A highly efficient approach to introduction of the single-base mutation in a plasmid containing adenovirus genome through Red recombination / 军事医学

Objective To construct a highly efficient approach to the introduction of the single-base mutation in a plasmid containing the adenovirus whole genome larger than 40 kb.Methods The target DNA with a mutation site was achieved by over-lapping PCR.The large plasmid with adenovirus genome and target DNA were co-transformed into Escherichia coli strain DY330 carrying a high rate Red recombination system.The positive clone was selected via colony PCR in combination with enzyme identification.The site-mutation large plasmid was transformed into E.coli strain DH10B in which the backbone of the large plasmid remained was stable.Results Two mutations were continuously introduced into the adenovirus genome,the location of which was pos.9171 and pos.24410 respectively.The integrality and stability of the plasmid backbone were verified by enzyme cutting identification.The two mutations on the plasmid were verified by DNA sequencing.Conclusion An efficient approach to the introduction of the single-base mutation in positions 9171 and 24410 from the adenovirus genome which was integrated into a plasmid is successfully established.The positive selection efficiency ranges from 5%to 15%.The construction of the approach will facilitate the study of adenovirus infection mechanism.

Effect of hyperbaric oxygen on the expression of nitric oxide synthase mRNA in cortex after acute traumatic cerebral injury / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of hyperbaric oxygen (HBO) treatment on the expression of nitric oxide synthase (NOS) mRNA in cortex after acute traumatic cerebral injury, and to study the mechanism of HBO on brain injury.</p><p><b>METHODS</b>Acute traumatic brain injury model was established with rest received free fall injury method in SD rats. 0.25 MPa HBO treatment was used 1 h or 12 h after brain injury and the cortex was isolated 6 h or 24 h after brain injury respectively. The expression of mRNA coding for nNOS, eNOS or iNOS were assayed using reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The expression of nNOS, eNOS and iNOS mRNA were significantly decreased in 0.25 MPa HBO treatment groups than those in acute cerebral injury groups (P < 0.01). The amount of nNOS, eNOS and iNOS mRNA was significantly lower in HBOT 24 h group than those in HBOT 6 h group (P < 0.05, P < 0.01). There was no significantly difference among nNOS, eNOS and iNOS mRNA in 0.25 MPa normoxic hyperbaric nitrogen groups and acute cerebral injury groups (P > 0.05).</p><p><b>CONCLUSION</b>HBO may exert significant effects on the expression of nNOS mRNA/iNOS mRNA and protect cortical neuronal from traumatic cerebral injury.</p>

Differential proteome analysis of conditioned medium of BPH-1 and LNCaP cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although the introduction of serum prostate-specific antigen (PSA) measurements into clinical practice has revolutionized the care of patients with prostate cancer, there are well-recognized limitations of PSA, and there is a critical need to identify additional prostate cancer biomarkers to assist in early detection and prognosis. In this regard, high resolution proteomic technology has the unexceptionable superiority to find those high abundance biomarkers. The purpose of this study was to search new tumor markers by proteomic technology.</p><p><b>METHODS</b>The proteins in conditioned medium (CM) of BPH-1 and LNCaP cells were profiled by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). The corresponding mRNA levels of some identified proteins were analyzed by RT-PCR.</p><p><b>RESULTS</b>Totally 11 differentially expressed proteins (6 up-regulated including creatine kinase, brain (CKB), triosephosphate isomerase 1 (TPI1), isocitrate dehydrogenase 2 (IDH2) and 5 down-regulated including glutathione S-transferase pi (GST-pi)) in the CM were identified using MALDI-TOF-MS and database search. The expression pattern between mRNA and CM protein levels of CKB, IDH2, TPI1 and GST-pi in BPH-1 and LNCaP was similar.</p><p><b>CONCLUSION</b>We proved a feasible and effective way to search new tumor markers by a proteomics-based strategy and identified 11 potentially useful proteins in CM of BPH-1 and LNCaP cells to distinguish prostate cancer from benign prostatic hypertrophy.</p>

