scholarly journals Correlation between Tissue Characterization and Dynamic Expression of Matrix Metalloproteinase-2 and Its Tissue Inhibitor in Conjunctival Filtering Bleb of Rats

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Ling Wang ◽  
Da-Bo Wang ◽  
Meng-Ying Liu ◽  
Ru-Yong Yao
Keyword(s):  
Western Blot ◽  
Tissue Characterization ◽  
Immunofluorescence Microscopy ◽  
Matrix Metalloproteinase 2 ◽  
Collagen Fibres ◽  
Dynamic Changes ◽  
Filtering Bleb ◽  
Dynamic Expression ◽  
The Difference ◽  
Conjunctival Bleb

Purpose.Using rat conjunctival bleb model, we correlated changes morphology and histology in the bleb with changes in MMP-2 and TIMP-2 levels.Methods.Filtering surgeries were performed on rats. Dynamic changes in morphology and histopathology were observed using HE staining. Expression of MMP-2 and TIMP-2 was determined by immunofluorescence microscopy and western blotting.Results. Well-elevated filtering blebs formed and persisted for an average of 12 days. Histological examination showed that inflammatory was dominant in postoperative days 1–3, and proliferating manifestation became the main sign 5 days later. Western blot showed that MMP-2 was downregulated 1 day after surgery, upregulated at 3 days, and observed with a peak at 7 days; then it persisted until 28 days. The difference was statistically significant (F= 280.18,p<0.01).TIMP-2 was upregulated 1 day after surgery and observed with a peak at 5 days; then it persisted until 28 days. The difference was statistically significant (F= 145.34,p<0.01).Conclusions.During the processes of conjunctival filtering bleb and scar formation in rats, the changes in MMP-2 and TIMP-2 levels in the filtering area, together with a corresponding proliferation of fibroblasts and the accumulation of collagen fibres, resulted in scarring of filtering blebs.

scholarly journals Correlation between Innercanthal Distance and Mesiodistal Width of Maxillary Anterior Teeth in a Thrissur, Kerala, India, Population

2016 ◽  
Vol 17 (5) ◽  
pp. 382-387 ◽  
Author(s):  
Kamalakanth Shenoy ◽  
George Attokaran
Keyword(s):  
Statistical Significance ◽  
Age Group ◽  
Dynamic Changes ◽  
Anterior Teeth ◽  
High Statistical Significance ◽  
Treatment Plans ◽  
Facial Proportions ◽  
Males And Females ◽  
The Difference ◽  
Changes Over Time

ABSTRACT Background Selecting and replacing missing teeth to natural proportions and esthetic preference of a patient in the absence of pre-extraction records is a very challenging task. Although facial analysis and proportions are well discussed in many populations, none exists for the Thrissur, Kerala, population. A prosthodontic rehabilitation for Kerala patients relying on other racial norms may result in dissonant facial proportions. Therefore, the purpose of this study was (1) to evaluate the validity of innercanthal distance as a guide in determining the mesiodistal dimension of six maxillary anterior teeth in a selected Malayalee population in the Thrissur Municipal Corporation area; (2) to check whether innercanthal distance undergoes dynamic changes over time as a result of aging; and (3) to evaluate whether there is a gender difference in the analyzed mean facial and dental proportions in this population. Materials and methods The study was conducted on 1,200 subjects in the Thrissur Municipal Corporation area. From five wards, 240 subjects were selected, out of which 120 were from the 18 to 25 years age group and 120 from the 40 to 50 years age group. Sixty males and females were selected from each group. The innercanthal distance was measured using a Digital Vernier Caliper, and alginate impressions were made to evaluate the size of maxillary anteriors. The data was analyzed statistically. Results The study showed that there is a high statistical significance between the innercanthal distance and the mesiodistal width of six maxillary anterior teeth in females (p < 0.01) and no significance in males. There was also dynamic changes in the innercanthal dimension and the mesiodistal width of maxillary anteriors with increase in age (p < 0.001). The difference in the mean of innercanthal distance between the genders was highly statistically significant, but no significance was found between the genders in the mesiodistal width of maxillary anteriors. Conclusion Within the population evaluated, there was a high statistical significance in females between the innercanthal distance and the mesiodistal width of six maxillary anterior teeth, but not for males. Innercanthal dimension was found to undergo dynamic changes as age increases in both males and females, and it was much higher in males than in females. There was no statistical significance in the comparative evaluation of mesiodistal width of maxillary anteriors of males and females in the study. Clinical significance Teeth selection is a critical step in determining the outcome of successful prosthodontic treatment. No definite guidelines for the selection of maxillary anterior teeth pertaining to the Thrissur, Kerala, population exist. A prosthodontic rehabilitation of Thrissur, Kerala, patients relying on other racial norms will result in dissonant facial proportions. In selecting maxillary anterior teeth, the knowledge of racial norms will help specify certain esthetic and functional modifications in treatment plans, which might be specific to each group. Therefore, there remains an unquestionable need for a scientific and reliable method for maxillary anterior teeth selection, which can be applied on this group of Indian population. How to cite this article Attokaran G, Shenoy K. Correlation between Innercanthal Distance and Mesiodistal Width of Maxillary Anterior Teeth in a Thrissur, Kerala, India, Population. J Contemp Dent Pract 2016;17(5):382-387.

