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In December 2019, the corona virus disease 2019 (COVID-19) caused by novel coronavirus (SARS-CoV-2) emerged in Wuhan, China and rapidly spread worldwide. Few information on clinical features and immunological profile of COVID-19 in paediatrics. The clinical features and treatment outcomes of twelve paediatric patients confirmed as COVID-19 were analyzed. The immunological features of children patients was investigated and compared with twenty adult patients. The median age was 14.5-years (range from 0.64 to 17), and six of the patients were male. The average incubation period was 8 days. Clinically, cough (9/12, 75%) and fever (7/12, 58.3%) were the most common symptoms. Four patients (33.3%) had diarrhea during the disease. As to the immune profile, children had higher amount of total T cell, CD8+ T cell and B cell but lower CRP levels than adults (P

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Spike protein that mediates coronavirus entry into host cells is a major target for COVID-19 vaccines and antibody therapeutics. However, multiple variants of SARS-CoV-2 have emerged, which may potentially compromise vaccine effectiveness. Using a pseudovirus-based assay, we evaluated SARS-CoV-2 cell entry mediated by the viral Spike B.1.617 and B.1.1.7 variants. We also compared the neutralization ability of monoclonal antibodies from convalescent sera and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine) and ZF2001 (RBD-subunit vaccine) against B.1.617 and B.1.1.7 variants. Our results showed that, compared to D614G and B.1.1.7 variants, B.1.617 shows enhanced viral entry and membrane fusion, as well as more resistant to antibody neutralization. These findings have important implications for understanding viral infectivity and for immunization policy against SARS-CoV-2 variants.

It is important to evaluate the durability of the protective immune response elicited by primary infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we systematically evaluated the SARS-CoV-2-specific memory B cell and T cell responses in healthy controls and individuals recovered from asymptomatic or symptomatic infection approximately 6 months prior. Comparatively low frequencies of memory B cells specific for the receptor-binding domain (RBD) of spike glycoprotein (S) persisted in the peripheral blood of individuals who recovered from infection (median 0.62%, interquartile range 0.48-0.69). The SARS-CoV-2 RBD-specific memory B cell response was detected in 2 of 13 individuals who recovered from asymptomatic infection and 10 of 20 individuals who recovered from symptomatic infection. T cell responses induced by S, membrane (M), and nucleocapsid (N) peptide libraries from SARS-CoV-2 were observed in individuals recovered from coronavirus disease 2019 (COVID-19), and cross-reactive T cell responses to SARS-CoV-2 were also detected in healthy controls.

SARS-CoV-2 Spike-specific antibodies contribute the majority of the neutralizing activity in most convalescent human sera. Two SARS-CoV-2 variants, N501Y.V1 (also known as B.1.1.7 lineage or VOC-202012/01) and N501Y.V2 (B.1.351 lineage), reported from the United Kingdom and South Africa, contain several mutations in the receptor binding domain of Spike and are of particular concern. To address the infectivity and neutralization escape phenotypes potentially caused by these mutations, we used SARS-CoV-2 pseudovirus system to compare the viral infectivity, as well as the neutralization activities of convalescent sera and monoclonal antibodies (mAbs) against SARS-CoV-2 variants. Our results showed that N501Y Variant 1 and Variant 2 increase viral infectivity compared to the reference strain (wild-type, WT) in vitro. At 8 months after symptom onset, 17 serum samples of 20 participants (85%) retaining titers of ID50 >40 against WT pseudovirus, whereas the NAb titers of 8 samples (40%) and 18 samples (90%) decreased below the threshold against N501Y.V1 and N501Y.V2, respectively. In addition, both N501Y Variant 1 and Variant 2 reduced neutralization sensitivity to most (6/8) mAbs tested, while N501Y.V2 even abrogated neutralizing activity of two mAbs. Taken together the results suggest that N501Y.V1 and N501Y.V2 reduce neutralization sensitivity to some convalescent sera and mAbs.

Excessive monocyte activation with the development of excessive or uncontrolled release of proinflammatory cytokines often results in host tissue injury and even death in patients with pneumonia caused by the 2019 novel coronavirus. However, the changes of cytokine profiles of coronavirus disease 2019 (COVID-19) patients, as well as the underlying mechanisms that are involved, remain unknown. Using a cytokine array containing 174 inflammation-related cytokines, we found significantly altered cytokine profiles in severe COVID-19 patients compared with those in mild patients or healthy controls, and identified leptin, CXCL-10, IL-6, IL-10, IL-12, and TNF-α as the top differentially expressed cytokines. Notably, leptin showed high consistency with CXCL-10 and TNF-α in predicting disease severity, and correlated with body mass index, decreased lymphocyte counts, and disease progression. Further analysis demonstrated that monocytes in severe patients with higher leptin levels were inclined toward M1 polarization. Mechanistic studies revealed that leptin synergistically up-regulated expression levels of inflammatory cytokines and surface markers with IL-6 in monocytes through STAT3 and NF-κB signaling pathways. Collectively, our results suggest that overweight COVID-19 patients were prone to have higher leptin levels, which further activated monocytes, resulting in amplified or dysregulated immune responses. Taken together, our findings argue that leptin correlates severity of COVID-19 and may indicate a possible mechanism by which overweight patients have a greater tendency to develop severe conditions.

