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1.
Neuroscience Bulletin ; (6): 57-68, 2023.
Article in English | WPRIM | ID: wpr-971536

ABSTRACT

PiT2 is an inorganic phosphate (Pi) transporter whose mutations are linked to primary familial brain calcification (PFBC). PiT2 mainly consists of two ProDom (PD) domains and a large intracellular loop region (loop7). The PD domains are crucial for the Pi transport, but the role of PiT2-loop7 remains unclear. In PFBC patients, mutations in PiT2-loop7 are mainly nonsense or frameshift mutations that probably cause PFBC due to C-PD1131 deletion. To date, six missense mutations have been identified in PiT2-loop7; however, the mechanisms by which these mutations cause PFBC are poorly understood. Here, we found that the p.T390A and p.S434W mutations in PiT2-loop7 decreased the Pi transport activity and cell surface levels of PiT2. Furthermore, we showed that these two mutations attenuated its membrane localization by affecting adenosine monophosphate-activated protein kinase (AMPK)- or protein kinase B (AKT)-mediated PiT2 phosphorylation. In contrast, the p.S121C and p.S601W mutations in the PD domains did not affect PiT2 phosphorylation but rather impaired its substrate-binding abilities. These results suggested that missense mutations in PiT2-loop7 can cause Pi dyshomeostasis by affecting the phosphorylation-regulated cell-surface localization of PiT2. This study helps understand the pathogenesis of PFBC caused by PiT2-loop7 missense mutations and indicates that increasing the phosphorylation levels of PiT2-loop7 could be a promising strategy for developing PFBC therapies.


Subject(s)
Humans , Cell Membrane , Mutation, Missense , Phosphates/metabolism , Sodium-Phosphate Cotransporter Proteins, Type III/genetics
2.
Chinese Journal of Biotechnology ; (12): 1373-1389, 2022.
Article in Chinese | WPRIM | ID: wpr-927787

ABSTRACT

The production efficiency of microbial cell factory is determined by the growth performance, product synthetic capacity, and stress resistance of the strain. Strengthening the stress resistance is the key point to improve the production efficiency of microbial cell factory. Tolerance engineering is based on the response mechanism of microbial cell factory to resist stress. Specifically, it consolidates the cell wall-cell membrane barrier to enhance the defense against stress, accelerates the stress response to improve the damage repair, and creates tolerance evolutionary tools to screen industrial microorganisms with enhanced robustness. We summarize the regulation strategies and forecast the prospects of tolerance engineering, which plays an important role in the microbial cell factories for sustainable production of natural products and bulk chemicals.


Subject(s)
Cell Membrane , Metabolic Engineering
3.
Chinese Journal of Biotechnology ; (12): 1173-1182, 2022.
Article in Chinese | WPRIM | ID: wpr-927772

ABSTRACT

Opsin3 (OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain. In recent years, it has been reported that OPN3 is also highly expressed in adipose tissue, and the protein is associated with the production of skin melanin. We found that the N82 site is the glycosylation site of OPN3. SNAP-tagTM has diverse functions and can be applied to a variety of different studies. By constructing a SNAP-tagged OPN3 recombinant protein, the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface® 549 and SNAP-Cell® OregonGreen®, which provides a new method for studying the function of OPN3. It also shows that SNAP-tag does not affect the function of OPN3. Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.


Subject(s)
Cell Membrane , Glycosylation , Melanins , Membrane Proteins , Skin
4.
Rev. Assoc. Med. Bras. (1992) ; 67(9): 1233-1239, Sept. 2021. tab, graf
Article in English | LILACS | ID: biblio-1351454

ABSTRACT

SUMMARY OBJECTIVE: To evaluate the association between muscle mass depletion and compromising of the cell membrane integrity and clinical-anthropometric characteristics in patients with nonalcoholic fatty liver disease. METHODS: This observational study evaluated waist circumference, body mass index, and waist-to-height ratio in patients with nonalcoholic fatty liver disease. Skeletal mass index corrected by weight and impairment of cell membrane integrity were assessed using bioelectrical impedance analysis. RESULTS: In 56 patients, muscle mass depletion was observed in 62.5% and cell membrane impairment in 28.6%. The metabolic syndrome and elevated aspartate aminotransferase were the only clinical factors associated with mass depletion (p<0.05). The linear regression analysis showed association between skeletal mass index and waist-to-height ratio and waist circumference, after adjustments (p<0.05). The phase angle value was not different between those with and without mass depletion, and also it did not have correlation with skeletal mass index and clinical parameters (p>0.05). CONCLUSIONS: The prevalence of mass depletion and cell membrane impairment was higher in patients with nonalcoholic fatty liver disease. The muscle mass depletion was associated with central obesity, aspartate aminotransferase elevated, and metabolic syndrome; however, the phase angle is not associated with clinical and anthropometric data.