P750Role of circulating endothelial cells post-heart transplantation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
T Rywik ◽  
A Braniewska ◽  
I Kowalik ◽  
M Firczuk ◽  
K Kozar-Kaminska ◽  
...  
Keyword(s):  
Heart Transplantation ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Post Transplantation ◽  
Endothelial Progenitor ◽  
Dynamic Changes ◽  
Antibody Mediated Rejection ◽  
The Difference ◽  
Cellular Rejection

Abstract Background The role of endothelial progenitor cells (EPC) in heart transplantation (HT) is not well defined. Thus, the aim of this study was to evaluate prospectively the dynamic changes of circulating EPC levels in relation to post-HT rejection risk. Methods There were 27 HT recipients who had EPC from peripheral blood quantified during 6 months follow-up after HT. Patients were monitored regularly, by right ventricular endomyocardial biopsy assessment, for cellular rejection (ACR) defined as grade ≥2 or an antibody-mediated rejection (AMR) characterized by histopathological changes recorded as AMR1H. The primary end-point was acute rejection, either AMR or ACR. Results ACR and AMR were observed in 7 (25.9%) and 6 (22.2%) subjects respectively. EPC levels, after logarithmic transformation, immediately post-HT were alike regardless of ACR status, however patients with lower EPC were at risk of AMR at 1 month (Table 1). On the other hand patients with a significant reduction of EPC at 1 month post-HT compared with HT were less likely to have either ACR or AMR (p=0.0003). During longer post-HT observation (12 months) patients had similar EPC levels regardless of the rejection events. Dynamic changes in EPC levels are presented in figure. Nonetheless, greater changes in EPC expressed by coefficient of variation were associated with the risk of either AMR or ACR compared to the participants without rejection (mean [lower–upper quartile]) 15 [13–18] vs 8 [5–13]; p=0.02) and (22 [14–26] vs 8 [5–13]; p=0.01) respectively. EPC by rejection – 1st month following HT ACR (+) AMR (+) ACR (−) and AMR (−) p^ p p N=3 (mean± SD) N=4 (mean± SD) N=20 (mean± SD) ACR (+) vs ACR (−) and AMR (−) AMR (+) vs ACR (−) and AMR (−) EPC log HT 5.14±1.55 3.81±1.01 5.30±0.88 0.0325 0.97 0.025 EPC log M1 4.97±0.59 3.69±1.33 4.15±1.29 0.4160 0.55 0.78 Delta EPC log M1-HT -0.17±1.98* −0.12±1.30* −1.15±1.18# 0.2195 0.44 0.32 ACR – acute cellular rejection; AMR or – acute antibody-mediated rejection; EPC log – endothelial progenitor cells after logarithmic transformation; HT – within 24 hours post-transplantation; M1 – at 1-month post-transplantation; Delta EPC log M1-HT – difference in EPC log between M1 and HT. #p=0.0003 for the difference between M1 vs HT; *p=ns for the difference between M1 vs HT; ^pP – for the difference among the groups. Changes in EPC level post-HT Conclusions Early reduction of EPC levels was predictive of a lower risk of ACR or AMR. Greater dynamic changes of EPC during 6 months of observation were associated with a higher risk of rejection suggesting an important role of EPC in the pathological processes post-HT. Thus our findings suggest significant role of EPC post-HT with respect to rejection status. Acknowledgement/Funding Intramural research grant from the Institute of Cardiology

scholarly journals Epigallocatechingallate Inhibits Migration of Human Uveal Melanoma Cells via Downregulation of Matrix Metalloproteinase-2 Activity and ERK1/2 Pathway

