Tandem mass spectrometry (MS/MS) molecular networking guided profiling of small molecules from Aquilaria sinensis (Lour.) Gilg leaves and their bioactivity evaluation.

INTRODUCTION: Agarwood, a fragrant resinous wood mainly formed by Aquilaria spp., is used worldwide as a natural fragrance and traditional medicine. A large amount of Aquilaria sinensis (Lour.) Gilg leaves are underutilised during the process of the agarwood industry, and the development of A. sinensis leaves as tea has recently attracted more and more attention. However, the small molecule profile of A. sinensis leaves and their bioactivities has been rarely reported. OBJECTIVE: To conduct a rapid untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of A. sinensis leaves with a molecular networking (MN) strategy and evaluate its antioxidant and antidiabetic value. METHOD: A MN-assisted tandem mass spectrometry (MS/MS) analysis strategy was used to investigate the small molecule profile of A. sinensis leaves. Additionally, the integration of antioxidant and α-glucosidase inhibitory assays with MN analysis was executed to expeditiously characterise the bioactive compounds for potential prospective application. RESULTS: Five main chemical groups including phenol C-glycosides, organic acids, 2-(2-phenylethyl) chromones, benzophenone O-glycosides and flavonoids were rapidly revealed from the A. sinensis leaves. Eighty-one compounds were provisionally identified by comparing their MS/MS fragments with canonical pathways. The featured xanthone C-glycosides and benzophenone C-glycosides were recognised as the primary components of A. sinensis leaves. Several dimers and a trimer of mangiferin were reported firstly in A. sinensis leaves. Furthermore, 17 and 14 potential bioactive molecules were rapidly annotated from antioxidant and α-glucosidase inhibitory fraction, respectively. CONCLUSION: Our findings will help expand the utilisation of A. sinensis leaves and thus promote the high-quality development of agarwood industry.

[Effects of Leptin and Leptin-associated Signaling Pathways on the Occurrence and Progression of Abdominal Aortic Aneurysms].

Abdominal aortic aneurysm(AAA) is a chronic dilated artery disease induced by atherosclerosis,infection,trauma and other related causes.The available studies about AAA mainly focus on the inflammatory response,senility,and microenvironmental changes,while the research on the metabolic changes such as glucose metabolism and lipid metabolism remains to be conducted.As a critical regulatory factor in endocrine,glucose,and lipid metabolisms,leptin is associated with a variety of signaling pathways such as adenosine monophosphate-activated protein kinase,Janus kinase/signal transducer and activator of transcription,and cytokine-cytokine receptor,as demonstrated by the KEGG pathway enrichment analysis.Moreover,these signaling pathways are generally involved in regulating the occurrence of AAA.In addition,leptin affects the occurrence of a variety of diseases such as obesity,diabetes,and hyperlipidemia,which contribute to the formation of AAA.Diabetes might be a protective factor for the formation of AAA,while the relationship of hyperlipidemia and obesity with the formation of AAA remains unclear.Therefore,leptin might play an essential role in the formation of AAA.Further studies about the effect of leptin on AAA may provide the potential research direction and facilitate the discovery of therapeutic targets.

[Screening of plant growth-promoting rhizobacteria and its effect on seed germination of Polygonum multiflorum].

In this study, the rhizobacteria and actinomycetes of Polygonum multiflorum were screened for the strains with indole acetic acid(IAA)-producing capacity by Salkowski method, the siderophore-producing strains by Chrome Azurol S(CAS) assay, and the strains with inorganic phosphorus-solubilizing capacity by PKO inorganic phosphorus medium. The strains were identified by morphological identification, physiological and biochemical characteristics, and 16 S rDNA sequences. Furthermore, the effect of growth-promoting strains on the seed germination and development of P. multiflorum was tested. The results showed that among 196 strains, two strains F17 and F42 were found to be capable of producing IAA and siderophore and solubilizing inorganic phosphorus simulta-neously. For F17 and F42, the results are listed below: 38.65 and 33.64 mg·L~(-1) for IAA production, 0.85 and 0.49 for siderophore-producing capacities(A_s/A_r), and 1.35 and 1.70 for inorganic phosphorus-solubilizing capacities(D/d), respectively. Comprehensive analysis revealed that strains F17 and F42 were identified as Pseudochrobactrum asacharolyticum and Bacillus aryabhattai, respectively, and both could significantly promote the seed germination of P. multiflorum.

