Study of concentration of amniotic fluid alpha-fetal protein in thalassemia fetus.

OBJECTIVES: To test the hypothesis that concentration of amniotic fluid alpha-fetal protein (AFAFP) is increased in thalassemia fetus. METHODS: A total of 135 cases of amniocentesis admitted from July 2013 to December 2014 were included in this study. Among them 98 cases of normal fetuses were assigned into control group and 37 cases of thalassemia fetus were included as thalassemia fetus group. Alpha-fetoprotein levels detected by enzyme linked immunosorbent assay and the alpha-fetoprotein concentration were compared between the two groups. There is no significant difference in gestational age between the two groups. RESULTS: 1. AFP concentration in thalassemia fetus group was significantly higher than that of normal control group [(1541.65 ± 734.78) µg/mL vs. (2728.84 ± 1539.97) µg/mL], and amniotic fluid AFP concentration was related to fetal thalassemia. 2. AFAFP concentration in pure α-thalassemia fetus was higher than that of ß-thalassemia fetus or mixed α- and ß-thalassemia fetus, but the difference was not significant. CONCLUSIONS: Concentration of amniotic fluid alpha-fetal protein is increased in thalassemia fetus. AFP concentration in α-thalassemia fetus was higher than that of ß-thalassemia or mixed α- and ß-thalassemia fetus but difference was not significance. Further studies are needed to explore the possible correlation between Down syndrome and biochemical markers of thalassemia.

Expression of soluble vascular endothelial growth factor receptor-1 and placental growth factor in fetal growth restriction cases and intervention effect of tetramethylpyrazine.

OBJECTIVE: To investigate the expression of soluble vascular endothelial growth factor receptor-1 (sFlt-1) and placental growth factor (PLGF) in the fetal growth restriction (FGR) cases and the intervention mechanism of tetramethylpyrazine. METHODS: A total of 60 fetal growth restriction cases that admitted to our hospital were randomly divided into ligustrazine intervention group (group A) and nutritional support group (group B). A total of 50 healthy pregnant women were also enrolled as control group (group C). Expression level of maternal serum sFlt1, PLGF and fetal growth parameters including HC, AC, FL, BPD, EFW as well as placenta PLGF, sFlt-1 mRNA expression were recorded and compared among the three groups. A total of 15 SD rats were selected and were divided into three groups, TMP group, alcohol and tobacco group and blank control group. Three groups of rats were dissected on the twentieth day of gestation. RESULTS: Expression level of sFlt-1 and PLGF in group A was not significantly different from that of group C (P>0.05); but significant difference in SFlt1 and PLGF expression level was observed between group C and group B (P<0.05). Before treatment, HC, AC, FL, BPD and EFW of group A and group B were significant lower than those of group C, but after treatment, those parameters in group A were significantly improved (P<0.05). In the animal experiment there was no significant difference in sFlt-1 between treatment group and FGR group without treatment or control group (P>0.05). There was significant difference in PLGF between FGR group with treatment and FGR group without treatment or control group (P<0.01). CONCLUSIONS: PLGF level is decreased and sFlt-1 increased in patients suffered from fetal growth restriction, and FGR rats show increased sFlt-1 and decreased PLGF, thus they can be indicator of the fetal growth restriction. Ligustrazine can effectively improve sFlt-1, PLGF expression level in fetal growth restriction cases, which can be used as treatment for FGR.

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant Lactococcus lactis for oral vaccination and its protective immune response in mice.

K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C(83549) challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice.

High-level secretion of a chimeric thermostable lichenase from Bacillus subtilis by screening of site-mutated signal peptides with structural alterations.

