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摘要:Background: Mesenchymal stromal cells (MSC) are being tested for the treatment of immune diseases. MSC are present in several adult tissues which milieu may influence MSC behavior particularly under inflammatory conditions. Additionally, culture conditions also can modify cell function or state of activation. Methods: To address the influence of the MSC source on its characteristics, we studied a xenofree, platelet lysate supplemented MSC from dental pulp, adipose tissue and bone marrow, co-cultured with isolated T cells and PBMC subset, and studied the effect of culture animal or human supplements immunomodulatory effect. Results: All three sources were efficient in inhibiting T cells. Among all MSC sources, as also described by others, adipose MSC was capable to significantly induce Treg phenotype and decrease T CD8+. Furthermore, comparing fetal bovine serum and platelet lysate, results demonstrate that platelet lysate alone is capable to induce immunomodulatory phenotype. Additional studies have to be made to elucidate the PL immunomodulatory effect....
[期刊论文] Afsana Munni
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2016年 03期
摘要:The glomerulonephritis disease is characterized by inflammation of glomeruli or small blood vessels in the kidney that causes kidney diseases. The reason of glomerulonephritis disease is to deposit the anti-GBM auto antibody in the glomerular basement membrane. The type IV collagen is the main component of glomerular basement membrane that has α3 chain of type (IV) collagen of non-collagenous domain which contains N-terminal 7S domain, a triple helical collagenous domain and C-terminal non-collagenous glomerular domain (NC1). The amino terminal of α3 (IV) NC1 that induces the Experimental Autoimmuno Glomerulonephritis (EAG) in rat model has been identified. The recombinant rat α3 (IV) NC1 antigen has nine amino acid spans that are consistent with antibody or T cell epitope that induces in EAG. The research is carried out on the recombinant rat α3 (IV) NC1 production, purification, quantification, and characterization. The circulation of anti-GBM antibody in glomerular basement membrane can be measured by the ELISA assay. In addition, the recombinant rat antigen is secreted in HEK293 cell supernatant that is purified by Anti-FLAG M2 monoclonal IgG antibody affinity column and characterized and quantified by SDS-PAGE gel electrophoresis and Western blotting techniques. ...
摘要:For today it is known, that primary and secondary disorders of the aerobic respiration, which are based on mitochondrial deficiency, lead to a wide spectrum of clinical manifestations and diseases. Therefore, the question about effective correction of various types of energy exchange disorders remains topical. Thus, the aim of our work was the study effect of the complex of biologically active substances (BAS) in ultra low concentrations on the activity of key enzymes of aerobic energy metabolism succinate dehydrogenase (EC 1.3.99.1) (SQR) and mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) (GPD2). The human lymphocytes assays were tested in vitro (22 donors). In negative control lymphocytes, cell culture normal saline solution was added. Normal saline solution with NaN3 was added in positive control lymphocytes cell culture. Experimental cell culture contained NaN3 and BAS. Our investigations had been revealed increase SQR activity in the experimental cell culture as compared with positive control culture throughout the time of experiment (measurements were carried out at 4, 24, 48, 72 h of incubation). The SQR activity of experimental cell culture and negative control lymphocytes cell culture was equal up to 24 h of experiment. It showed neutralization of NaN3 inhibitory effect (during 24 h) due to BAS influence. Activity of base glycerophosphate shunt ferment GPD2 of experimental lymphocyte cell culture was not different from GPD2 index in the negative control, but was lower than GPD2 activity in the positive control. It also indicated neutralization NaN3 inhibitory effect due to BAS influence. So we had found that extremely low concentrations of selected BAS with their complex impact on human lymphocytes in vitro could effectively neutralize the inhibitory effect of NaN3 on processes of aerobic energy metabolism link....