[Effects of drought stress on physiological and biochemical and chemical components of Cinnamomum cassia seedlings].

Six month old Cinnamomum cassia seedlings were used to simulate drought stress with polyethylene glycol(PEG 6000). The physiological indicators(osmotic substances, antioxidant enzymes, etc.) and chemical components of seedlings under different drought levels and the correlation between the two were studied. The results showed that the chlorophyll content and relative water content decreased gradually with the increase of PGE 6000(0, 5%, 10%, 15%) concentration and time(3, 5, 7 d), while the soluble protein content, soluble sugar content and catalase(CAT) activity increased, but the rising rate slowed down with the time. The activities of peroxidase(POD), superoxide dismutase(SOD), malondialdehyde(MDA) and proline content increased at first and then decreased. The content of coumarin, cinnamaldehyde, cinnamic acid and dimethoxycinnamaldehyde decreased, while the content of cinnamyl alcohol continued to increase.Under drought stress, the fluorescence signals of reactive oxygen species and no contents in roots of C. cassia seedlings were significantly stronger than those of the control.Further correlation analysis showed that coumarin content, di-methoxycinnamaldehyde content and osmoregulation substance content were significantly negatively correlated(P<0.05), cinnamic acid content was significantly negatively correlated with POD and SOD activities(P<0.01).It was found that C. cassia seedlings showed a certain degree of drought tolerance under short-term or mild drought stress, but if the drought exceeded a certain degree, the physiological metabolism of the seedlings would be unbalanced.

[Effect of Acupuncture on the Expression of Transcription Factor T-bet/GATA-3 in Plasma of Rats with Chronic Fatigue Syndrome].

OBJECTIVE: To observe the effect of acupuncture on the expression of T-box expressed in T cell (T-bet)/GATA binding factor-3 (GATA-3) in plasma of rats with chronic fatigue syndrome (CFS) and explore the mechanism of acupuncture treatment for CFS. METHODS: Forty-eight healthy male SD rats were randomly divided into blank control group, CFS model group, acupuncture group, and ginsenoside group (12 rats in each group). CFS rat model was established by combining restriction and cold water swimming. Acupuncture was applied to "Baihui"(GV 20), "Guanyuan" (CV 4) and "Zusanli" (ST 36, bilate-ral) acupoints, once a day for two weeks. The ginsenoside group was gavage administrated with ginsenoside, once a day for two weeks. After 14 days, behavioural changes were observed, and the expression levels of T-bet/GATA-3 genes in plasma were detected by RT-PCR. RESULTS: Compared with the blank control group, the time for immobility of forced suspensory test was signi-ficantly longer (P<0.05) and the time for exhaustive swimming was significantly shortened (P<0.05) in the CFS model group. Compared with the model group, the two indexes above-mentioned were reversed (P<0.05) both in the acupuncture group and the ginsenoside group, and the effects in the acupuncture group were more significant than those in the ginsenoside group (P<0.05). Compared with the blank control group, the expression level of T-cell transcription factor T-bet gene in plasma was higher in the CFS model group (P<0.05), companied with lower GATA-3 gene expression (P<0.05). The ratio of T-bet/GATA-3 was higher in the model group than in the blank control group(P<0.05). Compared with the CFS model group, all the indexes above-mentioned were reversed (P<0.05) in the two treatment groups. Acupuncture group showed a better effect on reducing T-bet gene expression than the ginsenoside group (P<0.05). CONCLUSIONS: Acupuncture can decrease the expression level of T-bet gene while increase the expression of GATA-3 gene, which may be associated with its role in treating CFS.

Expression of microRNA-29b2-c cluster is positively regulated by MyoD in L6 cells.

OBJECTIVES: To evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube of L6 myotube treated by glucose and insulin, and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters. METHODS: The expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis. The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search. The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay. Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence. Moreover, overexpression of myoD was achieved by a recombinant adenovirus system (Ad-myoD). L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c. RESULTS: The expression levels of myoD, miR-29a, miR-29b, and miR-29c were increased in L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube treated with glucose and insulin, but miR-29a depicted no significant change. Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster. Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells. CONCLUSION: MyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.

