Characterization of Two GAS1 Genes and Their Effects on Expression and Secretion of Heterologous Protein Xylanase B in Kluyveromyces lactis.

ß-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative ß-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of ß-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The ß-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

Peripheral nerve injury decreases the expression of metabolic glutamate receptor 7 in dorsal root ganglion neurons.

Group II and III metabolic glutamate receptors (mGluRs) are responsible for the glutamate-mediated postsynaptic excitation of neurons. Previous pharmacological evidences show that activation of mGluR7 could inhibit nociceptive reception. However, the distribution and expression patterns of mGluR7 after peripheral injury remain unclear. Herein we found that mGluR7 was expressed in the rat peptidergic dorsal root ganglion (DRG) neurons and large neurons, but rarely in isolectin B4 positive neurons. Sciatic nerve ligation experiment showed that mGluR7 was anterogradely transported from cell body to the peripheral site. Furthermore, after peripheral nerve injury, mGluR7 expression was down-regulated in both peptidergic and large DRG neurons. Our work suggests that mGluR7 might be involved in the regulation of pathological pain after peripheral nerve injury.

[Effects of Bushen Zhuangjin Decoction containing serum on the apoptosis of chondrocytes induced by mechanics stimulus].

OBJECTIVE: To study the effects of Bushen Zhuangjin Decoction (BZD) containing serum on the apoptosis of chondrocytes induced by mechanics stimulus. METHODS: The BZD containing serum was extracted. The chondrocyte nutritive media was divided into 3 groups, i.e., the common nutritive medium group, the blank rabbit serum medium group, and the BZD nutritive medium group. The apoptosis of chondrocytes was induced by continuing mechanics stimulus in 24 h. Then the chondrocytes were collected. The apoptosis rate of chondrocytes was determined by flow cytometry. The contents of interleukin 1beta (IL-1beta) and nitric oxide (NO) in the corresponding media were determined. RESULTS: The apoptosis of chondrocytes in the BZD nutritive medium group (19.55 +/- 7.98)% was lower than that of the common nutritive medium group (39.32 +/- 13.45)% and the blank rabbit serum medium group (37.87 +/- 9.67)%, showing statistical difference (P < 0.05). The contents of IL-1beta and NO were also lower in the BZD nutritive medium group with statistical difference when compared with those of the other two groups (P < 0.05). CONCLUSION: BZD containing serum could protect mechanics stimulus induced apoptosis of chondrocytes.

[Analysis of perioperation complications of total hip arthroplasty in treating Crowe type IV developmental dysplasia of the hip].

OBJECTIVE: To evaluate the clinical effects of total hip arthroplasty (THA) for Crowe type IV developmental dysplasia of the hip (DDH) and analyze perioperative complications. METHODS: From March 2000 to March 2010, 19 patients (23 hips, of them, 4 patients with bilateral hips) with Crowe type IV DDH underwent THA. There were 5 males and 14 females, with average age of 61.3 years (ranged, 41 to 72 years). All hips were treated with small acetabular components combined with medial protrusion technique in acetabular reconstruction, as well as subtrochanteric shortening osteotomy in femur. Joint function of hips were evaluated according to Harris scoring. RESULTS: All patients were followed up with an average of 4.2 years (ranged, 1 to 8 years). Postoperative X-ray films showed all acetabular prosthesis were in true acetabulum. No loosening and nonunion were found in all patients. Harris scoring improved from preoperative 34.0 +/- 6.9 to postoperative 85.0 +/- 7.5. Complications occurred in 11 cases in the patients, including femoral split fracture in 3 cases, nerve injury in 3 cases, delayed union in 2 cases, dislocation in 3 cases. CONCLUSION: Total hip arthroplasty using small acetabular component, medial protrusion, femoral subtrochanteric shortening osteotomy technique for the Crowe type IV DDH can effectively restore hip function and leg length. But incidence of complications is high. The long-term follow-up is necessary for further study.

Optimized medium improves expression and secretion of extremely thermostable bacterial xylanase, XynB, in Kluyveromyces lactis.

An extremely thermostable xylanase gene, xynB, from hyperthermophilic bacterium Thermotoga maritima MSB8 was successful expressed in Kluyveromyces lactis. Response surface methodology (RSM) was applied to optimize medium components for production of XynB secreted by the recombinant K. lactis. Secretion level (102 mg/L) and enzyme activity (49 U/ml) of XynB in the optimized medium (yeast extract, lactose, and urea; YLU) were much higher than those (56 mg/L, 16 U/ml) in original medium (yeast extract, lactose, and peptone; YLP). It was also observed that the secretory efficiency of mature XynB was improved by the YLU medium. mRNA levels of 13 characterized secretion-related genes between K. lactis cultured in YLP and YLU were detected using semi-quantitative RT-PCR method. It was found that unfolded protein response (UPR) related genes such as ero1, hac1, and kar2 were up-regulated in K. lactis cultured in YLU. Therefore, nutrient ingredient, especially nitrogen source had a significant influence on the XynB secretory efficiency in the host K. lactis.

