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Objective To explore the changes in serum energy metabolites in patients with peripheral T-cell lymphoma,and investigate serum biomarkers for monitoring peripheral T-cell lymphoma from the perspective of energy metabolism.Methods Multiple/selected reaction monitoring(MRM/SRM)was used to detect the energy-related metabolites in the sera of 16 patients with newly diagnosed peripheral T-cell lymphoma admitted in the Hematology Medical Center of the Second Affiliated Hospital of Army Medical University from November 2020 to December 2021,as well as 10 recruited healthy volunteers.The corresponding clinical data including medical history,laboratory results and image data were collected and retrospectively analyzed.Results Significant differences were seen in the contents and expression profiles of serum energy metabolism-related products between the patients and the healthy volunteers.The patients had significantly reduced serum contents of cyclic AMP,succinate,citrate and cis-aconitate(P<0.05),and elevated D-glucose 6-phosphate content(P<0.05).The serum contents of citrate and succinate were negatively correlated with the risk stratification(low-,moderate-and high-risk)and clinical stage of the disease(P<0.05).Meanwhile,there was a negative correlation between the contents of L-malic acid and citrate and the mid-term efficacy evaluation results,such as complete/partial response(CR/PR)or stable disease(SD)(P<0.05).For patients with extranodal NK/T cell lymphoma(n=10),there were also significant reductions in the contents of cyclic AMP,succinate,citrate,isocitrate and cis-aconitate in the sera of patients compared with healthy volunteers(P<0.05),and the contents of citrate and succinate were negatively correlated with the clinical stage(P<0.05)and were rather correlated with mid-term efficacy evaluation results(CR/PR or SD)(P<0.05).For patients with angioimmunoblastic T-cell lymphoma(n=6),the serum contents of cyclic AMP,citrate and succinate were significantly lower,while the content of D-glucose 6-phosphate was higher when compared with the healthy volunteers(P<0.05),and the content of succinate was negatively correlated with both clinical stage and risk grade of the patients(P<0.05).Conclusion There are 5 serum differential metabolites identified between patients with peripheral T-cell lymphoma and healthy controls,and succinate and citrate are expected to be serum biomarkers of peripheral T-cell lymphoma.
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BACKGROUND@#Exploring the underlying mechanism of rituximab resistance is critical to improve the outcomes of patients with diffuse large B-cell lymphoma (DLBCL). Here, we tried to identify the effects of the axon guidance factor semaphorin-3F (SEMA3F) on rituximab resistance as well as its therapeutic value in DLBCL.@*METHODS@#The effects of SEMA3F on the treatment response to rituximab were investigated by gain- or loss-of-function experiments. The role of the Hippo pathway in SEMA3F-mediated activity was explored. A xenograft mouse model generated by SEMA3F knockdown in cells was used to evaluate rituximab sensitivity and combined therapeutic effects. The prognostic value of SEMA3F and TAZ (WW domain-containing transcription regulator protein 1) was examined in the Gene Expression Omnibus (GEO) database and human DLBCL specimens.@*RESULTS@#We found that loss of SEMA3F was related to a poor prognosis in patients who received rituximab-based immunochemotherapy instead of chemotherapy regimen. Knockdown of SEMA3F significantly repressed the expression of CD20 and reduced the proapoptotic activity and complement-dependent cytotoxicity (CDC) activity induced by rituximab. We further demonstrated that the Hippo pathway was involved in the SEMA3F-mediated regulation of CD20. Knockdown of SEMA3F expression induced the nuclear accumulation of TAZ and inhibited CD20 transcriptional levels via direct binding of the transcription factor TEAD2 and the CD20 promoter. Moreover, in patients with DLBCL, SEMA3F expression was negatively correlated with TAZ, and patients with SEMA3F low TAZ high had a limited benefit from a rituximab-based strategy. Specifically, treatment of DLBCL cells with rituximab and a YAP/TAZ inhibitor showed promising therapeutic effects in vitro and in vivo .@*CONCLUSION@#Our study thus defined a previously unknown mechanism of SEMA3F-mediated rituximab resistance through TAZ activation in DLBCL and identified potential therapeutic targets in patients.