Many countries around the world have all seen a sharp rise in COVID-19 cases as the second wave since the beginning of October 2020. Decline of antibodies response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) that was reported exclusively in the early month increases the risk of reinfection for convalescent individuals. There is a current need to follow the maintenance of special antibodies against SARS-CoV-2. Here, we reported changes of antibodies against SARS-CoV-2 in convalescent patients over 8 months. Antibodies of all 20 participants targeting SARS-CoV-2 spike receptor binding-domain (RBD) had decreased from a mean OD450value 1.78 to 0.38 over 8 months. The neutralizing antibody (NAb) titers decreased from the mean ID50value 836 to 170. The NAb titers were significantly correlated with IgG level during 8 months (P

We used a new strategy to screen cytokines associated with SARS-CoV-2 infection. Cytokines that can classify populations in different states of SARS-CoV-2 infection were first screened in cross-sectional serum samples from 184 subjects by 2 statistical analyses. The resultant cytokines were then analyzed for their interrelationships and fluctuating features in sequential samples from 38 COVID-19 patients. Three cytokines, M-CSF, IL-8 and SCF, which were clustered into 3 different correlation groups and had relatively small fluctuations during SARS-CoV-2 infection, were selected for the construction of a multiclass classification model. This model discriminated healthy individuals and asymptomatic and nonsevere patients with accuracy of 77.4% but was not successful in classifying severe patients. Further searching led to a single cytokine, hepatocyte growth factor (HGF), which classified severe from nonsevere COVID-19 patients with a sensitivity of 84.6% and a specificity of 97.9% under a cutoff value of 1128 pg/ml. The level of this cytokine did not increase in nonsevere patients but was significantly elevated in severe patients. Considering its potent antiinflammatory function, we suggest that HGF might be a new candidate therapy for critical COVID-19. In addition, our new strategy provides not only a rational and effective way to focus on certain cytokine biomarkers for infectious diseases but also a new opportunity to probe the modulation of cytokines in the immune response.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel β-coronavirus, causes severe pneumonia and has spread throughout the globe rapidly. The disease associated with SARS-CoV-2 infection is named coronavirus disease 2019 (COVID-19). To date, real-time reverse-transcription polymerase chain reaction (RT-PCR) is the only test able to confirm this infection. However, the accuracy of RT-PCR depends on several factors; variations in these factors might significantly lower the sensitivity of detection. Methods In this study, we developed a peptide-based luminescent immunoassay that detected immunoglobulin (Ig)G and IgM. The assay cutoff value was determined by evaluating the sera from healthy and infected patients for pathogens other than SARS-CoV-2. Results To evaluate assay performance, we detected IgG and IgM in the sera from confirmed patients. The positive rate of IgG and IgM was 71.4% and 57.2%, respectively. Conclusions Therefore, combining our immunoassay with real-time RT-PCR might enhance the diagnostic accuracy of COVID-19.

BackgroundWe aim to investigate the profile of acute antibody response in COVID-19 patients, and provide proposals for the usage of antibody test in clinical practice.MethodsA multi-center cross-section study (285 patients) and a single-center follow-up study (63 patients) were performed to investigate the feature of acute antibody response to SARS-CoV-2. A cohort of 52 COVID-19 suspects and 64 close contacts were enrolled to evaluate the potentiality of the antibody test.ResultsThe positive rate for IgG reached 100% around 20 days after symptoms onset. The median day of seroconversion for both lgG and IgM was 13 days after symptoms onset. Seroconversion of IgM occurred at the same time, or earlier, or later than that of IgG. IgG levels in 100% patients (19/19) entered a platform within 6 days after seroconversion. The criteria of ‘IgG seroconversion’ and ‘> 4-fold increase in the IgG titers in sequential samples’ together diagnosed 82.9% (34/41) of the patients. Antibody test aided to confirm 4 patients with COVID-19 from 52 suspects who failed to be confirmed by RT-PCR and 7 patients from 148 close contacts with negative RT-PCR.ConclusionIgM and IgG should be detected simultaneously at the early phase of infection. The serological diagnosis criterion of seroconversion or the ‘>; 4-fold increase in the IgG titer’ is suitable for a majority of COVID-19 patients. Serologic test is helpful for the diagnosis of SARS-CoV-2 infection in suspects and close contacts.