Expression of high mobility group box chromosomal protein 1 in mice with lupus nephritis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the role of the novel proinflammatory cytokine high mobility group box chromosomal protein 1 (HMGB-1) in the pathogenesis of lupus nephritis.</p><p><b>METHODS</b>Serum levels of anti-dsDNA antibodies were determined by enzyme linked immunosorbent assay (ELISA). Renal morphologic features were examined by light microscopy, electron microscopy, and immunohistologic analyses. The mRNA expression of HMGB-1 and monocyte chemoattractant protein-1 (MCP-1) was detected by RT-PCR.</p><p><b>RESULT</b>MRL/lpr mice demonstrated characteristic alterations of serum immune parameters, with progressively increased anti-dsDNA antibodies with age, compared with age-matched C57BL/6J mice. MRL/lpr mice showed progressive development of renal damage, starting at 12 weeks of age and reached the peak at 20 weeks. The observed lesions included the presence of enlarged hypercellular glomeruli, with increased numbers of both resident cells and infiltrating leukocytes. Higher expression of HMGB-1 mRNA was found in MRL/lpr mice than what in C57BL/6J mice. Expression of HMGB-1 was positively correlated with that of MCP-1 mRNA.</p><p><b>CONCLUSION</b>The results demonstrate that the higher expression of HMGB-1 may contribute to the pathogenesis of lupus nephritis.</p>

Gene fusion of egfp & kan and recombinant plasmid construction by red mediated in vivo homologous recombination / 生物工程学报

Recombineering, a new genetic engineering technology based on high efficiency in vivo homologous recombination, can be used in target DNA knock-in, knock-out and gene cloning. In the process of gene subcloning mediated by Recombineering technique, high-quality target DNA fragments were difficult to obtain using in vitro overlapping PCR,therefore the efficiency of in vivo homologous recombination was severely interrupted. To solve this problem, some technology improvements have been established based on the principle of Red recombinases. The PCR DNA fragments of egfp and kan genes with complementary sequences on the end of each fragment were co-introduced into a pcDNA3.1 vector and Red recombinases containing E. coli DY331 host cells by electroporation. A recombinant plasmid pcDNA3.1-egfp-kan was screened directly by antibiotic marker. The positive rates can reach to 45%. The EGFP gene expression of pcDNA3.1-egfp-kan can be observed by transient transfection of 293 eukaryotic cells.

Role of HMGB1 in rheumatic diseases / 浙江大学学报·医学版

High mobility group box chromosomal protein 1 (HMGB1) is originally identified as a DNA-binding protein that functions as a structural co-factor. HMGB1 is actively secreted by macrophage/monocytes via inflammatory stimuli. The extracellular HMGB-1 acts as a mediator of acute and chronic systematic inflammation. In this article we briefly review its role in rheumatic diseases: arthritis, polymyositis and dermatomyositis, lupus erythematosus and Sjogren's syndrome. Increased cytoplasmic expression and extracellular deposition of HMGB1 are found in the affected tissues of those diseases, especially stronger in/around focal infiltrates of mononuclear cells. TNFalpha and IL-1beta are co-expressed in areas of extracellular HMGB1. HMGB1 together with TNF alpha and IL-1beta may form a proinflammatory loop promoting the chronic inflammations. The new findings of HMGB1 as a cytokine provide a better understanding of rheumatiod diseases, and could become a clinically relevant therapeutic target that might be more efficient than other known cytokines.

Anticancer effects of Chinese herbal medicine, science or myth? / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Currently there is considerable interest among oncologists to find anticancer drugs in Chinese herbal medicine (CHM). In the past, clinical data showed that some herbs possessed anticancer properties, but western scientists have doubted the scientific validity of CHM due to the lack of scientific evidence from their perspective. Recently there have been encouraging results, from a western perspective, in the cancer research field regarding the anticancer effects of CHM. Experiments showed that CHM played its anticancer role by inducing apoptosis and differentiation, enhancing the immune system, inhibiting angiogenesis, reversing multidrug resistance (MDR), etc. Clinical trials demonstrated that CHM could improve survival, increase tumor response, improve quality of life, or reduce chemotherapy toxicity, although much remained to be determined regarding the objective effects of CHM in human in the context of clinical trials. Interestingly, both laboratory experiments and clinical trials have demonstrated that when combined with chemotherapy, CHM could raise the efficacy level and lower toxic reactions. These facts raised the feasibility of the combination of herbal medicines and chemotherapy, although much remained to be investigated in this area.