Subject(s)
Humans , Animals , Mice , Rituximab/therapeutic use , Hippo Signaling Pathway , Lymphoma, Large B-Cell, Diffuse/pathology , Prognosis , Semaphorins/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Membrane Proteins/genetics , Nerve Tissue Proteins/geneticsABSTRACT
BACKGROUND@#Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown.@*METHODS@#Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca 2+ -related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance.@*RESULTS@#Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca 2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments.@*CONCLUSIONS@#Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.
Subject(s)
Animals , Humans , Mice , Apoptosis , Bone Marrow Cells , Cell Communication , Connexin 43/genetics , Gap Junctions/metabolism , Imatinib Mesylate/therapeutic use , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mesenchymal Stem Cells/metabolism , Tumor Microenvironment , Calcium/metabolismABSTRACT
@#AIM:To find out the pathogenesis of dry eye disease(DED)and the possible mechanisms of available effective treatments.<p>METHOD:Here we employed a non-targeted technology, ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry(UHPLC-Q-TOF-MS), to investigate metabolic characterizations for tear samples from 18 patients with DED upon drug or acupuncture treatment. <p>RESULTS:A total of 190 named metabolites were identified, which presented so far the broadest tear metabolome. Further analysis indicated a significantly distinct metabolomics profile among all patients, but very subtle metabolic differences upon drug or acupuncture treatment. On one hand, only six significantly changed metabolites were determined after drug treatment, five of which including inosine, monopalmitin, urate, propionylcarnitine, and nicotinamide were all increased and involved in inflammatory responses. On the other hand, merely four metabolites including alanine, serine, and homoserine were found to be significantly different. Further pathway analysis of those six and four significantly changed metabolites revealed that only one pathway, aminoacyl-tRNA biosynthesis was significantly influenced in acupuncture-treated patients, which were highly associated with the cause of the disease. The results here indicated acupuncture treatment may address the cause rather than the symptoms for dry eye disease, displaying partially better compared with drug treatment.<p>CONCLUSION: Collectively, this work extended our understanding on the key regulatory elements or pathways involved in the potential mechanisms of available effective treatments, and would be useful for providing novel potential targets and therapeutic strategies for DED.
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Objective: Chronic graft-versus-host disease (cGVHD) is a major long-term complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . It is important to study the changes of serum biomarkers expression in patients for early diagnosis and treatment. Methods: The expression levels of five serum protein markers (IL-1b, IL-16, CXCL9, CCL19, CCL17) in patients with or without cGVHD after allo-HSCT were detected by liquid suspension microarray. Results: Compared with the control group without cGVHD, the expression levels of CXCL9 and CCL17 in serum of patients with cGVHD were significantly increased (P<0.05) . CCL17 was correlated with the severity of cGVHD (P<0.001) . CXCL9 was significantly increased in the serum of patients with skin lesion (P<0.01) , and CCL17 was significantly expressed in cGVHD patients with liver as the target organ (P<0.01) . Conclusion: The combination of CXCL9 and CCL17 can be used as serum biomarkers of cGVHD, which has certain reference value in assisting the diagnosis and evaluation of cGVHD severity.
Subject(s)
Humans , Biomarkers , Chronic Disease , Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Transplantation, HomologousABSTRACT
Objective@#Chronic graft-versus-host disease (cGVHD) is a major long-term complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . It is important to study the changes of serum biomarkers expression in patients for early diagnosis and treatment.@*Methods@#The expression levels of five serum protein markers (IL-1b, IL-16, CXCL9, CCL19, CCL17) in patients with or without cGVHD after allo-HSCT were detected by liquid suspension microarray.@*Results@#Compared with the control group without cGVHD, the expression levels of CXCL9 and CCL17 in serum of patients with cGVHD were significantly increased (P<0.05) . CCL17 was correlated with the severity of cGVHD (P<0.001) . CXCL9 was significantly increased in the serum of patients with skin lesion (P<0.01) , and CCL17 was significantly expressed in cGVHD patients with liver as the target organ (P<0.01) .@*Conclusion@#The combination of CXCL9 and CCL17 can be used as serum biomarkers of cGVHD, which has certain reference value in assisting the diagnosis and evaluation of cGVHD severity.
