[Prognostic significance of the number of dissected lymph nodes in Siewert type â ¡adenocarcinoma of the esophagogastric junction without lymphatic metastasis].

OBJECTIVE: To investigate the correlation between the number of dissected lymph nodes (LNs) and the prognosis of patients with node-negative Siewert type ⅡAEG. METHODS: 248 patients with Siewert type Ⅱ AEG treated in our hospital between January 1998 and December 2008 were retrospectively assessed. All cases underwent left transthoracic subtotal esophagogastrectomy with conventional two-field lymphadenectomy, and were histopathologically proved to be without lymph node involvement. The prognostic impact of the number of dissected LNs was analyzed. RESULTS: The overall median survival time and the 1-, 3-, and 5-year overall survival rates were 64 months, 80.4%, 60.8% and 51.0%, respectively. Cox regression showed that the number of dissected LNs and the depth of tumor invasion were independent prognostic factors. Patients with a high number of negative LNs had better overall survival than patients with a low number of negative LNs (P<0.05). The patients had better long-term survival outcomes with more than 10 LN dissected for cases with pT1 tumor (P<0.001), and so did those with more than 15 LN dissected for cases with pT2-3 tumor (P=0.003, 0.018, respectively). CONCLUSION: The number of negative lymph nodes and the depth of tumor invasion are independent prognostic factors for node-negative Siewert type Ⅱ AEG, and adequate lymph node dissection can improve the long-term survival.

Influence of temperature on nucleus degradation of 4-androstene-3, 17-dione in phytosterol biotransformation by Mycobacterium sp.

UNLABELLED: One of the steroid intermediates, 4-androstene-3, 17-dione (AD), in the biotransformation of phytosterols is valuable for the production of steroid medicaments. However, its degradation during the conversion process is one of the main obstacles to obtain high yields. In this study, the effect of temperature on nucleus degradation during microbial biotransformation of phytosterol was investigated. The results indicated that microbial degradation of phytosterol followed the AD-ADD-'9-OH-ADD' pathway, and that two important reactions involved in nucleus degradation, conversions of AD to ADD and ADD to 9-OH-ADD, were inhibited at 37°C. With a change in the culture temperature from 30 to 37°C, nucleus degradation was reduced from 39·9% to 17·6%, due to inhibition of the putative KstD and Ksh. These results suggested a simple way to decrease the nucleus degradation in phytosterol biotransformation and a new perspective on the possibilities of modifying the metabolism of strains used in industrial applications. SIGNIFICANCE AND IMPACT OF THE STUDY: Nucleus degradation of products is one of the main problems encountered during phytosterol biotransformation. To solve this problem, the effect of temperature on nucleus degradation was investigated in the industrial production of steroid intermediates. The results are also helpful to the genetic modification of sterol-producing strains.

Reticulospinal plasticity after cervical spinal cord injury in the rat involves withdrawal of projections below the injury.

Restoring voluntary fine motor control of the arm and hand is one of the main goals following cervical spinal cord injury (SCI). Although the functional improvement achievable with rehabilitative training in rat models is frequently accompanied by corticospinal tract (CST) plasticity, CST rewiring alone seems insufficient to account for the observed recovery. Recent investigations in animal models of SCI have suggested that the reticulospinal tract (RtST) might contribute to mediating improved motor performance of the forelimb. Here we investigate whether the spared RtST can compensate for the loss of CST input and whether RtST projections rearrange in response to cervical SCI. Animals underwent unilateral ablation of the dorsal CST and rubrospinal tract at spinal level C4, while the ventral RtST projections were spared. At the end of the six-week recovery period, injured animals had made significant improvements in single pellet reaching. This was not accompanied by increased sprouting of the injured CST above the injury compared to uninjured control animals. Injury-induced changes in RtST fiber density within the gray matter, as well as in the number of RtST collaterals entering the gray matter or crossing the cord midline were minor above the injury. However, all analyses directly below the injured spinal level consistently point to a significant decrease of RtST projections. The mechanism and the functional relevance behind this new finding warrant further study. Our results also suggest that mechanisms other than anatomical plasticity, such as plastic changes on a cellular level, might be responsible for the observed spontaneous recovery.

Selective adsorption of Mycobacterium Phlei on pyrite and sphalerite.

The adsorption of Mycobacterium Phlei cells on the surfaces of pyrite and sphalerite was studied as functions of time and pH. The results indicated that a higher amount of cells adsorbing onto pyrite compared with that onto sphalerite under neutral and alkaline conditions, and it was also observed from photographs of scanning electron micrograph. To gain a better insight into the mechanisms of differential adsorption, the functional groups on cell surfaces and the chemical states of each element on mineral surfaces before and after interaction with bacterial cells were investigated. The results showed that many groups presented on cells surface, such as C-O-H, C-O-C, C=O, C-N, N-H and P=O. The change in state of each element on pyrite and sphalerite surfaces after interaction with bacterial cells revealed that there were chemical reactions between metal ions and S on mineral surface and atoms like N, O, P, etc. on cell surface, and the shifts in binding energy of each element on pyrite surface is larger than that of sphalerite. Possible mechanisms for selective adsorption of bacterial cells onto pyrite and sphalerite were discussed in the latter part of this paper.