摘要:P2Y receptors belong to the family of G protein-coupled receptors and are activated by nucleotides in the extracellular space. We showed that Xenopus P2Y1 and P2Y11 were expressed in the dorsal marginal zone from early gastrula stage and enriched in the central nervous system from neurula stages. They were expressed in the prospective head region during early development. Knockdown of P2Y1 and P2Y11 caused head malformation, such as small eyes, brain atrophy, and defect in cartilage tissues, as well as reduced expression of neural, placode, and neural crest markers. Furthermore, the expression of neural plate and epidermal markers was affected by P2Y1 or P2Y11 depletion at early neurula stage, suggesting that P2Y1 or P2Y11 might be required for the neural induction. Our findings suggested that P2Y receptors might be involved in distinguishing between neural and non-neural fates. The results also suggested that P2Y1 or P2Y11 could play a role in neural induction and/or maintenance of neural tissues in the head formation processes....
[期刊论文] Bor Luen Tang
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2015年 03期
摘要:Mutations in genes encoding a key component of cytotoxic granules, or the machinery for their release, underlie the systemic hyperiflammatory symptoms of familial hemophagocytic lymphohistiocytosis (FHL), a typically pediatric onset autosomal recessive disorder with five known genetic subtypes (FHL1 - 5). FHL1 mutations have been mapped to chromosome 9, while the respective genes mutated in FHL2 (PRF1), FHL3 (UNC13D/Munc13-4), FHL4 (STX11) and FHL5 (STXBP2/ Munc18b/Munc18-2) have been identified. Perforin gene mutation directly affected the cytolytic activity of the cytotoxic granules. All the other FHL mutations appear to affect some aspect of cytotoxic granule exocytosis, resulting in impaired target cell killing by cytolytic T lymphocytes (CTLs) and/or natural killer (NK) cells. Recent findings suggest that failure to kill and detach from target cells, and prolonged synapse connection time, promote cytokine hypersecretion by the defective CTLs and NKs, which in turn result in systemic inflammation. Deciphering the genetics of FHL has contributed towards our understanding of the cell biology of hyperinflammatory responses and hemophagocytic lymphohistiocytosis accompanying pathological conditions such as cancer and viral infections....
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2015年 02期
摘要:Being sessile, plants are continuously exposed to DNA-damaging agents presenting in the environment such as ultraviolet (UV). Sunlight acts as an energy source for photosynthetic plants; hence, avoidance of UV radiations (namely, UV-A, 315 - 400 nm; UV-B, 280 - 315 nm; and UV-C, <280 nm) is unpreventable. DNA in particular strongly absorbs UV-B; therefore, it is the most important target for UV-B induced damages. This paper mainly used different doses of the enhanced UV-B radiation (B1 group: 4.05 kJ•m-2•d-1, B2 group: 10.08 kJ•m-2•d-1, B3 group: 7.05 kJ•m-2•d-1, B4 group: 23.02 kJ•m-2•d-1) treatment wheat, then, explored on the growth of wheat root and wheat root tip cell of chromosome aberration effect. In wheat, root-tip cells were observed with confocal laser scanning microscopy (CLSM), the results showed that low doses of B1 group (4.05 kJ•m-2•d-1) promoted the growth of wheat root and cell mitosis frequency. But high dose of B2 group (10.08 kJ•m-2•d-1), B3 group (17.05 kJ•m-2•d-1), B4 group (23.02 kJ•m-2•d-1) inhibited the growth of wheat root tip, and made crooked growth of wheat root, and inhibited the wheat root tip cell mitotic frequency and processed that induce root tip cells of wheat produce all kinds of aberration of chromosome in the interphase containing “multiple nucleoli nuclei”, “incomplete nuclei”, “long round nuclei”, “bean sprouts nucleus”. In mitosis M period contains “dissociative chromosome”, “chromosome bridge”, “adhesion chromosome”, “multi-bundle divide”, “nuclear anomalies”. After, high doses of enhanced UV-B radiation treatment, most of the cell cycle anomaly concentrated in mitosis interphase. In mitosis M period, with UV-B radiation dose enhanced chromosome aberration rate was on the rise and the aberration types also increasing....