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Objective To study the method of emergency blood collection during a long-distance voyage to ensure blood transfusion treatment.Methods Ten voluntary blood donors were recruited, a base unit of blood was collected and preserved.Reactions of the blood donors were observed, and the blood quality was tested.Results The success rate of blood collection was 90% and the qualification rate was 100%.Conclusion Emergency blood collection during a long-distance voyage is feasible,which can help ensure blood supply in peace time or war time.
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This study aimed to explore the neuroprotective role of 6-hydroxy-1H-indazole on dopaminergic neurons in a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). Forty 12-week-old C57BL/6 male mice were were randomized divided into 4 groups. Mice were treated with 2mg/kg and 4mg/kg 6-hydroxy-1H-indazole (i.p.) 1d before the initiation of MPTP administration (30mg/kg), and the 6-hydroxy-1H-indazole were daily injected half an hour before MPTP treatment in the following 5 days. The MPTP group was given normal saline on day 1 (i.p.), followed by 30mg/kg MPTP treatment in the following 5 days. Control group received an equivalent volume of normal saline. Ten days after the final injection of MPTP, the mice were killed. The results showed that MPTP decreased the dopaminergic neurons in the substantia nigra and dopamine in the striatum, downregulated the expression of tyrosine hydroxylase (TH), induced the impairment of behavior and hyperphosphorylation of tau, However, 6-hydroxy-1-H-indazole decreased the loss of dopaminergic neurons, increased dopamine concentration and TH expression, alleviated the behavioral damage and level of phosphor-tau in the MPTP-induced model of PD in C57BL/6 mice. These findings showed that 6-hydroxy-1-H-indazole-mediated neuroprotection was related to the inactivation of tau. In addition, 6-hydroxy-1-H-indazole may be a potential drug candidate for PD.
Subject(s)
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacology , Indazoles/pharmacology , Parkinson Disease/etiology , Parkinson Disease/prevention & control , Animals , Behavior, Animal/drug effects , Disease Models, Animal , Dopamine/metabolism , Dopaminergic Neurons/drug effects , Male , Mesencephalon/drug effects , Mesencephalon/metabolism , Mice , Neostriatum/drug effects , Neostriatum/metabolism , Parkinson Disease/metabolism , Parkinson Disease/pathology , Phosphorylation/drug effects , Substantia Nigra/pathology , Tyrosine 3-Monooxygenase/metabolism , tau Proteins/metabolismABSTRACT
Objective To explore the clinical significance of combined detection of serum procalcitonin (PCT)and interleukin 6 (IL-6 )in patients with lower limbs long bone open fractures before and after emergency debridement fixation and postoperative reg-ular antibiotics treatment.Methods Patients with lower limbs long bone open fractures within 8-24 h after injury were divided in-to wounds fester group,wounds contamination group and wounds non-contamination group.Serum PCT and IL-6 levels were detec-ted and compared between patients group and control group.Results Before treatment,PCT and IL-6 levels in wounds contamina-tion group and wounds fester group were higher than control group (P0.05).After treatment,PCT and IL-6 levels in wounds fester group and wounds contamination group were decreased,but those in non-contamination group were with a trend of fluctuation.In the first day of treatment,PCT and IL-6 levels in contamination and fester group were higher than control group (P0.05).PCT and IL-6 levels in non-contamination group,compared with control group and levels before treatment,were not significantly different (P>0.05).In the third day of treatment,PCT and IL-6 levels in the three patient groups were higher than control group (P0.05).PCT and IL-6 levels in non-contamination group were significantly higher than the levels before treatment (P0.05). Conclusion Wounds infection could onset in patients combined with lower limbs long bone open fractures and soft tissue open inj u-ry.Dynamic monitoring of PCT and IL-6 levels might be significant for early prediction of bacteria infection,rational use of antibi-otics and promote healing of wounds and fractures.
