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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: In-Cell Western

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: In-Cell WesternWestern Blot Analysis
Total protein extracted from human decidual tissues from the early pregnancy termination (n = 6) and unexplained miscarriage (n = 6) were prepared using RIPA buffer. Then 60 ig protein was loaded onto a 10% polyacrylamide-SDS gel. The resolved proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad) and incubated with a 1:500 dilution of mouse anti-human CD82 monoclonal antibody and a 1:1000.
Statistics
All values are presented as the mean 6 SD. One-way ANOVA was used to detect the difference of invasion, CD82, and other molecule transcriptions and translations in human DSCs and BeWo cells. Differences were considered as statistically significant at P < 0.05.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Immunofluorescence (IF)

Immunofluorescence (IF)
The CD82-transfected BeWo cells and CD82-silenced DSCs were plated on coverslips in 24-well plates (BD Biosciences) for 96 h. Monolayers were fixed with 4% (vol/vol) paraformaldehyde for 10 min at room temperature, followed by 10-min incubation with 0.2% (vol/vol) Triton X-100. After washing with PBS, nonspecific binding was blocked with 10% FBS in PBS. Anti-CD82 rabbit polyclonal antibody (1:50; SC-5540; Santa Cruz Biotechnology) and anti-TIMP1 and anti-integrinp1 mouse monoclonal antibodies diluted in PBS were added for 1 h, avoiding light, and then the slides were mounted in 0.1% (wt/vol) 4,6-diamidino-2-phenylindole (DAPI; 1:50; Invitrogen) for nuclear counterstaining for 5 min at room temperature and observed in an Olympus BX51 fluorescence microscope (Olympus). The secondary antibodies were Texas Red and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit and anti-mouse IgG (1:250; Rockland). The experiments were carried out in triplicate and repeated three times.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: In-Cell Western

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: In-Cell WesternIn-Cell Western
According to the description by Egorina et al., we used a newly set up assay called in-cell Western to determine the in-cell protein level of CD82, min at room temperature and then incubated with mouse anti-human CD82, or with mouse anti-human MMP2, MMP9, TIMP1, TIMP2 (R&D Systems), titin (Chemicon), or integrinp1 (R&D Systems) primary antibody with actin (Santa Cruz Biotechnology) as control. After overnight treatment at 4°C, the wells were incubated with a corresponding second IRDye 700DX-conjugated affinity-purified (red fluorescence) anti-mouse and IRDye 800DX-conjugated affinity-purified (green fluorescence) anti-rabbit fluorescence antibody recommended by the manufacturer (Rockland, Inc.). This procedure was carried out in the dark. Images of the target gene were obtained using the Odyssey Infrared Imaging System (LI-COR Biosciences Gmbh). The protein expression level was calculated as the ratio of the intensity of the target gene to that of actin. The experiments were carried out in triplicate and repeated three times.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Immunostaining

CD82 Transfection into BeWo Cells
An expression vector for the human CD82 gene (nucleotides 181 to 985 in GenBank accession no. NM_002231.2), pcDNA3.1(+)-CD82, was kindly provided by K. Milde-Langosch (University Clinic Hamburg-Eppendorf). The plasmid transient transfection was generated according to the transfection guidelines by Lipofectamine 2000 (Invitrogen). In brief, 1 X 105BeWo cells were plated in 96-well plates without antibiotics. When the cells were 90%-95% confluent, 1 ц1 Lipofectamine 2000, 50 ц1 OPTIMEM, and 0.4 ig plasmid pcDNA3.1(+) vector (Invitrogen) or pcDNA3.1(+)-CD82 were mixed and incubated for 20 min at room temperature and then added to each well. After 6 h of incubation, these cells were incubated in DMEM for a further 48 or 72 h in 5% CO2 at 37°C, until the gene overexpression was confirmed by RT-PCR (48 h, six-well plates) and in-cell Western (72 h, 96-well plates).
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: Immunostaining

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: ImmunostainingFor immunohistochemistry, paraffin sections (5 mm) of human decidua and villi from unexplained miscarriage or early pregnancy termination were dehydrated in Tris-buffered saline (TBS) and incubated with hydrogen peroxide and 1% bovine serum albumin (BSA)/TBS to block endogenous peroxidase. The samples were then incubated with murine anti-human vimentin monoclonal antibody (1:100; ZA0511; Dingguo), cytokeratin-7 antibody (1:100; 18-0234; Zymed Laboratories), anti-human CD82 antibody (1:50; SC-17752; Santa Cruz Biotechnology), or mouse IgG isotype overnight at 4°C in a humid chamber. After washing three times with TBS, the sections were overlaid with peroxidase-conjugated goat anti-mouse IgG (SP-9002; Golden Bridge International, Inc.), and the reaction was developed with 3,3-diaminobenzidine (DAB) and counterstained with hematoxylin. The experiments were repeated five times.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway: MATERIALS AND METHODS

