Activation of Apoptosis in a βB1-CTGF Transgenic Mouse Model

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in βB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5–9/group) and quantitative real-time PCR analysis (n = 5–7/group) in 5- and 10-week-old βB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in βB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL+) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the βB1-CTGF mice a suitable model to study primary open-angle glaucoma.

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Regulation of Fas Ligand Expression by IL-8 in Human Endometrium

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.87.8.8713 ◽

2002 ◽

Vol 87 (8) ◽

pp. 3921-3927 ◽

Cited By ~ 36

Author(s):

Belgin Selam ◽

Umit A. Kayisli ◽

Juan A. Garcia-Velasco ◽

G. Eda Akbas ◽

Aydin Arici

Keyword(s):

T Lymphocytes ◽

Protein Expression ◽

Stromal Cells ◽

Fas Ligand ◽

Terminal Deoxynucleotidyl Transferase ◽

Pcr Analysis ◽

Mrna Levels ◽

Fasl Expression ◽

Endometrial Stromal Cells ◽

Endometrial Cells

Numerous cytokines and growth factors are synthesized in the endometrium. IL-8 is one of these cytokines regulating endometrial function. It is a neutrophil chemoattractant/ activating factor and a potent angiogenic agent. IL-8 is elevated in the peritoneal fluid of women with endometriosis. We have previously demonstrated a direct proliferative effect of IL-8 on endometrial stromal cells. We hypothesized that increased levels of IL-8 in the endometriotic environment could up- regulate Fas ligand (FasL) expression in endometrial cells and may be relevant for the development of a relative local immunotolerance in endometriosis by inducing apoptosis of cytotoxic T lymphocytes. To test our hypothesis, we studied the in vitro regulation of FasL expression and apoptosis by IL-8 in endometrial cells. Western blot analysis in endometrial stromal, glandular, and Ishikawa cells revealed that IL-8 up- regulated FasL protein expression in these cells. By semiquantitative RT-PCR analysis, IL-8 does not alter the expression of either Fas or FasL mRNA levels in these cells. Immunocytochemistry results from endometrial stromal cells treated with IL-8 demonstrated an up-regulation of FasL protein expression. IL-8 decreased apoptosis rate in endometrial stromal cells as evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. We observed an increased apoptotic rate in Jurkat (T lymphocyte line) cells plated on endometrial stromal cells previously treated with IL-8. We speculate that increased FasL expression by IL-8 may induce apoptosis of T lymphocytes and thus produce a local immunotolerant environment for the development of ectopic implants.

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Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Applied Sciences ◽

10.3390/app11135776 ◽

2021 ◽

Vol 11 (13) ◽

pp. 5776

Author(s):

Varvara G. Blinova ◽

Natalia S. Novachly ◽

Sofya N. Gippius ◽

Abdullah Hilal ◽

Yulia A. Gladilina ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Pcr Analysis ◽

Mrna Levels ◽

Rt Pcr ◽

Inflammatory Reactions ◽

Effector Cells ◽

Cycle Regulation ◽

Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

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Pulchinenosides from Pulsatilla Chinensis Increase P-Glycoprotein Activity and Induce P-Glycoprotein Expression

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2020/4861719 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

Yali Liu ◽

Ling Zhang ◽

Shaofeng Wei ◽

Jinyang Cai ◽

Zhenzhong Zang ◽

...

Keyword(s):

Membrane Vesicles ◽

Pcr Analysis ◽

Mrna Levels ◽

Intestinal Epithelial ◽

Glycoprotein P ◽

P Glycoprotein ◽

Human Intestinal Epithelial Cells ◽

Pulsatilla Chinensis ◽

Dependent Transport ◽

Cytotoxic Studies

Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 μM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 μM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.

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Cloning and functional analysis of the FAD2 gene family from desert shrub Artemisia sphaerocephala

BMC Plant Biology ◽

10.1186/s12870-019-2083-5 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

Xiumei Miao ◽

Lijing Zhang ◽

Xiaowei Hu ◽

Shuzhen Nan ◽

Xiaolong Chen ◽

...