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<p><b>OBJECTIVE</b>To compare the diagnostic value of (18)F-FDG PET-CT with abdomen contrast CT in the diagnosis of peritoneal metastases.</p><p><b>METHODS</b>Between January 2008 and May 2011, imaging results of 97 patients with suspicious peritoneal metastases were retrospectively reviewed, and all the patients underwent both abdomen contrast CT and (18)F-FDG PET-CT imaging. Final diagnosis was made by histopathology or follow up.</p><p><b>RESULTS</b>Seventy-seven patients were verified as peritoneal metastases after pathological examination(n=88) or follow up(n=9), while the other 20 patients were absent. The sensitivity of (18)F-FDG PET-CT was 90.9%(70/77), the specificity 85.0%(17/20), and the accuracy 89.7%(87/97). There were 3 false positive and 7 false negative. The sensitivity of contrast CT was 66.2%(51/77), the specificity 80.0%(16/20), and the accuracy 69.1%(67/97). There were 4 false positive and 26 false negative. The difference in diagnostic accuracy was statistically significantly between these two methods(P<0.05).</p><p><b>CONCLUSION</b>The diagnostic value of (18)F-FDG PET-CT is significantly higher than that of abdominal enhanced CT for peritoneal metastases.</p>
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , Peritoneal Neoplasms , Diagnostic Imaging , Positron-Emission Tomography , Retrospective Studies , Sensitivity and Specificity , Tomography, X-Ray ComputedABSTRACT
Objective To investigate the oncolytic effect of E1B mutant adenovirus (d11520) on human malignant gliomas. Methods Ad-βgal vector was used to investigate the infectibility of dl1520.U251,Hep3B (positive control) and T24 (negative control) cell lines were infected with dl1520respectively at 50,5,0.5,0.005 and 0 pfu of multiplicity of infection (MOI).The replication efficiency of d11520 in host cells was assessed by plaque assay.The cytopathic effect (CPE) was assessed by crystal violet staining in a panel of tumor cells. Results Crystal violet staining showed the Hep3B cell line was the most sensitive to dl1520 and had the fastest CPE,followed by the U251 cell line,while the T24cell line had no CEP.The replication and infection rates ofdl1520 in the U251 cell line were lower than in the Hep3B cell line but significantly higher than in the T24 cell line (P<0.05). Conclusion The E1B mutant adenovirus (dl1520) has a significant oncolytic effect on human malignant gliomas.
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<p><b>OBJECTIVE</b>MicroRNAs (miRNAs) play important roles in cell proliferation, differentiation and apoptosis. 1, 3, 4-tri-O-galloyl-6-O-caffeoyl-β-D-glucopyranose (BJA32515) is a new natural ellagitannin compound extracted from Balanophora Japonica MAKINO. The effect of BJA32515 on the expression of miRNAs in cancer cells has not yet been explored. Objective The present study was carried out to examine the changes in miRNA expression profiles in human HepG(2) hepatocarcinoma cells following BJA32515 exposure.</p><p><b>METHODS</b>The proliferation of BJA32515-exposed HepG(2) cells was assessed using a colorimetric assay (cell counting kit-8). The miRNA expression profile of the cancer cells was analyzed using a miRNA array and quantitative real-time PCR. Apoptosis was assessed by annexin V and propidium iodide staining.</p><p><b>RESULTS</b>BJA32515 inhibited the cell proliferation and increased apoptosis in HepG(2) cancer cells. The exposure to BJA32515 also caused alterations in the miRNA expression profile in the cells, with 33 miRNAs upregulated and 59 down-regulated. The up-regulation of let-7a and miR-29a and the down-regulation of miR-373 and miR-197 were verified by quantitative real-time PCR. CONCLSION: BJA32515-modifed miRNA expression may mediate the antiproliferative effect of this compound in HepG(2) cancer cells.</p>
Subject(s)
Humans , Antineoplastic Agents , Pharmacology , Apoptosis , Balanophoraceae , Chemistry , Caffeic Acids , Pharmacology , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glucosides , Pharmacology , Hep G2 Cells , Hydrolyzable Tannins , Pharmacology , MicroRNAs , Genetics , Metabolism , PolyphenolsABSTRACT