A respiratory illness has been spreading rapidly in China, since its outbreak in Wuhan city, Hubei province in December 2019. The illness was caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical manifestations related to SARS-CoV-2 infection ranged from no symptom to fatal pneumonia. World Health Organization (WHO) named the diseases associated with SARS-CoV-2 infection as COVID-19. Real time RT-PCR is the only laboratory test available till now to confirm the infection. However, the accuracy of real time RT-PCR depends on many factors, including sampling location and of methods, quality of RNA extraction and training of operators etc.. Variations in these factors might significantly lower the sensitivity of the detection. We developed a peptide-based luminescent immunoassay to detect IgG and IgM. Cut-off value of this assay was determined by the detection of 200 healthy sera and 167 sera from patients infected with other pathogens than SARS-CoV-2. To evaluate the performance of this assay, we detected IgG and IgM in the 276 sera from confirmed patients. The positive rate of IgG and IgM were 71.4% (197/276) and 57.2% (158/276) respectively. By combining with real time RT-PCR detection, this assay might help to enhance the accuracy of diagnosis of SARS-CoV-2 infection.

We previously reported that three point mutations in SASH1 and mutated SASH1 promote melanocyte migration in dyschromatosis universalis hereditaria (DUH) and a novel p53/POMC/Gαs/SASH1 autoregulatory positive feedback loop is regulated by SASH1 mutations to induce pathological hyperpigmentation phenotype. However, the underlying mechanism of molecular regulation to cause this hyperpigmentation disorder still remains unclear. In this study, we aimed to investigate the molecular mechanism undergirding hyperpigmentation in the dyschromatosis disorder. Our results revealed that SASH1 binds with MAP2K2 and is induced by p53‐POMC‐MC1R signal cascade to enhance the phosphorylation level of ERK1/2 and CREB. Moreover, increase in phosphorylated ERK1/2 and CREB levels and melanogenesis‐specific molecules is induced by mutated SASH1 alleles. Together, our results suggest that a novel SASH1/MAP2K2 crosstalk connects ERK1/2/CREB cascade with p53‐POMC‐MC1R cascade to cause hyperpigmentation phenotype of DUH.

Acute promyelocytic leukemia (APL) is a special subtype of acute myeloid leukemia that responds to treatment with all-trans retinoic acid and arsenic trioxide. However, severe side effects and drug resistance limit the effectiveness of these treatments. Hence, new drugs for APL are required urgently. Shikonin, an active naphthoquinone derived from the Chinese medical herb Zi Cao exerts antitumor activity in several cancers. In the present study, the effects of shikonin on proliferation and apoptosis in NB4 cells, as well as related mechanisms were assessed. Treatment of NB4 cells with shikonin inhibited proliferation in a concentration- and time-dependent manner. The cell cycle was arrested in the G1 phase. NB4 cells treated with shikonin exhibited more apoptosis and higher levels of cleaved caspase-3 and poly ADP-ribose polymerase than control cells. Western blotting results demonstrated that the expression of p-p38 mitogen-activated protein kinase (p-p38MAPK) and p-c-Jun N-terminal kinase (p-JNK) was increased significantly by shikonin treatment, while the expression of p-ERK and c-Myc was decreased. In summary, these findings indicated that shikonin inhibited cell proliferation and induced apoptosis partly through modulation of the MAPKs and downregulation of c-Myc.

In the majority of acute promyelocytic leukemia (APL) cases, translocons produce a promyelocytic leukemia protein-retinoic acid receptor α (PML-RARα) fusion gene. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RAα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. NLS-RARα promotes cell growth and inhibits differentiation in response to ATRA. However, the mechanisms by which NLS-RARα affects cell biological characteristics are yet to be fully elucidated. The present study found that the location of RARαwas altered after it was cleaved by NE. Firstly, NE was overexpressed during the preparation of recombinant plasmid NB-4/pCMV6-NE-Myc to cleave PML-RARα. The total protein expression levels of myc and NE and expression levels of NLS-RARα in nucleoprotein were detected by western blotting. Location of NLS-RARα protein was detected by immunofluorescence and confocal laser scanning. Secondly, a nude mice model was constructed and NE protein, NLS-RARα and RARα protein assays, and the location of NLS-RARα and RARα proteins were assessed as described. The present results showed that, compared with the control groups, the location of NLS-RARα protein was predominantly detected in the nucleus, whereas RARα was mainly distributed in the cytoplasm. These findings were consistent with those of the nude mice model, and these may be used as a foundation to explain the occurrence mechanism of APL.

Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with specificity for a broad range of substrates. NE has been reported to be associated with the pathogenesis of several conditions, particularly that of pulmonary diseases. Previous studies have shown that NE can cleave the pro-myelocyte - retinoic acid receptor-alpha chimeric protein and is important for the development of acute pro-myelocytic leukemia. To further elucidate the role of NE in acute pro-myelocytic leukemia, the present study successfully constructed a lentiviral vector containing the NE gene (LV5-NE), which was transfected into NB4 acute pro-myelocytic leukemia cells. The effects of NE overexpression in NB4 cells were detected using a Cell-Counting Kit-8 assay, flow cytometry and western blot analysis. The results showed that NE significantly promoted the proliferation of NB4 cells, inhibited cell apoptosis and apoptotic signaling, and led the activation of Akt. In an additional experiment, a vector expressing small hairpin RNA targeting NE was constructed to assess the effects of NE knockdown in U937 cells. Western blot analysis revealed that apoptotic signaling was increased, while Akt activation was decreased following silencing of NE. The results of the present study may indicate that NE activates the phosphoinositide-3 kinase/Akt signaling pathway in leukemia cells to inhibit apoptosis and enhance cell proliferation, and may therefore represent a molecular target for the treatment of pro-myelocytic leukemia.