Subject(s)
Humans , Non-alcoholic Fatty Liver Disease , Body Mass Index , Cell Membrane , Risk Factors , Waist Circumference , Muscles
5.
Int. j. morphol ; 39(1): 25-31, feb. 2021. tab
Article in Spanish | LILACS | ID: biblio-1385297

ABSTRACT

RESUMEN: En el semen criopreservado, los procesos de congelación/descongelación y posterior manipulación, dañan las células espermáticas provocando disminución de la capacidad fecundante de los espermatozoides descongelados. Estos procesos han sido asociados con el estado de estrés oxidativo (EO) inducido por altos niveles de especies reactivas de oxígeno (EROS), causando daño a la función y estructura espermática. Los espermatozoides descongelados pueden ser protegidos de este daño, con la adición de antioxidantes (AO) al medio de incubación. El fruto de Calafate (Berberis microphylla G. Forst.) posee una alta capacidad antioxidante, lo que hace interesante investigar el efecto de sus componentes antioxidantes en estos procesos biotecnológicos especialmente postdescongelación. El objetivo de este estudio fue determinar el efecto de la suplementación de extracto liofilizado de fruto de Calafate (ELC), sobre la calidad espermática post-descongelación. Previamente se caracterizó el ELC, determinando la actividad antioxidante y metabolitos como fenoles y antocianinas; posteriormente, espermatozoides de bovino descongelados fueron incubados en un medio base suplementado con diferentes concentraciones de ELC. Post-incubación se evaluó la motilidad progresiva; la viabilidad e integridad de la membrana plasmática (SYBR14- PI) y acrosomal (FITC-PNA/PI) y la peroxidación lipídica (BODIPY) por citometría de flujo. La caracterización de ELC demostró que tanto la actividad antioxidante como los fenoles y antocianinas incrementan concomitante con el aumento de la concentración de ELC. La adición de ELC al medio de incubación, dependiendo de la concentración y tiempo de incubación, sería eficaz en proteger la motilidad, viabilidad e integridad de la membrana plasmática y disminuir la lipoperoxidación en los espermatozoides de bovino descongelados.


SUMMARY: In cryopreserved semen, the freezing/thawing process following of manipulation, damage the sperm cell, decreasing the fertilizing capacity of the thawed sperm; being one of the main factors of this damage the oxidative stress. The sperm once thawed can be protected from this damage, with the addition of antioxidants to the incubation medium. The Calafate fruit (Berberis microphylla G. Forst.) has a high antioxidant capacity, making it an interesting resource for investigating the effect of its antioxidant components on biotechnological processes. The objective of this study was to determine the effect of supplementation of Calafate fruit lyophilized extract (ELC) on sperm quality. The lyophilized extract of the Calafate fruit was characterized, determining the antioxidant activity and metabolites such as phenols and anthocyanins; subsequently, thawed bovine sperm were incubated in a medium supplemented with different concentrations of ELC. Post-incubation, progressive motility was evaluated. By flow cytometry, the viability and integrity of the plasma (SYBR14-PI), and acrosomal (FITC-PNA / PI), as well as lipid peroxidation (BODIPY), was determined. The characterization of Calafate fruits lyophilized extract indicated that antioxidant activity, phenols and anthocyanins increased concomitantly with the increase of dose extract used. The addition of ELC to the incubation medium, depending on the concentration and incubation time, would be effective to protect motility, viability and integrity of the plasma membrane and decreased lipid peroxidation in thawed bovine sperm.


Subject(s)
Animals , Cattle , Semen/drug effects , Plant Extracts/pharmacology , Berberis/chemistry , Antioxidants/pharmacology , Phenols/analysis , Semen/physiology , Sperm Motility/physiology , Plant Extracts/chemistry , Lipid Peroxidation , Cryopreservation , Cell Membrane , Reactive Oxygen Species , Oxidative Stress , Incubators , Anthocyanins/analysis , Antioxidants/chemistry
6.
Malaysian Journal of Microbiology ; : 380-389, 2021.
Article in English | WPRIM | ID: wpr-972807

