The effects of tea polyphenols on the injury of fibrinolytic functions induced by high-methionine dietary in rats / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the protective impact of tea polyphenols (TP) on the injury of fibrinolytic functions induced by high-methionine dietary in rats.</p><p><b>METHODS</b>50 male Wistar rats were divided by stratified based on body weight into 5 groups with 10 in each group: namely control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group. The rats in model group and TP groups were fed with 3% methionine dietary, control group rats with routine diet. In addition, rats in low-dose, medium-dose and high-dose TP groups were treated with TP at 50, 100 and 200 mg/kg dosage respectively by gavages every day, control group and model group rats were given with same amount distilled water. The animals were sacrificed after 8 weeks. The levels of tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in plasma were determined by ELISA assays, mRNA levels of t-PA and PAI-1 in aortic arch were detected by RT-PCR, t-PA and PAI-1 expression in aortic arch were detected by immunohistochemistry strept-avidin-biotin complex (SABC).</p><p><b>RESULTS</b>After experiment, the t-PA expression of aortic arch in control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group were 133.03 ± 10.14, 95.46 ± 11.08, 111.97 ± 11.91, 130.23 ± 10.80, 139.39 ± 9.41 (F = 14.15, P < 0.01), respectively, and the PAI-1 expression were 90.91 ± 8.67, 166.76 ± 12.18, 139.63 ± 12.71, 134.66 ± 13.19, 109.49 ± 10.82 (F = 31.44, P < 0.01). The t-PA concentration of plasma were (10.69 ± 1.26), (6.13 ± 0.92), (8.56 ± 1.19), (9.69 ± 0.92), (11.97 ± 1.08) ng/ml, respectively (F = 41.98, P < 0.01), and the PAI-1 concentration of plasma were (6.31 ± 0.81), (16.98 ± 1.27), (11.39 ± 0.82), (8.46 ± 0.67), (8.08 ± 0.91) ng/ml, respectively (F = 207.74, P < 0.01). The mRNA levels of t-PA in aortic arch were 1.12 ± 0.02, 0.75 ± 0.14, 1.01 ± 0.09, 0.95 ± 0.08, 1.05 ± 0.13 (F = 5.77, P < 0.05), and the mRNA levels of PAI-1 in aortic arch were 1.25 ± 0.11, 1.74 ± 0.06, 1.23 ± 0.05, 1.09 ± 0.14, 1.23 ± 0.04 (F = 23.56, P < 0.01).</p><p><b>CONCLUSION</b>The results indicate that TP seems to have regulatory function on transcription and protein levels of t-PA and PAI-1, in addition to maintaining the balance between PAI-1 and t-PA and healing the injury of fibrinolytic functions in rats induced by high-methionine dietary.</p>

Effects of 1-bromopropane exposure on cognitive function in rats / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the effects of 1-bromopropane (1-BP) on the functions of learning-memory and the central cholinergic system in rats.</p><p><b>METHODS</b>Forty male Wistar rats were randomly divided into four groups: low 1-BP group (200 mg/kg), middle 1-BP group (400 mg/kg), high 1-BP group (800 mg/kg) and control group, and the exposure time was 7 days. The Morris water maze (MWM) test was applied to evaluate the learning-memory function in rats. After the MWM test, the rats were sacrificed, the cerebral cortex and hippocampus were quickly dissected and homogenized in ice bath. The activity of acetylcholine esterase (AChE) and choline acetyltransferase (ChAT) in supernatant of homogenate were detected.</p><p><b>RESULTS</b>The latency and swim path-length of rats in middle and high 1-BP groups prolonged significantly in place navigation test and the efficiency of searching strategy obviously decreased, as compared with control group (P < 0.05 or P < 0.01). In spatial probe test, the number of crossing platform in three 1-BP groups decreased significantly, as compared with control group (P < 0.05 or P < 0.01). The cortical AChE activity of rats in middle and high 1-BP groups was significantly higher than that of control and low 1-BP group (P < 0.05 or P < 0.01). The AChE activity in rat hippocampus of high 1-BP group obviously increased, as compared with control group as compared with control group (P < 0.05). There was no significant difference of cortical ChAT activity between three 1-BP groups and control group (P > 0.05). In the hippocampus, there was no difference of ChAT activity among the groups (P > 0.05).</p><p><b>CONCLUSION</b>1-BP exposure could significantly influence the learning-memory function in rats due to the increase of AChE activity.</p>

