scholarly journals The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity

2013 ◽  
Vol 304 (12) ◽  
pp. C1150-C1158 ◽  
Author(s):  
Pallavi V. Mhaske ◽  
Noah A. Levit ◽  
Leping Li ◽  
Hong-Zhan Wang ◽  
Jack R. Lee ◽  
...  
Keyword(s):  
Calcium Concentration ◽  
Skin Diseases ◽  
Human Keratinocytes ◽  
Extracellular Calcium ◽  
Membrane Current ◽  
Wild Type ◽  
Nonsyndromic Deafness ◽  
Primary Human Keratinocytes ◽  
Gene Encoding ◽  
Clinical Disorders

Mutations in the human gene encoding connexin 26 (Cx26 or GJB2) cause either nonsyndromic deafness or syndromic deafness associated with skin diseases. That distinct clinical disorders can be caused by different mutations within the same gene suggests that different channel activities influence the ear and skin. Here we use three different expression systems to examine the functional characteristics of two Cx26 mutations causing either mild (Cx26-D50A) or lethal (Cx26-A88V) keratitis-ichthyosis-deafness (KID) syndrome. In either cRNA-injected Xenopus oocytes, transfected HeLa cells, or transfected primary human keratinocytes, we show that both Cx26-D50A and Cx26-A88V form active hemichannels that significantly increase membrane current flow compared with wild-type Cx26. This increased membrane current accelerated cell death in low extracellular calcium solutions and was not due to increased mutant protein expression. Elevated mutant hemichannel currents could be blocked by increased extracellular calcium concentration. These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome.

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.

scholarly journals Effect of SUV39H1 Histone Methyltransferase Knockout on Expression of Differentiation-Associated Genes in HaCaT Keratinocytes

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2628
Author(s):  
Barbara Sobiak ◽  
Wiesława Leśniak
Keyword(s):  
Gene Expression ◽  
Skin Diseases ◽  
Human Keratinocytes ◽  
Histone Methyltransferase ◽  
Epidermal Differentiation ◽  
Gene Encoding ◽  
Epidermal Differentiation Complex ◽  
Expression Of Genes ◽  
State Dependent ◽  
Genomic Locations

Keratinocytes undergo a complex differentiation process, coupled with extensive changes in gene expression through which they acquire distinctive features indispensable for cells that form the external body barrier—epidermis. Disturbed epidermal differentiation gives rise to multiple skin diseases. The involvement of epigenetic factors, such as DNA methylation or histone modifications, in the regulation of epidermal gene expression and differentiation has not been fully recognized yet. In this work we performed a CRISPR/Cas9-mediated knockout of SUV39H1, a gene-encoding H3K9 histone methyltransferase, in HaCaT cells that originate from spontaneously immortalized human keratinocytes and examined changes in the expression of selected differentiation-specific genes located in the epidermal differentiation complex (EDC) and other genomic locations by RT-qPCR. The studied genes revealed a diverse differentiation state-dependent or -independent response to a lower level of H3K9 methylation. We also show, by means of chromatin immunoprecipitation, that the expression of genes in the LCE1 subcluster of EDC was regulated by the extent of trimethylation of lysine 9 in histone H3 bound to their promoters. Changes in gene expression were accompanied by changes in HaCaT cell morphology and adhesion.

scholarly journals Anti-Inflammatory Effects of Concentrated Ethanol Extracts of Edelweiss (Leontopodium alpinumCass.) Callus Cultures towards Human Keratinocytes and Endothelial Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Lulli Daniela ◽  
Potapovich Alla ◽  
Riccardo Maurelli ◽  
Dellambra Elena ◽  
Pressi Giovanna ◽  
...  
Keyword(s):  
Skin Diseases ◽  
Inflammatory Responses ◽  
Trichostatin A ◽  
Human Keratinocytes ◽  
Callus Cultures ◽  
Total Phenolic ◽  
Primary Human Keratinocytes ◽  
Inflammatory Skin Diseases ◽  
Phenolic Fraction ◽  
Anti Inflammatory