ABSTRACT

Aims@#Skin burns remain a noteworthy general medical issue throughout the world, as it boosts a condition of immuno-suppression. The present research aimed to evaluate the efficacy of Syzygium aromaticum extracts, silver sulphadiazine ointment, and different commercially available topical antibiotics against pathogenic bacteria, isolated from the skin of burn patients.@*Methodology and results@#A total of 124 clinical pus samples were collected from the skin of burn patients, admitted to two different tertiary care burn units at Peshawar, Pakistan. From these pus samples, 6 bacterial isolates from burned skin (Staphylococcus epidermidis, Streptococcus, Klebsiella, Enterobacter, Bacillus and Pseudomonas spp.) were isolated, while 4 different bacterial isolates (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus and Streptococcus spp.) were isolated from unburned skin via conventional culturing techniques. Further, antibacterial assays were performed to compare the efficacy of S. aromaticum extracts (methanolic and aqueous extract), silver sulphadiazine ointment, and different commercially available antibiotics against tested bacteria. It was observed that both methanolic and aqueous extracts of S. aromaticum were effective at all concentrations against all the tested bacteria. In addition, all the tested antibiotics expressed substantial activity against most of the bacterial isolates. While silver sulphadiazine ointment was observed to be less potent against isolated bacteria as compared to S. aromaticum extracts. @*Conclusion, significance and impact of study@#It was concluded that both aqueous and methanolic extracts of S. aromaticum were effective antimicrobial agents and could be used as an alternative to control bacterial infections of burn patients. This study would help to distinguish the risk factors of bacterial pathogenicity in burn patients and would also provide a guideline to utilize medicinal plants and their extracts to minimize the chances of antibiotic resistance phenomenon in burn patients.


Subject(s)
Anti-Bacterial Agents , Cell Membrane , Oxidative Stress , Permeability , Plant Extracts , Syzygium
7.
Journal of Biomedical Engineering ; (6): 695-702, 2021.
Article in Chinese | WPRIM | ID: wpr-888229

ABSTRACT

Patch clamp is a technique that can measure weak current in the level of picoampere (pA). It has been widely used for cellular electrophysiological recording in fundamental medical researches, such as membrane potential and ion channel currents recording, etc. In order to obtain accurate measurement results, both the resistance and capacitance of the pipette are required to be compensated. Capacitance compensations are composed of slow and fast capacitance compensation. The slow compensation is determined by the lipid bilayer of cell membrane, and its magnitude usually ranges from a few picofarads (pF) to a few microfarads (μF), depending on the cell size. The fast capacitance is formed by the distributed capacitance of the glass pipette, wires and solution, mostly ranging in a few picofarads. After the pipette sucks the cells in the solution, the positions of the glass pipette and wire have been determined, and only taking once compensation for slow and fast capacitance will meet the recording requirements. However, when the study needs to deal with the temperature characteristics, it is still necessary to make a recognition on the temperature characteristic of the capacitance. We found that the time constant of fast capacitance discharge changed with increasing temperature of bath solution when we studied the photothermal effect on cell membrane by patch clamp. Based on this phenomenon, we proposed an equivalent circuit to calculate the temperature-dependent parameters. Experimental results showed that the fast capacitance increased in a positive rate of 0.04 pF/℃, while the pipette resistance decreased. The fine data analysis demonstrated that the temperature rises of bath solution determined the kinetics of the fast capacitance mainly by changing the inner solution resistance of the glass pipette. This result will provide a good reference for the fine temperature characteristic study related to cellular electrophysiology based on patch clamp technique.


Subject(s)
Cell Membrane , Electric Capacitance , Membrane Potentials , Patch-Clamp Techniques , Temperature
8.
Acta Academiae Medicinae Sinicae ; (6): 620-627, 2021.
Article in Chinese | WPRIM | ID: wpr-887903

ABSTRACT

Multi-drug resistance(MDR)refers to the loss of sensitivity of tumor cells to traditional chemotherapeutics agents under the mediation of various mechanisms,resulting in the reduction of chemotherapy efficacy.Current studies suggest that a variety of factors,including cell membrane transporter-mediated efflux of anti-tumor drugs,special microenvironment in tumor tissue,DNA self-repair and anti-apoptotic process,and epithelial-mesenchymal cell transformation,may contribute to the formation of MDR.Cell membrane transporter-mediated drug efflux refers to an increase in the amount of anti-tumor drug pumped out of the cell through the up-regulation of the ATP-binding cassette transporter on tumor cell membrane,which reduces the concentration of the drug in the cell,thus forming MDR.An effective method to inhibit the efflux pump caused by overexpression of membrane transporters plays an important role in overcoming MDR.As a promising drug delivery system,multifunctional nanoparticles have demonstrated many advantages in antitumor therapy.Meanwhile,nanoparticles with tailored design are capable of overcoming MDR when combined with a variety of strategies.This paper described in detail the studies relevant to the use of multifunctional nano-sized drug delivery system combined with different strategies,such as co-delivery of agents,external responsiveness or target modification for intervention with efflux pump in order to reverse MDR.This paper provides reference for the development of nano-sized drug delivery system and the formulation of reversal strategy in the future.


