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A Practical and Efficient Method for The Retrieval of Ancient DNA Sequence

Ranran LIU ; Jing YUAN ; Xingbo ZHAO ; Ning LI

Progress in Biochemistry and Biophysics.2009;36(11):1495-1502. doi:10.3724/SP.J.1206.2009.00133

Retrieval of ancient DNA (aDNA) sequences from organism remains provide direct view of their evolutionary history. However, researches on aDNA have suffered from lots of technical problems. Specifically, discredited sequences were generated from damaged aDNA templates, and expensive and time-consuming methods were employed. Here, a method which could recover the endogenous aDNA as well as to reduce the cost and research period is described. This is achieved by improving the ancient DNA extraction method of isopropanol precipitation, and reevaluating the method of PCR after N-glycosylase (UNG) treatment, which could remove the damaged DNA from the aDNA extract. The efficiency of these methods were tested by comparing with traditional methods using ancient specimens of pig teeth aged between 4 300 years before present (BP) and 3 900 BP. The results showed that: firstly, the extraction efficiency of the improved method of isopropanol precipitation and current method with silica-based spin column were all 60%. Furthermore, the research period at least could be reduced by half with the application of the improved methods and the cost to 1/10 of the current method. Secondly, sequences obtained through the method of PCR after UNG treatment were 100% authentic. In contrast, 66%～ 88% sequences were authentic based on the results obtained with the method of multiple PCRs without UNG treatment. And the research cost and period needed by the method with UNG treatment were only half of the later one. These results demonstrate that the improved extraction method of isopropanol precipitation combined with the method of PCR after UNG treatment could increase the success rate of authentic DNA amplified and at least reduce the research cost and period by half. Therefore, this method can be applied in the large-scale detection of ancient specimens.

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Fast and Almost 100% Efficiency Site-directed Mutagenesis by The Megaprimer PCR Method

Zhenhua XIE ; Xiaojun SHI

Progress in Biochemistry and Biophysics.2009;36(11):1490-1494. doi:10.3724/SP.J.1206.2009.00139

A novel PCR-based mutagenesis method was reported, in which there is no need to purify megaprimers or design a special flanking primer. This method used one mutagenic primer and two sequencing primers (T_m≤58℃) as flanking primers. After first round PCR, 12.5 μl first PCR production was directly added into 50 μl second PCR system as template and megaprimer, and 10 rounds of asymmetrical PCR at high temperature of annealing (68 ℃ ) was to add in initiation of second PCR. This additional step greatly has increased the efficiency of mutagenesis via 600 bp or 800 bp long megaprimer. The results demonstrated that this method can achieve high fidelity, 97%～ 98% efficiency, high yield.

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Antibacterial Peptides of The Ovine Reproductive Tract

Chen CHEN ; Xinhua WANG ; Xinwen BO

Progress in Biochemistry and Biophysics.2009;36(11):1483-1489. doi:10.3724/SP.J.1206.2009.00011

The female sheep reproductive tracts were freshly collected from a local meat processing plant and used as experimental materials. Two antibacterial peptides were isolated and characterized from female sheep reproductive tracts by two consecutive chromatographic steps. The peptide isolation procedures included acetic acid extraction, dialyzed, gel filtration chromatography on Sephadex G-50, and reverse phase high-performance liquid chromatography (RP-HPLC). Their molecular mass were 4 820.47 u and 4 012.5 u, respectively, analyzed by MALDI-TOF-MS. The partial N-terminal amino acid sequences of two peptides were determined as AYVLDEPKP and YDSGA, respectively, by Edman degradation. The antimicrobial activity was tested during each purification step by the radial diffusion plate assay and broth microdilution method. These two peptides showed good antimicrobial activities against reference strains of G~+(S. Aureus ATCC2592 and Streptococcu ATCC55121), G~-(E. Coli ATCC25922) and fungi(C. Albicans ATCC2002). The peptides did not show active hemolytic activity against rabbit blood red cells and had no significant effects on human blood coagulation system. The discovery of antibacterial peptides in sheep reproductive system reveals that antibacterial peptides may play a role in innate immunity against microorganisms in a wide range of animal species.

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Suppression of ABCE1 Leads to Decreased Cell Proliferation and Increased Apoptosis in 95-D/NCI-H446 Lung Carcinoma Cells

Maogen ZHENG ; Ying GAO ; Bo HUANG ; Dali TIAN ; Chunlu YANG

Progress in Biochemistry and Biophysics.2009;36(11):1475-1482. doi:10.3724/SP.J.1206.2009.00223