[Effect of acupuncture intervention on learning-memory ability and cerebral superoxide dismutase activity and malonaldehyde concentration in chronic fatigue syndrome rats].

OBJECTIVE: To observe the effect of acupuncture intervention on learning-memory ability and cerebral superoxide dismutase (SOD) activity and malonaldehyde (MDA) content in chronic fatigure syndrome (CFS) rats so as to reveal its mechanism underlying improvement of clinical CFS. METHODS: Thirty-six male SD rats were randomly divided into control group, model group and acupuncture group (n = 12 in each group). CFS model was established by double stress stimulation of suspending (1.0 - 2.5 h increasing gradually) and forced swimming [Morris water maze tasks, 7 min in (10 +/- 1) degrees C water], once daily for 12 days. Manual acupuncture stimulation was applied to "Baihui" (CV 20), bilateral "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), once daily for 21 days (with 3 days' interval between every two weeks). Learning-memory ability was determined by Morris water maze tests, and SOD activity and MDA concentration in the brain tissues were detected by xanthine oxidase method and thiobarbiturif acid method, respectively. RESULTS: Compared with the control group, the escape latencies at time-points of day 1, 2, 3, 4 and 5 of Morris water maze tests were significantly longer, the target platform crossing times were markedly fewer and the target platform quadrant staying time obviously shorter, cerebral SOD activity was considerably decreased, and cerebral MDA content remarkably increased in the model group (P < 0.05, P < 0.01). Following acupuncture intervention, the escape latencies at time-points of day 1, 2, 3, 4 and 5 were significantly decreased, both target platform crossing times and staying time, and cerebral SOD activity were apparently increased, as well as cerebral MDA level was markedly lowered in comparison with the model group (P<0.05, P<0.01). CONCLUSION: Acupuncture intervention can improve the learning-memory ability in CFS rats, which may be related to its effect in regulating metabolism of free radicals in the brain tissues.

PTEN and PDCD4 are bona fide targets of microRNA-21 in human cholangiocarcinoma.

OBJECTIVE: To investigate the expression profile of microRNA-21 in human cholangiocarcinoma tissues and to validate its bona fide targets in human cholangiocarcinoma cells. METHODS: The expression profile of microRNA-21 in human cholangiocarcinoma tissues and cholangiocarcinoma cell line, QBC939, was evaluated by using real-time PCR analysis. The bona fide targets of microRNA-21 were analyzed and confirmed by dual luciferase reporter gene assay and western blot, respectively. The expressional correlation of microRNA-21 and its targets was probed in human cholangiocarcinoma tissues by using real-time PCR, locked nucleic acid in situ hybridization (LNA-ISH), and immunohistochemistry analysis. RESULTS: Real-time PCR analysis revealed that microRNA-21 expression depicted a significant up-regulation in human cholangiocarcinoma tissues about 5.6-fold as compared to the matched normal bile duct tissues (P<0.05). The dual luciferase reporter gene assay revealed endogenous microRNA-21 in cholangiocarcinoma cell line, QBC939, inhibited the luciferase reporter activities of wild-type PTEN (P<0.01) and PDCD4 (P<0.05) and had no this effect on mutated PTEN and PDCD4. Moreover, loss of microRNA-21 function led to a significant increase of PTEN and PDCD4 protein levels in QBC939 cells. Elevated microRNA-21 levels were accompanied by marked reductions of PTEN and PDCD4 expression in the same cholangiocarcinoma tissue. CONCLUSION: microRNA-21 expression is up-regulated in human cholangiocarcinoma and PTEN, PDCD4 are direct effectors of microRNA-21.

The clinicopathological and prognostic impact of 14-3-3 protein isoforms expression in human cholangiocarcinoma by immunohistochemistry.

The 14-3-3 proteins are highly conserved, ubiquitous molecules involved in a variety of biologic phenomena, such as cell cycle control, and apoptosis. However, their expression in cholangiocarcinoma has not been previously characterized. In this paper, immunohistochemistry using specific anti-14-3-3 monoclonal antibodies was performed on formalin-fixed;, paraffin embedded archival tissue from 86 patients of cholangiocarcinoma. We also examined the correlation between expression and survival rate and clinicopathologic factors such as tumor location, tumor size, pathologic differentiation, lymphatic permeation, lymph node metastasis, and tumor stage. Positive 14-3-3 proteins expression was observed for 6 isoforms (ß, σ, γ, θ, ß, η) of these proteins in 86 patients of cholangiocarcinoma. ß and σ isoform immunoreactivity was correlated with lymph node metastasis, tumor stage and patients' survival rate. In addition, δ isoform immunoreactivity showed trends with tumor location, tumor size, pathologic differentiation and tumor stage, while the θ isoform was correlated with pathologic differentiation. These results indicated that upregulated expression of some isoforms of 14-3-3 may be a common mechanism for evading apoptosis in cholangiocarcinoma, so that targeting 14-3-3 may be a novel promising strategy for the treatment of this tumor.

