scholarly journals Downregulation of Bax mRNA expression and protein stability by the E6 protein of human papillomavirus 16

2005 ◽  
Vol 86 (3) ◽  
pp. 611-621 ◽  
Author(s):  
Sharon Shnitman Magal ◽  
Anna Jackman ◽  
Shahar Ish-Shalom ◽  
Liat Edri Botzer ◽  
Pinhas Gonen ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Protein Stability ◽  
Mrna Expression ◽  
Human Keratinocytes ◽  
Dependent Manner ◽  
Primary Human Keratinocytes ◽  
Experimental Conditions ◽  
Human Papillomavirus 16 ◽  
Bax Protein

Previous studies have shown that human papillomavirus (HPV) 16 E6 inhibits apoptosis induced during terminal differentiation of primary human keratinocytes (PHKs) triggered by serum and calcium. E6 inhibition of apoptosis was accompanied with prolonged expression of Bcl-2 and reduced elevation of Bax levels. In the present study, the effect of E6 on Bax mRNA expression and protein stability was investigated. These studies indicate that stable E6 expression in differentiating keratinocytes reduced the steady-state levels of Bax mRNA and shortened the half-life of Bax protein. These results were confirmed in transiently transfected 293T cells where E6 degraded Bax in a dose-dependent manner. Bax degradation was also exhibited in Saos-2 cells that lack p53, indicating its p53 independence. E6 did not form complexes with Bax and did not induce Bax degradation in vitro under experimental conditions where p53 was degraded. Finally, E6 aa 120–132 were shown to be necessary for Bax destabilization and, more importantly, for abrogating the ability of Bax to induce cellular apoptosis, highlighting the functional consequences of the E6-induced alterations in Bax expression.

scholarly journals Expression of membrane type 1 matrix metalloproteinase in papillomavirus-positive cells: role of the human papillomavirus (HPV) 16 and HPV8 E7 gene products

2005 ◽  
Vol 86 (5) ◽  
pp. 1291-1296 ◽  
Author(s):  
Sigrun Smola-Hess ◽  
Jenny Pahne ◽  
Cornelia Mauch ◽  
Paola Zigrino ◽  
Hans Smola ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Cellular Proliferation ◽  
Human Keratinocytes ◽  
Hpv Infection ◽  
Host Cells ◽  
Primary Human Keratinocytes ◽  
E7 Gene ◽  
Membrane Type

Matrix metalloproteinases (MMPs) degrade extracellular matrix. They are involved in cellular proliferation, migration, angiogenesis, invasion and metastasis. MT-1 MMP, a membrane-bound MMP, is expressed in carcinomas of the uterine cervix in vivo. This type of cancer is associated with human papillomavirus (HPV) infection. Here it was shown that keratinocytes transformed with HPV16 or HPV18 in vitro, and HPV-positive cervical carcinoma cell lines, constitutively expressed MT-1 MMP. Expression of the E7 protein from the mucosal and cutaneous high-risk types HPV16 and HPV8, but not from the cutaneous low-risk type HPV1, was sufficient to induce MT-1 MMP expression in primary human keratinocytes and HaCaT cells. As a consequence, MMP-2 was activated. MT-1 MMP expression might play a role in the HPV life cycle by promoting proliferation of host cells and might contribute to their invasive phenotype during malignant progression.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

The Effect of Vaginal Microbes on in Vivo and in Vitro Expression of Human Papillomavirus 16 E6-E7 Genes

1999 ◽  
Vol 23 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Patricia J. McNicol ◽  
Maria Paraskevas ◽  
Fernando B. Guijon
Keyword(s):  
Human Papillomavirus ◽  
In Vitro Expression ◽  
Human Papillomavirus 16

scholarly journals Galanin inhibits leptin expression and secretion in rat adipose tissue and 3T3-L1 adipocytes

2004 ◽  
Vol 33 (1) ◽  
pp. 11-19 ◽  
Author(s):  
RY Li ◽  
HD Song ◽  
WJ Shi ◽  
SM Hu ◽  
YS Yang ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Mrna Expression ◽  
Vital Role ◽  
Dependent Manner ◽  
Protein Levels ◽  
Orexigenic Peptide ◽  
Fat Depot ◽  
Rat Adipose Tissue ◽  
Leptin Expression

