多巴胺D2受体基因多态性位点与海洛因依赖的关系

目的:探讨多巴胺D2受体(Dopamine D2 receptors,DRD2)基因3'非翻译区Taq ⅠA、启动子区-141 Ins/Del 2个多态性位点和海洛因依赖的相关性.方法:采用聚合酶链反应-限制性片段长度多态(PCR-RFLP)技术检测320例海洛因依赖患者及300例健康对照组的TaqⅠA和-141 Ins/Del2个多态性位点的基因型.采用HaploView4.0及SPSS11.5软件分析这2个多态性位点的基因型、等位基因频率及组间差异.结果:DRD2基因Taq ⅠA位点的基因型及等位基因频率分布在海洛因依赖组与正常对照组存在显著性差异(p<0.01),海洛因依赖组TaqⅠA位点的等位基因A1频率显著高于正常对照组(x2=11.156,p=0.001,OR=1.463,95%CI=1.170～1.830);DRD2基因-141 Ins/Del位点的基因型及等位基因频率分布在海洛因依赖组与正常对照组之间无统计学差异(p<0.05).结论:DRD2基因TaqⅠA位点多态性可能与海洛因依赖有关,携带有TaqⅠA多态性位点A1等位基因的个体可能更容易对海洛因产生依赖.     

酵母酶系合成三磷酸鸟苷的研究

研究了利用啤酒酵母细胞酶系催化鸟苷酸(GMP)合成三磷酸鸟苷(GTP)的工艺过程。采用分式析因及响应面实验设计对其工艺条件进行优化,分析了温度、pH、酵母、无机盐和表面活性剂等因素对GTP积累的影响,得到了一个可以较好预测实际反应的二次方模型以及优化条件即：GMP 7．16g/L,葡萄糖55g/L,酵母270g/L,硫酸氨1．5g/L,硫酸镁2g/L,磷酸二氢钾27．2g/L,表面活性剂8川L,氯化十六烷基吡啶1．5g/L,pH6．74,温度37．4℃。经过优化,反应得率从71．3％增至92．7％,较国内外报道的水平(80％)提高了12．7％.     

Human activities and climate variability drive fast‐paced change across the world's estuarine–coastal ecosystems

Time series of environmental measurements are essential for detecting, measuring and understanding changes in the Earth system and its biological communities. Observational series have accumulated over the past 2–5 decades from measurements across the world's estuaries, bays, lagoons, inland seas and shelf waters influenced by runoff. We synthesize information contained in these time series to develop a global view of changes occurring in marine systems influenced by connectivity to land. Our review is organized around four themes: (i) human activities as drivers of change; (ii) variability of the climate system as a driver of change; (iii) successes, disappointments and challenges of managing change at the sea‐land interface; and (iv) discoveries made from observations over time. Multidecadal time series reveal that many of the world's estuarine–coastal ecosystems are in a continuing state of change, and the pace of change is faster than we could have imagined a decade ago. Some have been transformed into novel ecosystems with habitats, biogeochemistry and biological communities outside the natural range of variability. Change takes many forms including linear and nonlinear trends, abrupt state changes and oscillations. The challenge of managing change is daunting in the coastal zone where diverse human pressures are concentrated and intersect with different responses to climate variability over land and over ocean basins. The pace of change in estuarine–coastal ecosystems will likely accelerate as the human population and economies continue to grow and as global climate change accelerates. Wise stewardship of the resources upon which we depend is critically dependent upon a continuing flow of information from observations to measure, understand and anticipate future changes along the world's coastlines.     

Determination of Parameters for the Supercritical Extraction of Antioxidant Compounds from Green Propolis Using Carbon Dioxide and Ethanol as Co-Solvent

The aim of this study was to determine the best processing conditions to extract Brazilian green propolis using a supercritical extraction technology. For this purpose, the influence of different parameters was evaluated such as S/F (solvent mass in relation to solute mass), percentage of co-solvent (1 and 2% ethanol), temperature (40 and 50°C) and pressure (250, 350 and 400 bar) using supercritical carbon dioxide. The Global Yield Isotherms (GYIs) were obtained through the evaluation of the yield, and the chemical composition of the extracts was also obtained in relation to the total phenolic compounds, flavonoids, antioxidant activity and 3,5-diprenyl-4-hydroxicinnamic acid (Artepillin C) and acid 4-hydroxycinnamic (p-coumaric acid). The best results were identified at 50°C, 350 bar, 1% ethanol (co-solvent) and S/F of 110. These conditions, a content of 8.93±0.01 and 0.40±0.05 g/100 g of Artepillin C and p-coumaric acid, respectively, were identified indicating the efficiency of the extraction process. Despite of low yield of the process, the extracts obtained had high contents of relevant compounds, proving the viability of the process to obtain green propolis extracts with important biological applications due to the extracts composition.     