Effect of polymorphism of human intestinal fatty acid binding protein gene on the therapeutic efficacy of fenofibrate / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the effect of polymorphism in codon Ala54Thr of human intestinal fatty acid-binding protein gene (IFABP) on the therapeutic efficacy of fenofibrate.</p><p><b>METHODS</b>Totally 147 patients with hyperlipidemia [72 men mean age: (56.2 +/- 8.63) years; 75 women mean age: (58.4 +/- 9.12) years] were enrolled. IFABP genotypes were detected by polymerase chain reaction, Hha I digestion, and sequencing. Four weeks before and after treatment, the levels of fasting serum total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), apolipoprotein A I (apoA I) and apolipoprotein B (apoB) were detected with biochemical techniques.</p><p><b>RESULTS</b>The frequency of IFABP genotype was 0.47 for A/A, 0.37 for A/T, and 0.16 for T/T, and the allelic frequency was 0.65 for A and 0.35 for T. No significant different was found in lipid levels in every genotype before treatment (P > 0.05). After 4 weeks of treatment, the levels of TC, TG, LDL-C, and apoB significantly decreased (P < 00.01), and the levels of HDH-C and apoA I significantly increased (P < 0.01). The total therapeutic efficacy on A54A and A54T were 97% and 95%, respectively. In the patients with T54T genotype after treatment, no significant difference in lipids levels was found except TG (P < 0.05), and the total efficacy was only 38%. The total therapeutic efficacies of fenofibrate on A54A and A54T were higher than those of T54T, and there was significant different between A54A and T54T (P < 0.01).</p><p><b>CONCLUSION</b>The polymorphism of human IFABP gene in hyperlipidemia is related with the therapeutic efficacy of fenofibrate, and the T54T IFABP genotype may have strong negative effect on such efficacy.</p>

Construction of recombinant plasmid using Neo/E Technology / 生物工程学报

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.

Impact of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2 / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the effect of PC-1 gene knockdown on the biological action of prostate cancer cell line C4-2.</p><p><b>METHODS</b>Recombinant plasmids of expressing short hairpin RNA targeting PC-1 mRNA were constructed using DNA recombinant technology and transfected into C4-2 cells via liposome. The positive cell clones were selected by G418. The expression of PC-1 gene was analyzed by RT-PCR and Western blotting technology. MTT and soft agar cloning formation were applied to observe the changes of the growth rate and independent anchor ability of C4-2 cells.</p><p><b>RESULTS</b>PC-1 RNA interference severely affected the expression of PC-1 gene and reduced the growth and colony formation ability of C4-2 cells.</p><p><b>CONCLUSION</b>RNA interference-mediated PC-1 gene knockdown can decrease the growth and cloning formation ability of C4-2 cells.</p>

Malignant transformation of NIH3T3 cells induced by ectopic expression of PC-1 gene / 中华病理学杂志

<p><b>OBJECTIVE</b>To establish a mouse fibroblastic cell line stably transfected with PC-1 gene, and using such cell line to investigate tumor development and progression imposed by the ectopic expression of PC-1 gene.</p><p><b>METHODS</b>Eukaryotic expression vector pcDNA3.1(-)/myc-his-pc-1 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfectants were selected by G418. The integration and expression of ectopic PC-1 gene were analyzed by PCR and RT-PCR. Cytomorphological analysis, MTT, soft agar colony formation and nude mice tumorigenesis assay were used to evaluate the effects of PC-1 gene expression on tumor development and progression.</p><p><b>RESULTS</b>NIH 3T3 cell lines stably expressing PC-1 gene were successfully established and confirmed by PCR and RT-PCR analyses of the integration and expression of ectopic PC-1 gene. Comparing with the parental cell line and cells transfected with control vector, the PC-1 gene transfectants acquired several phenotypes of transformed cells: increasing growth rate, ability to grow and form cell colonies on soft agar, and becoming tumorigenic in nude mice.</p><p><b>CONCLUSION</b>Ectopic expression of PC-1 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in vivo.</p>

Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system / 生物工程学报

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.

Skin-like structure generated from implantation of hair follicle bulb cells into collagen/chitosan porous scaffolds in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To observe the skin regeneration after hair follicle bulb cells were implanted into collagen/chitosan porous scaffolds in vitro.</p><p><b>METHODS</b>The cultured dorsal hair follicle bulb cells of 4d-old C57BL/6J mice were implanted into collagen/chitosan porous scaffolds in vitro. The skin regeneration was observed.</p><p><b>RESULT</b>The skin-like structure was formed on the collagen/chitosan porous scaffolds where were cultured the hair follicle bulb cells before 4th passages.</p><p><b>CONCLUSION</b>The skin-like structure is generated in vitro when early passages of cultured hair bulb cells are implanted into collagen/chitosan porous scaffolds.</p>