Subject(s)
Humans , Antineoplastic Agents/therapeutic use , Cell Membrane , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Membrane Transport Proteins/therapeutic use , Multifunctional Nanoparticles , Nanoparticles , Neoplasms/drug therapy , Tumor Microenvironment
9.
Arch. latinoam. nutr ; 70(2): 123-133, jun. 2020. tab, ilus
Article in Spanish | LIVECS, LILACS | ID: biblio-1140336

ABSTRACT

High intake of omega-3 fatty acids has been associated with synaptic plasticity, neurogenesis and memory in several experimental models. To assess the efficacy of fish oil supplementation on oxidative stress markers in patients diagnosed with probable Alzheimer´s disease (AD) we conducted a double blind, randomized, placebo controlled clinical trial. AD patients who met the inclusive criteria were given fish oil (containing 0.45 g eicosapentaenoic acid and 1 g docosahexaenoic acid) or placebo daily for 12 months. Oxidative stress markers [lipoperoxides, nitric oxide catabolites levels, oxidized/reduced glutathione ratio, and membrane fluidity] and fatty acid profile in erythrocytes were assessed at enrollment, and 6 and 12 months after the start of the testing period. At the end of the trial, in patients who received fish oil, we detected a decrease in the omega 6/omega 3 ratio in erythrocyte membrane phospholipids. This change was parallel with decreases in plasma levels of lipoperoxides and nitric oxide catabolites. Conversely, the ratio of reduced to oxidized glutathione was significantly increased. In addition, membrane fluidity was increased significantly in plasma membrane samples. In conclusion fish oil administration has a beneficial effect in decreasing the levels of oxidative stress markers and improving the membrane fluidity in plasma(AU)


El alto consumo de ácidos grasos omega-3 se asocia con la plasticidad sináptica, neurogénesis y memoria en varios modelos experimentales. Para evaluar la eficacia de la suplementación con aceite de pescado en los marcadores de estrés oxidativo en pacientes con diagnóstico de la enfermedad de Alzheimer (EA) probable realizamos un ensayo clínico doble ciego, aleatorizado, controlado con placebo. A los pacientes con la EA que cumplían los criterios de inclusión se les administró aceite de pescado (que contenía 0,45 g de ácido eicosapentaenoico y 1 g de ácido docosahexaenoico) o placebo diariamente durante 12 meses. Los marcadores de estrés oxidativo plasmático [niveles de lipoperóxidos y catabolitos del óxido nítrico, cociente de glutatión reducido a glutatiónoxidado) y fluidez de la membrana] y el perfil de ácidos grasos en los eritrocitos se evaluaron al inicio, 6 meses y alos 12 meses. Al final del ensayo, en pacientes que recibieron aceite de pescado detectamos una disminución en el cociente de ácidos grasos omega 6/omega 3 en los fosfolípidos de la membrana eritrocitaria. Este cambio ocurrió en paralelo a la disminución de los niveles plasmáticos de lipoperóxidos y catabolitos del óxido nítrico. Por el contrario, el cociente de glutatión reducido a glutatión oxidado se incrementó significativamente. Además, la fluidez de la membrana aumentó significativamente en las muestras analizadas. En conclusión, la administración de aceite de pescado tiene un efecto beneficioso al disminuir los niveles de marcadores de estrés oxidativo plasmático y mejorar la fluidez de la membrana plasmática(AU)


Subject(s)
Humans , Male , Female , Fish Oils , Fatty Acids, Omega-3 , Oxidative Stress , Alzheimer Disease , Cell Membrane , Chronic Disease , Neurogenesis
10.
Braz. dent. j ; 31(1): 32-36, Jan.-Feb. 2020. tab, graf
Article in English | LILACS | ID: biblio-1089272

