[Clinicopathologic and prognostic study of pediatric immature teratoma].

OBJECTIVE: To study the clinicopathologic features and biologic behavior of pediatric immature teratoma. METHODS: The clinical data, pathologic features, immunohistochemical findings (for cyclin D1, P27 and Ki-67) and follow-up information of 39 cases of pediatric immature teratoma were analyzed. RESULTS: Amongst the 39 cases studied, 12 arose in the sacrococcygeal region, 12 in testis, 5 in retroperitoneum, 4 in ovary, 4 in abdomen and 2 in mediastinum. Histologically, 16 cases were of grade 1, 8 cases of grade 2 and 15 cases of grade 3. Seven of the cases contained foci of yolk sac tumor. Immature neuroepithelial features used in histologic grading included the presence of primitive neural tubules, immature rosettes, undifferentiated neuroblastoma cells and primitive neuroectodermal structures. Immunohistochemical study showed that cyclin D1 was positive in 3 cases of grade 1 tumors, 4 cases of grade 2 tumors and 9 cases of grade 3 tumors. The positivity rates for p27 were 8, 3 and 6 cases respectively, while those for Ki-67 were 3, 4 and 13 cases respectively. Follow-up data were available in 30 cases. Three of them, including 2 cases with histologic grade 3 (with or without yolk sac tumor component), recurred after operation. CONCLUSIONS: The expression of cyclin D1 and Ki-67 is a useful adjunct in histologic grading. On the other hand, p27 overexpression shows little correlation with tumor grade. The prognosis of immature teratoma in children is different from that in adults. Sacrococcygeal immature teratoma occurring in patients younger than 1 year old and with low histologic grade do not require postoperative chemotherapy if the tumor is completely excised. Similarly, for testicular immature teratoma occurring in patients below 1 year of age, regardless of tumor grading, need no adjunctive therapy. On the other hand, ovarian immature teratoma with high histologic grade requires postoperative chemotherapy, regardless of age of the patients. The presence of microscopic foci of yolk sac tumor is a useful predictor of recurrence in pediatric immature teratoma.

Separation and determination of phthalates by micellar electrokinetic chromatography.

A method has been developed for the separation and determination of dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), di-(2-ethylhexyl) phthalate (DEHP) and di-n-octyl phthalate (DnOP) by micellar electrokinetic chromatography (MEKC). The baseline separation of phthalates was achieved by using a buffer of 100 mM sodium cholate, 50 mM borate and 15% methanol (pH 8.5). The optimized MEKC method was used to quantify the concentrations of phthalates in 11 soil samples from different regions of China. The contents of DEP, DBP and DEHP in soils were ranged 0-0.42, 0-1.43, and 0.24-2.35 mg/kg, respectively, and no DMP and DnOP was detected. The limits of detection for DMP, DEP, DBP, DEHP, and DnOP were found to be 0.050, 0.051, 0.052, 0.054, and 0.063 mg/kg, respectively. The results obtained by the MEKC method were compared with those obtained by gas chromatography with flame ionization detector (GC-FID), and a good agreement was achieved.

Separation and determination of phospholipids in plant seeds by nonaqueous capillary electrophoresis.

A method has been developed for the separation and determination of phospholipids by nonaqueous capillary electrophoresis in a separation medium of acetonitrile-2-proponol (3:2, v/v), 0.3% acetic acid and 60 mM ammonium acetate. To optimize the separation conditions, the composition of separation medium including alcohols, acetic acid, n-hexane and ammonium acetate was studied. The solvation interaction and ion-dipole interaction were also investigated. The contents of phospholipids in soybean, sunflower, peanut, apricot kernel, filbert and walnut were determined by the recommended method. The results obtained by the nonaqueous capillary electrophoreses were in good agreement with those determined by micellar electrokinetic chromatography.

[Cloning and expression of the alpha-amylase gene from a Bacillus sp. WS06, and characterization of the enzyme].

A Bacillus sp. WS06, which produces an extracellular alpha-amylase, was isolated from the cecum in a piglet. An amyF gene from this Bacillus strain was cloned and its nucleotide sequence was determined. An open reading frame composed of 1581 bases, which encodes 526 amino acid residues was found. The amyF gene shows high sequence homologies with other microbial amylase genes, such as Bacillus megaterium and Bacillus polymyxa (93% and 53% identity). The deduced amino acid sequence revealed that four highly conserved regions of the alpha-amylase family. The amyF gene was overepressed using the pET21a vector and Escherichia coli BL21 (DE3). The recombinant enzyme was purified 22.2 fold to electrophoretic homogeneity and had a molecular mass of 57kD (by SDS-PAGE). The enzyme was optimally active at pH 7 and 55 approximately 60 degrees C and showed stability at the temperature below 55 degrees C. This enzyme efficiently hydrolyzed various types of starch to yield a series of malto-oligosaccharides by endo-cleavage mode.

[Expression and purification of gene transfer vehicle mediated by epidermal growth factor receptor].

OBJECTIVE: To create a gene transfer vehicle for targeting gene therapy of cancer with epidermal growth factor receptor overexpressing. METHODS: Encoding sequences of the first domain of histone gene (H1) and EGF C-loop (EGFc) were obtained by PCR amplification. These two DNA fragments were ligated by EcoR I site, and cloned and sequenced. E. coli expression vector of the fusion gene was then constructed. The fusion protein H1EGFc was purified by specific band isolation from SDS-PAGE. RESULTS: The molecular weight of purified protein was consistent with the designed request. Its purity reached 94.02%. CONCLUSION: A fusion protein H1EGFc was expressed and purified.

[A strategy for targeting gene therapy against cancer mediated by epidermal growth factor receptor].

OBJECTIVE: To establish a protocol for the targeting gene therapy against cancer with rich epidermal growth factor receptor (EGFR). METHODS: A recombinant pcDNA3.1-PE III mut was constructed and combined with a non-viral vector, a fusion protein histone H1, epidermal growth factor C-loop previously expressed by us, to be a protein-DNA complex in vitro. Using the complex to treat BT-325 and Hela cancer cells with EGFR and JK cells without EGFR. The killing rates of the cells was calculated after 48 h of incubation at 37 degrees C. RESULTS: To BT-325 and Hela cells, the killing rates were 46.03% and 48.12% respectively. To JK cells, the complex had no killing function. CONCLUSION: The protocol for targeting gene therapy against cancer with EGFR has been established successfully.

[Cloning and expression of a thermostable beta-glycosidase gene from Thermus nonproteolyticus HG102].

The gene coding for beta-glycosidase (EC3.2.1.21) from Thermus nonproteolyticus HG102 has been cloned and expressed in E. coli. The gene open reading frame was 1311 bp and it codes for 436 amino acids. The deduced amino acid sequence of the enzyme showed identity with members of glycosyl hydrolase family I. The enzyme had high content of hydrophobic amino acid (Ala 12.8%, Leu 10.9%), Arg(9.6%), Glu(9.4%) and Pro(8.0%), but low content Cys(0.45%) and Met (0.9%). From the alignment of enzyme amino acid sequence with other glycosyl hydrolase family I members, Glu164 and Glu338 were predicated as the proton donor and nucleophile group. The DNASTAR program was used to predict the secondary structure. According to the Chou-Fasman model, the enzyme has 41.4% of alpha-helics, 16.2%, beta-strands, 14.4%, beta-turns. 14 of the 35 Pro were located at the second sites of beta-turns. Hydrophobic interaction, ion bond, alpha-helics and Pro had important contribution to Tn-gly thermostability.