Concise Total Synthesis and Antifungal Activities of Fusaric Acid, a Natural Product.

The total synthesis of a natural product alkaloid fusaric acid (FA), which exhibits herbicide, fungicide, insecticide and even diverse notable pharmacological activities, was accomplished in four steps using commercially available materials. The synthesis, based on a unified and flexible strategy using 6-bromonicotinaldehyde as a common intermediate, is concise, convergent, practical and can be carried out on a two-gram scale. This approach could be readily applicable to the synthesis of its analogues. In addition, FA had a wide range of inhibitory activities against 14 plant pathogenic fungi in this study, which demonstrated that as a leading compound, and it has great potential to be further developed as an agricultural fungicide.

Application of Solid Dispersion Technique to Improve Solubility and Sustain Release of Emamectin Benzoate.

The solid dispersion technique, which is widely used in the medical field, was applied to prepare a pesticide dosage form of emamectin benzoate (EM). The preparation, physicochemical characterization, aqueous solubility, release dynamics, photolytic degradation, bioactivity, and sustained-release effects of the prepared EM solid dispersions were studied by a solvent method, using polymer materials as the carriers. Water-soluble polyvinyl pyrrolidone (PVP) K30 and water-insoluble polyacrylic resin (PR)III were used as the carriers. The influence of various parameters, such as different EM:PVP-K30 and EM:PRIII feed ratios, solvent and container choices, rotational speed and mixing time effects on pesticide loading, and the entrapment rate of the solid dispersions were investigated. The optimal conditions for the preparation of EM-PVP-K30 solid dispersions required the use of methanol and a feed ratio between 1:1 and 1:50, along with a rotational speed and mixing time of 600 rpm and 60 min, respectively. For the preparation of EM-PRIII solid dispersions, the use of methanol and a feed ratio between 1:4 and 1:50 were required, in addition to the use of a porcelain mortar for carrying out the process. Under optimized conditions, the prepared EM-PVP-K30 solid dispersions resembled potato-like, round, and irregular structures with a jagged surface. In contrast, the EM-PRIII solid dispersions were irregular solids with a microporous surface structure. The results of X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), ultraviolet (UV) spectrometry, and infrared (IR) spectrometry showed that the solid dispersions were formed by intermolecular hydrogen bonding. The solid dispersion preparation in PVP-K30 significantly improved the solubility and dissolution rate of EM, particularly the aqueous solubility, which reached a maximum of 37.5-times the EM technical solubility, when the feed ratio of 1:10 was employed to prepare the dispersion. Importantly, the wettable powder of EM-PVP-K30 solid dispersion enhanced the insecticidal activity of EM against the Plutella xylostella larvae. Furthermore, the solid dispersion preparation in PRIII afforded a significant advantage by prolonging the EM technical release in water at a pH below 7.0, especially when the PRIII content in solid dispersions was high. While the amplified toxicity of the wettable powder of EM-PRIII solid dispersions against the P. xylostella larvae showed no significant differences from that of the EM technical, the long-term toxicity under the field condition was much better than that of the commercially available EM 1.5% emulsifiable concentrate. Notably, solid dispersions with both the PVP-K30 and PRIII carriers reduced the effect of UV photolysis.

Global mRNA and Long Non-Coding RNA Expression in the Placenta and White Adipose Tissue of Mice Fed a High-Fat Diet During Pregnancy.

BACKGROUND/AIMS: Gestational diabetes mellitus (GDM) is a common complication of pregnancy, but the mechanisms underlying the disorders remain unclear. The study aimed to identify mRNA and long non-coding RNA (lncRNA) profiles in placenta and gonadal fat of pregnant mice fed a high-fat diet and to investigate the transcripts and pathways involved in the development of gestational diabetes mellitus. METHODS: Deep and broad transcriptome profiling was performed to assess the expression of mRNAs and lncRNAs in placenta and gonadal fat from 3 mice fed an HFD and chow during pregnancy. Then, differentially expressed mRNAs and lncRNAs were validated by quantitative real-time PCR. The function of the differentially expressed mRNAs was determined by pathway and Gene Ontology (GO) analyses, and the physical or functional relationships between the lncRNAs and the corresponding mRNAs were determined. RESULTS: Our study revealed that 82 mRNAs and 52 lncRNAs were differentially expressed in the placenta of mice fed an HFD during pregnancy, and 202 mRNAs and 120 lncRNAs were differentially expressed in gonadal fat. GO and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed differentially expressed mRNAs of placenta were closely related to extracellular matrix interactions, digestion, adhesion, and metabolism, whereas the differentially expressed mRNAs in adipose tissue were related to metabolic and insulin signalling pathways. The gene network demonstrated that Actg2, Cnfn, Muc16, Serpina3k, NONMMUT068202, and NONMMUT068203, were the core of the network in placental tissue, and the genes Tkt, Acss2, and Elovl6 served as the core of the network in gonadal fat tissue. CONCLUSION: These newly identified key genes and pathways in mice might provide valuable information regarding the pathogenesis of GDM and might be used to improve early diagnosis, prevention, drug design, and clinical treatment.