Expression and purification of soluble B lymphocyte stimulator from recombinant Escherichia coli.

In this work, the expression conditions of fusion protein thioredoxin (Trx)-soluble B lymphocyte stimulator (sBLyS) in shake flask and bioreactor from the recombinant Escherichia coli BL21 (DE3) with a pET system encoding the fusion protein gene of Trx-sBLyS and the purification method of the sBLyS were optimized to effectively obtain the bioactive protein sBLyS with a high purity. A yield of about 250 mg Trx-sBLyS/g DWC (1686 mg Trx-sBLyS/L) and expression level of about 38.5% in soluble Trx-sBLyS were obtained in a 30 1 bioreactor after optimization of the fermentation conditions. After the completion of the optimized purification procedure in order of affinity chromatography, enzymatic cleavage with enterokinase and DEAE ion exchange chromatography, about 200 mg sBLyS per liter fermentation broth was obtained with a purity of about 95% and a yield of near 30%, respectively. Furthermore, the molecular weight (MW) and the isoelectric point (pI) of the purified sBLyS were determined by 2-D gel electrophoresis and SDS-PAGE analysis and estimated to be over 16 kDa and about pH 4.15, respectively. In addition, the bioactivities of the soluble Trx-sBLyS in fermentation broth and the purified sBLyS were tested by two kinds of analytical methods of bioactivity. The good fermentation yield and the satisfied, purified sBLyS product with high purity, yield and bioactivity demonstrated the sBLyS production procedure was promising in industry.

Inhibition of quorum sensing in Serratia marcescens H30 by molecular regulation.

Quorum sensing in Serratia marcescens, which uses two types of signaling molecules-N-acyl homoserine lactones and furanosyl borate diester-play important regulatory roles in the synthesis of 2,3-butanediol and prodigiosin. In the hope of understanding the effect of quorum sensing on physiologic metabolism, we established two molecular strategies, one to express acyl-homoserine lactone hydrolase to inactivate AI-1 signaling molecule using an expression vector with lactose as the inducer and the other to mutate luxS gene with a suicide plasmid pUTKm2 to inhibit the synthesis of AI-2 signaling molecule.

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer.

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.

Action mechanisms and structure-activity relationships of PI3Kgamma inhibitors on the enzyme: a molecular modeling study.

Action mechanisms of four types of PI3Kgamma inhibitors were investigated on the ligand-binding pocket (LBP) of PI3Kgamma with molecular modeling method. At first five compounds whose complex structures with PI3Kgamma were available experimentally were used to validate the reliability of docking program Autodock3.0. The results demonstrated that the program could reproduce the bound conformations of those compounds in crystal structures. Then the program was used to dock all the four types of PI3Kgamma inhibitors into the LBP of the enzyme. The predicted activities of these compounds were in agreement with their experimental activities, and a pharmacophore model was hence derived for these compounds, which consisted of one hydrophobic portion flanked by two symmetric hydrophilic portions. Furthermore, the structure-activity relationships of PI3Kgamma inhibitors were elucidated and the activity differences between them were discussed.

In vitro and in vivo reversal of cancer cell multidrug resistance by 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone.

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) isolated from the buds of Cleistocalyx operculatus, was investigated for its reversal effects on cancer cell multidrug resistance. DMC potentiated the cytotoxicity of the chemotherapeutic agent doxorubicin to drug-resistant KB-A1 cells. When 5 microM DMC was present simultaneously with doxorubicin, the IC50 of DOX on KB-A1 cells decreased from 13.9 +/- 0.7 microg/ml to 3.6 +/- 0.7 microg/ml. A human carcinoma xenograft model was established with the KB-A1 cell line. DMC could sensitize the tumors to doxorubicin as indicated by a considerable reduction in tumor weight. DMC increased the intracellular accumulation of doxorubicin in KB-A1 cells. When KB-A1 cells were exposed to 10 microg/ml doxorubicin combined with 5, 10, 20 microM DMC for 4 hours, the intracellular concentrations of doxorubicin were increased 1.4-, 1.8-, 3.1-fold, respectively, in comparison with doxorubicin alone treatment. All results indicated that DMC had reversal effects on the multidrug resistance phenotype.

Inhibition of liver cancer cell proliferation and migration by a combination of (-)-epigallocatechin-3-gallate and ascorbic acid.

