed

Ezzelarab, Mohamed B; Lu, Lien; Guo, Hao; Zahorchak, Alan F; Shufesky, William F; Cooper, David K C; Morelli, Adrian E; Thomson, Angus W

2016-01-01

Memory T cells (Tmem), particularly those resistant to costimulation blockade (CB), are a major barrier to transplant tolerance. The transcription factor Eomesodermin (Eomes) is critical for Tmem development and maintenance, but its expression by alloactivated T cells has not been examined in nonhuman primates. We evaluated Eomes and coinhibitory cytotoxic T lymphocyte antigen-4 (CTLA4) expression by alloactivated rhesus monkey T cells in the presence of CTLA4 immunoglobulin, both in vitro and in renal allograft recipients treated with CTLA4Ig, with or without regulatory dendritic cell (DCreg) infusion. In normal monkeys, CD8+ T cells expressed significantly more Eomes than CD4+ T cells. By contrast, CD8+ T cells displayed minimal CTLA4. Among T cell subsets, central Tmem (Tcm) expressed the highest levels of Eomes. Notably, Eomes(lo)CTLA4(hi) cells displayed higher levels of CD25 and Foxp3 than Eomes(hi)CTLA4(lo) CD8+ T cells. After allostimulation, distinct proliferating Eomes(lo)CTLA4(hi) and Eomes(hi)CTLA4(lo) CD8+ T cell populations were identified, with a high proportion of Tcm being Eomes(lo)CTLA4(hi). CB with CTLA4Ig during allostimulation of CD8+ T cells reduced CTLA4 but not Eomes expression, significantly reducing Eomes(lo)CTLA4(hi) cells. After transplantation with CB and rapamycin, donor-reactive Eomes(lo)CTLA4(hi) CD8+ T cells were reduced. However, in monkeys also given DCreg, absolute numbers of these cells were elevated significantly. Low Eomes and high CTLA4 expression by donor-reactive CD8+ Tmem is associated with prolonged renal allograft survival induced by DCreg infusion in CTLA4Ig-treated monkeys. Prolonged allograft survival associated with DCreg infusion may be related to maintenance of donor-reactive Eomes(lo)CTLA4(hi) Tcm.

Eomesoderminlo CTLA4hi Alloreactive CD8+ Memory T Cells Are Associated With Prolonged Renal Transplant Survival Induced by Regulatory Dendritic Cell Infusion in CTLA4Ig-Treated Non-Human Primates

PubMed Central

Ezzelarab, Mohamed B.; Lu, Lien; Guo, Hao; Zahorchak, Alan F.; Shufesky, William F.; Cooper, David K.C.; Morelli, Adrian E.; Thomson, Angus W.

2015-01-01

Background Memory T cells (Tmem), particularly those resistant to costimulation blockade (CB), are a major barrier to transplant tolerance. The transcription factor Eomesodermin (Eomes) is critical for Tmem development and maintenance, but its expression by alloactivated T cells has not been examined in non-human primates. Methods We evaluated Eomes and co-inhibitory cytotoxic T lymphocyte antigen-4 (CTLA4) expression by alloactivated rhesus monkey T cells in the presence of CTLA4 immunoglobulin (Ig), both in vitro and in renal allograft recipients treated with CTLA4Ig, with or without regulatory dendritic cell (DCreg) infusion. Results In normal monkeys, CD8+ T cells expressed significantly more Eomes than CD4+T cells. By contrast, CD8+T cells displayed minimal CTLA4. Among T cell subsets, central Tmem (Tcm) expressed the highest levels of Eomes. Notably, EomesloCTLA4hi cells displayed higher levels of CD25 and Foxp3 than EomeshiCTLA4lo CD8+ T cells. Following allostimulation, distinct proliferating EomesloCTLA4hi and EomeshiCTLA4lo CD8+ T cell populations were identified, with a high proportion of Tcm being EomesloCTLA4hi. CB with CTLA4Ig during allostimulation of CD8+T cells reduced CTLA4 but not Eomes expression, significantly reducing EomesloCTLA4hi cells. After transplantation with CB and rapamycin, donor-reactive EomesloCTLA4hi CD8+T cells were reduced. However, in monkeys also given DCreg, absolute numbers of these cells were elevated significantly. Conclusions Low Eomes and high CTLA4 expression by donor-reactive CD8+ Tmem is associated with prolonged renal allograft survival induced by DCreg infusion in CTLA4Ig-treated monkeys. Prolonged allograft survival associated with DCreg infusion may be related to maintenance of donor-reactive EomesloCTLA4hi Tcm. PMID:26680373

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells.

PubMed

Ju, Young Seok; Tubio, Jose M C; Mifsud, William; Fu, Beiyuan; Davies, Helen R; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J; Tan, Benita K T; Aparicio, Samuel; Span, Paul N; Martens, John W M; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Myklebost, Ola; Flanagan, Adrienne M; Foster, Christopher; Neal, David E; Cooper, Colin; Eeles, Rosalind; Bova, Steven G; Lakhani, Sunil R; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L; Purdie, Colin A; Thompson, Alastair M; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J; Stratton, Michael R

2015-06-01

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells. © 2015 Ju et al.; Published by Cold Spring Harbor Laboratory Press.

Frequent somatic transfer of mitochondrial DNA into the nuclear genome of human cancer cells

PubMed Central

Ju, Young Seok; Tubio, Jose M.C.; Mifsud, William; Fu, Beiyuan; Davies, Helen R.; Ramakrishna, Manasa; Li, Yilong; Yates, Lucy; Gundem, Gunes; Tarpey, Patrick S.; Behjati, Sam; Papaemmanuil, Elli; Martin, Sancha; Fullam, Anthony; Gerstung, Moritz; Nangalia, Jyoti; Green, Anthony R.; Caldas, Carlos; Borg, Åke; Tutt, Andrew; Lee, Ming Ta Michael; van't Veer, Laura J.; Tan, Benita K.T.; Aparicio, Samuel; Span, Paul N.; Martens, John W.M.; Knappskog, Stian; Vincent-Salomon, Anne; Børresen-Dale, Anne-Lise; Eyfjörd, Jórunn Erla; Flanagan, Adrienne M.; Foster, Christopher; Neal, David E.; Cooper, Colin; Eeles, Rosalind; Lakhani, Sunil R.; Desmedt, Christine; Thomas, Gilles; Richardson, Andrea L.; Purdie, Colin A.; Thompson, Alastair M.; McDermott, Ultan; Yang, Fengtang; Nik-Zainal, Serena; Campbell, Peter J.; Stratton, Michael R.

2015-01-01

Mitochondrial genomes are separated from the nuclear genome for most of the cell cycle by the nuclear double membrane, intervening cytoplasm, and the mitochondrial double membrane. Despite these physical barriers, we show that somatically acquired mitochondrial-nuclear genome fusion sequences are present in cancer cells. Most occur in conjunction with intranuclear genomic rearrangements, and the features of the fusion fragments indicate that nonhomologous end joining and/or replication-dependent DNA double-strand break repair are the dominant mechanisms involved. Remarkably, mitochondrial-nuclear genome fusions occur at a similar rate per base pair of DNA as interchromosomal nuclear rearrangements, indicating the presence of a high frequency of contact between mitochondrial and nuclear DNA in some somatic cells. Transmission of mitochondrial DNA to the nuclear genome occurs in neoplastically transformed cells, but we do not exclude the possibility that some mitochondrial-nuclear DNA fusions observed in cancer occurred years earlier in normal somatic cells. PMID:25963125

Reduced noradrenergic innervation of ventral midbrain dopaminergic cell groups and the subthalamic nucleus in MPTP-treated parkinsonian monkeys.

PubMed

Masilamoni, Gunasingh Jeyaraj; Groover, Olivia; Smith, Yoland

2017-04-01

There is anatomical and functional evidence that ventral midbrain dopaminergic (DA) cell groups and the subthalamic nucleus (STN) receive noradrenergic innervation in rodents, but much less is known about these interactions in primates. Degeneration of NE neurons in the locus coeruleus (LC) and related brainstem NE cell groups is a well-established pathological feature of Parkinson's disease (PD), but the development of such pathology in animal models of PD has been inconsistent across species and laboratories. We recently demonstrated 30-40% neuronal loss in the LC, A5 and A6 NE cell groups of rhesus monkeys rendered parkinsonian by chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study, we used dopamine-beta-hydroxylase (DβH) immunocytochemistry to assess the impact of this neuronal loss on the number of NE terminal-like varicosities in the substantia nigra pars compacta (SNC), ventral tegmental area (VTA), retrorubral field (RRF) and STN of MPTP-treated parkinsonian monkeys. Our findings reveal that the NE innervation of the ventral midbrain and STN of normal monkeys is heterogeneously distributed being far more extensive in the VTA, RRF and dorsal tier of the SNC than in the ventral SNC and STN. In parkinsonian monkeys, all regions underwent a significant (~50-70%) decrease in NE innervation. At the electron microscopic level, some DβH-positive terminals formed asymmetric axo-dendritic synapses in VTA and STN. These findings demonstrate that the VTA, RRF and SNCd are the main ventral midbrain targets of ascending NE inputs, and that these connections undergo a major break-down in chronically MPTP-treated parkinsonian monkeys. This severe degeneration of the ascending NE system may contribute to the pathophysiology of ventral midbrain and STN neurons in PD. Copyright © 2017 Elsevier Inc. All rights reserved.

Induction of immune response in macaque monkeys infected with simian-human immunodeficiency virus having the TNF-{alpha} gene at an early stage of infection

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

Shimizu, Yuya; Miyazaki, Yasuyuki; Ibuki, Kentaro

2005-12-20

TNF-{alpha} has been implicated in the pathogenesis of, and the immune response against, HIV-1 infection. To clarify the roles of TNF-{alpha} against HIV-1-related virus infection in an SHIV-macaque model, we genetically engineered an SHIV to express the TNF-{alpha} gene (SHIV-TNF) and characterized the virus's properties in vivo. After the acute viremic stage, the plasma viral loads declined earlier in the SHIV-TNF-inoculated monkeys than in the parental SHIV (SHIV-NI)-inoculated monkeys. SHIV-TNF induced cell death in the lymph nodes without depletion of circulating CD4{sup +} T cells. SHIV-TNF provided some immunity in monkeys by increasing the production of the chemokine RANTES andmore » by inducing an antigen-specific proliferation of lymphocytes. The monkeys immunized with SHIV-TNF were partly protected against a pathogenic SHIV (SHIV-C2/1) challenge. These findings suggest that TNF-{alpha} contributes to the induction of an effective immune response against HIV-1 rather than to the progression of disease at the early stage of infection.« less

Lethal canine distemper virus outbreak in cynomolgus monkeys in Japan in 2008.

PubMed

Sakai, Kouji; Nagata, Noriyo; Ami, Yasushi; Seki, Fumio; Suzaki, Yuriko; Iwata-Yoshikawa, Naoko; Suzuki, Tadaki; Fukushi, Shuetsu; Mizutani, Tetsuya; Yoshikawa, Tomoki; Otsuki, Noriyuki; Kurane, Ichiro; Komase, Katsuhiro; Yamaguchi, Ryoji; Hasegawa, Hideki; Saijo, Masayuki; Takeda, Makoto; Morikawa, Shigeru

2013-01-01

Canine distemper virus (CDV) has recently expanded its host range to nonhuman primates. A large CDV outbreak occurred in rhesus monkeys at a breeding farm in Guangxi Province, China, in 2006, followed by another outbreak in rhesus monkeys at an animal center in Beijing in 2008. In 2008 in Japan, a CDV outbreak also occurred in cynomolgus monkeys imported from China. In that outbreak, 46 monkeys died from severe pneumonia during a quarantine period. A CDV strain (CYN07-dV) was isolated in Vero cells expressing dog signaling lymphocyte activation molecule (SLAM). Phylogenic analysis showed that CYN07-dV was closely related to the recent CDV outbreaks in China, suggesting continuing chains of CDV infection in monkeys. In vitro, CYN07-dV uses macaca SLAM and macaca nectin4 as receptors as efficiently as dog SLAM and dog nectin4, respectively. CYN07-dV showed high virulence in experimentally infected cynomolgus monkeys and excreted progeny viruses in oral fluid and feces. These data revealed that some of the CDV strains, like CYN07-dV, have the potential to cause acute systemic infection in monkeys.

Bovine somatic cell nuclear transfer.

PubMed

Ross, Pablo J; Cibelli, Jose B

2010-01-01

Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes.

Cognitive Control Functions of Anterior Cingulate Cortex in Macaque Monkeys Performing a Wisconsin Card Sorting Test Analog

PubMed Central

Kuwabara, Masaru; Mansouri, Farshad A.; Buckley, Mark J.

2014-01-01

Monkeys were trained to select one of three targets by matching in color or matching in shape to a sample. Because the matching rule frequently changed and there were no cues for the currently relevant rule, monkeys had to maintain the relevant rule in working memory to select the correct target. We found that monkeys' error commission was not limited to the period after the rule change and occasionally occurred even after several consecutive correct trials, indicating that the task was cognitively demanding. In trials immediately after such error trials, monkeys' speed of selecting targets was slower. Additionally, in trials following consecutive correct trials, the monkeys' target selections for erroneous responses were slower than those for correct responses. We further found evidence for the involvement of the cortex in the anterior cingulate sulcus (ACCs) in these error-related behavioral modulations. First, ACCs cell activity differed between after-error and after-correct trials. In another group of ACCs cells, the activity differed depending on whether the monkeys were making a correct or erroneous decision in target selection. Second, bilateral ACCs lesions significantly abolished the response slowing both in after-error trials and in error trials. The error likelihood in after-error trials could be inferred by the error feedback in the previous trial, whereas the likelihood of erroneous responses after consecutive correct trials could be monitored only internally. These results suggest that ACCs represent both context-dependent and internally detected error likelihoods and promote modes of response selections in situations that involve these two types of error likelihood. PMID:24872558

Culture of prostate epithelial cells of the rhesus monkey on extracellular matrix substrate: influence of steroids and insulin-like growth factors.

PubMed

Udayakumar, T S; Jeyaraj, D A; Rajalakshmi, M; Sharma, R S

1999-09-01

Rhesus monkey prostate epithelial cells from the cranial lobe were isolated and cultured in flasks coated either with collagen IV or laminin. The effects of stromal cell medium, androgens and growth factors on cell number, thymidine incorporation and secretory activity were assessed. The results indicate that dihydrotestosterone (DHT) and androstenedione have stimulatory influences on cell proliferation and secretion in coated flasks. DHT was more effective in increasing cell number but the induction of secretory activity was similar with both steroids. The combination of IGF-I and -II resulted in inducing better cell proliferation and secretory activity than the individual IGFs but, of the two IGFs, IGF-I was more effective than IGF-II. DHT with IGFs was more potent in inducing proliferation, differentiation and secretion than androstenedione. Even in the absence of steroids or growth factors, colony formation and confluence occurred in coated flasks but cell differentiation and secretion only to a limited extent. In conclusion, we were able to establish an in vitro primary culture of prostate epithelial cells from rhesus monkey using extracellular matrix proteins, steroids and growth factors as additional supplements. This culture system may be useful to study prostate cell physiology and to identify drugs that can inhibit cell proliferation.

Chromosome microduplication in somatic cells decreases the genetic stability of human reprogrammed somatic cells and results in pluripotent stem cells.

PubMed

Yu, Yang; Chang, Liang; Zhao, Hongcui; Li, Rong; Fan, Yong; Qiao, Jie

2015-05-12

Human pluripotent stem cells, including cloned embryonic and induced pluripotent stem cells, offer a limitless cellular source for regenerative medicine. However, their derivation efficiency is limited, and a large proportion of cells are arrested during reprogramming. In the current study, we explored chromosome microdeletion/duplication in arrested and established reprogrammed cells. Our results show that aneuploidy induced by somatic cell nuclear transfer technology is a key factor in the developmental failure of cloned human embryos and primary colonies from implanted cloned blastocysts and that expression patterns of apoptosis-related genes are dynamically altered. Overall, ~20%-53% of arrested primary colonies in induced plurpotent stem cells displayed aneuploidy, and upregulation of P53 and Bax occurred in all arrested primary colonies. Interestingly, when somatic cells with pre-existing chromosomal mutations were used as donor cells, no cloned blastocysts were obtained, and additional chromosomal mutations were detected in the resulting iPS cells following long-term culture, which was not observed in the two iPS cell lines with normal karyotypes. In conclusion, aneuploidy induced by the reprogramming process restricts the derivation of pluripotent stem cells, and, more importantly, pre-existing chromosomal mutations enhance the risk of genome instability, which limits the clinical utility of these cells.

Health status and productive performance of somatic cell cloned cattle and their offspring produced in Japan.

PubMed

Watanabe, Shinya; Nagai, Takashi

2008-02-01

Since the first somatic cell cloned calves were born in Japan in 1998, more than 500 cloned cattle have been produced by somatic cell nuclear transfer and many studies concerning cloned cattle and their offspring have been conducted in this country. However, most of the results have been published in Japanese; thus, the data produced in this country is not well utilized by researchers throughout the world. This article reviews the 65 reports produced by Japanese researchers (62 written in Japanese and 3 written in English), which employed 171 clones and 32 offspring, and categorizes them according to the following 7 categories: (1) genetic similarities and muzzle prints, (2) hematology and clinical chemistry findings, (3) pathology, (4) growth performance, (5) reproductive performance, (6) meat production performance and (7) milk production performance. No remarkable differences in health status or reproductive performance were found among conventionally bred cattle, somatic cell cloned cattle surviving to adulthood and offspring of somatic cell cloned cattle. Similarities in growth performance and meat quality were observed between nuclear donor cattle and their clones. The growth curves of the offspring resembled those of their full siblings.

Estimation of Mineral and Trace Element Profile in Bubaline Milk Affected with Subclinical Mastitis.

PubMed

Singh, Mahavir; Yadav, Poonam; Sharma, Anshu; Garg, V K; Mittal, Dinesh

2017-04-01

The milk samples from buffaloes of Murrah breed at mid lactation stage, reared at an organised dairy farm, were screened for subclinical mastitis based on bacteriological examination and somatic cell count following International Dairy Federation criteria. Milk samples from subclinical mastitis infected and healthy buffaloes were analysed to evaluate physicochemical alterations in terms of protein, fat, pH, electrical conductivity, chloride, minerals (sodium, potassium and calcium) and trace elements (iron, zinc, copper and selenium). In the present study, protein, fat, zinc, iron, calcium and selenium content was significantly lower (P < 0.001), while pH and electrical conductivity were significantly higher in mastitic milk as compared to normal milk. Concentration of electrolytes mainly sodium and chloride significantly increased with higher somatic cell count in mastitic milk and to maintain osmolality; potassium levels decreased proportionately. Correlation matrix revealed significantly positive interdependences of somatic cell count with pH, electrical conductivity, sodium and chloride. However, protein, fat, calcium and potassium were correlated negatively with elevated somatic cell count in mastitic milk. It is concluded that udder infections resulting in elevated somatic cells may alter the mineral and trace element profile of milk, and magnitude of changes may have diagnostic and prognostic value.

Inducing somatic meiosis-like reduction at high frequency by caffeine in root-tip cells of Vicia faba.

PubMed

Chen, Y; Zhang, L; Zhou, Y; Geng, Y; Chen, Z

2000-07-20

Germinated seeds of Vicia faba were treated in caffeine solutions of different concentration for different durations to establish the inducing system of somatic meiosis-like reduction. The highest frequency of somatic meiosis-like reduction could reach up to 54.0% by treating the root tips in 70 mmol/l caffeine solution for 2 h and restoring for 24 h. Two types of somatic meiosis-like reduction were observed. One was reductional grouping, in which the chromosomes in a cell usually separated into two groups, and the role of spindle fibers did not show. The other type was somatic meiosis, which was analogous to meiosis presenting in gametogenesis, and chromosome pairing and chiasmata were visualized.

The nucleus pararaphales in the human, chimpanzee, and macaque monkey.

PubMed

Baizer, Joan S; Weinstock, Nadav; Witelson, Sandra F; Sherwood, Chet C; Hof, Patrick R

2013-03-01

The human cerebral cortex and cerebellum are greatly expanded compared to those of other mammals, including the great apes. This expansion is reflected in differences in the size and organization of precerebellar brainstem structures, such as the inferior olive. In addition, there are cell groups unique to the human brainstem. One such group may be the nucleus pararaphales (PRa); however, there is disagreement among authors about the size and location of this nucleus in the human brainstem. The name "pararaphales" has also been used for neurons in the medulla shown to project to the flocculus in the macaque monkey. We have re-examined the existence and status of the PRa in eight humans, three chimpanzees, and four macaque monkeys using Nissl-stained sections as well as immunohistochemistry. In the human we found a cell group along the midline of the medulla in all cases; it had the form of interrupted cell columns and was variable among cases in rostrocaudal and dorsoventral extent. Cells and processes were highly immunoreactive for non-phosphorylated neurofilament protein (NPNFP); somata were immunoreactive to the synthetic enzyme for nitric oxide, nitric oxide synthase, and for calretinin. In macaque monkey, there was a much smaller oval cell group with NPNFP immunoreactivity. In the chimpanzee, we found a region of NPNFP-immunoreactive cells and fibers similar to what was observed in macaques. These results suggest that the "PRa" in the human may not be the same structure as the flocculus-projecting cell group described in the macaque. The PRa, like the arcuate nucleus, therefore may be unique to humans.

Stochastic modeling indicates that aging and somatic evolution in the hematopoetic system are driven by non-cell-autonomous processes.

PubMed

Rozhok, Andrii I; Salstrom, Jennifer L; DeGregori, James

2014-12-01

Age-dependent tissue decline and increased cancer incidence are widely accepted to be rate-limited by the accumulation of somatic mutations over time. Current models of carcinogenesis are dominated by the assumption that oncogenic mutations have defined advantageous fitness effects on recipient stem and progenitor cells, promoting and rate-limiting somatic evolution. However, this assumption is markedly discrepant with evolutionary theory, whereby fitness is a dynamic property of a phenotype imposed upon and widely modulated by environment. We computationally modeled dynamic microenvironment-dependent fitness alterations in hematopoietic stem cells (HSC) within the Sprengel-Liebig system known to govern evolution at the population level. Our model for the first time integrates real data on age-dependent dynamics of HSC division rates, pool size, and accumulation of genetic changes and demonstrates that somatic evolution is not rate-limited by the occurrence of mutations, but instead results from aged microenvironment-driven alterations in the selective/fitness value of previously accumulated genetic changes. Our results are also consistent with evolutionary models of aging and thus oppose both somatic mutation-centric paradigms of carcinogenesis and tissue functional decline. In total, we demonstrate that aging directly promotes HSC fitness decline and somatic evolution via non-cell-autonomous mechanisms.

Somatic and germinal cells' interrelationship in the course of seminiferous tubule maturation in man.

PubMed

Kula, K; Romer, T E; Wlodarczyk, W P

1980-02-01

Certain successive phases of seminiferous tubule maturation were observed in a transsection of a Leydig cell adenoma-bearing testis of a boy with precocious puberty. Massively accumulated Leydig cells may stimulate the maturation of Sertoli cells, as indicated by progressive replacement of Sertoli cell precursors by mature Sertoli cells at a distance closer to the adenoma. On the other hand, tubules less advanced in maturation contained a higher number of somatic cells than those more advanced in maturation. Leydig-cell-dependent maturation of Sertoli cells may be in competition with Certoli cell multiplication, or numerous undifferentiated somatic cells may undergo a natural elimination in the course of tubular maturation. An inverse relation between the number of Sertoli cell precursors and the number of meiotic spermatocytes suggests that quantitative reduction of Sertoli cell precursors may be important for the intratubular milieu necessary for the onset of the first meiosis in man.

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

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

Watanabe, Masahito; Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571; Umeyama, Kazuhiro

2010-11-05

Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor themore » exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.« less

Fetal Neuropathology in Zika Virus-Infected Pregnant Female Rhesus Monkeys.

PubMed

Martinot, Amanda J; Abbink, Peter; Afacan, Onur; Prohl, Anna K; Bronson, Roderick; Hecht, Jonathan L; Borducchi, Erica N; Larocca, Rafael A; Peterson, Rebecca L; Rinaldi, William; Ferguson, Melissa; Didier, Peter J; Weiss, Deborah; Lewis, Mark G; De La Barrera, Rafael A; Yang, Edward; Warfield, Simon K; Barouch, Dan H

2018-05-17

The development of interventions to prevent congenital Zika syndrome (CZS) has been limited by the lack of an established nonhuman primate model. Here we show that infection of female rhesus monkeys early in pregnancy with Zika virus (ZIKV) recapitulates many features of CZS in humans. We infected 9 pregnant monkeys with ZIKV, 6 early in pregnancy (weeks 6-7 of gestation) and 3 later in pregnancy (weeks 12-14 of gestation), and compared findings with uninfected controls. 100% (6 of 6) of monkeys infected early in pregnancy exhibited prolonged maternal viremia and fetal neuropathology, including fetal loss, smaller brain size, and histopathologic brain lesions, including microcalcifications, hemorrhage, necrosis, vasculitis, gliosis, and apoptosis of neuroprogenitor cells. High-resolution MRI demonstrated concordant lesions indicative of deep gray matter injury. We also observed spinal, ocular, and neuromuscular pathology. Our data show that vascular compromise and neuroprogenitor cell dysfunction are hallmarks of CZS pathogenesis, suggesting novel strategies to prevent and to treat this disease. Copyright © 2018 Elsevier Inc. All rights reserved.

De novo DNA methylation during monkey pre-implantation embryogenesis.

PubMed

Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-04-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis.

De novo DNA methylation during monkey pre-implantation embryogenesis

PubMed Central

Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-01-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis. PMID:28233770

Human somatic cell nuclear transfer and reproductive cloning: an Ethics Committee opinion.

PubMed

2016-04-01

This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer and cloning," last published in Fertil Steril 2012;98:804-7. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Spontaneous Epithelioid Hemangiosarcoma in a Rhesus Monkey (Macaca mulatta)

PubMed Central

Tsuchiya, Takayuki; Gray, Tasha L; Gatto, Nicholas T; Forest, Thomas; Machotka, Sam V; Troth, Sean P; Prahalada, Srinivasa

2014-01-01

Epithelioid hemangiosarcoma is a rare malignant endothelial neoplasia with a unique, predominantly epithelioid morphology. A 4-y-old rhesus monkey from our laboratory had multiple neoplastic nodules in a digit, limb skin, hindlimb muscle, and visceral organs including lung, heart, and brain. The nodules were composed of pleomorphic, polygonal, epithelioid, neoplastic cells that were arranged in sheets, nests, and cords and supported by variably dense fibrovascular connective tissue. The morphologic features of this tumor were predominantly epithelioid. However, some regions contained cystic spaces, clefts, and channel-like structures, all of which were lined with morphologically distinct neoplastic endothelial cells. These neoplastic cells, with or without epithelioid morphology, were positive immunohistochemically for CD31, factor VIII-related antigen, and vimentin. The presence of multiple metastatic nodules, high mitotic rate, and extensive Ki67-positive staining were consistent with malignancy. This report is the first description of epithelioid hemangiosarcoma in a rhesus monkey. PMID:25296017

Spontaneous epithelioid hemangiosarcoma in a rhesus monkey (Macaca mulatta).

PubMed

Tsuchiya, Takayuki; Gray, Tasha L; Gatto, Nicholas T; Forest, Thomas; Machotka, Sam V; Troth, Sean P; Prahalada, Srinivasa

2014-08-01

Epithelioid hemangiosarcoma is a rare malignant endothelial neoplasia with a unique, predominantly epithelioid morphology. A 4-y-old rhesus monkey from our laboratory had multiple neoplastic nodules in a digit, limb skin, hindlimb muscle, and visceral organs including lung, heart, and brain. The nodules were composed of pleomorphic, polygonal, epithelioid, neoplastic cells that were arranged in sheets, nests, and cords and supported by variably dense fibrovascular connective tissue. The morphologic features of this tumor were predominantly epithelioid. However, some regions contained cystic spaces, clefts, and channel-like structures, all of which were lined with morphologically distinct neoplastic endothelial cells. These neoplastic cells, with or without epithelioid morphology, were positive immunohistochemically for CD31, factor VIII-related antigen, and vimentin. The presence of multiple metastatic nodules, high mitotic rate, and extensive Ki67-positive staining were consistent with malignancy. This report is the first description of epithelioid hemangiosarcoma in a rhesus monkey.

Synchronization of Somatic Embryogenesis in Date Palm Suspension Culture Using Abscisic Acid.

PubMed

Alwael, Hussain A; Naik, Poornananda M; Al-Khayri, Jameel M

2017-01-01

Somatic embryogenesis is considered the most effective method for commercial propagation of date palm. However, the limitation of obtaining synchronized development of somatic embryos remains an impediment. The synchronization of somatic embryo development is ideal for the applications to produce artificial seeds. Abscisic acid (ABA) is associated with stress response and influences in vitro growth and development. This chapter describes an effective method to achieve synchronized development of somatic embryos in date palm cell suspension culture. Among the ABA concentrations tested (0, 1, 10, 50, 100 μM), the best synchronized growth was obtained in response to 50-100 μM. Here we provide a comprehensive protocol for in vitro plant regeneration of date palm starting with shoot-tip explant, callus initiation and growth, cell suspension establishment, embryogenesis synchronization with ABA treatment, somatic embryo germination, and rooting as well as acclimatized plantlet establishment.

Somatic hybridization of sexually incompatible petunias: Petunia parodii, Petunia parviflora.

PubMed

Power, J B; Berry, S F; Chapman, J V; Cocking, E C

1980-01-01

Somatic hybrid plants were regenerated following the fusion of leaf mesophyll protoplasts of P. parodii with those isolated from a nuclear-albino mutant of P. parviflora. Attempts at sexual hybridization of these two species repeatedly failed thus confirming their previously established cross-incompatibility. Selection of somatic hybrid plants was possible since protoplasts of P. parodii would not develop beyond the cell colony stage, whilst those of the somatic hybrid and albino P. parviflora produced calluses. Green somatic hybrid calluses were visible against a background of albino cells/calluses, and upon transfer to regeneration media gave rise to shoots. Shoots and the resultant flowering plants were confirmed as somatic hybrids based on their growth habit, floral pigmentation and morphology, leaf hair structure, chromosome number and Fraction 1 protein profiles. The relevance of such hybrid material for the development of new, and extensively modified cultivars, is discussed.

Notch Signaling Regulates Ovarian Follicle Formation and Coordinates Follicular Growth

PubMed Central

Vanorny, Dallas A.; Prasasya, Rexxi D.; Chalpe, Abha J.; Kilen, Signe M.

2014-01-01

Ovarian follicles form through a process in which somatic pregranulosa cells encapsulate individual germ cells from germ cell syncytia. Complementary expression of the Notch ligand, Jagged1, in germ cells and the Notch receptor, Notch2, in pregranulosa cells suggests a role for Notch signaling in mediating cellular interactions during follicle assembly. Using a Notch reporter mouse, we demonstrate that Notch signaling is active within somatic cells of the embryonic ovary, and these cells undergo dramatic reorganization during follicle histogenesis. This coincides with a significant increase in the expression of the ligands, Jagged1 and Jagged2; the receptor, Notch2; and the target genes, Hes1 and Hey2. Histological examination of ovaries from mice with conditional deletion of Jagged1 within germ cells (J1 knockout [J1KO]) or Notch2 within granulosa cells (N2 knockout [N2KO]) reveals changes in follicle dynamics, including perturbations in the primordial follicle pool and antral follicle development. J1KO and N2KO ovaries also contain multi-oocytic follicles, which represent a failure to resolve germ cell syncytia, and follicles with enlarged oocytes but lacking somatic cell growth, signifying a potential role of Notch signaling in follicle activation and the coordination of follicle development. We also observed decreased cell proliferation and increased apoptosis in the somatic cells of both conditional knockout lines. As a consequence of these defects, J1KO female mice are subfertile; however, N2KO female mice remain fertile. This study demonstrates important functions for Jagged1 and Notch2 in the resolution of germ cell syncytia and the coordination of somatic and germ cell growth within follicles of the mouse ovary. PMID:24552588

Spontaneous generation of germline characteristics in mouse fibrosarcoma cells

NASA Astrophysics Data System (ADS)

Ma, Zhan; Hu, Yao; Jiang, Guoying; Hou, Jun; Liu, Ruilai; Lu, Yuan; Liu, Chunfang

2012-10-01

Germline/embryonic-specific genes have been found to be activated in somatic tumors. In this study, we further showed that cells functioning as germline could be present in mouse fibrosarcoma cells (L929 cell line). Early germline-like cells spontaneously appeared in L929 cells and further differentiated into oocyte-like cells. These germline-like cells can, in turn, develop into blastocyst-like structures in vitro and cause teratocarcinomas in vivo, which is consistent with natural germ cells in function. Generation of germline-like cells from somatic tumors might provide a novel way to understand why somatic cancer cells have strong features of embryonic/germline development. It is thought that the germline traits of tumors are associated with the central characteristics of malignancy, such as immortalization, invasion, migration and immune evasion. Therefore, germline-like cells in tumors might provide potential targets to tumor biology, diagnosis and therapy.

Changes in cell-cycle kinetics responsible for limiting somatic growth in mice

PubMed Central

Chang, Maria; Parker, Elizabeth A.; Muller, Tessa J. M.; Haenen, Caroline; Mistry, Maanasi; Finkielstain, Gabriela P.; Murphy-Ryan, Maureen; Barnes, Kevin M.; Sundaram, Rajeshwari; Baron, Jeffrey

2009-01-01

In mammals, the rate of somatic growth is rapid in early postnatal life but then slows with age, approaching zero as the animal approaches adult body size. To investigate the underlying changes in cell-cycle kinetics, [methyl-3H]thymidine and 5’-bromo-2’deoxyuridine were used to double-label proliferating cells in 1-, 2-, and 3-week-old mice for four weeks. Proliferation of renal tubular epithelial cells and hepatocytes decreased with age. The average cell-cycle time did not increase in liver and increased only 1.7 fold in kidney. The fraction of cells in S-phase that will divide again declined approximately 10 fold with age. Concurrently, average cell area increased approximately 2 fold. The findings suggest that somatic growth deceleration primarily results not from an increase in cell-cycle time but from a decrease in growth fraction (fraction of cells that continue to proliferate). During the deceleration phase, cells appear to reach a proliferative limit and undergo their final cell divisions, staggered over time. Concomitantly, cells enlarge to a greater volume, perhaps because they are relieved of the size constraint imposed by cell division. In conclusion, a decline in growth fraction with age causes somatic growth deceleration and thus sets a fundamental limit on adult body size. PMID:18535488

[Product safety analysis of somatic cell cloned bovine].

PubMed

Hua, Song; Lan, Jie; Song, Yongli; Lu, Chenglong; Zhang, Yong

2010-05-01

Somatic cell cloning (nuclear transfer) is a technique through which the nucleus (DNA) of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. It could be applied for the enhancement of reproduction rate and the improvement of food products involving quality, yield and nutrition. In recent years, the United States, Japan and Europe as well as other countries announced that meat and milk products made from cloned cattle are safe for human consumption. Yet, cloned animals are faced with a wide range of health problems, with a high death rate and a high incidence of disease. The precise causal mechanisms for the low efficiency of cloning remain unclear. Is it safe that any products from cloned animals were allowed into the food supply? This review focuses on the security of meat, milk and products from cloned cattle based on the available data.

Advances in reprogramming somatic cells to induced pluripotent stem cells.

PubMed

Patel, Minal; Yang, Shuying

2010-09-01

Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.

Somatic embryogenesis in ferns: a new experimental system.

PubMed

Mikuła, Anna; Pożoga, Mariusz; Tomiczak, Karolina; Rybczyński, Jan J

2015-05-01

Somatic embryogenesis has never been reported in ferns. The study showed that it is much easier to evoke the acquisition and expression of embryogenic competence in ferns than in spermatophytes. We discovered that the tree fern Cyathea delgadii offers an effective model for the reproducible and rapid formation of somatic embryos on hormone-free medium. Our study provides cyto-morphological evidence for the single cell origin and development of somatic embryos. Somatic embryogenesis (SE) in both primary and secondary explants was induced on half-strength micro- and macro-nutrients Murashige and Skoog medium without the application of exogenous plant growth regulators, in darkness. The early stage of SE was characterized by sequential perpendicular cell divisions of an individual epidermal cell of etiolated stipe explant. These resulted in the formation of a linear pro-embryo. Later their development resembled that of the zygotic embryo. We defined three morphogenetic stages of fern somatic embryo development: linear, early and late embryonic leaf stage. The transition from somatic embryo to juvenile sporophyte was quick and proceeded without interruption caused by dormancy. Following 9 weeks of culture the efficiency of somatic embryogenesis reached 12-13 embryos per responding explant. Spontaneous formation of somatic embryos and callus production, which improved the effectiveness of the process sevenfold in 10-month-long culture, occurred without subculturing. The tendency for C. delgadii to propagate by SE in vitro makes this species an excellent model for studies relating to asexual embryogenesis and the endogenous hormonal regulation of that process and opens new avenues of experimentation.

Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos.

PubMed

Sugimura, S; Narita, K; Yamashiro, H; Sugawara, A; Shoji, T; Terashita, Y; Nishimori, K; Konno, T; Yoshida, M; Sato, E

2009-09-01

Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (P<0.05). The use of donor cells of any type in later passages decreased the rate of blastocyst formation. Treatment with trichostatin-A did not improve the rate of blastocyst formation from cleaved dewclaw cell-derived embryos but did so in the embryos derived from the tail-tip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.

Visual motion integration by neurons in the middle temporal area of a New World monkey, the marmoset

PubMed Central

Solomon, Selina S; Tailby, Chris; Gharaei, Saba; Camp, Aaron J; Bourne, James A; Solomon, Samuel G

2011-01-01

Abstract The middle temporal area (MT/V5) is an anatomically distinct region of primate visual cortex that is specialized for the processing of image motion. It is generally thought that some neurons in area MT are capable of signalling the motion of complex patterns, but this has only been established in the macaque monkey. We made extracellular recordings from single units in area MT of anaesthetized marmosets, a New World monkey. We show through quantitative analyses that some neurons (35 of 185; 19%) are capable of signalling pattern motion (‘pattern cells’). Across several dimensions, the visual response of pattern cells in marmosets is indistinguishable from that of pattern cells in macaques. Other neurons respond to the motion of oriented contours in a pattern (‘component cells’) or show intermediate properties. In addition, we encountered a subset of neurons (22 of 185; 12%) insensitive to sinusoidal gratings but very responsive to plaids and other two-dimensional patterns and otherwise indistinguishable from pattern cells. We compared the response of each cell class to drifting gratings and dot fields. In pattern cells, directional selectivity was similar for gratings and dot fields; in component cells, directional selectivity was weaker for dot fields than gratings. Pattern cells were more likely to have stronger suppressive surrounds, prefer lower spatial frequencies and prefer higher speeds than component cells. We conclude that pattern motion sensitivity is a feature of some neurons in area MT of both New and Old World monkeys, suggesting that this functional property is an important stage in motion analysis and is likely to be conserved in humans. PMID:21946851

Genetic analysis of microglandular adenosis and acinic cell carcinomas of the breast provides evidence for the existence of a low-grade triple-negative breast neoplasia family.

PubMed

Geyer, Felipe C; Berman, Samuel H; Marchiò, Caterina; Burke, Kathleen A; Guerini-Rocco, Elena; Piscuoglio, Salvatore; Ng, Charlotte Ky; Pareja, Fresia; Wen, Hannah Y; Hodi, Zoltan; Schnitt, Stuart J; Rakha, Emad A; Ellis, Ian O; Norton, Larry; Weigelt, Britta; Reis-Filho, Jorge S

2017-01-01

Acinic cell carcinoma is an indolent form of invasive breast cancer, whereas microglandular adenosis has been shown to be a neoplastic proliferation. Both entities display a triple-negative phenotype, and may give rise to and display somatic genomic alterations typical of high-grade triple-negative breast cancers. Here we report on a comparison of previously published data on eight carcinoma-associated microglandular adenosis and eight acinic cell carcinomas subjected to targeted massively parallel sequencing targeting all exons of 236 genes recurrently mutated in breast cancer and/or DNA repair-related. Somatic mutations, insertions/ deletions, and copy number alterations were detected using state-of-the-art bioinformatic algorithms. All cases were of triple-negative phenotype. A median of 4.5 (1-13) and 4.0 (1-7) non-synonymous somatic mutations per carcinoma-associated microglandular adenosis and acinic cell carcinoma were identified, respectively. TP53 was the sole highly recurrently mutated gene (75% in microglandular adenosis versus 88% in acinic cell carcinomas), and TP53 mutations were consistently coupled with loss of heterozygosity of the wild-type allele. Additional somatic mutations shared by both groups included those in BRCA1, PIK3CA, and INPP4B. Recurrent (n=2) somatic mutations restricted to microglandular adenosis or acinic cell carcinomas included those affecting PTEN and MED12 or ERBB4, respectively. No significant differences in the repertoire of somatic mutations were detected between microglandular adenosis and acinic cell carcinomas, and between this group of lesions and 77 triple-negative carcinomas from The Cancer Genome Atlas. Microglandular adenosis and acinic cell carcinomas, however, were genetically distinct from estrogen receptor-positive and/or HER2-positive breast cancers from The Cancer Genome Atlas. Our findings support the contention that microglandular adenosis and acinic cell carcinoma are part of the same spectrum of lesions harboring frequent TP53 somatic mutations, and likely represent low-grade forms of triple-negative disease with no/minimal metastatic potential, of which a subset has the potential to progress to high-grade triple-negative breast cancer.

Genetic Analysis of Microglandular Adenosis and Acinic Cell Carcinomas of the Breast Provides Evidence for the Existence of a Low-grade Triple-Negative Breast Neoplasia Family

PubMed Central

Geyer, Felipe C; Berman, Samuel H.; Marchiò, Caterina; Burke, Kathleen A; Guerini-Rocco, Elena; Piscuoglio, Salvatore; Ng, Charlotte K Y; Pareja, Fresia; Wen, Hannah Y; Hodi, Zoltan; Schnitt, Stuart J; Rakha, Emad A; Ellis, Ian O; Norton, Larry; Weigelt, Britta; Reis-Filho, Jorge S

2016-01-01

Acinic cell carcinoma is an indolent form of invasive breast cancer, whereas microglandular adenosis has been shown to be a neoplastic proliferation. Both entities display a triple-negative phenotype, and may give rise to and display somatic genomic alterations typical of high-grade triple-negative breast cancers. Here we report on a comparison of previously published data on eight carcinoma-associated microglandular adenosis and eight acinic cell carcinomas subjected to targeted massively parallel sequencing targeting all exons of 236 genes recurrently mutated in breast cancer and/or DNA repair-related. Somatic mutations, insertions/deletions and copy number alterations were detected using state-of-the-art bioinformatic algorithms. All cases were of triple-negative phenotype. A median of 4.5 (1–13) and 4.0 (1–7) non-synonymous somatic mutations per carcinoma-associated microglandular adenosis and acinic cell carcinoma were identified, respectively. TP53 was the sole highly recurrently mutated gene (75% in microglandular adenosis versus 88% in acinic cell carcinomas), and TP53 mutations were consistently coupled with loss of heterozygosity of the wild-type allele. Additional somatic mutations shared by both groups included those in BRCA1, PIK3CA and INPP4B. Recurrent (n=2) somatic mutations restricted to microglandular adenosis or acinic cell carcinomas included those affecting PTEN and MED12, or ERBB4, respectively. No significant differences in the repertoire of somatic mutations were detected between microglandular adenosis and acinic cell carcinomas, and between this group of lesions and 77 triple-negative carcinomas from The Cancer Genome Atlas. Microglandular adenosis and acinic cell carcinomas, however, were genetically distinct from estrogen receptor-positive and/or HER2-positive breast cancers from The Cancer Genome Atlas. Our findings support the contention that microglandular adenosis and acinic cell carcinoma are part of the same spectrum of lesions harboring frequent TP53 somatic mutations, and likely represent low-grade forms of triple-negative disease with no/minimal metastatic potential, of which a subset has the potential to progress to high-grade triple-negative breast cancer. PMID:27713419

Relationship of milking rate to somatic cell count.

PubMed

Brown, C A; Rischette, S J; Schultz, L H

1986-03-01

Information on milking rate, monthly bucket somatic cell counts, mastitis treatment, and milk production was obtained from 284 lactations of Holstein cows separated into three lactation groups. Significant correlations between somatic cell count (linear score) and other parameters included production in lactation 1 (-.185), production in lactation 2 (-.267), and percent 2-min milk in lactation 2 (.251). Somatic cell count tended to increase with maximum milking rate in all lactations, but correlations were not statistically significant. Twenty-nine percent of cows with milking rate measurements were treated for clinical mastitis. Treated cows in each lactation group produced less milk than untreated cows. In the second and third lactation groups, treated cows had a shorter total milking time and a higher percent 2-min milk than untreated cows, but differences were not statistically significant. Overall, the data support the concept that faster milking cows tend to have higher cell counts and more mastitis treatments, particularly beyond first lactation. However, the magnitude of the relationship was small.

Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features

PubMed Central

Medrano, Jose V.; Martínez-Arroyo, Ana M.; Míguez, Jose M.; Moreno, Inmaculada; Martínez, Sebastián; Quiñonero, Alicia; Díaz-Gimeno, Patricia; Marqués-Marí, Ana I.; Pellicer, Antonio; Remohí, Jose; Simón, Carlos

2016-01-01

The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans. PMID:27112843

In vitro induction of protein complexes between bevacizumab, VEGF-A¹⁶⁵ and heparin: explanation for deposits observed on endothelial veins in monkey eyes.

PubMed

Julien, Sylvie; Biesemeier, Antje; Schraermeyer, Ulrich

2013-04-01

By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin. Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies. Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells. Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.

Phosphorylated α-Synuclein Accumulations and Lewy Body-like Pathology Distributed in Parkinson's Disease-Related Brain Areas of Aged Rhesus Monkeys Treated with MPTP.

PubMed

Huang, Baihui; Wu, Shihao; Wang, Zhengbo; Ge, Longjiao; Rizak, Joshua D; Wu, Jing; Li, Jiali; Xu, Lin; Lv, Longbao; Yin, Yong; Hu, Xintian; Li, Hao

2018-05-21

Phosphorylation of α-synuclein at serine 129 (P-Ser 129 α-syn) is involved in the pathogenesis of Parkinson's disease (PD) and Lewy body (LB) formation. However, there is no clear evidence indicates the quantitative relation of P-Ser 129 α-syn accumulation and dopaminergic cell loss, LBs pathology and the affected brain areas in PD monkeys. Here, pathological changes in the substantia nigra (SN) and PD-related brain areas were measured in aged monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) utilizing a modeling-recovery-remodeling strategy. Compared to age-matched controls, the MPTP-treated monkeys showed significantly reduced tyrosine hydroxylase (TH)-positive neurons and increased P-Ser 129 α-syn-positive aggregations in the SN. Double-labeling Immunofluorescence found some TH-positive neurons to be co-localized with P-Ser129 α-syn in the SN, suggesting the inverse correlation between P-Ser 129 α-syn aggregations and dopaminergic cell loss in the SN may represent an interactive association related to the progression of the PD symptoms in the model. P-Ser 129 α-syn aggregations or LB-like pathology was also found in the midbrain and the neocortex, specifically in the oculomotor nucleus (CN III), temporal cortex (TC), prefrontal cortex (PFC) and in cells surrounding the third ventricle. Notably, the occipital cortex (OC) was P-Ser 129 α-syn negative. The findings of LB-like pathologies, dopaminergic cell loss and the stability of the PD symptoms in this model suggest that the modeling-recovery-remodeling strategy in aged monkeys may provide a new platform for biomedical investigations into the pathogenesis of PD and potential therapeutic development. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Ultrastructural changes and the distribution of arabinogalactan proteins during somatic embryogenesis of banana (Musa spp. AAA cv. 'Yueyoukang 1').

PubMed

Pan, Xiao; Yang, Xiao; Lin, Guimei; Zou, Ru; Chen, Houbin; Samaj, Jozef; Xu, Chunxiang

2011-08-01

A better understanding of somatic embryogenesis in banana (Musa spp.) may provide a practical way to improve regeneration of banana plants. In this study, we applied scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to visualize the ultrastructural changes during somatic embryogenesis of banana (Musa AAA cv. 'Yueyoukang 1'). We also used histological and immunohistochemical techniques with 16 monoclonal antibodies to study the spatial distribution and cellular/subcellular localization of different arabinogalactan protein (AGP) components of the cell wall during somatic embryogenesis. Histological study with periodic acid-Schiff staining documented diverse embryogenic stages from embryogenic cells (ECs) to the late embryos. SEM revealed a mesh-like structure on the surface of proembryos which represented an early structural marker of somatic embryogenesis. TEM showed that ECs were rich in juvenile mitochondria, endoplasmic reticulum and Golgi stacks. Cells in proembryos and early globular embryos resembled ECs, but they were more vacuolated, showed more regular nuclei and slightly more developed organelles. Immunocytochemical study revealed that the signal of most AGP epitopes was stronger in starch-rich cells when compared with typical ECs. The main AGP component in the extracellular matrix surface network of banana proembryos was the MAC204 epitope. Later, AGP immunolabelling patterns varied with the developmental stages of the embryos. These results about developmental regulation of AGP epitopes along with developmental changes in the ultrastructure of cells are providing new insights into the somatic embryogenesis of banana. Copyright © Physiologia Plantarum 2011.

Analysis of allelic expression patterns in clonal somatic cells by single-cell RNA-seq.

PubMed

Reinius, Björn; Mold, Jeff E; Ramsköld, Daniel; Deng, Qiaolin; Johnsson, Per; Michaëlsson, Jakob; Frisén, Jonas; Sandberg, Rickard

2016-11-01

Cellular heterogeneity can emerge from the expression of only one parental allele. However, it has remained controversial whether, or to what degree, random monoallelic expression of autosomal genes (aRME) is mitotically inherited (clonal) or stochastic (dynamic) in somatic cells, particularly in vivo. Here we used allele-sensitive single-cell RNA-seq on clonal primary mouse fibroblasts and freshly isolated human CD8 + T cells to dissect clonal and dynamic monoallelic expression patterns. Dynamic aRME affected a considerable portion of the cells' transcriptomes, with levels dependent on the cells' transcriptional activity. Notably, clonal aRME was detected, but it was surprisingly scarce (<1% of genes) and mainly affected the most weakly expressed genes. Consequently, the overwhelming majority of aRME occurs transiently within individual cells, and patterns of aRME are thus primarily scattered throughout somatic cell populations rather than, as previously hypothesized, confined to patches of clonally related cells.

The actin-binding protein profilin is required for germline stem cell maintenance and germ cell enclosure by somatic cyst cells

PubMed Central

Shields, Alicia R.; Spence, Allyson C.; Yamashita, Yukiko M.; Davies, Erin L.; Fuller, Margaret T.

2014-01-01

Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. Here, we demonstrate that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia. PMID:24346697

Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory

PubMed Central

Kaji, Tomohiro; Ishige, Akiko; Hikida, Masaki; Taka, Junko; Hijikata, Atsushi; Kubo, Masato; Nagashima, Takeshi; Takahashi, Yoshimasa; Kurosaki, Tomohiro; Okada, Mariko; Ohara, Osamu

2012-01-01

One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell–dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell–dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen. PMID:23027924

Tumorigenicity assessment of human cell-processed therapeutic products.

PubMed

Yasuda, Satoshi; Sato, Yoji

2015-09-01

Human pluripotent stem cells (hPSCs) are expected to be sources of various cell types used for cell therapy, although hPSCs are intrinsically tumorigenic and form teratomas in immunodeficient animals after transplant. Despite the urgent need, no detailed guideline for the assessment of tumorigenicity of human cell-processed therapeutic products (hCTPs) has been issued. Here we describe our consideration on tumorigenicity and related tests of hCTPs. The purposes of those tests for hPSC-based products are classified into three categories: 1) quality control of raw materials; 2) quality control of intermediate/final products; and 3) safety assessment of final products. Appropriate types of tests need to be selected, taking the purpose(s) into consideration. In contrast, human somatic (and somatic stem) cells are believed to have little tumorigenicity. Therefore, GMP-compliant quality control is essential to avoid contamination of somatic cell-derived products with tumorigenic cells. Compared with in vivo tumorigenicity tests, in vitro cell proliferation assays may be more useful and reasonable for detecting immortalized cells that have a growth advantage in somatic cell-based products. The results obtained from tumorigenicity and related tests for hCTPs should meet the criteria for decisions on product development, manufacturing processes, and clinical applications. Copyright © 2015.

Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

PubMed Central

Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

2014-01-01

The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary. PMID:24658126

Intra-renal arterial injection of autologous bone marrow mesenchymal stromal cells ameliorates cisplatin-induced acute kidney injury in a rhesus Macaque mulatta monkey model.

PubMed

Moghadasali, Reza; Azarnia, Mahnaz; Hajinasrollah, Mostafa; Arghani, Hassan; Nassiri, Seyed Mahdi; Molazem, Mohammad; Vosough, Ahmad; Mohitmafi, Soroush; Najarasl, Mostafa; Ajdari, Zahra; Yazdi, Reza Salman; Bagheri, Mohsen; Ghanaati, Hossein; Rafiei, Behrooz; Gheisari, Yousof; Baharvand, Hossein; Aghdami, Nasser

2014-06-01

Clinically, acute kidney injury (AKI) is a potentially devastating condition for which no specific therapy improves efficacy of the repair process. Bone marrow mesenchymal stromal cells (BM-MSCs) are proven to be beneficial for the renal repair process after AKI in different experimental rodent models, but their efficacy in large animals and humans remains unknown. This study aims to assess the effect of autologous rhesus Macaque mulatta monkey BM-MSC transplantation in cisplatin-induced AKI. We chose a model of AKI induced by intravenous administration of 5 mg/kg cisplatin. BM-MSCs were transplanted through intra-arterial injection. The animals were followed for survival, biochemistry analysis and pathology. Transplantation of 5 × 10(6) cells/kg ameliorated renal function during the first week, as shown by significantly lower serum creatinine and urea values and higher urine creatinine and urea clearance without hyponatremia, hyperkalemia, proteinuria and polyuria up to 84 d compared with the vehicle and control groups. The superparamagnetic iron oxide nanoparticle-labeled cells were found in both the glomeruli and tubules. BM-MSCs markedly accelerated Foxp3+ T-regulatory cells in response to cisplatin-induced damage, as revealed by higher numbers of Foxp3+ cells within the tubuli of these monkeys compared with cisplatin-treated monkeys in the control and vehicle groups. These data demonstrate that BM-MSCs in this unique large-animal model of cisplatin-induced AKI exhibited recovery and protective properties. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Protein Equilibration through Somatic Ring Canals in Drosophila

PubMed Central

McLean, Peter F.; Cooley, Lynn

2013-01-01

Although intercellular bridges resulting from incomplete cytokinesis were discovered in somatic Drosophila tissues decades ago, the impact of these structures on intercellular communication and tissue biology is largely unknown. In this work, we demonstrate that the ~250 nm diameter somatic ring canals permit diffusion of cytoplasmic contents between connected cells and across mitotic clone boundaries, and enable the equilibration of protein between transcriptionally mosaic follicle cells in the Drosophila ovary. We obtained similar, though more restricted, results in the larval imaginal discs. Our work illustrates the lack of cytoplasmic autonomy in these tissues and suggests a role for somatic ring canals in promoting homogeneous protein expression within the tissue. PMID:23704373

PCR detection and DNA sequence analysis of the regulatory region of lymphotropic papovavirus in peripheral blood mononuclear cells of an immunocompromised rhesus macaque

NASA Technical Reports Server (NTRS)

Lednicky, John A.; Halvorson, Steven J.; Butel, Janet S.

2002-01-01

A lymphotropic papovavirus (LPV) archetypal regulatory region was amplified from DNA from the blood of an immunocompromised rhesus monkey. We believe this is the first nonserological evidence of LPV infection in rhesus monkeys.

Somatic cell hybrid mapping on mouse chromosome 11 (MMU11): Assignment of markers relative to two breakpoints in band D

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

Morris, D.J.; Robinson, T.J.; Adler, I.D.

1993-02-01

Mouse [times] rat somatic cell hybrids were generated by fusing mouse cell lines that are heterozygous for reciprocal translocations involving the T42H and T9Ad breakpoints on mouse chromosome 11 (MMU11) to a thymidine kinase-negative (Tk[sup [minus

Iron concentrations and distributions in the parkinsonian substantia nigra of aged and young primate models

NASA Astrophysics Data System (ADS)

Ren, M. Q.; Xie, J. P.; Wang, X. S.; Ong, W. Y.; Leong, S. K.; Watt, F.

2001-07-01

Parkinson's disease (PD) is a progressive neuronal degenerative brain disease of the elderly, and is caused by the selective degeneration of neurons in the substantia nigra (SN) region of the brain, resulting in a reduced production of the neurotransmitter dopamine. Iron has been linked to dopaminergic cell death in Parkinson's disease because of its potential to promote free radicals, leading to oxidative stress. The present study is aimed at using the techniques of nuclear microscopy to elucidate the iron concentrations and distributions in the SN of both young and old monkeys following unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioning. A group of three old monkeys (older than 7 years) and a group of three young monkeys (younger than 7 years) were unilaterally MPTP-lesioned (right side) to induce parkinsonism and sacrificed after 35 days. The left side SN was used as a control. This time interval was chosen to correspond to an average 50% loss of dopamine producing cells in the lesioned right side SN. We have observed a significant difference in iron concentrations between the SNs of the young and old monkeys (increasing from an average of 233 to 1092 parts per million dry weight). When comparing the lesioned and non-lesioned SNs of the same animal, we found no significant difference in iron levels for each young monkey. However we have found a slight increase in iron (approximately 10%) between the lesioned SN and control SN for old monkeys. We have also observed that in the SN of younger primates, there is a weak anti-correlation in the SN iron levels with the neuron distribution. In the older monkeys, however, we have observed a proliferation of iron-rich granules, which appear to be more strongly anti-correlated with the distribution of neurons. The iron-cell anti-correlation occurs both in the control as well as the lesioned SN. Our results suggest that iron, particularly in the form of iron-rich deposits, accumulates in specific sites in the SN with age. Since Parkinson's disease mainly occurs in the elderly, this may implicate iron as a factor in dopaminergic cell death through iron-catalysed free radical production.

The topography of primate retina: a study of the human, bushbaby, and new- and old-world monkeys.

PubMed

Stone, J; Johnston, E

1981-02-20

The distribution of ganglion cells has been studied in the retinas of four primates: the prosimian bushbaby, the New-World squirrel monkey, the Old-World crab-eating cynamolgous monkey, and the human. The sizes of ganglion cell somas were also measured at a number of retinal locations and compared with similar measurements in the cat retina to test for the presence in primates of retinal specializations such as the visual streak, and for gradients in retinal structure, such as that between temporal and nasal retina. In all four primates, ganglion cell somas in peripheral retina ranged considerably in diameter (6-16 micrometer in the bushbaby, 8-22 micrometer in the squirrel monkey, 8-23 micrometer in the cynamolgous monkey, 8-26 micrometer in the human). It seems likely that the strong physiological correlates of soma size which have been described among cat retinal ganglion cells and among the relay cells of the macaque lateral geniculate nucleus are generally present in primates. In all four primates, evidence was also obtained of a visual streak specialization; the isodensity lines in ganglion cell density maps were horizontally elongated, and small-bodied ganglion cells were relatively more common in the region of the proposed streak than in other areas of peripheral retina. However, the visual streak seems less well developed than in the cat; among the four primate species examined it was best developed in the bushbaby, at least as assessed by the shape of the isodensity lines. All four primates showed a clear foveal specialization, but this feature seemed least developed in the bushbaby. At the fovea, ganglion cells are smaller in soma size than in peripheral retina; they also seemed more uniform in size, although some distinctly larger cells persist in the human and bushbaby. Soma size measurements also provided evidence of a difference between nasal and temporal areas of peripheral retina comparable to that reported for the cat and other species. Thus the primate retinas examined show features, such as the foveal specialization, which seem unique to them among mammals. They also show features, such as nasal-temporal differences in ganglion cell size, and (though weakly developed) a visual streak, which they have in common with other mammals with widely different phylogenetic histories.

Dogs cloned from adult somatic cells.

PubMed

Lee, Byeong Chun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hossein, M Shamim; Shamim, M Hossein; Kim, Jung Ju; Kang, Sung Keun; Schatten, Gerald; Hwang, Woo Suk

2005-08-04

Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

76 FR 36078 - Milk for Manufacturing Purposes and Its Production and Processing; Requirements Recommended for...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-21

... that goats have a need for different regulatory limits for somatic cells than cows. DATES: Effective... producer herd goat milk. Due to inherent differences between cows and goats, goat milk with a somatic cell...

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

PubMed

Li, Lan; Shen, Wei; Pan, Qing-Yu; Min, Ling-Jiang; Sun, Yu-Jiang; Fang, Yong-Wei; Deng, Ji-Xian; Pan, Qing-Jie

2006-12-01

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

Stable transformation via particle bombardment in two different soybean regeneration systems.

PubMed

Sato, S; Newell, C; Kolacz, K; Tredo, L; Finer, J; Hinchee, M

1993-05-01

The Biolistics(®) particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for β-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.

Use of somatic cell banks in the conservation of wild felids.

PubMed

Praxedes, Érika A; Borges, Alana A; Santos, Maria V O; Pereira, Alexsandra F

2018-05-03

The conservation of biological resources is an interesting strategy for the maintenance of biodiversity, especially for wild felids who are constantly threatened with extinction. For this purpose, cryopreservation techniques have been used for the long-term storage of gametes, embryos, gonadal tissues, and somatic cells and tissues. The establishment of these banks has been suggested as a practical approach to the preservation of species and, when done in tandem with assisted reproductive techniques, could provide the means for reproducing endangered species. Somatic cell banks have been shown remarkable for the conservation of genetic material of felids; by merely obtaining skin samples, it is possible to sample a large group of individuals without being limited by factors such as gender or age. Thus, techniques for somatic tissue recovery, cryopreservation, and in vitro culture of different wild felids have been developed, resulting in a viable method for the conservation of species. One of the most notable conservation programs for wild felines using somatic samples was the one carried out for the Iberian lynx, the most endangered feline in the world. Other wild felids have also been studied in other continents, such as the jaguar in South America. This review aims to present the technical progress achieved in the conservation of somatic cells and tissues in different wild felids, as well address the progress that has been achieved in a few species. © 2018 Wiley Periodicals, Inc.

Cartilage regeneration by selected chondrogenic clonal mesenchymal stem cells in the collagenase-induced monkey osteoarthritis model.

PubMed

Jiang, Li; Ma, Anlun; Song, Lijun; Hu, Yanxin; Dun, Hao; Daloze, Pierre; Yu, Yonglin; Jiang, Jianyuan; Zafarullah, Muhammad; Chen, Huifang

2014-11-01

Osteoarthritis (OA) is the most common form of arthritis, in which cartilage is irreversibly degraded, causing severe pain and disability. Current therapeutic strategies cannot repair damaged cartilage. We evaluated the repair potential of selected chondrogenic clonal MSCs (sC-MSCs) by delivering them into the injured cartilage site in a collagenase-induced OA model in Cynomolgus monkeys. In vitro characterization showed that the isolated monkey sC-MSCs and polyclonal MSCs (P-MSCs) expressed mesenchymal stem cell markers and could differentiate into chondrocytes. The articular cartilage lesions in animals were treated with normal saline (NS), autologous P-MSCs and sC-MSCs, respectively, by direct delivery. The clinical parameters, radiographic images, histological and immunohistochemical examinations at weeks 8, 16 and 24 post-treatment demonstrated that the abrasions of articular cartilage were significantly improved and repaired by MSC-based treatment, particularly in the sC-MSC-treated group, which displayed consistently higher histological scores than those of other groups. In summary, treatment with sC-MSCs can effectively improve the healing of cartilage lesions in the Cynomolgus monkey collagenase-induced OA model. Due to the genetic proximity of monkey and human, the therapeutic strategy presented in this study will have broad applications in clinical practice. Copyright © 2013 John Wiley & Sons, Ltd.

Epstein-Barr virus-infected B cells expanding in germinal centers of infectious mononucleosis patients do not participate in the germinal center reaction.

PubMed

Kurth, Julia; Hansmann, Martin-Leo; Rajewsky, Klaus; Küppers, Ralf

2003-04-15

To assess the impact of the germinal center (GC) reaction on viral spread in Epstein-Barr virus (EBV) infection, we isolated EBV(+) GC B cells from the tonsils of two infectious mononucleosis patients, sequenced their rearranged V genes, and determined expression of the EBV latency genes EBV nuclear antigen 2 and latent membrane protein 1. Most EBV(+) GC B cells belonged to clones of cells harboring somatically mutated V gene rearrangements. Ongoing somatic hypermutation, the hallmark of the GC reaction, was seen only in uninfected GC B cell clones, not in EBV(+) B cell clones. Thus, in infectious mononucleosis, GC and/or memory B cells are directly infected by EBV and expand without somatic hypermutation, whereas the GC passage of EBV-infected naive B cells does not contribute detectably to the generation of infected memory B cells, the main reservoir of EBV during persistence. Most, if not all, EBV-infected cells in GCs exhibited an unusual EBV gene expression pattern in that they were positive for EBV nuclear antigen 2 but negative for latent membrane protein 1. Although the three main types of EBV-associated B cell lymphomas (Burkitt's, Hodgkin's, and posttransplant lymphomas) presumably are derived from GC B cells, EBV(+) GC B cells resembling these EBV(+) GC B cell lymphomas in terms of EBV gene expression and somatic hypermutation pattern could not be identified.

Effects of continuous nicotine treatment and subsequent termination on cocaine versus food choice in male rhesus monkeys.

PubMed

Schwienteck, Kathryn L; Negus, S Stevens; Poklis, Justin L; Banks, Matthew L

2015-10-01

One complicating factor in cocaine addiction may be concurrent exposure and potential dependence on nicotine. The aim of the present study was to determine the effects of continuous nicotine treatment and subsequent termination on cocaine versus food choice in rhesus monkeys (Macaca mulatta). For comparison, we also determined effects of the nicotinic receptor antagonist mecamylamine on cocaine versus food choice during continuous saline and nicotine treatment. Rhesus monkeys (N = 3) responded under a concurrent schedule of food pellet (1 g) and intravenous cocaine (0-0.1 mg/kg/injection) availability. Saline and ascending nicotine doses (0.1-1.0 mg/kg/hr, intravenous) were continuously infused for 7-day treatment periods and separated by 24-hr saline treatment periods. Acute effects of mecamylamine (0.32-1.8 mg/kg, intramuscular, 15 min pretreatment) were determined during continuous saline and 0.32-mg/kg/hr nicotine treatments. During saline treatment, cocaine maintained a dose-dependent increase in cocaine choice. Nicotine treatment did not alter cocaine versus food choice. In contrast, preference of 0.032 mg/kg/injection cocaine was attenuated 24 hr following termination of 0.32-mg/kg/hr nicotine treatment, despite no somatic abstinence signs being observed. Acute mecamylamine enhanced cocaine choice during saline treatment and mainly suppressed rates of behavior during nicotine treatment. Overall, continuous nicotine exposure, up to 1 mg/kg/hr, does not enhance cocaine choice and does not produce nicotine dependence, as demonstrated by the lack of abstinence signs. (c) 2015 APA, all rights reserved).

Effects of continuous nicotine treatment and subsequent termination on cocaine vs. food choice in male rhesus monkeys

PubMed Central

Schwienteck, Kathryn L.; Negus, S. Stevens; Poklis, Justin L.; Banks, Matthew L.

2015-01-01

One complicating factor in cocaine addiction may be concurrent exposure and potential dependence on nicotine. The aim of the present study was to determine the effects of continuous nicotine treatment and subsequent termination on cocaine vs. food choice in rhesus monkeys. For comparison, we also determined effects of the nicotinic receptor antagonist mecamylamine on cocaine vs. food choice during continuous saline and nicotine treatment. Rhesus monkeys (n=3) responded under a concurrent schedule of food pellet (1g) and intravenous cocaine (0 – 0.1 mg/kg/injection) availability. Saline and ascending nicotine doses (0.1 – 1.0 mg/kg/h, IV) were continuously infused for 7-day treatment periods and separated by 24 h saline treatment periods. Acute effects of mecamylamine (0.32 – 1.8 mg/kg, IM, 15 min pretreatment) were determined during continuous saline and 0.32 mg/kg/h nicotine treatments. During saline treatment, cocaine maintained a dose-dependent increase in cocaine choice. Nicotine treatment did not alter cocaine vs. food choice. In contrast, preference of 0.032 mg/kg/injection cocaine was attenuated 24 h following termination of 0.32 mg/kg/h nicotine treatment despite no somatic abstinence signs being observed. Acute mecamylamine enhanced cocaine choice during saline treatment and mainly suppressed rates of behavior during nicotine treatment. Overall, continuous nicotine exposure, up to 1 mg/kg/h, does not enhance cocaine choice and does not produce nicotine dependence as demonstrated by the lack of abstinence signs. PMID:26098473

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

PubMed Central

Keam, Simon P.; Young, Paul E.; McCorkindale, Alexandra L.; Dang, Thurston H.Y.; Clancy, Jennifer L.; Humphreys, David T.; Preiss, Thomas; Hutvagner, Gyorgy; Martin, David I.K.; Cropley, Jennifer E.; Suter, Catherine M.

2014-01-01

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals. PMID:25038252

Tracking cells implanted into cynomolgus monkeys (Macaca fascicularis) using MRI

PubMed Central

Ito-Fujishiro, Yasuyo; Koie, Hiroshi; Shibata, Hiroaki; Okabayashi, Sachi; Katakai, Yuko; Ohno, Chieko; Kanayama, Kiichi; Yasutomi, Yasuhiro; Ageyama, Naohide

2016-01-01

Regenerative therapy with stem cell transplantation is used to treat various diseases such as coronary syndrome and Buerger’s disease. For instance, stem-cell transplantation into the infarcted myocardium is an innovative and promising strategy for treating heart failure due to ischemic heart disease. Basic studies using small animals have shown that transplanted cells improve blood flow in the infarcted region. Magnetic resonance imaging (MRI) can noninvasively identify and track transplanted cells labeled with superparamagnetic iron oxide (SPIO). Although clinical regenerative therapies have been clinically applied to patients, the fate of implanted cells remains unknown. In addition, follow-up studies have shown that some adverse events can occur after recovery. Therefore, the present study evaluated the ability of MRI using a 3T scanner to track implanted peripheral blood mononuclear cells labeled with SPIO on days 0 and 7 after intramuscular (i.m.) and intravenous (i.v.) injection into a cynomolgus monkey. Labeled cells were visualized at the liver and triceps surae muscle on MR images using T1- and T2-weighted sequences and histologically localized by Prussian blue staining. The transplanted cells were tracked without abnormal clinical manifestations throughout this study. Hence, MRI of cynomolgus monkey transplanted SPIO-labeled cells is a safe and efficient method of tracking labeled cells that could help to determine the mechanisms involved in regenerative therapy. PMID:27062993

[Explantation method of isolating a persistent tick-borne encephalitis virus from the organs of infected monkeys].

PubMed

Levina, L S; Pogodina, V V

1981-01-01

The method of explantation was used to examine 63 organs from M. rhesus monkeys 92-783 days after intracerebral and subcutaneous inoculation with the Vasilchenko, Aina/1448 and 41/65 strains of tick-borne encephalitis virus. The optimal time for examination of the explants by tests of the hemagglutinating, cytopathogenic activity of the virus and its pathogenicity for mice was found to be the 15th day of cultivation. A comparative study of the properties of 3 isolates obtained from explants of the spleen, liver and subcortical cerebral ganglia 202 and 307 days after inoculation of monkeys was carried out. The isolates differed from the parental TBE virus strains by their capacity to form small plaques in PEKV cell cultures (pig embryo kidney cells in versen medium).

Spontaneous Findings in the Eyes of Cynomolgus Monkeys ( Macaca fascicularis) of Mauritian Origin.

PubMed

Woicke, Jochen; Haile, Solomon; Mysore, Jagannatha; Peden, W Michael; Lejeune, Typhaine; Sanderson, Thomas; Brodie, Thomas

2018-04-01

Spontaneous findings noted in the eyes of Mauritian cynomolgus monkeys are described and descriptions are supplemented with illustrations. Findings observed after extensive histopathologic examinations (20 to 44 sections per eye) from 20 control, 17 treatment-naive stock monkeys, and 2 findings noted in drug-treated monkeys that were considered to be spontaneous are included. Also included are findings from 361 control monkeys of routine toxicity studies performed at our laboratories, for most of which a standard histopathological examination of 1 section per eye was conducted. Common observations in monkeys examined extensively and in historical controls were limited to lymphocytic or mononuclear cell infiltrations of the uvea and/or conjunctiva/sclera and, less commonly observed, melanocytoma of the ciliary body or iris. Findings noted only in monkeys examined extensively consisted of inflammation of the conjunctiva, ora serrata cysts, glial nodules, focal degeneration of the retina, cystoid degeneration of the central retina, ballooning degeneration of the ciliary epithelium, cyst of the ciliary body, and decreased pigmentation of the retinal pigment epithelium. Changes recorded only in historical controls included retinal atrophy and nuclear displacement in the retina. Lesions are discussed and compared with pertinent literature.

Postnatal remodeling of the neural components of the epithelial-mesenchymal trophic unit in the proximal airways of infant rhesus monkeys exposed to ozone and allergen.

PubMed

Larson, Shawnessy D; Schelegle, Edward S; Walby, William F; Gershwin, Laural J; Fanuccihi, Michelle V; Evans, Michael J; Joad, Jesse P; Tarkington, Brian K; Hyde, Dallas M; Plopper, Charles G

2004-02-01

Nerves and neuroendocrine cells located within the airway epithelium are ideally situated to sample a changing airway environment, to transmit that information to the central nervous system, and to promote trophic interactions between epithelial and mesenchymal cellular and acellular components. We tested the hypothesis that the environmental stresses of ozone (O(3)) and house dust mite allergen (HDMA) in atopic infant rhesus monkeys alter the distribution of airway nerves. Midlevel bronchi and bronchioles from 6-month-old infant monkeys that inhaled filtered air (FA), house dust mite allergen HDMA, O(3), or HDMA + O(3) for 11 episodes (5 days each, 0.5 ppm O(3), 8 h/day followed by 9 days recovery) were examined using immunohistochemistry for the presence of Protein gene product 9.5 (PGP 9.5), a nonspecific neural indicator, and calcitonin gene-related peptide (CGRP). Along the axial path between the sixth and the seventh intrapulmonary airway generations, there were small significant (P < 0.05) decrements in the density of epithelial nerves in monkeys exposed to HDMA or O(3), while in monkeys exposed to HDMA + O(3) there was a greater significant (P < 0.05) reduction in epithelial innervation. In animals exposed to O(3) or HDMA + O(3) there was a significant increase in the number of PGP 9.5 positive/CGRP negative cells that were anchored to the basal lamina and emitted projections in primarily the lateral plain and often intertwined with projections and cell bodies of other similar cells. We conclude that repeated cycles of acute injury and repair associated with the episodic pattern of ozone and allergen exposure alter the normal development of neural innervation of the epithelial compartment and the appearance of a new population of undefined PGP 9.5 positive cells within the epithelium.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

PubMed

Tao, Yu; Zheng, Weisheng; Jiang, Yonghua; Ding, Guitao; Hou, Xinfeng; Tang, Yitao; Li, Yueying; Gao, Shuai; Chang, Gang; Zhang, Xiaobai; Liu, Wenqiang; Kou, Xiaochen; Wang, Hong; Jiang, Cizhong; Gao, Shaorong

2014-12-21

Nucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells. We generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs' tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs. The re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.

"Subpial Fan Cell" - A Class of Calretinin Neuron in Layer 1 of Adult Monkey Prefrontal Cortex.

PubMed

Gabbott, Paul L A

2016-01-01

Layer 1 of the cortex contains populations of neurochemically distinct neurons and afferent fibers which markedly affect neural activity in the apical dendritic tufts of pyramidal cells. Understanding the causal mechanisms requires knowledge of the cellular architecture and synaptic organization of layer 1. This study has identified eight morphological classes of calretinin immunopositive (CRet+) neurons (including Cajal-Retzius cells) in layer 1 of the prefrontal cortex (PFC) in adult monkey (Macaca fasicularis), with a distinct class - termed "subpial fan (SPF) cell" - described in detail. SPF cells were rare horizontal unipolar CRet+ cells located directly beneath the pia with a single thick primary dendrite that branched into a characteristic fan-like dendritic tree tangential to the pial surface. Dendrites had spines, filamentous processes and thorny branchlets. SPF cells lay millimeters apart with intralaminar axons that ramified widely in upper layer 1. Such cells were GABA immunonegative (-) and occurred in areas beyond PFC. Interspersed amidst SPF cells displaying normal structural integrity were degenerating CRet+ neurons (including SPF cells) and clumps of lipofuscin-rich cellular debris. The number of degenerating SPF cells increased during adulthood. Ultrastructural analyses indicated SPF cell somata received asymmetric (A - presumed excitatory) and symmetric (S - presumed inhibitory) synaptic contacts. Proximal dendritic shafts received mainly S-type and distal shafts mostly A-type input. All dendritic thorns and most dendritic spines received both synapse types. The tangential areal density of SPF cell axonal varicosities varied radially from parent somata - with dense clusters in more distal zones. All boutons formed A-type contacts with CRet- structures. The main post-synaptic targets were dendritic shafts (67%; mostly spine-bearing) and dendritic spines (24%). SPF-SPF cell innervation was not observed. Morphometry of SPF cells indicated a unique class of CRet+/GABA- neuron in adult monkey PFC - possibly a subtype of persisting Cajal-Retzius cell. The distribution and connectivity of SPF cells suggest they act as integrative hubs in upper layer 1 during postnatal maturation. The main synaptic output of SPF cells likely provides a transminicolumnar excitatory influence across swathes of apical dendritic tufts - thus affecting information processing in discrete patches of layer 1 in adult monkey PFC.

Simple Monitoring of Gene Targeting Efficiency in Human Somatic Cell Lines Using the PIGA Gene

PubMed Central

Karnan, Sivasundaram; Konishi, Yuko; Ota, Akinobu; Takahashi, Miyuki; Damdindorj, Lkhagvasuren; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2012-01-01

Gene targeting in most of human somatic cell lines has been labor-intensive because of low homologous recombination efficiency. The development of an experimental system that permits a facile evaluation of gene targeting efficiency in human somatic cell lines is the first step towards the improvement of this technology and its application to a broad range of cell lines. In this study, we utilized phosphatidylinositol glycan anchor biosynthesis class A (PIGA), a gene essential for the synthesis of glycosylphosphatidyl inositol (GPI) anchors, as a reporter of gene targeting events in human somatic cell lines. Targeted disruption of PIGA was quantitatively detected with FLAER, a reagent that specifically binds to GPI anchors. Using this PIGA-based reporter system, we successfully detected adeno-associated virus (AAV)-mediated gene targeting events both with and without promoter-trap enrichment of gene-targeted cell population. The PIGA-based reporter system was also capable of reproducing previous findings that an AAV-mediated gene targeting achieves a remarkably higher ratio of homologous versus random integration (H/R ratio) of targeting vectors than a plasmid-mediated gene targeting. The PIGA-based system also detected an approximately 2-fold increase in the H/R ratio achieved by a small negative selection cassette introduced at the end of the AAV-based targeting vector with a promoter-trap system. Thus, our PIGA-based system is useful for monitoring AAV-mediated gene targeting and will assist in improving gene targeting technology in human somatic cell lines. PMID:23056640

Postnatal changes in somatic gamma-aminobutyric acid signalling in the rat hippocampus.

PubMed

Tyzio, Roman; Minlebaev, Marat; Rheims, Sylvain; Ivanov, Anton; Jorquera, Isabelle; Holmes, Gregory L; Zilberter, Yuri; Ben-Ari, Yehezkiel; Khazipov, Rustem

2008-05-01

During postnatal development of the rat hippocampus, gamma-aminobutyric acid (GABA) switches its action on CA3 pyramidal cells from excitatory to inhibitory. To characterize the underlying changes in the GABA reversal potential, we used somatic cell-attached recordings of GABA(A) and N-methyl-D-aspartate channels to monitor the GABA driving force and resting membrane potential, respectively. We found that the GABA driving force is strongly depolarizing during the first postnatal week. The strength of this depolarization rapidly declines with age, although GABA remains slightly depolarizing, by a few millivolts, even in adult neurons. Reduction in the depolarizing GABA driving force was due to a progressive negative shift of the reversal potential of GABA currents. Similar postnatal changes in GABA signalling were also observed using the superfused hippocampus preparation in vivo, and in the hippocampal interneurons in vitro. We also found that in adult pyramidal cells, somatic GABA reversal potential is maintained at a slightly depolarizing level by bicarbonate conductance, chloride-extrusion and chloride-loading systems. Thus, the postnatal excitatory-to-inhibitory switch in somatic GABA signalling is associated with a negative shift of the GABA reversal potential but without a hyperpolarizing switch in the polarity of GABA responses. These results also suggest that in adult CA3 pyramidal cells, somatic GABAergic inhibition takes place essentially through shunting rather than hyperpolarization. Apparent hyperpolarizing GABA responses previously reported in the soma of CA3 pyramidal cells are probably due to cell depolarization during intracellular or whole-cell recordings.

29 CFR 1990.103 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Health and Human Services, or designee. Director of NCI means the Director of the National Cancer... means the induction of heritable changes in the genetic material of either somatic or germinal cells..., Neurospora or Drosophila melanogaster; (3) Mutagenesis in mammalian somatic cells; (4) Mutagenesis in...

29 CFR 1990.103 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Health and Human Services, or designee. Director of NCI means the Director of the National Cancer... means the induction of heritable changes in the genetic material of either somatic or germinal cells..., Neurospora or Drosophila melanogaster; (3) Mutagenesis in mammalian somatic cells; (4) Mutagenesis in...

29 CFR 1990.103 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Health and Human Services, or designee. Director of NCI means the Director of the National Cancer... means the induction of heritable changes in the genetic material of either somatic or germinal cells..., Neurospora or Drosophila melanogaster; (3) Mutagenesis in mammalian somatic cells; (4) Mutagenesis in...

29 CFR 1990.103 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Health and Human Services, or designee. Director of NCI means the Director of the National Cancer... means the induction of heritable changes in the genetic material of either somatic or germinal cells..., Neurospora or Drosophila melanogaster; (3) Mutagenesis in mammalian somatic cells; (4) Mutagenesis in...

Consequence for dairy herds in the United States of imposing different standards for somatic cell count

USDA-ARS?s Scientific Manuscript database

New European Union (E.U.) regulations may require that a somatic cell count (SCC) limit of 400,000 cells/mL for milk be met by every farm that contributes to pooled milk exported to Europe. In the United States, the standard is 750,000 cells/mL. Because bulk tank SCC is not readily available through...

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

Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were asmore » capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.« less

Donor cell differentiation, reprogramming, and cloning efficiency: elusive or illusive correlation?

PubMed

Oback, B; Wells, D N

2007-05-01

Compared to other assisted reproductive technologies, mammalian nuclear transfer (NT) cloning is inefficient in generating viable offspring. It has been postulated that nuclear reprogramming and cloning efficiency can be increased by choosing less differentiated cell types as nuclear donors. This hypothesis is mainly supported by comparative mouse cloning experiments using early blastomeres, embryonic stem (ES) cells, and terminally differentiated somatic donor cells. We have re-evaluated these comparisons, taking into account different NT procedures, the use of donor cells from different genetic backgrounds, sex, cell cycle stages, and the lack of robust statistical significance when post-blastocyst development is compared. We argue that while the reprogrammability of early blastomeres appears to be much higher than that of somatic cells, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic donor cell lineages. Copyright (c) 2006 Wiley-Liss, Inc.

Analysis of allelic expression patterns in clonal somatic cells by single-cell RNA-seq

PubMed Central

Ramsköld, Daniel; Deng, Qiaolin; Johnsson, Per; Michaëlsson, Jakob; Frisén, Jonas; Sandberg, Rickard

2016-01-01

Cellular heterogeneity can emerge from the expression of only one parental allele. However, it has remained controversial whether, or to what degree, random monoallelic expression of autosomal genes (aRME) is mitotically inherited (clonal) or stochastic (dynamic) in somatic cells, particularly in vivo. Here, we used allele-sensitive single-cell RNA-seq on clonal primary mouse fibroblasts and in vivo human CD8+ T-cells to dissect clonal and dynamic monoallelic expression patterns. Dynamic aRME affected a considerable portion of the cells’ transcriptomes, with levels dependent on the cells’ transcriptional activity. Importantly, clonal aRME was detected but was surprisingly scarce (<1% of genes) and affected mainly the most low-expressed genes. Consequently, the overwhelming portion of aRME occurs transiently within individual cells and patterns of aRME are thus primarily scattered throughout somatic cell populations rather than, as previously hypothesized, confined to patches of clonally related cells. PMID:27668657

Clock-like mutational processes in human somatic cells

DOE PAGES

Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; ...

2015-11-09

During the course of a lifetime, somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell's genome. Some processes generate mutations throughout life at a constant rate in all individuals, and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutationmore » rates in different tissues. However, their mutation rates are not correlated, indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This paper provides the first survey of clock-like mutational processes operating in human somatic cells.« less

Somatic cell cloning: the ultimate form of nuclear reprogramming?

PubMed

Piedrahita, Jorge A; Mir, Bashir; Dindot, Scott; Walker, Shawn

2004-05-01

With the increasing difficulties associated with meeting the required needs for organs used in transplantation, alternative approaches need to be considered. These include the use of stem cells as potential sources of specialized cells, the ability to transdifferentiate cell types in culture, and the development of complete organs that can be used in humans. All of the above goals will require a complete understanding of the factors affecting cell differentiation and nuclear reprogramming. To make this a reality, however, techniques associated with cloning and genetic modifications in somatic cells need to be continued to be developed and optimized. This includes not only an enhancement of the rate of homologous recombination in somatic cells, but also a thorough understanding of the nuclear reprogramming process taking place during nuclear transfer. The understanding of this process is likely to have an effect beyond the area of nuclear transfer and assist with better methods for transdifferentiation of mammalian cells.

Topographic and age-related changes of the retinal epithelium and Bruch's membrane of rhesus monkeys.

PubMed

Gouras, Peter; Ivert, Lena; Neuringer, Martha; Mattison, Julie A

2010-07-01

To examine structural differences in the retinal pigmented epithelium (RPE) and Bruch's membrane of rhesus monkeys (Macaca mulatta) as a function of topography and age. The retinas of two old (24 and 26 years old) and two young (1 and 6 years old) female monkeys were examined by light fluorescence and electron microscopy at the macula, equator, and ora serrata. All monkeys lacked fluorescence and lipofuscin granules in the RPE at the ora serrata where photoreceptors are absent. The equator and macula showed intense fluorescence and many lipofuscin granules in the RPE of the old but not the young monkeys. At the ora, the RPE contained many dense round melanin granules throughout the cell. At the equator and macula, melanin granules were more apical, less frequent, and often elongated. Mitochondria were clustered at the basal side of the RPE cell near infolds of the plasma membrane. Both mitochondria and infolds tended to increase toward the macula. In all regions, the basal lamina of the RPE did not penetrate the extracellular space adjacent to infolds. The elastin layer of Bruch's membrane was wide at the ora and equator and thinner at the macula. In the old monkeys, drusen were found at all retinal regions between the basal lamina and the internal collagen layer of Bruch's membrane. The drusen were often membrane-bound with a basal lamina and contained material resembling structures in the RPE. Lack of fluorescence and lipofuscin in the RPE at the ora serrata, where photoreceptors are absent, confirms that RPE fluorescence occurs only where outer segments are phagocytized. Mitochondrial clustering indicates that the basal side of the RPE cell uses the most energy and this becomes maximal at the macula. The presence of age-related degenerative changes and drusen at all retinal locations in the older monkeys, even at the ora where RPE lipofuscin was absent, indicates that these processes are not dependent on local lipofuscin accumulation. Therefore lipofuscin toxicity may not be the sole cause of age-related RPE degeneration.

Shigella sonnei Vaccine Candidates WRSs2 and WRSs3 Are as Immunogenic as WRSS1, a Clinically Tested Vaccine Candidate, in a Primate Model of Infection

DTIC Science & Technology

2011-01-01

Experiments in rabbit ileal loops and in rhesus monkeys have shown that shigellae are initially taken up by antigen sampling microfold (M) cells that are...2011) – Table 1 Shigella antigen-specific immune responses and colonization in rhesus monkeys after nasogastric administration of WRSS1, WRSs2...the respective antigens. 3.3. Mucosal responses in rhesus monkeys The mucosal immune response to Shigella LPS, and Invaplex- 50 antigens are summarized

Retinal ganglion cell dendritic fields in old-world monkeys are oriented radially.

PubMed

Schall, J D; Perry, V H; Leventhal, A G

1986-03-12

We analyzed the dendritic field morphology of 297 ganglion cells from peripheral regions of monkey retina. Most of the dendritic fields were elongated, and there was a significant tendency for the dendritic fields to be oriented radially, i.e., like the spokes of a wheel with the fovea at the hub. An overrepresentation of radial orientations in the peripheral retina of primates might explain why humans are best able to detect stimuli which are oriented radially using peripheral vision.

Somatization in the conceptualization of sickle cell disease.

PubMed

Wellington, Chanté; Edwards, Christopher L; McNeil, Janice; Wood, Mary; Crisp, Benjamin; Feliu, Miriam; Byrd, Goldie; McDougald, Camela; Edwards, Lekisha; Whitfield, Keith E

2010-11-01

The unpredictable nature of sickle cell disease (SCD) and its social and environmental consequences can produce an unhealthy and almost exclusive focus on physical functioning. At the upper range of this focus on health concerns is somatization. In the current study, using 156 adult patients (55.13% female, 86) with SCD, mean age 35.59 +/- 12.73, we explored the relationship of somatization to pain. We found somatization to be predictive of pain severity and current pain intensity as well as a range of averaged indices of pain over time (p < .0001). We further found somatization to be predictive of a range of negative psychological experiences to include depression, anxiety, and hostility (p < .0001). We interpret these data to suggest that patients with SCD who have a propensity to focus exclusively on their health or are more sensitive to minor changes in their health status (somatization) may also be more likely to report greater concerns about their health and higher ratings of pain.