Effects of tacrolimus (FK506) on heat-shock proteins 70, Bcl-2 and Bax expression in nervous tissue of acrylamide-induced rat / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of tacrolimus (FK506) on behavioral function and heat-shock proteins (HSP70) expression in nervous tissues of acrylamide (ACR)-induced rats.</p><p><b>METHODS</b>Totally 40 health Wistar rats were randomly divided into control group, model group, low and high doses of FK506 groups. All four groups were treated five times per week for four weeks. Gait score was measured every week. And rats were sacrificed on day 28, the cerebrum, spinal cord and sciatic nerve were dissected, and homogenized in ice bath, then the levels of HSP70 and Bcl-2, Bax were analyzed by western bloting.</p><p><b>RESULTS</b>Compared with the ACR model group, the gait score in low and high doses of FK506 groups decreased by 30.1% and 47.7% respectively in the 4th week. In the cerebrum and sciatic nerve pellet, the level of HSP70 in the FK506 groups increased by 11.6%, 33.3% and 56.3%, 58.5% (P < 0.01), but no significant changes existed in spinal cord. The level of Bcl-2 in the sciatic nerve pellet increased by 39.1% (P < 0.01) but no significant changes existed in the cerebrum and spinal cord from low dose of FK506 group. And the level of Bax in the spinal cord pellet markedly increased by 46.8% but not in cerebrum and sciatic nerve pellet; Whereas in the tissues mentioned above, the levels of Bcl-2 were enhanced remarkably by 16.3%, 14.8% and 56.0% (P < 0.01) in the high dose of FK506 group. And the level of Bax in the cerebrum and spinal cord pellet markedly increased by 16.4% and 40.2% but not in sciatic nerve. The values of Bcl-2/Bax in low and high doses of FK506 groups clearly increased by 15.9%, 33.3%, 36.9% and 30.1%, 49.1%, 60.1% (P < 0.01).</p><p><b>CONCLUSION</b>The administration of FK506 has dramatically neuroprotective effects against the development of ACR neuropathy, which may be related to up-regulating the expression of HSP70 and Bcl-2 with down-regulating the expression of Bax.</p>

Effects of garlic oil on n-hexane metabolized to 2, 5-hexanedione in mice serum / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the effects of garlic oil (GO) on n-hexane metabolized to 2, 5-hexanedione (2, 5-HD) in mice.</p><p><b>METHODS</b>Adult healthy Kunming-mice were treated with n-hexane and GO. The serum was obtained and extracted with ethyl acetate, and the levels of the serum 2, 5-HD were determined by gas chromatography.</p><p><b>RESULTS</b>(1) The concentration of 2, 5-HD in serum increased firstly after a single exposure to n-hexane (4 000 mg/kg). The peak value occurred at 10 hours after n-hexane treatment, but could hardly be detected at 20 h. (2) There was no 2, 5-HD in serum of control mice. The content of 2, 5-HD in serum increased along with the exposure dose of n-hexane. The serum 2, 5-HD contents of the 2000, 4000 and 6000 mg/kg groups mice were 8.04, 16.68 and 22.38 microg/ml at 8 h in pretreated mice, respectively, and showed significant dose-effect relationship. (3) When the different age mice were exposed to the same dose of n-hexane, the contents of 2, 5-HD in serum were significantly different after 8 hours (P<0.05). The serum 2, 5-HD level of the 5 weeks old mice (22.83 microg/ml) was much higher than the 4 (19.59 microg/ml) and 6 (16.42 microg/ml) weeks old mice. (4) When the different gender mice were exposed to the same dose of n-hexane, the concentration of 2, 5-HD in serum of female mice (13.22 microg/ml) was higher than that of the female mice (10.34 microg/ml, P<0.05). (5) GO significantly inhibited the increase of the serum 2, 5-HD levels of both the pretreatment and post-treatment groups treated with 80 mg/kg n-hexane respectively, but the pretreatment with GO exhibited the more suppressive effects than the post-treatment (P>0.05). Compared with the n-hexane group, the concentrations of serum 2, 5-HD in GO-pretreated groups mice decreased by 16.2%, 20.8%, 22.8% (P<0.05) and 32.1% (P<0.01), respectively, and showed significant dose-effect relationship.</p><p><b>CONCLUSION</b>The serum content of 2, 5-HD, the metabolite of n-hexane, is different in different genders and age mice after exposed to the same dose of n-hexane. GO can effectively inhibit the production of n-hexane metabolized to 2, 5-HD in mice serum.</p>