ABSTRACT

Abstract This study evaluated the cytotoxic effect and the ability to inhibit matrix metalloproteinases (MMP-2 and MMP-9) of 0.2% chitosan (CH) and 1% acetic acid (AA) compared with 17% ethylenediaminetetraacetic acid (EDTA). Cell viability assay was performed according to ISO 10993-5 with mouse fibroblasts (L929). The culture was exposed to 0.2% CH, 1% AA, and 17% EDTA. The chelating agents were evaluated immediately after contact with the cells and after 6 h, 12 h, and 24 h of incubation. Cell viability was analyzed using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Inhibition of the gelatinolytic activity of MMP-2 and MMP-9 was evaluated by gelatin zymography. Different concentrations of CH were evaluated: 50 mM, 5 mM, 0.5 mM, and 0.05 mM. EDTA (0.5 mM) was used as a positive control. The results demonstrated that CH and AA had an initial cytotoxic effect, which decreased after 6 h, 12 h, and 24 h, being statistically similar to EDTA (P > 0.05). Additionally, CH at concentrations of 50 mM, 5 mM, and 0.5 mM had an inhibitory effect on MMP-2 and MMP-9, similar to that of the control with EDTA. The chelating agents had no cytotoxic effects after 24 h. MMP-2 and MMP-9 were inhibited by the experimental solutions.


Resumo Este estudo avaliou o efeito citotóxico e a capacidade de inibição das metaloproteinases da matriz extracelular (MMP-2 e MMP-9) pela quitosana 0,2%(CH) e o ácido acético 1% (AA) em comparação com o ácido etilenodiaminotetracético 17% (EDTA). O ensaio de viabilidade celular foi realizado de acordo com a ISO 10993-5 com fibroblastos de camundongo (L929). A cultura foi exposta a CH 0,2%, AA 1% e EDTA 17%. Os agentes quelantes foram avaliados imediatamente após o contato com as células e após 6 h, 12 h e 24 h de incubação. A viabilidade celular foi analisada utilizando o ensaio de brometo de 3- (4,5-dimetitiazol-2-il) -2,5-difeniltetrazólio (MTT). A inibição da atividade gelatinolítica de MMP-2 e MMP-9 foi avaliada por zimografia de gelatina. Diferentes concentrações de CH foram avaliadas: 50 mM, 5 mM, 0,5 mM e 0,05 mM. EDTA (0,5 mM) foi usado como controlo positivo. Os resultados demonstraram que CH e AA apresentaram um efeito citotóxico inicial, que diminuiu após 6 h, 12 h e 24 h, sendo estatisticamente similar ao EDTA (P> 0,05). Adicionalmente, CH a concentrações de 50 mM, 5 mM e 0,5 mM tiveram um efeito inibidor sobre MMP-2 e MMP-9, semelhante ao controlo com EDTA. Os agentes quelantes apresentaram efeitos não citotóxicos após 24 h. MMP-2 e MMP-9 foram inibidas pelas soluções experimentais.


Subject(s)
Animals , Rabbits , Matrix Metalloproteinases , Endodontics , Cell Membrane , Chelating Agents , Matrix Metalloproteinase 2
11.
Biol. Res ; 53: 06, 2020. graf
Article in English | LILACS | ID: biblio-1089076

ABSTRACT

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma mem-brane showed features of high affinity/slow turnover ATPases, with enzymatic parametersKM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.


Subject(s)
Cell Membrane/metabolism , Copper-Transporting ATPases/metabolism , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/chemistry
12.
Journal of Korean Academy of Oral Health ; : 13-19, 2020.
Article in English | WPRIM | ID: wpr-820822

ABSTRACT

OBJECTIVES: Galla chinensis inhibited the adherence of planktonic oral bacteria and acid production by cariogenic bacteria. However, little is known about the relevant conditions of Galla Chinensis extract (GCE) exposure time and concentration and the effect of GCE on the structural and functional activity of cariogenic bacteria. The antibacterial effects of natural G. Chinensis extract on S. mutans , S. sanguinis, and S. oralis biofilms were evaluated in vitro.METHODS: Biofilms formed on glass surfaces were treated with different concentrations of GCE at different exposure times. The effects were assessed by examining the bactericidal activity, acidogenesis, minimum inhibitory concentration, and morphology.RESULTS: There was a statistically significant difference in the bacterial growth inhibition depending on the concentration of the GCE, with bacterial growth being inhibited as the concentration of GCE increased. A concentration of 1.0 mg/ml GCE had similar bactericidal effects against S. mutans and S. oralis biofilms to those produced by 2.0 mg/ml CHX. In the 1.0 mg/ml GCE group, incomplete septa were also observed in the outline of the cell wall, together with disruption of the cell membrane. In addition, there was also a slight exudation of the intracellular content from the bacteria in the 1.0 mg/ml GCE and 2 mg/ml CHX groups.CONCLUSIONS: These results indicate that GCE inhibits the growth of S. mutans, S. sanguinis, and S. oralis with increasing concentrations. It alters the microstructure of S. mutans biofilms. These results suggest that GCE might be a useful anti-bacterial agent for preventing dental caries.