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.     

Waterborne Risperidone Decreases Stress Response in Zebrafish

The presence of drugs and their metabolites in surface waters and municipal effluents has been reported in several studies, but its impacts on aquatic organisms are not yet well understood. This study investigated the effects of acute exposure to the antipsychotic risperidone on the stress and behavioral responses in zebrafish. It became clear that intermediate concentration of risperidone inhibited the hypothalamic-pituitary-interrenal axis and displayed anxiolytic-like effects in zebrafish. The data presented here suggest that the presence of this antipsychotic in aquatic environments can alter neuroendocrine and behavior profiles in zebrafish.     

Ultrasound‐mediated synthesis of camphoric acid‐based chiral salens for the enantioselective trimethylsilylcyanation of aldehydes

New chiral salen ligands were prepared by the ultrasound‐irradiated condensation of optically active (1R, 3S)‐1,2,2‐trimethyl‐1,3‐diaminocyclopentane with aromatic 1‐hydroxyaldehydes. The ultrasound‐mediated process is more convenient due to shorter reaction times, energy economy, and easier isolation of the products. The in situ formed Ti(IV)(salen) complexes, evaluated as catalysts in the enantioselective trimethylsilylcyanation of benzaldehyde, were found to be efficient for this process, originating the corresponding product in high yields (72–99%) and selectivities of up to 79%. The lowest energy transition states were determined by computational studies. These results were in qualitative agreement with the experimentally observed ones. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Superoxide dismutases—a review of the metal-associated mechanistic variations

Superoxide dismutases are enzymes that function to catalytically convert superoxide radical to oxygen and hydrogen peroxide. These enzymes carry out catalysis at near diffusion controlled rate constants via a general mechanism that involves the sequential reduction and oxidation of the metal center, with the concomitant oxidation and reduction of superoxide radicals. That the catalytically active metal can be copper, iron, manganese or, recently, nickel is one of the fascinating features of this class of enzymes. In this review, we describe these enzymes in terms of the details of their catalytic properties, with an emphasis on the mechanistic differences between the enzymes. The focus here will be concentrated mainly on two of these enzymes, copper, zinc superoxide dismutase and manganese superoxide dismutase, and some relatively subtle variations in the mechanisms by which they function.     