In addition to serving as a fat depot, adipose tissue is also considered as an important endocrine organ that synthesizes and secretes a number of factors. Leptin is an adipocyte-derived hormone that plays a vital role in energy balance. Expression of leptin is regulated by dietary status and hormones. In the present study, we report that galanin, an orexigenic peptide, inhibits leptin expression and secretion in rat adipose tissue and in 3T3-L1 adipocytes. Treatment with galanin (25 micro g/animal) induced approximately 46% down-regulation of leptin secretion at 15 min, followed by 40, 37 and 47% decreases in leptin secretion at 1, 2 and 4 h respectively. Although Northern blot analysis of adipose tissue from the same animals showed that leptin mRNA expression in adipose tissue was unaffected by galanin treatment for 2 h, galanin treatment for 4 h led to decline of leptin mRNA expression in a dose-dependent manner. Meanwhile, treating the rats with galanin had no effect on leptin mRNA expression in the hypothalamus. The inhibitory action of the galanin on leptin mRNA and protein levels was also observed in vitro. When incubated with 10 nM galanin for 48 h, leptin mRNA expression and protein secretion also decreased in 3T3-L1 adipocytes. On the other hand, galanin was found not only to express in rat adipose tissue, but also to increase about 8-fold after fasting. Based on these data, we speculate that increased galanin expression in rat adipose tissue after fasting may be involved in reducing leptin expression and secretion in fasting rats.

Protective effects of Phytohustil® on primary human keratinocytes in vitro

2021 ◽  
Author(s):  
I Gollin ◽  
G Bonaterra ◽  
J Müller ◽  
R Kinscherf
Keyword(s):  
Human Keratinocytes ◽  
Protective Effects ◽  
Primary Human Keratinocytes

scholarly journals Human TNF-α Affects the Egg-laying Dynamics and Glucose Metabolism of Schistosoma Mansoni Adult Worms in Vitro

2021 ◽  
Author(s):  
Ednilson Hilário Lopes-Junior ◽  
Gilbert de Oliveira Silveira ◽  
Camila Banca Guedes ◽  
Gratchela Dutra Rodrigues ◽  
Viviane Sousa Ribeiro ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Mrna Expression ◽  
Glucose Uptake ◽  
Lactate Production ◽  
Egg Laying ◽  
Dependent Manner ◽  
Tnf Α ◽  
Atp Metabolism ◽  
Fine Regulation

Abstract Several studies described the effect of human TNF-α on Schistosoma mansoni. It affects the worm’s development, metabolism, egg-laying, changes in the parasite´s gene expression and protein phosphorylation. Data available concerning the influence of hTNF-α on egg-laying are controversial and understanding the mechanism of egg-laying regulation is essential in combating schistosomiasis. We characterized the effects of in vitro treatment of S. mansoni adult worms with different doses of hTNF-α (5, 20 and 40ng/mL) for five days. We explored the effects on the egg-laying rate, glucose, ATP metabolism, mRNA expression levels of lactate dehydrogenase, of glucose transporters and of SmTNFR, the parasite gene for hTNF-α receptor. hTNF-α influenced egg-laying in a time and dose dependent manner: with 40ng/mL, egg-laying increased on day 2 and decreased on days 3 and 4; 20 ng/mL dose, egg-laying decreased on day 3, while at 5ng/mL dose, egg-laying decreased on day 4. The total number of eggs produced was not affected, but the egg-laying dynamic was altered; the median egg-laying time decreased significantly due to treatment. At 5 and 20ng/mL hTNF-alpha, lactate production diminished on days 3 up to 5, while glucose uptake increased on day 5. At 40ng/mL, glucose uptake diminished on days 1 up to 3, while ATP accumulation was detected on day 5. No significant changes in the mRNA expression were detected in all treatments. Crosstalk involving the hTNF-alpha and the parasite signaling play a role in the fine regulation of the worm´s metabolism and physiology and points to new strategies for disease control.

scholarly journals HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Irma Colombo ◽  
Enrico Sangiovanni ◽  
Roberta Maggio ◽  
Carlo Mattozzi ◽  
Stefania Zava ◽  
...  
Keyword(s):  
Cell Density ◽  
Skin Diseases ◽  
Inflammatory Responses ◽  
Human Keratinocytes ◽  
Suitable Model ◽  
Hacat Cells ◽  
Primary Human Keratinocytes ◽  
Proinflammatory Mediators ◽  
Cytokines And Chemokines

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

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