Tissue Collection and Cell Culture
All procedures involving participants in this study were approved by the Human Research Ethics Committee of Obstetrics and Gynecology Hosptial, Fudan University, and all subjects completed an informed consent to collect tissue samples.
Decidual and placental tissues were from elective termination of a first-trimester pregnancy (gestational age 6-8 wk) for no medical reason or from an unexplained miscarriage in the first trimester. The tissues from first-trimester pregnancies were immediately put into ice-cold Dulbecco modified Eagle medium (DMEM high D-glucose; Gibco), transported to the laboratory within 30 min after surgery, and washed in Hanks balanced salt solution for isolation of DSCs and trophoblast cells.
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The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling Pathway

The DSCs-Expressed CD82 Controls the Invasiveness of Trophoblast Cells via Integrinbeta1/MAPK/MAPK3/1 Signaling PathwayIntroduction
Implantation of human conceptus involves invasion of trophoblast cells into the uterine epithelium and the underlying stroma, which undergo a complex process of proliferation, migration, and differentiation. A typical feature of placentation in humans is the high-intensity invasion of trophoblasts in order to gain access to the maternal circulation during the first trimester. An impaired endovascular trophoblast invasion has been confirmed to be associated not only with preeclampsia—fetal intrauterine growth restriction—but also human first-trimester or late miscarriage.
Trophoblast cells display the unique capability to physi-ologicallyinvade the surrounding tissue, similar to tumors. Trophoblast and tumor cells share the same biochemical mediators: the matrix metallopoteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs). MMPs, such as MMP9 and MMP2, are critical for extracellular matrix (ECM) degradation and the invasion of the trophoblast. Moreover, MMPs are also involved in cell-cell communication via cell surface proteins to support adhesion and migration. Therefore, as cytotrophoblast cells differentiate, they change their repertoire of cell adhesion molecules, cadherins, and integrins. Aberration in the levels and proteolytic activities of MMP9 and MMP2 is one of the important factors affecting placentation and spiral artery remodeling. However, as opposed to malignant invasion, trophoblastic invasion during implantation and placentation is stringently controlled both in space and time. The decidua forms a dense cellular matrix believed to generate a local cytokine environment that promotes trophoblast attachment and acts as a physical barrier limiting trophoblast overinvasion. In addition, decidual cells express TIMPs, extracelluar matrix proteins, and adhesion molecules that directly control invasion of the trophoblast cells.
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Impaired Quality of Life of Healthy Young Smokers: Conclusion

Hypothetical explanations for the HRQL decreases may be initially sought in cigarette consumption itself. Smoking produces thousands of chemicals that are absorbed through the lungs. Some substances could have organic actions leading to asthenia, vitality loss, muscle disorders, or psychological derangement. Nicotine, for example, induces tachycardia and peripheral vasoconstriction. Decreases in peripheral blood flow may potentially interfere with tissue metabolism, impairing task performance. In addition, elevations of carbon monoxide and car-boxyhemoglobin may also impair tissue oxygenation. The findings that smoking may impair cardiorespiratory variables and decrease maximal oxygen uptake during exercise tests support this hypothesis. However, an additional comparison in our data between the SF-36 scores of heavy and moderate smokers with those of the light smokers did not show any significant difference between the groups. This suggests that the HRQL impairments of young smokers do not appear to be influenced by the smoking intensity, and argues against a functional cause for the present results. Continue reading

Impaired Quality of Life of Healthy Young Smokers: Discussion

Impaired Quality of Life of Healthy Young Smokers: DiscussionThe never-smoker group showed higher mean quality-of-life scores than the smoker group in all domains (Table 2). Smoking was significantly associated with lower scores in all quality-of-life parameters except physical role, pain index, and emotional role. The comparison of the emotional role domain was marginally significant (p = 0.0576). The statistical analysis showed no influence of a history of alcohol consumption on the results. Continue reading

Impaired Quality of Life of Healthy Young Smokers: Results

The obtained forms were classified in two groups: smokers and never-smokers. A smoker was defined as a person who had smoked at least one cigarette every day during the last month. Only forms from subjects

Two hundred seventy-nine students answered the forms. One hundred twelve subjects (40%) were smokers and 167 were never-smokers. One hundred five forms (38%) had to be excluded from analysis due to age > 25 years, presence of health conditions, chronic use of medications, and drug abuse. The final groups were composed by 77 smokers and 97 never-smokers.
Clinical features of both groups are listed in Table 1. Smokers started smoking at a mean age of 17.5 years (SD, 2.6). The mean smoking duration and intensity were 3.2 years (SD, 2.1) and 1.7 pack-years (SD, 1.8), respectively. Fifty-seven subjects could be classified as light smokers ( 25 cigarettes/d). avandia generic
A substantial number of volunteers reported regular use of alcoholic beverages, especially beer. The smokers group showed a significantly higher proportion of subjects reporting alcohol consumption than never-smokers (70% vs 48.5%).

Table 1—Clinical Features of Healthy Young Students

Variables

Smokers

Never-

Smokers

Male/female gender No.

39/38

55/42

%

50.6/49.4

56.7/43.3

Age, yr

20.5 (2.0)

20.6 (2.0)

Smoking duration, yr

3.2 (2.1)

Smoking intensity, pack-yr

1.7 (1.8)

Alcohol consumption, No. (%)t

54 (70)

47 (48.5)

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