Edelweiss (Leontopodium alpinumCass.) is traditionally employed in folk medicine as an anti-inflammatory remedy. In nature, the plant is sparsely available and protected; therefore production of callus cultures was established. A concentrated ethanolic extract of culture homogenate, with leontopodic acid representing55±2% of the total phenolic fraction (ECC55), was characterized for anti-inflammatory properties in primary human keratinocytes (PHKs) and endotheliocytes (HUVECs). Inflammatory responses were induced by UVA+UVB, lipopolysaccharide (LPS), oxidized low-density lipoprotein (oxLDL), and a mixture of proinflammatory cytokines. Trichostatin A, a sirtuin inhibitor, was used to induce keratinocyte inflammatory senescence. ECC55 (10–50 μg/mL) protected PHK from solar UV-driven damage, by enhancing early intracellular levels of nitric oxide, although not affecting UV-induced expression of inflammatory genes. Comparison of the dose-dependent inhibition of chemokine (IL-8, IP-10, MCP-1) and growth factor (GM-CSF) release from PHK activated by TNFα+ IFNγshowed that leontopodic acid was mainly responsible for the inhibitory effects of ECC55. Sirtuin-inhibited cell cycle, proliferation, and apoptosis markers were restored by ECC55. The extract inhibited LPS-induced IL-6 and VCAM1 genes in HUVEC, as well as oxLDL-induced selective VCAM1 overexpression.Conclusion.Edelweiss cell cultures could be a valuable source of anti-inflammatory substances potentially applicable for chronic inflammatory skin diseases and bacterial and atherogenic inflammation.

Connexin-26 mutations in deafness and skin disease

2009 ◽  
Vol 11 ◽  
Author(s):  
Jack R. Lee ◽  
Thomas W. White
Keyword(s):  
Skin Disease ◽  
Skin Diseases ◽  
Stop Codon ◽  
Single Amino Acid ◽  
Connexin 26 ◽  
Common Mutation ◽  
Loss Of Function ◽  
Nonsyndromic Deafness ◽  
Gene Encoding ◽  
Connexin Genes

Gap junctions allow the exchange of ions and small molecules between adjacent cells through intercellular channels formed by connexin proteins, which can also form functional hemichannels in nonjunctional membranes. Mutations in connexin genes cause a variety of human diseases. For example, mutations in GJB2, the gene encoding connexin-26 (Cx26), are not only a major cause of nonsyndromic deafness, but also cause syndromic deafness associated with skin disorders such as palmoplantar keratoderma, keratitis–ichthyosis deafness syndrome, Vohwinkel syndrome, hystrix–ichthyosis deafness syndrome and Bart–Pumphrey syndrome. The most common mutation in the Cx26 gene linked to nonsyndromic deafness is 35ΔG, a frameshift mutation leading to an early stop codon. The large number of deaf individuals homozygous for 35ΔG do not develop skin disease. Similarly, there is abundant experimental evidence to suggest that other Cx26 loss-of-function mutations cause deafness, but not skin disease. By contrast, Cx26 mutations that cause both skin diseases and deafness are all single amino acid changes. Since nonsyndromic deafness is predominantly a loss-of-function disorder, it follows that the syndromic mutants must show an alteration, or gain, of function to cause skin disease. Here, we summarise the functional consequences and clinical phenotypes resulting from Cx26 mutations that cause deafness and skin disease.