Keyword(s):

Fatty Acid ◽

Linoleic Acid ◽

Gene Family ◽

Fatty Acid Biosynthesis ◽

Family Members ◽

Pcr Analysis ◽

Mrna Levels ◽

Acid Biosynthesis ◽

Desert Shrub ◽

Artemisia Sphaerocephala

Abstract Background Linoleic acid is an important polyunsaturated fatty acid, required for all eukaryotes. Microsomal delta-12 (Δ12) oleate desaturase (FAD2) is a key enzyme for linoleic acid biosynthesis. Desert shrub Artemisia sphaerocephala is rich in linoleic acid, it has a large FAD2 gene family with twenty-six members. The aim of this work is to unveil the difference and potentially functionality of AsFAD2 family members. Results Full-length cDNAs of twenty-one AsFAD2 genes were obtained from A. sphaerocephala. The putative polypeptides encoded by AsFAD2 family genes showed a high level of sequence similarity and were relatively conserved during evolution. The motif composition was also relatively conservative. Quantitative real-time PCR analysis revealed that the AsFAD2–1 gene was strongly expressed in developing seeds, which may be closely associated with the high accumulating ability of linoleic acid in A. sphaerocephala seeds. Although different AsFAD2 family members showed diverse response to salt stress, the overall mRNA levels of the AsFAD2 family genes was stable. Transient expression of AsFAD2 genes in the Nicotiana benthamiana leaves revealed that the encoded proteins were all located in the endoplasmic reticulum. Heterologous expression in Saccharomyces cerevisiae suggested that only three AsFAD2 enzymes, AsFAD2–1, − 10, and − 23, were Δ12 oleate desaturases, which could convert oleic acid to linoleic acid, whereas AsFAD2–1 and AsFAD2–10 could also produce palmitolinoleic acid. Conclusions This research reported the cloning, expression studies, subcellular localization and functional identification of the large AsFAD2 gene family. These results should be helpful in understanding fatty acid biosynthesis in A. sphaerocephala, and has the potential to be applied in the study of plant fatty acids traits.

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Enhanced activity of ventricular Na+-HCO3− cotransport in pressure overload hypertrophy

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00964.2006 ◽

2007 ◽

Vol 293 (2) ◽

pp. H1254-H1264 ◽

Cited By ~ 24

Author(s):

Taku Yamamoto ◽

Takeshi Shirayama ◽

Tomohiko Sakatani ◽

Tomosaburo Takahashi ◽

Hideo Tanaka ◽

...

Keyword(s):

Ventricular Myocytes ◽

Single Cells ◽

Ischemia Reperfusion ◽

Pressure Overload ◽

Pcr Analysis ◽

Mrna Levels ◽

Aortic Constriction ◽

Ph Indicator ◽

Adult Rat ◽

Acid Extrusion

The Na+-HCO3− cotransporter (NBC) plays a key role in intracellular pH (pHi) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pHi was measured in single cells using the fluorescent pH indicator 2′,7′-bis(2-carboxyethyl)5-( 6 )carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC ( JNBC) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na+/H+ exchange was also increased in hypertrophied myocytes, the relative enhancement of JNBC was larger, 6) membrane depolarization markedly increased JNBC in hypertrophied myocytes, and 7) losartan, an ANG II AT1 receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na+ overload, which may render the ventricles more vulnerable to Ca2+ overload during ischemia-reperfusion.

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Receptive field of the retinal bipolar cell: a pharmacological study in the tiger salamander

Journal of Neurophysiology ◽

10.1152/jn.1996.76.3.2005 ◽

1996 ◽

Vol 76 (3) ◽

pp. 2005-2019 ◽

Cited By ~ 42

Author(s):

W. A. Hare ◽

W. G. Owen

Keyword(s):