Neutrophil elastase (NE) is an early myeloid-specific serine protease, which is predominantly produced by promyelocytes. A previous study demonstrated that NE has an important role in the development of acute promyelocytic leukemia (APL). The process of APL was shown to be accelerated in animals that expressed abundant NE, whereas NE‑deficient mice were protected from APL development; thus suggesting an important role for NE in the development of APL. The present study aimed to investigate the effects and possible mechanisms of NE. Up- and downregulation of NE in various leukemia cell lines was conducted in order to explore its significance in the occurrence and procession of leukemia, with the aim of identifying novel targeted therapeutic drugs for the treatment of leukemia. NE was overexpressed in cells following infection with an adenovirus, and Cell Counting kit‑8 and flow cytometry results demonstrated that cell proliferation was promoted, and cell apoptosis was inhibited, as compared with the untreated cells. NE was downregulated in the cells by both RNA interference and treatment with GW311616A, a specific inhibitor of NE, following which cell growth was shown to be inhibited and apoptosis was induced. These results suggested that NE may promote the development of APL, therefore, NE may be a therapeutic target and its inhibitor GW311616A may be a potential therapeutic drug for leukemia. Furthermore, the apoptosis‑associated protein B‑cell lymphoma 2 (Bcl‑2)‑associated X protein was significantly increased, whereas Bcl‑2 was markedly decreased in the cells with downregulated NE. Further experiments revealed that the probable apoptosis‑associated signaling pathway was the phosphoinositide 3‑kinase/AKT pathway. The present study is the first, to the best of our knowledge, to demonstrate that GW311616A, a specific NE inhibitor, may act as a potential targeted drug for leukemia, which may have a profound impact on the future of leukemia-targeted therapy.

The BCR-ABL kinase inhibitor, imatinib mesylate, is the front-line treatment for chronic myeloid leukemia, but the emergence of imatinib resistance has led to the search for alternative drug treatments. There is a pressing need, therefore, to develop and test novel drugs. Natural products including plants, microorganisms, and halobios provide rich resources for discovery of anticancer drugs. In this article, we demonstrate that emodin inhibited the growth of K562 cells harboring BCR-ABL in vitro and in vivo, and induced abundant apoptosis, which was correlated with the inhibition of PETN/PI3K/Akt level and deletion of BCR-ABL. These findings suggest that emodin is a promising agent to kill K562 cells harboring BCR-ABL.

p53‐Transcriptional‐regulated proteins interact with a large number of other signal transduction pathways in the cell, and a number of positive and negative autoregulatory feedback loops act upon the p53 response. P53 directly controls the POMC/α‐MSH productions induced by ultraviolet (UV) and is associated with UV‐independent pathological pigmentation. When identifying the causative gene of dyschromatosis universalis hereditaria (DUH), we found three mutations encoding amino acid substitutions in the gene SAM and SH3 domain containing 1 (SASH1), and SASH1 was associated with guanine nucleotide‐binding protein subunit‐alpha isoforms short (Gαs). However, the pathological gene and pathological mechanism of DUH remain unknown for about 90 years. We demonstrate that SASH1 is physiologically induced by p53 upon UV stimulation and SASH and p53 is reciprocally induced at physiological and pathophysiological conditions. SASH1 is regulated by a novel p53/POMC/α‐MSH/Gαs/SASH1 cascade to mediate melanogenesis. A novel p53/POMC/Gαs/SASH1 autoregulatory positive feedback loop is regulated by SASH1 mutations to induce pathological hyperpigmentation phenotype. Our study demonstrates that a novel p53/POMC/Gαs/SASH1 autoregulatory positive feedback loop is regulated by SASH1 mutations to induce pathological hyperpigmentation phenotype.