Improved conditions for periodate/Schiff's base-based fluorescent staining of glycoproteins with dansylhydrazine in SDS-PAGE.

An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins.

Proteomic study on the protective mechanism of fibroblast growth factor 21 to ischemia-reperfusion injury.

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. In this study, we found that FGF-21 can significantly attenuate ischemia-reperfusion (I/R) induced damage in H9c2 cells (rat heart). However, it is unclear which signal transduction pathway is involved in the cardioprotective effect of FGF-21. Thus, this study was designed to investigate the potential mechanism induced by FGF-21. The results showed that FGF-21 treatment prevented the oxidative stress and apoptosis associated with I/R damage by reducing the levels of superoxide anions, inhibiting glycogen synthase kinase (GSK) 3ß by activating Akt phosphorylation, and recovering the levels of ATP synthase pyruvate kinase isozymes M1 and protein kinase C, thereby improving energy supply. In summary, we conclude that FGF-21 protects H9c2 cells against I/R injury mainly through the Akt-GSK-3ß-caspase-3 dependent pathway, preventing oxidative stress, and recovery of the energy supply.

Design, Synthesis, Structure-Activity Relationship, and Three-Dimensional Quantitative Structure-Activity Relationship of Fusarium Acid Derivatives and Analogues as Potential Fungicides.

In research related to fungicides, the development of compounds from natural products with high antifungal activity has attracted considerable attention. Fusaric acid (FA), an alkaloid isolated from the metabolites of Fusarium oxysporum, is an important precursor for developing pharmacologically active herbicides. In our previous work, we reported that FA has a wide range of inhibitory activities against 14 plant pathogenic fungi. In particular, it exhibited excellent antifugal effects on Colletotrichum higginsianum (EC50 = 31.7 µg/mL). Herein, to explore the practical application in the agricultural field, the design and synthesis of three series of FA derivatives and their inhibitory activities against plant pathogenic fungi were examined. Results demonstrated that the optimized FA derivatives had excellent inhibitory activities against C. higginsianum, Helminthosporium (Harpophora maydis), and Pyricularia grisea. In particular, the inhibitory activities were considerably improved when the 5-butyl groups of FA were substituted. The EC50 of C. higginsianum and P. grisea was only 1.2 and 12.0 µg/mL when 5-butylalkyl groups were substituted with 5-([1,1'-biphenyl]-4-yl) and 5-phenyl, respectively. Moreover, the safety index of target compounds, which was obtained from the treatment index of medicines, on rice seeds was evaluated. Finally, 16 leading compounds (H4, H22-H24, H27, H29, H30-H34, H37, H45, H50, H52, and H53) were obtained; they had considerable potential for additional modification and optimization as agricultural fungicides. Moreover, three-dimensional quantitative structure-activity relationship models were developed for obtaining a systematic structure-activity relationship profile to explore the possibility of more potent FA derivatives as novel fungicides.

[Preliminary study of biological mechanism and apoptotic effect induced by YM155 in SUDHL-4 cell line].