Subject(s)
Bacteria , Biofilms , Cell Membrane , Cell Wall , Dental Caries , Glass , In Vitro Techniques , Microbial Sensitivity Tests , Plankton , Streptococcus mutans
13.
Journal of Peking University(Health Sciences) ; (6): 214-220, 2020.
Article in Chinese | WPRIM | ID: wpr-941990

ABSTRACT

OBJECTIVE@#To identify the chaperone of polypyrimidine tractor-binding protein-associated splicing factor (PSF) in myeloid leukemia cells, and to explore the mechanism and redistributive pattern to cell surface of PSF in chemo-sensitive HL60 cells and resistant HL60/DOX cells.@*METHODS@#The eukaryotic expression vector of PSF was transfected with liposomes transiently, then flow cytometry was used to detect the expression level of PSF on the cell surface 24 h, 48 h and 72 h after vector transfections. We constructed a chimeric expression vector, streptavidin binding peptide (SBP)-PSF, meanwhile this vector was transfected and made SBP-PSF fusion protein overexpress. In addition, we used streptavidin magnetic beads to precipitate the cellular chaperonin of PSF and then identified its chaperonin by mass spectrometry (MS). Lentiviral vectors containing cytokeratin18 (K18) interference sequences were transfected into 293T cells to prepare lentivirus. HL60 and HL60/DOX cells were infected with lentivirus to obtain stable interfering K18 cell lines. Next, flow cytometry was used to test the membrane relocation level of PSF. Together, these methods confirmed the similar or different mechanisms of the PSF redistributing to membrane synergistically mediated by K18 in HL60 and HL60/DOX cells.@*RESULTS@#The expression of membrane relocated PSF was detected every day for three days (at the end of 24 h, 48 h and 72 h) after transient overexpression. The expressing rate of PSF on the cell surface was 22.4%±3.5%, 37.9%±6.0%, 58.3%±8.8%, respectively in sensitive HL60 cells, while that was 4.7%±0.5%, 3.9%±0.6%, 2.9%±0.6% , respectively in resistant HL60/DOX cells. The difference of expressing rate on each day was significant, P<0.01. We identified K18 detected by co-immunoprecipitation and mass spectrum assay which was the cellular chaperone of PSF. We found that K18 knockdown decreased the PSF expression level which redistributed on cell surface from 48.9%±5.4% to 6.2%±1.0% in sensitive HL60 cells, and from 9.11%±1.2% to 2.21%±0.51% in resistant HL60/DOX cells, respectively.@*CONCLUSION@#K18 is the intracellular chaperonin of PSF. The interaction of PSF and K18 mediates its redistribution to cell membrane in sensitive cells. While in resistant cells, PSF failed to relocate at the cell surface and accumulated in cells, which mediated resistance to chemotherapeutics.


Subject(s)
Humans , Cell Membrane , Doxorubicin , Drug Resistance, Multiple , Keratin-18/metabolism , Leukemia, Myeloid
14.
Chonnam Medical Journal ; : 20-26, 2020.
Article in English | WPRIM | ID: wpr-787278

ABSTRACT

We examined the effect of fluoxetine, a selective serotonin reuptake inhibitor antidepressant, on neuronal viability in mouse cortical near-pure neuronal cultures. Addition of fluoxetine to the media for 24 hours induced neuronal death in a concentration-dependent manner. To delineate the mechanisms of fluoxetine-induced neuronal death, we investigated the effects of trolox, cycloheximide (CHX), BDNF, z-VAD-FMK, and various metal-chelators on fluoxetine-induced neuronal death. Neuronal death was assessed by MTT assay. The addition of 20 µM fluoxetine to the media for 24 hours induced 60–70% neuronal death, which was associated with the hallmarks of apoptosis, chromatin condensation and DNA laddering. Fluoxetine-induced death was significantly attenuated by CHX, BDNF, or z-VAD-FMK. Treatment with antioxidants, trolox and ascorbate, also markedly attenuated fluoxetine-induced death. Interestingly, some divalent cation chelators (EGTA, Ca-EDTA, and Zn-EDTA) also markedly attenuated the neurotoxicity. Fluoxetine-induced reactive oxygen species (ROS) generation was measured using the fluorescent dye 2′,7′-dichlorofluorescin diacetate. Trolox and bathocuproine disulfonic acid (BCPS), a cell membrane impermeable copper ion chelator, markedly attenuated the ROS production and neuronal death. However, deferoxamine, an iron chelator, did not affect ROS generation or neurotoxicity. We examined the changes in intracellular copper concentration using a copper-selective fluorescent dye, Phen Green FL, which is quenched by free copper ions. Fluoxetine quenched the fluorescence in neuronal cells, and the quenching effect of fluoxetine was reversed by co-treatment with BCPS, however, not by deferoxamine. These findings demonstrate that fluoxetine could induce apoptotic and oxidative neuronal death associated with an influx of copper ions.