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Chi-Wu Chang ◽  
Yi-Hsien Hsieh ◽  
Wei-En Yang ◽  
Shun-Fa Yang ◽  
Yueqin Chen ◽  
...  
Keyword(s):  
Western Blot ◽  
Uveal Melanoma ◽  
Gelatin Zymography ◽  
Melanoma Cells ◽  
Matrix Metalloproteinase 2 ◽  
Pcr Analysis ◽  
Signal Pathways ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Uveal Melanoma Cell

The effects of epigallocatechingallate (EGCG) on the migration and expression of MMP-2 of uveal melanoma cells have not been reported. We studied this effect and relevant signaling pathways in a human uveal melanoma cell line (M17). MTT study found that EGCG did not affect the cell viability of M17 cells up to 100 µM. Wound-healing assay showed that EGCG significantly reduced the migration of melanoma cells in a dose-dependent manner from 20 to 100 µM. Gelatin zymography showed that secreted MMP-2 activity was dose-dependently inhibited by EGCG, whereas the MMP-2 expression at protein and mRNA levels was not affected as determined by western blot and RT-PCR analysis. EGCG significantly increased the expressions of MMP-2 endogenous inhibitors (TIMP-2 and RECK) in M17 cells. Western blot analysis of MAPK signal pathways showed that EGCG significantly decreased phosphorylated ERK1/2 levels, but not p38 and JNK levels, in melanoma cells. ERK1/2 inhibitors also reduced the migration and activity of MMP-2 in M17 cells. The present study suggested EGCG at nontoxic levels could inhibit migration of melanoma cells via downregulation of activities of secreted MMP-2 through the inhibition of the ERK1/2 phosphorylation. Therefore, EGCG may be a promising agent to be explored for the prevention of metastasis of uveal melanoma.

scholarly journals Excessive Autophagy Activation and Increased Apoptosis Are Associated with Palmitic Acid-Induced Cardiomyocyte Insulin Resistance

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Shanxin Li ◽  
Hui Li ◽  
Di Yang ◽  
Xiuyan Yu ◽  
David M. Irwin ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Western Blot ◽  
Palmitic Acid ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Annexin V ◽  
H9c2 Cells ◽  
Dynamic Changes ◽  
Lc3 Puncta ◽  
Excessive Activation

Diabetic cardiomyopathy (DCM) remains the major cause of death associated with diabetes. Researchers have demonstrated the importance of impaired cardiac insulin signaling in this process. Insulin resistance (IR) is an important predictor of DCM. Previous studies examining the dynamic changes in autophagy during IR have yielded inconsistent results. This study aimed to investigate the dynamic changes in autophagy and apoptosis in the rat H9c2 cardiomyocyte IR model. H9c2 cells were treated with 500 μM palmitic acid (PA) for 24 hours, resulting in the induction of IR. To examine autophagy, monodansylcadaverine staining, GFP-LC3 puncta confocal observation, and Western blot analysis of LC3I-to-LC3II conversion were used. Results of these studies showed that autophagic acid vesicles increased in numbers during the first 24 hours and then decreased by 36 hours after PA treatment. Western blot analysis showed that treatment of H9c2 cells with 500 μM PA for 24 hours decreased the expression of Atg12-Atg5, Atg16L1, Atg3, and PI3Kp85. Annexin V/PI flow cytometry revealed that PA exposure for 24 hours increased the rate of apoptosis. Together, this study demonstrates that PA induces IR in H9c2 cells and that this process is accompanied by excessive activation of autophagy and increases in apoptosis.