[Effect of acupuncture on serum malonaldehyde content, superoxide dismutase and glutathione peroxidase activity in chronic fatigue syndrome rats].

OBJECTIVE: To study the effect of acupuncture on blood oxygen free radical metabolism in rats with chronic fatigue syndrome (CFS). METHODS: Thirty male SD rats were randomly divided into control group (n = 10), model group (n = 10) and acupuncture group (n = 10). CFS model was established by repeated suspension (1.0-2.5 h) and forced cold water swimming (7 min), once daily continuously for 12 days. For rats in the acupuncture group, bilateral "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) were stimulated by manipulating the acupuncture needles intermittently for 20 min, once daily, and with 7 days being a treatment course. The treatment was conducted for three courses with an interval of 3 days between two courses. Serum malonaldehyde (MDA) content, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-PX) activity were detected by thiobarbituric acid chromatometry (TBA), xanthine oxidase (XOD) and dithio-bis-nitrobenzoic acid (DTNB), respectively. RESULTS: In comparison with the control group, serum MDA content was up-regulated significantly, while serum SOD activity and GSH-PX activity were decreased considerably in the model group (P < 0.01). Compared with the model group, serum MDA level was down-regulated apparently, and serum SOD activity and GSH-PX activity were up-regulated remarkably in the acupuncture group (P < 0.01). CONCLUSION: Acupuncture can adjust metabolism of serum oxygen free radicals in CFS rats, which probably contributes to its effect in relieving CFS in clinic.

Hypoxia-inducible factor-1 (HIF-1) promotes LDL and VLDL uptake through inducing VLDLR under hypoxia.

Metabolism under hypoxia is significantly different from that under normoxia. It has been well elucidated that HIF-1 (hypoxia-inducible factor-1) plays a central role in regulating glucose metabolism under hypoxia; however, the role of HIF-1 in lipid metabolism has not yet been well addressed. In the present study we demonstrate that HIF-1 promotes LDL (low-density lipoprotein) and VLDL (very-LDL) uptake through regulation of VLDLR (VLDL receptor) gene expression under hypoxia. Increased VLDLR mRNA and protein levels were observed under hypoxic or DFO (deferoxamine mesylate salt) treatment in MCF7, HepG2 and HeLa cells. Using dual-luciferase reporter and ChIP (chromatin immunoprecipitation) assays we confirmed a functional HRE (hypoxia-response element) which is localized at +405 in exon 1 of the VLDLR gene. Knockdown of HIF1A (the α subunit of HIF-1) and VLDLR, but not HIF2A (the α subunit of HIF-2), attenuated hypoxia-induced lipid accumulation through affecting LDL and VLDL uptake. Additionally we also observed a correlation between HIF-1 activity and VLDLR expression in hepatocellular carcinoma specimens. The results of the present study suggest that HIF-1-mediated VLDLR induction influences intracellular lipid accumulation through regulating LDL and VLDL uptake under hypoxia.

[Bio panning of human stem cell factor(2) mimetic peptides from phage displayed random peptide library].

OBJECTIVE: To screen human stem cell factor (hSCF) mimetic peptides in vitro with a phage-display random peptide library. METHODS: Phage clones with high hSCF receptor (rc-kit/Ig 1-3)-binding activity was screened from phage-displayed random hepta/dodecapeptide library by phage enzyme-linked immunosorbent assay (ELISA). Phage single DNA was extracted and sequenced. Four kinds of peptide with higher c-Kit/Ig 1-3 binding activity were chosen for synthesis and characterized by using cell proliferation assay with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) method in UT-7 cells. RESULTS: Eleven Ph.D.-C7C clones and eight Ph.D-12 phage clones with high hSCF receptor-binding activity were selected from phage-displayed random hepta/dodecapeptide library, respectively. Sequence analysis showed there were no homologous sequence between hSCF and these screened mimetic peptides except one homologous sequence DPSPHTH found in heptapeptide library. All these four synthesized peptides (CE3, CE16, LE4, and LE20), particularly CE16 and LE20, stimulated UT-7 cell proliferation. CONCLUSION: Four hSCF mimetic peptides were successfully isolated from phage-displayed random peptide library..