Acute promyelocytic leukemia (APL) is characterized by the presence of the promyelocytic leukemia (PML)-retinoic acid receptor-α (RAR-α) fusion protein. PML-RARα can be cleaved by neutrophil elastase (NE) in several positions in cells in the promyelocytic stage, nuclear location signal (NLS)-negative PML and NLS-RARα may be the products of PML-RARα by NE. The function of NLS-RARα may be affected by the addition of NLS, which would alter its localization in cells, as the role of NLS is to identify proteins for transport to the nucleus. Preliminary experiments demonstrated that the overexpression of NLS-RARα in HL-60 cells could promote cellular proliferation and inhibit cellular differentiation. Following treatment with all-trans retinoic acid (ATRA), the degree of cellular differentiation was enhanced. In the present study, the localization of NLS-RARα was identified and its activity as a novel transcriptional factor was assessed, which may be critical in the development of APL. The location of NLS-RARα was detected in the nucleus and cytoplasm by indirect immunofluorescence and western blot analysis, with expression in the nucleus revealed to be increased compared with that in the cytoplasm. Next, native-PAGE was performed and NLS-RARα and RXRα were revealed to form heterodimers in the nucleus. In addition, co-immunoprecipitation revealed an interaction between NLS-RARα and retinoid X receptor-α (RXRα). An electrophoresis mobility shift assay (EMSA) indicated that NLS-RARα could bind retinoic acid response elements (RAREs) in the presence of ATRA. Indeed, NLS-RARα could bind RAREs just as WTRARα could, including the RAREs direct repeat-2 (DR-2) and DR-5. In addition, results from a luciferase reporter gene assay demonstrated that NLS-RARα could mediate the activity of RAREs that it bound. Together, these results indicated that NLS-RARα may be a novel transcription factor that contributes to leukemogenesis by competitively binding RAREs as heterodimers with RXRα, just as PML-RARα does, thus repressing the gene transcription essential for myeloid differentiation. These findings indicate the potential role of NLS-RARα targeted therapy in APL.

Objective To investigate the effect of tumor-suppressing gene JTV1 on proliferation and apoptosis of leukemic K562 cells,and the changes in apoptosis factors Bcl-2,C-myc and Bax genes.Methods The recombinate vector pcDNA3.1-JTV1,and the empty vector pcDNA3.1 were transfected into K562 cells as control.The cell proliferation of K562 cells was evaluated by colony formation assay;the cell cycle and apoptosis rate were assessed by flow cytometry(FCM);the mRNA levels of apoptosis related genes Bax,Bcl-2 and C-myc were determined by RT-PCR;the protein levels of Bax,Bcl-2 and C-myc were assayed by Western Blotting.Results The colony formation assay showed that the proliferation of K562 cells decreased when the expression of JTV1 gene was up-regulated.FCM assay showed that the G phase cells in pcDNA3.1-JTV1 positive transfection group increased compared with that of the control group and the pcDNA3.1 empty vector transfected group,and the differences were statistically significant(P < 0.05).Compared with the control group and the empty vector group,the mRNA transcription level and the protein translation level of Bax gene increased significantly,and the mRNA transcription level and the protein translation level of Bcl-2 and C-myc gene were reduced significantly(P < 0.05).Conclusions The expressions of Bcl-2 and C-myc gene are inhibited when the gene JTV1 is up-regulated,leading to an increase in Bax gene expression,inhibition of K562 cell proliferation,and promotion of tumor cells apoptosis.Over expression of JTV1 gene can inhibit the proliferation of K562 cells and promote cell apoptosis by inhibiting Bcl-2 and C-myc expression and up-regulating that of Bax.

The natural product caffeic acid is a specific inhibitor of 5-lipoxygenase (5-LOX); it also possesses antioxidant and antiinflammatory properties. The current study was designed to determine whether the neuroprotective properties of caffeic acid are due to inhibition of 5-LOX. Cerebral damage was induced in mice by intracerebroventricular microinjection of aluminum (5.0 microg aluminum in 2.0 microL, once a day, for 5 days). Caffeic acid was administered intragastrically at 30 min prior to aluminum and repeated daily for an additional 10 days. The brain injury was determined by observation of behavioral changes in mice, as well as by measuring biochemical and pathological changes in the cerebral tissue. The levels of 5-LOX proteins and 5-LOX mRNA expression were measured in brain tissue. Aluminum impaired learning and memory in mice produced neuronal death in hippocampi, elevated brain malondialdehyde levels, increased protein expression of amyloid precursor protein (APP), amyloid beta, and 5-LOX. It also increased 5-LOX mRNA expression and decreased choline acetyl transferase (ChAT) protein expression in the brain tissue of mice. Caffeic acid prevented brain damage as well as behavioral and biochemical changes caused by aluminum overload. The results of this study suggest that overexpression of 5-LOX accompanies the cerebral injury induced by aluminum overload in mice, and that selective inhibitors of 5-LOX may have potential value in the treatment of aluminum neurotoxicity and conceivably of diseases associated with neuronal injury.