[期刊论文] Bor Luen Tang
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2015年 02期
摘要:Other than the respiratory chain components, most mitochondrial proteins are synthesized in the cytosol and imported into the mitochondria. Many mitochondrial proteins therefore have at least a transient cytosolic appearance, and several have a dual mitochondrial-cytosol functional localization. However, recent work has revealed several proteins, one of which is a large protein complex, with dual mitochondrial and nuclear localizations. The enzyme fumarase which catalyzes the reversible hydration/dehydration of fumarate to malate is part of the mitochondria matrix tricarboxylic acid (TCA) cycle. It could, however, be recruited from the cytosol to the nucleus in response to DNA damage, where it is important for DNA repair. The pyruvate dehydrogenase complex (PDC) generates acetyl-CoA from pyruvate, and is recently shown to translocate from the mitochondrial matrix into the nuclear under mitogenic and stress conditions to generate acetyl–CoA within the nucleus. The mitochondrial monooxygenase CLK-1/COQ7 responsible for the synthesis of ubiquinone is most recently found to have a nuclear isoform with an uncleaved amino terminus, where it affects transcriptional changes associated with mitochondrial reactive oxygen species (ROS) generation. In this review, we highlight these unusual cases of nuclear localization of classically mitochondrial proteins, and discuss their possible functions in the nucleus....
摘要:Common additives in plastics such as bisphenol A (BPA) or phthalates like di-(2-ethylhexyl) phthalate (DEHP) are environmental estrogens that have been shown to be endocrine disruptors in some experimental animal models. This project used the C2C12 cell culture model to examine how exposure to BPA or DEHP affects two aspects of skeletal muscle development, the fusion of myoblasts into myotubes and agrin-induced clustering of acetylcholine receptors (AChRs). During myotube formation AChRs cluster spontaneously. Treatment with motor neuron derived agrin increases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. For this project C2C12 cells were exposed to BPA or DEHP while myoblasts fused into myotubes. After exposure to 10 μM BPA or 100 μM DEHP the frequency of agrin-induced AChR clusters decreased. In addition, myotube formation decreased as a higher percentage of nuclei remained in myoblasts. Furthermore, BPA or DEHP reduced the amount of the myogenic regulatory factor myogenin. This suggests that BPA and DEHP decrease AChR clustering by reducing myogenin. Moreover, plastic additives like BPA and DEHP may pose a risk for skeletal muscle development in humans....
摘要:Macrophages play a crucial role in detecting, regulating, and resolving immune crises, requiring migration through complex extracellular matrices. Unwarranted macrophage inflammatory activity potentiates kidney disease, rheumatoid arthritis, and transplant rejection. Proper remodeling of the actin cytoskeleton, especially at adhesion structures, is essential to the translocation of macrophages. Macrophages form actin-rich adhesions termed “podosomes”, giving them the capacity to make contacts with the substratum for traction through interstitial tissues. Macrophages express multiple formins, including FMNL1, Dia1, and Fhod1, with potential to impact actin remodeling involved in migration. Formins are a family of proteins that are best known for modifying the actin cytoskeleton via nucleation, elongation, bundling, and/or severing actin filaments. In this study we demonstrate that the formin FMNL1 is a key regulator of podosomes and is required for normal macrophage migration. Additionally, this is the first study to demonstrate defects in primary human cell migration resulting from specific formin silencing. Pharmacologic inhibition of all formin activity results in a significant decrease in podosome formation and normal macrophage migration. Furthermore, targeted suppression of FMNL1 results in decreases in macrophage migration similar to inhibition of all expressed macrophage formins. These novel findings suggest FMNL1 as a possible chemotherapeutic target to hinder macrophage migration, which could offer an innovative method for limiting unnecessary macrophage-mediated inflammation. We hypothesize that formins are required in podosome actin dynamics to support macrophage migration....