[Effect of Tuina on oxygen free radicals metabolism in patients with chronic fatigue syndrome].

OBJECTIVE: To explore the mechanism of Tuina for treatment of chronic fatigue syndrome. METHODS: A total of 90 patients were randomly divided into a Tuina group, a Taijiquan (take exercise) group and a Fluoxetine group, 30 cases in each group. They were treated with Tuina, Taijiquan and Fluoxetine, respectively. After a month, the therapeutic effects and the changes of malondialdehyde (MDA) content and the activity of serum superoxide dismutases (SOD) and serum glutathione peroxidase (GSH-Px) were ohserved. RESULTS: The total effective rate of 93.3% (28/30) in the Tuina group was better than 80.0% (24/30) in the Taijiquan group and 73.3% (22/30) in the Fluoxetine group (both P < 0.05). After treatment, MDA contents in the three groups were all decreased (P < 0.01, P < 0.05), and the activity of SOD. GSH-Px in both the Tuina group and the Fluoxetine group were increased (P < 0.01, P < 0.05), and especially in the Tuina group with a significant difference as compared with the other two groups (P < 0.05, P < 0.01). CONCLUSION: The therapeutic effect of the Tuina group is superior to that of the Taijiquan group and the Fluoxetine group. Tuina can regulate oxygen free radicals metabolism and clean superfluous oxygen free radicals to alleviate fatigue, which may be one of the mechanisms of Tuina in treating chronic fatigue syndrome.

[Full-length of human papillomavirus type 18L1 gene optimized using the plant preferred codons and synthesized by overlapping PCR].

OBJECTIVE: The Human papillomavirus type 18L1 (HPV18L1) gene was synthesized by overlapping PCR after optimization using plant preferred codons. METHODS: The gene sequences of HPV18L1 were obtained from GenBank and analyzed using DNAMAN, Lasergene, Vector NTI and BLAST. The target sequence was selected and modified using plant preferred codons by the Synthetic Gene Designer and JCat (Java Codon Adaptation Tool) with the addition of a His-tag to the C-terminus to construct the full-length modified HPV18L1 (mHPV18L1). mHPV18L1 was divided into 5 large segments, namely LS1 to LS5, with sizes ranging from 204 to 477 bp. Forty-three small oligonucleotide fragments with sizes of 57-59 bp and 6 pairs of primers were designed and synthesized. mHPV18L1 was amplified by overlapping PCR and subcloned into pMD18-T vector. The recombinant plasmid was identified by restriction enzymes digestion and sequencing. RESULTS: mHPV18L1 was successfully assembled using overlapping PCR. The results of digestion with restriction enzymes and PCR amplification confirmed that the recombinant vector pMD18T- mHPV18L1 contained the inserts with expected size of 1749 bp. mHPV18L1 sequence was confirmed by sequencing. CONCLUSION: mHPV18L1 with plant preferred codons and the recombinant vector pMD18T- mHPV18L1 have been obtained.

Inhibition of human prostate cancer xenograft growth by 125I labeled triple-helix forming oligonucleotide directed against androgen receptor.