<p><b>OBJECTIVE</b>To evaluate the bioequivalence of orally disintegrating tablets of pentoxyverine citrate (tested preparation) in healthy male volunteers.</p><p><b>METHODS</b>A single oral dose of the tested and reference preparations at 25 mg were given to 20 healthy volunteers in a randomized two-period cross-over design. Plasma pentoxyverine citrate concentrations were determined by HPLC-MS/ESI+ method. The pharmacokinetic parameters were calculated and the bioequivalence of the two preparations were evaluated using DAS program.</p><p><b>RESULTS</b>The Tmax, Cmax, AUC0 15 and AUC0infinity of tested and reference preparations were 1.62-/+0.75 h and 2.52-/+1.21 h, 62.28-/+33.06 microg/L and 59.72-/+33.25 microg/L, 234.44-/+130.01 microg.h.L(-1) and 228.77-/+129.24 microg.h.L(-1), 246.80-/+136.19 microg.h.L(-1) and 244.11-/+140.73 microg.h.L(-1), respectively. The 90% confidence interval of C(max), AUC0 15 and AUC0infinity of tested preparations were 81.4%-138.4%, 86.0%-123.3% and 86.5%-121.2%, respectively.</p><p><b>CONCLUSION</b>The tested and reference preparations are bioequivalent.</p>
Subject(s)
Adult , Humans , Male , Young Adult , Area Under Curve , Biological Availability , Citric Acid , Pharmacokinetics , Cross-Over Studies , Cyclopentanes , Pharmacokinetics , Tablets , Therapeutic EquivalencyABSTRACT
<p><b>OBJECTIVE</b>To investigate the cough-relieving, analgesic and antibiotic effects of durian shell extract (DSE) in relieving cough and its analgesic and antibiotic effects.</p><p><b>METHODS</b>The effect of DSE in relieving cough was assessed in mice challenged with ammonia and SO(2) to induce coughing. The analgesic and antibiotic effects of DSE in mice were evaluated by hot plate test and twisting reaction induced by acetic acid, and by minimal inhibitory concentration (MIC) and disc-agar diffusion tests, respectively.</p><p><b>RESULTS</b>Compared with the control group, the mice treated with 300 and 900 mg/kg DSE showed significantly prolonged latency with decreased number of coughing induced by ammonia and SO(2), and the effect was dose-dependent. DSE markedly prolonged the latency and decreased the twisting number of the mice induced by acetic acid without affecting the pain threshold in hot plate test. DSE produced no significant inhibitory effects against Staphylococcus aureus, Staphylococcus epidermidis, or E. coli, and showed a week inhibition against Bacillus aeruginosus.</p><p><b>CONCLUSION</b>DSE shows obvious effect in relieving cough and produces better analgesic effect against chemical factor-induced pain than against physical agent-induced pain sensation. DSE has a moderate inhibitory effect against Bacillus aeruginosus.</p>
Subject(s)
Animals , Male , Mice , Analgesics , Pharmacology , Anti-Bacterial Agents , Pharmacology , Antitussive Agents , Pharmacology , Bombacaceae , Chemistry , Plant Extracts , Pharmacology , Random AllocationABSTRACT
<p><b>OBJECTIVE</b>To investigate the inhibitory effect of naringin on monocyte adhesion to high glucose-induced human umbilical vein endothelial cells (HUVECs).</p><p><b>METHODS</b>Cultured HUVECs isolated from human umbilical cords were pretreated with or without naringin and induced with high glucose (33 mmol/L) for 48 h. Human monocyte THP-1 cells, after labeling with BCECF-AM, were co-cultured with the HUVECs for 30 min. The labeled THP-1 cells adhering to HUVECs were observed under fluoroscence microscope, and the inhibitory effect of naringin on the cell adhesion was evaluated by measuring the adhering cell density. Western blot analysis was used to detect the expressions of the adhesion molecules in the HUVECs, and reactive oxygen species (ROS) production in the HUVECs was measured using an oxidation-sensitive fluorescent probe (DCFH-DA). The nuclear extracts of the HUVECs were prepared to examine the expression of nuclear factor-kappa B (NF-kappaB) in the cell nuclei by Western blotting.</p><p><b>RESULTS</b>HUVECs in high-glucose culture showed increased adhesion to THP-1 cells and enhanced expressions of the cell adhesion molecules, which were significantly attenuated by pretreatment with naringin (10-50 microg/ml). High glucose induced DCF-sensitive intracellular ROS production in the HUVECs, and this effect was inhibited by naringin pretreatment of the cells. Naringin also suppressed high glucose-induced increment of NF-kappaB expression in the cell nuclei of HUVECs.</p><p><b>CONCLUSION</b>Naringin can suppress high glucose-induced vascular inflammation possibly by inhibiting ROS production and NF-kappaB activation in HUVECs.</p>
