Brain contusion as the main risk factor of memory or emotional complaints in chronic complicated mild traumatic brain injury.

OBJECTIVE: To investigate the risk factors for memory or emotional complaints in patients with complicated mild traumatic brain injury (mTBI). METHODS: Retrospective analysis of medical records was conducted by physicians in a teaching hospital in Southern Taiwan, and complicated mTBI had been identified by means of computed tomography. Psychological complaints, including problems with memory and emotions, were collected by structured telephone interviews, 10-15 minutes long, and were held with subjects who agreed to participate in our study. Among 327 patients who were injured for more than two years, 190 agreed to join this study (mean age: 41.6 years; male: 60.5%; stably employed: 50.0%). We used demographic data and neurological factors to predict memory or emotional complaints without muscle power or response speed (MEMR) complaints. RESULTS: Only the presence or absence of cerebral contusions predicted memory or emotional complaints without MEMR complaints in different employed status, and the odds ratio was 4.82-13.50 times higher for those with cerebral contusions than for those without. CONCLUSIONS: Cerebral contusions were the primary risk factor for MEMR complaints in chronic complicated mTBI. Early preventive psychological intervention might be necessary for patients with complicated mTBI and cerebral contusions.

Cloning and Optimized Expression of Human Angiogenin in E.coli.

Angiogenin(ANG) is an important factor of angiogenesis during different stage of tumor development and exists widely in various tumors. To study the biological funcption and find the antagonistic drugs of angiogenin, the angiogenin was allowed to be expressed by E.coli. By the aid of computer, the sequence around the start codon of angiogenin gene was modified according to local secondary structure. The modified human ang gene was amplified by reverse-transcription polymerase chain reaction from the human lung cancer cell line A549, and inserted into the prokaryotic expression vector pLDH99. After screening, high expression recombinants were obtained, and the expression level of the hANG was about 30% of total bacteria protein by SDS-PAGE. Biological assays indicated that the rhANG could induce new blood vessel formation in CAM in vitro. Our data showed that the recombinant hANG was active and the optimized expression of ang gene was practicable.

[Soluble expression, purification and bioactivity of recombinant human Era protein].

AIM: To prepare a soluble human Era(hEra) protein and to measure its bioactivity. METHODS: Human era cDNA gene from pUC19 plasmid was subcloned into the expression plasmid pMAL-p2x. pMAL-hEra was transducted to E.coli TB1 and the strain was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). RESULTS: The expressed MBP-fused protein existed in a soluble form. The fused protein made up 23.9% of the total cell lysate. It was purified by amylose affinity chromotography and digested with Factor X. Although the fused segment was dissected, the remained hEra protein was unstable in the solution with the passage of time. The activity assay showed that hEra was a GTPase that could bind GTP and hydrolyze GTP to GDP. CONCLUSION: Human Era protein can be expressed in a soluble form and it has been proved to be a kind of G protein by the experiments in vitro. The study is important to further research into the function of human era gene.

[Construction of shRNA expression vector pWH1 and its function in silencing the HIF1 gene].

AIM: To construct the expression vector of small hairpin RNA(shRNA) and to test its efficacy in silencing the Hypoxia-inducible Factor-1 (HIF1) gene. METHODS: The H1 gene promoter was amplified from the genome of the human blood cells by PCR. Then the promoter was cloned into the pEGFP-C1 vector digested with the restriction enzyme. The constructed vector was named pWH1. The primer was designed to target the human HIF1 cDNA gene. The annealed primer fragment was cloned into pWH1. The new constructed plasmid was transfected into the SGC7901 cell line. Then the expression level of HIF1 gene was assayed by RT-PCR and Western blot. RESULTS: The newly constructed plasmid expressed shRNA to target the HIF1 gene.The results of RT-PCR and Western blot showed the expression of HIF1 gene was reduced dramatically in mRNA and at protein level. CONCLUSION: The successful construction of shRNA expression vector (pWH1) provides a tool for further research into the function of a novel gene.

[Expression of mouse lipopolysaccharide response protein LRP in E. coli and preparation of rabbit anti-mLRP serum].

AIM: To express mouse lipopolysaccharide response protein (mLRP) and prepare rabbit anti-mLRP serum. METHODS: The predicted mouse lrp cDNA sequence was obtained by splicing homologous ESTs by comparing human lrp cDNA with mouse ESTs. Then the primers were designed. mlrp cDNA from NIH3T3 cells stimulated with lipopolysaccharide (LPS) was amplified by RT-PCR and was cloned into prokaryotic expression vector pTAT to construct recombinant expression vector pTAT-mlrp. The His-TAT-mLRP fusion protein was expressed in E. coli BL21(DE3) and was used to immunize the rabbits to get rabbit anti-mLRP serum. The anti-serum was purified by the acetone precipitation method. The specificity of the rabbit anti-mLRP serum was determined by Western blot. RESULTS: The predicted length of mlrp cDNA was 1905 bp. The encoding region of the cloned mlrp cDNA, 1554 bp, was inserted into pTAT. The His-TAT-mLRP fusion protein was expressed successfully in E. coli. The rabbit anti-mLRP serum was prepared by immunizing the rabbit with mLRP protein. CONCLUSION: The successful expression of mLRP and the preparation of rabbit anti-mLRP serum lays the foundation for further study of the function of mLRP.