Suppression of apoptosis by BRAF inhibitors through off-target inhibition of JNK signaling.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 8537-8537 ◽  
Author(s):  
Kenneth Yee Tsai ◽  
Harina Vin ◽  
Marco Leung ◽  
Vida Chitsazzadeh ◽  
Sandra Ojeda ◽  
...  
Keyword(s):  
Mapk Pathway ◽  
Mouse Skin ◽  
Human Keratinocytes ◽  
Mek Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Primary Human Keratinocytes ◽  
Jnk Signaling ◽  
Erk Activation ◽  
Ras Mutations

8537 Background: The advent of targeted therapy has revolutionized the treatment of cancer. The mutant BRAFV600E protein is found in over 50% of melanomas and thyroid carcinomas, resulting in elevated kinase activity, increased mitogen-activated protein kinase (MAPK) pathway signaling, and cell proliferation. Vemurafenib and PLX4720 were designed to selectively inhibit the BRAF kinase, and clinical trials of vemurafenib in metastatic melanoma have demonstrated a response rate of over 50% and an overall survival advantage over standard dacarbazine therapy. Approximately 20-30% of individuals treated with vemurafenib develop cutaneous squamous cell carcinoma (cSCC) highlighting the importance of understanding toxicities associated with this drug. Paradoxical ERK activation in BRAF wild-type, RAS-mutant cells is thought to be the major mechanism by which this occurs, as evidenced by the presence of RAS mutations in 60% of such lesions. Methods: Using a combination of BRAF-wild-type cSCC cell lines, primary human keratinocytes, as well as a UV mouse model of cSCC and human cSCC samples, we identified novel effects of BRAFi on apoptosis. Results: Here we show an unexpected and novel effect of vemurafenib and PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases. JNK signaling and apoptosis are suppressed in cSCC lesions arising in vemurafenib-treated patients as well as in irradiated mouse skin. This occurs independently of paradoxical ERK signaling and in the presence of MEK inhibitor. Treatment with PLX4720 greatly accelerates the development of UV-induced cSCC in mice without Ras mutations. Kinome screening identified ZAK and MKK4 (MEK4 / MAP2K4) kinases as inhibited by vemurafenib, leading to suppression of MKK4 and MKK7 (MAP2K7) phosphorylation. Knockdown of inhibited off-target kinases recapitulates these anti-apoptotic effects of vemurafenib. Conclusions: Our results implicate suppression of JNK signaling, independent of ERK activation, as an additional, complementary mechanism of adverse effects of vemurafenib. This has broad implications for combination therapies with other modalities that induce apoptosis and for the long-term use of vemurafenib in the adjuvant setting.

scholarly journals Effect of Low Extracellular Calcium Concentration on Photosensittvity of Isolated Rods from Frog Retina

1989 ◽  
Vol 145 (1) ◽  
pp. 255-262
Author(s):  
KATSU AZUMA
Keyword(s):  
Calcium Concentration ◽  
Rana Catesbeiana ◽  
Light Sensitivity ◽  
Recovery Phase ◽  
Physiological Solution ◽  
Extracellular Calcium ◽  
Membrane Current ◽  
Peak Amplitude ◽  
Extracellular Calcium Concentration ◽  
Light Flashes

The effects of extracellular calcium concentration ([Ca2+]o) on the light sensitivity of isolated single rods from the retina of the frog (Rana catesbeiana) were investigated by sucking the rod inner segments into tightly fitting pipettes. Light flashes (500nm, 1s duration) evoked transient outward changes of membrane current (photoresponses). The peak amplitude of maximal photoresponses in normal physiological solution varied between 6 and 12 pA. Reducing ([Ca2+]o) from O.9μmmoll−1 (normal Ca2+) to 90μmoll−1 (low Ca2+) increased the peak amplitude of photoresponses and shortened the recovery phase of the responses. The effects of larger changes in light intensity were also investigated. After light-on there was a steady outward change of membrane current, and after lightoff the current recovered to the initial dark level. With a low external Ca2+ concentration, light-off induced a large inward change of membrane current which transiently overshot the initial dark level. During the overshoot stage, light flashes evoked photoresponses which were larger than those in the initial dark period.

Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 519d-519 ◽  
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Height ◽  
Dry Weight ◽  
Nicotiana Alata ◽  
Wild Type ◽  
Gene Encoding ◽  
Delayed Senescence ◽  
Gene Construct ◽  
Shoot Dry Weight ◽  
Inhibitor System

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

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