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1.
J. venom. anim. toxins incl. trop. dis ; 27: e20200182, 2021. tab, graf, ilus
Article in English | ID: biblio-1250254

ABSTRACT

The accessory ß1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by ß1 subunits. Methods: Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in ß1 subunits. Results: The results show that deglycosylation of ß1 subunits through double-site mutations (ß1 N80A/N142A or ß1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+ß1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation. Conclusion: The present study reveals that glycosylation is an indispensable determinant of the modulation of ß1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of ß1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.(AU)


Subject(s)
Glycosylation , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase , Mutation , Pharmacology
2.
Article in Chinese | WPRIM | ID: wpr-817693

ABSTRACT

@#【Objective】Due to the tough nature of skin tissue and a high presence of RNases,the isolation of skin RNA by the classical Trizol method presents a challenge. Therefore,we adapted and tested different sample treatment protocols to improve the Trizol method for high- quality extraction of skin RNA.【Methods】In this study,normal skin of mice processed by different treatments(Tri:submersion Trizol;Pro:RNA sample protector;Cry:cryopreservation in liquid nitrogen frozen and then - 80 ℃ refrigerator;LNG:liquid nitrogen grinding;Cut:scissor cutting)were used as the experimental groups. Spinal cord tissue(Sp)was used as the reference group,and skin tissue of mouse psoriasis model induced by imiquimod(IMQ)was used as the validation group. We compared skin RNA concentration,purity and integrity, as well as IL- 1β mRNA expression extracted by conventional Trizol methods(1-Tri,Nor)and modified Trizol methods(2-Tri,LNG-Tri,Tri-Cut,Pro),which were determined by UV spectrophotometry,agar gel electrophoresis and quantitative reverse transcription PCR(qRT- PCR).【Results】① Compared with spinal cord(Sp),the total RNA of normal skin tissue extracted with the same classical Trizol method(1-Tri)was with lower yields,more obvious DNA contamination and 5S RNA bands,and higher IL-1β mRNA relative expression,suggesting that skin tissue was relatively special and the classical Trizol methods of skin RNA extraction should be improved. ② Among the different treatment methods of skin tissue,2-Tri and LNG-Tri methods resulted in higher RNA concentrations,lower RNA degradation and lower DNA contamination,and the expression of IL-1β mRNA was closer to normal levels. More importantly,the skin RNA samples extracted by the 2-Tri method can reflect more realistically the variation of IL-1β mRNA expression between normal and psoriasiform groups.【Conclusion】Improved 2-Tri or LNG-Tri method has the advantage of high quality of total RNA,and 2-Tri can more reliably reflect the mRNA expression pattern under physiological and pathological conditions.

3.
Acta Physiologica Sinica ; (6): 883-893, 2019.
Article in Chinese | WPRIM | ID: wpr-781386

ABSTRACT

In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2-4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec's Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-β, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1β and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo.


Subject(s)
Animals , Cell Culture Techniques , Methods , Cell Separation , Methods , Cells, Cultured , Hippocampus , Magnetics , Mice , Mice, Inbred C57BL , Microglia , Cell Biology
4.
Article in English | WPRIM | ID: wpr-776893

ABSTRACT

Resistance to cisplatin (DDP)-based chemotherapy is a major cause of treatment failure in human gastric cancer (GC). It is necessary to identify the drugs to re-sensitize GC cells to DDP. In our previous research, Zuo Jin Wan Formula (ZJW) has been proved could increase the mitochondrial apoptosis via cofilin-1 in a immortalized cell line, SGC-7901/DDP. Due to the immortalized cells may still difficult highly recapitulate the important molecular events in vivo, primary GC cells model derived from clinical patient was constructed in the present study to further evaluate the effect of ZJW and the underlying molecular mechanism. Immunofluorescent staining was used to indentify primary cultured human GC cells. Western blotting was carried out to detect the protein expression. Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation. Flow cytometry analysis was performed to assess cell apoptosis. ZJW inhibited proliferation and induced apoptosis in primary DDP-resistant GC cells. Notably, the apoptosis in GC cells was mediated by inducing cofilin-1 mitochondrial translocation, down-regulating Bcl-2 and up-regulating Bax expression. Surprisingly, the level of p-AKT protein was higher in DDP-resistant GC cells than that of the DDP-sensitive GC cells, and the activation of AKT could attenuate ZJW-induced sensitivity to DDP. These data revealed that ZJW can increase the chemosensitivity in DDP-resistant primary GC cells by inducing mitochondrial apoptosis and AKT inactivation. The combining chemotherapy with ZJW may be an effective therapeutic strategy for GC chemoresistance patients.


Subject(s)
Adult , Aged , Aged, 80 and over , Apoptosis , Cell Proliferation , Cisplatin , Pharmacology , Therapeutic Uses , Cofilin 1 , Metabolism , Drug Resistance, Neoplasm , Drugs, Chinese Herbal , Pharmacology , Female , Humans , Male , Middle Aged , Mitochondria , Metabolism , Pathology , Proto-Oncogene Proteins c-akt , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Stomach Neoplasms , Drug Therapy , Metabolism , Pathology , Tumor Cells, Cultured
5.
Journal of Experimental Hematology ; (6): 1415-1419, 2017.
Article in Chinese | WPRIM | ID: wpr-301714