OBJECTIVE: To explore the apoptotic effect of follicular lymphoma and related mechanism induced by YM155 in vitro and provide laboratory rationales for the clinical treatment of follicular of lymphoma with YM155 in the future. METHODS: SUDHL-4 cells were cultured to logarithmic phase and transferred to 96-well plates. There were a series of YM155 concentration gradients: 100, 10, 1, 0.1 and 0 ng/ml and cultured for 24, 48 and 72 h. After the addition of CCK-8 reagent for 2 h at each time point, optical density values were obtained from the cell growth inhibition curves depending on time and drug concentration and the half growth inhibition concentration (IC(50)) values calculated. SUDHL-4 cells were co-cultured with YM155 (1 ng/ml) for 0, 24, 48 and 72 h respectively. Then flow cytometry (FCM) was used to detect apoptosis. SUDHL-4 cell line was treated with YM155 for 24 and 48 h to extract the total RNA. The mRNA expressions of bcl-2, bcl-xl, bid, bax and survivin gene at the time point of 48 h and the survivin mRNA expression at 24 h were detected by reverse transcription-PCR (RT-PCR). The protein expressions of survivin, caspase-9, cleaved caspase-9, caspase-3 and cleaved caspase-3 were detected at each time point with Western blot respectively. RESULTS: SUDHL-4 cell line showed significant growth inhibition effect depending on time and dose. And the 24, 48, 72 h IC(50) was 6.1, 2.7 and 1.2 ng/ml respectively. SUDHL-4 cells stained AnnexinV-FITC and PI examined by FCM demonstrated that the proportion of AnnexinV-FITC positive cells gradually increased with time (17.3% ± 2.1%, 35.7% ± 3.3%, 54.6% ± 4.3% vs 2.1% ± 0.3%, all P < 0.05). And the results of real-time fluorescent PCR proved that YM155 decreased the expression of survivin gene obviously (24 h: 0.72 ± 0.02, 48 h: 0.56 ± 0.01 vs 1.00, both P < 0.05) but had little effects on the gene expressions of bax, bid, bcl-2 and bcl-xl. The Western blot results further confirmed that the protein expressions of survivin and caspase-3 decreased with time while caspase-9 and cleaved caspase-9 showed no obvious changes. But cleaved caspase-3 increased significantly. CONCLUSIONS: YM155 displays significant apoptotic effects in SUDHL-4 cell lines. The mechanism may be the direct activation of caspase-3 through the down-regulation of survivin. And the apoptotic pathway is probably not regulated by bcl-2 family.

The Effectiveness and Safety of Ropivacaine and Medium-Dose Dexmedetomidine in Cesarean Section.

Objective: To study the effects of epidural anesthesia with different doses of dexmedetomidine and ropivacaine on postoperative hemodynamics and neonatal outcome of cesarean section parturients. Methods. A total of 90 parturients who underwent cesarean section admitted to our hospital from January 2019 to January 2020 were selected as the research objects and were divided into groups A, B, and C according to different dosages of dexmedetomidine, with 30 cases in each group. Groups A, B, and C were given dexmedetomidine 0.5 µg/kg, 0.8 µg/kg, 1.0 µg/kg, respectively, combined with 0.2% ropivacaine. The anesthesia effect, traction response, hemodynamic indexes, and neonatal Apgar score of the three groups were compared; the "Numerical Rating Scale (NRS) Score" was used to assess the postoperative pain of the parturients, and the "Ramsay Sedation Scale" was used to assess the sedation state of the parturients. Results. The superior anesthesia effect of group B was obtained compared with groups A and C (P < 0.05). Group B witnessed a lower degree of grade III stretching response, as compared to group A (P < 0.05). In comparison with groups A and C, superior results of the heart rate and mean artery pressure (MAP) of group B at T1 and T2 were obtained (P < 0.05). The neonatal Apgar score in group B was lower than those in groups A and C (P < 0.05), and the NRS score of group B was also lower than that of group A (P < 0.05). Compared with groups A and C, group B yielded a more favorable outcome in terms of the Ramsay score (P < 0.05). Conclusion. The use of medium-dose dexmedetomidine in cesarean section parturients is safer and can effectively reduce the impact on maternal hemodynamics, which is worthy of promotion and application.

Infliximab is effective in the treatment of ulcerative colitis with dermatomyositis: A case report.