A chimeric gene mHG (669 bp) was constructed by substitution of Clostridium thermocellum ZJL4 lichenase (CG) N-terminal fragment (except its signal sequence) for the counterpart of Bacillus sp. A3 lichenase (BG). To acquire high-level secretion of the chimeric lichenase (mHG) in Bacillus subtilis, a series of site-mutated signal peptides were designed. The activity of mHG, which was directed by an artificial hydrophobic signal peptide H1 (MMARKIAGMATSLLVIFSSSAVA) from cytoplasm into growth medium, reached 80.56 U/ml after 22-h incubation, indicating that signal peptide hydrophobicity appears to be critical for early stages in mHG export. By purification of the mHG (approximately 25.3 kDa) from cultures of B. subtilis (pBSG-H1), enzymatic property assays showed that the common optima for mHG were 70 degrees C and pH 5.0. The residual activity of mHG at 90 degrees C for 10 min was 83.45% of its maximum activity, which was almost similar to that of CG (90 degrees C, 10 min, 84.33%). This constructed shuttle expression vector with a novel signal peptide exhibited its applicability for high-level production of heterologous proteins from B. subtilis. Moreover, the high-level secreted mHG with relatively high thermostability could be a potential candidate for feed industrial applications.

[Effect of blocking of ROCK-1, an effector of small G protein Rho, on the malignant behavior of ovarian tumor cells in vitro].

OBJECTIVE: To investigate the possible role of ROCK-1 in ovarian cancer invasion and metastasis. METHODS: ROCK-1 ASODN was transfected into SW626 and Caov-3 cell lines mediated by Lipofectamine 2000. The expressions of ROCK-1 mRNA and protein were detected by RT-PCR and Western-blot assay. Boyden chamber was used to assess the effect of ROCK-1 ASODN on the invasion and migration of the cell lines. The changes in the adhesion and proliferation of the transfected cells were detected by MTT assay. RESULTS: The expressions level of ROCK-1 mRNA and protein in the cell lines were decreased significantly after transfection at doses of 10 micromol/L and 20 micromol/L ROCK-1 ASODN. When compared with the control group, the invasion capability of transfected cells was inhibited to an extent of 75.6% +/- 3.8% and 54.7% +/- 2.9%, respectively, for SW626 cell line, and 68.8% +/- 4.7% and 50.0% +/- 4.5% for Caov-3 cell line, respectively. The random migratory activity of these two cell lines was inhibited by 80.0% +/- 1.3%, 63.7% +/- 1.9%, 72.5% +/- 3.4% and 55.9% +/- 2.5%, respectively, and the inhibition of chemotaxis activity of the two cell lines was 83.9% +/- 1.4%, 64.1% +/- 1.3%, 72.5% +/- 3.4% and 54.5% +/- 1.9%, respectively. No significant difference was found in the adhesion and proliferation of the cells transfected with ROCK-1 ASODN and control cells. CONCLUSION: The expression of ROCK-1 was closely related to the invasion capability and migratory activity of ovarian cancer cells. ROCK-1 may play a crucial role in invasion and metastasis of ovarian cancer.

Construction and characterization of a bifunctional fusion enzyme of Bacillus-sourced beta-glucanase and xylanase expressed in Escherichia coli.

A chimeric gene, Glu-Xyl, encoding Bacillus amyloliquefaciens glucanase (Glu, 24.4 kDa) and Bacillus subtilis xylanase (Xyl, 21.2 kDa), was constructed via end-to-end fusion and expressed successfully in Escherichia coli. The purified fusion protein (46.1 kDa) exhibited both glucanase and xylanase activities. Compared with parental enzymes, the Glu moiety was characterized by kinetic parameters of decreased K(m) (0.66-fold) and increased K(cat) (2.75-fold), whereas the Xyl moiety had an increased K(m) (1.37-fold) and decreased K(cat) (0.79-fold). These indicate a 3.15-fold net increase and a 31% decrease in catalytic efficiency (K(cat)/K(m)) of the Glu and Xyl moieties. Activities and stabilities of both moieties at 40-90 degrees C or pH 3.0-10.0 were compared with those of the parental enzymes. Despite some variations, common optima were 40 degrees C and pH 9.0 for the Glu moiety and parent, and 50-60 degrees C and pH 9.0 for the Xyl counterparts. Thus, the fusion enzyme Glu-Xyl was bifunctional, with greatly enhanced glucanase activity associated with a decrease in xylanase activity.