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81.
The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function 总被引:6,自引:0,他引:6
Dr. Nancy L. Parenteau Patrick Bilbo Cynthia J. M. Nolte Valerie S. Mason Mireille Rosenberg 《Cytotechnology》1992,9(1-3):163-171
We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal
fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required
the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to
maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters
were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural
morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation
markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal
layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct
also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid
interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications. 相似文献
82.
Robert?P. Johnson Aaron?M. Fleming Qian Jin Cynthia?J. Burrows Henry?S. White 《Biophysical journal》2014,107(4):924-931
The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12–35°C) and KCl concentration (0.15–1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼8 kJ mol−1 decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes. 相似文献
83.
84.
Alisha J. Beirl Tor H. Linbo Marea J. Cobb Cynthia D. Cooper 《Pigment cell & melanoma research》2014,27(2):178-189
We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410‐kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2‐deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar‐type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore‐specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post‐fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish. 相似文献
85.
DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway. 相似文献
86.
Kennedy RC Shearer MH Lowe DB Jumper CA Chiriva-Internati M Bright RK 《Cancer immunology, immunotherapy : CII》2004,53(11):987-994
Purpose: Immunologic-based cancer treatment modalities represent an active area of investigation. Included in these strategies are passive administration of monoclonal antibodies which recognize tumor-associated antigens and active vaccination with identified tumor antigens. However, several problems associated with these types of treatment strategies have been identified. Methods: In this report, we address certain issues by employing a murine model for experimental pulmonary metastasis and a tumor antigen vaccination strategy that induces complete tumor immunity in this system. Utilizing this model, we attempt to address issues related to unresponsiveness to tumor antigen immunization induced by passive administration of a rat monoclonal anti-CD4 and the induction of anti-idiotype responses to a passively administered monoclonal antibody and the effects on the induction of tumor immunity. Results: The results presented indicate that passive administration of rat monoclonal anti-CD4 exhibits immunosuppressive effects that inhibit the production of antibodies to the tumor antigen immunization and abolishes tumor immunity. Repeated administration of the rat monoclonal anti-CD4 results in an anti-idiotype response that can abrogate unresponsiveness to tumor antigen immunization and promote systemic tumor immunity. Conclusions: The data examine a number of potential problems associated with immunologic-based treatments for cancer. These problems include the potential for tolerance to the tumor antigen and establishing an immunocompromised state where immunization with a tumor antigen failed to generate tumor immunity. Approaches to eliminate tolerant T cells by targeting anti-CD4 via anti-idiotype responses that could be generated in vivo without CD4+ T cells allowed for recovery of nontolerant T cells, and an antibody response to the tumor antigen that results in tumor immunity.Abbreviations CTL
Cytotoxic T lymphocyte
- FITC
Fluorescein isothiocyanate
- OD
Optical density
- PBS
Phosphate-buffered saline
- SV40
Simian virus 40 相似文献
87.
CXC chemokine ligand 10 controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells 下载免费PDF全文
Trifilo MJ Montalto-Morrison C Stiles LN Hurst KR Hardison JL Manning JE Masters PS Lane TE 《Journal of virology》2004,78(2):585-594
How chemokines shape the immune response to viral infection of the central nervous system (CNS) has largely been considered within the context of recruitment and activation of antigen-specific lymphocytes. However, chemokines are expressed early following viral infection, suggesting an important role in coordinating innate immune responses. Herein, we evaluated the contributions of CXC chemokine ligand 10 (CXCL10) in promoting innate defense mechanisms following coronavirus infection of the CNS. Intracerebral infection of RAG1(-/-) mice with a recombinant CXCL10-expressing murine coronavirus (mouse hepatitis virus) resulted in protection from disease and increased survival that correlated with a significant increase in recruitment and activation of natural killer (NK) cells within the CNS. Accumulation of NK cells resulted in a reduction in viral titers that was dependent on gamma interferon secretion. These results indicate that CXCL10 expression plays a pivotal role in defense following coronavirus infection of the CNS by enhancing innate immune responses. 相似文献
88.
Christopher J. Vinyard Kenneth E. Glander Mark F. Teaford Cynthia L. Thompson Max Deffenbaugh Susan H. Williams 《International journal of primatology》2012,33(3):611-631
We lack a general understanding of how primates perform physiologically during feeding to cope with the challenges of their natural environments. We here discuss several methods for studying the ecological physiology of feeding in mantled howlers (Alouatta palliata) at La Pacifica, Costa Rica. Our initial physiological effort focuses on recording electromyographic activity (EMG) from the jaw muscles in free-ranging howlers while they feed in their natural forest habitat. We integrate these EMG data with measurements of food material properties, dental wear rates, as well as spatial analyses of resource use and food distribution. Future work will focus on incorporating physiological measures of bone deformation, i.e., bone strain; temperatures; food nutritional data; and hormonal analyses. Collectively, these efforts will help us to better understand the challenges that howlers face in their environment and the physiological mechanisms they employ during feeding. Our initial efforts provide a proof of concept demonstrating the methodological feasibility of studying the physiology of feeding in free-ranging primates. Although howlers offer certain advantages to in vivo field research, many of the approaches described here can be applied to other primates in natural habitats. By collecting physiological data simultaneously with ecological and behavioral data, we will promote a more synthetic understanding of primate feeding and its evolutionary history. 相似文献
89.
90.
Soil amendment interacts with invasive grass and drought to uniquely influence aboveground versus belowground biomass in aridland restoration 总被引:2,自引:0,他引:2
Water‐holding soil amendments such as super‐absorbent polymer (SAP) may improve native species establishment in restoration but may also interact with precipitation or invasive species such as Bromus tectorum L. (cheatgrass or downy brome) to influence revegetation outcomes. We implemented an experiment at two sites in Colorado, U.S.A., in which we investigated the interactions of drought (66% reduction of ambient rainfall), B. tectorum seed addition (BRTE, 465 seeds/m2), and SAP soil amendment (25 g/m2) on initial plant establishment and 3‐year aboveground and belowground biomass and allocation. At one site, SAP resulted in higher native seeded species establishment but only with ambient precipitation. However, by the third year, we detected no SAP effects on native seeded species biomass. Treatments interacted to influence aboveground and belowground biomass and allocation differently. At one site, a SAP × precipitation interaction resulted in lower belowground biomass in plots with SAP and drought (61.7 ± 7.3 g/m2) than plots with drought alone (91.6 ± 18.1 g/m2). At the other site, a SAP × BRTE interaction resulted in higher belowground biomass in plots with SAP and BRTE (56.6 ± 11.2 g/m2) than BRTE alone (35.0 ± 3.7 g/m2). These patterns were not reflected in aboveground biomass. SAP should be used with caution in aridland restoration because initial positive effects may not translate to long‐term benefits, SAP may uniquely influence aboveground versus belowground biomass, and SAP can interact with environmental variables to impact developing plant communities in positive and negative ways. 相似文献