Subject(s)
Humans , Cell Adhesion , Cell Adhesion Molecules , Metabolism , Cell Line , Cells, Cultured , Coculture Techniques , Endothelial Cells , Cell Biology , Flavanones , Pharmacology , Glucose , Pharmacology , Monocytes , Cell Biology , NF-kappa B , Metabolism , Reactive Oxygen Species , Metabolism , Umbilical Veins , Cell BiologyABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of hydrocamptothecin on the expression of Wnt signaling pathway inhibitor DKK-1 in tumor cells.</p><p><b>METHODS</b>Human HepG2, Hep3B, LoVo and U251 cells were treated with the antitumor drug Hydrocamptothecin. DKK-1 mRNA expression in the cells was detected with RT-PCR, and beta-catenin expression was measured by fluorescence-activated cell sorting (FACS).</p><p><b>RESULTS</b>DKK mRNA in Hep3B, HepG2, LoVo and U251 cells was significantly increased after hydrocamptothecin treatment for 24 h, and the percentage of beta-catenin-positive cells and fluorescence intensity for beta-catenin expression was lowered in the cells after the treatment.</p><p><b>CONCLUSION</b>Hydrocamptothecin promotes mRNA expression of Wnt signaling pathway inhibitor DKK-1 in Hep3B, HepG2, LoVo and U251 cells.</p>
Subject(s)
Humans , Antineoplastic Agents, Phytogenic , Pharmacology , Camptothecin , Pharmacology , Cell Line, Tumor , Colorectal Neoplasms , Genetics , Metabolism , Pathology , Flow Cytometry , Intercellular Signaling Peptides and Proteins , Genetics , Liver Neoplasms , Genetics , Metabolism , Pathology , RNA, Messenger , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Wnt Proteins , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To compare the in vitro inhibitory effect of expolysaccharides from Streptomyces, polysaccharides of Ganoderma lucidum and rice bran on six-alpha-helix bundle formation of HIV gp41 protein.</p><p><b>METHODS</b>The amount of six-alpha-helix bundle formed in the presence of N36 and C34 was tested by ELISA in response to treatments with different doses of polysaccharides.</p><p><b>RESULTS</b>Expolysaccharides from Streptomyces potentially inhibited six-alpha-helix bundle formation with the effective concentration (IC(50)) of 145.48-/+7.25 mg /L. Polysaccharides of Ganoderma lucidum and rice bran showed no effect on the six-alpha-helix bundle formation.</p><p><b>CONCLUSION</b>Expolysaccharides from Streptomyces can inhibit the six-alpha-helix bundle formation of HIV gp41, whereas polysaccharides of Ganoderma lucidum and rice bran do not exhibit such activity.</p>
Subject(s)
HIV Envelope Protein gp41 , Chemistry , Kinetics , Oryza , Chemistry , Polysaccharides , Pharmacology , Protein Structure, Secondary , Reishi , Chemistry , Streptomyces , ChemistryABSTRACT
Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Genes, Synthetic , Genetics , Physiology , Models, Theoretical , Peptide Library , Polymerase Chain Reaction , Promoter Regions, Genetic , Genetics , Transcription Factors , Chemistry , Metabolism , Zinc Fingers , Genetics , PhysiologyABSTRACT
A high efficiency phenol-degrading bacterial strain PS1 was isolated from the drainage ditch of chemical laboratory of East China Institute of Technology.PS1 is a coccus,Gram negative and can live on phenol as its sole carbon and energy source.PS1 to identified as a strain of Raoultella sp.by 16S rRNA gene sequence analysis,which can degrade and tolerate more than 3500mg/L phenol.When phenol concentration is 500mg/L and 1000mg/L,PS1 can completely degrade it in 22 h and 32h,respectively.And while it is between 1500mg/L~3000mg/L,all phenol can be degraded by PS1 in 32h~50h.When phenol concentration is 2500mg/L,the phenol-degrading rate is the biggest and can reach to 78.1mg/h.The optimum growth and phenol-degrading conditions were obtained by orthogonal experiment,which are 25℃,pH6.5,glucose concentration 500mg/L and 20℃,pH7.0,glucose concentration 500mg/L,respectively.