BACKGROUND: The failure of hormone treatment for advanced prostate cancer might be related to aberrant activation of the androgen receptor. We have shown that (125)I labeled triple-helix forming oligonucleotide (TFO) against the androgen receptor gene inhibits androgen receptor expression and cell proliferation of LNCaP prostate cancer cells in vitro. This study aimed at exploring the effects of the (125)I-TFO on prostate tumor growth in vivo using a nude mouse xenograft model. METHODS: TFO was labeled with (125)I by the iodogen method. Thirty-two nude mice bearing LNCaP xenograft tumors were randomized into 4 groups and were intratumorally injected with (125)I-TFO, unlabeled TFO, Na(125)I and normal saline. Tumor size was measured weekly. The tumor growth inhibition rate (RI) was calculated by measurement of tumor weight. The expression of the androgen receptor gene was performed by RT-PCR and immunohistochemical study. The prostate specific antigen (PSA) serum levels were measured by enzyme linked immunosorbent assay. The tumor cell apoptosis index (AI) was detected by TUNEL assay. RESULTS: Tumor measurements showed that tumor development was significantly inhibited by either (125)I-TFO or TFO, with tumor RIs of 50.79% and 32.80% respectively. (125)I-TFO caused greater inhibition of androgen receptor expression and higher AIs in tumor tissue than TFO. Both the tumor weight and the PSA serum levels in (125)I-TFO treated mice ((0.93 +/- 0.15) g and (17.43 +/- 1.85) ng/ml, respectively) were significantly lower than those ((1.27 +/- 0.21) g and (28.25 +/- 3.41) ng/ml, respectively) in TFO treated mice (all P < 0.05). Na(125)I did not significantly affect tumor growth and androgen receptor expression in tumor tissue. CONCLUSIONS: The (125)I-TFO can effectively inhibit androgen receptor expression and tumor growth of human prostate cancer xenografts in vivo. The inhibitory efficacy of (125)I-TFO is more potent than that of TFO, providing a reference for future studies of antigen radiotherapy.

[Establishment of the genotype-specific targets of botulinum neurotoxins types B and E based on reverse genetics].

OBJECTIVE: To establish the genotype-specific targets plasmids and engineered E.coli strains of botulinum neurotoxins (BoNT) types B and E based on reverse genetics. METHODS: The gene sequences of BoNT were obtained from GenBank and analyzed using DNAMAN, Lasergene, Vector NTI and BLAST. Two target fragments of BoNT/B and BoNT/E were anchored and then synthesized as 5 and 10 short DNA single strands, respectively. The full-length target sequences were amplified by overlapping PCR and subcloned into pMD 18-T vector, and the recombinant plasmids were identified by restriction enzyme digestion and sequencing. RESULTS: Sixty full-length sequences of 4 types of BoNT, namely types A, B, E, and F, were available in GenBank. Two target fragments, BoNT/B of 215 bp and BoNT/E of 360 bp, and their specific primer pairs were anchored after sequence analysis. pMD 18-T-BoNT/B and pMD 18-T-BoNT/E containing these two target sequences were confirmed. CONCLUSION: The engineered plasmids and E.coli stains containing the genotype-specific target fragments of BoNT/B and BoNT/E have been constructed successfully.

[Inhibitory effects of immunotargeting of Chinese cobra cytotoxin and iodine-131 against nasopharyngeal carcinoma cells in vitro].

OBJECTIVE: To explore a new specific therapy of nasopharyngeal carcinoma (NPC) using an anti-nasopharyngeal carcinoma (NPC) monoclonal antibody BAC5 conjugate with Chinese cobra (CT) and iodine-131(131I). METHODS: BAC5 was labeled with 131I by chloramine-T method, CT was labeled with 125I using iodogen method, and BAC5 and 125I-CT were conjugated by SPDP method. The inhibitory effect of the conjugate on cultured human NPC CNE2 cells was observed using MTT assay. RESULTS: The IC50 of 125I-CT-BAC5 conjugate was 9.17x10(-8) mol/L, and that of 131I-BAC5 was 5.83x10(8) Bq/L, and their combined administration showed obvious inhibitory effect on the NPC cells. CONCLUSION: Both 125I-CT-BAC5 and 131I-BAC5 have obvious inhibition effects against NPC cells, but their combined use shows stronger effects.

[Randomized control study on the treatment of 26 cases of acne conglobata with encircling acupuncture combined with venesection and cupping].