Moderately Thermophilic Magnetotactic Bacteria from Hot Springs in Nevada

Populations of a moderately thermophilic magnetotactic bacterium were discovered in Great Boiling Springs, Nevada, ranging from 32 to 63°C. Cells were small, Gram-negative, vibrioid to helicoid in morphology, and biomineralized a chain of bullet-shaped magnetite magnetosomes. Phylogenetically, based on 16S rRNA gene sequencing, the organism belongs to the phylum Nitrospirae.Magnetotactic bacteria are a metabolically, morphologically, and phylogenetically heterogeneous group of prokaryotes that passively align and actively swim along magnetic field lines (3). This behavior, called magnetotaxis, is due to the presence of intracellular, membrane-bounded, single-magnetic-domain crystals of magnetite (Fe₃O₄) and/or greigite (Fe₃S₄) (3).Most known cultured magnetotactic bacteria are mesophilic and do not grow much above 30°C (e.g., Magnetospirillum species and Desulfovibrio magneticus strains MV-1 and MC-1 [D. A. Bazylinski, unpublished data]). Uncultured magnetotactic bacteria have been observed in numerous habitats that were mostly at 30°C and below. There is only one report describing thermophilic magnetotactic bacteria despite a number of efforts to look for them (e.g., in hydrothermal vents [D. A. Bazylinski, unpublished data]). Nash (12) reported the presence of thermophilic magnetotactic bacteria in microbial mats at about 45 to 55°C adjacent to the main flow in Little Hot Creek (but not in other springs in the same area at 40 to 80°C) and in microbial mats of other springs in central California at up to 58°C, all on the east side of the Sierra Nevada mountains. Cells biomineralized bullet-shaped crystals of magnetite and were phylogenetically affiliated with the phylum Nitrospirae (12). Few additional details were provided regarding the organisms and their habitat.In this study, water and surface sediment samples were taken from the Great Boiling Springs (GBS) geothermal field in Gerlach, NV. GBS is a series of hot springs that range from ambient temperature to ∼96°C (2, 5). The geology, chemistry, and microbial ecology of the springs have been described in some detail (2, 5). The pHs of the samples ranged from 6.4 to 7.5, while the salinities were about 4 to 5 ppt, as determined with a handheld Palm Abbe PA203 digital refractometer (MISCO Refractometer, Cleveland, OH). Samples were examined for the presence of magnetotactic bacteria using the hanging drop technique on-site and in the laboratory at room temperature with and without magnetic enrichment of the sample (15). Some samples taken back to the laboratory were kept at an elevated temperature (∼62°C), while others were kept at ambient temperature. There did not appear to be a significant difference in the number of magnetotactic cells in samples taken back to the laboratory and kept at these two temperatures. Only one morphotype of magnetotactic bacteria was found in samples from nine springs whose temperatures ranged from 32 to 63°C, and we estimate their numbers to be between 10³ to 10⁵ cells ml⁻¹ in surface sediments in sample bottles. We did not observe magnetotactic cells of this type in a large number of springs or pools that were at <32°C. Only one spring positive for the presence of these magnetotactic bacteria had sediment that was partially covered with a microbial mat, while sediment at most of the springs was dark gray in color. Cells were small (1.8 ± 0.4 by 0.4 ± 0.1 μm; n = 59), Gram negative, vibrioid to helicoid in morphology, and possessed a single polar flagellum (Fig. ​(Fig.1A).1A). Magnetotactic bacteria were not observed in springs that were at 67°C and above, suggesting the maximum survival and perhaps growth temperature for the organism is about 63°C. In the lab, cells remained viable and motile in samples kept at 25 to 62°C for several months. We refer to this organism as strain HSMV-1.Open in a separate windowFIG. 1.Transmission electron microscope (TEM) images of cells and magnetosomes of strain HSMV-1. (A) TEM image of unstained cell of HSMV-1 showing a single polar flagellum and a single chain of bullet-shaped magnetosomes. The electron-dense structures at the poles were found to be phosphorus-rich based on energy-dispersive X-ray analysis (data not shown) and therefore likely represent polyphosphate granules. (B) Higher-magnification TEM image of the magnetosome chain. (C) High-magnification TEM image of magnetosomes from which a selected area electron diffraction (SAED) pattern was obtained (inset of B). The SAED pattern corresponds to the [1 0−1] zone of magnetite, Fe₃O₄: reflection o, (0 0 0); reflection a, (1 −1 1) (0.48 nm); reflection b, (1 1 1) (0.48 nm); reflection c, (2 0 2) (0.30 nm); angle a-o-b, 70.5°. (D) Iron, sulfur, and oxygen elemental maps, derived from energy-filtering transmission electron microscopy (EFTEM), showing that the positions of the magnetosome crystals correlate with increased concentrations of Fe and O, but not S, consistent with the iron oxide magnetite (Fe₃O₄).Cells of HSMV-1 biomineralized a single chain of magnetosomes that traversed the cells along their long axis (Fig. 1A to C). Selected area electron diffraction (SAED) and energy-filtering transmission electron microscopy (EFTEM) elemental maps were determined on magnetosome crystals using a Tecnai model G2 F30 Super-Twin transmission electron microscope (FEI Company, Hillsboro OR). SAED patterns of HSMV-1 magnetosome crystals (Fig. ​(Fig.1B,1B, inset) indicated that they consisted of magnetite, while EFTEM elemental maps (Fe, O and S) (Fig. ​(Fig.1D)1D) clearly showed that the crystals consisted of an iron oxide and not an iron sulfide, again consistent with the mineral magnetite. Cells contained an average of 12 ± 6 magnetosome crystals per cell (n = 15 cells) that averaged 113 ± 34 by 40 ± 5 nm in size (n = 179). A plot of the length of the crystals as a function of the shape factor (width/length ratio) is provided in Figure S1 in the supplemental material and shows that the crystals fit in the theoretical single-magnetic-domain