[Comparison of embryotoxicity of di(2-ethylhexyl) phthalate using mouse and human embryonic stem cell test models in vitro].

OBJECTIVE: To establish a mouse embryonic stem cell test (mEST) model and human embryonic stem cell test (hEST) model, to evaluate the embryotoxicity of di(2-ethylhexyl) phthalate (DEHP). METHODS: We developed mEST and hEST models according to the European Centre for the Validation of Alternative METHODS (ECVAM). We used penicillin G (PN-G) as the standard negative reference and 5-fluorouracil (5-FU) as the standard positive reference, respectively, to verify validity of the models. Based on model validity, mouse embryonic stem cells D3 (mESC-D3), mouse Balb/c-3T3 (3T3), and human embryonic stem cells H9 (hESC-H9) were administered different concentrations of DEHP (15.6, 31.2, 62.5, 125.0, 250.0, 500.0, and 1 000.0 µg/ml) for 7 days. A cell counting Kit-8 was used to detect the 50% inhibitory proliferation concentration (IC50) of mESC-D3 cells, 3T3 cells, and hESC-H9 with DEHP. mESC-D3 and hESC-H9 were treated with DEHP (15.6, 31.2, 62.5, 125.0, 250.0 µg/ml, and 500.0 µg/ml) for 10 days based on the cytotoxicity results. At day 10, the expression of cardiomyocyte differentiation gene alpha-myosin heavy chain (α-MHC) was detected by real-time PCR and the 50% inhibition of cardiomyocycte differentiation (ID50) determined. Based on the values of IC50 and ID50, functions Ⅰ, Ⅱ and Ⅱ could be calculated by three linear discriminant functions in the EST model and the embryotoxicity of DEHP described by comparing the three functions. RESULTS: Nontrophoblast lineage both ES cells were cultured under optimal conditions and highly expressed hESC markers OCT4 , SSEA4, and TRA-1-60. The embryoid bodies formed were uniform in size and shape, and these results were highly repeatable. The PN-G and 5-FU results coincided with the prediction by ECVAM. Validation of our EST models was satisfactory. RESULTS of the three endpoints of DEHP in mEST were 197.3 µg/ml (IC50 3T3), 210.0 µg/ml (IC50 D3) and 246.8 µg/ml (ID50 D3). DEHP was evaluated to be a nonembryotoxic compound based on values of function Ⅰ (7.78), function Ⅱ (7.58) and function Ⅲ (-7.79). The three endpoints of DEHP in hEST were 195.4 µg/ml (IC50 3T3), 184.8 µg/ml (IC50 D3), and 84.3 µg/ml (ID50). By comparing the values of function Ⅰ (3.21), function Ⅱ (5.77), and function Ⅲ (-6.46), DEHP was evaluated to be weakly embryotoxic. CONCLUSION: DEHP was determined to be a nonembryotoxic compound by mEST and weakly embryotoxic by hEST. Therefore, hEST is a more sensible model for the evaluation of DEHP embryotoxicity.

Orthotopic abdominal multivisceral transplantation without venovenous bypass in pigs.

BACKGROUND: Because venovenous bypass (VVB) can cause specific complications, a simplified orthotopic abdominal multivisceral transplantation (MVTX) technique without VVB in pigs has been evaluated in terms of morbidity and mortality. MATERIALS AND METHODS: Outbred large-white pigs weighing 25 to 40 kg of random sex underwent MVTX operation. After in situ cold perfusion through the aorta and superior mesenteric vein, the multivisceral allograft was procured from the donor and tailored at the back table. The multivisceral allograft, including liver, pancreas, stomach, duodenum, and proximal 2 m of jejunum, was en bloc transplanted into recipient after resection of entire foregut and midgut; VVB was not used. We analyzed the hemodynamic change, arterial blood gas data, and fluid requirements intraoperatively. RESULTS: Among 25 MVTXs, 19 (76%) animals survived after the operation. Without using an immunosuppressant, postoperative survival time was 2 to 146 hours. Ten recipient pigs died within 24 hours. Seven animals were lost between postoperative days 2 and 5. Two pigs survived for more than 5 days. The recipient pigs were mostly in a state of hypovolemic shock and metabolic acidosis during the reperfusion phase. CONCLUSIONS: Despite a high morbidity and mortality, the simplified technique without VVB is feasible to successfully achieve MVTX in the pig.

A stable glucose biosensor prepared by co-immobilizing glucose oxidase into poly(p-chlorophenol) at a platinum electrode.