To explore the impact of extracellular acidic environment on the expression and activity of P-glycoprotein (P-gp) and on the P-gp-mediated cytotoxicity of daunomycin in cancer cells by using microfluidic chip technology.The A549 cells cultured on a microfluidic chip were divided into experiment group and control group. The experiment group was exposed to an acidic cell culture medium (pH 6.6), while the control group was treated with a neutral cell culture medium (pH 7.4). The expression of P-gp was detected by cell immunofluorescense analysis and the activity of P-gp was evaluated by Rhodamine 123 efflux experiment. Meanwhile, the cytotoxicity of daunomycin was analyzed by cell live/dead fluorescence staining method.Microfluidic chip designed in this study could provide a suitable microenvironment for the growth of A549 cells and the A549 cells reached the confluence of 90% after inoculation for 72 h. Treatment of the acidic cell culture media on A549 cells did not make a significant difference on the expression level of P-gp. However, the activity of P-gp was significantly enhancement and peaked at 6 h after treatment with acidic cell culture media. Meanwhile, the cytotoxicity of daunomycin reduced significantly after treatment with acidic cell culture medium for 6 h,and a reversal effect was obtained when synergy with verapamil.Microfluidic chip technology can shorten the analysis time and reduce the reagent consumption. It can be used as a new technology platform for understanding the mechanisms of multi-drug resistance and for screening highly efficient multi-drug resistance reversal agents.

To explore the effect and mechanism of recombined adenovirus carrying NLS-RARalpha gene on proliferation of HL-60 cells and the differentiation of HL-60 cells induced by ATRA.HL-60 cells was infected with Ad-NLS-RARalpha and control virus Ad-KZ. The efficiency of infection was detected by FCM. The mRNA and protein levels of NLS-RARalpha were assessed by Real-time PCR (RT-PCR) and Western blot, respectively. MTT assay were applied to determine proliferation of HL-60 cells. Cell surface differentiation antigen CD11b of infected HL-60 cell induced by ATRA was examined by FCM. The mRNA and protein levels of C-MYC of infected HL-60 cell induced by ATRA were determined by Real-time PCR (RT-PCR) and Western blot assay.The efficiency of infection of Ad-NLS-RARalpha and Ad-KZ on HL-60 cell was 70%-80%. The mRNA and protein levels of NLS-RARalpha gene of HL-60 cells which infected with Ad-NLS-RARalpha were both obviously higher than that of the cells which infected with Ad-KZ and non-infected (P0.05). The proliferation ability of HL-60 cell infected with Ad-NLS-RARalpha was significantly increased (P0.05). The level of CD11b of HL-60 cell infected with Ad-NLS-RARalpha and induced by ATRA was clearly decreased than control groups (P0.05). The mRNA and protein levels of C-MYC gene of HL-60 cells infected with Ad-NLS-RARalpha and induced by ATRA were both obviously higher than that of the cells which infected with Ad-KZ and non-infected (P0.05).The recombined adenovirus Ad-NLS-RARalpha can increase the proliferation ability of HL-60 cell, and inhibit the differentiation of HL-60 cell through reduce the expression level of C-MYC gene.

To determine the effect and mechanism of action of PML(NLS-) gene on emodin-induced apoptosis of human HL-60 cells.HL-60 cells were infected with recombinant adenovirus Ad-PML (NLS-) and Ad-KZ, respectively. The PML(NLS-) gene was detected by Real-time PCR(RT-PCR) and Western blot. The proliferation level of the HL-60 cells was determined by MTT method. The HL-60 cells were treated with 60 micromol/L emodin for 72 h and then analyzed by flow cytometry for their cell cycle and apoptosis rate. The transcription levels of apoptosis-related BCL-2, BAX and C-MYC genes were determined by RT-PCR. The translation levels of those genes were determined by Western blot.Compared with normal controls and the HL-60 cells infected with Ad-KZ, the mRNA and protein expression levels of PML(NLS-) gene increased significantly in the HL-60 cells infected with Ad-PML( NLS-). Increased proliferation levels of the Ad-PML (NLS-) infected HL-60 cells were observed in those treated with 60 pmol/L emodin, which showed decreased percentage of cells at Gx phase, increased percentage of cells at S phase, and decreased emodin-induced apoptosis. The levels of mRNA transcription and protein expression of BAX gene decreased, while those of BCL-2 and C-MYC genes increased significantly.The over-expression of PML(NLS-) gene might promote the proliferation and arrest the apoptosis of HL-60 cells by up-regulating the expressions of BCL-2 and C-MYC genes and down-regulating the expression of BAX gene.