Subject(s)
Animals , Mice , Antioxidants , Apoptosis , Brain-Derived Neurotrophic Factor , Cell Death , Cell Membrane , Chelating Agents , Chromatin , Copper , Cycloheximide , Deferoxamine , DNA , Fluorescence , Fluoxetine , Ions , Iron , Neurons , Reactive Oxygen Species , Serotonin
15.
Biol. Res ; 52: 6, 2019. tab, graf
Article in English | LILACS | ID: biblio-1011409

ABSTRACT

BACKGROUND: Pollen development is an energy-consuming process that particularly occurs during meiosis. Low levels of adenosine triphosphate (ATP) may cause cell death, resulting in CMS (cytoplasmic male sterility). DNA sequence differences in ATP synthase genes have been revealed between the N- and S-cytoplasms in the cotton CMS system. However, very few data are available at the RNA level. In this study, we compared five ATP synthase genes in the H276A, H276B and fertile F1 (H276A/H268) lines using RNA editing, RNA blotting and quantitative real time-PCR (qRT-PCR) to explore their contribution to CMS. A molecular marker for identifying male sterile cytoplasm (MSC) was also developed. RESULTS: RNA blotting revealed the absence of any novel orf for the ATP synthase gene sequence in the three lines. Forty-one RNA editing sites were identified in the coding sequences. RNA editing showed that proteins had 32.43% higher hydrophobicity and that 39.02% of RNA editing sites had proline converted to leucine. Two new stop codons were detected in atp6 and atp9 by RNA editing. Real-time qRT-PCR data showed that the atp1, atp6, atp8, and atp9 genes had substantially lower expression levels in H276A compared with those in H276B. By contrast, the expression levels of all five genes were increased in F1 (H276A/H268). Moreover, a molecular marker based on a 6-bp deletion upstream of atp8 in H276A was developed to identify male sterile cytoplasm (MSC) in cotton. CONCLUSIONS: Our data substantially contributes to the understanding of the function of ATP synthase genes in cotton CMS. Therefore, we suggest that ATP synthase genes might be an indirect cause of cotton CMS. Further research is needed to investigate the relationship among ATP synthase genes in cotton CMS.


Subject(s)
Cell Membrane/genetics , RNA Editing , Adenosine Triphosphatases/genetics , Gossypium/enzymology , Plant Infertility/genetics , DNA, Mitochondrial/genetics , Polymerase Chain Reaction , Gene Expression Regulation, Plant/genetics , Gossypium/genetics , Cytoplasm/metabolism , RNA, Mitochondrial/genetics
16.
Mycobiology ; : 242-249, 2019.
Article in English | WPRIM | ID: wpr-760535

ABSTRACT

Betaine derivatives are considered major ingredients of shampoos and are commonly used as antistatic and viscosity-increasing agents. Several studies have also suggested that betaine derivatives can be used as antimicrobial agents. However, the antifungal activity and mechanism of action of betaine derivatives have not yet been fully understood. In this study, we investigated the antifungal activity of six betaine derivatives against Malassezia restricta, which is the most frequently isolated fungus from the human skin and is implicated in the development of dandruff. We found that, among the six betaine derivatives, lauryl betaine showed the most potent antifungal activity. The mechanism of action of lauryl betaine was studied mainly using another phylogenetically close model fungal organism, Cryptococcus neoformans, because of a lack of available genetic manipulation and functional genomics tools for M. restricta. Our genome-wide reverse genetic screening method using the C. neoformans gene deletion mutant library showed that the mutants with mutations in genes for cell membrane synthesis and integrity, particularly ergosterol synthesis, are highly sensitive to lauryl betaine. Furthermore, transcriptome changes in both C. neoformans and M. restricta cells grown in the presence of lauryl betaine were analyzed and the results indicated that the compound mainly affected cell membrane synthesis, particularly ergosterol synthesis. Overall, our data demonstrated that lauryl betaine influences ergosterol synthesis in C. neoformans and that the compound exerts a similar mechanism of action on M. restricta.