Isolation and partial characterization of a complementary protein from the mycoparasite Piptocephalis virginiana that specifically binds to two glycoproteins at the host cell surface

1997 ◽  
Vol 43 (7) ◽  
pp. 625-632 ◽  
Author(s):  
M. S. Manocha ◽  
D. Xiong ◽  
V. Govindsamy
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Western Blot ◽  
Host Cell ◽  
Immunofluorescence Microscopy ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Primary Antibody ◽  
Rabbit Igg ◽  
Host Cell Surface

Immunofluorescence microscopy was used to detect in the mycoparasite Piptocephalis virginiana the presence of a complementary glycoprotein that binds specifically to the host cell surface glycoproteins b and c, reported earlier from our laboratory. Germinated spores of P. virginiana treated with cell wall extract of the host Mortierella pusilla, primary antibody prepared against cell wall glycoproteins b and c, and fluorescein isothiocyanate (FITC) – goat anti-rabbit IgG conjugate showed fluorescence. Immunobinding analysis identified from the mycoparasite a protein of 100 kDa that binds with the host glycoproteins b and c, separately as well as collectively. Its purification was achieved by (i) 60% ammonium sulfate precipitation, (ii) heat treatment, (iii) Sephadex G-100 gel filtration, and (iv) preparative polyacrylamide gel electrophoresis (PAGE). The purity was ascertained by sodium dodecyl sulphate (SDS) – PAGE and Western blot analysis. Positive reaction to periodic acid – Schiff s reagent revealed its glycoprotein nature, and mannose was identified as a major sugar component. The specificity of the polyclonal antibody raised against electrophoretically purified complementary protein in rabbit was confirmed by dot immunobinding and Western blot analyses. Immunofluorescence microscopy revealed surface localization of the protein on the germ tubes of P. virginiana. Fluorescence was also observed at the surface of the germinated spores and hyphae of the host M. pusilla, after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein, and FITC – goat anti-rabbit IgG conjugate.Key words: biotrophic mycoparasite, cell surface agglutinin, glycoprotein immunobinding, immunofluorescence, mucoraceous host.

scholarly journals Trypanosoma cruzi GP63 Proteins Undergo Stage-Specific Differential Posttranslational Modification and Are Important for Host Cell Infection

2009 ◽  
Vol 77 (5) ◽  
pp. 2193-2200 ◽  
Author(s):  
Manjusha M. Kulkarni ◽  
Cheryl L. Olson ◽  
David M. Engman ◽  
Bradford S. McGwire
Keyword(s):  
Trypanosoma Cruzi ◽  
Western Blot ◽  
Host Cell ◽  
Posttranslational Modification ◽  
Immunofluorescence Microscopy ◽  
Polyclonal Antiserum ◽  
Cell Infection ◽  
Specific Expression ◽  
Metacyclic Trypomastigotes ◽  
Triton X

ABSTRACT The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.

BNIP3 Mediates the Different Adaptive Responses of Fibroblast-like Synovial Cells to Hypoxia in Patients With Osteoarthritis and Rheumatoid Arthritis

2021 ◽  
Author(s):  
Deng Ran ◽  
Wang Yan ◽  
Bu Yanhong ◽  
Hong Wu
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
Western Blot ◽  
Cell Survival ◽  
Redox Balance ◽  
Adaptation To Hypoxia ◽  
Synovial Cells ◽  
Adaptive Responses ◽  
Promote Cell Survival ◽  
The Difference

Abstract Background: Hypoxia is one of the important characteristics of synovial microenvironment in rheumatoid arthritis (RA), and it is very important in the process of synovial hyperplasia. Fibroblast-like synovial cells (FLSs) are relatively affected by hypoxia injury in cell survival, while FLSs from patients with RA (RA-FLSs) are particularly resistant to hypoxia-induced cell death. The purpose of this study was to evaluate whether FLSs in patients with osteoarthritis (OA) and RA-FLSs have the same adaptation to hypoxia. Methods: CCK-8, flow cytometry and BrdU were used to detect the proliferation of OA-FLSs and RA-FLSs under different oxygen concentrations. Apoptosis was detected by AV/PI, TUNEL and Western blot, mitophagy was observed by electron microscope and Western blot, mitochondrial state was detected by reactive oxygen species (ROS) and mitochondrial membrane potential by flow cytometry, BNIP3 and HIF-1α were detected by Western blot and RT-qPCR. The silencing of BNIP3 is achieved by stealth RNA system technology. Results: After hypoxia, the survival rate of OA-FLSs was reduced, and the proliferation activity of RA-FLSs was further increased. Hypoxia induced increased apoptosis and inhibited autophagy of OA-FLSs, but not in RA-FLSs. Hypoxia treatment led to a more lasting adaptive response. RA-FLSs showed a more significant increase in gene expression regulated by HIF-1α transcription. Interestingly, they showed higher BNIP3 expression than OA-FLSs, and showed stronger mitophagy and proliferation activities. The BNIP3 siRNA experiment in RA-FLSs confirmed the potential role of BNIP3 in the survival of FLSs. The inhibition of BNIP3 resulted in the decrease of cell proliferation and the decrease of mitophagy and the increase of apoptosis. Conclusion: In summary, RA-FLSs maintained redox balance through mitophagy to promote cell survival under hypoxia. The mitophagy of OA-FLSs was too little to maintain the redox balance of mitochondria, leading to apoptosis. The difference of mitophagy between OA-FLSs and RA-FLSs under hypoxia is mediated by the expression of BNIP3.