size range (4), along with all known mature magnetosome magnetite crystals from magnetotactic bacteria (3).Whole-cell PCR amplification of the 16S rRNA gene was performed by first magnetically purifying cells of HSMV-1 using the “capillary racetrack” described by Wolfe et al. (18). Purity of the collected cells was determined by microscopic examination, and contaminating cells were never observed. The 16S rRNA gene was amplified using bacteria-specific primers 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5′-TACGGHTACCTTGTTACGACTT-3′ (11). PCR products were cloned into pGEM-T Easy vector (Promega Corporation, Madison, WI) and sequenced (Functional Biosciences, Inc., Madison, WI). Six of eight clones sequenced had identical inserts.Alignment of 16S rRNA gene sequences was performed using the CLUSTAL W multiple alignment accessory application in the BioEdit sequence alignment editor (7). Phylogenetic trees were constructed using MEGA version 4 (17) by applying the neighbor-joining method (14). Bootstrap values were calculated with 1,000 replicates. The 16S rRNA gene sequence of strain HSMV-1 places the organism in the phylum Nitrospirae (Fig. ​(Fig.2),2), with its closest relative in culture being Thermodesulfovibrio hydrogeniphilus (87.2% identity) (8). Two other uncultured magnetotactic bacteria are phylogenetically affiliated with the phylum Nitrospirae, including the unnamed rod-shaped bacterium strain MHB-1 (86.5% identity) (6) and the very large Candidatus Magnetobacterium bavaricum (86.4% identity) (16). Interestingly, all the magnetotactic bacteria associated with the phylum Nitrospirae thus far (e.g., Candidatus Magnetobacterium bavaricum) contain bullet-shaped magnetite crystals in their magnetosomes.Open in a separate windowFIG. 2.Phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strain HSMV-1 in the phylum Nitrospirae. Bootstrap values at nodes are percentages of 1,000 replicates. The magnetotactic bacteria Desulfovibrio magneticus and Candidatus Magnetoglobus multicellularis (outgroup; deltaproteobacteria) were used to root the tree. GenBank accession numbers are given in parentheses. Bar represents 2% sequence divergence.Fluorescent in situ hybridization (FISH) was used to authenticate the 16S rRNA gene sequence. A specific Alexa594-labeled probe for HSMV-1 was designed (HSMVp, 5′-CCTTCGCCACAGGCCTTCTA-3′, complementary to nucleotides 690 to 709 of the 16S rRNA molecule) based on the alignment of 10 of the most similar 16S rRNA gene sequences found in GenBank after BLAST analysis (1) and on cultivated members of the phylum Nitrospirae. FISH with the Alexa594-labeled probe was carried out after fixation of magnetically concentrated cells directly on the wells of gelatin-coated hydrophobic microscope slides with 4% paraformaldehyde. FISH was performed according to the work of Pernthaler et al. (13). The hybridization solution contained 10 ng/ml of the probe, 20% formamide, 0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM Na₂EDTA, and 0.01% sodium dodecyl sulfate (SDS). Cells of HSMV-1 hybridized to the HSMVp probe, while other cells in the sample did not (Fig. ​(Fig.3),3), indicating that the 16S rRNA gene sequence we obtained is from the magnetotactic bacterium under study. Strain HSMV-1 clearly represents a new genus (Fig. ​(Fig.2),2), and based on the phylogeny and what we currently know phenotypically about strain HSMV-1, we propose the name Candidatus Thermomagnetovibrio paiutensis (the GBS site was originally occupied by the Paiute Indian Tribe).Open in a separate windowFIG. 3.Fluorescent in situ hybridization (FISH) of cells of strain HSMV-1 using an HSMV-1-specific oligonucleotide rRNA probe (HSMVp). Cells used for FISH were magnetically concentrated by placing a magnet next to the side of the sample bottle for 30 min and then removed with a Pasteur pipette. This technique was used rather than the magnetic racetrack method in order to have many HSMV-1 cells as well as some other cells that could be used as a negative control. (A) Differential interference contrast (DIC) image of HSMV-1 cells (filled arrows) and other cells (negative control; empty arrows) from hot spring samples; (B) cells stained with 4′,6-diamidino-2-phenylindole (DAPI); (C) cells hybridized with the specific probe HSMVp.Nash (12) first reported thermophilic magnetotactic bacteria phylogenetically affiliated with the Nitrospirae phylum in hot springs, and it would be interesting and important to compare these organisms and their habitats. However, little can be compared at this time due to lack of information. Nash (12) reported that the one spring at Little Hot Creek was freshwater and that microbial mats were present at all springs where thermophilic magnetotactic bacteria were found. The water at our sampling sites was brackish, not freshwater, and microbial mats were not an important feature of our springs. Thus, it is difficult to determine without knowing the relationship between the organisms found by Nash (12) and strain HSMV-1 what environmental parameters are important to the growth and survival of these bacteria.It is also difficult to determine the temperature ranges for the survival and growth for strain HSMV-1 without having a pure culture. Data presented here suggest that the temperature range for both is quite wide, and this would be important for the continued presence of HSMV-1 at GBS, as temperatures in the hot springs are known to fluctuate greatly (2). Even if the maximum growth temperature of HSMV-1 is slightly lower than the maximum survival temperature (a conservative estimate) that we know of (63°C), it would still be considered a moderately thermophilic bacterium.The results presented here clearly show that some magnetotactic bacteria can be considered at least moderately thermophilic. They extend the upper temperature limit for environments where magnetotactic bacteria exist and likely grow (∼63°C) and where magnetosome magnetite is deposited, a finding that may prove significant in the study and interpretation of magnetofossils (9, 10).