Dynamic study of neurofilament contents in rat's spinal cord induced by 2, 5-hexanedione / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic changes of neurofilament contents in rat's spinal cord induced by 2, 5-hexanedione (2, 5-HD), and explore the molecular mechanism of n-hexane neuropathy.</p><p><b>METHODS</b>Male Wistar rats were administered at a dosage of 400 mg/kg/day 2, 5-HD for 2, 4 and 8 weeks respectively. HD-induced neurological defects were detected and quantified using gait score, and the relative lev-els of NF-H, NF-M, and NF-L in spinal cords of rats were determined by Western Blotting.</p><p><b>RESULTS</b>Exposure to 2, 5-HD produced progressive gait abnormalities, which suggested that the rat model of 2, 5-HD-induced neurotoxicity was established successfully. Western-Blotting results showed that NFs content in spinal cord demonstrated a progressive decline as the intoxication continued. In the supernatant fraction, compared to the controls, NF-H con-tent decreased by 15.7%, 57.0%, and 58.0% respectively after 2, 4, and 8-week treatment with 2, 5-HD (P < 0.01); accordingly, NF-M decreased by 36.0%, 61.3%, and 65.2% respectively (P < 0.01); NF-L decreased by 20.8%, 43.9%, and 44.3% respectively (P < 0.01). In the pellet fraction, the contents of NF-H in groups of 4 and 8 weeks' exposure to HD decreased by 35.6% and 43.2%, respectively (P < 0.01), and those of NF-L decreased by 26.4% and 42.1%, respectively (P < 0.01) when compared to the control. Further-more, NF-M contents in groups of 2, 4 and 8 weeks' exposure decreased by 23.3%, 33.9%, and 63.7% respectively (P < 0.01). The NFs level in spinal cords was highly correlated with gait abnormality of treated rats as the intoxication went on. Multiple correlation coefficients of NF-H, NF-M, and NF-L content with gait score of HD-treated rat were 0.8912, 0.9282 and 0.8981 (P < 0.01) respectively.</p><p><b>CONCLUSION</b>The declines of NFs are high-ly related to neurobehavioral abnormality of 2, 5-HD-treated animals, and involved in the development of n-hexane neuropathy.</p>

Effects of methamidophos on microtubule and microfilament proteins in sciatic nerve of hens / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic changes of alpha-tubulin, beta-tubulin and beta-actin in sciatic nerve of hen with organophosphorus ester-induced delayed neuropathy (OPIDN).</p><p><b>METHODS</b>OPIDN was induced in 10-month-old Roman hens by daily subcutaneous administration of 30 mg/kg methamidophos for 15 days. Hens were sacrificed 2, 10, and 23 days respectively after manifesting neuropathy. The sciatic nerves were dissected, homogenized and used for the determination of the alpha-tubulin, beta-tubulin and beta-actin levels by western blotting.</p><p><b>RESULTS</b>The levels of alpha-tubulin in supernatant of sciatic nerves were decreased by 6%, 15% and 25% respectively on day 2, 10 and 23 respectively, while those in pellet remained almost unchanged. beta-tubulin were decreased by 27%, 6%, 19% in pellet and 1%, 21%, 22% in supernatant of sciatic nerves on 2, 10 and 23 days. Beta-actin level in pellet of sciatic nerve increased by 24%, 48% and 17% on day 2, 10 and 23, and little changes were observed in supernatant.</p><p><b>CONCLUSION</b>Methamidophos may induced changes of alpha-tubulin, beta-tubulin and beta-actin levels in sciatic nerve of hen, which may be one of the mechanism of the contribution to the occurrence and development of OPIDN.</p>