To study and compare the effect of neutrophil elastase inhibitors (GW311616A and sivelestat) on the proliferation and apoptosis of U937 cells.Inhibitory effects of GW311616A and sivelestat on the proliferation of U937 cells were assayed by MTT assay. The morphologic changes of U937 cells were detected by transmission electron microscope, and apoptosis was observed by AnnexinV-FITC/PI staining. The changes of cell cycle and apoptosis were detected by flow cytometry. The expression of NE in U937 cells was observed by indirect immunofluorescence, the variations of content and activity of NE in U937 cells were measured through ELISA assay and colorimetric method.MTT showed that both NE inhibitors could inhibit the proliferation of U937 cells in a dose dependent manner. The IC50 of GW311616A and sivelestat were 150 and 214 μmol/L respectively. The inhibition effect of GW311616A was significantly higher than of sivelestat (P0.01). Typical apoptosis morphological changes of U937 cells was observed through electron microscope. AnnexinV-FITC/PI staining showed that U937 cells could be induced to undergo apoptosis by the two inhibitors, the apoptosis ratio of 150μmol/L GW311616A group (13.60%) was significantly higher than that of 150μmol/L sivelestat group (3.69%)(P0.01). The result of flow cytometry indicated that the apoptosis ratio of 150 μmol/L GW311616A group was 14.61%, U937 cell cycle was mainly blocked in G2/M phase; meanwhile 150 μmol/L sivelestat group as 4.25% with cell cycle in S phase. The fluorescence intensity of GW311616A group obviously decreased than of sivelestat group. And the two inhibitors could reduce the content and activity of NE in U937 cells, but the effect of GW311616A was significantly higher than of sivelestat (P0.01).GW311616A and sivelestat could inhibit the proliferation and cause apoptosis of U937 cells. Furthermore, GW311616A was more effective and harmful to cells than sivelestat.

To explore the interaction between BPHL and PML-C by co-immunoprecipitation and yeast two-hybird system.The recombination expression plasmids pGBKT7-PML-C and pACT2-BPHL were cotransformed into yeast AH109, to investigate their interaction in vivo. The expression vector of HA-tagged fusion protein (pCMV-HA-PML-C) and the expression vector of myc-tagged fusion protein (pCMV-myc-BPHL) were constructed and identified respectively, and cotransfected into human embryo kidney 293 (HEK293) cells. The interaction between PML-C and BPHL was investigated by co-immunoprecipitation in vitro.Blue clones were found in QDO/5-bromo-4-chloro-3-indolyl-alpha-D-galactoside (X-alpha-gal) plate, eukaryotic expression vectors named as pCMV-HA-PML-C and pCMV-myc-BPHL were constructed and confirmed with double restriction enzyme digestion and co-transfected into HEK 293 cells successfully. After immunoprecipitation of HA-PML-C with anti-HA polyclonal antibody, expressed myc-BPHL protein was identified by Western blot with anti-c-myc monoclonal antibody from immunoprecipitated complex.The eukaryotic expression vector of PCMV-HA-PML-C and PCMV-myc-BPHL were constructed successfully. The interaction between PML-C and BPHL was identified by co-immunoprecipitation and yeast two-hybird technique.

This paper developed a temperature control system in order to carry out a real time and continuous observation on living cells under microscope. In this system, transparent indium tin oxid (ITO) conductive film was used as a heater, temperature was measured by the temperature dependence of resistance behavior of the ITO film, C8051F340 single-chip was used as the control hardware core, the modified PID algorithm and pulse width modulation(PWM) were adopt as the key control software programs. The temperature system had a simple structure without added temperature sensor, an uniform temperature distribution within ±1 °C on the ITO film surface and temperature control precision of ±0.2 °C could be obtained. The system could maintain a good growth state for BHK-21 living cell under microscope for 48 hours.

To identify the interactions between JTV1 and NLS-RAR alpha by Yeast two-hybrid and co-immunoprecipitation.The plasmids of bait-protein and JTV1 protein were cotransformed into yeast AH109 to investigate their interactions in vivo. Tagged fusion protein eukaryotic expression vectors were constructed and then cotransfected into human embryo kidney 293 cells. Co-immunoprecipitation was used to investigate the interactions between NLS-RAR alpha and JTV1 in vitro.Blue clones were found in QDO/X-alpha-gal plates. Eukaryotic expression vectors were co-transfected into HEK 293 cells. The HA-NLS-RAR alpha protein was immunoprecipitated by anti-HA polyclonal antibody. Myc-JTV1 protein was detected by western blotting with anti-Myc monoclonal antibody from the immunoprecipitared complex.The interactions between NLS-RAR alpha and JTV1 are identified by Yeast two-hybrid and co-immunoprecipitation.

The anti-tumor activity of dexamethasone derivatives (9-fluoro-16alpha-methyl-11,17-dihydroxy-3-oxo-1,4-androsladiene-17beta-carboxylic acid) is superior to that of dexamethasone. This study was to screen the proteins interacting with dexamethasone derivates, thus to explore the anti-tumor mechanism of dexamethasone derivates in vivo.The bait plasmid pGBKT7-GRalpha-LBD was constructed. Screening of the target proteins interacting with dexamethasone derivatives was performed by yeast three-hybrid technique using human K562 cell cDNA library.The bait plasmid was successfully constructed. It produced a 31 ku bait protein with no toxicity, leakage and self-activation. Thirty-seven positive clones which interacted with dexamethasone derivatives were obtained from human K562 cell cDNA library, 20 of which were identified by re-transforming into yeast AH109 cells.Twenty positive clones interacting with dexamethasone derivates are identified in vivo.