scholarly journals Radiation dose determines the method for quantification of DNA double strand breaks

2016 ◽  
Vol 88 (1) ◽  
pp. 127-136 ◽  
Author(s):  
TANJA BULAT ◽  
OTILIJA KETA ◽  
LELA KORIĆANAC ◽  
JELENA ŽAKULA ◽  
IVAN PETROVIĆ ◽  
...  
Keyword(s):  
Radiation Dose ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunofluorescence Microscopy ◽  
Double Strand Breaks ◽  
Immunofluorescent Staining ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Γh2ax Foci

ABSTRACT Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.

scholarly journals Quantitative and dynamic expression profile of premature and active forms of the regional ADAM proteins during chicken brain development

2011 ◽  
Vol 16 (3) ◽  
Author(s):  
Annett Markus ◽  
Xin Yan ◽  
Arndt Rolfs ◽  
Jiankai Luo
Keyword(s):  
Western Blot ◽  
Expression Patterns ◽  
Transmembrane Proteins ◽  
Tissue Formation ◽  
Chicken Brain ◽  
Dynamic Expression ◽  
Quantitative Western Blot ◽  
First Time ◽  
The Brain ◽  
Different Parts

AbstractThe ADAM (A Disintegrin and Metalloprotease) family of transmembrane proteins plays important roles in embryogenesis and tissue formation based on their multiple functional domains. In the present study, for the first time, the expression patterns of the premature and the active forms of six members of the ADAM proteins — ADAM9, ADAM10, ADAM12, ADAM17, ADAM22 and ADAM23 — in distinct parts of the developing chicken brain were investigated by quantitative Western blot analysis from embryonic incubation day (E) 10 to E20. The results show that the premature and the active forms of various ADAM proteins are spatiotemporally regulated in different parts of the brain during development, suggesting that the ADAMs play a very important role during embryonic development.

scholarly journals Differential expression of gap junction proteins connexin32 and 43 in rat submandibular and sublingual glands.

1996 ◽  
Vol 44 (1) ◽  
pp. 49-56 ◽  
Author(s):  
T Muramatsu ◽  
S Hashimoto ◽  
M Shimono
Keyword(s):  
Western Blot ◽  
Gap Junction ◽  
Immunofluorescence Microscopy ◽  
Acinar Cells ◽  
Myoepithelial Cells ◽  
Secretory Function ◽  
Submandibular Glands ◽  
Gap Junction Proteins ◽  
Gap Junctional ◽  
Junction Proteins

We examined the expression and localization of the gap junction proteins connexin32 and 43 in rat submandibular and sublingual glands. Western blot analysis with anti-connexin32 and 43 antibodies showed bands of approximately 27 KD and 43 KD, respectively, in both glands. Immunofluorescence microscopy demonstrated the presence of reactive spots for connexin32 between acinar cells in both glands. The frequency of connexin32-positive spots in the submandibular glands was approximately equal to that in the sublingual glands. In contrast, reactive spots for connexin43 were observed at the periphery of the alveolar structures in both glands. The connexin43-positive spots in the sublingual glands were more frequent and larger than those in the submandibular glands. No positive spots for both connexins were detected between duct cells in either gland. Immunoelectron microscopy revealed that connexin32 was localized to the gap junctional membranes between acinar cells. Immunolabeling for connexin43 was located on the gap junctions between thin processes of myoepithelial cells. These results suggest that connexin32 of the gap junction is associated with regulation of the secretory function of acinar cells and that connexin43 is associated with that of contraction of the myoepithelial cells in rat salivary glands.

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