[Expression and purification of two kinds of alternative splicing mouse Era].

AIM: To express and purify two alternative splicing mouse Era proteins and detect whether anti-human Era antibody can be used in the study of mouse Era proteins. METHODS: Two fusion protein expression vectors, pMAL-meraW and pMAL-meraS, were constructed, then the MBP-mEra proteins were expressed in E. coli. The target proteins were purified by amylose affinity chromatography. The specificity of rabbit anti-human Era antibody to the proteins was identified by Western blot. RESULTS: The expressed MBP-mEraW and MBP-mEraS proteins constituted approximately 17% and 19% of the total bacterial proteins. The purity of the fused proteins was 67% and 61% respectively after amylose affinity chromatography. Rabbit anti-human Era antibody had high specificity to these two kinds of splicing mouse Era proteins. CONCLUSION: Two fusion mera genes could be expressed in E. coli by using gene recombination technique. The high specificity of rabbit anti-human Era antibody to the two splicing mouse ERA proteins indicates that this antibody can be used to study the function of these two kinds of splicing mouse Era.

[Expression of a candidate human Era binding protein A19 and the preparation of its polyclonal antibody].

AIM: To express a candidate hEra binding protein A19 in Escherichia coli and to prepare anti-A19 antibody. METHODS: A19 gene was amplified by PCR from the plasmid containing A19 gene and was cloned into the expression vector pGEX-4T3 which was then transformed into E.coli. The A19 protein was expressed under IPTG induction. Antiserum was prepared by immunizing rabbits with the expressed A19 protein. The titer and specificity of polyclonal antibody were detected by Western blot. RESULTS: The expressed A19 accounted for about 30.2% of total bacterial protein. The titer of the antiserum was about 1:4 000. Western blot analysis indicated that the antiserum had high specificity. CONCLUSION: A19 fusion protein was highly expressed. The specific anti-A19 antiserum was prepared successfully.

[Preparation of anti-Red antisera and its subcellular localization].

AIM: To prepare rabbit anti-Red antisera. METHODS: The bet, exo and gam genes of lambda phage were amplified by PCR from genomic DNA and cloned into the expression vector pDH2, respectively. Red proteins were induced to express at 42 degrees C. The expressed proteins were analyzed by PAGE and thin-layer scanning. The antisera were prepared by immunizing rabbits with the three Red proteins, respectively. The titers and specificities of the antisera were detected by Western blot. RESULTS: Beta, Exo and Gam proteins accounted for about 40.3%, 49.2% and 73.4% of total bacterial protein, respectively. The titers of the antisera were about 1:2,000. Western blot analysis indicated that the three antisera all had good specificities to the corresponding proteins. CONCLUSION: Specific anti-Red antisera are prepared successfully.

[High density fed-batch culture of Escherichia coli DH5 alpha/pDH-B2m with DO feed-back control of nutrient feeding].

Optimization of cultivation condition of recombinant E. coli DH5 alpha/pDH-B2m and the condition suitable for expression of recombinant mature peptide of human bone morphogenetic protein-2 was carried out in 500 mL shaking flasks and then transferred to NBS Bioflo IV, a 20 L DO feed-back fed-batch culture system, to obtain rhBMP-2. The results indicate that keeping dissolved oxygen at 40% and controlling nutrient feeding rate with DO feed back strategy can obtain theoretically 3.59 g recombinant mature peptide of hBMP-2 per liter of broth, the final cell density OD600 reaches 57(22.8 g dry cell weight/L), and the expression of rhBMP-2 amounts to 30% of the total protein in E. coli.

[The expression of truncated YggG protein and preparation of its polyclonal antibody].

AIM: To express truncated YggG protein (TYP) in Escherichia coli and to prepare anti-TYPE antibody. METHODS: Truncated yggg gene was amplified by PCR from the plasmid containing full length yggg gene and was cloned into the expression vector pDH2. The expression of TYP was achieved by thermal induction. Antiserum was prepared by immunizing rabbit with TYP,The titer and specificity of polyclonal antibody were detected by Western blot. RESULTS: Thin-layer scan analysis showed that TYP accounted for about 37.1% of total bacterial protein. The antiserum was about 1:2,000 in titier and highly specific. Full-length rggG protein could also be recognized by the antiserum. CONCLUSION: The TYP was highly expressed, and specific anti-TYP antiserum was prepared successfully.