摘要:
Raw264.7 cells are monocytic cells that can differentiate to activated macrophages after lipopoly-saccharide (LPS) stimulation. Here, we analyzed the factors secreted by Raw264.7 cells in response to LPS. The culture media of LPS-treated Raw264.7 cells was able to stimulate growth in MEF1F2 and NIH3T3 mouse fibroblast cell lines. We identified five secreted and LPS-induced chemokines, CCL2, CCL5, CCL12, CxCL2, and CxCL10, by microarray analysis and tested their stimulatory activity. We used commercially available bacterially expressed proteins, and found only CCL12, CxCL2 and CxCL10 stimulated growth in MEF1F2 and NIH3T3 cells. The saturation density of the cells was also increased. They were not able to stimulate growth in v-Src transformed MEF1F2 or SWAP-70 transformed NIH3T3 cells. We examined signaling pathways activated by these three factors. We found that ERK and p38 MAP kinase were activated and were required for the activity to stimulate the cell growth. Other pathways including phosophatidylinositol-3 kinase (PI3K), NFκB pathways were not activated. These results suggest that Raw264.7 cells secretes growth stimulation factors for fibroblasts when differentiated to macrophages implicating that fast growth of them is related to inflamation although the reason is still unclear.
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摘要:
A battery of sex steroids were used to check their effect on mouse melanoma (B16F10) cell growth in-vitro. Progesterone and its synthetic receptor antagonist RU-486 showed maximum inhibition on in-vitro melanoma cell growth [1]. Further research work showed that the inhibition by progesterone was not a toxic, spurious or non-specific effect on mouse melanoma cell growth and the inhibition by progesterone was not mediated through progesterone receptor.
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摘要:Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells....
摘要:Lipofuscin-like pigments (LFPs) are highly oxidized cross-linked aggregates of oxidized protein and lipids which are formed under oxidative state conditions by free radicals produced. The present study aimed to evaluate the probable ameliorative effects of some of the Mn-salens namely EUKs 8, 134, 15, 115, 122 and 132 (compounds 1-6) and vitamin C against carbon tetrachloride (CCl4)-induced acute damage on rats' livers and brains. Exposure to CCl4 is believed to induce oxidative stress and cause tissue damage due to the formation of trichloromethyl (·CCl3) and peroxy trichloromethyl (·OOCCl3) radicals. In this study, 54 rats were randomly divided into 9 groups of six each: normal group received only vehicle (olive oil; 2 ml/kg b.w.) for 6 consecutive days; CCl4- intoxicated group received the vehicle and CCl4 (50% solution of CCl4 in olive oil, 2 ml/kg b.w.) on the first and second days and the vehicle on the third to sixth days; test rats received Mn-salens or vitamin C (20 mg/kg b.w.) and CCl4 (2 ml/kg b.w.) on the first and second days and Mn-salens or vitamin C (20 mg/kg b.w.) on the third to sixth days. Mn-salens administration ameliorated the effects of CCl4 by decreasing the levels of ROS, lipid and protein oxidations and LFPs formation on liver and brain as well as cholesterol and triglycerides, aminotransferases and alkaline phosphatase contents in sera of rats whereas increased the activities of catalase, superoxide dismutase,glutathione reductase, glutathione peroxidase and reduced glutathione in liver and brain tissues. Histopathological studies confirmed the toxic effects of CCl4 and ameliorative action of Mn-salens on tissues. These results suggest that the evaluated EUKs were able to attenuate LFPs accumulation and morphological changes caused by CCl4 in rats and thus, confirming the ameliorative role of Mn-salens against CCl4-induced oxidative damage and age-related diseases....