Pre-screening method for somatic cell contamination in human sperm epigenetic studies.

PubMed

Jenkins, Timothy G; Liu, Lihua; Aston, Kenneth I; Carrell, Douglas T

2018-04-01

Sperm epigenetic profiles are frequently studied and are of great interest in many fields. One major technical concern when assessing these marks is the potential for somatic cell contamination. Because somatic cells have dramatically different epigenetic signatures, even small levels of contamination can result in significant problems in analysis and interpretation of data. In this study we evaluate an assay, which we designed to offer a reliable 'pre-screen' for somatic cell contamination that directly assesses the DNA being used in the study to determine tissue purity. In brief, we designed an inexpensive and simple assay that utilizes the strong differential methylation between sperm and somatic cells at four genomic loci to assess the general purity of samples prior to performing expensive and time intensive assays. The assay is able to reliably detect contamination qualitatively by running the sample on an agarose gel, or quantitatively with the use of a bioanalyzer. With this technique we have found that we can detect potentially contaminating signals in samples of many different types, including those from patients with poor sperm phenotypes (oligozoospermia, asthenozoospermia, and teratozoospermia). We also have found that the use of multiple sites to determine potential contamination is key, as some conditions (asthenozoospermia specifically) appear at one site to reflect a somatic-like profile, while at all other sites it appears to have very typical sperm DNA methylation signatures. Taken together, the use of the assay described herein was effective at identifying contamination and could be implemented in many labs to quickly and inexpensively pre-screen samples prior to performing far more expensive and labor intensive procedures. Additionally, the principles applied to the development of this assay could be easily adapted for the development of other assays to pre-screen different tissue/cell types or model organisms.

Epithalon peptide induces telomerase activity and telomere elongation in human somatic cells.

PubMed

Khavinson, V Kh; Bondarev, I E; Butyugov, A A

2003-06-01

Addition of Epithalon peptide in telomerase-negative human fetal fibroblast culture induced expression of the catalytical subunit, enzymatic activity of telomerase, and telomere elongation, which can be due to reactivation of telomerase gene in somatic cells and indicates the possibility of prolonging life span of a cell population and of the whole organism.

Evaluation of a killed Rocky Mountain spotted fever vaccine in cynomolgus monkeys.

PubMed Central

Gonder, J C; Kenyon, R H; Pedersen, C E

1979-01-01

A nonhuman primate model of Rocky Mountain spotted fever infection was developed in cynomolgus monkeys (Macaca fascicularis) infected by the subcutaneous route or by aerosol. Clinical responses, hematology and serum chemistry values, and pathological findings were similar to those found in humans ill with Rocky Mountain spotted fever. The clinical model was then used to test the efficacy of a killed Rocky Mountain spotted fever vaccine grown in chicken embryo cells. Monkeys were immunized with varying dilutions of the vaccine with a two-dose schedule and then challenged at 2 months with virulent Rickettsia rickettsii by the subcutaneous route or by aerosol. The undiluted vaccine totally protected monkeys against both challenges, even at extremely high doses. PMID:120877

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

PubMed Central

Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

2017-01-01

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

Ultrastructural Analysis of the Cristae Ampullares in the Squirrel Monkey (Saimiri sciureus)*

PubMed Central

Lysakowski, Anna; Goldberg, Jay M.

2008-01-01

Type I hair cells outnumber type II hair cells (HCs) in squirrel monkey (Saimiri sciureus) cristae by a nearly 3:1 ratio. Associated with this type I HC preponderance, calyx fibers make up a much larger fraction of the afferent innervation than in rodents (Fernández et al., 1995). To study how this affected synaptic architecture, we used disector methods to estimate various features associated with type I and type II HCs in central (CZ) and peripheral (PZ) zones of monkey cristae. Each type I HC makes, on average, 5–10 ribbon synapses with the inner face of a calyx ending. Inner-face synapses outnumber those on calyx outer faces by a 40:1 ratio. Expressed per afferent, there are, on average, 15 inner-face ribbon synapses, 0.38 outer-face ribbons and 2.6 efferent boutons on calyx-bearing endings. Calyceal invaginations per type I HC range from 19 in CZ to 3 in PZ. For type II HCs, there are many more ribbons and afferent boutons in PZ than in CZ, whereas efferent innervation is relatively uniform throughout the neuroepithelium. Despite outer-face ribbons being more numerous in chinchilla than in squirrel monkey, afferent discharge properties are similar (Lysakowski et al., 1995), reinforcing the importance of inner-face ribbons in synaptic transmission. Comparisons across mammalian species suggest the prevalence of type I HCs is a primate characteristic, rather than an arboreal lifestyle adaptation. Unlike cristae, type II HCs predominate in monkey maculae. Differences in hair-cell counts may reflect the stimulus magnitudes handled by semicircular canals and otolith organs. PMID:18729176

Somatic cells initiate primordial follicle activation and govern the development of dormant oocytes in mice.

PubMed

Zhang, Hua; Risal, Sanjiv; Gorre, Nagaraju; Busayavalasa, Kiran; Li, Xin; Shen, Yan; Bosbach, Benedikt; Brännström, Mats; Liu, Kui

2014-11-03

The majority of oocytes in the mammalian ovary are dormant oocytes that are enclosed in primordial follicles by several somatic cells, which we refer to as primordial follicle granulosa cells (pfGCs). Very little is known, however, about how the pfGCs control the activation of primordial follicles and the developmental fates of dormant oocytes. By targeting molecules in pfGCs with several mutant mouse models, we demonstrate that the somatic pfGCs initiate the activation of primordial follicles and govern the quiescence or awakening of dormant oocytes. Inhibition of mTORC1 signaling in pfGCs prevents the differentiation of pfGCs into granulosa cells, and this arrests the dormant oocytes in their quiescent states, leading to oocyte death. Overactivation of mTORC1 signaling in pfGCs accelerates the differentiation of pfGCs into granulosa cells and causes premature activation of all dormant oocytes and primordial follicles. We further show that pfGCs trigger the awakening of dormant oocytes through KIT ligand (KITL), and we present an essential communication network between the somatic cells and germ cells that is based on signaling between the mTORC1-KITL cascade in pfGCs and KIT-PI3K signaling in oocytes. Our findings provide a relatively complete picture of how mammalian primordial follicles are activated. The microenvironment surrounding primordial follicles can activate mTORC1-KITL signaling in pfGCs, and these cells trigger the awakening of dormant oocytes and complete the process of follicular activation. Such communication between the microenvironment, somatic cells, and germ cells is essential to maintaining the proper reproductive lifespan in mammals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Immunoregulation.

DTIC Science & Technology

1981-05-01

variety of antigens, KLH, GAT, TGAL and antigens from pathogenic bacteria such as Streptococcus mutans . Furthermore, we now have these systems...histocompatibility complex; PBL, peripheral blood lymphocytes; SAI/II, Streptococcus mutans antigen I/II complex; MHFSAI/II, monkey helper factor specific...from Streptococcus mutans . Helper activity was removed from supernatants of monkey cells by affinity chromatography on Sepharose 4B insolubilized

Cryopreservation, Culture, and Transplantation of Human Fetal Mesencephalic Tissue into Monkeys

NASA Astrophysics Data System (ADS)

Redmond, D. E.; Naftolin, F.; Collier, T. J.; Leranth, C.; Robbins, R. J.; Sladek, C. D.; Roth, R. H.; Sladek, J. R.

1988-11-01

Studies in animals suggest that fetal neural grafts might restore lost neurological function in Parkinson's disease. In monkeys, such grafts survive for many months and reverse signs of parkinsonism, without attendant graft rejection. The successful and reliable application of a similar transplantation procedure to human patients, however, will require neural tissue obtained from human fetal cadavers, with demonstrated cellular identity, viability, and biological safety. In this report, human fetal neural tissue was successfully grafted into the brains of monkeys. Neural tissue was collected from human fetal cadavers after 9 to 12 weeks of gestation and cryopreserved in liquid nitrogen. Viability after up to 2 months of storage was demonstrated by cell culture and by transplantation into monkeys. Cryopreservation and storage of human fetal neural tissue would allow formation of a tissue bank. The stored cells could then be specifically tested to assure their cellular identity, viability, and bacteriological and virological safety before clinical use. The capacity to collect and maintain viable human fetal neural tissue would also facilitate research efforts to understand the development and function of the human brain and provide opportunities to study neurological diseases.

Parallel evolution of a self-signal: humans and new world monkeys independently lost the cell surface sugar Neu5Gc.

PubMed

Springer, Stevan A; Diaz, Sandra L; Gagneux, Pascal

2014-11-01

Human sialic acid biology is unusual and thought to be unique among mammals. Humans lack a functional cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) protein and cannot synthesize the sugar Neu5Gc, an innate mammalian signal of self. Losing this sugar changed how humans interact with some of our deadliest pathogens: malaria, influenza, and streptococcus among others. We show that the New World monkeys, comprising the third of all primate species, have human-like sialic acid biology. They have lost Neu5Gc because of an independent CMAH inactivation ~30 million years ago (mya) (compared to ~3 mya in hominids). This parallel loss of Neu5Gc opens sialic acid biology to comparative phylogenetic analysis and reveals an unexpected conservation priority. New World monkeys risk infection by human pathogens that can recognize cells in the absence of Neu5Gc. This striking molecular convergence provides a mechanism that could explain the long-standing observation that New World monkeys are susceptible to some human diseases that cannot be transmitted to other primates.

PRC2 Represses Hormone-Induced Somatic Embryogenesis in Vegetative Tissue of Arabidopsis thaliana

PubMed Central

Mozgová, Iva

2017-01-01

Many plant cells can be reprogrammed into a pluripotent state that allows ectopic organ development. Inducing totipotent states to stimulate somatic embryo (SE) development is, however, challenging due to insufficient understanding of molecular barriers that prevent somatic cell dedifferentiation. Here we show that Polycomb repressive complex 2 (PRC2)-activity imposes a barrier to hormone-mediated transcriptional reprogramming towards somatic embryogenesis in vegetative tissue of Arabidopsis thaliana. We identify factors that enable SE development in PRC2-depleted shoot and root tissue and demonstrate that the establishment of embryogenic potential is marked by ectopic co-activation of crucial developmental regulators that specify shoot, root and embryo identity. Using inducible activation of PRC2 in PRC2-depleted cells, we demonstrate that transient reduction of PRC2 activity is sufficient for SE formation. We suggest that modulation of PRC2 activity in plant vegetative tissue combined with targeted activation of developmental pathways will open possibilities for novel approaches to cell reprogramming. PMID:28095419

Genetic improvement of mastitis resistance: validation of somatic cell score and clinical mastitis as selection criteria.

PubMed

Odegård, J; Klemetsdal, G; Heringstad, B

2003-12-01

Mean daughter deviations for clinical mastitis among second-crop daughters were regressed on predicted transmitting abilities for clinical mastitis and lactation mean somatic cell score in first-crop daughters to validate the predictive ability of these traits as selection criteria for reduced incidence of clinical mastitis. A total of 321 sires had 684,897 second-crop daughters, while predicted transmitting abilities were calculated for 2159 sires, based on 495,681 records of first-crop daughters. Predictive ability, as a measure of efficiency of selection, was 23 to 43% higher for clinical mastitis than for lactation mean somatic cell score. Compared to single-trait selection, predictive ability improved 8 to 13% from utilizing information on both traits. The relative weight that should be assigned to standardized predicted transmitting abilities from univariate genetic analyses were 60 to 67% for clinical mastitis and 33 to 40% for lactation mean somatic cell score. No significant nonlinear genetic relationship between the two traits was found.

Effects of copper and arsenic stress on the development of Norway spruce somatic embryos and their visualization with the environmental scanning electron microscope.

PubMed

Đorđević, Biljana; Neděla, Vilém; Tihlaříková, Eva; Trojan, Václav; Havel, Ladislav

2018-05-18

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 μM and 10-50 μM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce. Copyright © 2018 Elsevier B.V. All rights reserved.

Somatic Cell Nuclear Transfer in the Mouse

NASA Astrophysics Data System (ADS)

Kishigami, Satoshi; Wakayama, Teruhiko

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

The Xenopus Maternal-to-Zygotic Transition from the Perspective of the Germline.

PubMed

Yang, Jing; Aguero, Tristan; King, Mary Lou

2015-01-01

In Xenopus, the germline is specified by the inheritance of germ-plasm components synthesized at the beginning of oogenesis. Only the cells in the early embryo that receive germ plasm, the primordial germ cells (PGCs), are competent to give rise to the gametes. Thus, germ-plasm components continue the totipotent potential exhibited by the oocyte into the developing embryo at a time when most cells are preprogrammed for somatic differentiation as dictated by localized maternal determinants. When zygotic transcription begins at the mid-blastula transition, the maternally set program for somatic differentiation is realized. At this time, genetic control is ceded to the zygotic genome, and developmental potential gradually becomes more restricted within the primary germ layers. PGCs are a notable exception to this paradigm and remain transcriptionally silent until the late gastrula. How the germ-cell lineage retains full potential while somatic cells become fate restricted is a tale of translational repression, selective degradation of somatic maternal determinants, and delayed activation of zygotic transcription. © 2015 Elsevier Inc. All rights reserved.

Clock-like mutational processes in human somatic cells

PubMed Central

Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; Sale, Julian E.; Campbell, Peter J.; Nik-Zainal, Serena; Stratton, Michael R.

2016-01-01

During the course of a lifetime somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell’s genome. Some processes generate mutations throughout life at a constant rate in all individuals and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutation rates in different tissues. However, their mutation rates are not correlated indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This study provides the first survey of clock-like mutational processes operative in human somatic cells. PMID:26551669

Advanced glycation end products of bovine serum albumin-induced endothelial-to-mesenchymal transition in cultured human and monkey endothelial cells via protein kinase B signaling cascades.

PubMed

Ma, Jianli; Liu, Ting; Dong, Xiaoguang

2010-12-09

Advanced glycation end products of BSA (AGE-BSA) participate in the pathogenesis of diabetic vascular disease. However, the role of AGE-BSA in diabetic retinopathy, especially in retinal neovascularization, remains incomplete. This study aimed to determine the contributions of AGE-BSA in the endothelial-to-mesenchymal transition (EnMT) of cultured human and monkey endothelial cell lines and the mechanism that may be related with the transition. Monkey choroid-retinal endothelial cells (RF/6A) and human umbilical vein endothelial cells (HUVEC) were cultured in Dulbecco's modified Eagle's Medium (DMEM) and Ham's F12 medium containing 200 mg/l AGE-BSA. The expression of VE-cadherin, β-catenin, vimentin, N-cadherin, and protein kinase B (AKT2) was observed by immunocytochemistry and flow cytometry. Cell motility was determined by migration assays; the endothelial function of the formatting tube was measured by tube formation assays, while the change in the polarity was measured using resistance instruments. The characteristics of EnMT included loss of endothelial markers of VE-cadherin and β-catenin, which were replaced by mesenchymal markers of vimentin and N-cadherin, enhanced migration and tube formation, and diminished polarity. AGE-BSA contributed to upregulation of the protein expression of VE-cadherin and β-catenin and downregulation of protein expression of vimentin and N-cadherin, leading to enhanced migration and tube formation and diminished polarity. During this process, expression of AKT2 was upregulated. AGE-BSA can induce EnMT of cultured human and monkey endothelial cells. The signal pathway involving AKT2 may play a role in this process.

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

PubMed Central

Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

2015-01-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

Attentional modulation of cell-class specific gamma-band synchronization in awake monkey area V4

PubMed Central

Vinck, Martin; Womelsdorf, Thilo; Buffalo, Elizabeth A.; Desimone, Robert; Fries, Pascal

2013-01-01

Summary Selective visual attention is subserved by selective neuronal synchronization, entailing precise orchestration among excitatory and inhibitory cells. We tentatively identified these as broad (BS) and narrow spiking (NS) cells and analyzed their synchronization to the local field potential in two macaque monkeys performing a selective visual attention task. Across cells, gamma phases scattered widely but were unaffected by stimulation or attention. During stimulation, NS cells lagged BS cells on average by ~60° and gamma synchronized twice as strongly. Attention enhanced and reduced the gamma locking of strongly and weakly activated cells, respectively. During a pre-stimulus attentional cue period, BS cells showed weak gamma synchronization, while NS cells gamma synchronized as strongly as with visual stimulation. These analyses reveal the cell-type specific dynamics of the gamma cycle in macaque visual cortex and suggest that attention affects neurons differentially depending on cell type and activation level. PMID:24267656

Species-specific inflammatory responses as a primary component for the development of glomerular lesions in mice and monkeys following chronic administration of a second-generation antisense oligonucleotide.

PubMed

Frazier, Kendall S; Sobry, Cécile; Derr, Victoria; Adams, Mike J; Besten, Cathaline Den; De Kimpe, Sjef; Francis, Ian; Gales, Tracy L; Haworth, Richard; Maguire, Shaun R; Mirabile, Rosanna C; Mullins, David; Palate, Bernard; Doorten, Yolanda Ponstein-Simarro; Ridings, James E; Scicchitano, Marshall S; Silvano, Jérémy; Woodfine, Jennie

2014-07-01

Chronic administration of drisapersen, a 2'-OMe phosphorothioate antisense oligonucleotide (AON) to mice and monkeys resulted in renal tubular accumulation, with secondary tubular degeneration. Glomerulopathy occurred in both species with species-specific characteristics. Glomerular lesions in mice were characterized by progressive hyaline matrix accumulation, accompanied by the presence of renal amyloid and with subsequent papillary necrosis. Early changes involved glomerular endothelial hypertrophy and degeneration, but the chronic glomerular amyloid and hyaline alterations in mice appeared to be species specific. An immune-mediated mechanism for the glomerular lesions in mice was supported by early inflammatory changes including increased expression of inflammatory cytokines and other immunomodulatory genes within the renal cortex, increased stimulation of CD68 protein, and systemic elevation of monocyte chemotactic protein 1. In contrast, kidneys from monkeys given drisapersen chronically showed less severe glomerular changes characterized by increased mesangial and inflammatory cells, endothelial cell hypertrophy, and subepithelial and membranous electron-dense deposits, with ultrastructural and immunohistochemical characteristics of complement and complement-related fragments. Lesions in monkeys resembled typical features of C3 glomerulopathy, a condition described in man and experimental animals to be linked to dysregulation of the alternative complement pathway. Thus, inflammatory/immune mechanisms appear critical to glomerular injury with species-specific sensitivities for mouse and monkey. The lower observed proinflammatory activity in humans as compared to mice and monkeys may reflect a lower risk of glomerular injury in patients receiving AON therapy. © 2014 by The Author(s).

Developmental sources of conservation and variation in the evolution of the primate eye.

PubMed

Dyer, Michael A; Martins, Rodrigo; da Silva Filho, Manoel; Muniz, José Augusto P C; Silveira, Luiz Carlos L; Cepko, Constance L; Finlay, Barbara L

2009-06-02

Conserved developmental programs, such as the order of neurogenesis in the mammalian eye, suggest the presence of useful features for evolutionary stability and variability. The owl monkey, Aotus azarae, has developed a fully nocturnal retina in recent evolution. Description and quantification of cell cycle kinetics show that embryonic cytogenesis is extended in Aotus compared with the diurnal New World monkey Cebus apella. Combined with the conserved mammalian pattern of retinal cell specification, this single change in retinal progenitor cell proliferation can produce the multiple alterations of the nocturnal retina, including coordinated reduction in cone and ganglion cell numbers, increase in rod and rod bipolar numbers, and potentially loss of the fovea.

Production of Reference Enteroviruses

PubMed Central

Kalter, S. S.; Rodriguez, A. R.; Armour, V.

1968-01-01

Forty-five human enterovirus reagents of certified purity and quality were prepared for use as seed viruses and as immunizing antigens. One of the reagents was ampouled as “untreated” seed virus, whereas 14 were ampouled as “MgCl2-stabilized” reagents. The remaining 30 reagents were ampouled as “untreated” seed viruses and as “MgCl2-stabilized” reagents. Thirty of the reagents were propagated on primary African green monkey kidney cells, 3 on primary baboon kidney cells, 3 on primary rhesus monkey kidney cells, and the remaining 9 on human amnion cells. Forty-two of the viral antigens were concentrated for use in the production of high-titered specific antisera in large animals. PMID:4300898

Carbohydrate-mediated responses during zygotic and early somatic embryogenesis in the endangered conifer, Araucaria angustifolia

PubMed Central

Elbl, Paula; De Souza, Amanda P.; Jardim, Vinicius; de Oliveira, Leandro F.; Macedo, Amanda F.; dos Santos, André L. W.; Buckeridge, Marcos S.; Floh, Eny I. S.

2017-01-01

Three zygotic developmental stages and two somatic Araucaria angustifolia cell lines with contrasting embryogenic potential were analyzed to identify the carbohydrate-mediated responses associated with embryo formation. Using a comparison between zygotic and somatic embryogenesis systems, the non-structural carbohydrate content, cell wall sugar composition and expression of genes involved in sugar sensing were analyzed, and a network analysis was used to identify coordinated features during embryogenesis. We observed that carbohydrate-mediated responses occur mainly during the early stages of zygotic embryo formation, and that during seed development there are coordinated changes that affect the development of the different structures (embryo and megagametophyte). Furthermore, sucrose and starch accumulation were associated with the responsiveness of the cell lines. This study sheds light on how carbohydrate metabolism is influenced during zygotic and somatic embryogenesis in the endangered conifer species, A. angustifolia. PMID:28678868

Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

PubMed

Gao, Fang; Li, Jingyu; Zhang, Heng; Yang, Xu; An, Tiezhu

2017-08-01

Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

Genome engineering in cattle: recent technological advancements.

PubMed

Wang, Zhongde

2015-02-01

Great strides in technological advancements have been made in the past decade in cattle genome engineering. First, the success of cloning cattle by somatic cell nuclear transfer (SCNT) or chromatin transfer (CT) is a significant advancement that has made obsolete the need for using embryonic stem (ES) cells to conduct cell-mediated genome engineering, whereby site-specific genetic modifications can be conducted in bovine somatic cells via DNA homologous recombination (HR) and whereby genetically engineered cattle can subsequently be produced by animal cloning from the genetically modified cells. With this approach, a chosen bovine genomic locus can be precisely modified in somatic cells, such as to knock out (KO) or knock in (KI) a gene via HR, a gene-targeting strategy that had almost exclusively been used in mouse ES cells. Furthermore, by the creative application of embryonic cloning to rejuvenate somatic cells, cattle genome can be sequentially modified in the same line of somatic cells and complex genetic modifications have been achieved in cattle. Very recently, the development of designer nucleases-such as zinc finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-has enabled highly efficient and more facile genome engineering in cattle. Most notably, by employing such designer nucleases, genomes can be engineered at single-nucleotide precision; this process is now often referred to as genome or gene editing. The above achievements are a drastic departure from the traditional methods of creating genetically modified cattle, where foreign DNAs are randomly integrated into the animal genome, most often along with the integrations of bacterial or viral DNAs. Here, I review the most recent technological developments in cattle genome engineering by highlighting some of the major achievements in creating genetically engineered cattle for agricultural and biomedical applications.

Can Metabolic Mechanisms of Stem Cell Maintenance Explain Aging and the Immortal Germline?

PubMed

Snoeck, Hans-Willem

2015-06-04

The mechanisms underlying the aging process are not understood. Even tissues endowed with somatic stem cells age while the germline appears immortal. I propose that this paradox may be explained by the pervasive use of glycolysis by somatic stem cells as opposed to the predominance of mitochondrial respiration in gametes. Copyright © 2015 Elsevier Inc. All rights reserved.

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells.

PubMed

Keam, Simon P; Young, Paul E; McCorkindale, Alexandra L; Dang, Thurston H Y; Clancy, Jennifer L; Humphreys, David T; Preiss, Thomas; Hutvagner, Gyorgy; Martin, David I K; Cropley, Jennifer E; Suter, Catherine M

2014-08-01

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterization of Bacteroides forsythus Strains from Cat and Dog Bite Wounds in Humans and Comparison with Monkey and Human Oral Strains

PubMed Central

Hudspeth, M. K.; Gerardo, S. Hunt; Maiden, M. F. J.; Citron, D. M.; Goldstein, E. J. C.

1999-01-01

Bacteroides forsythus strains recovered from cat and dog bite wound infections in humans (n = 3), monkey oral strains (n = 3), and the human oral ATCC 43037 type strain were characterized by using phenotypic characteristics, enzymatic tests, whole cell fatty acid analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, PCR fingerprinting, and 16S rDNA (genes coding for rRNA) sequencing. All three bite wound isolates grew on brucella agar supplemented with 5% sheep blood, vitamin K1, and hemin. These strains, unlike the ATCC strain and previously described monkey oral and human clinical strains, did not require N-acetylmuramic acid supplementation for growth as pure cultures. However, their phenotypic characteristics, except for catalase production, were similar to those of previously identified strains. PCR fingerprinting analysis showed differences in band patterns from the ATCC strain. Also, SDS-PAGE and whole cell fatty acid analysis indicated that the dog and cat bite wound strains were similar but not identical to the human B. forsythus ATCC 43037 type strain and the monkey oral strains. The rDNA sequence analysis indicated that the three bite wound isolates had 99.93% homology with each other and 98.9 and 99.22% homology with the human ATCC 43037 and monkey oral strains, respectively. These results suggest that there are host-specific variations within each group. PMID:10325363

40 CFR 799.4360 - Tributyl phosphate.

Code of Federal Regulations, 2013 CFR

2013-07-01

... in somatic cells in culture test shall be conducted with TBP in accordance with § 798.5300 of this... requirements. (A) The somatic cells in culture assay shall be completed and the final report submitted to EPA...) Required testing. (A) An in vitro mammalian cytogenetics test shall be conducted with TBP in accordance...

40 CFR 799.4360 - Tributyl phosphate.

Code of Federal Regulations, 2012 CFR

2012-07-01

... in somatic cells in culture test shall be conducted with TBP in accordance with § 798.5300 of this... requirements. (A) The somatic cells in culture assay shall be completed and the final report submitted to EPA...) Required testing. (A) An in vitro mammalian cytogenetics test shall be conducted with TBP in accordance...

40 CFR 799.4360 - Tributyl phosphate.

Code of Federal Regulations, 2014 CFR

2014-07-01

... in somatic cells in culture test shall be conducted with TBP in accordance with § 798.5300 of this... requirements. (A) The somatic cells in culture assay shall be completed and the final report submitted to EPA...) Required testing. (A) An in vitro mammalian cytogenetics test shall be conducted with TBP in accordance...

Atherosclerosis in the Erythrocebus patas, an old world monkey.

PubMed Central

Mahley, R. W.; Johnson, D. K.; Pucak, G. J.; Fry, D. L.

1980-01-01

Fifty monkeys of the species Erythrocebus patas were fed a control monkey chow, a semi-synthetic diet containing 25% lard, or a semisynthetic diet containing 25% lard and 0.5% cholesterol for 2 years. The patas monkeys had naturally occurring atherosclerosis that was greatly accelerated by feeding a diet containing cholesterol. The atherosclerosis involved the aorta, predominantly the abdominal portion, the coronary arteries, and various peripheral vessels. Histologically, the atherosclerosis was characterized by intimal proliferative lesions associated with intra- and extracellular lipid deposition. Complicated lesions that developed after 2 years on the cholesterol-containing diet were associated with lipid crystals, necrosis, mineralization, and encroachment upon the media. Adventitial reactions characterized by increased vascularity and the presence of inflammatory cells were seen. All of these observations have been described as components of the human atherosclerotic disease process. The similarity of the patas monkey atherosclerosis to human atherosclerosis, the relatively large size and easy handling of the animals, and the fact that previous studies have shown the lipoproteins of both control and cholesterol-fed monkeys to resemble human lipoproteins all contribute to making the patas monkey a useful model for the study of experimental atherosclerosis. Images Figure 1-5 Figure 6 Figure 7-10 Figure 11 Figure 12 PMID:6766672

An Overview of Direct Somatic Reprogramming: The Ins and Outs of iPSCs

PubMed Central

Menon, Siddharth; Shailendra, Siny; Renda, Andrea; Longaker, Michael; Quarto, Natalina

2016-01-01

Stem cells are classified into embryonic stem cells and adult stem cells. An evolving alternative to conventional stem cell therapies is induced pluripotent stem cells (iPSCs), which have a multi-lineage potential comparable to conventionally acquired embryonic stem cells with the additional benefits of being less immunoreactive and avoiding many of the ethical concerns raised with the use of embryonic material. The ability to generate iPSCs from somatic cells provides tremendous promise for regenerative medicine. The breakthrough of iPSCs has raised the possibility that patient-specific iPSCs can provide autologous cells for cell therapy without the concern for immune rejection. iPSCs are also relevant tools for modeling human diseases and drugs screening. However, there are still several hurdles to overcome before iPSCs can be used for translational purposes. Here, we review the recent advances in somatic reprogramming and the challenges that must be overcome to move this strategy closer to clinical application. PMID:26805822

Bovine oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP).

PubMed

Tani, Tetsuya; Shimada, Hiroaki; Kato, Yoko; Tsunoda, Yukio

2007-01-01

Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary.

Direct reprogramming of somatic cells into neural stem cells or neurons for neurological disorders.

PubMed

Hou, Shaoping; Lu, Paul

2016-01-01

Direct reprogramming of somatic cells into neurons or neural stem cells is one of the most important frontier fields in current neuroscience research. Without undergoing the pluripotency stage, induced neurons or induced neural stem cells are a safer and timelier manner resource in comparison to those derived from induced pluripotent stem cells. In this prospective, we review the recent advances in generation of induced neurons and induced neural stem cells in vitro and in vivo and their potential treatments of neurological disorders.

Relationships Among Microbial Communities, Maternal Cells, Oligosaccharides, and Macronutrients in Human Milk.

PubMed

Williams, Janet E; Price, William J; Shafii, Bahman; Yahvah, Katherine M; Bode, Lars; McGuire, Mark A; McGuire, Michelle K

2017-08-01

Human milk provides all essential nutrients necessary for early life and is rich in nonnutrients, maternally derived (host) cells, and bacteria, but almost nothing is known about the interplay among these components. Research aim: The primary objective of this research was to characterize relationships among macronutrients, maternal cells, and bacteria in milk. Milk samples were collected from 16 women and analyzed for protein, lipid, fatty acid, lactose, and human milk oligosaccharide concentrations. Concentrations of maternal cells were determined using microscopy, and somatic cell counts were enumerated. Microbial ecologies were characterized using culture-independent methods. Absolute and relative concentrations of maternal cells were mostly consistent within each woman as were relative abundances of bacterial genera, and there were many apparent relationships between these factors. For instance, relative abundance of Serratia was negatively associated with somatic cell counts ( r = -.47, p < .0001) and neutrophil concentration ( r = -.38, p < .0006). Concentrations of several oligosaccharides were correlated with maternally derived cell types as well as somatic cell counts; for example, lacto-N-tetraose and lacto-N-neotetraose were inversely correlated with somatic cell counts ( r = -.64, p = .0082; r = -.52, p = .0387, respectively), and relative abundance of Staphylococcus was positively associated with total oligosaccharide concentration ( r = .69, p = .0034). Complex relationships between milk nutrients and bacterial community profile, maternal cells, and milk oligosaccharides were also apparent. These data support the possibility that profiles of maternally derived cells, nutrient concentrations, and the microbiome of human milk might be interrelated.

Defined three-dimensional microenvironments boost induction of pluripotency

NASA Astrophysics Data System (ADS)

Caiazzo, Massimiliano; Okawa, Yuya; Ranga, Adrian; Piersigilli, Alessandra; Tabata, Yoji; Lutolf, Matthias P.

2016-03-01

Since the discovery of induced pluripotent stem cells (iPSCs), numerous approaches have been explored to improve the original protocol, which is based on a two-dimensional (2D) cell-culture system. Surprisingly, nothing is known about the effect of a more biologically faithful 3D environment on somatic-cell reprogramming. Here, we report a systematic analysis of how reprogramming of somatic cells occurs within engineered 3D extracellular matrices. By modulating microenvironmental stiffness, degradability and biochemical composition, we have identified a previously unknown role for biophysical effectors in the promotion of iPSC generation. We find that the physical cell confinement imposed by the 3D microenvironment boosts reprogramming through an accelerated mesenchymal-to-epithelial transition and increased epigenetic remodelling. We conclude that 3D microenvironmental signals act synergistically with reprogramming transcription factors to increase somatic plasticity.

Stem cell fusion as an ultimate line of defense against xenobiotics.

PubMed

Padron Velazquez, Julio Lazaro

2006-01-01

There are several indications that the potential of stem cells to fuse with somatic cells is extremely high and, what's more exciting, in some instances goes as far as reprogramming and/or rescuing altered cells. It remains unclear, however, how frequent this mechanism is and what patho-physiological role it might play in nature. A plausible hypothesis, discussed in this paper, suggests that stem cell niches might provide a safeguard for the intact genome and epigenome. By fusing with somatic de-differentiated cells, stem cells might consent epigenetic reprogramming and/or genetic recovery of genes which otherwise could drive altered cells to malignancy. If the many sophisticated mechanisms of metabolism, cell repair, programmed cell death and tissue regeneration should fail, stem cells might represent a final attempt to recover dedifferentiated cells to avoid inflowing in cancer. In the current reappraisal of the different mechanisms of defense against xenobiotics, even the incidence of cancer itself is considered an evolving mechanism which, through a kind of programmed death of individuals exhibiting defective mutations, favors advancement of the phenotypes which adapt best. Additionally, with regard to the mechanisms of transmitting somatic mutations, based on stem cells' capacity to migrate and to fuse, here it is speculated that stem cells might be capable of carrying acquired somatic mutations from peripheral tissues to the gonads, and transmit that information into the germinal line. If appropriately demonstrated, these mechanisms might delineate a novel therapeutic area to be explored. The use of stem cells to reprogram/recover irreversibly damaged cells or to transmit beneficial mutations might be a valuable therapeutic approach in the future.

Is the replication of somatic coliphages in water environments significant?

PubMed

Jofre, J

2009-04-01

Somatic coliphages are amid several groups of bacteriophages that have been suggested as indicators in water quality assessment. One of the limitations frequently endorsed to somatic coliphages as indicators is that they can replicate in the water environment. This review intends to evaluate the significance of this potential replication. In view of: the threshold densities of somatic coliphages and host bacteria needed for productive infection to occur, the densities of both host cells supporting somatic coliphages replication and these phages in water environments, and the poor contribution of lysogenic induction to the free somatic coliphage numbers in water, it can be concluded that replication of somatic coliphages in waters is very unlikely. Consequently, the contribution of replication in the environment of somatic coliphages is expected to have a non-noticeable influence on the numbers of somatic coliphages detected in water environments. Thus, the replication in the environment should not be argued as a limitation to the use of somatic coliphages as indicators.

One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs.

PubMed

Zuo, Erwei; Cai, Yi-Jun; Li, Kui; Wei, Yu; Wang, Bang-An; Sun, Yidi; Liu, Zhen; Liu, Jiwei; Hu, Xinde; Wei, Wei; Huo, Xiaona; Shi, Linyu; Tang, Cheng; Liang, Dan; Wang, Yan; Nie, Yan-Hong; Zhang, Chen-Chen; Yao, Xuan; Wang, Xing; Zhou, Changyang; Ying, Wenqin; Wang, Qifang; Chen, Ren-Chao; Shen, Qi; Xu, Guo-Liang; Li, Jinsong; Sun, Qiang; Xiong, Zhi-Qi; Yang, Hui

2017-07-01

The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.

Comparative Infectivity Determinations of Dengue Virus Vaccine Candidates in Rhesus Monkeys, Mosquitoes, and Cell Cultures

DTIC Science & Technology

1993-01-28

34 are required for the evaluation of these vaccine candidates. RE: DAMDI7-89-C-9175 Page 16 REFERENCES 1. Sabin AB, Sclesinger RW, 1945. Production of...AD-A261 892 CONTRACT NO: DAMD17-89-C-9 175 \\II\\IllI\\I\\I1\\\\~il\\ TITLE: COMPARATIVE INFECTIVITY DETERMINATIONS OF DENGUE VIRUS VACCINE CANDIDATES IN... Vaccine Candidates in Rhesus Monkeys, 63002A Mosquitoes, and Cell Cultures 3M263002D870 AC 6. AUTHOR(S) DA335475 Edmundo Kraiselburd 7. PERFORMING

A soma-to-germline transformation in long-lived C. elegans mutants

PubMed Central

Curran, Sean P.; Wu, Xiaoyun; Riedel, Christian G.; Ruvkun, Gary

2009-01-01

Unlike the soma which ages during the lifespan of multicellular organisms, the germline traces an essentially immortal lineage. Genomic instability in somatic cells increases with age, and this decline in somatic maintenance might be regulated to facilitate resource reallocation toward reproduction at the expense of cellular senescence. We report here that C. elegans mutants with increased longevity exhibit a soma-to-germline transformation of gene expression programs normally limited to the germline. Decreased insulin-like signaling causes the somatic misexpression of germline-limited pie-1 and pgl family of genes in intestinal and ectodermal tissues. DAF-16/FoxO, the major transcriptional effector of insulin-like signaling, regulates pie-1 expression by directly binding to the pie-1 promoter. The somatic tissues of insulin-like mutants are more germline-like and protected from genotoxic stress. Gene inactivation of components of the cytosolic chaperonin complex that induce increased longevity also cause somatic misexpression of PGL-1. These results suggest that the acquisition of germline characteristics by the somatic cells of C. elegans mutants with increased longevity contributes to their increased health and survival. PMID:19506556

Evolutionary Origin of OwlRep, a Megasatellite DNA Associated with Adaptation of Owl Monkeys to Nocturnal Lifestyle

PubMed Central

Nishihara, Hidenori; Stanyon, Roscoe; Kusumi, Junko; Hirai, Hirohisa

2018-01-01

Abstract Rod cells of many nocturnal mammals have a “non-standard” nuclear architecture, which is called the inverted nuclear architecture. Heterochromatin localizes to the central region of the nucleus. This leads to an efficient light transmission to the outer segments of photoreceptors. Rod cells of diurnal mammals have the conventional nuclear architecture. Owl monkeys (genus Aotus) are the only taxon of simian primates that has a nocturnal or cathemeral lifestyle, and this adaptation is widely thought to be secondary. Their rod cells were shown to exhibit an intermediate chromatin distribution: a spherical heterochromatin block was found in the central region of the nucleus although it was less complete than that of typical nocturnal mammals. We recently demonstrated that the primary DNA component of this heterochromatin block was OwlRep, a megasatellite DNA consisting of 187-bp-long repeat units. However, the origin of OwlRep was not known. Here we show that OwlRep was derived from HSAT6, a simple repeat sequence found in the centromere regions of human chromosomes. HSAT6 occurs widely in primates, suggesting that it was already present in the last common ancestor of extant primates. Notably, Strepsirrhini and Tarsiformes apparently carry a single HSAT6 copy, whereas many species of Simiiformes contain multiple copies. Comparison of nucleotide sequences of these copies revealed the entire process of the OwlRep formation. HSAT6, with or without flanking sequences, was segmentally duplicated in New World monkeys. Then, in the owl monkey linage after its divergence from other New World monkeys, a copy of HSAT6 was tandemly amplified, eventually forming a megasatellite DNA. PMID:29294004

A framework for evaluating developmental defects at the cellular level: An example from ten maize anther mutants using morphological and molecular data.

PubMed

Egger, Rachel L; Walbot, Virginia

2016-11-01

In seed plants, anthers are critical for sexual reproduction, because they foster both meiosis and subsequent pollen development of male germinal cells. Male-sterile mutants are analyzed to define steps in anther development. Historically the major topics in these studies are meiotic arrest and post-meiotic gametophyte failure, while relatively few studies focus on pre-meiotic defects of anther somatic cells. Utilizing morphometric analysis we demonstrate that pre-meiotic mutants can be impaired in anticlinal or periclinal cell division patterns and that final cell number in the pre-meiotic anther lobe is independent of cell number changes of individual differentiated somatic cell types. Data derived from microarrays and from cell wall NMR analyses allow us to further refine our understanding of the onset of phenotypes. Collectively the data highlight that even minor deviations from the correct spatiotemporal pattern of somatic cell proliferation can result in male sterility in Zea mays. Copyright © 2016 Elsevier Inc. All rights reserved.

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

PubMed

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2015-07-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

PubMed Central

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H.

2015-01-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods. PMID:26098448

Predatory stem cells in the non-zebrafish chordate, Botryllus schlosseri.

PubMed

Laird, Diana J; De Tomaso, Anthony W

2005-01-01

Botryllus schlosseri is a primitive marine chordate which provides a new model organism to study stem cell biology for several reasons. First, B. schlosseri is a colonial organism that undergoes continuous and regular asexual development. Botryllus adults regenerate themselves, including all somatic tissues and the germline, every week. Second, under natural conditions the cells responsible can mobilize and transplant between two individuals. Once transplanted, these cells can proliferate, differentiate, and often completely replace the cells of the host in both the germline and/or somatic tissues. These processes are called germ cell parasitism (gcp), or somatic cell parasitism (scp), respectively, and we have shown that there are winners and losers in this process, implying that the competitive ability of stem cells is a genetically-determined trait. Fundamental characteristics of stem cell biology, such as self-renewal capacity, homing, or differentiation kinetics must underlie the ability of a stem cell of one genotype to out-compete a stem cell of another genotype, and we are using this system prospectively to isolate the cells responsible and to analyze the molecular mechanisms underlying gcp and scp phenotypes.

Changes in the Plasma Proteome Follows Chronic Opiate Administration In Simian Immunodeficiency Virus Infected Rhesus Macaques*

PubMed Central

Wiederin, Jayme L.; Yu, Fang; Donahoe, Robert M.; Fox, Howard S.; Ciborowski, Pawel; Gendelman, Howard E.

2011-01-01

Background Substantive plasma proteomic changes follow lentiviral infection and disease pathobiology. We posit that such protein alterations are modified during drug abuse, further serving to affect the disease. To this end, we investigated the effect of opiate administration on the plasma proteome of Indian-strain rhesus monkeys infected with simian immunodeficiency virus (SIV) strain smm9. Methods Whole blood was collected at 7 weeks prior to and 1.4 and 49 weeks after viral infection. Viral load, CD4+ T cell subsets, and plasma protein content were measured from monkeys that did or did not receive continuous opiate administrations. The plasma proteome was identified and quantified by isobaric tags for relative and absolute quantitation labeling (iTRAQ) and mass spectrometry. Results While substantive changes in plasma proteins were seen during SIV infection, the addition of opiates led to suppression of these changes as well as increased variance of the proteome. These changes demonstrate that opiates induce broad but variant immune suppression in SIV-infected monkeys. Conclusion The broad suppressive changes seen in plasma of SIV-infected monkeys likely reflect reduced multisystem immune homeostatic responses induced by opiates. Such occur as a consequence of complex cell-to-cell interactions operative between the virus and the host. We conclude that such changes in plasma proteomic profiling may be underappreciated and as such supports the need for improved clinical definitions. PMID:21821369

A New, Dynamic Era for Somatic Cell Nuclear Transfer?

PubMed

Loi, Pasqualino; Iuso, Domenico; Czernik, Marta; Ogura, Atsuo

2016-10-01

Cloning animals by somatic cell nuclear transfer (SCNT) has remained an uncontrollable process for many years. High rates of embryonic losses, stillbirths, and postnatal mortality have been typical outcomes. These developmental problems arise from abnormal genomic reprogramming: the capacity of the oocyte to reset the differentiated memory of a somatic cell. However, effective reprogramming strategies are now available. These target the whole genome or single domains such as the Xist gene, and their effectiveness has been validated with the ability of experimental animals to develop to term. Thus, SCNT has become a controllable process that can be used to 'rescue' endangered species, and for biomedical research such as therapeutic cloning and the isolation of induced pluripotent stem cells (iPSCs). Copyright © 2016 Elsevier Ltd. All rights reserved.

Somatic Point Mutation Calling in Low Cellularity Tumors

PubMed Central

Kassahn, Karin S.; Holmes, Oliver; Nones, Katia; Patch, Ann-Marie; Miller, David K.; Christ, Angelika N.; Harliwong, Ivon; Bruxner, Timothy J.; Xu, Qinying; Anderson, Matthew; Wood, Scott; Leonard, Conrad; Taylor, Darrin; Newell, Felicity; Song, Sarah; Idrisoglu, Senel; Nourse, Craig; Nourbakhsh, Ehsan; Manning, Suzanne; Wani, Shivangi; Steptoe, Anita; Pajic, Marina; Cowley, Mark J.; Pinese, Mark; Chang, David K.; Gill, Anthony J.; Johns, Amber L.; Wu, Jianmin; Wilson, Peter J.; Fink, Lynn; Biankin, Andrew V.; Waddell, Nicola; Grimmond, Sean M.; Pearson, John V.