Receptive Field ◽

Gaba Receptors ◽

Bipolar Cell ◽

Horizontal Cell ◽

Pharmacological Study ◽

Bipolar Cells ◽

Horizontal Cells ◽

Gaba Uptake ◽

Gamma Aminobutyric Acid ◽

Gabaergic Synapse

1. It is widely believed that signals contributing to the receptive field surrounds of retinal bipolar cells pass from horizontal cells to bipolar cells via GABAergic synapses. To test this notion, we applied gamma-aminobutyric acid (GABA) agonists and antagonists to isolated, perfused retinas of the salamander Ambystoma tigrinum while recording intracellularly from bipolar cells, horizontal cells, and photoreceptors. 2. As we previously reported, administration of the GABA analogue D-aminovaleric acid in concert with picrotoxin did not block horizontal cell responses or the center responses of bipolar cells but blocked the surround responses of both on-center and off-center bipolar cells. 3. Surround responses were not blocked by the GABA, antagonists picrotoxin or bicuculline, the GABAB agonist baclofen or the GABAB antagonist phaclofen, and the GABAC antagonists picrotoxin or cis-4-aminocrotonic acid. Combinations of these drugs were similarly ineffective. 4. GABA itself activated a powerful GABA uptake mechanism in horizontal cells for which nipecotic acid is a competitive agonist. It also activated, both in horizontal cells and bipolar cells, large GABAA conductances that shunted light responses but that could be blocked by picrotoxin or bicuculline. 5. GABA, administered together with picrotoxin to block the shunting effect of GABAA activation, did not eliminate bipolar cell surround responses at concentrations sufficient to saturate the known types of GABA receptors. 6. Surround responses were not blocked by glycine or its antagonist strychnine, or by combinations of drugs designed to eliminate GABAergic and glycinergic pathways simultaneously. 7. Although we cannot fully discount the involvement of a novel GABAergic synapse, the simplest explanation of our findings is that the primary pathway mediating the bipolar cell's surround is neither GABAergic nor glycinergic.

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Age-Related Alterations in the Behavior and Serotonin-Related Gene mRNA Levels in the Brain of Males and Females of Short-Lived Turquoise Killifish (Nothobranchius furzeri)

Biomolecules ◽

10.3390/biom11101421 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1421

Author(s):

Valentina S. Evsiukova ◽

Elizabeth A. Kulikova ◽

Alexander V. Kulikov

Keyword(s):

Locomotor Activity ◽

Body Mass ◽

Mrna Level ◽

Model Organism ◽

The Body ◽

Suitable Model ◽

Mrna Levels ◽

Age Related ◽

Nothobranchius Furzeri ◽

Males And Females

Short-lived turquoise killifish (Nothobranchius furzeri) have become a popular model organism for neuroscience. In the present paper we study for the first time their behavior in the novel tank diving test and the levels of mRNA of various 5-HT-related genes in brains of 2-, 4- and 6-month-old males and females of N. furzeri. The marked effect of age on body mass, locomotor activity and the mRNA level of Tph1b, Tph2, Slc6a4b, Mao, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes in the brains of N. furzeri males was shown. Locomotor activity and expression of the Mao gene increased, while expression of Tph1b, Tph2, Slc6a4b, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes decreased in 6-month-old killifish. Significant effects of sex on body mass as well as on mRNA level of Tph1a, Tph1b, Tph2, Slc6a4b, Htr1aa, 5-HT2a, Htr3a, Htr3b, Htr4, and Htr6 genes were revealed: in general both the body mass and the expression of these genes were higher in males. N. furzeri is a suitable model with which to study the fundamental problems of age-related alterations in various mRNA levels related with the brains 5-HT system.

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Expression patterns of endothelial and inducible nitric oxide synthase isoforms in corpora lutea of pseudopregnant rabbits at different luteal stages

Journal of Endocrinology ◽

10.1677/joe.0.1730285 ◽

2002 ◽

Vol 173 (2) ◽

pp. 285-296 ◽

Cited By ~ 11

Author(s):

C Boiti ◽

D Zampini ◽

G Guelfi ◽

F Paolocci ◽

M Zerani ◽

...

Keyword(s):

Nitric Oxide ◽

Expression Patterns ◽

Physiological Role ◽

Total Activity ◽

Pcr Analysis ◽

Inos Mrna ◽

Corpora Lutea ◽

Mrna Levels ◽

Isolated Cells ◽

Protein Levels

Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P</=0.05) by day 13. By contrast, luteal eNOS proteins increased 2-fold (P</=0.05) from the early- to late-luteal phase. Independently of CL age, iNOS mRNA was very poorly expressed while protein levels gradually declined from the early- to late-luteal stage. Intense eNOS-like immunoreactivity was detected in large luteal cells, while iNOS staining was targeted to a few, isolated cells, probably macrophages. Basal NOS activity was greater in day 4 CL than in both day 9 and day 13 CL. These data are the first to characterize in rabbit CL the temporal expression patterns of NOS isoforms across different luteal stages of pseudopregnancy and, collectively, suggest the existence of an expressional control for this constitutive isoform, which might have a physiological role in regulating CL function during development.

