皮肤细胞复合改性聚乳酸构建组织工程皮肤

目的:探讨以改性聚乳酸为细胞外基质网架构建组织工程皮肤的可行性。方法:采用盐溶法制备机械性能得到部分改进的聚乳酸多孔泡沫网架,向改进的聚乳酸网架接种真皮成纤维细胞和表皮角质形成细胞,以普通聚乳酸支架作为对照,构建组织工程皮肤。体外培养一周,对网架进行形态学观察。主要观察指标:①一般形态观察②组织学观察。结果:复层组织工程皮肤在结构上与正常皮肤相似,具有真皮、表皮双层结构。改性聚乳酸网架上有双层细胞生长,生长的细胞与网架接触,并且在其表面形成较为明显而连续的细胞层。随着培养时间的延长,发生了一系列变化:表皮部分细胞层数逐渐增多,真皮部分细胞也逐渐增多,并向表皮层深入,位于表皮与网架之间。结论:双醛淀粉作为良好的增柔剂在改善聚乳酸网架的机械性能的同时,也具有良好的细胞相容性,不影响细胞的生长增殖和代谢,可以进一步用作组织工程皮肤的支架材料。     

辽宁碱蓬胆碱单加氧酶(CMO)基因启动子分离及功能元件分析

根据已知的辽宁碱蓬CMO cDNA 5′端序列设计两个基因特异的反向引物(CR1,CR2),通过衔接头PCR获得了CMO基因起始密码子上游498 bp的序列。根据所获得的序列设计两个基因特异的反向引物(CR3,CR4),用CR2、CR3、CR4分别与4个简并引物配对,通过TAIL-PCR扩增,获得了约2 kb的序列。经Sequencer软件拼接上述两段序列,获得了CMO基因起始密码子上游2,332 bp的序列。用TSSP-TCM软件分析此序列,预测出转录起始点(C)位于起始密码子上游128 bp处,由此我们获得了2,204 bp的SlCMO启动子序列。用PLACE软件分析此序列,发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如盐诱导元件GAAAAA,冷胁迫诱导元件CANNTG,ABA 响应因子NAACAA,水胁迫元件CGGTTG和伤害诱导元件GTTAGGTTC等,是一个强的胁迫诱导启动子。辽宁碱蓬胆碱单加氧酶基因盐诱导启动子的获得,为盐诱导启动子功能元件分析提供了可能,为进一步研究启动子结构与功能的相互关系、CMO基因的表达调控机制奠定了基础。     

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of D-amphetamine and diphenhydramine in beagle dog plasma and its application to a pharmacokinetic study

A new drug, quick-acting anti-motion capsule (QAAMC) composed of d-amphetamine sulfate, dimenhydrinate and ginger extraction has been studied for anti-motion-sickness use. We have developed a sensitive, specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative determination of d-amphetamine and diphenhydramine, the main effective components of the QAAMC, using pseudoephedrine as the internal standard. The analytes and internal standard were isolated from 200 microL plasma samples by a simple liquid-liquid extraction (LLE). Reverse-phase HPLC separation was accomplished on a Zorbax SB-C18 column (100 mm x 3.0 mm, 3.5 microm) with a mobile phase composed of methanol-water-formic acid (65:35:0.5, v/v/v) at a flow rate of 0.2 mL/min. The method had a chromatographic total run time of 5 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 136.0-->91.0 (D-amphetamine), 256.0-->167.0 (diphenhydramine) and 166.1-->148.0 (IS) used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.5 ng/mL for d-amphetamine and 1 ng/mL for diphenhydramine, with good linearity in the range 0.5-200 ng/mL for D-amphetamine and 1-500 ng/mL for diphenhydramine (r(2)> or =0.9990). All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic study of the QAAMC in beagle dogs.     

Labdane diterpenoid glycosides from Aster veitchianus

Four new labdane-type rhamnopyranosides derived from 13-epimanool, compounds 1-4, with differently acetylated sugar moieties, were isolated from A. veitchianus. Their structures and absolute configurations were elucidated by chemical transformation, spectroscopic and mass-spectrometric analyses (IR, 1D- and 2D-NMR, HR-ESI-MS), as well as by single-crystal X-ray diffraction (compound 1). The isolates 2-4 were investigated for their cytotoxic properties against cultured human hepatoma (SMMC-7721), ovarian neoplasm (HO-8910), and leukemia (HL-60) cells, and for their antibacterial activities against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus.     

Plasmodium strain determines dendritic cell function essential for survival from malaria

The severity of malaria can range from asymptomatic to lethal infections involving severe anaemia and cerebral disease. However, the molecular and cellular factors responsible for these differences in disease severity are poorly understood. Identifying the factors that mediate virulence will contribute to developing antiparasitic immune responses. Since immunity is initiated by dendritic cells (DCs), we compared their phenotype and function following infection with either a nonlethal or lethal strain of the rodent parasite, Plasmodium yoelii, to identify their contribution to disease severity. DCs from nonlethal infections were fully functional and capable of secreting cytokines and stimulating T cells. In contrast, DCs from lethal infections were not functional. We then transferred DCs from mice with nonlethal infections to mice given lethal infections and showed that these DCs mediated control of parasitemia and survival. IL-12 was necessary for survival. To our knowledge, our studies have shown for the first time that during a malaria infection, DC function is essential for survival. More importantly, the functions of these DCs are determined by the strain of parasite. Our studies may explain, in part, why natural malaria infections may have different outcomes.     