BACKGROUND: Joint, skin, oral cavity, and eye lesions are the most common extraintestinal manifestations of ulcerative colitis that can occur before or after its onset. The cases of ulcerative colitis with dermatomyositis (DM) are rare. In this study, we report a rare case of ulcerative colitis with DM that was effectively treated with infliximab. CASE SUMMARY: The patient was a 57-year-old female with a 2-year history of DM. The patient was admitted to hospital with abdominal pain, diarrhea, and blood in stool lasting for more than 2 mo. Colonoscopy revealed multiple erosions and ulcers in the entire colon and rectum. Pathological sections showed chronic inflammatory cell infiltration, especially neutrophil infiltration, in the colonic mucosa; therefore, the patient was diagnosed with ulcerative colitis. Preparations of 5-aminosalicylic acid was added to her treatment based on the original treatment for DM, but its effect was unsatisfactory. The patient's discomfort was relieved after infliximab treatment. CONCLUSION: Infliximab can improve DM in the treatment of ulcerative colitis. Specialists need to raise awareness about patients with inflammatory bowel disease who have rare extraintestinal manifestations.

Effect of High-Fat Diet on Immature Female Mice and Messenger and Noncoding RNA Expression Profiling in Ovary and White Adipose Tissue.

Obesity is a chronic multifactorial disease prevalent in many areas of the world and is a major cause of morbidity and mortality. In women, obesity increases the risks of both metabolic and reproductive diseases, such as diabetes and infertility. The mechanisms underlying these effects, especially in young women, are largely unknown. To explore these mechanisms, we established a high-fat diet (HFD) model of obesity in immature female mice. Microarray analysis of gene expression in ovaries and white adipose tissue identified a large number of differentially expressed genes (>1.3-fold change) in both tissues. In ovaries of the HFD group, there were 208 differentially expressed messenger RNAs (mRNAs), including 98 upregulated and 110 downregulated, and 295 differentially expressed lncRNAs (long non coding RNAs), including 63 upregulated and 232 downregulated. In white adipose tissue, there were 625 differentially expressed mRNAs, including 220 upregulated and 605 downregulated in the HFD group, and 1595 differentially expressed lncRNAs, including 1320 and 275 downregulated in the HFD group. Our results reveal significant differences between the transcriptomes of the HFD and control groups in both ovaries and white adipose tissue that provide clues to the molecular mechanisms of diet-induced female reproductive dysfunction and metabolic disorders, as well as biomarkers of risk for these disorders.

Release and Degradation of Microencapsulated Spinosad and Emamectin Benzoate.

The dynamics of release and degradation of the microencapsulation formulation containing spinosad (SP) and emamectin benzoate (EM) were evaluated in the present study. SP and EM were microencapsulated using biodegradable poly-lactic acid (PLA) as the wall material. Their release from and degradation within the prepared SP and EM microspheres (SP-EM-microspheres) were studied. It was found that the encapsulation significantly prolonged the insecticide release. The release could be further extended if the external aqueous phase was pre-saturated with the insecticides and the microspheres were additionally coated with gelatin. On the other hand, increasing the water content of the emulsion or the hydrophilic polycaprolactone (PCL) content in the PLA/PCL mixture accelerated the release. Due to the photolysis and hydrolysis of SP and EM by sunlight, the toxicity of the non-encapsulated insecticides in water declined continuously from 0 through the 9th day (d), and dissipated in 13 d. In contrast, an aqueous suspension containing 5% SP-EM-microspheres maintained a mostly constant toxicity to Plutella xylostella for 17 d. The biodegradable SP-EM-microspheres showed significantly higher long-term toxicity to P. xylostella due to lower release, reduced photolysis and hydrolysis of the encapsulated insecticides, which were affected by the varied preparation conditions.

Sensitive Electrochemical Capsaicin Sensor Based on a Screen Printed Electrode Modified with Poly(sodium 4-styrenesulfonate) Functionalized Graphite.

A sensitive capsaicin sensor was constructed based on a poly(sodium 4-styrenesulfonate) functionalized graphite modified screen printed electrode (PSS-Grp/SPE) in this study. The PSS-Grp and poly(diallyldimethylammonium chloride) functionalized graphite (PDDA-Grp) were easily synthesized by interacting Grp with PSS and PDDA through sonication, and resulted in negative and with positive charges on the surface, respectively. The prepared PSS-Grp and PDDA-Grp were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and ultraviolet and visible spectroscopy (UV-vis). The electrochemical performance of PSS-Grp in a 50 µM capsaicin solution presented a current density of 33 µA cm-2, which was much higher than the PDDA-Grp of 1.5 µA cm-2. Our study showed that capsaicin could interact better with strong negative charges on the PSS-Grp/SPE surface to give a higher electrochemical response. The direct electrochemical sensing of capsaicin was achieved at PSS-Grp/SPE using differential pulse stripping voltammetry (DPSV) under the optimized conditions.