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GmGSTU13 is related to the development of mosaic symptoms in soybean plants infected with Soybean mosaic virus

Phytopathology ◽

10.1094/phyto-11-20-0498-r ◽

2021 ◽

Author(s):

Kai Zhang ◽

Yingchao Shen ◽

Tao Wang ◽

Yu Wang ◽

Song Xue ◽

...

Keyword(s):

Mosaic Virus ◽

Soybean Mosaic Virus ◽

Positive Control ◽

Pcr Analysis ◽

Mrna Levels ◽

Protein Levels ◽

Cp Gene ◽

Viral Coat ◽

Soybean Plants ◽

Different Strains

The leaves of soybean cv. ZheA8901 show various symptoms (necrosis, mosaic and symptomless) when infected with different strains of Soybean mosaic virus (SMV). Based on a proteomic analysis performed with tandem mass tags (TMT), 736 proteins were differentially expressed from soybean samples that showed asymptomatic, mosaic and necrosis symptoms induced by SMV strains SC3, SC7, and SC15, respectively. Among these, GmGSTU13 and APX (ascorbate peroxidase) were only upregulated in mosaic and symptomless leaves, respectively. The protein level of GmGSTU13 determined by Western blot was consistent with TMT analysis, qRT-PCR analysis showed that GmGSTU13 mRNA levels in mosaic plants was 5.26- and 3.75-fold higher than that in necrotic and symptomless plants, respectively. Additionally, the expression of viral coat protein (CP) gene was increased, and serious mosaic symptoms were observed in GmGSTU13-overexpressing plants inoculated with all three SMV strains. These results showed that GmGSTU13 is associated with the development of SMV-induced mosaic symptoms in soybean and that APX is upregulated in symptomless leaves at both the transcriptional and protein levels. In APX gene-silenced soybean plants, the relative expression of the viral CP gene was 1.50, 7.59 and 1.30 times higher than in positive control plants inoculated with the three SMV strains, suggesting that the upregulation of APX may be associated with lack of symptoms in soybean infected with SMV. This work provides a useful dataset for identifying key proteins responsible for symptom development in soybean infected with different SMV strains.

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Investigation of the effect of Prunus Amygdalus Amara on the expression of some genes of apoptosis and immortality in breast cancer cells (MCF-7)

Current Drug Research Reviews ◽

10.2174/2589977513666211202094433 ◽

2021 ◽

Vol 13 ◽

Author(s):

Maryam Abdolahi-Majd ◽

Gholamhossein Hassanshahi ◽

Mahboubeh Vatanparast ◽

Mojgan Noroozi Karimabad

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Analysis ◽

Mrna Levels ◽

Prunus Amygdalus ◽

Mcf7 Cells ◽

Significant Difference ◽

Anti Cancer ◽

Bitter Almond ◽

Mcf 7

Background: Anti-cancer effects of almond nuts or oil have been approved, but there are a few pieces of research that have evaluated, in detail, almond and other seeds' effects on cancer. Therefore, in the present project, the aim was to explore the regulatory effect of the bitter almond extract (Prunus amygdalus Batsch) on the apoptotic and anti-cancer potency of MCF-7 cells. Objectives: In the current experimental research, the Almond effect on MCF7 cells was evaluated by investigating the expression and the balance between Bcl-2, Bax genes to unmark the potential molecular mechanism. Methods: For 24 and 48h, the MCF7 cells were treated with the bitter almond extract (187.5-3000 µg/mL). MTT assay was used to assess the viability, and Real-time-PCR was applied to determine the expression of Bax and Bcl-2, facing β-actin. Results: Our results revealed a significant difference between different extract concentrations on the viability of MCF7 cell lines in 24 and 48 h; cell viability decreased time-dependently (P < 0.05). After 24 and 48h of extract facing MCF7 cells, the evaluated IC50 value was 3000 and 1500 µg/mL, respectively. Based on Real Time-PCR analysis, after 24 and 48 h, the mRNA levels of BCL-2 decreased by the extract, whereas BAX was in the MCF-7 cell line. Conclusion: From the results, it can be concluded that bitter almond extract has anti-cancer properties that may influence the apoptotic pathways by regulating relative gene expression.

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