摘要:
Transporter associated with antigen processing (TAP)-like (TAPL; ABCB9) is a half-type ABC transporter with sequence similarity to TAP1 and TAP2 that function in the ER membrane. To determine the cellular localization, TAPL and truncated forms of it were tagged with GFP at their car-boxyl termini. Intracellular localization of these fusion proteins was compared between transient and stable expression in CHO-K1 cells. When they were expressed transiently, the fluorescence of the fusion proteins was detected on the intracellular membrane, mainly in the ER, and all the fusion proteins, i.e., TAPL(M1 -A766)-GFP, TAPL(M1-S275)-GFP, TAPL(M1-K182 )-GFP, TAPL(M 1-R141)-GFP and TAPL(M1-G75), were co-localized with an ER marker, PDI. However, the fluorescence of all of them except for TAPL(M1-G 75)-GFP and TAPL(M1-S275)-GFP overlapped with a lysosome marker, cathepsin D, upon stable expression. Lysosomal localization was similarly observed with TAPL(M1- A766)-DsRed, which was stably expressed. These results suggest that TAPL is sorted to the lysosomal membrane when expressed stably in CHO-K1 cells. Furthermore, the lysosomal targeting signal may comprise the N-terminal four transmembrane helices since the N-terminal two transmembrane helices may not be enough to function as such a signal.
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摘要:The purpose of the work was to assess the combined effect of drought and salinity (50, 100, 200 mМ NaCl) on the meso- and ultrastructure of mesophyll cells of wheat seedlings. Stress development was estimated by a decrease in the relative water content (RWC) and CO2-dependent O2 evolution (An) in leaves. The decrease in the RWC and in An occurred rapidly in the absence of salt in the substrate and slowly in the presence of salt, especially at a treatment of 100 mM NaCl. The resumption of watering led to the recovery of the both parameters in all variants except one with 200 mM NaCl. Structural studies showed that a weak drought stress (RWC 60%) without salinity led to the destruction of cell membranes and hyaloplasm, which did not occur in all salt treatments. By contrast, the ultrastructure of nuclei in weak drought without salinity remained unchanged, whereas in all salt treatments chromatin changed substantially. Heterochromatin underwent a strong condensation followed by the fusion into a united mass with the simultaneous loss of electron density. A strong water stress (RWC 40%) in all variants led to cell destruction and the hydrolysis of cell compounds. Under the drought without salinity, vacuoles disappeared, whereas in salt-treated samples they were retained and filled with organelles being at different degrees of degradation. Cell nuclei under strong drought stress lost their rounded shape, nuclear envelopes were destroyed, and at the end only a finely dispersed substance remained. Thus, under the combined action of drought and salt, there is some critical level of salt concentration in substrate above which the effect of NaCl changes to the adverse, which enhances the action of drought. Among structural components of mesophyll cells, the most sensitive parts to NaCl are nuclei and their chromatin....
[期刊论文] Bor Luen Tang
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2014年 04期
摘要:Pyruvate is a key intermediate at the branchpoint of anaerobic and aerobic energy metabolism. Its transport into the mitochondrial matrix is necessary prior to its decarboxylation into acetyl-CoA, which feeds the reducing equivalent-generating tricarboxylic acid (TCA) cycle. Although the existence of specific carrier transport of cytosolic pyruvate into the mitochondria has been inferred from a myriad of studies, the identities of the mitochondrial pyruvate carrier (MPC) were only confirmed very recently. Identification of the MPC facilitated several other recent advances. These include the finding of MPC's inhibition by the insulin-sensitizing drug family thiazolidinediones, how cells respond flexibly to a reduction in MPC functionality, as well as insights into how changes in MPC levels affect oncogenic potential of cancer cells. These new findings, discussed here in this brief review, have important implications in therapeutic approaches towards metabolic disorders and cancer....