OBJECTIVE: To observe the curative effect of acne conglobata treated by encircling acupuncture combined with ventouse and cupping. METHODS: A total of 52 acne conglobata patients were randomly divided into acupuncture group (n=26) and Western medicine group (n=26). Patients of acupuncture group were treated with encircling acupuncture around the affected focus. Common acupuncture was applied to Hegu (LI 4), Xuehai (SP 10), Fenglong (ST 40) and Sanyinjiao (SP 6), once daily. Dazhui (GV 14) and Feishu (BL 13) were used for venesection and cupping (twice a week). Patients of medication group were treated with oral administration of Isotretinoin Capsules (10 mg, t.i.d.). The treatment duration of 2 groups was 4 weeks. Serum IL-6 content was detected with double-antibody sandwich elisa enzyme linked immunosorbent assay (ELISA). RESULTS: After the treatment, in acupuncture group and Western medicine group, 3 (11.5%) and 4 (15.4%) cases experienced remarkable relief in their signs, 14 (53.8%) and 11 (42.3%) had marked improvement, 6 (23. 1%) and 7 (26.9%) had improvement, 3 (11.5%) and 4 (15.4%) failed, with the effective rates being 88.5% and 84.6%, respectively. No significant difference was found between two groups in the therapeutic effect (P>0.05). Self-comparison showed that after the treatment, IL-6 in both groups decreased significantly (P<0.01). The therapeutic effect of acupuncture group was significantly superior to that of Western medicine group in lowering serum IL-6 (P<0.05). CONCLUSION: Both acupuncture and medication can effectively promote the recovery of the affected skin, and lower serum IL-6 level in acne conglobata patients. The effect of acupuncture is stronger than that of Isotretinoin Capsules in lowering serum IL-6 content and has fewer adverse effects.

[Expression, purification, and characterization of the first three immunoglobulin-like domains of human stem cell factor receptor].

OBJECTIVE: To express the first three immunoglobulin-like domains of human stem cell factor receptor (c-Kit/Ig1-3) in E. coli and HEK293 ET cells and study their binding activity for stem cell factor (SCF). METHODS: In prokaryotic expression system, a double mutant form of c-Kit /Ig1-3 (c-Kit /Ig1-3(DM) was produced by overlap PCR and cloned into pET16b. The recombinant protein was expressed in E. coli BL21 (DE3) and refolded by dilution. In eukaryotic expression system, the gene of c-Kit/Igl13 with eight histidine segments was cloned into pEAK12 and the recombinant plasmid was transfected into HEK293 ET cells. The fusion protein was harvested from the growth medium and purified on Ni-NTA agarose column. The recombinant protein was tested for the receptor binding activity with his-tag pull-down and enzyme-linked immunosorbent binding assay. RESULTS: In E. coli c-Kit /Ig1-3(DM) as produced as an inclusion body and showed low binding activity for SCF after refolding. Two HEK293 ET cell clones that express high levels of c-Kit/Ig1-3 were produced and each clone secreted 2p micro/ml of recombinant protein, whose relative molecular mass was about 58,000. Eukaryotically expressed c-Kit/Ig1-3 had specific binding activity for SCF, and the dissociation constant (Kd) was 9.39 nmol/L. CONCLUSION: c-Kit/Ig1-3 with high receptor binding activity is successfully produced in HEK293 ET cells.

Human Flt3 ligand from Pichia pastoris inhibits growth of lymphoma and colon adenocarcinoma in mice.

UNLABELLED: Potent effects of Flt3 ligand (FL) on the development of the immune system have generated much interest in application of FL in cancer immunotherapy. OBJECTIVE: To evaluate the effects of Pichia pastoris secreted rhFL on the growth of mouse EL-4 lymphoma and C26 colon adenocarcinoma injected in syngeneic mice for the first time. METHODS: Mice were placed into one of two treatment groups. 2 x 10(5) EL-4 or C26 cells were injected subcutaneously (SC.) into mice on day 0. Group 1 received subcutaneous PBS injections from Day -7 to Day 14 and group 2 received subcutaneous rhFL injections at 30 microg/day from Day -7 to Day 14. Serial tumor areas were measured. On Day 22, mice from each group were sacrificed, and weight of tumors and spleens were evaluated. Data analysis used Student t tests. RESULTS: Pichia pastoris secreted rhFL resulted in tumor growth delay for both EL-4 lymphoma and C26 colon adenocarcinoma compared with control (P < 0.01). Tumors from rhFL-treated mice were smaller (P < 0.01) than controls while spleens larger (P < 0.01) than controls. Histological examination of tumor sections revealed an obvious increase in regions composed largely of infiltrating cells in the rhFL-treated tumors. Infiltrating cells could be detected in clusters among tumors from mice treated with rhFL whereas these cells were only occasionally detected in sections of control tumors. CONCLUSION: Treatment of rhFL expressed from Pichia pastoris resulted in an antitumor response against EL-4 and C26 tumors injected in syngeneic mice.