Effect of genistein on voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells.

AIM: To investigate the action of genistein (GST), a broad spectrum tyrosine kinase inhibitor, on voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells. METHODS: Smooth muscle cells in guinea pig proximal colon were enzymatically isolated. Nystatin-perforated whole cell patch clamp technique was used to record potassium currents including fast transient outward current (IKto) and delayed rectifier current (IKdr), two of which were isolated pharmacologically with 10 mmol/L tetraethylammonium or 5 mmol/L 4-aminopyridine. Contamination of calcium-dependent potassium currents was minimized with no calcium and 0.2 mmol/L CdCl2 in an external solution. RESULTS: GST (10-100 micromol/L) reversibly and dose-dependently reduced the peak amplitude of IKto with an IC50 value of 22.0+/-6.9 micromol/L. To a lesser extent, IKdr was also inhibited in both peak current and sustained current. GST could not totally block the outward potassium current as a fraction of the outward potassium current, which was insensitive to GST. GST had no effect on the steady-state activation (n=6) and inactivation kinetics (n=6) of IKto. Sodium orthovanadate (1 mmol/L), a potent inhibitor of tyrosine phosphatase, significantly inhibited GST-induced inhibition (P<0.05). CONCLUSION: GST can dose-dependently and reversibly block voltage-gated potassium channels in guinea pig proximal colon smooth muscle cells.

Impact of humic/fulvic acid on the removal of heavy metals from aqueous solutions using nanomaterials: a review.

Nowadays nanomaterials have been widely used to remove heavy metals from water/wastewater due to their large surface area and high reactivity. Humic acid (HA) and fulvic acid (FA) exist ubiquitously in aquatic environments and have a variety of functional groups which allow them to complex with metal ions and interact with nanomaterials. These interactions can not only alter the environmental behavior of nanomaterials, but also influence the removal and transportation of heavy metals by nanomaterials. Thus, the interactions and the underlying mechanisms involved warrant specific investigations. This review outlined the effects of HA/FA on the removal of heavy metals from aqueous solutions by various nanomaterials, mainly including carbon-based nanomaterials, iron-based nanomaterials and photocatalytic nanomaterials. Moreover, mechanisms involved in the interactions were discussed and potential environmental implications of HA/FA to nanomaterials and heavy metals were evaluated.

High-glucose inhibits human fibroblast cell migration in wound healing via repression of bFGF-regulating JNK phosphorylation.

One of the major symptoms of diabetes mellitus (DM) is delayed wound healing, which affects large populations of patients worldwide. However, the underlying mechanism behind this illness remains elusive. Skin wound healing requires a series of coordinated processes, including fibroblast cell proliferation and migration. Here, we simulate DM by application of high glucose (HG) in human foreskin primary fibroblast cells to analyze the molecular mechanism of DM effects on wound healing. The results indicate that HG, at a concentration of 30 mM, delay cell migration, but not cell proliferation. bFGF is known to promote cell migration that partially rescues HG effects on cell migration. Molecular and cell biology studies demonstrated that HG enhanced ROS production and repressed JNK phosphorylation, but did not affect Rac1 activity. JNK and Rac1 activation were known to be important for bFGF regulated cell migration. To further confirm DM effects on skin repair, a type 1 diabetic rat model was established, and we observed the efficacy of bFGF on both normal and diabetic rat skin repair. Furthermore, proteomic studies identified an increase of Annexin A2 protein nitration in HG-stressed fibroblasts and the nitration was protected by activation of bFGF signaling. Treatment with FGFR1 and JNK inhibitors delayed cell migration and increased Annexin A2 nitration levels, indicating that Annexin A2 nitration is modulated by bFGF signaling via activation of JNK. Together with these results, our data suggests that the HG-mediated delay of cell migration is linked to the inhibition of bFGF signaling, specifically through JNK suppression.

[Silencing of survivin gene in Jeko-1 cell line with small interfering RNA].