Effect of monoclonal antibody against extracellular domain III of vascular endothelial growth factor receptor KDR on proliferation of vascular endothelial cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To prepare a neutralizing monoclonal antibody (McAb) against vascular endothelial growth factor receptor KDR and study its biological activity.</p><p><b>METHODS</b>Extracellular immunoglobulin (Ig)-like domain III of KDR (KDR III) was expressed in E. coli and purified by affinity chromatograph. Monoclonal antibody against KDR III was prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [(3)H]-TdR incorporation assay were also used to detect the activity of anti-KDR McAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on VEGF-induced mitogenesis of human endothelial cells.</p><p><b>RESULTS</b>McAb Ycom1D3 against KDR III was prepared which bound specifically to both the soluble KDR III and the cell-surface expressed KDR. It effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated activation of KDR expression on human endothelial cells. Furthermore, Ycom1D3 efficiently neutralized VEGF-induced mitogenesis of human umbilical vascular endothelial cells.</p><p><b>CONCLUSION</b>McAb Ycom1D3 against KDR III may suppress the action of VEGF by blocking native vascular endothelial growth factor receptor KDR. It has potential clinical applications in the treatment of cancers and other diseases where pathological angiogenesis is involved.</p>

Cloning and expression of the extracellular domain of 4-1BBL / 生物工程学报

RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.

Alterations of neurofilament proteins in sciatic nerve of hens induced the delayed neurotoxicity by methamidophos / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the dynamic alterations of neurofilament subunits (NF) in sciatic nerve of hens with organophosphorus ester induced the delayed neurotoxicity or neuropathy (OPIDN).</p><p><b>METHODS</b>Hens with OPIDN were produced by giving 30 mg/kg methamidophos subcutaneously to the 10-month-old Roman hens daily for 15 days, and sacrificed after manifesting neurotoxic clinical signs on the 2nd, 10th, and 23rd day respectively. The sciatic nerves were dissected, homogenized and centrifuged. The levels of NF in supernatant and pellet of sciatic nerves were examined by Western blotting respectively at different time from 2 to 23 days.</p><p><b>RESULTS</b>Integrated optional density (IOD) of high molecular weight neurofilament (NF-H) in sciatic nerve pellet of hens on the day 2, 10, 23 after appearance of OPIDN were 145,117 +/- 17,038, 55,917 +/- 17,333 and 45,038 +/- 6,662 respectively. As compared with the control group (78,875 +/- 22,569), the contents of NF-H in pellet were increased by 84% on day 2, and decreased by 29% and 43% on day 10 and 23 respectively. IOD of NF-H in supernatant of sciatic nerves were 4,709 +/- 1,739, 12,337 +/- 3,205 and 16,745 +/- 931, which were reduced significantly as compared with the control (44,083 +/- 6,895) at three different times. There was no significant difference in IOD of middle molecular weight neurofilament (NF-M) between control group (27,925 +/- 2,660) and on day 2 (31,493 +/- 4,625) in pellet. Those were 19,367 +/- 2,746 and 6,612 +/- 1,119 respectively on day 10 and day 23 in pellet of hen's sciatic nerve, which were much less than that in control. Little were detected in supernatant on day 10, and the IOD of NF-M were 3,196 +/- 269 and 5,206 +/- 1,292 on day 2 and day 23 respectively, which were lessened by 81% and 70% as compared with the control (17,243 +/- 3,232). In sciatic nerve pellet of hens, IOD of low molecular weight neurofilament (NF-L) on day 2 was 39,211 +/- 3,800, which was much higher than that in the control (28,749 +/- 9,319). There were no significant differences between IOD on day 10 (27,974 +/- 3,611), day 23 (21,507 +/- 2,286) and the control. There was no detection both on day 2 and 10 in supernatant of sciatic nerve, and IOD of NF-L were 5,962 +/- 1,929 on day 23, which were reduced significantly compared with the control (11,897 +/- 352).</p><p><b>CONCLUSION</b>The alterations of NF in sciatic nerve might contribute to the occurrence and development of OPIDN.</p>