ATP-binding cassette protein E (ABCE1) has been annotated as an Rnase L inhibitor in eukaryotes. Previous study showed that the overexpression of ABCE1 was related with the occurrence and clinical stage of lung adenocarcinoma. As an initial investigation into the novel functions of ABCE1, siRNA-expressing vectors targeting sites of the ABCE1 gene were constructed from RNAi-Ready pSIREN-DNR-DsRed-Express vector. Cultured 95-D and NCI-H446 lung carcinoma cells were transfected with the siRNA-expressing vectors using FuGENE 6 and transfection efficiency was determined by using fluorescence microscopy. The expression level of ABCE1 protein was determined by Western blot and immunofluorescence staining. Cell viability was determined by MTT, cell cycle was analysed by flow cytometry.The apoptotic rate was observed by ELISA. Fluorescence microscopy showed a satisfactory transfection efficiency which was about 42.70%. Cell viability and the growth fraction were markedly suppressed, whereas the apoptosis was significantly increased in SiRNA-95-D and SiRNA-NCI-H446 cells than controls(P< 0.05). It can be concluded that the siRNA targeting ABCE1 gene shows a dramatic inhibitory effect on RNA transcription and protein expression and a promoting effect on the apoptosis in 95-D/NCI-H446 cells, which offers a reliable base for the further in vivo experiment.

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The Effects of BHC80 Down Regulation on Embryonic Erythropoiesis in Zebrafish

Jiayun HOU ; Qiu JIANG ; Jie ZHANG ; Li TAN ; Houyan SONG

Progress in Biochemistry and Biophysics.2009;36(11):1469-1474. doi:10.3724/SP.J.1206.2009.00301

Whole mount in situ hybridization with BHC80 RNA probe showed that BHC80 was expressed in zebrafish central nervous system. Morpholino-modified antisense oligonucleotide was injected into zebrafish zygotes to knock down BHC80 expression. BHC80 knockdown resulted in striking decrease of erythrocytes and accumulation of erythrocytes at PBI. Further investigation of embryonic erythrocytes marker βe3 globin and hematopoiesis transcription factors gata1, c-myb and lmo2 by in situ hybridization showed that the erythroid progenitors marked with gata1 in BHC80 knockdown embryos were high proliferation and their differentiation were delayed, which led to decrease of erythrocytes and accumulation of erythrocytes at PBI. Both in situ hybridization and microangiography indicated that vasculature pattern of BHC80 knockdown embryos were almost normal.

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Aqueous Polymer Two-phase Partition for The Proteomic Analysis of Plasma Membrane From Rat Dorsal Root Ganglion Neurons

Xia XIONG ; Jianying SHEN ; Jianjun LI ; Hai ZHANG ; Xianchun WANG ; Songping LIANG

Progress in Biochemistry and Biophysics.2009;36(11):1458-1468. doi:10.3724/SP.J.1206.2009.00276

Dorsal root ganglion (DRG) neurons are primary sensory neurons that conduct neuronal impulses related to pain, touch and temperature senses. To comprehensively identify proteins of plasma membrane (PM) from small amount of dorsal root ganglion (DRG) neurons, a proteomics strategy that utilizes aqueous polymer two-phase partition in combination with differential velocity centrifugation was adopted to enrich the PM, followed by SDS-PAGE, CapLC-MS/MS and bioinformatics analysis. Western blot analysis showed that the concentration of PM in purified plasma membrane(PPM) was 2.3 times higher than that in crude plasma membrane(CPM), 15 times higher than that in whole tissue lysate (WTL). By searching against the rat IPI protein sequence database, a total of 729 non-redundant proteins were identified from the PM preparation, of which 547 had a gene ontology (GO) annotation indicating a cellular component, and 159 (21.8%) were unambiguously identified as PM proteins. A data set of plasma membrane proteins of DRG as well as a tool to study PM proteins were provided in a small amounts of sample.

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Using FRET to Study The Interaction Domain of TLR4 Binding to MD-2 in Living Cells

Tianyu ZHONG ; Jing TANG ; Dengyu CHEN ; Yawei LIU ; Wei WANG ; Jinghua LIU ; Yong JIANG

Progress in Biochemistry and Biophysics.2009;36(11):1451-1457. doi:10.3724/SP.J.1206.2009.00246

TLR4-MD-2 complex plays a key role in LPS recognition and its signal transduction. These steps are the vital elements of the host's defensive reaction. Studying the functional domain of TLR4 and MD-2 is very important to further understand the mechanism of LPS signal transduction. It was studied the interaction domain of TLR4 and MD-2 in living cells based on gene mutation, gene transfection and fluorescence resonance energy tramsfer(FRET) which is considered as one of the best methods used for intracellular protein-protein interaction study. CY-15P which was fused by CFP and YFP through 15 neutral amino acids was used as positive control, while co-expressed CFP and YFP proteins were used as negative control. The results showed that the ability of TLR4 binding to MD-2 decreased dramatically after the deletion of Glu~(24) ～ Met~(41) in N terminal of TLR4. Aggregation of TLR4 to LPS stimulation was observed, however, TLR4 without the Glu~(24)～ Met~(41) mutation did not aggregate. All these results indicated that TLR4 Glu~(24)～ Met~(41) might be the interaction domain of TLR4 binding to MD-2 and participate in the aggregation effect of TLR4 upon LPS stimulation.