This study was aimed to investigate the impact of specific siRNA on survivin gene in transfected lymphoma cell line and provide experimental evidences for future treatment of mantle cell lymphoma. The small interfering RNA (siRNA) targeted survivin mRNA was synthesized in vitro and was transfected into Jeko-1 that showed high survivin expression in mRNA level. The levels of survivin mRNA and protein expression were detected by quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively. The apoptosis effect was examined by calculating the ratio of Annexin V-FITC/PI positive cells using flow cytometry. The inhibition of cell proliferation was assayed with CCK-8 reagent after transfection. The results showed that expression of survivin mRNA was markedly suppressed by the siRNA. The relative expression levels were 0.49 ± 0.03, 0.38 ± 0.02 and 0.17 ± 0.02 at time points of 24, 48 and 72 h respectively, compared with the control group; the inhibitive rates of cell proliferation were (31.2 ± 2.1)%, (43.3 ± 3.4)% and (52.6 ± 2.5)%; the apoptotic rates of cells were (6.3 ± 0.5)%, (13.5 ± 1.1)% and (23.6 ± 1.6)% respectively; survivin protein expression levels were gradually reduced. It is concluded that the siRNA targeting survivin down-regulates the expressions of survivin mRNA and protein evidently. The siRNA of survivin displays the potent ability to inhibit the proliferation of lymphoma cell line Jeko-1; survivin may become a potential molecular target for the therapy of lymphoma in the future.

[The effect of apoptosis induced by IL-21 in SUDHL-4 cell line and its mechanism].

OBJECTIVE: To investigate the apoptosis effect of diffuse large B-cell lymphoma cell line (DLBCL) SUDHL-4 induced by IL-21 and its related mechanism. METHODS: SUDHL-4 cells were treated with IL-21 at different concentration (1000 ng/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml) for 24 h, 48 h, 72 h, respectively. The inhibitory rate of cell proliferation was detected by CCK-8 assay. The cell growth curves were drawn and half inhibitory concentration (IC(50)) values were calculated. The cell apoptosis were detected by flow cytometry (FCM), the expression of the caspase-9, caspase-3, cleaved caspase-3, Bcl-2, Bcl-XL, Bid, Bax and c-myc protein in SUDHL-4 cells treated with IL-21 by western blot, the mRNA expression of Bcl-2, Bcl-XL, Bid, Bax, c-myc by Survivin gene with RT-PCR. RESULTS: IL-21 markedly inhibited SUDHL-4 cell growth in a time- and dose-dependent manner. The 48 hIC(50) was 140.9ng/ml; The FCM showed that the apoptosis proportion of SUDHL-4 cells treated with 100 ng/ml of IL-21 apoptosis (AnnexinV-FITC(+) positive cells) gradually increased (48 h: 19.7 ± 2.3%). The protein expression of caspase-9, caspase-3, Bcl-2 and Bcl-XL decreased in a time-dependent manner. The Bax and c-myc protein markedly increased, but the Bid protein level did not change. IL-21 up regulated c-myc and Bax gene expression, however down regulated Bcl-2 and BCL-XL gene expression, but the gene expression of Bid and Survivin hadn't been changed significantly. CONCLUSIONS: IL-21 can inhibit proliferation and induce apoptosis of SUDHL-4 cell. The mechanism may involve in endogenous mitochondrial pathway mediated by the c-myc and the Bcl-2 genes.

Down-regulation of SENP1 expression increases apoptosis of Burkitt lymphoma cells.