High prevalence of NTDs in Shanxi Province: a combined epidemiological approach

BACKGROUND: Shanxi Province has historically reported a high prevalence of NTDs. In order to establish baseline rates for NTDs and discuss the risk factors associated with sociodemographic, maternal characteristics, and geographic factors, we performed the present study using an approach combining population and hospital-based methodologies. METHODS: We used chi(2) and Fisher's exact tests to evaluate variation in the prevalence by selected covariates and computed crude ORs and 95% CIs. Adjusted odds ratios (AORs) were performed using logistic regression with all the covariates included in the model. RESULTS: The overall NTD prevalence during the 3 year study period was 199.38 per 10,000 births, with a higher NTD prevalence clustered in 46 villages within this geographic area. However, no statistical significance was found between NTD prevalence and the elevation of the villages or their distance from coal plants. AORs revealed women aged 20 and above had a lower risk of NTDs compared to those younger than 20 (AOR range 0.4-0.5). A higher risk of NTDs was observed among female infants (AOR 1.50; 95% CI: 1.04-2.17), women with four or more previous births (AOR 2.80; 95% CI: 1.20-6.52), and a previous history of birth defects (AOR 3.23; 95% CI: 1.46-7.12). CONCLUSIONS: This study has documented a high prevalence of NTDs in Shanxi. Similar variations to other reports were found in the risk of NTDs by maternal demographic characteristics and a clustering of NTDs in certain villages that require further exploration.     

Dissecting metal ion-dependent folding and catalysis of a single DNAzyme

Protein metalloenzymes use various modes for functions for which metal-dependent global conformational change is required in some cases but not in others. In contrast, most ribozymes require a global folding that almost always precedes enzyme reactions. Herein we studied metal-dependent folding and cleavage activity of the 8-17 DNAzyme using single-molecule fluorescence resonance energy transfer. Addition of Zn2+ and Mg2+ induced folding of the DNAzyme into a more compact structure followed by a cleavage reaction, which suggests that the DNAzyme may require metal-dependent global folding for activation. In the presence of Pb2+, however, the cleavage reaction occurred without a precedent folding step, which suggests that the DNAzyme may be prearranged to accept Pb2+ for the activity. Neither ligation reaction of the cleaved substrates nor dynamic changes between folded and unfolded states was observed. These features may contribute to the unusually fast Pb2+-dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use.     

An unconventionally secreted effector from the root knot nematode Meloidogyne incognita,Mi-ISC-1, promotes parasitism by disrupting salicylic acid biosynthesis in host plants

Plant-parasitic nematodes need to deliver effectors that suppress host immunity for successful parasitism. We have characterized a novel isochorismatase effector from the root-knot nematode Meloidogyne incognita, named Mi-ISC-1. The Mi-isc-1 gene is expressed in the subventral oesophageal glands and is up-regulated in parasitic-stage juveniles. Tobacco rattle virus-induced gene silencing targeting Mi-isc-1 attenuated M. incognita parasitism. Enzyme activity assays confirmed that Mi-ISC-1 can catalyse hydrolysis of isochorismate into 2,3-dihydro-2,3-dihydroxybenzoate in vitro. Although Mi-ISC-1 lacks a classical signal peptide for secretion at its N-terminus, a yeast invertase secretion assay showed that this protein can be secreted from eukaryotic cells. However, the subcellular localization and plasmolysis assay revealed that the unconventional secretory signal present on the Mi-ISC-1 is not recognized by the plant secretory pathway and that the effector was localized within the cytoplasm of plant cells, but not apoplast, when transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Ectopic expression of Mi-ISC-1 in N. benthamiana reduced expression of the PR1 gene and levels of salicylic acid (SA), and promoted infection by Phytophthora capsici. The cytoplasmic localization of Mi-ISC-1 is required for its function. Moreover, Mi-ISC-1 suppresses the production of SA following the reconstitution of the de novo SA biosynthesis via the isochorismate pathway in the cytoplasm of N. benthamiana leaves. These results demonstrate that M. incognita deploys a functional isochorismatase that suppresses SA-mediated plant defences by disrupting the isochorismate synthase pathway for SA biosynthesis to promote parasitism.     

Metagenomic insights into the environmental adaptation and metabolism of Candidatus Haloplasmatales,one archaeal order thriving in saline lakes

The KTK 4A-related Thermoplasmata thrives in the sediment of saline lakes; however, systematic research on its taxonomy, environmental adaptation and metabolism is lacking. Here, we detected this abundant lineage in the sediment of five artificially separated ponds (salinity 7.0%–33.0%) within a Chinese soda-saline lake using culture-independent metagenomics and archaeal 16S rRNA gene amplicons. The phylogenies based on the 16S rRNA gene, and 122 archaeal ubiquitous single-copy proteins and genome-level identity analyses among the metagenome-assembled genomes demonstrate this lineage forming a novel order, Candidatus Haloplasmatales, comprising four genera affiliated with the identical family. Isoelectric point profiles of predicted proteomes suggest that most members adopt the energetically favourable ‘salt-in’ strategy. Functional prediction indicates the lithoheterotrophic nature with the versatile metabolic potentials for carbohydrate and organic acids as well as carbon monoxide and hydrogen utilization. Additionally, hydrogenase genes hdrABC-mvhADG are linked with incomplete reductive citrate cycle genes in the genomes, suggesting their functional connection. Comparison with the coupling of HdrABC-MvhADG and methanogenesis pathway provides new insights into the compatibility of laterally acquired methanogenesis with energy metabolism in the related order Methanomassiliicoccales. Globally, our research sheds light on the taxonomy, environmental adaptative mechanisms, metabolic potentials and evolutional significance of Ca. Haloplasmatales.