2013-01-01

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform. PMID:24250782

Segregation of short-wavelength-sensitive (S) cone signals in the macaque dorsal lateral geniculate nucleus

PubMed Central

Roy, Sujata; Jayakumar, Jaikishan; Martin, Paul R; Dreher, Bogdan; Saalmann, Yuri B; Hu, Daping; Vidyasagar, Trichur R

2009-01-01

An important problem in the study of the mammalian visual system is whether functionally different retinal ganglion cell types are anatomically segregated further up along the central visual pathway. It was previously demonstrated that, in a New World diurnal monkey (marmoset), the neurones carrying signals from the short-wavelength-sensitive (S) cones [blue–yellow (B/Y)-opponent cells] are predominantly located in the koniocellular layers of the dorsal lateral geniculate nucleus (LGN), whereas the red–green (R/G)-opponent cells carrying signals from the medium- and long-wavelength-sensitive cones are segregated in the parvocellular layers. Here, we used extracellular single-unit recordings followed by histological reconstruction to investigate the distribution of color-selective cells in the LGN of the macaque, an Old World diurnal monkey. Cells were classified using cone-isolating stimuli to identify their cone inputs. Our results indicate that the majority of cells carrying signals from S-cones are located either in the koniocellular layers or in the ‘koniocellular bridges’ that fully or partially span the parvocellular layers. By contrast, the R/G-opponent cells are located in the parvocellular layers. We conclude that anatomical segregation of B/Y- and R/G-opponent afferent signals for color vision is common to the LGNs of New World and Old World diurnal monkeys. PMID:19821840

Selfish cells in altruistic cell society - a theoretical oncology.

PubMed

Chigira, M

1993-09-01

In multicellular organisms, internal evolution of individual cells is strictly forbidden and 'evolutional' DNA replication should be performed only by the sexual reproduction system. Wholistic negative control system called 'homeostasis' serves all service to germ line cells. All somatic cells are altruistic to the germ line cells. However, in malignant tumors, it seems that individual cells replicate and behave 'selfishly' and evolve against the internal microenvironment. Tumor cells only express the occult selfishness which is programmed in normal cells a priori. This phenomenon is based on the failure of identical DNA replication, and results in 'autonomy' and 'anomie' of cellular society as shown in tumor cells. Genetic programs of normal cells connote this cellular autonomy and anomie introduced by the deletion of regulators on structure genes. It is rather paradoxical that the somatic cells get their freedom from wholistic negative regulation programmed internally. However, this is not a true paradox, since multicellular organisms have clearly been evolved from 'monads' in which cells proliferate without wholistic regulation. Somatic cells revolt against germ cell DNA, called 'selfish replicator' by Dawkins. It is an inevitable destiny that the 'selfishness' coded in genome should be revenged by itself. Selfish replicator in germ cell line should be revolted by its selfishness in the expansion of somatic cells, since they have an orthogenesis to get more selfishness in order to increase their genome. Tumor heterogeneity and progression can be fully explained by this self-contradictory process which produces heterogeneous gene copies different from the original clone in the tumor, although 'selfish' gene replication is the final target of being. Furthermore, we have to discard the concept of clonality of tumor cells since genetic instability is a fundamental feature of tumors. Finally, tumor cells and proto-oncogenes can be considered as the ultimate parasite to germ line cells.

Consequence of alternative standards for bulk tank somatic cell count of dairy herds in the United States

USDA-ARS?s Scientific Manuscript database

Comparison of dairy operations failing compliance with current US and European Union (EU) standards for bulk-tank somatic cell count (BTSCC) as well as BTSCC standards proposed by 3 national organizations were evaluated using 2 populations of US dairy herds: Dairy Herd Improvement Association (DHI) ...

Multitrait modeling of first vs. later parities for US yield, somatic cell score, and fertility traits

USDA-ARS?s Scientific Manuscript database

Genetic merits in first vs. later parity with correlations <1 were compared to official repeatability models using 88 million lactation records of 34 million cows for yield traits and fewer records for somatic cell score (SCS) and 2 cow fertility traits. Estimated genetic correlations of first with ...

[Investigations on the pathogenesis of changes in somatic growth of Lymnaea stagnalis (Gastropoda: Pulmonata) experimentally infected with parthenites Opisthioglyphe ranae (Digenea: Plagiorchiida). I. Relative weight of accessory sex organs and synthetic activity of neurosecretory cells].

PubMed

Pokora, Z

1996-01-01

In the paper an attempt to define pathogenesis of changes in somatic growth of juvenile individuals of the popular freshwater snail Lymnaea stagnalis experimentally infected with parthenites of the trematode Opisthioglyphe ranae was undertaken. Significant enlargement of relative wet weight of examined accessory sex organs (albumen gland, oothecal gland, prostate, male copulatory organ) observed in infected snails permits to explain increase of their somatic growth basing on the hypothesis of disturbances in energetistic budget of the host-as a consequence of reduction by the parasite activity of the snail's reproductive system. Pathogenesis of this phenomenon has probably a complicated character, including also effect of parthenites on activity of the neurosecretory cells that control somatic growth in examined species of the snail. An argument for this standpoint is, observed in infected snails, increase of amount of neurosecretory material and RNA in cytoplasm of these cells (the light green cells of cerebral ganglia), as well as amount of the loose fraction of chromatine in their nuclei.

Resolving rates of mutation in the brain using single-neuron genomics

PubMed Central

Evrony, Gilad D; Lee, Eunjung; Park, Peter J; Walsh, Christopher A

2016-01-01

Whether somatic mutations contribute functional diversity to brain cells is a long-standing question. Single-neuron genomics enables direct measurement of somatic mutation rates in human brain and promises to answer this question. A recent study (Upton et al., 2015) reported high rates of somatic LINE-1 element (L1) retrotransposition in the hippocampus and cerebral cortex that would have major implications for normal brain function, and suggested that these events preferentially impact genes important for neuronal function. We identify aspects of the single-cell sequencing approach, bioinformatic analysis, and validation methods that led to thousands of artifacts being interpreted as somatic mutation events. Our reanalysis supports a mutation frequency of approximately 0.2 events per cell, which is about fifty-fold lower than reported, confirming that L1 elements mobilize in some human neurons but indicating that L1 mosaicism is not ubiquitous. Through consideration of the challenges identified, we provide a foundation and framework for designing single-cell genomics studies. DOI: http://dx.doi.org/10.7554/eLife.12966.001 PMID:26901440

Prestin and the cholinergic receptor of hair cells: positively-selected proteins in mammals

PubMed Central

Elgoyhen, Ana Belén; Franchini, Lucía F.

2010-01-01

The hair cells of the vertebrate inner ear posses active mechanical processes to amplify their inputs. The stereocilia bundle of various vertebrate animals can produce active movements. Though standard stereocilia-based mechanisms to promote amplification persist in mammals, an additional radically different mechanism evolved: the so called somatic electromotility which refers to the elongation/contraction of the outer hair cells’ (OHC) cylindrical cell body in response to membrane voltage changes. Somatic electromotility in OHCs, as the basis for cochlear amplification, is a mammalian novelty and it is largely dependent upon the properties of the unique motor protein prestin. We review recent literature which has demonstrated that although the gene encoding prestin is present in all vertebrate species, mammalian prestin has been under positive selective pressure to acquire motor properties, probably rendering it fit to serve somatic motility in outer hair cells. Moreover, we discuss data which indicates that a modified α10 nicotinic cholinergic receptor subunit has coevolved in mammals, most likely to give the auditory feedback system the capability to control somatic electromotility. PMID:20056140

Comparative analysis of the oral mucosae from rodents and non-rodents: Application to the nonclinical evaluation of sublingual immunotherapy products

PubMed Central

Thirion-Delalande, Catherine; Gervais, Frédéric; Fisch, Cécile; Cuiné, Jean; Baron-Bodo, Véronique; Moingeon, Philippe

2017-01-01

Background A comparative characterization of the oral mucosa in various animals is needed to identify the best animal model(s) for nonclinical evaluation of sublingual immunotherapy products. With this aim, we studied the histological characteristics and immune cell infiltrates of oral mucosae from common animal species. Methods Three oral regions (i.e. ventral surface of the tongue, mouth floor and cheek) obtained from eight animal species, including rodents (i.e. mice, rats, hamsters, guinea pigs) and non-rodents (i.e. rabbits, dogs, minipigs and monkeys) were characterized by histology and immunohistology in comparison with a human tongue. Results Rodents exhibit a thin keratinized epithelium with low epithelial extensions, whereas non-rodents, most particularly minipigs and monkeys, display a non-keratinized epithelium with larger rete ridges, similarly to humans. Glycogen-rich cells in the superficial epithelial layers are observed in samples from both minipigs, monkeys and humans. Comparable immune subpopulations detected in the 3 oral regions from rodent and non-rodent species include MHC-II+ antigen presenting cells, mostly CD163+ macrophages, located in the lamina propria (LP) and muscle tissue in the vicinity of resident CD3+CD4+ T cells. Limited numbers of mast cells are also detected in the LP and muscle tissue from all species. Conclusion The oral mucosae of minipigs and monkeys are closest to that of humans, and the immune networks are quite similar between all rodents and non-rodents. Taking into account the ethical and logistical difficulties of performing research in the latter species, rodents and especially mice, should preferentially be used for pharmacodynamics/efficacy studies. Our data also support the use of minipigs to perform biodistribution and safety studies of sublingual immunotherapy products. PMID:28886055

Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

PubMed

Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

2013-01-01

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

Keith's MAGIC: Cloning and the Cell Cycle.

PubMed

Wells, D N

2013-10-01

Abstract Professor Keith Campbell's critical contribution to the discovery that a somatic cell from an adult animal can be fully reprogrammed by oocyte factors to form a cloned individual following nuclear transfer (NT)(Wilmut et al., 1997 ) overturned a dogma concerning the reversibility of cell fate that many scientists had considered to be biologically impossible. This seminal experiment proved the totipotency of adult somatic nuclei and finally confirmed that adult cells could differentiate without irreversible changes to the genetic material.

Perspectives for induced pluripotent stem cell technology: new insights into human physiology involved in somatic mosaicism.

PubMed

Nagata, Naoki; Yamanaka, Shinya

2014-01-31

Induced pluripotent stem cell technology makes in vitro reprogramming of somatic cells from individuals with various genetic backgrounds possible. By applying this technology, it is possible to produce pluripotent stem cells from biopsy samples of arbitrarily selected individuals with various genetic backgrounds and to subsequently maintain, expand, and stock these cells. From these induced pluripotent stem cells, target cells and tissues can be generated after certain differentiation processes. These target cells/tissues are expected to be useful in regenerative medicine, disease modeling, drug screening, toxicology testing, and proof-of-concept studies in drug development. Therefore, the number of publications concerning induced pluripotent stem cells has recently been increasing rapidly, demonstrating that this technology has begun to infiltrate many aspects of stem cell biology and medical applications. In this review, we discuss the perspectives of induced pluripotent stem cell technology for modeling human diseases. In particular, we focus on the cloning event occurring through the reprogramming process and its ability to let us analyze the development of complex disease-harboring somatic mosaicism.

Active tactile exploration using a brain-machine-brain interface.

PubMed

O'Doherty, Joseph E; Lebedev, Mikhail A; Ifft, Peter J; Zhuang, Katie Z; Shokur, Solaiman; Bleuler, Hannes; Nicolelis, Miguel A L

2011-10-05

Brain-machine interfaces use neuronal activity recorded from the brain to establish direct communication with external actuators, such as prosthetic arms. It is hoped that brain-machine interfaces can be used to restore the normal sensorimotor functions of the limbs, but so far they have lacked tactile sensation. Here we report the operation of a brain-machine-brain interface (BMBI) that both controls the exploratory reaching movements of an actuator and allows signalling of artificial tactile feedback through intracortical microstimulation (ICMS) of the primary somatosensory cortex. Monkeys performed an active exploration task in which an actuator (a computer cursor or a virtual-reality arm) was moved using a BMBI that derived motor commands from neuronal ensemble activity recorded in the primary motor cortex. ICMS feedback occurred whenever the actuator touched virtual objects. Temporal patterns of ICMS encoded the artificial tactile properties of each object. Neuronal recordings and ICMS epochs were temporally multiplexed to avoid interference. Two monkeys operated this BMBI to search for and distinguish one of three visually identical objects, using the virtual-reality arm to identify the unique artificial texture associated with each. These results suggest that clinical motor neuroprostheses might benefit from the addition of ICMS feedback to generate artificial somatic perceptions associated with mechanical, robotic or even virtual prostheses.

The protein DIIIC-2, aggregated with a specific oligodeoxynucleotide and adjuvanted in alum, protects mice and monkeys against DENV-2.

PubMed

Gil, Lázaro; Marcos, Ernesto; Izquierdo, Alienys; Lazo, Laura; Valdés, Iris; Ambala, Peris; Ochola, Lucy; Hitler, Rikoi; Suzarte, Edith; Álvarez, Mayling; Kimiti, Prisilla; Ndung'u, James; Kariuki, Thomas; Guzmán, María Guadalupe; Guillén, Gerardo; Hermida, Lisset

2015-01-01

Previously, we reported the ability of the chimeric protein DIIIC-2 (domain III of the dengue envelope protein fused to the capsid protein of dengue-2 virus), to induce immunity and protection in mice, when it is highly aggregated with a non-defined oligodeoxynucleotide (ODN) and adjuvanted in alum. In this work, three different defined ODNs were studied as aggregating agents. Our results suggest that the nature of the ODN influences the capacity of protein DIIIC-2 to activate cell-mediated immunity in mice. Consequently, the ODN 39M was selected to perform further experiments in mice and nonhuman primates. Mice receiving the preparation 39M-DIIIC-2 were solidly protected against dengue virus (DENV) challenge. Moreover, monkeys immunized with the same preparation developed neutralizing antibodies, as measured by four different neutralization tests varying the virus strains and the cell lines used. Two of the immunized monkeys were completely protected against challenge, whereas the third animal had a single day of low-titer viremia. This is the first work describing the induction of short-term protection in monkeys by a formulation that is suitable for human use combining a recombinant protein from DENV with alum.

Cloning endangered gray wolves (Canis lupus) from somatic cells collected postmortem.

PubMed

Oh, H J; Kim, M K; Jang, G; Kim, H J; Hong, S G; Park, J E; Park, K; Park, C; Sohn, S H; Kim, D Y; Shin, N S; Lee, B C

2008-09-01

The objective of the present study was to investigate whether nuclear transfer of postmortem wolf somatic cells into enucleated dog oocytes, is a feasible method to produce a cloned wolf. In vivo-matured oocytes (from domestic dogs) were enucleated and fused with somatic cells derived from culture of tissue obtained from a male gray wolf 6h after death. The reconstructed embryos were activated and transferred into the oviducts of naturally synchronous domestic bitches. Overall, 372 reconstructed embryos were transferred to 17 recipient dogs; four recipients (23.5%) were confirmed pregnant (ultrasonographically) 23-25 d after embryo transfer. One recipient spontaneously delivered two dead pups and three recipients delivered, by cesarean section, four cloned wolf pups, weighing 450, 190, 300, and 490g, respectively. The pup that weighed 190g died within 12h after birth. The six cloned wolf pups were genetically identical to the donor wolf, and their mitochondrial DNA originated from the oocyte donors. The three live wolf pups had a normal wolf karyotype (78, XY), and the amount of telomeric DNA, assessed by quantitative fluorescence in situ hybridization, was similar to, or lower than, that of the nuclear donor. In conclusion, the present study demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells, isolated postmortem from a wolf, and transferred into enucleated dog oocytes. Therefore, somatic cell nuclear transfer has potential for preservation of canine species in extreme situations, including sudden death.

A novel method for detecting IgA endomysial antibodies by using human umbilical vein endothelial cells.

PubMed

Castellino, F; Scaglione, N; Grosso, S B; Sategna-Guidetti, C

2000-01-01

Although tissue transglutaminase was recently identified as the main autoantigen recognized by endomysial antibodies in coeliac patients, anti-endomysium antibody detection still persists as the gold standard for coeliac disease screening and diagnosis. (1) To evaluate human umbilical vein cells (HUVEC) as an alternative source of endomysial antigen and to assess their suitability in the diagnosis of coeliac disease. (2) To verify whether tissue transglutaminase is one target antigen eliciting the endomysial antibody fraction of coeliac serum IgA. University teaching hospital. Sera from 123 untreated adults with biopsy-proven coeliac disease and 84 controls (40 healthy and 44 diseased) were assessed by indirect immunofluorescence, using HUVEC on glass slides prepared by cytocentrifugation and permeabilized by using Triton X (0.5%). Indirect immunofluorescence was performed: (1) using coeliac disease serum samples on HUVEC with or without prior incubation with tissue transglutaminase; and (2) incubating both HUVEC and monkey oesophagus with goat anti-guinea pig tissue transglutaminase antibody. All the coeliac patients, who were also positive on monkey oesophagus, showed the typical fluorescent homogeneous cytoplasmic stain on HUVEC. All control sera were negative both on HUVEC and on monkey oesophagus. IgA antibodies did not react with non-permeabilized cells, with intact membrane. Preincubation of coeliac sera with tissue transglutaminase abolished the typical fluorescent pattern. The incubation of anti-tissue transglutaminase antibody with monkey oesophagus and HUVEC resulted in an immunofluorescence staining pattern identical to that obtained with positive coeliac sera. (1) As a substrate for anti-endomysial antibody, HUVEC may provide the same diagnostic accuracy as monkey oesophagus, thus bypassing economical and ethical problems. The HUVEC antigen reacting with IgA from coeliac disease sera is an intracellular rather than a cell-surface antigen, as IgA antibodies reacted only with permeabilized cells. (2) Pretreatment of untreated coeliac sera with tissue transglutaminase abolished almost completely the specific staining; incubation with anti-tissue transglutaminase antibody elicited the characteristic fluorescent pattern, thus confirming that tissue transglutaminase represents the prominent autoantigen in coeliac disease.

Effect of chronic antipsychotic exposure on astrocyte and oligodendrocyte numbers in macaque monkeys

PubMed Central

Konopaske, Glenn T.; Dorph-Petersen, Karl-Anton; Sweet, Robert A.; Pierri, Joseph N.; Zhang, Wei; Sampson, Allan R.; Lewis, David A.

2008-01-01

Background Both in vivo and post-mortem studies suggest that oligodendrocyte and myelination alterations are present in individuals with schizophrenia. However, it is unclear whether prolonged treatment with antipsychotic medications contributes to these disturbances. We recently reported that chronic exposure of macaque monkeys to haloperidol or olanzapine was associated with a 10−18% lower glial cell number in the parietal grey matter. Consequently, in this study we sought to determine whether the lower glial cell number was due to fewer oligodendrocytes as opposed to lower numbers of astrocytes. Methods Using fluorescent immunocytochemical techniques, we optimized the visualization of each cell type throughout the entire thickness of tissue sections, while minimizing final tissue shrinkage. As a result, we were able to obtain robust stereological estimates of total oligodendrocyte and astrocyte numbers in the parietal grey matter using the optical fractionator method. Results We found a significant 20.5% lower astrocyte number with a non-significant 12.9% lower oligodendrocyte number in the antipsychotic-exposed monkeys. Similar effects were seen in both the haloperidol and olanzapine groups. Conclusion These findings suggest that studies investigating glial cell alterations in schizophrenia must take into account the effect of antipsychotic treatment. PMID:17945195

Induction, management, and complications of streptozotocin-induced diabetes mellitus in rhesus monkeys.

PubMed

Kim, Jong-Min; Shin, Jun-Seop; Min, Byoung-Hoon; Kim, Hyun-Je; Kim, Jung-Sik; Yoon, Il-Hee; Jeong, Won-Young; Lee, Ga-Eul; Kim, Min-Sun; Kim, Ju-Eun; Jin, Sang-Man; Park, Chung-Gyu

2016-11-01

Diabetes mellitus (DM) model using streptozotocin (STZ) which induces chemical ablation of β cell in the pancreas has been widely used for various research purposes in non-human primates. However, STZ has been known to have a variety of adverse effects such as nephrotoxicity, hepatotoxicity, and even mortality. The purpose of this study is to report DM induction by STZ, toxicity associated with STZ and procedure and complication of exogenous insulin treatment for DM management in rhesus monkeys (Macaca mulatta) that are expected to be transplanted with porcine islets within 2 months. Streptozotocin (immediately dissolved in normal saline, 110 mg/kg) was slowly infused via central catheter for 10 minutes in 22 rhesus monkeys. Clinical signs, complete blood count and blood chemistry were monitored to evaluate toxicity for 1 week after STZ injection. Monkey basal C-peptides were measured and intravenous glucose tolerance test was performed to confirm complete induction of DM. Exogenous insulin was subcutaneously injected to maintain blood glucose in diabetic rhesus monkeys and the complications were recorded while in insulin treatment. Severe salivation and vomiting were observed within 1 hour after STZ injection in 22 rhesus monkeys. One monkey died at 6 hours after STZ injection and the reason for the death was unknown. Pancreatitis was noticed in one monkey after STZ injection, but the monkey recovered after 5 days by medical treatment. Serum total protein and albumin decreased whereas the parameters for the liver function such as aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase significantly increased (P<.05) after STZ injection, but they were resolved within 1 week. Azotemia was not observed. Monkey fasting C-peptide levels after STZ injection were <0.1 ng/mL in 18 rhesus monkeys, but 0.34, 0.22, 0.16 ng/mL in three monkeys, respectively. The value of daily insulin requirement was 0.92±0.26IU/kg/d (range=0.45-1.29) in the monkeys. Diabetic ketoacidosis was observed in one rhesus monkeys, but the monkey recovered after 24 hours by fluid and insulin treatment. Streptozotocin was effective for inducing DM in rhesus monkeys, but various adverse effects such as pancreatitis, liver toxicity or death were observed. Therefore, careful and suitable medical managements should be implemented to eliminate the risks of mortality and severe adverse effects. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus).

PubMed

Du, Fuliang; Shen, Perng-Chih; Xu, Jie; Sung, Li-Ying; Jeong, B-Seon; Lucky Nedambale, Tshimangadzo; Riesen, John; Cindy Tian, X; Cheng, Winston T K; Lee, Shan-Nan; Yang, Xiangzhong

2006-02-01

One of the several factors that contribute to the low efficiency of mammalian somatic cloning is poor fusion between the small somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following somatic cell nuclear transfer (Experiment 3). Cloning with somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

Technical note: Selection of suitable reference genes for studying gene expression in milk somatic cell of yak (Bos grunniens) during the lactation cycle.

PubMed

Bai, W L; Yin, R H; Zhao, S J; Jiang, W Q; Yin, R L; Ma, Z J; Wang, Z Y; Zhu, Y B; Luo, G B; Yang, R J; Zhao, Z H

2014-02-01

Quantitative real-time PCR is the most sensitive technique for gene expression analysis. Data normalization is essential to correct for potential errors incurred in all steps from RNA isolation to PCR amplification. The commonly accepted approach for normalization is the use of reference gene. Until now, no suitable reference genes have been available for data normalization of gene expression in milk somatic cells of lactating yaks across lactation. In the present study, we evaluated the transcriptional stability of 10 candidate reference genes in milk somatic cells of lactating yak, including ACTB, B2M, GAPDH, GTP, MRPL39, PPP1R11, RPS9, RPS15, UXT, and RN18S1. Four genes, RPS9, PPP1R11, UXT, and MRPL39, were identified as being the most stable genes in milk somatic cells of lactating yak. Using the combination of RPS9, PPP1R11, UXT, and MRPL39 as reference genes, we further assessed the relative expression of 4 genes of interest in milk somatic cells of yak across lactation, including ELF5, ABCG2, SREBF2, and DGAT1. Compared with expression in colostrum, the overall transcription levels of ELF5, ABCG2, and SREBF2 in milk were found to be significantly upregulated in early, peak, and late lactation, and significantly downregulated thereafter, before the dry period. A similar pattern was observed in the relative expression of DGAT1, but no significant difference was revealed in its expression in milk from late lactation compared with colostrum. Based on these results, we suggest that the geometric mean of RPS9, PPP1R11, UXT, and MRPL39 can be used for normalization of real-time PCR data in milk somatic cells of lactating yak, if similar experiments are performed. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cells Comprising the Prostate Cancer Microenvironment Lack Recurrent Clonal Somatic Genomic Aberrations

PubMed Central

Bianchi-Frias, Daniella; Basom, Ryan; Delrow, Jeffrey J; Coleman, Ilsa M; Dakhova, Olga; Qu, Xiaoyu; Fang, Min; Franco, Omar E.; Ericson, Nolan G.; Bielas, Jason H.; Hayward, Simon W.; True, Lawrence; Morrissey, Colm; Brown, Lisha; Bhowmick, Neil A.; Rowley, David; Ittmann, Michael; Nelson, Peter S.

2017-01-01

Prostate cancer-associated stroma (CAS) plays an active role in malignant transformation, tumor progression, and metastasis. Molecular analyses of CAS have demonstrated significant changes in gene expression; however, conflicting evidence exists on whether genomic alterations in benign cells comprising the tumor microenvironment (TME) underlie gene expression changes and oncogenic phenotypes. This study evaluates the nuclear and mitochondrial DNA integrity of prostate carcinoma cells, CAS, matched benign epithelium and benign epithelium-associated stroma by whole genome copy number analyses, targeted sequencing of TP53, and fluorescence in situ hybridization. Comparative genomic hybridization (aCGH) of CAS revealed a copy-neutral diploid genome with only rare and small somatic copy number aberrations (SCNAs). In contrast, several expected recurrent SCNAs were evident in the adjacent prostate carcinoma cells, including gains at 3q, 7p, and 8q, and losses at 8p and 10q. No somatic TP53 mutations were observed in CAS. Mitochondrial DNA (mtDNA) extracted from carcinoma cells and stroma identified 23 somatic mtDNA mutations in neoplastic epithelial cells but only one mutation in stroma. Finally, genomic analyses identified no SCNAs, no loss of heterozygosity (LOH) or copy-neutral LOH in cultured cancer-associated fibroblasts (CAFs), which are known to promote prostate cancer progression in vivo. PMID:26753621

Mitochondrial DNA transmission and confounding mitochondrial influences in cloned cattle and pigs.

PubMed

Takeda, Kumiko

2013-04-01

Although somatic cell nuclear transfer (SCNT) is a powerful tool for production of cloned animals, SCNT embryos generally have low developmental competency and many abnormalities. The interaction between the donor nucleus and the enucleated ooplasm plays an important role in early embryonic development, but the underlying mechanisms that negatively impact developmental competency remain unclear. Mitochondria have a broad range of critical functions in cellular energy supply, cell signaling, and programmed cell death; thus, affect embryonic and fetal development. This review focuses on mitochondrial considerations influencing SCNT techniques in farm animals. Donor somatic cell mitochondrial DNA (mtDNA) can be transmitted through what has been considered a "bottleneck" in mitochondrial genetics via the SCNT maternal lineage. This indicates that donor somatic cell mitochondria have a role in the reconstructed cytoplasm. However, foreign somatic cell mitochondria may affect the early development of SCNT embryos. Nuclear-mitochondrial interactions in interspecies/intergeneric SCNT (iSCNT) result in severe problems. A major biological selective pressure exists against survival of exogenous mtDNA in iSCNT. Yet, mtDNA differences in SCNT animals did not reflect transfer of proteomic components following proteomic analysis. Further study of nuclear-cytoplasmic interactions is needed to illuminate key developmental characteristics of SCNT animals associated with mitochondrial biology.

Parthenogenesis and somatic cell nuclear transfer in sheep oocytes using Polscope.

PubMed

Nandedkar, Pandit; Chohan, Parul; Patwardhan, Archana; Gaikwad, Santosh; Bhartiya, Deepa

2009-07-01

Parthenogenesis and Somatic cell nuclear transfer (SCNT) techniques, offer a unique approach to manipulate the genetic composition of derived human embryonic stem cells - an essential step if the full opportunities for disease modeling, drug discovery or individualized stem cell therapy are to be realized. The present study describes the use of sheep oocytes to acquire expertise and establish methods to reconstruct embryos for obtaining blastocysts before venturing into human SCNT where the oocytes are a very precious starting material. Maturation of sheep eggs in vitro for 20-24 hr resulted in 65% metaphase II (MII) eggs which were either parthenogenetically activated using calcium ionomycin or ethanol or subjected to SCNT using cumulus cell as somatic cell. Sixteen blastocysts were produced by parthenogenetic activation of 350 eggs whereas reconstructed embryos, after SCNT carried out in 139 eggs, progressed only up to morula stage. The procedure of parthenogenesis and SCNT will be useful to generate autologous ES cells using human eggs.

Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

PubMed

Han, Chengquan; Deng, Ruizhi; Mao, Tingchao; Luo, Yan; Wei, Biao; Meng, Peng; Zhao, Lu; Zhang, Qing; Quan, Fusheng; Liu, Jun; Zhang, Yong

2018-05-23

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency. © 2018 Federation of European Biochemical Societies.

Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

PubMed

Song, Jie; Hu, Yajie; Hu, Yunguang; Wang, Jingjing; Zhang, Xiaolong; Wang, Lichun; Guo, Lei; Wang, Yancui; Ning, Ruotong; Liao, Yun; Zhang, Ying; Zheng, Huiwen; Shi, Haijing; He, Zhanlong; Li, Qihan; Liu, Longding

2016-03-02

Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus. Copyright © 2016 Elsevier B.V. All rights reserved.

STUDIES ON THE PROPAGATION IN VITRO OF POLIOMYELITIS VIRUSES

PubMed Central

Scherer, William F.; Syverton, Jerome T.

1952-01-01

The growth of poliomyelitis virus, Type 2, Yale-SK strain, in cultures of monkey testicular tissue was observed to occur in discrete cycles. Growth curves showed that each cycle was composed of (a) an initial lag phase when little or no virus was released from the cells, (6) a phase of viral production, and (c) a plateau which represented a decrement in the rate of viral production. This pattern of viral multiplication occurred in monkey testicular tissue cultures which have as the liquid phase either ox serum ultrafiltrate or monkey serum-chicken embryonic extract medium. The presence of a solid medium composed of chicken plasma, clotted either with chicken embryonic extract or bovine thrombin, did not alter the pattern of viral multiplication. The shape of the growth curve as established by any of four different techniques for tissue cultivation, was shown to be independent of the cultural technique employed. For cultures of monkey testicular tissue, the amount of virus in the tissue was as much as tenfold greater than that in the liquid of the same cultures. Moreover, viral production was evident earlier and was detectable for a longer period of time in the tissue than in the liquid phase. The rapidly incremental phase of the growth cycle, when large quantities of virus were released into the liquid phase, coincided in time with the destruction of the spindle-shaped cells, which extended from the explants. Although destruction of outgrowth cells was marked, there remained cells within the explants capable of supporting the growth of poliomyelitis virus. PMID:12981221

EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation

PubMed Central

Béguelin, Wendy; Popovic, Relja; Teater, Matt; Jiang, Yanwen; Bunting, Karen L.; Rosen, Monica; Shen, Hao; Yang, Shao Ning; Wang, Ling; Ezponda, Teresa; Martinez-Garcia, Eva; Zhang, Haikuo; Zhang, Yupeng; Verma, Sharad K.; McCabe, Michael T.; Ott, Heidi M.; Van Aller, Glenn S.; Kruger, Ryan G.; Liu, Yan; McHugh, Charles F.; Scott, David W.; Chung, Young Rock; Kelleher, Neil; Shaknovich, Rita; Creasy, Caretha L.; Gascoyne, Randy D.; Wong, Kwok-Kin; Cerchietti, Leandro C.; Levine, Ross L.; Abdel-Wahab, Omar; Licht, Jonathan D.; Elemento, Olivier; Melnick, Ari M.

2013-01-01

The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B-cells and targeted by somatic mutations in B-cell lymphomas. Here we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions in mice. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B-cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets in B-cells, and in human B-cell lymphomas. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GCB-type DLBCLs are mostly addicted to EZH2, regardless of mutation status, but not the more differentiated ABC-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting. PMID:23680150

Genotype x environment interactions in milk yield and quality in Angus, Brahman, and reciprocal-cross cows on different forage systems.

PubMed

Brown, M A; Brown, A H; Jackson, W G; Miesner, J R

2001-07-01

Milk yield and quality were observed on 93 Angus, Brahman, and reciprocal-cross cows over 3 yr to evaluate the interactions of direct and maternal breed effects and heterosis with forage environment. Forage environments were common bermudagrass (BG), endophyte-infected tall fescue (E+), and a rotational system (ROT) of both forages, in which each forage (BG or E+) was grazed during its appropriate season, usually June through October for BG and November through May for E+. Milk yield was estimated each of 6 mo (April through September) via milking machine and converted to a 24-h basis. Milk fat, milk protein, and somatic cell count were analyzed by a commercial laboratory. Heterosis for milk yield was similar among forages, averaging 2.4 kg (P < 0.01). Expressed as percentages of purebred means, heterosis for milk yield was largest on E+ (52.8%), intermediate on ROT (39.3%), and smallest on BG (23.7%). Direct breed effects for milk yield favored Brahman, and they were similar among forages but tended to be larger for E+ (2.5 kg) and ROT (2.8 kg) than for BG (1.3 kg). Direct breed effects for milk fat favored Brahman and were similar among forages but tended to be larger for E+ (1.0%) and ROT (1.0%) than for BG (0.6%). Purebred cows exceeded crossbreds in milk protein by 0.1% on ROT (P < 0.10). Crossbred cows had lower somatic cell counts than purebreds on BG (P < 0.05), E+ (P < 0.01), or ROT (P > 0.30). Heterosis for somatic cell counts as percentages of purebred means was similar for BG (-68.3%) and E+ (-68.9%) and less favorable for ROT (-31.6%). Maternal breed effects for somatic cell count favored Angus on ROT (P < 0.10) with a similar nonsignificant trend on BG and E+. Direct breed effects for somatic cell count favored Brahman on ROT (P < 0.10) with similar nonsignificant trends on BG and E+. These results suggested that a rotation of cows from E+ to BG in the summer can partially alleviate negative effects of E+ on milk yield. Conclusions also indicated an advantage to crossbred cows in somatic cell count and provided evidence of both direct and maternal breed effects for this trait. The results also suggested that direct breed effects for milk yield, milk fat, and somatic cell count and heterosis for milk yield and somatic cell count (as percentages of purebred means) tended to vary with forage environment, indicating a potential for genotype x environment interaction for these traits.

“Subpial Fan Cell” — A Class of Calretinin Neuron in Layer 1 of Adult Monkey Prefrontal Cortex

PubMed Central

Gabbott, Paul L. A.

2016-01-01

Layer 1 of the cortex contains populations of neurochemically distinct neurons and afferent fibers which markedly affect neural activity in the apical dendritic tufts of pyramidal cells. Understanding the causal mechanisms requires knowledge of the cellular architecture and synaptic organization of layer 1. This study has identified eight morphological classes of calretinin immunopositive (CRet+) neurons (including Cajal-Retzius cells) in layer 1 of the prefrontal cortex (PFC) in adult monkey (Macaca fasicularis), with a distinct class — termed “subpial fan (SPF) cell” — described in detail. SPF cells were rare horizontal unipolar CRet+ cells located directly beneath the pia with a single thick primary dendrite that branched into a characteristic fan-like dendritic tree tangential to the pial surface. Dendrites had spines, filamentous processes and thorny branchlets. SPF cells lay millimeters apart with intralaminar axons that ramified widely in upper layer 1. Such cells were GABA immunonegative (-) and occurred in areas beyond PFC. Interspersed amidst SPF cells displaying normal structural integrity were degenerating CRet+ neurons (including SPF cells) and clumps of lipofuscin-rich cellular debris. The number of degenerating SPF cells increased during adulthood. Ultrastructural analyses indicated SPF cell somata received asymmetric (A — presumed excitatory) and symmetric (S — presumed inhibitory) synaptic contacts. Proximal dendritic shafts received mainly S-type and distal shafts mostly A-type input. All dendritic thorns and most dendritic spines received both synapse types. The tangential areal density of SPF cell axonal varicosities varied radially from parent somata — with dense clusters in more distal zones. All boutons formed A-type contacts with CRet- structures. The main post-synaptic targets were dendritic shafts (67%; mostly spine-bearing) and dendritic spines (24%). SPF-SPF cell innervation was not observed. Morphometry of SPF cells indicated a unique class of CRet+/GABA- neuron in adult monkey PFC — possibly a subtype of persisting Cajal-Retzius cell. The distribution and connectivity of SPF cells suggest they act as integrative hubs in upper layer 1 during postnatal maturation. The main synaptic output of SPF cells likely provides a transminicolumnar excitatory influence across swathes of apical dendritic tufts — thus affecting information processing in discrete patches of layer 1 in adult monkey PFC. PMID:27147978

THE EDINGER-WESTPHAL NUCLEUS: A HISTORICAL, STRUCTURAL AND FUNCTIONAL PERSPECTIVE ON A DICHOTOMOUS TERMINOLOGY

PubMed Central

Kozicz, Tamás; Bittencourt, Jackson C.; May, Paul J.; Reiner, Anton; Gamlin, Paul D. R.; Palkovits, Miklós; Horn, Anja K.E.; Toledo, Claudio A. B.; Ryabinin, Andrey E.

2013-01-01

The eponymous term nucleus of Edinger-Westphal (EW) has come to be used to describe two juxtaposed and somewhat intermingled cell groups of the midbrain that differ dramatically in their connectivity and neurochemistry. On one hand, the classically defined EW is the part of the oculomotor complex that is the source of the parasympathetic preganglionic motoneuron input to the ciliary ganglion (CG), through which it controls pupil constriction and lens accommodation. On the other hand, EW is applied to a population of centrally projecting neurons involved in sympathetic, consumptive and stress-related functions. This terminology problem arose because the name EW has historically been applied to the most prominent cell collection above or between the somatic oculomotor nuclei (III), an assumption based on the known location of the preganglionic motoneurons in monkeys. However, in many mammals, the nucleus designated as EW is not made up of cholinergic, preganglionic motoneurons supplying the CG, and instead contains neurons using peptides, such as urocortin 1, with diverse central projections. As a result, the literature has become increasingly confusing. To resolve this problem, we suggest that the term EW be supplemented with terminology based on connectivity. Specifically, we recommend that: 1. The cholinergic, preganglionic neurons supplying the CG be termed the Edinger-Westphal preganglionic (EWpg) population, and 2. The centrally projecting, peptidergic neurons be termed the Edinger-Westphal centrally projecting (EWcp) population. The history of this nomenclature problem and the rationale for our solutions are discussed in this review. PMID:21452224

Administration of high doses of copper to capuchin monkeys does not cause liver damage but induces transcriptional activation of hepatic proliferative responses.

PubMed

Araya, Magdalena; Núñez, Héctor; Pavez, Leonardo; Arredondo, Miguel; Méndez, Marco; Cisternas, Felipe; Pizarro, Fernando; Sierralta, Walter; Uauy, Ricardo; González, Mauricio

2012-02-01

Liver cells respond to copper loading upregulating protective mechanisms. However, to date, except for liver content, there are no good indicators that identify individuals with excess liver copper. We hypothesized that administering high doses of copper to young (5.5 mg Cu · kg⁻¹ . d⁻¹) and adult (7.5 mg Cu · kg⁻¹ . d⁻¹) capuchin monkeys would induce detectable liver damage. Study groups included adult monkeys (2 females, 2 males) 3-3.5 y old at enrollment treated with copper for 36 mo (ACu); age-matched controls (1 female, 3 males) that did not receive additional copper (AC); young monkeys (2 female, 2 males) treated from birth with copper for 36 mo (YCu); and young age-matched controls (2 female, 2 males) that did not receive additional copper (YC). We periodically assessed clinical, blood biochemical, and liver histological indicators and at 36 mo the hepatic mRNA abundance of MT2a, APP, DMT1, CTR1, HGF, TGFβ, and NFκΒ only in adult monkeys. After 36 mo, the liver copper concentration was 4-5 times greater in treated monkeys relative to controls. All monkeys remained healthy with normal routine serum biochemical indices and there was no evidence of liver tissue damage. Relative mRNA abundance of HGF, TGFβ and NFκB was significantly greater in ACu than in AC monkeys. In conclusion, capuchin monkeys exposed to copper at doses up to 50 times the current upper level enhanced expression of genes related to inflammation and injury without clinical, blood biochemical, or histological evidence of liver damage.

[New possibilities will open up in human gene therapy].

PubMed

Portin, Petter

2016-01-01

Gene therapy is divided into somatic and germ line therapy. The latter involves reproductive cells or their stem cells, and its results are heritable. The effects of somatic gene therapy are generally restricted to a single tissue of the patient in question. Until now, all gene therapies in the world have belonged to the regime of somatic therapy, germ line therapy having been a theoretical possibility only. Very recently, however, a method has been developed which is applicable to germ line therapy as well. In addition to technical challenges, severe ethical problems are associated with germ line therapy, demanding opinion statement.

Sodium Orthovanadate Effect on Outflow Facility and Intraocular Pressure in Live Monkeys

PubMed Central

Tan, James C.H.; Kiland, Julie A.; Gonzalez, Jose M.; Gabelt, B’Ann T.; Peters, Donna M.; Kaufman, Paul L.

2010-01-01

Sodium orthovanadate (Na3VO4) is reported to reduce IOP by affecting aqueous formation, but whether it also affects outflow facility (OF) is unclear. We tested the effect of Na3VO4 on OF and intraocular pressure (IOP) in live cynomolgus monkeys, and on actin and cell adhesion organization in cultured human trabecular meshwork (HTM) cells. Total OF (n = 12) was measured by 2-level constant pressure perfusion of the monkey anterior chamber (AC) before and after exchange with 1 mM Na3VO4 or vehicle in opposite eyes. Topical 1% Na3VO4 or vehicle only was given twice daily (each 2×20 μL drops) for 4 days to opposite eyes (n = 8), and Goldmann IOP was measured before and hourly after treatment for 6 hours on Days 1 and 4. Filamentous actin and vinculin-containing cell adhesions were examined by epifluorescence microscopy after the cells had been incubated with 1 mM Na3VO4 for 24 hours. A) In monkeys, Na3VO4 increased OF by 29.3 ± 8.8% (mean ± s.e.m.) over the perfusion interval when adjusted for baseline and contralateral eye washout (p = 0.01; n = 12). B) Day 1 baseline IOP was 16.2 ± 1.5 mmHg in treated eyes and 15.9 ± 1.3 mmHg in the contralateral control eyes. Following treatment on Day 1, IOP was no different (p>0.05) between treated eyes and control eyes at any time-point or compared to baseline. Day 4 mean IOP averaged over hours 2–6 was 13.5 ± 0.8 mmHg in treated eyes and 16.1 ± 0.2 mmHg in control eyes. Treated eye IOP was lower than its Day 4 baseline (p<0.005), lower than control eyes for the same Day 4 interval (p = 0.009), and lower than the Day 1 baseline (p = 0.0000). Control eye IOP on Day 4 was not significantly different from baseline on Day 1. C) Incubation of HTM cells with 1 mM Na3VO4 for 24 hours caused a loss of actin stress fibers and vinculin-containing adhesions. Cell retraction and separation was also observed in vanadate-treated cultures. Reformation of actin stress fibers, vinculin-containing adhesions and confluent monolayers occurred within 24 hours after Na3VO4-containing culture medium was replaced with Na3VO4-free medium. Ocular administration of Na3VO4 to live monkeys significantly increases OF and reduces IOP. Na3VO4 reversibly disrupts actin and cell adhesion organization and causes retraction and separation of cultured HTM cells. Na3VO4 increases pressure-dependent outflow in live monkeys. Altered actin architecture in the TM may play a part in this increased OF. PMID:20620138

Fungal Infection Increases the Rate of Somatic Mutation in Scots Pine (Pinus sylvestris L.).

PubMed

Ranade, Sonali Sachin; Ganea, Laura-Stefana; Razzak, Abdur M; García Gil, M R

2015-01-01

Somatic mutations are transmitted during mitosis in developing somatic tissue. Somatic cells bearing the mutations can develop into reproductive (germ) cells and the somatic mutations are then passed on to the next generation of plants. Somatic mutations are a source of variation essential to evolve new defense strategies and adapt to the environment. Stem rust disease in Scots pine has a negative effect on wood quality, and thus adversely affects the economy. It is caused by the 2 most destructive fungal species in Scandinavia: Peridermium pini and Cronartium flaccidum. We studied nuclear genome stability in Scots pine under biotic stress (fungus-infected, 22 trees) compared to a control population (plantation, 20 trees). Stability was assessed as accumulation of new somatic mutations in 10 microsatellite loci selected for genotyping. Microsatellites are widely used as molecular markers in population genetics studies of plants, and are particularly used for detection of somatic mutations as their rate of mutation is of a much higher magnitude when compared with other DNA markers. We report double the rate of somatic mutation per locus in the fungus-infected trees (4.8×10(-3) mutations per locus), as compared to the controls (2.0×10(-3) mutations per locus) when individual samples were analyzed at 10 different microsatellite markers. Pearson's chi-squared test indicated a significant effect of the fungal infection which increased the number of mutations in the fungus-infected trees (χ(2) = 12.9883, df = 1, P = 0.0003134). © The American Genetic Association 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

UV-induced somatic mutations elicit a functional T cell response in the YUMMER1.7 mouse melanoma model.

PubMed

Wang, Jake; Perry, Curtis J; Meeth, Katrina; Thakral, Durga; Damsky, William; Micevic, Goran; Kaech, Susan; Blenman, Kim; Bosenberg, Marcus

2017-07-01

Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single-cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1 -/- mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti-CTLA-4 and anti-PD-1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hair-cell counts and afferent innervation patterns in the cristae ampullares of the squirrel monkey with a comparison to the chinchilla

NASA Technical Reports Server (NTRS)

Fernandez, C.; Lysakowski, A.; Goldberg, J. M.