OBJECTIVE: To investigate the effect of down-regulation of Sentrin/SUMO-specific protease 1 (SENP1) expression on the apoptosis of human Burkitt lymphoma cells (Daudi cells) and potential mechanisms. METHODS: Short hairpin RNA (shRNA) targeting SENP1 was designed and synthesized and then cloned into a lentiviral vector. A lentiviral packaging plasmid was used to transfect Daudi cells (sh-SENP1-Daudi group). Daudi cells without transfection (Daudi group) and Daudi cells transfected with blank plasmid (sh-NC-Daudi group) served as control groups. Flow cytometry was performed to screen GFP positive cells and semiquantitative PCR and Western blot assays were employed to detect the inference efficiency. The morphology of cells was observed under a microscope before and after transfection. Fluorescence quantitative PCR and Western blot assays were conducted to measure the mRNA and protein expression of apoptosis related molecules (caspase-3, 8 and 9). After treatment with COCl2 for 24 h, the mRNA and protein expression of hypoxia inducible factor -1α (HIF-1α) was determined. RESULTS: Sequencing showed the expression vectors of shRNA targeting SENP1 to be successfully constructed. Following screening of GFP positive cells by FCM, semiqualitative PCR showed the interference efficiency was 79.2±0.026%. At 48 h after transfection, the Daudi cells became shrunken, had irregular edges and presented apoptotic bodies. Western blot assay revealed increase in expression of caspase-3, 8 and 9 with prolongation of transfection (P<0.05). Following hypoxia treatment, mRNA expression of HIF-1α remained unchanged in three groups (P>0.05) but the protein expression of HIF-1α markedly increased (P<0.05). However, in the sh-SENP1-Daudi group, the protein expression of HIF-1α remained unchanged. CONCLUSION: SENP1-shRNA can efficiently inhibit SENP1 expression in Daudi cells. SENP1 inhibition may promote cell apoptosis. These findings suggest that SENP1 may serve as an important target in the gene therapy of Burkitts lymphoma.

[Study of hypermethylation of SOCS gene in typical myeloproliferative disease].

OBJECTIVE: To investigate the clinical role of hypermethylation of suppressor of cytokine signaling (SOCS) on typical myeloproliferative disease (MPD) patients and its mechanism. METHODS: Methylation specific PCR was used to detect SOCS1, 2, 3 methylation, direct DNA sequencing was performed to detect JAK2V617F mutation, real-time fluorescence quantitative PCR were applied to evaluate transcriptional activity of SOCS1, 2, 3. RESULTS: Among 100 MPD patients, hypermethylation of SOCS1 was detected in 27 (27%), hypermethylation of SOCS2 in 9 (9%), hypermethylation of SOCS3 in 34 (34%); JAK2V617F mutation in 64 (64%). Hypermethylation of SOCS1, 3 greatly inhibited gene expression compared with unmethylated ones (P < 0.05). Presence of JAK2V617F mutation markedly down-regulated SOCS1, 3 gene mRNA expression compared with wild JAK2V617F (P < 0.05). CONCLUSION: Hypermethylation of SOCS1, 3 and JAK2V617F mutation exist in MPD, which inhibited SOCS1, 3 gene expression. SOCS hypermethylation and JAK2V617F mutation can activate JAK-STAT signaling pathways, these observations may provide a potential therapeutic direction.

[Effect of VEGFR1 gene silencing by shRNA on proliferation, migration and apoptosis of U937 line].

OBJECTIVE: To investigate the effects of lentivirus-mediated RNA interference targeting vascular endothelial growth factor receptor 1 (VEGFR1) gene on the proliferation, migration and apoptosis of leukemic cell line U937. METHODS: Short hairpin RNAs (shRNA) targeting VEGFR-1 was synthesized and cloned into pRNAT-U6.2 lentiviral vector. The expression vectors were transfected into 293T cell line to produce packaged lentivirus. After infected with the packaged lentivirus, the expression of VEGFR-1 gene of U937 cells at mRNA and protein level was detected by real-time PCR and Western blot. VEGF production by the cells was determined by ELISA. Cell proliferation and survival under regular culture and in the presence of cytarabine (Ara-C) was determined by CCK-8 assay. Migration assays were performed by 5 µm pore transwell inserts. RESULTS: The lentiviral shRNA vector targeting VEGFR-1 was successfully constructed and transfected into U937 cells. The shRNA vector effectively inhibited the expression of VEGFR-1 gene in U937 cell line at mRNA and protein levels. As compared to that of the control, the proliferation rate of U937-shVEGFR-1 cells reduced; The VEGF production and migrated cell number of U937-shVEGFR-1 cells decreased dramatically. After treated with Ara-C, the inhibition rate and apoptotic rate of U937-shVEGFR-1 cells increased significantly. The number of migrated cells in the KD group under regular culture and in the presence of VEGF was markedly lower than that in the NC group and CON group. Bevacizumab could decrease the number of migrated cells in the NC group and CON group, but could not in the KD group. CONCLUSIONS: Lentivirus-mediated RNA interference targeting VEGFR1 gene reduces the proliferation, migration of U937 cell line and enhances its sensitivity to Ara-C.