摘要:Mesenchymal stromal cells(MSC)have shown their benefits in graft-versus-host disease(GVHD), with five unsettled matters: 1) MSCs expansion in medium with Fetal Bovine Serum(FBS)and its risk of xenoreaction; 2) The number of cells indicated for therapy that is relatively high, with the need to optimize the expansion, number and time wise; 3) The utilization of third party donors; 4) Culture passage number(P);and 5) Source of the cells. This study was designed to determine the superiority of the Platelet Lysates(PL)over FBS on the expansion of MSC, the optimal cell' plating density and days between each pass, and to investigate if donor total nucleated cells(TNC)obtained from the washouts of discharged bags and filters of hematopoietic stem cell transplantation(HSCT)can be expanded to be used at clinical grade. TNC were removed, plated and after the first passage were cultivated in different concentrations with FBS or PL, and the number of days to reach 80% of confluence was observed. Next, cultures with the same plating density were fed either with PL or with FBS and after seven days counted to analyze how much they had grown in that period. The proliferation of mesenchymal stromal cells in the presence of PL and SFB was averaged 11.88 and 2.5 times, respectively, in a period of 7 days. The highest concentration of plating cells using PL took less time to reach confluence as compared with the three lower ones. This study suggests that the PL is the best choice as a supplement to expand MSC and to allow the proliferation of enough number of MSC at P2 for clinical use....
摘要:
The primary culture bone marrow (PCBM) cells are often used in different cytological investigations. Here we study the behavior of porcine unstimulated PCBM cells during the cultivation. DNA cytophotometry analyses revealed that immature white blood cells (WBC) such as lymphoblasts, monoblasts and myeloid cells during cultivation process reduced the DNA content in the nuclei. This resulted in displacement of the DNA histogram to the left and increased the content of diploid nuclei. This is a result of cells unblocking which are in G2 cell cycle phase. We also observed diploid pathological cells with accessory or fragmentized nuclei. However, the data with erythroid cells (EC) somehow demonstrate the opposite tendency. During the late stage of cultivation, the number of immature EC with the tetraploid DNA is increased.
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摘要:
Oxidative stress has been implicated to play a crucial role in the pathogenesis of Alzheimer's disease (AD). Currently, it is known that numerous signaling pathways involved in neurodegenerative disorders are activated in response to oxidative stress. Recent directions on AD treatments have focused on the use of antioxidants including Curcumin, a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa, to augment the intracellular antioxidant defences. In the present study, hydrogen peroxide (H2O2) was used to evaluate the effects of oxidative stress on apoptotic SK-N-MC cells death with focus on changes in activity of Notch signaling pathway. The extent of lipid peroxidation, protein oxidation and intracellular ROS (Reactive Oxygen Species) levels was investigated as oxidative stress biomarkers. Here, we showed that H2O2 reduced GSH levels and activity of antioxidant enzymes and also influenced Notch signaling activation. The present data concluded that Curcumin protected cells against oxidative stress-induced apoptosis.
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摘要:Atypical PKC (aPKC) plays a role in establishing cell polarity and has been indicated in neuronal differentiation and polarization, including neurite formation in rat pheochromocytoma PC12 cells, albeit by unclear mechanisms. Here, the role of the aPKC isoform, PKC iota (PKCι), in the early neuronal differentiation of PC12 cells, was investigated. NGF-treated PC12 cells with stably expressed exogenous wild-type PKCι showed decreased expression of a neuroendocrine marker, increased expression of a neuronal marker and increased neurite formation. Stable expression of a kinase-inactive PKCι, but not constitutively active PKCι lacking a regulatory domain, had similar though less potent effects. Pharmacological inhibition of endogenous aPKC kinase activity in parental PC12 cells did not inhibit neurite formation, suggesting that some of the observed effects of PKCι expression on neuronal differentiation are kinase-independent. Interestingly, exogenous expression of wild-type and kinase-inactive PKCι had little effect on overall PKCι activity, but caused a decrease in PKC zeta (PKCζ) kinase activity, suggesting an interplay between the two isoforms that may underlie the observed results. Overall, these findings suggest that in PC12 and perhaps other neuroendocrine precursor cells, PKCι influences an early differentiation decision between the neuroendocrine (chromaffin) and sympathetic neuron cell lineages, potentially by affecting PKCζ function....
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