An amperometric glucose biosensor was successfully developed by electrochemical polymerization of p-chlorophenol (4-CP) at a Pt electrode in the presence of glucose oxidase. The amperometric response of this biosensor to hydrogen peroxide, formed as the product of enzymatic reaction, was measured at a potential of 0.6 V (vs. SCE) in phosphate buffer solution. The performances of sensors, prepared at different monomer concentrations and polymerization potentials, were investigated in detail. The biosensor prepared under optimal conditions had a linear response to glucose ranging from 2.5 x 10(-4) to 1.5 x 10(-2) mol L(-1) with a correlation coefficient of 0.997 and a response time of less than 2 s. Substrate selectivity of the polymer-based enzyme electrode was tested for coexisting interferents such as uric acid and ascorbic acid, and no discernible response was observed. After 90 days, the response of the biosensor remained almost unchanged, indicating very good stability.

[Overproduction of cholera toxin B subunit by recombinant Escherichia coli MM2 in lactate-containing medium].

The expression of ctb gene in recombinant E. coli MM2 strain is affected by temperature, pH and carbon source. High level production of cholera toxin B subunit (CTB) was investigated in lactate-containing medium which designed by experiments of orthogonal test. Upshifting temperature from 30 degrees C to 37 degrees C could increase the production of CTB by 4 fold, upshifting pH value from 7.2 to 8.4 at the later culture stage could increase the specific expression level of CTB by 2.14 fold, and adding sodium acetate increased the production of CTB by 65%. In 5 L fermentor the cell density was reached at OD600 30, and the 186.7 mg/L of CTB was obtained. The CTB in the culture supernatants was in the form of polymer like the native CTB from Sigma, and also possessed the same antigenity.

The electrochemical copolymerization of 3,4-dihydroxybenzoic acid and aniline at microdisk gold electrode and its amperometric determination for ascorbic acid.

The electrochemical copolymerization of 3,4-dihydroxybenzoic acid (3,4-DHBA) and aniline was carried out at microdisk gold electrodes by means of cyclic voltammetric sweep. The polymer obtained on the electrode shows good electrochemical activity and high stability even though in neutral and weakly basic media. It was found that the response current of ascorbic acid was greatly enhanced at this composite polymer electrode. Moreover, the anodic overpotential was significantly reduced for about 200 mV (vs. SCE) compared with that obtained at bare gold electrodes. The electrode exhibits a rapid current response (less than 2 s) and a high sensitivity (0.21 AM(-1) cm(-2)). The dependence of response currents on the concentration of ascorbic acid was linear in the range of 1.0x10(-4)-1.0x10(-2) M. In addition this composite polymer modified electrode exhibits a high electrode stability for a long-term use.

Amperometric glucose enzyme electrode by immobilizing glucose oxidase in multilayers on self-assembled monolayers surface.

A novel and robust amperometric enzyme electrode for the determination of glucose was constructed by immobilizing glucose oxidase (GOD) and Os(bpy)(2)Cl-poly(4-vinyl)pyridine (Os-PVP) complex multilayers on thiol self-assembled monolayers surface. The apparent Michaelis-Menton constant K(m)' increased with increasing the number of Os-PVP/GOD multilayers. The concentration range of linear response and detection limit were 0.1-10 and 0.05 mM, the interference of ascorbic acid and uric acid were eliminated by the presence of SAMs and the enzyme electrodes were stable over 3 weeks. The preparation technique may be useful for controlling the performance of multilayer enzyme electrodes by changing the enzyme content.

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family.

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis-trans peptidyl-prolyl isomerases (PPlases). slyD mutations affect plaque formation by the phage phiX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of phiX174 on these E(R) strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.

The impact of the neonatal resuscitation program guidelines (NRPG) on the neonatal mortality in a hospital in Zhuhai, China.

AIM: The neonatal resuscitation program (NRPG) was first introduced in our hospital to replace the traditional resuscitation (TR) program in 1993. TR has been in existence in China for a long time. The implementation of NRPG was timely in reducing the number of infant mortality and also to disseminate to the many hospitals in China which are still practising TR. METHOD: A perspective study of 4,751 newborns with 366 asphyxiated babies in a period of 2 years was carried out. A previous sample of 1,722 live births under the TR program was compared as a controlled group statistically. RESULTS: From August 1993 to August 1995, when NRPG was exclusively implemented in our hospital, only 16 newborns died within 7 days, out of 4,751 births (3.4%) with 2 deaths in the delivery room. Seventeen newborns died within 7 days out of 1,722 births (9.9+) in the TR group, with 10 deaths in the delivery room. From the data shown, it can be clearly seen that perinatal neonatal mortality rate was reduced almost 3 times after NRPG was implemented (chi(2) = 10.54, p < 0.01). The follow-up results of 21 cases of severe asphyxia at 2 months--1 year of age were normal except for one with cerebral palsy. CONCLUSIONS: Our study showed that NRPG was indeed a very effective and feasible technique during the delivery process in the reduction of neonatal mortality. It is important to disseminate widely the knowledge and technique of NRPG in places where TR is still being widely practiced especially in developing countries.