1995-01-01

1. The numbers of type I and type II hair cells were estimated by dissector techniques applied to semithin, stained sections of the horizontal, superior, and posterior cristae in the squirrel monkey and the chinchilla. 2. The crista in each species was divided into concentrically arranged central, intermediate, and peripheral zones of equal areas. The three zones can be distinguished by the sizes of individual hair cells and calyx endings, by the density of hair cells, and by the relative frequency of calyx endings innervating single or multiple type I hair cells. 3. In the monkey crista, type I hair cells outnumber type II hair cells by a ratio of almost 3:1. The ratio decreases from 4-5:1 in the central and intermediate zones to under 2:1 in the peripheral zone. For the chinchilla, the ratio is near 1:1 for the entire crista and decreases only slightly between the central and peripheral zones. 4. Nerve fibers supplying the cristae in the squirrel monkey were labeled by extracellular injections of horseradish peroxidase (HRP) into the vestibular nerve. Peripheral terminations of individual fibers were reconstructed and related to the zones of the cristae they innervated and to the sizes of their parent axons. Results were similar for the horizontal, superior, and posterior cristae. 5. Axons seldom bifurcate below the neuroepithelium. Most fibers begin branching shortly after crossing the basement membrane. Their terminal arbors are compact, usually extending no more than 50-100 microns from the parent exon. A small number of long intraepithelial fibers enter the intermediate and peripheral zones of the cristae near its base, then run unbranched for long distances through the neuroepithelium to reach the central zone. 6. There are three classes of afferent fibers innervating the monkey crista. Calyx fibers terminate exclusively on type I hair cells, and bouton fibers end only on type II hair cells. Dimorphic fibers provide a mixed innervation, including calyx endings to type I hair cells and bouton endings to type II hair cells. Long intraepithelial fibers are calyx and dimorphic units, whose terminal fields are similar to those of other fibers. The central zone is innervated by calyx and dimorphic fibers; the peripheral zone, by bouton and dimorphic fibers; and the intermediate zone, by all three kinds of fibers. Internal (axon) diameters are largest for calyx fibers and smallest for bouton fibers. Of the entire sample of 286 labeled fibers, 52% were dimorphic units, 40% were calyx units, and 8% were bouton units.(ABSTRACT TRUNCATED AT 400 WORDS).

Control of female gamete formation by a small RNA pathway in Arabidopsis.

PubMed

Olmedo-Monfil, Vianey; Durán-Figueroa, Noé; Arteaga-Vázquez, Mario; Demesa-Arévalo, Edgar; Autran, Daphné; Grimanelli, Daniel; Slotkin, R Keith; Martienssen, Robert A; Vielle-Calzada, Jean-Philippe

2010-03-25

In the ovules of most sexual flowering plants female gametogenesis is initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore mother cell (MMC). Because some mutants and certain sexual species exhibit more than one MMC, and many others are able to form gametes without meiosis (by apomixis), it has been suggested that somatic cells in the ovule are competent to respond to a local signal likely to have an important function in determination. Here we show that the Arabidopsis protein ARGONAUTE 9 (AGO9) controls female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in cytoplasmic foci of somatic companion cells. Mutations in SUPPRESSOR OF GENE SILENCING 3 and RNA-DEPENDENT RNA POLYMERASE 6 exhibit an identical defect to ago9 mutants, indicating that the movement of small RNA (sRNAs) silencing out of somatic companion cells is necessary for controlling the specification of gametic cells. AGO9 preferentially interacts with 24-nucleotide sRNAs derived from transposable elements (TEs), and its activity is necessary to silence TEs in female gametes and their accessory cells. Our results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA-dependent silencing in plant gametes.

Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

PubMed

Wakayama, Teruhiko

2007-02-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

Intrinsic and extrinsic molecular determinants or modulators for epigenetic remodeling and reprogramming of somatic cell-derived genome in mammalian nuclear-transferred oocytes and resultant embryos.

PubMed

Samiec, M; Skrzyszowska, M

2018-03-01

The efficiency of somatic cell cloning in mammals remains disappointingly low. Incomplete and aberrant reprogramming of epigenetic memory of somatic cell nuclei in preimplantation nuclear- transferred (NT) embryos is one of the most important factors that limit the cloning effectiveness. The extent of epigenetic genome-wide alterations, involving histone or DNA methylation and histone deacetylation, that are mediated by histone-lysine methyltransferases (HMTs) or DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) can be modulated/reversed via exogenous inhibitors of these enzymes throughout in vitro culture of nuclear donor cells, nuclear recipient oocytes and/or cloned embryos. The use of the artificial modifiers of epigenomically-conditioned gene expression leads to inhibition of both chromatin condensation and transcriptional silencing the genomic DNA of somatic cells that provide a source of nuclear donors for reconstruction of enucleated oocytes and generation of cloned embryos. The onset of chromatin decondensation and gene transcriptional activity is evoked both through specific/selective inactivating HMTs by BIX-01294 and through non-specific/non-selective blocking the activity of either DNMTs by 5-aza-2'-deoxycytidine, zebularine, S-adenosylhomocysteine or HDACs by trichostatin A, valproic acid, scriptaid, oxamflatin, sodium butyrate, m-carboxycinnamic acid bishydroxamide, panobinostat, abexinostat, quisinostat, dacinostat, belinostat and psammaplin A. Epigenomic modulation of nuclear donor cells, nuclear recipient cells and/or cloned embryos may facilitate and accelerate the reprogrammability for gene expression of donor cell nuclei that have been transplanted into a host ooplasm and subsequently underwent dedifferentiating and re-establishing the epigenetically dependent status of their transcriptional activity during pre- and postimplantation development of NT embryos. Nevertheless, a comprehensive additional work is necessary to determine whether failures in the early-stage reprogramming of somatic cell-inherited genome are magnified downstream in development of cloned conceptuses and neonates. Copyright© by the Polish Academy of Sciences.

CD4 T Cell Depletion Exacerbates Acute Mycobacterium tuberculosis While Reactivation of Latent Infection Is Dependent on Severity of Tissue Depletion in Cynomolgus Macaques

PubMed Central

Lin, Philana Ling; Rutledge, Tara; Green, Angela M.; Bigbee, Matthew; Fuhrman, Carl; Klein, Edwin

2012-01-01

Abstract CD4 T cells are believed to be important in protection against Mycobacterium tuberculosis, but the relative contribution to control of initial or latent infection is not known. Antibody-mediated depletion of CD4 T cells in M. tuberculosis-infected cynomolgus macaques was used to study the role of CD4 T cells during acute and latent infection. Anti-CD4 antibody severely reduced levels of CD4 T cells in blood, airways, and lymph nodes. Increased pathology and bacterial burden were observed in CD4-depleted monkeys during the first 8 weeks of infection compared to controls. CD4-depleted monkeys had greater interferon (IFN)-γ expression and altered expression of CD8 T cell activation markers. During latent infection, CD4 depletion resulted in clinical reactivation in only three of six monkeys. Reactivation was associated with lower CD4 T cells in the hilar lymph nodes. During both acute and latent infection, CD4 depletion was associated with reduced percentages of CXCR3+ expressing CD8 T cells, reported to be involved in T cell recruitment, regulatory function, and effector and memory T cell maturation. CXCR3+ CD8 T cells from hilar lymph nodes had more mycobacteria-specific cytokine expression and greater coexpression of multiple cytokines compared to CXCR3− CD8 T cells. CD4 T cells are required for protection against acute infection but reactivation from latent infection is dependent on the severity of depletion in the draining lymph nodes. CD4 depletion influences CD8 T cell function. This study has important implications for human HIV–M. tuberculosis coinfection. PMID:22480184

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis.

PubMed

Marum, Liliana; Loureiro, João; Rodriguez, Eleazar; Santos, Conceição; Oliveira, M Margarida; Miguel, Célia

2009-09-25

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

Somatic Host Cell Alterations in HPV Carcinogenesis

PubMed Central

Litwin, Tamara R.; Clarke, Megan A.; Dean, Michael; Wentzensen, Nicolas

2017-01-01

High-risk human papilloma virus (HPV) infections cause cancers in different organ sites, most commonly cervical and head and neck cancers. While carcinogenesis is initiated by two viral oncoproteins, E6 and E7, increasing evidence shows the importance of specific somatic events in host cells for malignant transformation. HPV-driven cancers share characteristic somatic changes, including apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-driven mutations and genomic instability leading to copy number variations and large chromosomal rearrangements. HPV-associated cancers have recurrent somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and phosphatase and tensin homolog (PTEN), human leukocyte antigen A and B (HLA-A and HLA-B)-A/B, and the transforming growth factor beta (TGFβ) pathway, and rarely have mutations in the tumor protein p53 (TP53) and RB transcriptional corepressor 1 (RB1) tumor suppressor genes. There are some variations by tumor site, such as NOTCH1 mutations which are primarily found in head and neck cancers. Understanding the somatic events following HPV infection and persistence can aid the development of early detection biomarkers, particularly when mutations in precancers are characterized. Somatic mutations may also influence prognosis and treatment decisions. PMID:28771191

Somatic Host Cell Alterations in HPV Carcinogenesis.

PubMed

Litwin, Tamara R; Clarke, Megan A; Dean, Michael; Wentzensen, Nicolas

2017-08-03

High-risk human papilloma virus (HPV) infections cause cancers in different organ sites, most commonly cervical and head and neck cancers. While carcinogenesis is initiated by two viral oncoproteins, E6 and E7, increasing evidence shows the importance of specific somatic events in host cells for malignant transformation. HPV-driven cancers share characteristic somatic changes, including apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC)-driven mutations and genomic instability leading to copy number variations and large chromosomal rearrangements. HPV-associated cancers have recurrent somatic mutations in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA ) and phosphatase and tensin homolog ( PTEN ), human leukocyte antigen A and B ( HLA-A and HLA-B ) -A/B , and the transforming growth factor beta (TGFβ) pathway, and rarely have mutations in the tumor protein p53 ( TP53 ) and RB transcriptional corepressor 1 ( RB1 ) tumor suppressor genes. There are some variations by tumor site, such as NOTCH1 mutations which are primarily found in head and neck cancers. Understanding the somatic events following HPV infection and persistence can aid the development of early detection biomarkers, particularly when mutations in precancers are characterized. Somatic mutations may also influence prognosis and treatment decisions.

Milk somatic cells, factors influencing their release, future prospects, and practical utility in dairy animals: An overview

PubMed Central

Alhussien, Mohanned Naif; Dang, Ajay Kumar

2018-01-01

Milk somatic cells (SCs) are a mixture of milk-producing cells and immune cells. These cells are secreted in milk during the normal course of milking and are used as an index for estimating mammary health and milk quality of dairy animals worldwide. Milk SC is influenced by cow productivity, health, parity, lactation stage, and breed of an animal. Any change in environmental conditions, poor management practices, and also stressful conditions significantly increases the amount of SC coming in milk. Better hygiene and proper nutrition help in reducing milk SC. Milk with low SC means better milk products with a longer shelf life. The present review describes the role of SCs (both secretory and immune) in milk, their role in maintaining the integrity of the mammary gland, and factors affecting their release in milk. This information may help to reduce milk somatic cell counts (SCCs) and to establish differential SCC standards. PMID:29915493

Somatic cell nuclear transfer in Oregon: expanding the pluripotent space and informing research ethics.

PubMed

Lomax, Geoffrey P; DeWitt, Natalie D

2013-12-01

In May, Oregon Health and Science University (OHSU) announced the successful derivation, by the Mitalipov laboratory, of embryonic stem cells by somatic cell nuclear transfer. This experiment was recognized as a "formidable technical feat" and potentially a key step toward developing cell-based therapies. The OHSU report is also an example of how a scientific breakthrough can inform research ethics. This article suggests ways that nuclear transfer embryonic stem cell lines may contribute to research ethics by adding rigor to studies addressing pressing research questions important to the development of cell-based therapies.

Somatic cell reprogramming informed by the oocyte.

PubMed

Gonzalez-Munoz, Elena; Cibelli, Jose B

2018-05-08

The successful production of animals and embryonic stem cells (ESCs) using somatic cell nuclear transfer (SCNT) has demonstrated the unmatched nuclear reprogramming capacity of the oocyte and helped prove the degree of plasticity of differentiated cells. The introduction of transcription factors to generate induced pluripotent stem cells (iPSCs) displaced SCNT and, due to its ease of implementation, became the method of choice for cell reprogramming. Nonetheless, iPSC derivation remains inefficient and stochastic. This review article focuses on using the oocyte as a source of reprogramming factors, comparing the SCNT and iPSC mechanisms for remodeling chromatin and acquiring pluripotency.

Stem Cells in Mammalian Gonads.

PubMed

Wu, Ji; Ding, Xinbao; Wang, Jian

Stem cells have great value in clinical application because of their ability to self-renew and their potential to differentiate into many different cell types. Mammalian gonads, including testes for males and ovaries for females, are composed of germline and somatic cells. In male mammals, spermatogonial stem cells maintain spermatogenesis which occurs continuously in adult testis. Likewise, a growing body of evidence demonstrated that female germline stem cells could be found in mammalian ovaries. Meanwhile, prior studies have shown that somatic stem cells exist in both testes and ovaries. In this chapter, we focus on mammalian gonad stem cells and discuss their characteristics as well as differentiation potentials.

CENH3-GFP: a visual marker for gametophytic and somatic ploidy determination in Arabidopsis thaliana.

PubMed

De Storme, Nico; Keçeli, Burcu Nur; Zamariola, Linda; Angenon, Geert; Geelen, Danny

2016-01-05

The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell's chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo. By modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events. This study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.

Pathology of experimental Ebola virus infection in African green monkeys. Involvement of fibroblastic reticular cells.

PubMed

Davis, K J; Anderson, A O; Geisbert, T W; Steele, K E; Geisbert, J B; Vogel, P; Connolly, B M; Huggins, J W; Jahrling, P B; Jaax, N K

1997-08-01

Ebola virus has been responsible for explosive lethal outbreaks of hemorrhagic fever in both humans and nonhuman primates. Previous studies showed a predilection of Ebola virus for cells of the mononuclear phagocyte system and endothelial cells. To examine the distribution of lesions and Ebola virus antigen in the tissues of six adult male African green monkeys (Cercopithecus aethiops) that died 6 to 7 days after intraperitoneal inoculation of Ebola-Zaire (Mayinga) virus. Tissues were examined histologically, immunohistochemically, and ultrastructurally. A major novel finding of this study was that fibroblastic reticular cells were immunohistochemically and ultrastructurally identified as targets of Ebola virus infection. The role of Ebola virus-infected fibroblastic reticular cells in the pathogenesis of Ebola hemorrhagic fever warrants further investigation. This is especially important because of recent observations indicating that fibroblastic reticular cells, along with the reticular fibers they produce, maximize the efficiency of the immune response.

Chinmo prevents transformer alternative splicing to maintain male sex identity.

PubMed

Grmai, Lydia; Hudry, Bruno; Miguel-Aliaga, Irene; Bach, Erika A

2018-02-01

Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. We demonstrate this feminization occurs by transcriptional and post-transcriptional regulation of traF. We find that chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, we show that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. Our work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility.

Chinmo prevents transformer alternative splicing to maintain male sex identity

PubMed Central

Hudry, Bruno; Miguel-Aliaga, Irene

2018-01-01

Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform traF. In turn, TraF alternatively splices dsx to the female isoform dsxF. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs) of the testis causes them to “feminize”, resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. We demonstrate this feminization occurs by transcriptional and post-transcriptional regulation of traF. We find that chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). traF splicing in chinmo-deficient CySCs leads to the production of DsxF at the expense of the male isoform DsxM, and both TraF and DsxF are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, we show that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. Our work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of traF production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility. PMID:29389999

A Novel Class of Somatic Small RNAs Similar to Germ Cell Pachytene PIWI-interacting Small RNAs*

PubMed Central

Ortogero, Nicole; Schuster, Andrew S.; Oliver, Daniel K.; Riordan, Connor R.; Hong, Annie S.; Hennig, Grant W.; Luong, Dickson; Bao, Jianqiang; Bhetwal, Bhupal P.; Ro, Seungil; McCarrey, John R.; Yan, Wei

2014-01-01

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3′-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs. PMID:25320077

Potential role of centrioles in determining the morphogenetic status of animal somatic cells.

PubMed

Tkemaladze, J; Chichinadze, K

2005-05-01

Irreversible differentiation (change of morphogenetic status) and programmed death (apoptosis) are observed only in somatic cells. Cell division is the only way by which the morphogenetic status of the offspring cells may be modified. It is known that there is a fixed limit to the number of possible cell divisions, the so-called 'Hayflick limit'. Existing links between cell division, differentiation and apoptosis make it possible to conclude that all these processes could be controlled by a single self-reproducing structure. Potential candidates for this replicable structure in a somatic cell are chromosomes, mitochondria (both contain DNA), and centrioles. Centrioles (diplosome) are the most likely unit that can fully regulate the processes of irreversible differentiation, determination and modification of the morphogenetic status. It may contain differently encoded RNA molecules stacked in a definite order. During mitosis, these RNA molecules are released one by one into the cytoplasm. In the presence of reverse transcriptase and endonuclease, RNA can be embedded in nuclear DNA. This process presumably changes the status of repressed and potentially active genes and, subsequently, the morphogenetic status of a cell.

Prestin-based outer hair cell electromotility in knockin mice does not appear to adjust the operating point of a cilia-based amplifier

PubMed Central

Gao, Jiangang; Wang, Xiang; Wu, Xudong; Aguinaga, Sal; Huynh, Kristin; Jia, Shuping; Matsuda, Keiji; Patel, Manish; Zheng, Jing; Cheatham, MaryAnn; He, David Z.; Dallos, Peter; Zuo, Jian

2007-01-01

The remarkable sensitivity and frequency selectivity of the mammalian cochlea is attributed to a unique amplification process that resides in outer hair cells (OHCs). Although the mammalian-specific somatic motility is considered a substrate of cochlear amplification, it has also been proposed that somatic motility in mammals simply acts as an operating-point adjustment for the ubiquitous stereocilia-based amplifier. To address this issue, we created a mouse model in which a mutation (C1) was introduced into the OHC motor protein prestin, based on previous results in transfected cells. In C1/C1 knockin mice, localization of C1-prestin, as well as the length and number of OHCs, were all normal. In OHCs isolated from C1/C1 mice, nonlinear capacitance and somatic motility were both shifted toward hyperpolarization, so that, compared with WT controls, the amplitude of cycle-by-cycle (alternating, or AC) somatic motility remained the same, but the unidirectional (DC) component reversed polarity near the OHC's presumed in vivo resting membrane potential. No physiological defects in cochlear sensitivity or frequency selectivity were detected in C1/C1 or C1/+ mice. Hence, our results do not support the idea that OHC somatic motility adjusts the operating point of a stereocilia-based amplifier. However, they are consistent with the notion that the AC component of OHC somatic motility plays a dominant role in mammalian cochlear amplification. PMID:17640919

Sequential monitoring and stability of ex vivo-expanded autologous and non-autologous regulatory T cells following infusion in non-human primates

PubMed Central

Zhang, H.; Guo, H.; Lu, L.; Zahorchak, A. F.; Wiseman, R. W.; Raimondi, G.; Cooper, D. K. C.; Ezzelarab, M. B.; Thomson, A. W.

2016-01-01

Ex vivo-expanded cynomolgus monkey CD4+CD25+CD127− regulatory T cells (Treg) maintained Foxp3 demethylation status at the Treg-Specific Demethylation Region (TSDR), and potently suppressed T cell proliferation through 3 rounds of expansion. When CFSE- or VPD450-labeled autologous (auto) and non-autologous (non-auto) expanded Treg were infused into monkeys, the number of labeled auto-Treg in peripheral blood declined rapidly during the first week, but persisted at low levels in both normal and anti-thymocyte globulin plus rapamycin-treated (immunosuppressed; IS) animals for at least 3 weeks. By contrast, MHC-mismatched non-auto-Treg could not be detected in normal monkey blood or in blood of two out of the three IS monkeys by day 6 post-infusion. They were also more difficult to detect than auto-Treg in peripheral lymphoid tissue. Both auto- and non-auto-Treg maintained Ki67 expression early after infusion. Sequential monitoring revealed that adoptively-transferred auto-Treg maintained similarly high levels of Foxp3 and CD25 and low CD127 compared with endogenous Treg, although Foxp3 staining diminished over time in these non-transplanted recipients. Thus, infused ex vivo-expanded auto-Treg persist longer than MHC-mismatched non-auto-Treg in blood of non-human primates and can be detected in secondary lymphoid tissue. Host lymphodepletion and rapamycin administration did not consistently prolong the persistence of non-auto-Treg in these sites. PMID:25783759

Stearidonic and γ-linolenic acids in echium oil improves glucose disposal in insulin resistant monkeys.

PubMed

Kavanagh, K; Flynn, D M; Jenkins, K A; Wilson, M D; Chilton, F H

2013-07-01

Echium oil (EO) contains stearidonic acid (18:4), a n-3 polyunsaturated fatty acids (PUFAs), and gamma-linolenic acids (18:3), a n-6 PUFA that can be converted to long chain (LC)-PUFAs. We aimed to compare a safflower oil (SO)-enriched diet to EO- and fish oil (FO)-enriched diets on circulating and tissue PUFAs levels and glycemic, inflammatory, and cardiovascular health biomarkers in insulin resistant African green monkeys. In a Latin-square cross-over study, eight monkeys consumed matched diets for 6 weeks with 3-week washout periods. Monkeys consuming FO had significantly higher levels of n-3 LC-PUFAs and EO supplementation resulted in higher levels of circulating n-3 LC-PUFAs and a significant increase in dihomo-gamma linolenic acid (DGLA) in red blood cells and muscle. Glucose disposal was improved after EO consumption. These data suggest that PUFAs in EO supplementation have the capacity to alter circulating, RBC and muscle LC-PUFA levels and improve glucose tolerance in insulin-resistant monkeys. Copyright © 2013 Elsevier Ltd. All rights reserved.

Angiotensin II receptors in testes

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

Millan, M.A.; Aguilera, G.

Receptors for angiotensin II (AII) were identified and characterized in testes of rats and several primate species. Autoradiographic analysis of the binding of 125I-labeled (Sar1,Ile8)AII to rat, rhesus monkey, cebus monkey, and human testicular slide-mounted frozen sections indicated specific binding to Leydig cells in the interstitium. In rat collagenase-dispersed interstitial cells fractionated by Percoll gradient, AII receptor content was parallel to that of hCG receptors, confirming that the AII receptors are in the Leydig cells. In rat dispersed Leydig cells, binding was specific for AII and its analogs and of high affinity (Kd, 4.8 nM), with a receptor concentration ofmore » 15 fmol/10(6) cells. Studies of AII receptors in rat testes during development reveals the presence of high receptor density in newborn rats which decreases toward the adult age (4934 +/- 309, 1460 +/- 228, 772 +/- 169, and 82 +/- 12 fmol/mg protein at 5, 15, 20, and 30 days of age, respectively) with no change in affinity. At all ages receptors were located in the interstitium, and the decrease in binding was parallel to the decrease in the interstitial to tubular ratio observed with age. AII receptor properties in membrane-rich fractions from prepuberal testes were similar in the rat and rhesus monkey. Binding was time and temperature dependent, reaching a plateau at 60 min at 37 C, and was increased by divalent cations, EGTA, and dithiothreitol up to 0.5 mM. In membranes from prepuberal monkey testes, AII receptors were specific for AII analogs and of high affinity (Kd, 4.2 nM) with a receptor concentration of 7599 +/- 1342 fmol/mg protein. The presence of AII receptors in Leydig cells in rat and primate testes in conjunction with reports of the presence of other components of the renin-angiotensin system in the testes suggests that the peptide has a physiological role in testicular function.« less

Climbing Mount Efficiency--small steps, not giant leaps towards higher cloning success in farm animals.

PubMed

Oback, Björn

2008-07-01

Despite more than a decade of research efforts, farm animal cloning by somatic cell nuclear transfer (SCNT) is still frustratingly inefficient. Inefficiency manifests itself at different levels, which are currently not well integrated. At the molecular level, it leads to widespread genetic, epigenetic and transcriptional aberrations in cloned embryos. At the organismal level, these genome-wide abnormalities compromise development of cloned foetuses and offspring. Specific molecular defects need to be causally linked to specific cloned phenotypes, in order to design specific treatments to correct them. Cloning efficiency depends on the ability of the nuclear donor cell to be fully reprogrammed into an embryonic state and the ability of the enucleated recipient cell to carry out the reprogramming reactions. It has been postulated that reprogrammability of the somatic donor cell epigenome is influenced by its differentiation status. However, direct comparisons between cells of divergent differentiation status within several somatic lineages have found no conclusive evidence for this. Choosing somatic stem cells as donors has not improved cloning efficiency, indicating that donor cell type may be less critical for cloning success. Different recipient cells, on the other hand, vary in their reprogramming ability. In bovine, using zygotes instead of oocytes has increased cloning success. Other improvements in livestock cloning efficiency include better coordinating donor cell type with cell cycle stage and aggregating cloned embryos. In the future, it will be important to demonstrate if these small increases at every step are cumulative, adding up to an integrated cloning protocol with greatly improved efficiency.

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

PubMed Central

Starich, Todd A.; Hall, David H.; Greenstein, David

2014-01-01

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans. PMID:25195067

Transcript levels of several epigenome regulatory genes in bovine somatic donor cells are not correlated with their cloning efficiency.

PubMed

Zhou, Wenli; Sadeghieh, Sanaz; Abruzzese, Ronald; Uppada, Subhadra; Meredith, Justin; Ohlrichs, Charletta; Broek, Diane; Polejaeva, Irina

2009-09-01

Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.

Measurement of telomerase activity in dog tumors.

PubMed

Yazawa, M; Okuda, M; Setoguchi, A; Nishimura, R; Sasaki, N; Hasegawa, A; Watari, T; Tsujimoto, H

1999-10-01

Telomeres are specific structures present at the end of liner chromosomes. DNA polymerase can not synthesize the end of liner DNA and, as a result, the telomeres become progressively shortened by successive cell divisions. To overcome the end replication problem, telomerase adds new telomeric sequences to the end of chromosomal DNA. The enzyme activity is undetectable in most normal human adult somatic cells, in which shortening of the telomere is thought to limit the somatic-cell life span. In contrast to normal somatic cells, many human tumors possess telomerase activity. The present study looked at whether telomerase activity might serve as a marker for canine tumors. Telomerase activity was measured using the telomeric repeat amplification protocol assay. Normal dog somatic tissues showed little or no telomerase activity, while normal testis exhibited a high level of telomerase activity. We measured telomerase activity in tumor samples from 45 dogs; 21 mammary gland tumors, 16 tumors developed in the skin and oral cavity, 7 vascular tumors and 1 Sertoli cell tumor. Greater than 95% of the tumor samples contained telomerase activity (3-924 U/2 micrograms protein). The results obtained in this study indicated that telomerase should be a useful diagnostic marker for a variety of dog tumors, and it may serve as a target for antitumor chemotherapy.

[PIWI protein as a nucleolus visitor in Drosophila melanogaster].

PubMed

Mikhaleva, E A; Iakushev, E Iu; Stoliarenko, A D; Klenov, M S; Pozovskiĭ, Ia M; Gvozdev, V A

2015-01-01

The evolutionarily conserved nuclear Piwi protein of Drosophila melanogaster is a representative of the Argonaute small RNA binding protein family. Guided by small piRNAs, Piwi functions in transposon silencing in somatic and germ cells of the gonad. We found that in ovarian somatic and germ cells, as well as in the established ovarian somatic cell line, Piwi is concentrated predominantly in the nucleolus--the main nuclear compartment, participating not only in rRNA synthesis, but also in various cell stress responses. We demonstrated the colocalization of Piwi with nucleolar marker proteins--fibrillarin and Nopp140. A mutation preventing Piwi transport to the nucleus and disturbing transposon silencing (piwi(Nt)) leads to 6-8-fold upregulation of rRNA genes expression, as evaluated by the level of transcripts of transposon insertions in 28S rRNA genes. RNase treatment of live cultured ovarian somatic cells depletes Piwi from the nucleolus. The same effect is observed upon inhibiting RNA polymerase I which transcribes rRNA, but not RNA polymerase II. In contrast, upon heat shock Piwi is concentrated in the nucleolus and is depleted from the nucleoplasm. These results implicate Piwi in RNA polymerase activity modulation and stress response in the nucleolus. We discuss possible noncanonical Piwi functions along with its canonical role in transposon silencing by piRNAs.

Somatic cell nuclear transfer cloning: practical applications and current legislation.

PubMed

Niemann, H; Lucas-Hahn, A

2012-08-01

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved. © 2012 Blackwell Verlag GmbH.

Polyamine and ethylene biosynthesis in relation to somatic embryogenesis in carrot (Daucus carota L.) cell cultures

Treesearch

Subhash C. Minocha; Cheryl A. Robie; Akhtar J. Khan; Nancy S. Papa; Andrew I. Samuelsen; Rakesh Minocha

1990-01-01

Carrot cell cultures provide a model experimental system for the analysis of biochemical and molecular events associated with morphogenesis in plants (3, 4, 5, 14). Among the biochemical changes accompanying somatic embryogenesis in this tissue is an increased biosynthesis ofpolyamines (1, 2, 7, 10, 11, 13). A variety of inhibitors of polyamine biosynthetic enzymes...

Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells.

PubMed Central

Harlow, E; Pim, D C; Crawford, L V

1981-01-01

Mouse cells transformed by simian virus 40 (SV40) have been shown to contain a complex of the virus-coded large-T antigen with a host 53,000-molecular-weight (53K) protein. Initial attempts to detect a similar complex in lytically infected cells were unsuccessful, and it therefore seemed that the complex might be peculiar to transformed or abortively transformed nonpermissive cells. Immunoprecipitation of [32P]phosphate-labeled extracts of SV40-infected CV-1 African green monkey kidney cells with antibodies specific for large-T or the 53K protein revealed that the large-T-53K protein complex was formed during lytic infections. Only a minor fraction of the large-T present was associated with 53K protein, and large-T and the 53K host protein cosedimented during centrifugation through sucrose gradients. We used monospecific sera and monoclonal antibodies to study the rate of synthesis and phosphorylation of the 53K protein during lytic infections. Infection of CV-1 cells with SV40 increased the rate of synthesis of the 53K protein fivefold over that in mock-infected cells. At the same time, the rate of phosphorylation of the 53K protein increased more than 30-fold compared with control cultures. Monkey cells transformed by UV-irradiated SV40 (Gluzman et al., J. Virol. 22:256-266, 1977) also contained the large-T-53K protein complex. The formation of the complex is therefore not a peculiarity of SV40-transformed rodent cells but is a common feature of SV40 infections. Images PMID:6163871

The BABY BOOM Transcription Factor Activates the LEC1-ABI3-FUS3-LEC2 Network to Induce Somatic Embryogenesis1[OPEN

PubMed Central

Weemen, Mieke

2017-01-01

Somatic embryogenesis is an example of induced cellular totipotency, where embryos develop from vegetative cells rather than from gamete fusion. Somatic embryogenesis can be induced in vitro by exposing explants to growth regulators and/or stress treatments. The BABY BOOM (BBM) and LEAFY COTYLEDON1 (LEC1) and LEC2 transcription factors are key regulators of plant cell totipotency, as ectopic overexpression of either transcription factor induces somatic embryo formation from Arabidopsis (Arabidopsis thaliana) seedlings without exogenous growth regulators or stress treatments. Although LEC and BBM proteins regulate the same developmental process, it is not known whether they function in the same molecular pathway. We show that BBM transcriptionally regulates LEC1 and LEC2, as well as the two other LAFL genes, FUSCA3 (FUS3) and ABSCISIC ACID INSENSITIVE3 (ABI3). LEC2 and ABI3 quantitatively regulate BBM-mediated somatic embryogenesis, while FUS3 and LEC1 are essential for this process. BBM-mediated somatic embryogenesis is dose and context dependent, and the context-dependent phenotypes are associated with differential LAFL expression. We also uncover functional redundancy for somatic embryogenesis among other Arabidopsis BBM-like proteins and show that one of these proteins, PLETHORA2, also regulates LAFL gene expression. Our data place BBM upstream of other major regulators of plant embryo identity and totipotency. PMID:28830937

Uncoupling the Trade-Off between Somatic Proteostasis and Reproduction in Caenorhabditis elegans Models of Polyglutamine Diseases

PubMed Central

Shemesh, Netta; Shai, Nadav; Meshnik, Lana; Katalan, Rotem; Ben-Zvi, Anat

2017-01-01

Caenorhabditis elegans somatic protein homeostasis (proteostasis) is actively remodeled at the onset of reproduction. This proteostatic collapse is regulated cell-nonautonomously by signals from the reproductive system that transmit the commitment to reproduction to somatic cells. Here, we asked whether the link between the reproductive system and somatic proteostasis could be uncoupled by activating downstream effectors in the gonadal longevity cascade. Specifically, we examined whether over-expression of lipl-4 (lipl-4(oe)), a target gene of the gonadal longevity pathway, or increase in arachidonic acid (AA) levels, associated with lipl-4(oe), modulated proteostasis and reproduction. We found that lipl-4(oe) rescued somatic proteostasis and postponed the onset of aggregation and toxicity in C. elegans models of polyglutamine (polyQ) diseases. However, lipl-4(oe) also disrupted fatty acid transport into developing oocytes and reduced reproductive success. In contrast, diet supplementation of AA recapitulated lipl-4(oe)-mediated proteostasis enhancement in wild type animals but did not affect the reproductive system. Thus, the gonadal longevity pathway mediates a trade-off between somatic maintenance and reproduction, in part by regulating the expression of genes, such as lipl-4, with inverse effects on somatic maintenance and reproduction. We propose that AA could uncouple such germline to soma crosstalk, with beneficial implications protein misfolding diseases. PMID:28503130

Induced adult stem (iAS) cells and induced transit amplifying progenitor (iTAP) cells-a possible alternative to induced pluripotent stem (iPS) cells?

PubMed

Heng, Boon Chin; Richards, Mark; Ge, Zigang; Shu, Yimin

2010-02-01

The successful derivation of iPSC lines effectively demonstrates that it is possible to reset the 'developmental clock' of somatic cells all the way back to the initial embryonic state. Hence, it is plausible that this clock may instead be turned back half-way to a less immature developmental stage that is more directly applicable to clinical therapeutic applications or for in vitro pharmacology/toxicology screening assays. Such a suitable developmental state is postulated to be either the putative transit amplifying progenitor stage or adult stem cell stage. It is hypothetically possible to reprogram mature and terminally differentiated somatic cells back to the adult stem cell or transit amplifying progenitor stage, in a manner similar to the derivation of iPSC. It is proposed that the terminology 'Induced Adult Stem Cells' (iASC) or 'Induced Transit Amplifying Progenitor Cells' (iTAPC) be used to described such reprogrammed somatic cells. Of particular interest, is the possibility of resetting the developmental clock of mature differentiated somatic cells of the mesenchymal lineage, explanted from adipose tissue, bone marrow and cartilage. The putative adult stem cell sub-population from which these cells are derived, commonly referred to as 'mesenchymal stem cells', are highly versatile and hold much therapeutic promise in regenerative medicine, as attested to by numerous human clinical trials and animal studies. Perhaps it may be appropriate to term such reprogrammed cells as 'Induced Mesenchymal Stem Cells' (iMSC) or as 'Induced Mesenchumal Progenitor Cells' (iMPC). Given that cells from the same organ/tissue will share some commonalities in gene expression, we hypothesize that the generation of iASC or iTAPC would be more efficient as compared to iPSC generation, since a common epigenetic program must exist between the reprogrammed cells, adult stem cell or progenitor cell types and terminally differentiated cell types from the same organ/tissue.

Studies on the propagation in vitro of poliomyelitis viruses. II. A description of the growth curve and its relation to the cytopathogenicity of poliomyelitis virus.

PubMed

SCHERER, W F; SYVERTON, J T

1952-05-01

The growth of poliomyelitis virus, Type 2, Yale-SK strain, in cultures of monkey testicular tissue was observed to occur in discrete cycles. Growth curves showed that each cycle was composed of (a) an initial lag phase when little or no virus was released from the cells, (6) a phase of viral production, and (c) a plateau which represented a decrement in the rate of viral production. This pattern of viral multiplication occurred in monkey testicular tissue cultures which have as the liquid phase either ox serum ultrafiltrate or monkey serum-chicken embryonic extract medium. The presence of a solid medium composed of chicken plasma, clotted either with chicken embryonic extract or bovine thrombin, did not alter the pattern of viral multiplication. The shape of the growth curve as established by any of four different techniques for tissue cultivation, was shown to be independent of the cultural technique employed. For cultures of monkey testicular tissue, the amount of virus in the tissue was as much as tenfold greater than that in the liquid of the same cultures. Moreover, viral production was evident earlier and was detectable for a longer period of time in the tissue than in the liquid phase. The rapidly incremental phase of the growth cycle, when large quantities of virus were released into the liquid phase, coincided in time with the destruction of the spindle-shaped cells, which extended from the explants. Although destruction of outgrowth cells was marked, there remained cells within the explants capable of supporting the growth of poliomyelitis virus.

Inhibition of microtubules and dynein rescues human immunodeficiency virus type 1 from owl monkey TRIMCyp-mediated restriction in a cellular context-specific fashion.

PubMed

Pawlica, Paulina; Dufour, Caroline; Berthoux, Lionel

2015-04-01

IFN-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. TRIM5α (tripartite motif protein 5, isoform α) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5α maps to the N-terminal domain of the retroviral capsid proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA)-coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits human immunodeficiency virus type 1 (HIV-1). Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5α. Here, we showed that efficient restriction of HIV-1 by omkTRIMCyp was similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by small interfering RNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggested the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp. © 2015 The Authors.

Clinical study report on milk production in the offspring of a somatic cell cloned Holstein cow.

PubMed

Takahashi, Masahiro; Tsuchiya, Hideki; Hamano, Seizo; Inaba, Toshio; Kawate, Noritoshi; Tamada, Hiromichi

2013-12-17

This study examined two female offspring of a somatic cell cloned Holstein cow that had reproduction problems and milk production performance issues. The two offspring heifers, which showed healthy appearances and normal reproductive characteristics, calved on two separate occasions. The mean milk yields of the heifers in the first lactation period were 9,037 kg and 7,228 kg. The relative mean milk yields of these cows were 111.2% and 88.9%, respectively, when compared with that of the control group. No particular clinical abnormalities were revealed in milk yields and milk composition rate [e.g., fat, protein and solids-not-fat (SNF)], and reproductive characteristics of the offspring of the somatic cell cloned Holstein cow suggested that the cloned offspring had normal milk production.

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming.

PubMed

No, Jin-Gu; Choi, Mi-Kyung; Kwon, Dae-Jin; Yoo, Jae Gyu; Yang, Byoung-Chul; Park, Jin-Ki; Kim, Dong-Hoon

2015-01-01

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot(TM) reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.

Germline Proliferation Is Regulated by Somatic Endocytic Genes via JNK and BMP Signaling in Drosophila.

PubMed

Tang, Yaning; Geng, Qing; Chen, Di; Zhao, Shaowei; Liu, Xian; Wang, Zhaohui

2017-05-01

Signals derived from the microenvironment contribute greatly to tumorigenesis . The underlying mechanism requires thorough investigation. Here, we use Drosophila testis as a model system to address this question, taking the advantage of the ease to distinguish germline and somatic cells and to track the cell numbers. In an EMS mutagenesis screen, we identified Rab5 , a key factor in endocytosis, for its nonautonomous role in germline proliferation. The disruption of Rab5 in somatic cyst cells, which escort the development of germline lineage, induced the overproliferation of underdifferentiated but genetically wild-type germ cells. We demonstrated that this nonautonomous effect was mediated by the transcriptional activation of Dpp [the fly homolog of bone morphogenetic protein (BMP)] by examining the Dpp-reporter expression and knocking down Dpp to block germline overgrowth. Consistently, the protein levels of Bam, the germline prodifferentiation factor normally accumulated in the absence of BMP/Dpp signaling, decreased in the overproliferating germ cells. Further, we discovered that the JNK signaling pathway operated between Rab5 and Dpp, because simultaneously inhibiting the JNK pathway and Rab5 in cyst cells prevented both dpp transcription and germline tumor growth. Additionally, we found that multiple endocytic genes, such as avl , TSG101 , Vps25 , or Cdc42 , were required in the somatic cyst cells to restrict germline amplification. These findings indicate that when the endocytic state of the surrounding cells is impaired, genetically wild-type germ cells overgrow. This nonautonomous model of tumorigenesis provides a simple system to dissect the relation between tumor and its niche. Copyright © 2017 by the Genetics Society of America.

Production of cloned mice by somatic cell nuclear transfer.

PubMed

Kishigami, Satoshi; Wakayama, Sayaka; Thuan, Nguyen Van; Ohta, Hiroshi; Mizutani, Eiji; Hikichi, Takafusa; Bui, Hong-Thuy; Balbach, Sebastian; Ogura, Atsuo; Boiani, Michele; Wakayama, Teruhiko

2006-01-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains < 5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cell's genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in > or = 3 months.

CD105+-mesenchymal stem cells migrate into osteoarthritis joint: An animal model.

PubMed

Fernandez-Pernas, Pablo; Rodríguez-Lesende, Iván; de la Fuente, Alexandre; Mateos, Jesús; Fuentes, Isaac; De Toro, Javier; Blanco, Fco J; Arufe, M C

2017-01-01

Mesenchymal stem cells are being the focus of connective tissue technology and regenerative medicine, presenting a good choice cell source for improving old and well recognized techniques of cartilage defect repair. For instance, the autologous chondrocyte transplantation using new concepts of regenerative medicine. The present study investigated the risk of xenogenicity of human synovial membrane-derived MSCs, injected into the monkeys using intravenous and intra-articular administration. The animal models used were adult monkeys Rhesus which had been injured into the left knee to create an Osteoarthritis (OA) animal model. CD105+-MSCs were injected twice into the OA monkeys with an interval of one week between them. The animals were euthanized one month after treatment. Immunohistochemistry analysis of different organs: spleen, heart, fat, liver, gut, pancreas, lung, skeletal muscle and kidney from the animals revealed that CD105+-MSCs migrated towards the injured knee joint. MSCs naive were found statistically significant increased in the injured knee in front of healthy one. CD105+-MSCs were negatives for CD68 and the area where CD105+-MSCs were found presented SDF-1 increased levels in front of healthy knee. We concluded that a characterized MSCs subset could be a safe alternative for cell therapy in clearly localized pathologies.

CD105+-mesenchymal stem cells migrate into osteoarthritis joint: An animal model

PubMed Central

Fernandez-Pernas, Pablo; Rodríguez-Lesende, Iván; de la Fuente, Alexandre; Mateos, Jesús; Fuentes, Isaac; De Toro, Javier; Blanco, Fco J.

2017-01-01

Mesenchymal stem cells are being the focus of connective tissue technology and regenerative medicine, presenting a good choice cell source for improving old and well recognized techniques of cartilage defect repair. For instance, the autologous chondrocyte transplantation using new concepts of regenerative medicine. The present study investigated the risk of xenogenicity of human synovial membrane-derived MSCs, injected into the monkeys using intravenous and intra-articular administration. The animal models used were adult monkeys Rhesus which had been injured into the left knee to create an Osteoarthritis (OA) animal model. CD105+-MSCs were injected twice into the OA monkeys with an interval of one week between them. The animals were euthanized one month after treatment. Immunohistochemistry analysis of different organs: spleen, heart, fat, liver, gut, pancreas, lung, skeletal muscle and kidney from the animals revealed that CD105+-MSCs migrated towards the injured knee joint. MSCs naive were found statistically significant increased in the injured knee in front of healthy one. CD105+-MSCs were negatives for CD68 and the area where CD105+-MSCs were found presented SDF-1 increased levels in front of healthy knee. We concluded that a characterized MSCs subset could be a safe alternative for cell therapy in clearly localized pathologies. PMID:29190645

Disruptions in follicle cell functions in the ovaries of rhesus monkeys during summer

PubMed Central

VandeVoort, Catherine A.; Mtango, Namdori R.; Midic, Uros

2015-01-01

Oocytes isolated from female rhesus monkeys following standard ovarian stimulation protocols during the summer months displayed a reduced capacity to mature compared with stimulation during the normal breeding season. Because the gene expression profiles of oocyte-associated cumulus cells and mural granulosa cells (CCs and GCs) are indicative of altered oocyte quality and can provide insight into intrafollicular processes that may be disrupted during oogenesis, we performed array-based transcriptome comparisons of CCs and GCs from summer and normal breeding season stimulation cycles. Summer CCs and GCs both display deficiencies in expression of mRNAs related to cell proliferation, angiogenesis, and endocrine signaling, as well as reduced expression of glycogen phosphorylase. Additionally, CCs display deficiencies in expression of mRNAs related to stress response. These results provide the first insight into the specific molecular pathways and processes that are disrupted in the follicles of rhesus macaque females during the summer season. Some of the changes seen in summer GCs and CCs have been reported in humans and in other model mammalian species. This suggests that the seasonal effects seen in the rhesus monkey may help us to understand better the mechanisms that contribute to reduced oocyte quality and fertility in humans. PMID:25586978

Differential arousal regulation by prokineticin 2 signaling in the nocturnal mouse and the diurnal monkey.