ABSTRACT

<p><b>OBJECTIVE</b>To explore the clinical efficacy of GEMOX regimen on patients with refractory non-hodgkin's lymphoma.</p><p><b>METHODS</b>Eighty-two cases of non-Hodgkin's lymphoma were divided into 2 groups: gemcitabine+oxaliplation(Gem+Oxa) group (42 cases) and vinorelbine+oxaliplatin(Vin+Oxa) group (40 cases) according to chemotherapy regimens. The clinical efficacy, side effects, progression-free survival situation in 2 groups were compared.</p><p><b>RESULTS</b>There was no significant difference on the clinical effects of 2 groups (P>0.05); The therapeutic efficacy for B cell lymphoma was higher than that for T cell lymphoma(P<0.05); The therapeutic effects for I-II stages was lower than that for III-IV stages(P<0.05); The incidences of platelet decline, nausea and vomit, peripheral nerve symptoms in Gem+Oxa group were lower than those in Vin+Oxa group(P<0.05); There was no significant difference in the median progression free survival(P>0.05).</p><p><b>CONCLUSION</b>The efficacy of GEMOX regimen for refractory non-Hodgkin's lymphoma has been confirmed to be good, it has distinct clinical curative effect, it can prolong the progression-free survival time in patients with B cell lymphoma, specially for III-IV stages. It can be used as the preferred method for the treatment of patients with refractory NHL.</p>

6.
J. venom. anim. toxins incl. trop. dis ; 21: 5-5, 31/03/2015. tab, graf
Article in English | LILACS-Express | LILACS | ID: biblio-954758

ABSTRACT

AbstractBackground BmK I, a site-3-specific modulator of voltage-gated sodium channels (VGSCs), causes pain and hyperalgesia in rats, while BmK IT2, a site-4-specific modulator of VGSCs, suppresses pain-related responses. A stronger pain-related effect has been previously attributed to Buthus martensi Karsch (BmK) venom, which points out the joint pharmacological effect in the crude venom.Methods In order to detect the joint effect of BmK I and BmK IT2 on ND7-23 cells, the membrane current was measured by whole cell recording. BmK I and BmK IT2 were applied successively and jointly, and the synergistic modulations of VGSCs on ND7-23 cells were detected.Results Larger peak I Na and more negative half-activation voltage were elicited by joint application of BmK I and BmK IT2 than by application of BmK I or BmK IT2 alone. Compared to the control, co-applied BmK I and BmK IT2 also significantly prolonged the time constant of inactivation.Conclusions Our results indicated that site-4 toxin (BmK IT2) could enhance the pharmacological effect induced by site-3 toxin (BmK I), suggesting a stronger effect elicited by both toxins that alone usually exhibit opposite pharmacological effects, which is related to the allosteric interaction between receptor site 3 and site 4. Meanwhile, these results may bring a novel perspective for exploring the underlying mechanisms of scorpion sting-induced pain.

7.
Chinese Journal of Cardiology ; (12): 737-742, 2015.
Article in Chinese | WPRIM | ID: wpr-351611

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the distribution and major influencing factors of annual systolic blood pressure variability from a large population cohort.</p><p><b>METHODS</b>In this prospective cohort study, data from Kailuan Group employees who attended all 4 physical examinations ( taken in June 2006 to October 2007, June 2008 to October 2009, June 2010 to October 2011, June 2012 to October 2013, respectively) were analyzed (32 959 males and 10 401 females, mean age: (48.2 ± 11.5) years old). Systolic blood pressure variability was defined as the standard deviation (SSD) and the coefficient of variation (SCV) of systolic blood pressure of 4 physical examinations. Multivariate linear regression analysis was used to determine the related influencing factors of SSD and SCV.</p><p><b>RESULTS</b>(1) The mean of SSD and SCV for this cohort was 10.91 mmHg (1 mmHg = 0.133 kPa) and 8.34%, respectively. SSD and SCV increased in male and female with increasing age (both P < 0.001). (2) Multiple linear regression analysis showed that systolic blood pressure (β = 0.225, P < 0.001), age (β = 0.163, P < 0.001), fasting blood glucose (β = 0.038, P < 0.001), the use of anti-hypertensive drugs (β = 0.038, P < 0.001), sex (β = 0.038, P < 0.001), smoking (β = 0.025, P < 0.001), alcohol drinking (P = -0.022, P < 0.001), physical exercise (β = -0.018, P = 0.001), high-sensitivity c-reactive protein (β = 0.016, P = 0.001) body mass index (β = -0.011, P = 0.018) were related to SSD. Age (β = 0.139, P < 0.001), sex (β = 0.055, P < 0.001), systolic blood pressure (β = 0.047, P < 0.001), fasting blood glucose (P = 0.033, P < 0.001), drinking (β = -0.030, P < 0.001), body mass index (β = -0.026, P < 0.001), the use of anti- hypertensive drugs (β = 0.026, P < 0.001), smoking (β = 0.024, P < 0.001), physical exercise (β = -0.015, P = 0. 001), high-sensitivity c-reactive protein (β = 0. 014, P = 0. 001) were related to SCV.</p><p><b>CONCLUSIONS</b>SSD and SCV increase with increasing age. Systolic blood pressure, age, fasting blood glucose, the use of anti-hypertensive drugs, sex, smoking, drinking, physical exercise, high-sensitivity c-reactive protein, body mass index are major influencing factors for SSD. Age, sex, systolic blood pressure, fasting blood glucose, alcohol drinking, body mass index, the use of anti-hypertensive drugs, smoking, physical exercise, high-sensitivity c-reactive protein are major influencing factors for SCV.</p>


Subject(s)
Alcohol Drinking , Antihypertensive Agents , Blood Pressure , Body Mass Index , C-Reactive Protein , Cohort Studies , Exercise , Female , Humans , Male , Middle Aged , Prospective Studies , Smoking
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