[Surgical treatment and the fixator selection for Pilon fractures].

OBJECTIVE: To explore the internal fixation selection and the operative method of Pilon fracture. METHODS: From Aug. 2004 to Aug. 2007,there were 40 patients with Pilon fractures involving 30 males and 10 females. Thirty cases were closed fracture and 10 were open fracture. Seven patients were treated by emergency operation, 3 patients were debrided and suture at first. Twenty-three patients were treated with open reduction and internal fixation (ORIF),the other seventeen patients were treated with LC-DCP,reconstruct plate for fixation or screw, Kirschner wire combine screw for fixation. RESULTS: All patients were followed up for 6 to 32 months (means 19 months). The time of union of fracture was about 3 to 5 months. The outcome was evaluated according to Baired-Jackson criteria, the results were excellent in 27 cases,good in 7, fair in 5, poor in 1. Seven cases were found the operative incision and wound surface difficult to heal,among them 5 cases came from the emergency operation patients, 1 case was closed fracture patient. They were be healed by change of dressing or dermatoplasty or skin flap. CONCLUSION: It is the key factor of treatment for Pilon fractures to correct evaluate before operation and select correct operation time. It is also important to selected suitable internal fixation and good reduction.

[High-level expression of an extreme-thermostable xylanase B from Thermotoga maritima MSB8 in Escherichia coli and Pichia pastoris].

The family 10 xylanase (XynB) from Thermotoga maritima MSB8 is extremely thermostable and great potential in the applications of various fields of industry. The gene mxynB(64) was amplified by the method of PCR with the template of the genomic DNA of Thermotoga maritima MSB8, and cloned into the expression vectors of Escherichia coli and Pichia pastoris respectively. Xylanase B(40kD) was successfully expressed by the two heterologous protein expression systems with high-level production. The recombinant protein of XynB expressed in Pichia pastoris showed extreme thermostability and pH stability, which was optimally active at 90 degrees C and quite stable over the pH range of pH 5.0-10.8 at 70 degrees C. After incubation of the enzyme at 100 degrees C for 30 min, XynB retained 70% higher residual activity. The recombinant XynB expressed in Pichia pastoris is of great use in a variety of industrial and agricultural applications.

[Transcriptional differences between a heterokaryon and its segregants of Fusarium oxysporum f. sp. vasinfectum].

In order to elucidate the mechanism of fungal heterokayosis, a wild type strain of Fusarium oxysporum f. sp. vasinfectum was isolated from the cotton field in Anyang, Henan Province. Through single hyphal-tip isolation, a heterokaryon, Ag149, was obtained, and its two different phenotypic segregants, Ag149-I and Ag149-III, were separated from the mutated sectors on colony of the heterokaryon. They have remarkable differences on color of colony, morphology of hypha and pathogenicity. After analyzing by RAPD on their nuclear DNA with 100 random primers, no polymorphic difference was found among them. On going to find the different expressed gene, the mRNA differential display method was performed. Two kinds of reverse transcriptase AMV and MMLV, and a kit which consists of three kinds of 3' terminal anchor primers and eight kinds of 5' terminal arbitrary primers were used in differential display PCR (DD-PCR). Total RNA as template was reverse transcribed into corresponding cDNA by 3' terminal anchor primers, and the cDNA were amplified by polymerase chain reaction with a set of one same 3' anchor primer and one 5' arbitrary primer. The PCR products were then resolved on denaturing polyacylamimide gel, and the cDNA bands were visualized by silver staining. Among the 144 PCR products, 19 differentially expressed cDNA fragments ranged from 300 bp to 700 bp were purified. All of them were ligated to pGEM-T vector respectively for sequencing and Rev-Northern blotting. Two cDNA fragments (G5 and C6) were observed to be positive after Rev-Northern blotting. The C6 was highly expressed in the heterokaryon Ag149 and its segregant Ag149-I. It is 564 bp in length and can be predicted 77 amino acids from the beginning of the 3rd to 233th base, and then searched from GenBank. The amino acid sequence of C6 shared homologies with the 6th subunit of NADH dehydrogenase found in some bacteria, plants and animals at 30% - 70% level. While the G5 was highly expressed in Ag149 and its segregant Ag149-III. It is 432 bp in length and can be predicted 101 amino acids from the beginning of the 2nd to 304th base, and the amino acid sequence is 35% homologies comparing with the tetracycline efflux protein (OtrB) of Streptomyces rimosus. We also checked these two kinds of the pGEM-T vector harboring cDNA fragments (C6 and G5) by Southern blotting with their nuclear DNA and mitochondrial DNA (mtDNA) separately. The positive identification signals only appeared from nuclear DNA,and it addressed that C6 and G5 locate on their nuclear genome. The result indicated that the difference between the heterokaryon and its segregants is distinct on gene transcriptional level. Thus a molecular evidence for the formation of heterokaryon in filamentous fungi was provided.