PubMed

Zhou, Qun-Yong; Burton, Katherine J; Neal, Matthew L; Qiao, Yu; Kanthasamy, Anumantha G; Sun, Yanjun; Xu, Xiangmin; Ma, Yuanye; Li, Xiaohan

2016-08-18

The temporal organization of activity/rest or sleep/wake rhythms for mammals is regulated by the interaction of light/dark cycle and circadian clocks. The neural and molecular mechanisms that confine the active phase to either day or night period for the diurnal and the nocturnal mammals are unclear. Here we report that prokineticin 2, previously shown as a circadian clock output molecule, is expressed in the intrinsically photosensitive retinal ganglion cells, and the expression of prokineticin 2 in the intrinsically photosensitive retinal ganglion cells is oscillatory in a clock-dependent manner. We further show that the prokineticin 2 signaling is required for the activity and arousal suppression by light in the mouse. Between the nocturnal mouse and the diurnal monkey, a signaling receptor for prokineticin 2 is differentially expressed in the retinorecipient suprachiasmatic nucleus and the superior colliculus, brain projection targets of the intrinsically photosensitive retinal ganglion cells. Blockade with a selective antagonist reveals the respectively inhibitory and stimulatory effect of prokineticin 2 signaling on the arousal levels for the nocturnal mouse and the diurnal monkey. Thus, the mammalian diurnality or nocturnality is likely determined by the differential signaling of prokineticin 2 from the intrinsically photosensitive retinal ganglion cells onto their retinorecipient brain targets.

Morphological analyses and variation in carbohydrate content during the maturation of somatic embryos of Carica papaya.

PubMed

Vale, Ellen Moura; Reis, Ricardo Souza; Passamani, Lucas Zanchetta; Santa-Catarina, Claudete; Silveira, Vanildo

2018-03-01

Efficient protocols for somatic embryogenesis of papaya ( Carica papaya L.) have great potential for selecting elite hybrid genotypes. Addition of polyethylene glycol (PEG), a nonplasmolyzing osmotic agent, to a maturation medium increases the production of somatic embryos in C . papaya . To study the effects of PEG on somatic embryogenesis of C . papaya , we analyzed somatic embryo development and carbohydrate profile changes during maturation treatments with PEG (6%) or without PEG (control). PEG treatment (6%) increased the number of normal mature somatic embryos followed by somatic plantlet production. In both control and PEG treatments, pro-embryogenic differentiation to the cotyledonary stage was observed and was significantly higher with PEG treatment. Histomorphological analysis of embryonic cultures with PEG revealed meristematic centers containing small isodiametric cells with dense cytoplasm and evident nuclei. Concomitant with the increase in the differentiation of somatic embryos in PEG cultures, there was an increase in the endogenous content of sucrose and starch, which appears to be related to a rising demand for energy, a key point in the conversion of C . papaya somatic embryos. The endogenous carbohydrate profile may be a valuable parameter for developing optimized protocols for the maturation of somatic embryos in papaya.

DNMT1 maintains progenitor function in self-renewing somatic tissue.

PubMed

Sen, George L; Reuter, Jason A; Webster, Daniel E; Zhu, Lilly; Khavari, Paul A

2010-01-28

Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

Evaluation of Macaca mulatta as a model for genotoxicity studies.

PubMed

Dobrovolsky, Vasily N; Shaddock, Joseph G; Mittelstaedt, Roberta A; Manjanatha, Mugimane G; Miura, Daishiro; Uchikawa, Makoto; Mattison, Donald R; Morris, Suzanne M

2009-02-19

We have investigated the use of peripheral blood from the nonhuman primate (NHP) rhesus monkey (Macaca mulatta) as a model system for mutation detection. The rhesus monkey is metabolically closer to humans than most common laboratory animals, and therefore may be a relevant model for hazard identification and human risk assessment. To validate the model, conditions were determined for in vitro selection and expansion of 6-thioguanine-resistant (6-TGr) HPRT mutant and proaerolysin-resistant (ProAERr) PIG-A mutant lymphocytes from peripheral blood obtained by routine venipuncture. Also, flow cytometric methods were developed for the rapid detection of PIG-A mutant erythrocytes. The flow cytometric analysis of PIG-A mutant erythrocytes was based on enumerating cells deficient in surface markers attached to the cellular membrane via glycosylphosphatidyl inositol (GPI) anchors. Mutant cells were enumerated over an extended period of time in peripheral blood of male monkeys receiving daily doses of the electrolyte replenisher Prangtrade mark (a common carrier for oral delivery of drugs in NHPs), and in the blood of one male monkey treated with a single i.p. dose of 50mg/kg of N-ethyl-N-nitrosourea at approximately 2 years of age and another similar injection at approximately 3.5 years of age. The spontaneous PIG-A and HPRT T-cell mutant frequency (MF) was low in animals receiving Prang (0-8x10(-6)), and treatment with ENU resulted in a clearly detectable increase in the frequency of ProAERr and 6-TGr lymphocytes (up to approximately 28x10(-6) and approximately 30x10(-6), respectively). Also, the ENU-treated animal had higher frequency of GPI-deficient erythrocytes (46.5x10(-6) in the treated animal vs. 7.8+/-4.2x10(-6) in control animals). Our results indicate that the rhesus monkey can be a valuable model for the identification of agents that may impact upon human health as mutagens and that the PIG-A gene can be a useful target for detection of mutation in both white and red blood cells.

Bridging the divide

PubMed Central

McLean, Peter F; Cooley, Lynn

2014-01-01

Ring canals are made from arrested cleavage furrows, and provide direct cytoplasmic connections among sibling cells. They are well documented for their participation in Drosophila oogenesis, but little is known about their role in several somatic tissues in which they are also found. Using a variety of genetic tools in live and fixed tissue, we recently demonstrated that rapid intercellular exchange occurs through somatic ring canals by diffusion, and presented evidence that ring canals permit equilibration of protein among transcriptionally mosaic cells. We also used a novel combination of markers to evaluate the extent of protein movement within and across mitotic clones in follicle cells and imaginal discs, providing evidence of robust movement of GFP between the 2 sides of mitotic clones and frequently into non-recombined cells. These data suggest that, depending on the experimental setup and proteins of interest, inter-clonal diffusion of protein may alter the interpretation of clonal data in follicle cells. Here, we discuss these results and provide additional insight into the impact of ring canals in Drosophila somatic tissues. PMID:24406334

Animal studies on Spacelab-3

NASA Technical Reports Server (NTRS)

Schatte, C.; Grindeland, R.; Callahan, P.; Berry, W.; Funk, G.; Lencki, W.

1987-01-01

The flight of two squirrel monkeys and 24 rats on Spacelab-3 was the first mission to provide hands-on maintenance on animals in a laboratory environment. With few exceptions, the animals grew and behaved normally, were free of chronic stress, and differed from ground controls only for gravity dependent parameters. One of the monkeys exhibited symptoms of space sickness similar to those observed in humans, which suggests squirrel monkeys may be good models for studying the space adaptation syndrome. Among the wide variety of parameters measured in the rats, most notable was the dramatic loss of muscle mass and increased fragility of long bones. Other interesting rat findings were those of suppressed interferom production by spleen cells, defective release of growth hormone by somatrophs, possible dissociation of circadian pacemakers, changes in hepatic lipid and carbohydrate metabolism, and hypersensitivity of marrow cells to erythropoietin. These results portend a strong role for animals in identifying and elucidating the physiological and anatomical responses of mammals to microgravity.

Movement Limitation and Immune Responses of Rhesus Monkeys

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Morton, Darla S.; Swiggett, Jeanene P.; Hakenewerth, Anne M.; Fowler, Nina A.

1993-01-01

The effects of restraint on immunological parameters was determined in an 18 day ARRT (adult rhesus restraint test). The monkeys were restrained for 18 days in the experimental station for the orbiting primate (ESOP), the chair of choice for Space Shuttle experiments. Several immunological parameters were determined using peripheral blood, bone marrow, and lymph node specimens from the monkeys. The parameters included: response of bone marrow cells to GM-CSF (granulocyte-macrophage colony stimulating factor), leukocyte subset distribution, and production of IFN-alpha (interferon-alpha) and IFN-gamma (interferon-gamma). The only parameter changed after 18 days of restraint was the percentage of CDB+ T cells. No other immunological parameters showed changes due to restraint. Handling and changes in housing prior to the restraint period did apparently result in some restraint-independent immunological changes. Handling must be kept to a minimum and the animals allowed time to recover prior to flight. All experiments must be carefully controlled. Restraint does not appear to be a major issue regarding the effects of space flight on immune responses.

Spaceflight and immune responses of rhesus monkeys

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Morton, Darla S.; Swiggett, Jeanene P.; Hakenewerth, Anne M.; Fowler, Nina A.

1995-01-01

The effects of restraint on immunological parameters was determined in an 18 day ARRT (adult rhesus restraint test). The monkeys were restrained for 18 days in the experimental station for the orbiting primate (ESOP), the chair of choice for Space Shuttle experiments. Several immunological parameters were determined using peripheral blood, bone marrow, and lymph node specimens from the monkeys. The parameters included: response of bone marrow cells to GM-CSF (granulocyte-macrophage colony stimulating factor), leukocyte subset distribution, and production of IFN-a (interferon-alpha) and IFN-gamma (interferon-gamma). The only parameter changed after 18 days of restraint was the percentage of CD8+ T cells. No other immunological parameters showed changes due to restraint. Handling and changes in housing prior to the restraint period did apparently result in some restraint-independent immunological changes. Handling must be kept to a minimum and the animals allowed time to recover prior to flight. All experiments must be carefully controlled. Restraint does not appear to be a major issue regarding the effects of space flight on immune responses.

Animal studies on Spacelab-3

NASA Technical Reports Server (NTRS)

Schatte, C.; Grindeland, R.; Callahan, P.; Funk, G.; Lencki, W.; Berry, W.

1986-01-01

The flight of two squirrel monkeys and 24 rates on Spacelab-3 was the first mission to provide hand-on maintenance on animals in a laboratory environment. With few exceptions, the animals grew and behaved normally, were free of chronic stress, and differed from ground controls only for gravity-dependent parameters. One of the monkeys exhibited symptoms of space sickness similar to those observed in humans, which suggests squirrel monkeys may be good models for studying the space-adaptation syndrome. Among the wide variety of parameters measured in the rats, most notable was the dramatic loss of muscle mass and increased fragility of long bones. Other interesting rat findings were those of suppressed interferon production by spleen cells, defective release of growth hormone by somatotrophs, possible dissociation of circadian pacemakers, changes in hepatic lipid and carbohydrate metabolism, and hypersensitivity of marrow cells to erythopoietin. These results portend a strong role for animals in identifying and elucidating the physiological and anatomical responses of mammals to microgravity.

The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model

PubMed Central

Kataoka, Chikako; Suzuki, Tadaki; Kotani, Osamu; Iwata-Yoshikawa, Naoko; Nagata, Noriyo; Ami, Yasushi; Wakita, Takaji; Nishimura, Yorihiro; Shimizu, Hiroyuki

2015-01-01

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, occasionally causes severe neurological symptoms. We identified P-selectin glycoprotein ligand-1 (PSGL-1) as an EV71 receptor and found that an amino acid residue 145 in the capsid protein VP1 (VP1-145) defined PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) phenotypes of EV71. However, the role of PSGL-1-dependent EV71 replication in neuropathogenesis remains poorly understood. In this study, we investigated viral replication, genetic stability, and the pathogenicity of PB and non-PB strains of EV71 in a cynomolgus monkey model. Monkeys were intravenously inoculated with cDNA-derived PB and non-PB strains of EV71, EV71-02363-EG and EV71-02363-KE strains, respectively, with two amino acid differences at VP1-98 and VP1-145. Mild neurological symptoms, transient lymphocytopenia, and inflammatory cytokine responses, were found predominantly in the 02363-KE-inoculated monkeys. During the early stage of infection, viruses were frequently detected in clinical samples from 02363-KE-inoculated monkeys but rarely in samples from 02363-EG-inoculated monkeys. Histopathological analysis of central nervous system (CNS) tissues at 10 days postinfection revealed that 02363-KE induced neuropathogenesis more efficiently than that induced by 02363-EG. After inoculation with 02363-EG, almost all EV71 variants detected in clinical samples, CNS, and non-CNS tissues, possessed a G to E amino acid substitution at VP1-145, suggesting a strong in vivo selection of VP1-145E variants and CNS spread presumably in a PSGL-1-independent manner. EV71 variants with VP1-145G were identified only in peripheral blood mononuclear cells in two out of four 02363-EG-inoculated monkeys. Thus, VP1-145E variants are mainly responsible for the development of viremia and neuropathogenesis in a non-human primate model, further suggesting the in vivo involvement of amino acid polymorphism at VP1-145 in cell-specific viral replication, in vivo fitness, and pathogenesis in EV71-infected individuals. PMID:26181772

A preclinical evaluation of alternative site for islet allotransplantation

PubMed Central

He, Sirong; Yuan, Yujia; Han, Pengfei; Wang, Dan; Chen, Younan; Liu, Jingping; Tian, Bole; Yang, Guang; Yi, Shounan; Gao, Fabao; Zhong, Zhihui; Li, Hongxia; Cheng, Jingqiu; Lu, Yanrong

2017-01-01

The bone marrow cavity (BMC) has recently been identified as an alternative site to the liver for islet transplantation. This study aimed to compare the BMC with the liver as an islet allotransplantation site in diabetic monkeys. Diabetes was induced in Rhesus monkeys using streptozocin, and the monkeys were then divided into the following three groups: Group1 (islets transplanted in the liver with immunosuppressant), Group 2 (islets transplanted in the tibial BMC), and Group 3 (islets transplanted in the tibial BMC with immunosuppressant). The C-peptide and blood glucose levels were preoperatively measured. An intravenous glucose tolerance test (IVGTT) was conducted to assess graft function, and complete blood cell counts were performed to assess cell population changes. Cytokine expression was measured using an enzyme-linked immune sorbent assay (ELISA) and MILLIPLEX. Five monkeys in Group 3 exhibited a significantly increased insulin-independent time compared with the other groups (Group 1: 78.2 ± 19.0 days; Group 2: 58.8 ± 17.0 days; Group 3: 189.6 ± 26.2 days) and demonstrated increases in plasma C-peptide 4 months after transplantation. The infusion procedure was not associated with adverse effects. Functional islets in the BMC were observed 225 days after transplantation using the dithizone (DTZ) and insulin/glucagon stains. Our results showed that allogeneic islets transplanted in the BMC of diabetic Rhesus monkeys remained alive and functional for a longer time than those transplanted in the liver. This study was the first successful demonstration of allogeneic islet engraftment in the BMC of non-human primates (NHPs). PMID:28358858

The effect of recombinant GM-CSF on the recovery of monkeys transplanted with autologous bone marrow.

PubMed

Monroy, R L; Skelly, R R; MacVittie, T J; Davis, T A; Sauber, J J; Clark, S C; Donahue, R E

1987-11-01

The regulatory function of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) on granulocyte production in vivo was evaluated in an autologous bone marrow transplantation model using rhesus monkeys. Monkeys were exposed to 9.0 Gy total body irradiation and then transplanted with 5.0 x 10(7) low-density bone marrow cells/kg. Alzet miniosmotic pumps were subcutaneously implanted to deliver rhGM-CSF at a rate of 50,400 U/kg/d. Minipumps, containing either rhGM-CSF or saline, were implanted between zero and five days after transplantation for seven days. Kinetic recoveries of peripheral blood cells after either saline or rhGM-CSF treatment were compared. Treatment with rhGM-CSF accelerated the recovery of neutrophils. Neutrophils in rhGM-CSF-treated animals recovered to 80% (3.4 x 10(3)/mm3) pre-irradiation control levels by day 20, in comparison with only 33% (0.9 x 10(3)/mm3) recovery for saline control monkeys. In addition, the recovery of neutrophils was enhanced over that of the controls, reaching 140% v 70% on day 30. Another prominent feature of rhGM-CSF-treated monkeys was the accelerated recovery of platelets, reaching near 50% normal levels by day 24 in comparison with 20% of normal levels for controls. The infusion of rhGM-CSF was shown to be an effective regulator of early hematopoietic regeneration, leading to the accelerated recovery of both neutrophils and platelets and then providing a consistent sustained increase of neutrophils even in the absence of rhGM-CSF.

COMPARISON OF EFFECTS OF DEUTERIUM OXIDE AND X-RAY IRRADIATION ON MULTIPLICATION OF POLIOVIRUS

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

Kritchevsky, D.; Manson, L.A.; Hartzell, R.W. Jr.

1963-01-01

An attenuated strain of poliomyelitis virus (CHAT) will not grow in monkey kidney cells at 40 un. Concent 85% C. When deuterium oxide (25 to 40%) is present in the medium, replication of CHAT virus will take place at 40 un. Concent 85% C. Since both deuterium oxide treatment and irradiation with x rays yield giant cells, the 2 treatments have been compared for their ability to support the growth of CHAT poliovirus at 40 un. Concent 85% C. At several levels of x irradiation, monkey kidney cells will not support the growth of CHAT virus at 40 un. Concentmore » 85% C. When D/sub 2/O is added to he medium of the x- irradiated cells at 40 un. Concent 85% C, replication of CHAT virus is observed. The effect is not due to cell size or number. (auth)« less

Differences in reward processing between putative cell types in primate prefrontal cortex

PubMed Central

Fan, Hongwei; Wang, Rubin; Sakagami, Masamichi

2017-01-01

Single-unit studies in monkeys have demonstrated that neurons in the prefrontal cortex predict the reward type, reward amount or reward availability associated with a stimulus. To examine contributions of pyramidal cells and interneurons in reward processing, single-unit activity was extracellularly recorded in prefrontal cortices of four monkeys performing a reward prediction task. Based on their shapes of spike waveforms, prefrontal neurons were classified into broad-spike and narrow-spike units that represented putative pyramidal cells and interneurons, respectively. We mainly observed that narrow-spike neurons showed higher firing rates but less bursty discharges than did broad-spike neurons. Both narrow-spike and broad-spike cells selectively responded to the stimulus, reward and their interaction, and the proportions of each type of selective neurons were similar between the two cell classes. Moreover, the two types of cells displayed equal reliability of reward or stimulus discrimination. Furthermore, we found that broad-spike and narrow-spike cells showed distinct mechanisms for encoding reward or stimulus information. Broad-spike neurons raised their firing rate relative to the baseline rate to represent the preferred reward or stimulus information, whereas narrow-spike neurons inhibited their firing rate lower than the baseline rate to encode the non-preferred reward or stimulus information. Our results suggest that narrow-spike and broad-spike cells were equally involved in reward and stimulus processing in the prefrontal cortex. They utilized a binary strategy to complementarily represent reward or stimulus information, which was consistent with the task structure in which the monkeys were required to remember two reward conditions and two visual stimuli. PMID:29261734

Differences in reward processing between putative cell types in primate prefrontal cortex.

PubMed

Fan, Hongwei; Pan, Xiaochuan; Wang, Rubin; Sakagami, Masamichi

2017-01-01

Single-unit studies in monkeys have demonstrated that neurons in the prefrontal cortex predict the reward type, reward amount or reward availability associated with a stimulus. To examine contributions of pyramidal cells and interneurons in reward processing, single-unit activity was extracellularly recorded in prefrontal cortices of four monkeys performing a reward prediction task. Based on their shapes of spike waveforms, prefrontal neurons were classified into broad-spike and narrow-spike units that represented putative pyramidal cells and interneurons, respectively. We mainly observed that narrow-spike neurons showed higher firing rates but less bursty discharges than did broad-spike neurons. Both narrow-spike and broad-spike cells selectively responded to the stimulus, reward and their interaction, and the proportions of each type of selective neurons were similar between the two cell classes. Moreover, the two types of cells displayed equal reliability of reward or stimulus discrimination. Furthermore, we found that broad-spike and narrow-spike cells showed distinct mechanisms for encoding reward or stimulus information. Broad-spike neurons raised their firing rate relative to the baseline rate to represent the preferred reward or stimulus information, whereas narrow-spike neurons inhibited their firing rate lower than the baseline rate to encode the non-preferred reward or stimulus information. Our results suggest that narrow-spike and broad-spike cells were equally involved in reward and stimulus processing in the prefrontal cortex. They utilized a binary strategy to complementarily represent reward or stimulus information, which was consistent with the task structure in which the monkeys were required to remember two reward conditions and two visual stimuli.

Variation of mutational burden in healthy human tissues suggests non-random strand segregation and allows measuring somatic mutation rates.

PubMed

Werner, Benjamin; Sottoriva, Andrea

2018-06-01

The immortal strand hypothesis poses that stem cells could produce differentiated progeny while conserving the original template strand, thus avoiding accumulating somatic mutations. However, quantitating the extent of non-random DNA strand segregation in human stem cells remains difficult in vivo. Here we show that the change of the mean and variance of the mutational burden with age in healthy human tissues allows estimating strand segregation probabilities and somatic mutation rates. We analysed deep sequencing data from healthy human colon, small intestine, liver, skin and brain. We found highly effective non-random DNA strand segregation in all adult tissues (mean strand segregation probability: 0.98, standard error bounds (0.97,0.99)). In contrast, non-random strand segregation efficiency is reduced to 0.87 (0.78,0.88) in neural tissue during early development, suggesting stem cell pool expansions due to symmetric self-renewal. Healthy somatic mutation rates differed across tissue types, ranging from 3.5 × 10-9/bp/division in small intestine to 1.6 × 10-7/bp/division in skin.

Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide, a Histone Deacetylase Inhibitor*

PubMed Central

Dai, Xiangpeng; Hao, Jie; Hou, Xiao-jun; Hai, Tang; Fan, Yong; Yu, Yang; Jouneau, Alice; Wang, Liu; Zhou, Qi

2010-01-01

Somatic cell nuclear transfer (SCNT) has shown tremendous potential for understanding the mechanisms of reprogramming and creating applications in the realms of agriculture, therapeutics, and regenerative medicine, although the efficiency of reprogramming is still low. Somatic nucleus reprogramming is triggered in the short time after transfer into recipient cytoplasm, and therefore, this period is regarded as a key stage for optimizing SCNT. Here we report that CBHA, a histone deacetylase inhibitor, modifies the acetylation status of somatic nuclei and increases the developmental potential of mouse cloned embryos to reach pre- and post-implantation stages. Furthermore, the cloned embryos treated by CBHA displayed higher efficiency in the derivation of nuclear transfer embryonic stem cell lines by promoting outgrowths. More importantly, CBHA increased blastocyst quality compared with trichostatin A, another prevalent histone deacetylase inhibitor reported previously. Use of CBHA should improve the productivity of SCNT for a variety of research and clinical applications, and comparisons of cells with different levels of pluripotency and treated with CBHA versus trichostatin A will facilitate studies of the mechanisms of reprogramming. PMID:20566633

Bloom syndrome: a mendelian prototype of somatic mutational disease.

PubMed

German, J

1993-11-01

Spontaneous mutations in human somatic cells occur far more often than normal in individuals with Bloom syndrome. The basis for understanding these mutations and their developmental consequences emerges from examination of BS at the molecular, cellular, and clinical levels. The major clinical feature of BS, proportional dwarfism, as well as its major clinical complication, an exceptionally early emergence of neoplasia of the types and sites that affect the general population, are attributable to the excessive occurrence of mutations in somatic cells. Here, the following aspects of BS are discussed: (i) the BS phenotype; (ii) neoplasia in BS, including the means--the Bloom's Syndrome Registry--by which the significant risk for diverse sites and types of cancer in these patients was revealed; (iii) the biological basis for the cancer proneness of BS; and, finally, (iv) the significance for both basic human biology and clinical medicine of BS as the prototype of somatic mutational disease.

Nectin-4 Interactions Govern Measles Virus Virulence in a New Model of Pathogenesis, the Squirrel Monkey (Saimiri sciureus).

PubMed

Delpeut, Sébastien; Sawatsky, Bevan; Wong, Xiao-Xiang; Frenzke, Marie; Cattaneo, Roberto; von Messling, Veronika

2017-06-01

In addition to humans, only certain nonhuman primates are naturally susceptible to measles virus (MeV) infection. Disease severity is species dependent, ranging from mild to moderate for macaques to severe and even lethal for certain New World monkey species. To investigate if squirrel monkeys ( Saimiri sciureus ), which are reported to develop a course of disease similar to humans, may be better suited than macaques for the identification of virulence determinants or the evaluation of therapeutics, we infected them with a green fluorescent protein-expressing MeV. Compared to cynomolgus macaques ( Macaca fascicularis ) infected with the same virus, the squirrel monkeys developed more-severe immunosuppression, higher viral load, and a broader range of clinical signs typical for measles. In contrast, infection with an MeV unable to interact with the epithelial receptor nectin-4, while causing immunosuppression, resulted in only a mild and transient rash and a short-lived elevation of the body temperature. Similar titers of the wild-type and nectin-4-blind MeV were detected in peripheral blood mononuclear cells and lymph node homogenates, but only the wild-type virus was found in tracheal lavage fluids and urine. Thus, our study demonstrates the importance of MeV interactions with nectin-4 for clinical disease in the new and better-performing S. sciureus model of measles pathogenesis. IMPORTANCE The characterization of mechanisms underlying measles virus clinical disease has been hampered by the lack of an animal model that reproduces the course of disease seen in human patients. Here, we report that infection of squirrel monkeys ( Saimiri sciureus ) fulfills these requirements. Comparative infection with wild-type and epithelial cell receptor-blind viruses demonstrated the importance of epithelial cell infection for clinical disease, highlighting the spread to epithelia as an attractive target for therapeutic strategies. Copyright © 2017 American Society for Microbiology.

Single inverted terminal repeats of the Junonia coenia Densovirus promotes somatic chromosomal integration of vector plasmids in insect cells and supports high efficiency expression

USDA-ARS?s Scientific Manuscript database

Plasmids that contain a disrupted genome of the Junonia coenia densovirus (JcDNV) integrate into the chromosomes of the somatic cells of insects. When subcloned individually, both the P9 inverted terminal repeat (P9-ITR) and the P93-ITR promote the chromosomal integration of vector plasmids in insec...

Adult Mammalian Neural Stem Cells and Neurogenesis: Five Decades Later

PubMed Central

Bond, Allison M.; Ming, Guo-li; Song, Hongjun

2015-01-01

Summary Adult somatic stem cells in various organs maintain homeostatic tissue regeneration and enhance plasticity. Since its initial discovery five decades ago, investigations of adult neurogenesis and neural stem cells have led to an established and expanding field that has significantly influenced many facets of neuroscience, developmental biology and regenerative medicine. Here we review recent progress and focus on questions related to adult mammalian neural stem cells that also apply to other somatic stem cells. We further discuss emerging topics that are guiding the field toward better understanding adult neural stem cells and ultimately applying these principles to improve human health. PMID:26431181

Repeated episodes of ozone inhalation amplifies the effects of allergen sensitization and inhalation on airway immune and structural development in Rhesus monkeys.

PubMed

Schelegle, Edward S; Miller, Lisa A; Gershwin, Laurel J; Fanucchi, Michelle V; Van Winkle, Laura S; Gerriets, Joan E; Walby, William F; Mitchell, Valerie; Tarkington, Brian K; Wong, Viviana J; Baker, Gregory L; Pantle, Lorraine M; Joad, Jesse P; Pinkerton, Kent E; Wu, Reen; Evans, Michael J; Hyde, Dallas M; Plopper, Charles G

2003-08-15

Twenty-four infant rhesus monkeys (30 days old) were exposed to 11 episodes of filtered air (FA), house dust mite allergen aerosol (HDMA), ozone (O3), or HDMA + O3 (5 days each followed by 9 days of FA). Ozone was delivered for 8 h/day at 0.5 ppm. Twelve of the monkeys were sensitized to house dust mite allergen (Dermatophagoides farinae) at ages 14 and 28 days by subcutaneous inoculation (SQ) of HDMA in alum and intraperitoneal injection of heat-killed Bordetella pertussis cells. Sensitized monkeys were exposed to HDMA aerosol for 2 h/day on days 3-5 of either FA (n = 6) or O3 (n = 6) exposure. Nonsensitized monkeys were exposed to either FA (n = 6) or O3 (n = 6). During the exposure regimen, parameters of allergy (i.e., serum IgE, histamine, and eosinophilia), airways resistance, reactivity, and structural remodeling were evaluated. Eleven repeated 5-day cycles of inhaling 0.5 ppm ozone over a 6-month period had only mild effects on the airways of nonsensitized infant rhesus monkeys. Similarly, the repeated inhalation of HDMA by HDMA-sensitized infant monkeys resulted in only mild airway effects, with the exception of a marked increase in proximal airway and terminal bronchiole content of eosinophils. In contrast, the combined cyclic inhalation of ozone and HDMA by HDMA sensitized infants monkeys resulted in a marked increase in serum IgE, serum histamine, and airways eosinophilia. Furthermore, combined cyclic inhalation of ozone and HDMA resulted in even greater alterations in airway structure and content that were associated with a significant elevation in baseline airways resistance and reactivity. These results suggest that ozone can amplify the allergic and structural remodeling effects of HDMA sensitization and inhalation.

Cloning Endangered Felids by Interspecies Somatic Cell Nuclear Transfer.

PubMed

Gómez, Martha C; Pope, C Earle

2015-01-01

In 2003, the first wild felid was produced by interspecies somatic cell nuclear transfer. Since then other wild felid clone offspring have been produced by using the same technique with minor modifications. This chapter describes detailed protocols used in our laboratory for (1) the isolation, culture, and preparation of fibroblast cells as donor nucleus, and (2) embryo reconstruction with domestic cat enucleated oocytes to produce cloned embryos that develop to the blastocyst stage in vitro and, after transfer into synchronized recipients, establish successful pregnancies.

Experimentally-induced immune activation in natural hosts of SIV induces significant increases in viral replication and CD4+ T cell depletion

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

Ribeiro, Ruy M

2008-01-01

Chronically SIVagm-infected African green monkeys (AGMs) have a remarkably stable non-pathogenic disease course, with levels of immune activation in chronic SIVagm infection similar to those observed in uninfected monkeys and stable viral loads (VLs) for long periods of time. In vivo administration of lipopolysaccharide (LPS) or an IL-2/diphtheria toxin fusion protein (Ontak) to chronically SIVagm-infected AGMs triggered increases in immune activation and subsequently of viral replication and depletion of intestinal CD4{sup +} T cells. Our study indicates that circulating microbial products can increase viral replication by inducing immune activation and increasing the number of viral target cells, thus demonstrating thatmore » immune activation and T cell prolifeation are key factors in AIDS pathogenesis.« less

Visual responses of ganglion cells of a New-World primate, the capuchin monkey, Cebus apella.

PubMed

Lee, B B; Silveira, L C; Yamada, E S; Hunt, D M; Kremers, J; Martin, P R; Troy, J B; da Silva-Filho, M

2000-11-01

1. The genetic basis of colour vision in New-World primates differs from that in humans and other Old-World primates. Most New-World primate species show a polymorphism; all males are dichromats and most females trichromats. 2. In the retina of Old-World primates such as the macaque, the physiological correlates of trichromacy are well established. Comparison of the retinae in New- and Old-World species may help constrain hypotheses as to the evolution of colour vision and the pathways associated with it. 3. Ganglion cell behaviour was recorded from trichromatic and dichromatic members of a New-World species (the capuchin monkey, Cebus apella) and compared with macaque data. Despite some differences in quantitative detail (such as a temporal response extended to higher frequencies), results from trichromatic animals strongly resembled those from the macaque. 4. In particular, cells of the parvocellular (PC) pathway showed characteristic frequency-dependent changes in responsivity to luminance and chromatic modulation, cells of the magnocellular (MC) pathway showed frequency-doubled responses to chromatic modulation, and the surround of MC cells received a chromatic input revealed on changing the phase of heterochromatically modulated lights. 5. Ganglion cells of dichromats were colour-blind versions of those of trichromats. 6. This strong physiological homology is consistent with a common origin of trichromacy in New- and Old-World monkeys; in the New-World primate the presence of two pigments in the middle-to-long wavelength range permits full expression of the retinal mechanisms of trichromatic vision.

Elevated Levels of Somatic Mutation as a Biomarker of Environmental Effects Contributing to Breast Carcinogenesis

DTIC Science & Technology

2001-07-01

and hepatocellular carcinoma patients have been shown to exhibit elevated somatic mutation frequencies with the GPA assay (Okada et al., 1997...T, Kyogoku A, Yoshimori M (1997) Evidence for increased somatic cell mutations in patients with hepatocellular carcinoma . Carcinogenesis 18: 445-449...significant increase in mutation at the GPA locus has been reported for a population of hepatocellular carcinoma patients (Okada et al., 1997

Genomic individuality and its biological implications.

PubMed

Zhao, J

1996-06-01

It is a widely accepted fundamental concept that all somatic genomes of a human individual are identical to each other. The theoretical basis of this concept is that all of these somatic genomes are the descendants of the genome of a single fertilized cell as well as the simple replicated products of asexual reproduction, thus not forming any new recombined genomes. The question here is whether such a concept might only represent one side of somatic genome biology and, even worse, whether it has perhaps already led to a very prevalent misconception that within the organism body, there exists no variability among individual somatic genomes. A hypothesis, called genomic individuality, is proposed, simply saying that every individual somatic genome, perhaps with rare exceptions, has its own unique or individual 'genetic identity' or 'fingerprint', which is characterized by its distinctive sequences or patterns of deoxyribonucleic acid molecules, or both. Thus, no two somatic genomes can be identical to each other in every or all aspects, and consequently, there must be a great deal of genomic variation present within the body of any multicellular organism. The concept or hypothesis of genomic individuality would not only provide a more complete understanding of genome biology, but also suggest a new insight into the studies of the biology of cells and organisms.

Effects of formaldehyde gas on the respiratory tract of rhesus monkeys. Pathology and cell proliferation.

PubMed Central

Monticello, T. M.; Morgan, K. T.; Everitt, J. I.; Popp, J. A.

1989-01-01

Formaldehyde is a nasal carcinogen in rats but it remains to be determined what cancer risk this chemical poses in humans. Molecular dosimetry studies of formaldehyde and cellular proliferative responses to formaldehyde-induced cytotoxicity have been studied in the rodent and are important components of the authors' ongoing research, which has now been extended to nonhuman primates, a species more analogous to humans. The present study was designed to characterize formaldehyde injury in the respiratory tract of nonhuman primates to provide a direct comparison to the toxic effects of formaldehyde in rodents. Groups of three rhesus monkeys were exposed to room air, or 6 ppm formaldehyde for 5 days per week for 1 or 6 weeks, and the respiratory tract was assessed for nature and extent of histologic responses, and changes in epithelial cell proliferation rate. Lesions were characterized by mild degeneration and early squamous metaplasia confined to specific regions of the transitional and respiratory epithelia of the nasal passages and the respiratory epithelium of the trachea and major bronchi. There was minimal progression of histologic changes between 1 and 6 weeks; however, the percent of nasal surface area affected significantly increased in the 6-week exposure group. Formaldehyde-induced lesions were associated with increases in cell proliferation rates up to 18-fold over controls, which remained significantly elevated after 6 weeks of exposure. Histologic lesions and increases in cell proliferation were most extensive in the nasal passages and were minimal in the lower airways, whereas the maxillary sinuses exhibited no evidence of a response to formaldehyde exposure. Based on the extent of lesions and cell proliferation data, it appears that the monkey is more sensitive than the rat to the acute and subacute effects of formaldehyde at 6 ppm. The absence of response in the maxillary sinuses in the monkey suggests that combining tumors of the nasal cavity and sinuses in epidemiologic studies may not be appropriate for formaldehyde cancer risk assessment. Results of this study also have provided important information for tissue sample site selection in the monkey respiratory tract for ongoing molecular dosimetry studies. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 9 PMID:2923182

Plant regeneration via direct somatic embryogenesis from leaf explants of Tolumnia Louise Elmore 'Elsa'.

PubMed

Shen, Hui-Ju; Chen, Jen-Tsung; Chung, Hsiao-Hang; Chang, Wei-Chin

2018-01-22

Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l -1 in darkness. TDZ at 3 mg l -1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95-100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore 'Elsa', was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells.

PubMed

Preite, Silvia; Baumjohann, Dirk; Foglierini, Mathilde; Basso, Camilla; Ronchi, Francesca; Fernandez Rodriguez, Blanca M; Corti, Davide; Lanzavecchia, Antonio; Sallusto, Federica

2015-11-01

We previously reported that Cd3e-deficient mice adoptively transferred with CD4(+) T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B-cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation-induced cytidine deaminase. Furthermore, GC B cells from Cd3e(-/-) mice accumulate fewer somatic mutations as compared with GC B cells from wild-type mice, and exhibit impaired affinity maturation and reduced differentiation into long-lived plasma cells. Reconstitution of Cd3e(-/-) mice with regulatory T (Treg) cells restored Tfh-cell numbers, GC B-cell numbers and B-cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh-cell numbers and GC B-cell numbers and dynamics were also restored by pre-reconstitution of Cd3e(-/-) mice with Cxcr5(-/-) Treg cells or non-regulatory, memory CD4(+) T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh-cell response for an efficient and long-lasting serological response. © 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two classes of gap junction channels mediate soma-germline interactions essential for germline proliferation and gametogenesis in Caenorhabditis elegans.

PubMed

Starich, Todd A; Hall, David H; Greenstein, David

2014-11-01

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma-germline interactions in C. elegans. Copyright © 2014 by the Genetics Society of America.

Analyses of spontaneous pink mutant events in the stamen hairs of Tradescantia clone BNL 4430 cultivated in a nutrient solution circulating growth chamber.

PubMed

Ichikawa, S; Wushur, S

2000-12-20

In order to obtain more fundamental data on Tradescantia clone BNL 4430, one of the most suitable testers for environmental mutagens, the occurrences of spontaneous somatic pink mutations in the stamen hairs were scored for 52 weeks from 12 December 1998 to 10 December 1999, cultivating the young inflorescence-bearing shoots with roots in a nutrient solution circulating (NSC) growth chamber. The environmental conditions in the chamber were 22.0+/-0.5 degrees C during the 16h day with the light intensity of 7.5klx from white fluorescent tubes, and 20.0+/-0.5 degrees C at night. During the scoring period, 697,443 stamen hairs with an average cell number of 25.36 were observed and 2642 pink mutant events (PMEs) were detected. The overall spontaneous mutation frequency was 1.56+/-0.03 PMEs per 10(4) hair-cell divisions, and the frequency was significantly lower in May, July and August and significantly higher in November and December. By analyzing the sectoring patterns of 1856 PMEs (70.25% of PMEs detected), the most of 172 cases of multiple (two to five) pink sectors observed in the same hairs (scored as 232 PMEs for calculating mutation frequency) were found to be the results of events involving somatic recombinations occurred in single cells or cell lineages, rather than those of two or more independent somatic mutations occurred in different cells. This finding clearly shows the significance of somatic recombinations in producing such multiple sectors (382 sectors in total) which occupied 19.0% of the 2006 pink sectors in total analyzed. Somatic recombinations were considered to be playing a significant role also in producing single PMEs in the stamen hairs.

The effect of season on somatic cell count and the incidence of clinical mastitis.

PubMed

Olde Riekerink, R G M; Barkema, H W; Stryhn, H

2007-04-01

Bulk milk somatic cell count (BMSCC), individual cow somatic cell count (ICSCC), and incidence rate of clinical mastitis (IRCM) are all udder health parameters. So far, no studies have been reported on the effect of season on BMSCC, IRCM, and ICSCC in the same herds and period over multiple years. The objectives of this study were to determine the seasonal pattern over a 4-yr period of 1) BMSCC, 2) elevated ICSCC, 3) IRCM, and 4) pathogen-specific IRCM. Bulk milk somatic cell count, ICSCC, and pathogen-specific clinical mastitis data were recorded in 300 Dutch dairy farms. For the analyses of BMSCC, ICSCC, and IRCM, a mixed, a transitional, and a discrete time survival analysis model were used, respectively. Sine and cosine were included in the models to investigate seasonal patterns in the data. For all parameters, a seasonal effect was present. Bulk milk somatic cell count peaked in August to September in all 4 years. The probability of cows getting or maintaining a high ICSCC was highest in August and May, respectively. Older and late-lactation cows were more likely to develop or maintain a high ICSCC. Incidence rate of clinical mastitis was highest in December to January, except for Streptococcus uberis IRCM, which was highest in August. Totally confined herds had a higher Escherichia coli IRCM in summer than in winter. Compared with the major mastitis pathogens, the seasonal differences in IRCM were smaller for the minor pathogens. Distinguishing between Strep. uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, and other streptococci is essential when identifying Streptococcus spp. because each of them has a unique epidemiology. Streptococcus uberis IRCM seemed to be associated with being on pasture, whereas E. coli IRCM was more housing-related.

Interactions of Corticotropin-Releasing Factor, Urocortin and Citalopram in a Primate Model of Stress-Induced Amenorrhea

PubMed Central

Weissheimer, Karin V.; Herod, Skyla M.; Cameron, Judy L.; Bethea, Cynthia L.

2010-01-01

Background/Aims We established a cynomolgus macaque model of stress-induced amenorrhea in which the application of combined metabolic and psychosocial stress suppressed ovulation in stress-sensitive (SS) individuals, but not in highly stress-resilient (HSR) individuals. We previously reported that SS monkeys have deficits in global serotonin release and serotonin-related gene expression in the raphe nucleus, and that administration of the selective serotonin reuptake inhibitor S-citalopram increased estrogen and progesterone production in SS monkeys. In this study, we questioned whether there was a difference in corticotropin-releasing factor (CRF) or urocortin (UCN) stress-related peptide systems in the midbrain raphe region when HSR and SS monkeys treated with placebo or S-citalopram are compared. Methods Monkeys characterized as HSR or SS were administered placebo or S-citalopram for 15 weeks. CRF fibers in the dorsal raphe were detected with an antibody against human CRF. UCN1 fibers were immunostained in an area rostral to the dorsal raphe. The fibers were quantified by stereology and analyzed by two-way ANOVA. UCN1 cell bodies were counted in the supraoculomotor area near the Edinger-Westphal nucleus. Results S-citalopram significantly decreased the CRF fiber density in SS animals, but not in HSR animals. SS monkeys had a significantly lower UCN1 fiber density compared to HSR monkeys, but S-citalopram treatment did not alter the UCN1 fiber density. SS animals treated with S-citalopram tended to have a higher number of UCN1-positive cell bodies than the other groups. Conclusion S-citalopram decreased CRF fiber density and appears to increase the production of UCN1 in SS individuals, indicating the likelihood that serotonin is involved in regulating CRF and UCN1 in individuals who are sensitive to the effects of serotonin. PMID:20714124

Phenobarbital increases monkey in vivo nicotine disposition and induces liver and brain CYP2B6 protein

PubMed Central

Lee, Anna M; Miksys, Sharon; Tyndale, Rachel F

2006-01-01

CYP2B6 is a drug-metabolizing enzyme expressed in the liver and brain that can metabolize bupropion (Zyban®, a smoking cessation drug), activate tobacco-smoke nitrosamines, and inactivate nicotine. Hepatic CYP2B6 is induced by phenobarbital and induction may affect in vivo nicotine disposition, while brain CYP2B6 induction may affect local levels of centrally acting substrates. We investigated the effect of chronic phenobarbital treatment on induction of in vivo nicotine disposition and CYP2B6 expression in the liver and brain of African Green (Vervet) monkeys. Monkeys were split into two groups (n=6 each) and given oral saccharin daily for 22 days; one group was supplemented with 20 mg kg−1 phenobarbital. Monkeys were given a 0.1 mg kg−1 nicotine dose subcutaneously before and after treatment. Phenobarbital treatment resulted in a significant, 56%, decrease (P=0.04) in the maximum nicotine plasma concentration and a 46% decrease (P=0.003) in the area under the concentration–time curve. Phenobarbital also increased hepatic CYP2B6 protein expression. In monkey brain, significant induction (P<0.05) of CYP2B6 protein levels was observed in all regions tested (caudate, putamen, hippocampus, cerebellum, brain stem and frontal cortex) ranging from 2-fold to 150-fold. CYP2B6 expression was induced in specific cells, such as frontal cortical pyramidal cells and thalamic neurons. In conclusion, chronic phenobarbital treatment in monkeys resulted in increased in vivo nicotine disposition, and induced hepatic and brain CYP2B6 protein levels and cellular expression. This induction may alter the metabolism of CYP2B6 substrates including peripherally acting drugs such as cyclophosphamide and centrally acting drugs such as bupropion, ecstasy and phencyclidine. PMID:16751792

Knockdown of Progesterone Receptor (PGR) in Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production.

PubMed

Bishop, Cecily V; Hennebold, Jon D; Kahl, Christoph A; Stouffer, Richard L

2016-05-01

Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGR mRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4-6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGR mRNA and P levels. Addition of hCG increased (P < 0.05) PGR mRNA and medium P levels in controls. However, a time-dependent decline (P < 0.05) in PGR mRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P < 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization. © 2016 by the Society for the Study of Reproduction, Inc.

[Raman spectra of monkey cerebral cortex tissue].

