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211.
Bussenius J Anand NK Blazey CM Bowles OJ Bannen LC Chan DS Chen B Co EW Costanzo S DeFina SC Dubenko L Engst S Franzini M Huang P Jammalamadaka V Khoury RG Kim MH Klein RR Laird D Le DT Mac MB Matthews DJ Markby D Miller N Nuss JM Parks JJ Tsang TH Tsuhako AL Wang Y Xu W Rice KD 《Bioorganic & medicinal chemistry letters》2012,22(6):2283-2286
212.
Purpose
Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in–vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures.Methods
Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA.Results
Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15–20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed ‘spherules’ with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons).Conclusion
The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future. 相似文献213.
C Miaskowski BA Cooper A Dhruva LB Dunn DJ Langford JK Cataldo CR Baggott JD Merriman M Dodd K Lee C West SM Paul BE Aouizerat 《PloS one》2012,7(7):e40560
The purposes of this study were to identify distinct latent classes of individuals based on subjective reports of sleep disturbance; to examine differences in demographic, clinical, and symptom characteristics between the latent classes; and to evaluate for variations in pro- and anti-inflammatory cytokine genes between the latent classes. Among 167 oncology outpatients with breast, prostate, lung, or brain cancer and 85 of their FCs, growth mixture modeling (GMM) was used to identify latent classes of individuals based on General Sleep Disturbance Scale (GSDS) obtained prior to, during, and for four months following completion of radiation therapy. Single nucleotide polymorphisms (SNPs) and haplotypes in candidate cytokine genes were interrogated for differences between the two latent classes. Multiple logistic regression was used to assess the effect of phenotypic and genotypic characteristics on GSDS group membership. Two latent classes were identified: lower sleep disturbance (88.5%) and higher sleep disturbance (11.5%). Participants who were younger and had a lower Karnofsky Performance status score were more likely to be in the higher sleep disturbance class. Variation in two cytokine genes (i.e., IL6, NFKB) predicted latent class membership. Evidence was found for latent classes with distinct sleep disturbance trajectories. Unique genetic markers in cytokine genes may partially explain the interindividual heterogeneity characterizing these trajectories. 相似文献
214.
215.
Heterogeneity in the content and function of subcellular organelles on the intercellular and intracellular level plays an important role in determining cell fate. These variations extend to normal-state and disease-state cellular functions and responses to environmental stimuli, such as oxidative stress and therapeutic drugs. Analytical tools to characterize variation in all types of organelles are essential to provide insights that can lead to advances in medicine, such as therapies targeted to specific subcellular regions. In this review, we discuss analytical techniques for interrogating individual intact organelles (e.g. mitochondria and synaptic vesicles) and lysates in a high-throughput manner, including a recently developed nanoscale fluorescence-activated subcellular sorter and techniques based on capillary electrophoresis with laser-induced fluorescence detection. We then highlight the advantages that droplet microfluidics offers for probing subcellular heterogeneity. 相似文献
216.
Bhandaru M Pasham V Yang W Bobbala D Rotte A Lang F 《Cellular physiology and biochemistry》2012,29(3-4):533-542
Azathioprine is a powerful immunosuppressive drug, which is partially effective by interfering with the maturation and function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs are stimulated by bacterial lipopolysaccharides (LPS), which trigger the formation of reactive oxygen species (ROS), paralleled by activation of the Na(+)/H(+) exchanger. The carrier is involved in the regulation of cytosolic pH, cell volume and migration. The present study explored whether azathioprine influences Na(+)/H(+) exchanger activity in DCs. DCs were isolated from murine bone marrow, cytosolic pH (pH(i)) was estimated utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) fluorescence, Na(+)/H(+) exchanger activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNFα release utilizing ELISA, and migration utilizing transwell migration assays. Exposure of DCs to lipopolysaccharide (LPS, 1 μg/ml) led to a transient increase of Na(+)/H(+) exchanger activity, an effect paralleled by ROS formation, increased cell volume, TNFα production and stimulated migration. Azathioprine (10 μM) did not significantly alter the Na(+)/H(+) exchanger activity, cell volume and ROS formation prior to LPS exposure but significantly blunted the LPS-induced stimulation of Na(+)/H(+) exchanger activity, ROS formation, cell swelling, TNFα production and cell migration. In conclusion, azathioprine interferes with the activation of dendritic cell Na(+)/H(+) exchanger by bacterial lipopolysaccharides, an effect likely participating in the anti-inflammatory action of the drug. 相似文献
217.
Sitaram A Dennis MK Chaudhuri R De Jesus-Rojas W Tenza D Setty SR Wood CS Sviderskaya EV Bennett DC Raposo G Bonifacino JS Marks MS 《Molecular biology of the cell》2012,23(16):3178-3192
Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine-based sorting signal in the pigment cell-specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1- and AP-3-favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs. 相似文献
218.
V Anand N Dogra S Singh SN Kumar MK Jena D Malakar AK Dang BP Mishra TK Mukhopadhyay JK Kaushik AK Mohanty 《PloS one》2012,7(7):e40469
Background
The objective of this study was to establish the buffalo mammary epithelial cell line (BuMEC) and characterize its mammary specific functions.Methodology
Buffalo mammary tissue collected from the slaughter house was processed enzymatically to obtain a heterogenous population of cells containing both epithelial and fibroblasts cells. Epithelial cells were purified by selective trypsinization and were grown in a plastic substratum. The purified mammary epithelial cells (MECs) after several passages were characterized for mammary specific functions by immunocytochemistry, RT-PCR and western blot.Principal Findings
The established buffalo mammary epithelial cell line (BuMEC) exhibited epithelial cell characteristics by immunostaining positively with cytokeratin 18 and negatively with vimentin. The BuMEC maintained the characteristics of its functional differentiation by expression of β-casein, κ-casein, butyrophilin and lactoferrin. BuMEC had normal growth properties and maintained diploid chromosome number (2n = 50) before and after cryopreservation. A spontaneously immortalized buffalo mammary epithelial cell line was established after 20 passages and was continuously subcultured for more than 60 passages without senescence.Conclusions
We have established a buffalo mammary epithelial cell line that can be used as a model system for studying mammary gland functions. 相似文献219.
Role of the non‐haem, manganese catalase (Mn‐catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn‐catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat+. The Alr0998 protein could be immunodetected in AnKat+ cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat+ cells but not in the wild‐type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn‐catalase. In response to oxidative stress, the AnKat+ showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress‐inducible 2‐Cys‐Prx protein. Treatment of wild‐type Anabaena PCC7120 with H2O2 caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO2 fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat+ strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn‐catalase. 相似文献
220.
Arjun Chatterjee Manan Shah Anna O D'Souza Benno Bechtel Glenn Crater Anand A Dalal 《Respiratory research》2012,13(1):15