Subject(s)
Humans , Anti-Infective Agents , Betaine , Cell Membrane , Cryptococcus , Cryptococcus neoformans , Dandruff , Ergosterol , Fungi , Gene Deletion , Genetic Testing , Genomics , Malassezia , Methods , Skin , Transcriptome
17.
Chinese Journal of Biotechnology ; (12): 1162-1173, 2019.
Article in Chinese | WPRIM | ID: wpr-771812

ABSTRACT

Cell-penetrating peptides (CPPs) are short peptides that can penetrate the cell membrane or tissue barrier. CPPs can deliver a variety of biomacromolecules, such as proteins, RNA and DNA, into cells to produce intracellular functional effects. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms for CPPs-mediated cargo delivery. Compared with other non-natural chemical molecules-based delivery reagents, the CPPs have better biocompatibility, lower cytotoxicity, are easily degraded after cargo delivery, and can be fused and recombined expressed with bioactive proteins. Because of these advantages, the CPPs have become an important potential tool for delivery of developing drugs which targets intracellular factors. As a novel delivery tool, the CPPs also show promising application prospects in biomedical researches. This review summarized recent advances regarding the classification characteristics, the cellular uptake mechanisms and therapeutic application potentials of CPPs.


Subject(s)
Biological Transport , Cell Membrane , Cell-Penetrating Peptides , Metabolism , Endocytosis
18.
Chinese Journal of Biotechnology ; (12): 1463-1468, 2019.
Article in Chinese | WPRIM | ID: wpr-771783

ABSTRACT

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.


Subject(s)
Cell Membrane , DNA Primers , Escherichia coli , Gene Expression , Gene Products, tat , Genetic Vectors , Recombinant Fusion Proteins
19.
Biomolecules & Therapeutics ; : 562-569, 2019.
Article in English | WPRIM | ID: wpr-763045

ABSTRACT

Niacinamide (NIA) is a water-soluble vitamin that is widely used in the treatment of skin diseases. Moreover, NIA displays antioxidant effects and helps repair damaged DNA. Recent studies showed that particulate matter 2.5 (PM(2.5)) induced reactive oxygen species (ROS), causing disruption of DNA, lipids, and protein, mitochondrial depolarization, and apoptosis of skin keratinocytes. Here, we investigated the protective effects of NIA on PM(2.5)-induced oxidative stress in human HaCaT keratinocytes. We found that NIA could inhibit the ROS generation induced by PM(2.5), as well block the PM(2.5)-induced oxidation of molecules, such as lipids, proteins, and DNA. Furthermore, NIA alleviated PM(2.5)-induced accumulation of cellular Ca²⁺, which caused cell membrane depolarization and apoptosis, and reduced the number of apoptotic cells. Collectively, the findings show that NIA can protect keratinocytes from PM(2.5)-induced oxidative stress and cell damage.


Subject(s)
Humans , Antioxidants , Apoptosis , Cell Membrane , DNA , Keratinocytes , Mitochondrial Proteins , Niacinamide , Oxidative Stress , Particulate Matter , Reactive Oxygen Species , Skin Diseases , Skin , Vitamins
20.
Biomolecules & Therapeutics ; : 276-282, 2019.
Article in English | WPRIM | ID: wpr-763017

ABSTRACT

β-amyloid precursor protein (APP) can be cleaved by α-, and γ-secretase at plasma membrane producing soluble ectodomain fragment (sAPPα). Alternatively, following endocytosis, APP is cleaved by β-, and γ-secretase at early endosomes generating β-amyloid (Aβ), the main culprit in Alzheimer's disease (AD). Thus, APP endocytosis is critical for Aβ production. Recently, we reported that Monsonia angustifolia, the indigenous vegetables consumed in Tanzania, improved cognitive function and decreased Aβ production. In this study, we examined the underlying mechanism of justicidin A, the active compound of M. angustifolia, on Aβ production. We found that justicidin A reduced endocytosis of APP, increasing sAPPα level, while decreasing Aβ level in HeLa cells overexpressing human APP with the Swedish mutation. The effect of justicidin A on Aβ production was blocked by endocytosis inhibitors, indicating that the decreased APP endocytosis by justicidin A is the underlying mechanism. Thus, justicidin A, the active compound of M. angustifolia, may be a novel agent for AD treatment.


Subject(s)
Humans , Alzheimer Disease , Cell Membrane , Cognition , Endocytosis , Endosomes , HeLa Cells , Tanzania , Vegetables
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