PubMed

Zhu, Ji-chun; Guo, Jian-yu; Cai, Wei-ying; Wang, Zu-geng; Sun, Zhen-rong

2010-01-01

Monkey cerebral cortex, an important part in the brain to control action and thought activities, is mainly composed of grey matter and nerve cell. In the present paper, the in situ Raman spectra of the cerebral cortex of the birth, teenage and aged monkeys were achieved for the first time. The results show that the Raman spectra for the different age monkey cerebral cortex exhibit most obvious changes in the regions of 1000-1400 and 2800-3000 cm(-1). With monkey growing up, the relative intensities of the Raman bands at 1313 and 2885 cm(-1) mainly assigned to CH2 chain vibrational mode of lipid become stronger and stronger whereas the relative intensities of the Raman bands at 1338 and 2932 cm(-1) mainly assigned to CH3 chain vibrational mode of protein become weaker and weaker. In addition, the two new Raman bands at 1296 and 2850 cm(-1) are only observed in the aged monkey cerebral cortex, therefore, the two bands can be considered as a character or "marker" to differentiate the caducity degree with monkey growth In order to further explore the changes, the relative intensity ratios of the Raman band at 1313 cm(-1) to that at 1338 cm(-1) and the Raman band at 2885 cm(-1) to that at 2 932 cm(-1), I1313/I1338 and I2885/I2932, which are the lipid-to-protein ratios, are introduced to denote the degree of the lipid content. The results show that the relative intensity ratios increase significantly with monkey growth, namely, the lipid content in the cerebral cortex increases greatly with monkey growth. So, the authors can deduce that the overmuch lipid is an important cause to induce the caducity. Therefore, the results will be a powerful assistance and valuable parameter to study the order of life growth and diagnose diseases.

In-vivo genotoxicity of the alkaloid drug pilocarpine nitrate in bone marrow cells and male germ cells of mice.

PubMed

Hegde, M J; Sujatha, T V

1995-10-01

Pilocarpine nitrate, an alkaloid drug of plant origin induces spindle disfunction in bone marrow cells of mice. Further studies were carried out to investigate its mutagenic effects in somatic and germ cells of mice by assessing chromosome aberrations at mitotic metaphase and as micronuclei in bone marrow cells and sperm-shape abnormality in cauda epididymides. The dose and time yield effects of the drug were investigated. The statistically significant results that were obtained for both chromosomal aberrations and micronucleus test but not for the sperm-shape abnormality test, indicated the genotoxicity of this compound in somatic cells but not in germ cells.

Sequencing thousands of single-cell genomes with combinatorial indexing.

PubMed

Vitak, Sarah A; Torkenczy, Kristof A; Rosenkrantz, Jimi L; Fields, Andrew J; Christiansen, Lena; Wong, Melissa H; Carbone, Lucia; Steemers, Frank J; Adey, Andrew

2017-03-01

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.

Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

PubMed

Rao, Shengbin; Fujimura, Tatsuya; Matsunari, Hitomi; Sakuma, Tetsushi; Nakano, Kazuaki; Watanabe, Masahito; Asano, Yoshinori; Kitagawa, Eri; Yamamoto, Takashi; Nagashima, Hiroshi

2016-01-01

Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future. © 2015 Wiley Periodicals, Inc.

Structural and functional localization of airway effects from episodic exposure of infant monkeys to allergen and/or ozone

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

Joad, Jesse P.; Kott, Kayleen S.; Bric, John M.

2006-08-01

Both allergen and ozone exposure increase asthma symptoms and airway responsiveness in children. Little is known about how these inhalants may differentially modify airway responsiveness in large proximal as compared to small distal airways. We evaluated whether bronchi and respiratory bronchioles from infant monkeys exposed episodically to allergen and/or ozone differentially develop intrinsic hyperresponsiveness to methacholine and whether eosinophils and/or pulmonary neuroendocrine cells play a role. Infant monkeys were exposed episodically for 5 months to: (1) filtered air, (2) aerosolized house dust mite allergen, (3) ozone 0.5 ppm, or (4) house dust mite allergen + ozone. Studying the function/structure relationshipmore » of the same lung slices, we evaluated methacholine airway responsiveness and histology of bronchi and respiratory bronchioles. In bronchi, intrinsic responsiveness was increased by allergen exposure, an effect reduced by bombesin antagonist. In respiratory bronchioles, intrinsic airway responsiveness was increased by allergen + ozone exposure. Eosinophils were increased by allergen and allergen + ozone exposure in bronchi and by allergen exposure in respiratory bronchioles. In both airways, exposure to allergen + ozone resulted in fewer tissue eosinophils than did allergen exposure alone. In bronchi, but not in respiratory bronchioles, the number of eosinophils and neuroendocrine cells correlated with airway responsiveness. We conclude that episodically exposing infant monkeys to house dust mite allergen with or without ozone increased intrinsic airway responsiveness to methacholine in bronchi differently than in respiratory bronchioles. In bronchi, eosinophils and neuroendocrine cells may play a role in the development of airway hyperresponsiveness.« less

Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens

PubMed Central

Valansi, Clari; Moi, David; Leikina, Evgenia; Matveev, Elena; Chernomordik, Leonid V.

2017-01-01

Cell–cell fusion is inherent to sexual reproduction. Loss of HAPLESS 2/GENERATIVE CELL SPECIFIC 1 (HAP2/GCS1) proteins results in gamete fusion failure in diverse organisms, but their exact role is unclear. In this study, we show that Arabidopsis thaliana HAP2/GCS1 is sufficient to promote mammalian cell–cell fusion. Hemifusion and complete fusion depend on HAP2/GCS1 presence in both fusing cells. Furthermore, expression of HAP2 on the surface of pseudotyped vesicular stomatitis virus results in homotypic virus–cell fusion. We demonstrate that the Caenorhabditis elegans Epithelial Fusion Failure 1 (EFF-1) somatic cell fusogen can replace HAP2/GCS1 in one of the fusing membranes, indicating that HAP2/GCS1 and EFF-1 share a similar fusion mechanism. Structural modeling of the HAP2/GCS1 protein family predicts that they are homologous to EFF-1 and viral class II fusion proteins (e.g., Zika virus). We name this superfamily Fusexins: fusion proteins essential for sexual reproduction and exoplasmic merger of plasma membranes. We suggest a common origin and evolution of sexual reproduction, enveloped virus entry into cells, and somatic cell fusion. PMID:28137780

Composition, proteolysis indices and coagulating properties of ewe milk as affected by bulk tank somatic cell count.

PubMed

Martí-De Olives, Ana; Navarro-Ríos, María Jesús; Rubert-Alemán, Joaquín; Fernández, Nemesio; Molina, Maria Pilar

2015-08-01

The aim of this study was to assess the effect of ovine bulk tank somatic cell count (BTSCC) on composition, proteose-peptone (p-p) content and casein fractions as indicating parameters for proteolysis and coagulating properties of milk. A total of 97 samples of bulk tank milk from Manchega breed ewe flocks were grouped according to somatic cell count (SCC) into four classes: fewer than 500,000 cells/ml, from 500,000 to 10,00000 cells/ml, from 10,00000 to 15,00000 and more than 15,00000 cells/ml. The casein : protein ratio and lactose content decreased with BTSCC. Proteolysis increased with BTSCC, causing a drop in β-casein and an increase in the γ-caseins from a concentration of 500,000 cells/ml. Regarding coagulation behaviour, the rennet clotting time (RCT) and firming time (k20) rose from 10,00000-15,00000 cells/ml of milk. The results showed that the impairment of milk quality and milk ability to make cheese as affected by intramammary infection (IMI) can be inferred from the bulk tank milk of flocks with poor udder health.

Metabolome Profiling of Partial and Fully Reprogrammed Induced Pluripotent Stem Cells.

PubMed

Park, Soon-Jung; Lee, Sang A; Prasain, Nutan; Bae, Daekyeong; Kang, Hyunsu; Ha, Taewon; Kim, Jong Soo; Hong, Ki-Sung; Mantel, Charlie; Moon, Sung-Hwan; Broxmeyer, Hal E; Lee, Man Ryul

2017-05-15

Acquisition of proper metabolomic fate is required to convert somatic cells toward fully reprogrammed pluripotent stem cells. The majority of induced pluripotent stem cells (iPSCs) are partially reprogrammed and have a transcriptome different from that of the pluripotent stem cells. The metabolomic profile and mitochondrial metabolic functions required to achieve full reprogramming of somatic cells to iPSC status have not yet been elucidated. Clarification of the metabolites underlying reprogramming mechanisms should enable further optimization to enhance the efficiency of obtaining fully reprogrammed iPSCs. In this study, we characterized the metabolites of human fully reprogrammed iPSCs, partially reprogrammed iPSCs, and embryonic stem cells (ESCs). Using capillary electrophoresis time-of-flight mass spectrometry-based metabolomics, we found that 89% of analyzed metabolites were similarly expressed in fully reprogrammed iPSCs and human ESCs (hESCs), whereas partially reprogrammed iPSCs shared only 74% similarly expressed metabolites with hESCs. Metabolomic profiling analysis suggested that converting mitochondrial respiration to glycolytic flux is critical for reprogramming of somatic cells into fully reprogrammed iPSCs. This characterization of metabolic reprogramming in iPSCs may enable the development of new reprogramming parameters for enhancing the generation of fully reprogrammed human iPSCs.

A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila.

PubMed

Saito, Kuniaki; Inagaki, Sachi; Mituyama, Toutai; Kawamura, Yoshinori; Ono, Yukiteru; Sakota, Eri; Kotani, Hazuki; Asai, Kiyoshi; Siomi, Haruhiko; Siomi, Mikiko C

2009-10-29

PIWI-interacting RNAs (piRNAs) silence retrotransposons in Drosophila germ lines by associating with the PIWI proteins Argonaute 3 (AGO3), Aubergine (Aub) and Piwi. piRNAs in Drosophila are produced from intergenic repetitive genes and piRNA clusters by two systems: the primary processing pathway and the amplification loop. The amplification loop occurs in a Dicer-independent, PIWI-Slicer-dependent manner. However, primary piRNA processing remains elusive. Here we analysed piRNA processing in a Drosophila ovarian somatic cell line where Piwi, but not Aub or AGO3, is expressed; thus, only the primary piRNAs exist. In addition to flamenco, a Piwi-specific piRNA cluster, traffic jam (tj), a large Maf gene, was determined as a new piRNA cluster. piRNAs arising from tj correspond to the untranslated regions of tj messenger RNA and are sense-oriented. piRNA loading on to Piwi may occur in the cytoplasm. zucchini, a gene encoding a putative cytoplasmic nuclease, is required for tj-derived piRNA production. In tj and piwi mutant ovaries, somatic cells fail to intermingle with germ cells and Fasciclin III is overexpressed. Loss of tj abolishes Piwi expression in gonadal somatic cells. Thus, in gonadal somatic cells, tj gives rise simultaneously to two different molecules: the TJ protein, which activates Piwi expression, and piRNAs, which define the Piwi targets for silencing.

Induction of pluripotency by defined factors

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

Okita, Keisuke, E-mail: okita@cira.kyoto-u.ac.jp; Yamanaka, Shinya; Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507

2010-10-01

Somatic cells can be reprogrammed into pluripotent stem cells by introducing a combination of several transcription factors. The induced pluripotent stem (iPS) cells from a patient's somatic cells could be useful source of cells for drug discovery and cell transplantation therapies. However, most human iPS cells are made by viral vectors, such as retrovirus and lentivirus, which integrate the reprogramming factors into host genomes and may increase the risk of tumor formation. Studies of the mechanisms underlying the reprogramming and establishment of non-integration methods contribute evidence to resolve the safety concerns associated with iPS cells. On the other hand, patient-specificmore » iPS cells have already been established and used for recapitulating disease pathology.« less

Genotoxicity testing of different types of beverages in the Drosophila wing Somatic Mutation And Recombination Test.

PubMed

Graf, U; Moraga, A A; Castro, R; Díaz Carrillo, E

1994-05-01

Five wines and one brandy of Spanish origin as well as three herbal teas and ordinary black tea were tested for genotoxicity in the wing Somatic Mutation And Recombination Test (SMART) which makes use of the two recessive wing cell markers multiple wing hairs (mwh) and flare (flr3) on the left arm of chromosome 3 of Drosophila melanogaster. 3-day-old larvae trans-heterozygous for these two markers were fed the beverages at different concentrations and for different feeding periods using Drosophila instant medium. Somatic mutations or mitotic recombinations induced in the cells of the wing imaginal discs give rise to mutant single or twin spots on the wing blade of the emerging adult flies showing either the mwh phenotype or/and the flr phenotype. One of the red wines showed a clear genotoxic activity that was not due to its ethanol content. Two herbal teas (Urtica dioica, Achillea millefolium) and black tea (Camellia sinensis) proved to be weakly genotoxic as well. Furthermore, it was shown that quercetin and rutin, two flavonols present in beverages of plant origin, also exhibited weak genotoxic activity in the somatic cells of Drosophila. These results demonstrate that Drosophila in vivo somatic assays can detect the genotoxicity of complex mixtures such as beverages. In particular, it is possible to administer these test materials in the same form as that in which they are normally consumed.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

PubMed

Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

2010-02-18

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Coordinated tissue-specific regulation of adjacent alternative 3′ splice sites in C. elegans

PubMed Central

Ragle, James Matthew; Katzman, Sol; Akers, Taylor F.; Barberan-Soler, Sergio; Zahler, Alan M.

2015-01-01

Adjacent alternative 3′ splice sites, those separated by ≤18 nucleotides, provide a unique problem in the study of alternative splicing regulation; there is overlap of the cis-elements that define the adjacent sites. Identification of the intron's 3′ end depends upon sequence elements that define the branchpoint, polypyrimidine tract, and terminal AG dinucleotide. Starting with RNA-seq data from germline-enriched and somatic cell-enriched Caenorhabditis elegans samples, we identify hundreds of introns with adjacent alternative 3′ splice sites. We identify 203 events that undergo tissue-specific alternative splicing. For these, the regulation is monodirectional, with somatic cells preferring to splice at the distal 3′ splice site (furthest from the 5′ end of the intron) and germline cells showing a distinct shift toward usage of the adjacent proximal 3′ splice site (closer to the 5′ end of the intron). Splicing patterns in somatic cells follow C. elegans consensus rules of 3′ splice site definition; a short stretch of pyrimidines preceding an AG dinucleotide. Splicing in germline cells occurs at proximal 3′ splice sites that lack a preceding polypyrimidine tract, and in three instances the germline-specific site lacks the AG dinucleotide. We provide evidence that use of germline-specific proximal 3′ splice sites is conserved across Caenorhabditis species. We propose that there are differences between germline and somatic cells in the way that the basal splicing machinery functions to determine the intron terminus. PMID:25922281

Defined tuberculosis vaccine, Mtb72F/AS02A, evidence of protection in cynomolgus monkeys

PubMed Central

Reed, Steven G.; Coler, Rhea N.; Dalemans, Wilfried; Tan, Esterlina V.; DeLa Cruz, Eduardo C.; Basaraba, Randall J.; Orme, Ian M.; Skeiky, Yasir A. W.; Alderson, Mark R.; Cowgill, Karen D.; Prieels, Jean-Paul; Abalos, Rodolfo M.; Dubois, Marie-Claude; Cohen, Joe; Mettens, Pascal; Lobet, Yves

2009-01-01

The development of a vaccine for tuberculosis requires a combination of antigens and adjuvants capable of inducing appropriate and long-lasting T cell immunity. We evaluated Mtb72F formulated in AS02A in the cynomolgus monkey model. The vaccine was immunogenic and caused no adverse reactions. When monkeys were immunized with bacillus Calmette–Guérin (BCG) and then boosted with Mtb72F in AS02A, protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology, and survival. We observed long-term survival and evidence of reversal of disease progression in monkeys immunized with the prime-boost regimen. Antigen-specific responses from protected monkeys receiving BCG and Mtb72F/AS02A had a distinctive cytokine profile characterized by an increased ratio between 3 Th1 cytokines, IFN-γ, TNF, and IL-2 and an innate cytokine, IL-6. To our knowledge, this is an initial report of a vaccine capable of inducing long-term protection against tuberculosis in a nonhuman primate model, as determined by protection against severe disease and death, and by other clinical and histopathological parameters. PMID:19188599

Species and sex differences in susceptibility to olfactory lesions among the mouse, rat and monkey following an intravenous injection of vincristine sulphate.

PubMed

Kai, Kiyonori; Sahto, Hiroshi; Yoshida, Mitsuyoshi; Suzuki, Takami; Shikanai, Yukari; Kajimura, Tetsuyo; Furuhama, Kazuhisa

2006-01-01

Species and sex differences in susceptibility to vincristine sulphate (VCR)-induced olfactory epithelial lesions were investigated among the BALB/c mice, Crj: CD(SD) IGS rats and common marmoset monkeys following a single intravenous administration on day 1. As dosage levels, the 0.17-fold LD10, 0.6-fold LD10 and LD10 were used for mice and rats, and a maximum tolerated dose (MTD) was chosen only for monkeys. The order of strength of VCR action on peripheral neuropathic signs, body weight gain, and hematological parameters was mice > rats > monkeys, without clear sex differences. Histopathologically, on day 2, single cell death in the olfactory epithelium and vomeronasal organ was observed only in male mice at LD10, and in female mice at 0.6-fold LD10 or more. On day 5, the olfactory epithelium in these mice showed regenerative proliferation suggesting a sign of recovery. On day 10, axonopathy and demyelination in the sciatic and trigeminal nerves were noted in mice of both sexes at 0.6-fold LD10 or more. In rats and monkeys of either sex, however, no morphological changes were observed at any dose level. In conclusion, mice, particularly females, were shown to be more susceptible to VCR-induced apoptosis in the olfactory epithelium than rats and monkeys.

Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

PubMed

Secher, Jan O; Liu, Ying; Petkov, Stoyan; Luo, Yonglun; Li, Dong; Hall, Vanessa J; Schmidt, Mette; Callesen, Henrik; Bentzon, Jacob F; Sørensen, Charlotte B; Freude, Kristine K; Hyttel, Poul

2017-03-01

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines. The porcine stem cells used were: Induced pluripotent stem cells (iPSCs) created by lentiviral doxycycline-dependent reprogramming and cultered with a GSK3β- and MEK-inhibitor (2i) and leukemia inhibitor factor (LIF) (2i LIF DOX-iPSCs), iPSCs created by a plasmid-based reprogramming and cultured with 2i and fibroblast growth factor (FGF) (2i FGF Pl-iPSCs) and embryonic germ cells (EGCs), which have earlier been characterized as being multipotent. The SCNT efficiencies of these stem cell lines were compared with that of the two fibroblast cell lines from which the iPSC lines were derived. The blastocyst rates for the 2i LIF DOX-iPSCs were 14.7%, for the 2i FGF Pl-iPSC 10.1%, and for the EGCs 34.5% compared with the fibroblast lines yielding 36.7% and 25.2%. The fibroblast- and EGC-derived embryos were used for embryo transfer and produced live offspring at similar low rates of efficiency (3.2 and 4.0%, respectively) and with several instances of malformations. In conclusion, potentially pluripotent porcine stem cells resulted in lower rates of embryonic development upon SCNT than multipotent stem cells and differentiated somatic cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Cow's milk quality and energy value during different lactation stages.

PubMed

Salamończyk, Ewa

2013-01-01

The quality of dairy products, raw milk in particular, depends on many factors. Low bacterial and somatic cell counts are basic determinants of the appropriate raw milk quality. The objective of the work was to assess the effect of selected factors, that is, the age of cows and their daily milk performance, on cytological quality (somatic cell count) and energy value of milk produced at individual stages of lactation. Somatic cell count and energy value of cow's milk were assessed. A total of 229 792 milk samples were examined. Data for analysis were taken from milk records of 350 dairy herds. It was demonstrated that, of all the lactations studied, the fi rst lactation (from calving to the 100th day of lactation) was characterised by the highest daily milk performance (25.1 kg) and the lowest somatic cell count (356 thous./1 ml), fat, protein and dry matter contents (4.06, 2.96 and 12.41%, respectively) and milk calorific value (732 kcal/kg). The highest energy value was recorded in cow's milk produced towards the end of lactation, that is from day 300 till the end of lactation (842 kcal/kg). High milk calorific value in late lactation and high fat and protein contents were accompanied by low raw milk quality.

Localization of alkali-labile sites in donkey (Equus asinus) and stallion (Equus caballus) spermatozoa.

PubMed

Cortés-Gutiérrez, Elva I; Dávila-Rodríguez, Martha I; López-Fernández, Carmen; Fernández, José Luis; Crespo, Francisco; Gosálvez, Jaime

2014-01-15

The presence of constitutive alkali-labile sites (ALS) has been investigated using a protocol of DNA breakage detection-fluorescence in situ hybridization and comet assay in spermatozoa of donkey (Equus asinus) and stallion (Equus caballus). These results were compared with those obtained using a similar experimental approach using somatic cells. The relative abundance of ALS was of the order of four times more in spermatozoa than in somatic cells. Alkali-labile sites showed a tendency to cluster localized at the equatorial-distal regions of the sperm. The amount of hybridized signal in the ALS in the sperm of donkey (Equus asinus) was 1.3 times greater than in stallion (Equus caballus), and the length of the comet tail obtained in donkey sperm was 1.6 times longer than that observed in stallion (P < 0.05); however, these differences were not appreciated in somatic cells. In conclusion, ALS localization in sperm is not a randomized event and a different pattern of ALS distribution occurs for each species. These results suggest that ALS represents a species-specific issue related to chromatin organization in sperm and somatic cells in mammalian species, and they might diverge even with very short phylogenetic distances. Copyright © 2014 Elsevier Inc. All rights reserved.

Primary cutaneous B-cell lymphoma is associated with somatically hypermutated immunoglobulin variable genes and frequent use of VH1-69 and VH4-59 segments.

PubMed

Perez, M; Pacchiarotti, A; Frontani, M; Pescarmona, E; Caprini, E; Lombardo, G A; Russo, G; Faraggiana, T

2010-03-01

Accurate assessment of the somatic mutational status of clonal immunoglobulin variable region (IgV) genes is relevant in elucidating tumour cell origin in B-cell lymphoma; virgin B cells bear unmutated IgV genes, while germinal centre and postfollicular B cells carry mutated IgV genes. Furthermore, biases in the IgV repertoire and distribution pattern of somatic mutations indicate a possible antigen role in the pathogenesis of B-cell malignancies. This work investigates the cellular origin and antigenic selection in primary cutaneous B-cell lymphoma (PCBCL). We analysed the nucleotide sequence of clonal IgV heavy-chain gene (IgVH) rearrangements in 51 cases of PCBCL (25 follicle centre, 19 marginal zone and seven diffuse large B-cell lymphoma, leg-type) and compared IgVH sequences with their closest germline segment in the GenBank database. Molecular data were then correlated with histopathological features. We showed that all but one of the 51 IgVH sequences analysed exhibited extensive somatic hypermutations. The detected mutation rate ranged from 1.6% to 21%, with a median rate of 9.8% and was independent of PCBCL histotype. Calculation of antigen-selection pressure showed that 39% of the mutated IgVH genes displayed a number of replacement mutations and silent mutations in a pattern consistent with antigenic selection. Furthermore, two segments, VH1-69 (12%) and VH4-59 (14%), were preferentially used in our case series. Data indicate that neoplastic B cells of PBCBL have experienced germinal centre reaction and also suggest that the involvement of IgVH genes is not entirely random in PCBCL and that common antigen epitopes could be pathologically relevant in cutaneous lymphomagenesis.

Mutant Alpha-Synuclein Causes Age-Dependent Neuropathology in Monkey Brain

PubMed Central

Yang, Weili; Wang, Guohao; Wang, Chuan-En; Guo, Xiangyu; Yin, Peng; Gao, Jinquan; Tu, Zhuchi; Wang, Zhengbo; Wu, Jing; Hu, Xintian; Li, Shihua

2015-01-01

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2–3, 7–8, and >15 years of age to examine the expression of Parkin, PINK1, and α-synuclein, which are known to cause PD via loss- or gain-of-function mechanisms. We found that α-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of α-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant α-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD. PMID:26019347

9 CFR 113.55 - Detection of extraneous agents in Master Seed Virus.

Code of Federal Regulations, 2010 CFR

2010-01-01

... MSV that is found unsatisfactory by any prescribed test. (a) At least a 1.0 ml aliquot per cell... monkey kidney) cell line; (2) Embryonic cells, neonatal cells, or a cell line of the species for which...) and (a)(2) of this section. Cell lines used shall have been found satisfactory when tested as...

Chemical compound-based direct reprogramming for future clinical applications

PubMed Central

Takeda, Yukimasa; Harada, Yoshinori; Yoshikawa, Toshikazu; Dai, Ping

2018-01-01

Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy. PMID:29739872

In Vitro Interleukin-1 and 2 Production and Interleukin 2 Receptor Expression in the Rhesus Monkey

NASA Technical Reports Server (NTRS)

Schmitt, Didier A.; Sonnenfeld, Gerald; Husson, David; Tkaczuk, Jean; Andre, Eric; Schaffar, Laurance

1996-01-01

Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.

DNMT1 Maintains Progenitor Function in Self-Renewing Somatic Tissue

PubMed Central

Sen, George L.; Reuter, Jason A.; Webster, Daniel E.; Zhu, Lilly; Khavari, Paul A.

2010-01-01

Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation1,2. DNA methylation3,4,5 provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1)6,7 maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance,8 a clear role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unknown. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis revealed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, we show that UHRF1,9,10 a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A11,12 and B13, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue. PMID:20081831

Cloned ferrets produced by somatic cell nuclear transfer.

PubMed

Li, Ziyi; Sun, Xingshen; Chen, Juan; Liu, Xiaoming; Wisely, Samantha M; Zhou, Qi; Renard, Jean-Paul; Leno, Gregory H; Engelhardt, John F

2006-05-15

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.

Single-cell transcriptome of early embryos and cultured embryonic stem cells of cynomolgus monkeys

PubMed Central

Nakamura, Tomonori; Yabuta, Yukihiro; Okamoto, Ikuhiro; Sasaki, Kotaro; Iwatani, Chizuru; Tsuchiya, Hideaki; Saitou, Mitinori

2017-01-01

In mammals, the development of pluripotency and specification of primordial germ cells (PGCs) have been studied predominantly using mice as a model organism. However, divergences among mammalian species for such processes have begun to be recognized. Between humans and mice, pre-implantation development appears relatively similar, but the manner and morphology of post-implantation development are significantly different. Nevertheless, the embryogenesis just after implantation in primates, including the specification of PGCs, has been unexplored due to the difficulties in analyzing the embryos at relevant developmental stages. Here, we present a comprehensive single-cell transcriptome dataset of pre- and early post-implantation embryo cells, PGCs and embryonic stem cells (ESCs) of cynomolgus monkeys as a model of higher primates. The identities of each transcriptome were also validated rigorously by other way such as immunofluorescent analysis. The information reported here will serve as a foundation for our understanding of a wide range of processes in the developmental biology of primates, including humans. PMID:28649393

Hemopoiesis in the pig-tailed monkey Macaca nemestrina during chronic altitude exposure.

NASA Technical Reports Server (NTRS)

Buderer, M. C.; Pace, N.

1972-01-01

Study of monkeys for 180 days at 3800 m altitude to examine their hemopoietic response. Plasma volume was found to be reduced while red cell volume increased steadily for four to five months. Reduction in mean corpuscular hemoglobin content was observed from day 30 to day 120 at altitude. Total plasma protein concentration was unchanged at altitude, but marked reduction in the albumin/globulin ratio occurred. Total circulating plasma protein and albumin were reduced in amount, whereas nonalbumin protein was unchanged. These results imply loss of albumin coupled with a corresponding loss of water from the blood and maintenance of normal plasma osmotic pressure. The body/venous hematocrit ratio was found to be reduced at altitude, possibly as a consequence of the expanded capillary volume of the body. The hemopoietic responses of the pig-tailed monkey at altitude require at least several months for completion, and closely resemble those seen in man; thus, the monkey can serve well for long-term studies of high-altitude acclimatization.

Telemetric recordings of single neuron activity and visual scenes in monkeys walking in an open field.

PubMed

Lei, Yanlin; Sun, Ninglei; Wilson, Fraser A W; Wang, Xiusong; Chen, Nanhui; Yang, Jianzhen; Peng, Yanping; Wang, Jianhong; Tian, Shaohua; Wang, Maohua; Miao, Yingda; Zhu, Weina; Qi, Hua; Ma, Yuanye

2004-05-30

This paper describes a portable recording system and methods for obtaining chronic recordings of single units and tracking rhesus monkey behavior in an open field. The integrated system consists of four major components: (1) microelectrode assembly; (2) head-stage; (3) recording station; and (4) data storage station, the first three of which are carried by the monkey and weigh 800 g. Our system provides synchronized video and electrophysiological signals, which are transmitted by a wireless system to a distance of 50 m. Its major advantages are that neuronal recordings are made in freely moving monkeys, and well-separated action potentials with amplitude five times higher than the background noise are usually recorded and readily kept for many hours. Using this system, we were able to study "place cells" in non-human primate brains. The described methods provide a new way to examine correlations between single neuron activity and primate behaviors, and can also be used to study the cellular basis of social behaviors in non-human primates.

Compartmentalized beta subunit distribution determines characteristics and ethanol sensitivity of somatic, dendritic, and terminal large-conductance calcium-activated potassium channels in the rat central nervous system.

PubMed

Wynne, P M; Puig, S I; Martin, G E; Treistman, S N

2009-06-01

Neurons are highly differentiated and polarized cells, whose various functions depend upon the compartmentalization of ion channels. The rat hypothalamic-neurohypophysial system (HNS), in which cell bodies and dendrites reside in the hypothalamus, physically separated from their nerve terminals in the neurohypophysis, provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins. Using electrophysiological and immunohistochemical techniques, we characterized the large-conductance calcium-activated potassium (BK) channel in each of the three primary compartments (soma, dendrite, and terminal) of HNS neurons. We found that dendritic BK channels, in common with somatic channels but in contrast to nerve terminal channels, are insensitive to iberiotoxin. Furthermore, analysis of dendritic BK channel gating kinetics indicates that they, like somatic channels, have fast activation kinetics, in contrast to the slow gating of terminal channels. Dendritic and somatic channels are also more sensitive to calcium and have a greater conductance than terminal channels. Finally, although terminal BK channels are highly potentiated by ethanol, somatic and dendritic channels are insensitive to the drug. The biophysical and pharmacological properties of somatic and dendritic versus nerve terminal channels are consistent with the characteristics of exogenously expressed alphabeta1 versus alphabeta4 channels, respectively. Therefore, one possible explanation for our findings is a selective distribution of auxiliary beta1 subunits to the somatic and dendritic compartments and beta4 to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate beta1 or beta4 channel clusters in the membrane of somatic and dendritic or nerve terminal compartments, respectively.

Ovarian folliculogenesis: detrimental effect of prenatal exposure to cyclophosphamide: a preliminary study.

PubMed

Ray, B; Potu, B K

2010-01-01

To investigate whether cyclophosphamide interferes with ovarian folliculogenesis. In this experiment, pregnant rats (n=12) were randomly assigned into two groups, control group (n=6) and cyclophosphamide treatment group (n=6). In the cyclophosphamide treatment group cyclophosphamide was injected intraperitoneally from day 10 of gestation till 20th day, at 2 mg/kg of body weight. The pregnant rats were sacrificed on gestation day 20 and the fetus was collected. The collected fetuses were processed for sectioning and stained with haematoxyline and eosin for microscopic observation of the ovaries. A meshwork-like appearance of mesenchyme with decreased number of somatic cells and absence of the majority of the germ cells in the ovarian follicles were found in treated fetus. Non-availability of primordial germ cells stopped the interaction between primordial germ cells and somatic supporting cells leading to nonproliferation and degeneration of somatic cells and fluid-filled vacant spaces in the meshwork -like arrangement of mesenchymal cells. We conclude that cyclophosphamide exposure prevents folliculogenesis by causing anovulation and results in infertility. The same detrimental effect might be seen in human fertility with environmental pollutants which are also metabolites of the drug (Fig. 2, Ref. 25).

[Construction and characterization of liposomal magnetofection system in pig kidney cells].

PubMed

Chen, Wenjie; Cui, Haixin; Zhao, Xiang; Cui, Jinhui; Wang, Yan; Sun, Changjiao

2014-06-01

Magnetic nano gene vector is one of the non-viral gene vectors, modified by functional group to bind cationic transfect reagents. Coupling magnetofection with the universal lipofection we developed a novel somatic cell transfection method as the so-called liposomal magnetofection (LMF). This approach is potential to provide somatic cell cloning with stable genetic cell lines to cultivate transgenic animals. In order to construct such liposomal magnetic gene vectors complexes system, we used nano magnetic gene vector to combine with liposomal cationic transfect reagents by molecular self-assembly. This vectors system successfully carried exogenous gene and then transfected animal somatic cells. Here, we conducted atomic force microscopy (AFM), zeta potential-diameter analysis and other characterization experiments to investegate the size distribution and morphology of magnetic nanoparticles, the way of the vectors to load and concentrate DNA molecules. Our data reveal that, the LMF of Pig Kidney cells exhibited higher transfection efficiency comparing with the transfection mediated by the commercial lipofectamine2000. Moreover, LMF method overcomes the constraint of transient expression mediated by lipofection. Meanwhile, MTT assay showed low cytotoxicity of LMF. Hence, LMF is a feasible, low cytotoxic and effective method of cell transfection.

Role of human oocyte-enriched factors in somatic cell reprograming.

PubMed

El-Gammal, Zaynab; AlOkda, Abdelrahman; El-Badri, Nagwa

2018-06-08

Cellular reprograming paves the way for creating functional patient-specific tissues to eliminate immune rejection responses by applying the same genetic profile. However, the epigenetic memory of a cell remains a challenge facing the current reprograming methods and does not allow transcription factors to bind properly. Because somatic cells can be reprogramed by transferring their nuclear contents into oocytes, introducing specific oocyte factors into differentiated cells is considered a promising approach for mimicking the reprograming process that occurs during fertilization. Mammalian metaphase II oocyte possesses a superior capacity to epigenetically reprogram somatic cell nuclei towards an embryonic stem cell-like state than the current factor-based reprograming approaches. This may be due to the presence of specific factors that are lacking in the current factor-based reprograming approaches. In this review, we focus on studies identifying human oocyte-enriched factors aiming to understand the molecular mechanisms mediating cellular reprograming. We describe the role of oocyte-enriched factors in metabolic switch, chromatin remodelling, and global epigenetic transformation. This is critical for improving the quality of resulting reprogramed cells, which is crucial for therapeutic applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Induction of pluripotent stem cells from fibroblast cultures.

PubMed

Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

2007-01-01

Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

Frequency of histopathological changes in Howler monkeys ( Alouatta sp.) naturally infected with yellow fever virus in Brazil.

PubMed

Leal, Silvana Gomes; Romano, Alessandro Pecego Martins; Monteiro, Rafael Veríssimo; Melo, Cristiano Barros de; Vasconcelos, Pedro Fernando da Costa; Castro, Márcio Botelho de

2016-02-01

Due to the importance that Howler monkeys have on the yellow fever (YF) epidemiological sylvatic cycle in Brazil, more accurate morphological diagnostic criteria needs to be established, especially considering the differences that may exist between the genera of Brazilian non-human primates (NHPs) involved in yellow fever virus (YFV) epizootics. Records of YF epizootics in NHPs in Brazil between 2007 and 2009 were obtained from the Brazilian Ministry of Health database to select YF positive (n=98) Howler monkeys (Alouatta sp.) for this study. The changes described in the histopathological reports were categorized by organ and their frequencies calculated. The most frequent lesions observed in the animals with YF were hepatocyte apoptosis (Councilman body formation), midzonal hepatocyte necrosis, steatosis, liver hemorrhage, inflammatory mononuclear cell infiltration of the liver, renal acute tubular necrosis and interstitial nephritis. Midzonal hepatocyte necrosis, steatosis and hemorrhage presented positive correlations with apoptosis of hepatocytes, suggesting strong YFV pathogenic effect association; they were also the main histopathological changes in the Alouatta sp. A pronounced negative correlation between apoptosis of hepatocytes and hepatic mononuclear cell infiltration pointed to significant histopathological differences between YFV infection in Howler monkeys and humans. The results warn that NHPs may exhibit different response patterns following YFV infection and require a more careful diagnosis. Presumptive diagnosis based on primate histopathological lesions may contribute to public health service control.

Donor-Derived Regulatory Dendritic Cell Infusion Maintains Donor-Reactive CD4+CTLA4hi T Cells in Non-Human Primate Renal Allograft Recipients Treated with CD28 Co-Stimulation Blockade.

PubMed

Ezzelarab, Mohamed B; Lu, Lien; Shufesky, William F; Morelli, Adrian E; Thomson, Angus W

2018-01-01

Donor-derived regulatory dendritic cell (DCreg) infusion before transplantation, significantly prolongs renal allograft survival in non-human primates. This is associated with enhanced expression of the immunoregulatory molecules cytotoxic T-lymphocyte-associated antigen (Ag) 4 (CTLA4) and programmed cell death protein 1 (PD1) by host donor-reactive T cells. In rodents and humans, CD28 co-stimulatory pathway blockade with the fusion protein CTLA4:Ig (CTLA4Ig) is associated with reduced differentiation and development of regulatory T cells (Treg). We hypothesized that upregulation of CTLA4 by donor-reactive CD4 + T cells in DCreg-infused recipients treated with CTLA4Ig, might be associated with higher incidences of donor-reactive CD4 + T cells with a Treg phenotype. In normal rhesus monkeys, allo-stimulated CD4 + CTLA4 hi , but not CD4 + CTLA4 med/lo T cells exhibited a regulatory phenotype, irrespective of PD1 expression. CTLA4Ig significantly reduced the incidence of CD4 + CTLA4 hi , but not CD4 + CTLA4 med/lo T cells following allo-stimulation, associated with a significant reduction in the CD4 + CTLA4 hi /CD4 + CTLA4 med/lo T cell ratio. In CTLA4Ig-treated renal allograft recipient monkeys, there was a marked reduction in circulating donor-reactive CD4 + CTLA4 hi T cells. In contrast, in CTLA4Ig-treated monkeys with DCreg infusion, no such reduction was observed. In parallel, the donor-reactive CD4 + CTLA4 hi /CD4 + CTLA4 med/lo T cell ratio was reduced significantly in graft recipients without DCreg infusion, but increased in those given DCreg. These observations suggest that pre-transplant DCreg infusion promotes and maintains donor-reactive CD4 + CTLA4 hi T cells with a regulatory phenotype after transplantation, even in the presence of CD28 co-stimulation blockade.

Donor-Derived Regulatory Dendritic Cell Infusion Maintains Donor-Reactive CD4+CTLA4hi T Cells in Non-Human Primate Renal Allograft Recipients Treated with CD28 Co-Stimulation Blockade

PubMed Central

Ezzelarab, Mohamed B.; Lu, Lien; Shufesky, William F.; Morelli, Adrian E.; Thomson, Angus W.

2018-01-01

Donor-derived regulatory dendritic cell (DCreg) infusion before transplantation, significantly prolongs renal allograft survival in non-human primates. This is associated with enhanced expression of the immunoregulatory molecules cytotoxic T-lymphocyte-associated antigen (Ag) 4 (CTLA4) and programmed cell death protein 1 (PD1) by host donor-reactive T cells. In rodents and humans, CD28 co-stimulatory pathway blockade with the fusion protein CTLA4:Ig (CTLA4Ig) is associated with reduced differentiation and development of regulatory T cells (Treg). We hypothesized that upregulation of CTLA4 by donor-reactive CD4+ T cells in DCreg-infused recipients treated with CTLA4Ig, might be associated with higher incidences of donor-reactive CD4+ T cells with a Treg phenotype. In normal rhesus monkeys, allo-stimulated CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells exhibited a regulatory phenotype, irrespective of PD1 expression. CTLA4Ig significantly reduced the incidence of CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells following allo-stimulation, associated with a significant reduction in the CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio. In CTLA4Ig-treated renal allograft recipient monkeys, there was a marked reduction in circulating donor-reactive CD4+CTLA4hi T cells. In contrast, in CTLA4Ig-treated monkeys with DCreg infusion, no such reduction was observed. In parallel, the donor-reactive CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio was reduced significantly in graft recipients without DCreg infusion, but increased in those given DCreg. These observations suggest that pre-transplant DCreg infusion promotes and maintains donor-reactive CD4+CTLA4hi T cells with a regulatory phenotype after transplantation, even in the presence of CD28 co-stimulation blockade. PMID:29520267

Isolation, molecular cloning and in vitro expression of rhesus monkey (Macaca mulatta) prominin-1.s1 complementary DNA encoding a potential hematopoietic stem cell antigen.

PubMed

Husain, S M; Shou, Y; Sorrentino, B P; Handgretinger, R

2006-10-01

Human prominin-1 (CD133 or AC133) is an important cell surface marker used to isolate primitive hematopoietic stem cells. The commercially available antibody to human prominin-1 does not recognize rhesus prominin-1. Therefore, we isolated, cloned and characterized the complementary DNA (cDNA) of rhesus prominin-1 gene and determined its coding potential. Following the nomenclature of prominin family of genes, we named this cDNA as rhesus prominin-1.s1. The amino acid sequence data of the putative rhesus prominin-1.s1 could be used in designing antigenic peptides to raise antibodies for use in isolation of pure populations of rhesus prominin-1(+) hematopoietic cells. To the best of our knowledge, there has been no previously published report about the isolation of a prominin-1 cDNA from rhesus monkey (Macaca mulatta).

Stability of hematologic analytes in monkey, rabbit, rat, and mouse blood stored at 4°C in EDTA using the ADVIA 120 hematology analyzer.

PubMed

Ameri, Mehrdad; Schnaars, Henry A; Sibley, John R; Honor, David J

2011-06-01

The time from sampling to analysis can be delayed when blood samples are shipped to distant reference laboratories or when analysis cannot be readily performed. The objective of this study was to evaluate the stability of hematologic analytes in blood samples from monkeys, rabbits, rats, and mice when samples were stored for up to 72 hours at 4°C. Blood samples from 30 monkeys, 15 rabbits, 20 rats, and 30 mice were collected into EDTA-containing tubes and were initially analyzed within 1 hour of collection using the ADVIA 120 analyzer. The samples were then stored at 4°C and reanalyzed at 24, 48, and 72 hours after collection. Significant (P<.0003) changes in hematologic analytes and calculations included increased HCT and MCV and decreased MCHC and cell hemoglobin concentration mean (CHCM) at 72 hours and increased MPV at 24 hours in monkeys; increased MCV at 72 hours and MPV at 48 hours and decreased monocyte count at 24 hours in rabbits; increased MCV and decreased MCHC, CHCM, and monocyte count at 24 hours in rats; increased MCV, red cell distribution width, and MPV and decreased MCHC, CHCM, and monocyte count at 24 hours in mice. Although most of the changes in the hematologic analytes in blood from monkeys, rabbits, rats, and mice when samples were stored at 4°C were analytically acceptable and clinically negligible, the best practice in measuring hematologic analytes in these animals is timely processing of blood samples, preferably within 1 hour after collection. ©2011 American Society for Veterinary Clinical Pathology.

Accelerated Episodic LH Release Accompanies Blunted Progesterone Regulation in PCOS-like Female Rhesus Monkeys (Macaca mulatta) Exposed to Testosterone During Early-to-Mid Gestation.

PubMed

Abbott, David H; Vepraskas, Sarah H; Horton, Teresa H; Terasawa, Ei; Levine, Jon E

2018-06-15

Ovarian theca cell hyperandrogenism in women with PCOS is compounded by androgen receptor-mediated impairment of estradiol and progesterone negative feedback regulation of episodic LH release. The resultant LH hypersecretion, likely the product of accelerated episodic release of GnRH from the median eminence of the hypothalamus, hyperstimulates ovarian theca cell steroidogenesis, enabling testosterone (T) and androstenedione excess. Prenatally androgenized female monkeys (PA) exposed to fetal male levels of T during early-to-mid gestation, when adult, demonstrate PCOS-like traits, including high T and LH levels. This study tests the hypothesis that progesterone resistance-associated acceleration in episodic LH release contributes to PA monkey LH excess. 4 PA and 3 regularly cycling, healthy control adult female rhesus monkeys of comparable age and body mass index underwent (1) a 10 h, frequent intravenous sampling assessment for LH episodic release, immediately followed by (2) IV infusion of exogenous GnRH to quantify continuing pituitary LH responsiveness, and subsequently (3) an SC injection of a progesterone receptor antagonist, mifepristone, to examine LH responses to blockade of progesterone-mediated action. Compared to controls, the relatively hyperandrogenic PA females exhibited ~100% increase (p = 0.037) in LH pulse frequency, positive correlation of LH pulse amplitude (p = 0.017) with androstenedione, ~100% greater increase (p = 0.034) in acute (0--10 min) LH responses to exogenous GnRH, and an absence (p = 0.008) of modest LH elevation following acute progesterone receptor blockade suggestive of diminished progesterone negative feedback. Such dysregulation of LH release in PCOS-like monkeys implicates impaired feedback control of episodic release of hypothalamic GnRH reminiscent of PCOS neuroendocrinopathy. 2018 S. Karger AG, Basel.

A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses.