Construction and expression of single chain Fv gene against domain II of human VEGF receptor II / 生物工程学报

The genes encoding for the light and heavy chain variable regions (V(H) and V(L)) has been cloned by RT-PCR from a murine hybridoma that produced monoclonal antibody (mAb) Ycom1D3, which was against domain III of human vascular endothelial growth factor receptor II (KDRIII) and were then connected to each other by a short peptide linker containing 15 amino acids (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant Ycom1D3-ScFv gene was cloned into the expression vector pAYZ and induced to express in E. coli 16C9. SDS-PAGE and Western blot analysis showed that the recombinant Ycom1D3-ScFv gene was expressed in E. coli 16C9 and the relative molecular weight of the fusion protein is 30kD which was consistent with the theoretically predicted value. ScFv expression was in the form of an inclusion body and the purified fusion protein was obtained after a series of purification steps including cell breakage, inclusion body solubilization, TALON metal affinity chromatography and protein refolding. Flow cytometric analysis showed that the ScFv fragment can react with human umbilical vein endothelial cells (HUVECs) which express KDR on the cell surface. In Conclusion, Recombinant Ycom1D3-ScFv gene has been successfully constructed and expressed in E. coli 16C9, which could be useful in both diagnostic and therapeutic applications.

Construction and expression of anti-CD3/ anti-Pgp Diabody / 生物工程学报

The use of tumor antigen specific antibodies for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. However, several factors restrict the use of anti-PGP monoclonal antibodies(Mabs). First, Pgp is expressed in normal tissues, particularly in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroids plexus and other organs. It plays a significant role to transport drugs and toxins in these organs. Therefore, anti-PGP antibodies in combination with cytotoxic compounds or radiolabelled antibodies should neither inhibit the activity of PGP, nor harm the cells which expressed PGP normally. BiMab exploit the specificity of Mab and ensures activation of cellular cytotoxic mechanisms which kill tumor cells only, but not harm normal cells. It will provide a strategy for resistant cancer therapy using anti-PGP antibodies. Second, Repeated administration of murine antibodies generates a strong human anti-mouse immune (HAMA) response in up to 50% of patients after the first dose, and appro ximately 90% following a second treatment. In an effort to reduce the toxicity and antigenicity, we focus to produce anti-PGP antibodies which have the binding activity only, but not inhibit the function of the "pump", and to construct a small and partially humanized recombinant molecule with dual specificity for both PGP and CD3 complex to activate the host immune response toward the tumour. PCR and overlap PCR were used to construct anti-CD3/ anti-Pgp Diabody. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by both the detection of western blot and size exclusion chromatography; its antigen-binding activity was examined by FACS, cellular RIA. Plasmid pAYZDCP which expressed the anti-CD3/anti-Pgp Diabody was constructed correctly. The diabody was recovered in high yield( up to 2mg/ L) after E-taq purification and predominantly(90%) as a dimer. The diabody can bind to Jurkat cells (CD3+) and K562/A02 cells(Pgp+). The affinities of the diabody were similar with the anti-CD3 ScFv or anti-Pgp ScFv, respectively. The anti-CD3/ anti-Pgp BsF(ab')2 was first recast into the diabody format and succeeded to obtain high level expression. The results of some biological activity experiments indicated that the diabody could bind to Jurkat cells and K562/A02 cells. Multidrug resistance can be reversed experimentally by a variety of drugs, among which the best known are verapamil and trifluoperazine, which unfortunately are of limited use in practice due to severe collateral cardiac toxicity. Anti-PGP x anti-CD3 diabody will provide another therapeutic strategy against multidrug resistance cancer.

One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity / 生物工程学报

Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future.