A mixture of (-)-epigallocatechin-3-gallate (EGCG) and ascorbic acid exhibited 73.2% inhibition of SMMC-7721 cell proliferation in a soft agar colony formation assay, which was much higher than EGCG (40.4%) or ascorbic acid (12.4%) alone. In the cell migration assay, the mixture also significantly suppressed the migration of SMMC-7721 cells by 65.9% while EGCG and/or ascorbic acid did by 28.9% and 18.7%, respectively. Ascorbic acid was able to enhance the antioxidant activity of EGCG by decreasing the intracellular oxidative stress according to fluorographic analysis of oxidative stress. In conclusion, the combination of EGCG and ascorbic acid can strongly suppress the proliferation and metastasis of liver cancer cells, possibly with a mechanism associated with the scavenging of reactive oxygen species. All these events add to our knowledge of liver cancer chemotherapy.

[Studies on synthesis of glutathione by E. coli BL21 (pTrc-gsh) coupled with Saccharomyces cerevisiae].

During synthesis of GSH by the engineered strain E. coli BL21(pTrc-gsh) coupled with Saccharomyces cerevisiae producing ATP from adenosin, the inconsistency of two systems in the concentration of phosphate buffer was solved by decreasing concentration to 250 mmol/L. The conditions under 250 mmol/L phosphate buffer were optimized and the yield of GSH was 1.6 g/L, which was higher than that of summation by two systems under the same conditions respectively. Addition of glycine later after glutamate and cysteine weakened the inhibition of GSH to GSHI. It made the yield of GSH reach to 2.13 g/L which was 30.7% higher than the control.

[Studies on lipase production from Candida rugosa].

We studied some factors affecting the lipase production from candida rugosa, they mainly included medium compositions and culture condition. The result showed that the optimal medium compositions for lipase production are 0.1% glucose 4.0% olive oil (carbon source), 0.3% NH4NO3(nitrogen source), 1.2% K2HPO4 and 0.4% MgSO4.7H2O. And the optimal culture condition is initial pH6.5, temperature 30 degrees C, agitation 180 r/min and time 60 h. As a result, and the lipase activity could reach 19.5 u/mL. Meanwhile we found that the surfactant could be helpful to the lipase production, and the optimal surfactant concentration was 0.03% GPE. The lipase activity was improved by more than 170% after we optimized the medium compositions and culture condition. While in a 5L fermentator, the lipase activity of fermentation broth could reach 33.5 u/mL within 48 hours.

[Cloning and expression of the genes of glutathione synthetases].

The genes(gsh-I,gsh-II) for gamma-glutamyl-cysteine synthetase(GSH-I) and glutathione synthetase(GSH-II) from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively. The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc-gsh. E. coli BL21 was transformed by pTrc-gsh for expression of the related enzymes. Analysis of SDS-PAGE showed that the expected products were expressed. E. coli BL21(pTrc-gsh) were incubated at 37 degrees C and pH 7.2 to OD550 = 0.5. The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/L IPTG. The expressed products were up to 25% of the total protein of the bacteria. Acetone-treated cells of the engineered strain could synthesize GSH efficiently.

[Study on continuous synthesis of galacto-oligosaccharide by immobilized Bacillus stearothermophilus].

The galacto-oligosaccharide was synthesized continuously by immobilized Bacillus stearothermophilu producing beta-galactosidase in fibrous bed reactor. The effect of substrate concentration, pH, reaction temperature and retention time on production of GOS was investigated. The optimal reaction conditions were determined. Substrate concentration were 450 g/L; Reaction temperature was 55 degrees C; pH was 7.0; Residence time was 100 min. The product yield reached up to 50.7%. GOS synthesis was promoted by feeding 1.5% D-glactose after 24 h. The immobilized cell reactor can work stably for 120 h.

Correlations of blood lipids and lipoproteins with coronary artery disease documented by angiography.

A study of the relationships between serum TC, TG and HDL.C and selective coronary arteriography was carried out on 117 patients who were divided into groups according to the extent of artery stenosis. Normal control group consisted of 153 healthy subjects. There were no statistical differences in TC, TG, HDL.C, LDL.C, TC/HDL.C, LDL.C/HDL.C and TC-HDL.C/HDL.C between normal control group and the group with normal coronary arteriogram. LDL.C, TC/HDL.C, LDL.C/HDL.C, TC HDL.C/HDL.C rose and HDL.C decreased as the degree of coronary artery stenosis and the extent of stenosis increased, besides the medium and severe stenosis group. Analyses based on the correlation coefficients indicate that 3 compound indexes (TC-HDL.C/HDL.C, TC-HDL.C, LDL.C/HDL.C) are better in assessing CAD than single index such as LDL.C, HDL.C and TC. The results of our study showed that the 3 compound indexes might be regarded as important risk factors for CAD.