PubMed

de Carvalho Dominguez Souza, Breno Frederico; König, Alexander; Rasche, Andrea; de Oliveira Carneiro, Ianei; Stephan, Nora; Corman, Victor Max; Roppert, Pia Luise; Goldmann, Nora; Kepper, Ramona; Müller, Simon Franz; Völker, Christof; de Souza, Alex Junior Souza; Gomes-Gouvêa, Michele Soares; Moreira-Soto, Andrés; Stöcker, Andreas; Nassal, Michael; Franke, Carlos Roberto; Rebello Pinho, João Renato; Soares, Manoel do Carmo Pereira; Geyer, Joachim; Lemey, Philippe; Drosten, Christian; Netto, Eduardo Martins; Glebe, Dieter; Drexler, Jan Felix

2018-06-01

All known hepatitis B virus (HBV) genotypes occur in humans and hominoid Old World non-human primates (NHPs). The divergent woolly monkey HBV (WMHBV) forms another orthohepadnavirus species. The evolutionary origins of HBV are unclear. We analysed sera from 124 Brazilian monkeys collected during 2012-2016 for hepadnaviruses using molecular and serological tools, and conducted evolutionary analyses. We identified a novel orthohepadnavirus species in capuchin monkeys (capuchin monkey hepatitis B virus [CMHBV]). We found CMHBV-specific antibodies in five animals and high CMHBV concentrations in one animal. Non-inflammatory, probably chronic infection was consistent with an intact preCore domain, low genetic variability, core deletions in deep sequencing, and no elevated liver enzymes. Cross-reactivity of antisera against surface antigens suggested antigenic relatedness of HBV, CMHBV, and WMHBV. Infection-determining CMHBV surface peptides bound to the human HBV receptor (human sodium taurocholate co-transporting polypeptide), but preferentially interacted with the capuchin monkey receptor homologue. CMHBV and WMHBV pseudotypes infected human hepatoma cells via the human sodium taurocholate co-transporting polypeptide, and were poorly neutralised by HBV vaccine-derived antibodies, suggesting that cross-species infections may be possible. Ancestral state reconstructions and sequence distance comparisons associated HBV with humans, whereas primate hepadnaviruses as a whole were projected to NHP ancestors. Co-phylogenetic analyses yielded evidence for co-speciation of hepadnaviruses and New World NHP. Bayesian hypothesis testing yielded strong support for an association of the HBV stem lineage with hominoid ancestors. Neither CMHBV nor WMHBV was likely the ancestor of the divergent human HBV genotypes F/H found in American natives. Our data suggest ancestral co-speciation of hepadnaviruses and NHP, and an Old World origin of the divergent HBV genotypes F/H. The identification of a novel primate hepadnavirus offers new perspectives for urgently needed animal models of chronic hepatitis B. The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

The Drosophila BCL6 homolog Ken and Barbie promotes somatic stem cell self-renewal in the testis niche.

PubMed

Issigonis, Melanie; Matunis, Erika

2012-08-15

Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Here, we find that ken and barbie (ken) is autonomously required for the self-renewal of CySCs but not GSCs. Furthermore, Ken misexpression in the CySC lineage induces the cell-autonomous self-renewal of somatic cells as well as the nonautonomous self-renewal of germ cells outside the niche. Thus, Ken, like Stat92E and its targets ZFH1 (Leatherman and Dinardo, 2008) and Chinmo (Flaherty et al., 2010), is necessary and sufficient for CySC renewal. However, ken is not a JAK-STAT target in the testis, but instead acts in parallel to Stat92E to ensure CySC self-renewal. Ken represses a subset of Stat92E targets in the embryo (Arbouzova et al., 2006) suggesting that Ken maintains CySCs by repressing differentiation factors. In support of this hypothesis, we find that the global JAK-STAT inhibitor Protein tyrosine phosphatase 61F (Ptp61F) is a JAK-STAT target in the testis that is repressed by Ken. Together, our work demonstrates that Ken has an important role in the inhibition of CySC differentiation. Studies of ken may inform our understanding of its vertebrate orthologue B-Cell Lymphoma 6 (BCL6) and how misregulation of this oncogene leads to human lymphomas. Copyright © 2012 Elsevier Inc. All rights reserved.

Somatic Embryogenesis in Peach Palm Using the Thin Cell Layer Technique: Induction, Morpho-histological Aspects and AFLP Analysis of Somaclonal Variation

PubMed Central

Steinmacher, D. A.; Krohn, N. G.; Dantas, A. C. M.; Stefenon, V. M.; Clement, C. R.; Guerra, M. P.

2007-01-01

Background and Aims The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. Methods TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0–600 µm Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. Key Results Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150–600 µm Picloram (83–97 %, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43 % embryogenic callus production from shoot meristem TCL on 300 µm Picloram. In maturation conditions, 34 ± 4 somatic embryos per embryogenic callus were obtained, and 45·0 ± 3·4 % of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80 % survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92 % of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. Conclusions The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees. PMID:17670751

Growth and development of cultured carrot cells and embryos under spaceflight conditions

NASA Technical Reports Server (NTRS)

Krikorian, A. D.; Dutcher, F. R.; Quinn, C. E.; Steward, F. C.

1981-01-01

Morphogenetically competent proembryonic cells and well-developed somatic embryos of carrot at two levels of organization were exposed for 18.5 days to a hypogravity environment aboard the Soviet Biosatellite Cosmos 1129. It was confirmed that cultured totipotent cells of carrot can give rise to embryos with well-developed roots and minimally developed shoots. It was also shown that the space hypogravity environment could support the further growth of already-organized, later somatic embryonic stages and give rise to fully developed embryo-plantlets with roots and shoots.

Co-evolution of MHC class I and variable NK cell receptors in placental mammals.

PubMed

Guethlein, Lisbeth A; Norman, Paul J; Hilton, Hugo G; Parham, Peter

2015-09-01

Shaping natural killer (NK) cell functions in human immunity and reproduction are diverse killer cell immunoglobulin-like receptors (KIRs) that recognize polymorphic MHC class I determinants. A survey of placental mammals suggests that KIRs serve as variable NK cell receptors only in certain primates and artiodactyls. Divergence of the functional and variable KIRs in primates and artiodactyls predates placental reproduction. Among artiodactyls, cattle but not pigs have diverse KIRs. Catarrhine (humans, apes, and Old World monkeys) and platyrrhine (New World monkeys) primates, but not prosimians, have diverse KIRs. Platyrrhine and catarrhine systems of KIR and MHC class I are highly diverged, but within the catarrhines, a stepwise co-evolution of MHC class I and KIR is discerned. In Old World monkeys, diversification focuses on MHC-A and MHC-B and their cognate lineage II KIR. With evolution of C1-bearing MHC-C from MHC-B, as informed by orangutan, the focus changes to MHC-C and its cognate lineage III KIR. Evolution of C2 from C1 and fixation of MHC-C drove further elaboration of MHC-C-specific KIR, as exemplified by chimpanzee. In humans, the evolutionary trajectory changes again. Emerging from reorganization of the KIR locus and selective attenuation of KIR avidity for MHC class I are the functionally distinctive KIR A and KIR B haplotypes. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Assignment of electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) to human chromosome 4q33 by fluorescence in situ hybridization and somatic cell hybridization.

PubMed

Spector, E B; Seltzer, W K; Goodman, S I

1999-08-01

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a nuclear-encoded protein located in the inner mitochondrial membrane. Inherited defects of ETF-QO cause glutaric acidemia type II. We here describe the localization of the ETF-QO gene to human chromosome 4q33 by somatic cell hybridization and fluorescence in situ hybridization. Copyright 1999 Academic Press.

Somatic mosaicism caused by monoallelic reversion of a mutation in T cells of a patient with ADA-SCID and the effects of enzyme replacement therapy on the revertant phenotype.

PubMed

Moncada-Vélez, M; Vélez-Ortega, A; Orrego, J; Santisteban, I; Jagadeesh, J; Olivares, M; Olaya, N; Hershfield, M; Candotti, F; Franco, J

2011-11-01

Patients with adenosine deaminase (ADA) deficiency exhibit spontaneous and partial clinical remission associated with somatic reversion of inherited mutations. We report a child with severe combined immunodeficiency (T-B- SCID) due to ADA deficiency diagnosed at the age of 1 month, whose lymphocyte counts including CD4+ and CD8+ T and NK cells began to improve after several months with normalization of ADA activity in Peripheral blood lymphocytes (PBL), as a result of somatic mosaicism caused by monoallelic reversion of the causative mutation in the ADA gene. He was not eligible for haematopoietic stem cell transplantation (HSCT) or gene therapy (GT); therefore he was placed on enzyme replacement therapy (ERT) with bovine PEG-ADA. The follow-up of metabolic and immunologic responses to ERT included gradual improvement in ADA activity in erythrocytes and transient expansion of most lymphocyte subsets, followed by gradual stabilization of CD4+ and CD8+ T (with naïve phenotype) and NK cells, and sustained expansion of TCRγδ+ T cells. This was accompanied by the disappearance of the revertant T cells as shown by DNA sequencing from PBL. Although the patient's clinical condition improved marginally, he later developed a germinal cell tumour and eventually died at the age of 67 months from sepsis. This case adds to our current knowledge of spontaneous reversion of mutations in ADA deficiency and shows that the effects of the ERT may vary among these patients, suggesting that it could depend on the cell and type in which the somatic mosaicism is established upon reversion. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

Somatic mosaicism due to monoallelic reversion of a mutation in T cells of an ADA-SCID patient and the effects of enzyme replacement therapy on the revertant phenotype

PubMed Central

Moncada-Vélez, Marcela; Vélez-Ortega, Alejandra C.; Orrego, Julio C.; Santisteban, Inés; Jagadeesh, Jayashree; Olivares, Margarita; Olaya, Natalia; Hershfield, Michael S.; Candotti, Fabio; Franco, Jose L.

2011-01-01

Patients with adenosine deaminase (ADA) deficiency exhibit spontaneous and partial clinical remission associated with somatic reversion of inherited mutations. We report a child with severe combined immunodeficiency (T-B-NK- SCID) due to ADA deficiency diagnosed at the age of 1 month, whose lymphocyte counts including CD4+ and CD8+ T and NK cells began to improve after several months with normalization of ADA activity in PBL, as a result of somatic mosaicism due to monoallelic reversion of the causative mutation in the ADA gene. Our patient was not eligible for hematopoietic stem cell transplantation (HSCT) or gene therapy (GT); therefore enzyme replacement therapy (ERT) with bovine PEG-ADA was initiated. The follow up of metabolic and immunologic responses to ERT included gradual improvement in ADA activity in erythrocytes and transient expansion of most lymphocyte subsets, followed by gradual stabilization of CD4+ and CD8+ T (with naïve phenotype) and NK cells, with sustained expansion of TCRγδ+ T cells. This was accompanied by disappearance of the revertant T cells as shown by DNA sequencing from PBL. Although the patient’s clinical condition improved marginally, he later developed a germinal cell tumor and eventually died at the age of 67 months from sepsis. This case adds to our current knowledge of spontaneous reversion of mutations in ADA deficiency and shows that the effects of the ERT may vary among these patients, suggesting that it could depend on the cell and type in which the somatic mosaicism is established upon reversion. PMID:21671975

Transmissible [corrected] dog cancer genome reveals the origin and history of an ancient cell lineage.

PubMed

Murchison, Elizabeth P; Wedge, David C; Alexandrov, Ludmil B; Fu, Beiyuan; Martincorena, Inigo; Ning, Zemin; Tubio, Jose M C; Werner, Emma I; Allen, Jan; De Nardi, Andrigo Barboza; Donelan, Edward M; Marino, Gabriele; Fassati, Ariberto; Campbell, Peter J; Yang, Fengtang; Burt, Austin; Weiss, Robin A; Stratton, Michael R

2014-01-24

Canine transmissible venereal tumor (CTVT) is the oldest known somatic cell lineage. It is a transmissible cancer that propagates naturally in dogs. We sequenced the genomes of two CTVT tumors and found that CTVT has acquired 1.9 million somatic substitution mutations and bears evidence of exposure to ultraviolet light. CTVT is remarkably stable and lacks subclonal heterogeneity despite thousands of rearrangements, copy-number changes, and retrotransposon insertions. More than 10,000 genes carry nonsynonymous variants, and 646 genes have been lost. CTVT first arose in a dog with low genomic heterozygosity that may have lived about 11,000 years ago. The cancer spawned by this individual dispersed across continents about 500 years ago. Our results provide a genetic identikit of an ancient dog and demonstrate the robustness of mammalian somatic cells to survive for millennia despite a massive mutation burden.

Analysis of a four generation family reveals the widespread sequence-dependent maintenance of allelic DNA methylation in somatic and germ cells

PubMed Central

Tang, Aifa; Huang, Yi; Li, Zesong; Wan, Shengqing; Mou, Lisha; Yin, Guangliang; Li, Ning; Xie, Jun; Xia, Yudong; Li, Xianxin; Luo, Liya; Zhang, Junwen; Chen, Shen; Wu, Song; Sun, Jihua; Sun, Xiaojuan; Jiang, Zhimao; Chen, Jing; Li, Yingrui; Wang, Jian; Wang, Jun; Cai, Zhiming; Gui, Yaoting

2016-01-01

Differential methylation of the homologous chromosomes, a well-known mechanism leading to genomic imprinting and X-chromosome inactivation, is widely reported at the non-imprinted regions on autosomes. To evaluate the transgenerational DNA methylation patterns in human, we analyzed the DNA methylomes of somatic and germ cells in a four-generation family. We found that allelic asymmetry of DNA methylation was pervasive at the non-imprinted loci and was likely regulated by cis-acting genetic variants. We also observed that the allelic methylation patterns for the vast majority of the cis-regulated loci were shared between the somatic and germ cells from the same individual. These results demonstrated the interaction between genetic and epigenetic variations and suggested the possibility of widespread sequence-dependent transmission of DNA methylation during spermatogenesis. PMID:26758766

Sexual Fate Change of XX Germ Cells Caused by the Deletion of SMAD4 and STRA8 Independent of Somatic Sex Reprogramming

PubMed Central

Wu, Quan; Fukuda, Kurumi; Kato, Yuzuru; Zhou, Zhi; Deng, Chu-Xia; Saga, Yumiko

2016-01-01

The differential programming of sperm and eggs in gonads is a fundamental topic in reproductive biology. Although the sexual fate of germ cells is believed to be determined by signaling factors from sexually differentiated somatic cells in fetal gonads, the molecular mechanism that determines germ cell fate is poorly understood. Herein, we show that mothers against decapentaplegic homolog 4 (SMAD4) in germ cells is required for female-type differentiation. Germ cells in Smad4-deficient ovaries respond to retinoic acid signaling but fail to undergo meiotic prophase I, which coincides with the weaker expression of genes required for follicular formation, indicating that SMAD4 signaling is essential for oocyte differentiation and meiotic progression. Intriguingly, germline-specific deletion of Smad4 in Stra8-null female germ cells resulted in the up-regulation of genes required for male gonocyte differentiation, including Nanos2 and PLZF, suggesting the initiation of male-type differentiation in ovaries. Moreover, our transcriptome analyses of mutant ovaries revealed that the sex change phenotype is achieved without global gene expression changes in somatic cells. Our results demonstrate that SMAD4 and STRA8 are essential factors that regulate the female fate of germ cells. PMID:27606421

A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin.

PubMed

Watanabe, Satoshi; Iwamoto, Masaki; Suzuki, Shun-ichi; Fuchimoto, Daiichiro; Honma, Daisuke; Nagai, Takashi; Hashimoto, Michiko; Yazaki, Satoko; Sato, Masahiro; Onishi, Akira

2005-02-01

Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 microg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.

Expansion of somatically reverted memory CD8+ T cells in patients with X-linked lymphoproliferative disease caused by selective pressure from Epstein-Barr virus

PubMed Central

Low, Carol; Bell, Andrew I.; Abbott, Rachel J.M.; Phan, Tri Giang; Riminton, D. Sean; Choo, Sharon; Smart, Joanne M.; Lougaris, Vassilios; Giliani, Silvia; Buckley, Rebecca H.; Grimbacher, Bodo; Alvaro, Frank; Klion, Amy D.; Nichols, Kim E.; Adelstein, Stephen; Rickinson, Alan B.

2012-01-01

Patients with the primary immunodeficiency X-linked lymphoproliferative disease (XLP), which is caused by mutations in SH2D1A, are highly susceptible to Epstein-Barr virus (EBV) infection. Nonetheless, some XLP patients demonstrate less severe clinical manifestations after primary infection. SH2D1A encodes the adaptor molecule SLAM-associated protein (SAP), which is expressed in T and natural killer cells and is required for cytotoxicity against B cells, the reservoir for EBV. It is not known why the clinical presentation of XLP is so variable. In this study, we report for the first time the occurrence of somatic reversion in XLP. Reverted SAP-expressing cells resided exclusively within the CD8+ T cell subset, displayed a CD45RA−CCR7− effector memory phenotype, and were maintained at a stable level over time. Importantly, revertant CD8+ SAP+ T cells, but not SAP− cells, proliferated in response to EBV and killed EBV-infected B cells. As somatic reversion correlated with EBV infection, we propose that the virus exerts a selective pressure on the reverted cells, resulting in their expansion in vivo and host protection against ongoing infection. PMID:22493517

Sexual Fate Change of XX Germ Cells Caused by the Deletion of SMAD4 and STRA8 Independent of Somatic Sex Reprogramming.

PubMed

Wu, Quan; Fukuda, Kurumi; Kato, Yuzuru; Zhou, Zhi; Deng, Chu-Xia; Saga, Yumiko

2016-09-01

The differential programming of sperm and eggs in gonads is a fundamental topic in reproductive biology. Although the sexual fate of germ cells is believed to be determined by signaling factors from sexually differentiated somatic cells in fetal gonads, the molecular mechanism that determines germ cell fate is poorly understood. Herein, we show that mothers against decapentaplegic homolog 4 (SMAD4) in germ cells is required for female-type differentiation. Germ cells in Smad4-deficient ovaries respond to retinoic acid signaling but fail to undergo meiotic prophase I, which coincides with the weaker expression of genes required for follicular formation, indicating that SMAD4 signaling is essential for oocyte differentiation and meiotic progression. Intriguingly, germline-specific deletion of Smad4 in Stra8-null female germ cells resulted in the up-regulation of genes required for male gonocyte differentiation, including Nanos2 and PLZF, suggesting the initiation of male-type differentiation in ovaries. Moreover, our transcriptome analyses of mutant ovaries revealed that the sex change phenotype is achieved without global gene expression changes in somatic cells. Our results demonstrate that SMAD4 and STRA8 are essential factors that regulate the female fate of germ cells.

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium.

PubMed

Smith, D L; Krikorian, A D

1989-01-01

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.

Release of somatic embryogenic potential from excised zygotic embryos of carrot and maintenance of proembryonic cultures in hormone-free medium

NASA Technical Reports Server (NTRS)

Smith, D. L.; Krikorian, A. D.

1989-01-01

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and multiplication of globular somatic proembryos. The sequence of events leading from excised broken zygotic embryos to the formation of somatic embryos and the maintenance of somatic proembryos are demonstrated by scanning electron microscopy and histological preparations. Germination levels from intact zygotic embryos on media with varying levels and ratios of unreduced vs. reduced inorganic nitrogen were determined as well and provided baseline or control data on the type of response obtained from nonwounded material.

Prevalence of somatic mitochondrial mutations and spatial distribution of mitochondria in non-small cell lung cancer.

PubMed

Kazdal, Daniel; Harms, Alexander; Endris, Volker; Penzel, Roland; Kriegsmann, Mark; Eichhorn, Florian; Muley, Thomas; Stenzinger, Albrecht; Pfarr, Nicole; Weichert, Wilko; Warth, Arne

2017-07-11

Mitochondria are considered relevant players in many tumour entities and first data indicate beneficial effects of mitochondria-targeted antioxidants in both cancer prevention and anticancer therapies. To further dissect the potential roles of mitochondria in NSCLC we comprehensively analysed somatic mitochondrial mutations, determined the spatial distribution of mitochondrial DNA within complete tumour sections and investigated the mitochondrial load in a large-scale approach. Whole mitochondrial genome sequencing of 26 matched tumour and non-neoplastic tissue samples extended by reviewing published data of 326 cases. Systematical stepwise real-time PCR quantification of mitochondrial DNA covering 16 whole surgical tumour sections. Immunohistochemical determination of the mitochondrial load in 171 adenocarcinoma and 145 squamous cell carcinoma. Our results demonstrate very low recurrences (max. 1.7%) and a broad distribution of 456 different somatic mitochondrial mutations. Large inter- and intra-tumour heterogeneity were seen for mitochondrial DNA copy numbers in conjunction with a correlation to the predominant histological growth pattern. Furthermore, tumour cells had significantly higher mitochondrial level compared to adjacent stroma, whereas differences between tumour entities were negligible. Non-evident somatic mitochondrial mutations and highly varying mitochondrial DNA level delineate challenges for the approach of mitochondria-targeted anticancer therapies in NSCLC.

Loss of CXCR6 coreceptor usage characterizes pathogenic lentiviruses

PubMed Central

Wetzel, Katherine S.; Yi, Yanjie; Bauer, Anya M.; Bibollet-Ruche, Frederic; Hahn, Beatrice H.; Paiardini, Mirko; Silvestri, Guido; Peeters, Martine; Collman, Ronald G.

2018-01-01

Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with env contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is a major coreceptor for SIV in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for entry and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of multiple primate species. Testing lymphocytes from SM, the best-studied natural host, we found that CXCR6 is restricted to CD4+ effector memory cells, and is expressed by a sub-population distinct from those expressing CCR5. Thus, efficient CXCR6 use, previously identified in SM and AGM infection, also characterizes a member of the SIV lineage that gave rise to SIVcpz/HIV-1. Loss of CXCR6 usage by SIVcpz may have altered its cell tropism, shifting virus from CXCR6-expressing cells that may support replication without disrupting immune function or homeostasis, towards CCR5-expressing cells with pathogenic consequences. PMID:29659623

Loss of CXCR6 coreceptor usage characterizes pathogenic lentiviruses.

PubMed

Wetzel, Katherine S; Yi, Yanjie; Yadav, Anjana; Bauer, Anya M; Bello, Ezekiel A; Romero, Dino C; Bibollet-Ruche, Frederic; Hahn, Beatrice H; Paiardini, Mirko; Silvestri, Guido; Peeters, Martine; Collman, Ronald G

2018-04-01

Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with env contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is a major coreceptor for SIV in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for entry and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of multiple primate species. Testing lymphocytes from SM, the best-studied natural host, we found that CXCR6 is restricted to CD4+ effector memory cells, and is expressed by a sub-population distinct from those expressing CCR5. Thus, efficient CXCR6 use, previously identified in SM and AGM infection, also characterizes a member of the SIV lineage that gave rise to SIVcpz/HIV-1. Loss of CXCR6 usage by SIVcpz may have altered its cell tropism, shifting virus from CXCR6-expressing cells that may support replication without disrupting immune function or homeostasis, towards CCR5-expressing cells with pathogenic consequences.

Multiple independent second-site mutations in two siblings with somatic mosaicism for Wiskott-Aldrich syndrome.

PubMed

Boztug, K; Germeshausen, M; Avedillo Díez, I; Gulacsy, V; Diestelhorst, J; Ballmaier, M; Welte, K; Maródi, L; Chernyshova, Li; Klein, C

2008-07-01

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disorder associated with microthrombocytopenia, eczema, autoimmunity and predisposition to malignant lymphoma. Although rare, few cases of somatic mosaicism have been published in WAS patients to date. We here report on two Ukrainian siblings who were referred to us at the age of 3 and 4 years, respectively. Both patients suffered from severe WAS caused by a nonsense mutation in exon 1 of the WAS gene. In both siblings, flow cytometric analysis revealed the presence of Wiskott-Aldrich syndrome protein (WASp)-positive and WASp-negative cell populations among T and B lymphocytes as well as natural killer (NK) cells. In contrast to previously described cases of revertant mosaicism in WAS, molecular analyses in both children showed that the WASp-positive T cells, B cells, and NK cells carried multiple different second-site mutations, resulting in different missense mutations. To our knowledge, this is the first report describing somatic mosaicism in WAS patients caused by several independent second-site mutations in the WAS gene.

Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci

PubMed Central

Philippe, Claude; Vargas-Landin, Dulce B; Doucet, Aurélien J; van Essen, Dominic; Vera-Otarola, Jorge; Kuciak, Monika; Corbin, Antoine; Nigumann, Pilvi; Cristofari, Gaël

2016-01-01

LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants. DOI: http://dx.doi.org/10.7554/eLife.13926.001 PMID:27016617

Bottlenecks in bog pine multiplication by somatic embryogenesis and their visualization with the environmental scanning electron microscope.

PubMed

Vlašínová, Helena; Neděla, Vilem; Đorđević, Biljana; Havel, Ladislav

2017-07-01

Somatic embryogenesis (SE) is an important biotechnological technique used for the propagation of many pine species in vitro. However, in bog pine, one of the most endangered tree species in the Czech Republic, limitations were observed, which negatively influenced the development and further germination of somatic embryos. Although initiation frequency was very low-0.95 %, all obtained cell lines were subjected to maturation. The best responding cell line (BC1) was used and subjected to six different variants of the maturation media. The media on which the highest number of early-precotyledonary/cotyledonary somatic embryos was formed was supplemented with 121 μM abscisic acid (ABA) and with 6 % maltose. In the end of maturation experiments, different abnormalities in formation of somatic embryos were observed. For visualization and identification of abnormalities in meristem development during proliferation and maturation processes, the environmental scanning electron microscope was used. In comparison to the classical light microscope, the non-commercial environmental scanning electron microscope AQUASEM II has been found as a very useful tool for the quick recognition of apical meristem disruption and abnormal development. To our knowledge, this is the first report discussing somatic embryogenesis in bog pine. Based on this observation, the cultivation procedure could be enhanced and the method for SE of bog pine optimized.

Somatic stem cell heterogeneity: diversity in the blood, skin and intestinal stem cell compartments

PubMed Central

Goodell, Margaret A.; Nguyen, Hoang; Shroyer, Noah

2017-01-01

Somatic stem cells replenish many tissues throughout life to repair damage and to maintain tissue homeostasis. Stem cell function is frequently described as following a hierarchical model in which a single master cell undergoes self-renewal and differentiation into multiple cell types and is responsible for most regenerative activity. However, recent data from studies on blood, skin and intestinal epithelium all point to the concomitant action of multiple types of stem cells with distinct everyday roles. Under stress conditions such as acute injury, the surprising developmental flexibility of these stem cells enables them to adapt to diverse roles and to acquire different regeneration capabilities. This paradigm shift raises many new questions about the developmental origins, inter-relationships and molecular regulation of these multiple stem cell types. PMID:25907613

Somatic hybrid plants from sexually incompatible woody species: Citrus reticulata and Citropsis gilletiana.

PubMed

Grosser, J W; Gmitter, F G; Tusa, N; Chandler, J L

1990-04-01

Allotetraploid intergeneric somatic hybrid plants between Citrus reticulata Blanco cv. Cleopatra mandarin and Citropsis gilletiana Swing. & M. Kell. (common name Gillet's cherry orange) were regenerated following protoplast fusion. Cleopatra protoplasts were isolated from an ovule-derived embryogenic suspension culture and fused chemically with leaf-derived protoplasts of Citropsis gilletiana. Cleopatra mandarin and somatic hybrid plants were regenerated via somatic embryogenesis. Hybrid plant identification was based on differential leaf morphology, root-tip cell chromosome number, and electrophoretic analyses of phosphoglucose mutase (PGM) and phosphohexose isomerase (PHI) isozyme banding patterns. This is the first somatic hybrid within the Rutaceae reported that does not have Citrus sinensis (sweet orange) as a parent, and the first produced with a commercially important citrus rootstock and a complementary but sexually incompatible, related species.

Pre-procambial cells are niches for pluripotent and totipotent stem-like cells for organogenesis and somatic embryogenesis in the peach palm: a histological study.

PubMed

de Almeida, Marcilio; de Almeida, Cristina Vieira; Mendes Graner, Erika; Ebling Brondani, Gilvano; Fiori de Abreu-Tarazi, Monita

2012-08-01

The direct induction of adventitious buds and somatic embryos from explants is a morphogenetic process that is under the influence of exogenous plant growth regulators and its interactions with endogenous phytohormones. We performed an in vitro histological analysis in peach palm (Bactris gasipaes Kunth) shoot apexes and determined that the positioning of competent cells and their interaction with neighboring cells, under the influence of combinations of exogenously applied growth regulators (NAA/BAP and NAA/TDZ), allows the pre-procambial cells (PPCs) to act in different morphogenic pathways to establish niche competent cells. It is likely that there has been a habituation phenomenon during the regeneration and development of the microplants. This includes promoting the tillering of primary or secondary buds due to culturing in the absence of NAA/BAP or NAA/TDZ after a period in the presence of these growth regulators. Histological analyses determined that the adventitious roots were derived from the dedifferentiation of the parenchymal cells located in the basal region of the adventitious buds, with the establishment of rooting pole, due to an auxin gradient. Furthermore, histological and histochemical analyses allowed us to characterize how the PPCs provide niches for multipotent, pluripotent and totipotent stem-like cells for vascular differentiation, organogenesis and somatic embryogenesis in the peach palm. The histological and histochemical analyses also allowed us to detect the unicellular or multicellular origin of somatic embryogenesis. Therefore, our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells. Our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells.

Germline contamination and leakage in whole genome somatic single nucleotide variant detection.

PubMed

Sendorek, Dorota H; Caloian, Cristian; Ellrott, Kyle; Bare, J Christopher; Yamaguchi, Takafumi N; Ewing, Adam D; Houlahan, Kathleen E; Norman, Thea C; Margolin, Adam A; Stuart, Joshua M; Boutros, Paul C

2018-01-31

The clinical sequencing of cancer genomes to personalize therapy is becoming routine across the world. However, concerns over patient re-identification from these data lead to questions about how tightly access should be controlled. It is not thought to be possible to re-identify patients from somatic variant data. However, somatic variant detection pipelines can mistakenly identify germline variants as somatic ones, a process called "germline leakage". The rate of germline leakage across different somatic variant detection pipelines is not well-understood, and it is uncertain whether or not somatic variant calls should be considered re-identifiable. To fill this gap, we quantified germline leakage across 259 sets of whole-genome somatic single nucleotide variant (SNVs) predictions made by 21 teams as part of the ICGC-TCGA DREAM Somatic Mutation Calling Challenge. The median somatic SNV prediction set contained 4325 somatic SNVs and leaked one germline polymorphism. The level of germline leakage was inversely correlated with somatic SNV prediction accuracy and positively correlated with the amount of infiltrating normal cells. The specific germline variants leaked differed by tumour and algorithm. To aid in quantitation and correction of leakage, we created a tool, called GermlineFilter, for use in public-facing somatic SNV databases. The potential for patient re-identification from leaked germline variants in somatic SNV predictions has led to divergent open data access policies, based on different assessments of the risks. Indeed, a single, well-publicized re-identification event could reshape public perceptions of the values of genomic data sharing. We find that modern somatic SNV prediction pipelines have low germline-leakage rates, which can be further reduced, especially for cloud-sharing, using pre-filtering software.

Developmental localization and the role of hydroxyproline rich glycoproteins during somatic embryogenesis of banana (Musa spp. AAA)

PubMed Central

2011-01-01

Background Hydroxyproline rich glycoproteins (HRGPs) are implicated to have a role in many aspects of plant growth and development but there is limited knowledge about their localization and function during somatic embryogenesis of higher plants. In this study, the localization and function of hydroxyproline rich glycoproteins in embryogenic cells (ECs) and somatic embryos of banana were investigated by using immunobloting and immunocytochemistry with monoclonal JIM11 and JIM20 antibodies as well as by treatment with 3,4-dehydro-L-proline (3,4-DHP, an inhibitor of extensin biosynthesis), and by immunomodulation with the JIM11 antibody. Results Immunofluorescence labelling of JIM11 and JIM20 hydroxyproline rich glycoprotein epitopes was relatively weak in non-embryogenic cells (NECs), mainly on the edge of small cell aggregates. On the other hand, hydroxyproline rich glycoprotein epitopes were found to be enriched in early embryogenic cells as well as in various developmental stages of somatic embryos. Embryogenic cells (ECs), proembryos and globular embryos showed strong labelling of hydroxyproline rich glycoprotein epitopes, especially in their cell walls and outer surface layer, so-called extracellular matrix (ECM). This hydroxyproline rich glycoprotein signal at embryo surfaces decreased and/or fully disappeared during later developmental stages (e.g. pear-shaped and cotyledonary stages) of embryos. In these later developmental embryogenic stages, however, new prominent hydroxyproline rich glycoprotein labelling appeared in tri-cellular junctions among parenchymatic cells inside these embryos. Overall immunofluorescence labelling of late stage embryos with JIM20 antibody was weaker than that of JIM11. Western blot analysis supported the above immunolocalization data. The treatment with 3,4-DHP inhibited the development of embryogenic cells and decreased the rate of embryo germination. Embryo-like structures, which developed after 3,4-DHP treatment showed aberrant non-compact epidermis with discontinuous ECM at the outer surface as well as much less immunolabelling with the JIM11 antibody. This treatment also decreased the plant regeneration capacity in embryogenic banana cultures. Finally, immunomodulation of surface hydroxyproline rich glycoproteins by co-culture of embryos with the JIM11 antibody resulted in a much lower germination capacity of these embryos. Conclusions These results suggest that hydroxyproline rich glycoproteins play an important developmental role, especially in the process of regeneration and germination of embryos during plant regeneration via somatic embryogenesis. Proper content and localization of hydroxyproline rich glycoproteins seem to be essential for the formation and regeneration of banana somatic embryos. PMID:21349190

STUDIES ON VARIANTS OF POLIOMYELITIS VIRUS

PubMed Central

Sabin, Albert B.; Hennessen, Walter A.; Winsser, Johan

1954-01-01

Attempts were made to "convert" highly virulent strains of the 3 immunologic types of poliomyelitis virus (Mahoney, Y-SK, and Leon) into avirulent variants. Tests involving intracerebral, intramuscular, or oral administration of virus to cynomolgus monkeys indicated that mere propagation in cultures of kidney tissue of cynomolgus monkeys had no effect on virulence when single or small numbers of virus particles were used as seed, and harvests were delayed for 24 hours or more after the appearance of cytopathogenic change. On the other hand, passages at 24 hour intervals with large inocula (105 to 106 TCD60) produced culture fluids with diminished virulence and unusual patterns of response in cynomolgus monkeys. Purification of such culture fluids by the terminal dilution technique yielded modified strains which proved to be avirulent after administration by the intracerebral, intramuscular, or oral routes in cynomolgus monkeys. Neither paralysis nor CNS lesions were found in any of more than 80 monkeys inoculated intracerebrally with various amounts of virus. However, focal neuronal lesions were found in the spinal cord of 3 of 48 monkeys inoculated intramuscularly with various amounts of the Mahoney variant, in 2 of 20 receiving the Y-SK variant, though in none of 40 inoculated with various amounts of the Leon variant. Virus recovered from the spinal cord of one of the monkeys in the Mahoney group produced no paralysis on intracerebral passage in monkeys. It is assumed that all 3 modified viruses possess a limited capacity to affect lower motor neurones of cynomolgus monkeys when these are directly exposed to them by accidental intraneural or traumatic intracerebral injection. On propagation in cynomolgus kidney cultures the modified viruses reached titers of approximately 107 TCD50 per ml., as measured by cytopathogenic activity on renal epithelial cells in vitro, yet produced no perceptible pathologic changes in the muscles, kidneys, testes, ovaries, heart, pancreas, adrenals, liver, or spleen of cynomolgus monkeys inoculated intramuscularly. The modified viruses were immunogenic after intramuscular injection, but a large proportion of cynomolgus monkeys failed to develop antibody after small doses, indicating that in this host the experimentally produced variants multiplied less readily in non-nervous tissue than the virulent parent strains. Tests with the Type 1 virus showed that the orally administered avirulent variant can induce the formation of antibody and bring about resistance to the occurrence of paralysis such as results from ingestion of the virulent, parent strain. The Types 1 and 2 modified viruses are paralytogenic in mice after direct spinal inoculation whereas the Type 3 virus is not. The Type 1 virus became paralytogenic for mice when it lost its virulence for cynomolgus monkeys by the indicated routes. The Type 2 virus lost its virulence for mice by the intracerebral but not intraspinal routes when it was still fully virulent for cynomolgus monkeys, and retained its paralytogenic activity in intraspinally inoculated mice after it had lost its virulence for cynomolgus monkeys by the indicated routes. The parent Type 3 virus was paralytogenic in intraspinally inoculated mice when it was still fully virulent for cynomolgus monkeys, but this property disappeared in the modified virus when it became avirulent for monkeys. PMID:13163327

Effect of animal sera on Bacillus anthracis Sterne spore germination and vegetative cell growth.

PubMed

Bensman, M D; Mackie, R S; Minter, Z A; Gutting, B W

2012-08-01

 The aims of this work were to investigate the effects of sera on B. anthracis Sterne germination and growth. Sera examined included human, monkey and rabbit sera, as well as sera from eight other species.  Standard dilution plate assay (with and without heat kill) was used as a measure of germination, and spectroscopy was used to measure growth. In addition, a Coulter Counter particle counter was used to monitor germination and growth based on bacterial size. Spores germinated best in foetal bovine and monkey sera, moderately with human sera and showed limited germination in the presence of rabbit or rat sera. Vegetative bacteria grew best in foetal bovine sera and moderately in rabbit sera. Human and monkey sera supported little growth of vegetative bacteria.  The data suggested sera can have a significant impact on germination and growth of Sterne bacteria.  These data should be considered when conducting in vitro cell culture studies and may aid in interpreting in vivo infection studies. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Recovery of neurological functions in non-human primate model of Parkinson's disease by transplantation of encapsulated neonatal porcine choroid plexus cells.

PubMed

Luo, Xian-Ming; Lin, Hai; Wang, Wei; Geaney, Marilyn S; Law, Lee; Wynyard, Shaun; Shaikh, Shamim B; Waldvogel, Henry; Faull, Richard L M; Elliott, Robert B; Skinner, Stephen J M; Lee, Jacqueline E; Tan, Paul L-J

2013-01-01

Parkinson's disease (PD) is a neurodegenerative disease that is primarily characterized by degeneration of dopaminergic (DA) neurons in the substantia nigra (SN) and a loss of their fibre projections in the striatum. We utilized the neonatal porcine choroid plexus (CP), an organ that secretes cerebrospinal fluid containing various types of neurotrophic and neuroprotective factors, to ameliorate the Parkinsonian symptoms in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated rhesus monkeys without requiring immunosuppression. We demonstrate that transplanted encapsulated CP clusters (eCPs) significantly improved neurological functions in MPTP-treated monkeys during the course of six months after transplantation (p < 0.001) when compared with monkeys implanted with empty capsules or subjected to sham surgery. The improvement in neurological scores was accompanied by a corresponding improvement in apomorphine-induced circling behaviour (p < 0.001) as well as increased tyrosine hydroxylase (TH) staining in the striatum. Our results suggest that eCPs are a promising cell therapeutic agent to treat Parkinson's disease.

Assessment of ixekizumab, an interleukin-17A monoclonal antibody, for potential effects on reproduction and development, including immune system function, in cynomolgus monkeys.

PubMed

Clarke, D O; Hilbish, K G; Waters, D G; Newcomb, D L; Chellman, G J

2015-12-01

The reproductive and developmental toxicity of ixekizumab, a selective inhibitor of interleukin-17A (IL-17A), was assessed in the following studies in cynomolgus monkeys: fertility (3-month dosing), embryo-fetal development (EFD; dosing from gestation day (GD) 20 through 139), and pre-postnatal development (PPND; dosing from GD 20 through parturition). Because IL-17A has functional roles in innate and humoral immunity, immune system modulation was evaluated in the EFD and PPND studies; immunological evaluations in infants comprised peripheral blood immunophenotyping, Natural Killer cell cytolytic activity, and T-cell-dependent antibody (IgG and IgM) primary and secondary responses to antigen challenge. Ixekizumab exposure was sustained during the dosing periods in most adult monkeys. Fetal exposure at Cesarean section (GD 140-142; EFD study) was 18-25% of maternal exposure and ixekizumab was present in infants for up to 29 weeks postpartum. There were no adverse effects attributed to ixekizumab in any study. Importantly, immune system development and maturation were unaffected. Copyright © 2015 Elsevier Inc. All rights reserved.

Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey

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

Bernemann, Inga, E-mail: bernemann@imp.uni-hannover.de; Mueller, Thomas; Blasczyk, Rainer

Highlights: {yields} Marmoset bone marrow-derived MSCs differentiate in suspension into adipogenic, osteogenic and chondrogenic lineages. {yields} Marmoset MSCs integrate in collagen type I scaffolds and differentiate excellently into adipogenic cells. {yields} Common marmoset monkey is a suitable model for soft tissue engineering in human regenerative medicine. -- Abstract: In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 {mu}m were optimal for cell seeding and cultivating. However, before clinical application andmore » transplantation of MSC-derived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human. Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential potential of marmoset MSCs in adipogenic, osteogenic and chondrogenic lineages and the suitability of collagen scaffolds as carrier material undisturbing differentiation of primate mesenchymal stem cells.« less

High-frequency oscillations in human and monkey neocortex during the wake–sleep cycle

PubMed Central

Le Van Quyen, Michel; Muller, Lyle E.; Telenczuk, Bartosz; Halgren, Eric; Cash, Sydney; Hatsopoulos, Nicholas G.; Dehghani, Nima; Destexhe, Alain

2016-01-01

Beta (β)- and gamma (γ)-oscillations are present in different cortical areas and are thought to be inhibition-driven, but it is not known if these properties also apply to γ-oscillations in humans. Here, we analyze such oscillations in high-density microelectrode array recordings in human and monkey during the wake–sleep cycle. In these recordings, units were classified as excitatory and inhibitory cells. We find that γ-oscillations in human and β-oscillations in monkey are characterized by a strong implication of inhibitory neurons, both in terms of their firing rate and their phasic firing with the oscillation cycle. The β- and γ-waves systematically propagate across the array, with similar velocities, during both wake and sleep. However, only in slow-wave sleep (SWS) β- and γ-oscillations are associated with highly coherent and functional interactions across several millimeters of the neocortex. This interaction is specifically pronounced between inhibitory cells. These results suggest that inhibitory cells are dominantly involved in the genesis of β- and γ-oscillations, as well as in the organization of their large-scale coherence in the awake and sleeping brain. The highest oscillation coherence found during SWS suggests that fast oscillations implement a highly coherent reactivation of wake patterns that may support memory consolidation during SWS. PMID:27482084

High-frequency oscillations in human and monkey neocortex during the wake-sleep cycle.

PubMed

Le Van Quyen, Michel; Muller, Lyle E; Telenczuk, Bartosz; Halgren, Eric; Cash, Sydney; Hatsopoulos, Nicholas G; Dehghani, Nima; Destexhe, Alain

2016-08-16

Beta (β)- and gamma (γ)-oscillations are present in different cortical areas and are thought to be inhibition-driven, but it is not known if these properties also apply to γ-oscillations in humans. Here, we analyze such oscillations in high-density microelectrode array recordings in human and monkey during the wake-sleep cycle. In these recordings, units were classified as excitatory and inhibitory cells. We find that γ-oscillations in human and β-oscillations in monkey are characterized by a strong implication of inhibitory neurons, both in terms of their firing rate and their phasic firing with the oscillation cycle. The β- and γ-waves systematically propagate across the array, with similar velocities, during both wake and sleep. However, only in slow-wave sleep (SWS) β- and γ-oscillations are associated with highly coherent and functional interactions across several millimeters of the neocortex. This interaction is specifically pronounced between inhibitory cells. These results suggest that inhibitory cells are dominantly involved in the genesis of β- and γ-oscillations, as well as in the organization of their large-scale coherence in the awake and sleeping brain. The highest oscillation coherence found during SWS suggests that fast oscillations implement a highly coherent reactivation of wake patterns that may support memory consolidation during SWS.

Prevalence of antibodies to soluble antigen of Epstein-Barr virus-producing cells (P3HR-1) in the sera of hamadryas baboons of the high lymphoma incidence stock.

PubMed

Voevodin, A F; Lapin, B A; Yakovleva, L A; Ponomarjova, T I; Agrba, V Z

1979-01-01

Soluble antigen of P3HR-1 cells (SA-P3HR-1) was identified in indirect double immunodiffusion enhanced with tannic acid using serum of a nasopharyngeal carcinoma patient containing high-titer antibodies to Epstein-Barr virus (EBV) antigens. SA-P3HR-1 was nonidentical to soluble antigen of Raji cells. Human and baboon sera containing antibodies to all the known antigen of EBV and HVP respectively were anti-SA-3HR-1-positive. Human and baboon sera containing antibodies to all the known antigens of EBV and herpesvirus Papio (HVP) were also anti-SA-P3HR-1-negative. Prevalence of anti-SA-P3HR-1 was very high in two groups of the high-lymphoma incidence stock of hamadryas baboons of the Sukhumi monkey colony. 54% (15 of 28) and 38% (13 of 34) of clinically lymphomatous and clinical normal monkeys, respectively, were anti-SA-P3HR-1-positive.. Only 1 of 30 normal baboons studied, living in the forest and having no contacts with the baboons in the main stock of the Sukhumi monkey colony, was anti-SA-P3HR-1-positive (3%).