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Perceptual Learning and Transfer Study of First-and Second-order Motion Direction Discrimination

Rong CHEN ; Zhuping QIU ; Yang ZHANG ; Yifeng ZHOU

Progress in Biochemistry and Biophysics.2009;36(11):1442-1450. doi:10.3724/SP.J.1206.2009.00224

In order to investigate the interactions between first- and second-order motion perception systems, 14 adult subjects with normal or correct to normal visual acuity were recruited and divided into two groups. Then these two groups were trained, in their parafovea, to discriminate the direction of first- and second-order motion gratings respectively. The spatial frequency of the gratings used in study was fixed at 2 cycles/degree and the temporal frequency was fixed at 8 Hz during training. Contrast sensitivity of subjects in these two training groups for first- and second- order motion was measured before and after 7 days' training to estimate the effects of training. In addition, the differences between the two training groups(14 subjects) and another control group (11 subjects) were studied. The results showed that: 1) the training with first-order motion gratings can improve subjects' performance in first-order motion direction discrimination but the improvement can't be transferred to the performance in second-order motion task; 2) the training with second-order motion gratings can improve subjects' performance in both first- and second-order motion direction discrimination tasks. In conclusion, an " asymmetric transfer" occurred between the training effect of first- and second-order motion gratings. These results indicate that there are two mechanisms for perceiving first- and second-order motion respectively. However, they are not completely different from each other but only partly separated.

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Variation of Monoamine Neurotransmitter in Forebrain of Presenilin-1/Presenilin-2 Double Knockout Mice

Dongli ZHANG ; Liyun LIANG ; Tingting JI ; Yiqun CHEN ; Tianshu ZHOU ; Bing MEI

Progress in Biochemistry and Biophysics.2009;36(11):1436-1441. doi:10.3724/SP.J.1206.2009.00196

Conditional forebrain-specific presenilin-1 and presenilin-2 double knockout mice (dKO mice) exhibit several neurodegenerative phenotypes of Alzheimer's disease (AD) pathology, such as tau hyperphosphorylation, neuron loss, forebrain cortical shrinkage and memory impairment. By using capillary electrophoresis assay, monoamine neurotransmitters in forebrain cortex, hippocampus and other forebrain region of dKO mice aged at 6, 9 and 12 months were measured to illustrate the relationship among presenilins function deficiency, neurodegenerative phenotypes and monoamine neurotransmitters. Data showed that levels of monoamine neurotransmitters in forebrain cortex of dKO mice were significantly decreased at 6 months when compared to controls, while as mice getting older, levels of monoamine neurotransmitters increased to that of controls, or even higher. In hippocampus, 5-hydroxytryptamin and epinephrine in dKO mice had a significant increase at 6 months, followed with a significant increase of each monoamine neurotransmitter at 12 months age. In other forebrain region, 5-hydroxytryptamin and dopamine had a similar level between control and dKO mice at 6 and 9 months but a significant decrease at 12 months; however, level of norepinephrine and epinephrine were significantly decreased at 6 and 12 months except epinephrine of 6 months. These results demonstrated that knockout of presenilins genes could lead to the variation of monoamine neurotransmitters, and the variation profiles were different among forebrain cortex, hippocampus and other forebrain region. However, whether presenilins deficiency caused the variation of monoamine neurotransmitter directly or not, and how about the effects of variation of monoamine neurotransmitters on AD-like pathology need to be further analyzed.

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In vitro Efficacy of mda-7 Gene for Hepatocellular Carcinoma Gene Therapy Mediated by Human Ribosomal DNA Targeting Vector

Jinfeng XUE ; Xionghao LIU ; Qiang HE ; Zhigang XUE ; Youjin HI ; Zhuo LI ; Junlin YANG ; Ting GAO ; Qian PAN ; Zhigao LONG ; Lingqian WU ; Kun XIA ; Desheng LIANG ; Jiahui XIA

Progress in Biochemistry and Biophysics.2009;36(11):1429-1435. doi:10.3724/SP.J.1206.2009.00255

Human ribosomal DNA (hrDNA) targeting vector pHr is a homologous recombinant plasmid for human genome which developed in the State Key Laboratory of Medical Genetics. pHr was used to construct a recombinant plasmid pHr-CMG expressing mda-7/GFP fusion gene and its efficacy in the hepatocellular carcinoma cell line Bel-7402 was investigated. The expression of mda-7/GFP fusion gene was detected by fluorescent microscope, RT-PCR and Western blotting, and its function was detected by cell-cycle analyses, MTT assay and Hoechst33258 staining. The results demonstrated that pHr-CMG vector could express MDA-7/GFP fusion protein effectually and the mda-7 gene could induce cell apoptosis and proliferation suppression in Bel-7402 cell line, which might be caused by the G2/M cell cycle arrest. These results also suggested that human ribosomal DNA targeting vector system and the pHr-CMG vector may be applied in further gene therapy researches for hepatocellular carcinoma.

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