To investigate the protective effects of the total base from rhizoma coptis chinensis (CTB) and berberine (Ber) on neurodegeneration induced by aluminum overload in rats.The study took place in the Department of Pharmacology, Chongqing Medical University, Chongqing, China, between February 2005 and May 2007. Wistar rats were divided into control group, model group, Ber-treated group, CTB (55 mg/kg and 110 mg/kg)-treated group, and nimodipine-treated group (n=20). A rat brain damage model was established via intragastric administration of 400 mg/kg element aluminum once a day, 5 days a week for 12 weeks. The CTB, Ber, and nimodipine were intragastrically administered 4 hours after each aluminum administration for 12 weeks. The morphological changes of the neurons of the rat hippocampus and the changes of rat learning and memory functions were observed. The superoxide dismutase (SOD), choline acetyltransferase (ChAT), acetylcholinesterase (AchE), and monoamine oxidase-B (MAO-B) activities and malondialdehyde (MDA) content, as well as the MAO-B expression in the rat brain were examined.The CTB, Ber, and nimodipine significantly improved the learning and memory ability impairment and hippocampal neuronal death. The CTB, Ber, and nimodipine also significantly blunted the decrease of SOD and ChAT activities, and the increase of MDA content, AchE activities, and MAO-B expressions and activity in the aluminum-overload rats.The CTB and Ber have protective effects on neurodegeneration induced by aluminum overload. The CTB (110 mg/kg) has more powerful neuroprotection than Ber.

Objective: To investigate the effects of Ro13-7410 on telomerase activity and cell cycle distribution. Methods: Telomerase activity of HL-60 cells induced by retinoid Ro13-7410 was detected by telomerase PCR-ELISA-kit. The cell cycle was analyzed by flow cytometry. Results: Telomerase activity declined gradually after 10−6 mol/L Ro13-7410 treatment, and the inhibition of telomerase activity at day 5 of treatment with Ro13-7410 was less effective than with Retinoid Acid (RA). DNA flow cytofluorimetric analysis revealed that Ro13-7410 caused partial cells arrest in the G2/M phase after 4-days treatment. Conclusion: Telomerase activity declined gradually and partial cells were arrested in the G2/M phase after Ro13-7410 treatment.

To screen the protein interacting with retinoic acid receptor variant protein (RAR alpha-V) via the yeast two-hybrid technique (YTHT) and to find out the targets protein and study its biological function.The bait vector of pGBKT7-RAR alpha-V was constructed for screening proteins interacting with RAR alpha-V in K562 cell cDNA expression library via YTHT. The protein-protein interaction was confirmed with re-transformation in yeast and GST pull-down in vitro.The bait vector was successfully constructed without toxicity, leakage and self-activation. Sixteen proteins were screened by YTHT and eight positive clones were identified by re-transformation in yeast. The interaction between RAR alpha-V and JTV-1 was confirmed by GST pull-down in vitro.There are some kinds of proteins interacting with RAR alpha-V in cell. The biological dysfunction caused by certain protein-protein interaction may be involved in the pathogenesis of leukemia.

The differentiation of K562 cells can be induced by a given concentration of matrine. This study was designed to investigate the mechanism of signal transduction in induced differentiation of K562 cells.ELISA coupled with streptavidin-biotin system was used to dynamically detect the activity of protein tyrosine kinase and phosphatase in the cytoplasm and the membrane of K562 cells treated by matrine.Matrine inhibited the activity of protein tyrosine kinase in K562 cells, and the inhibitive effect depended on matrine of the concentration within 0.1 mg/ml. The effects of different concentration of matrine on the protein tyrosine phosphatase activity had no significant difference. Activity of the protein tyrosine phosphatase decreased transiently accompanied with the activity of the protein tyrosine kinase in K562 cells treated with 0.1 mg/ml matrine.The change of protein tyrosine kinase activity is involved in the differentiation of K562 cells induced by matrine. The tyrosine kinase activity in cell membrane decreased more rapidly than that in cell cytoplasm, suggesting there be a transmembrane signal transduction. The change of tyrosine phosphatase activity following the kinase indicates the real time regulation of phosphorylation and dephosphorylation. Moreover, the inhibitory effect of matrine on the protein tyrosine kinase shows the characters of specificity and saturation, suggesting the exist of matrine-associated receptor in K562 cells.

It has been reported that matrine had the anti-tumor activity, and our previous study have provided that matrine had the effects of inhibiting proliferation and inducing differentiation in K562 cell line, but its molecular mechanism is unknown yet.The expression of several proto-oncogenes(c-myc, c-jun, H-ras, p21, and HNF-1 alpha) in K562 cell line treated by 0.2 mg/ml matrine were measured by RT-PCR.The c-myc, c-jun, and HNF-1 alpha mRNA of K562 cells treated by 0.2 mg/ml matrine were dramatically decreased at the early stage (3 h), while the H-ras and p21 mRNA were increased obviously at the same time.The changes of several proto-oncogenes in K562 cells treated by 0.2 mg/ml matrine may be related to inhibiting proliferation and inducing differentiation.