酪氨酸酶的应用研究进展

酪氨酸酶具有重要的生理生化特性,在医药、环境、食品、精细化工等领域具有广泛的用 途。酪氨酸酶可以氧化L-酪氨酸合成L-多巴和黑色素,L-多巴用于帕金森症的治疗,黑色素能够 杀死HIV病毒。酪氨酸酶可用于环境工程领域处理含苯酚及胺类废水,用于精细化工领域催化 有机合成反应。综述了酪氨酸酶在各个领域的应用概况,阐明了其在工业生产领域的应用前景。     

Pathological interactions of bacteria and toxins with the gastrointestinal epithelial tight junctions and/or the zonula adherens: an update.

Communication between bacteria and the gastrointestinal tight junctions (TJs) and zonula adherens is examined. Bacteial-epithelial TJs "crosstalk" can be mediated by many virulence factors, mainly secreted toxins, or can be induced by direct contact of the pathogen with epithelial membrane. Moreover, there are several mechanisms by which bacteria may act on gastrointestinal TJs. First, bacteria can act indirectely at the TJs level by inducing cell transepithelial migration. More particularly, neutrophil or dendritic cells can cross the epithelium by a paracellular pathway. Secondly, bacteria and/or toxins can trigger actin cytoskeleton reorganization (depolymerization or hyperpolymerization). Thirdly, some enteric pathogens are susceptible to act on TJs by activation of cellular signal transduction. Finally, cleavage or modification of TJs proteins can be used by bacteria. New therapeutic strategies may result from a deeper knowledge of the cellular and molecular processes induced by bacteria at the TJ level. Moreover, studies of action of the different bacterial virulence factors on the molecules comprising the TJs and zonula adherens allow us an interesting approach on our understanding of TJ complex regulation.     

Regulation of pancreatic exocrine secretion in vitro: the action of secretagogues

Pancreatic acinar cells possess two functionally distinct mechanisms by which secretagogues can increase enzyme secretion. One mechanism is mediated by mobilization of cellular calcium and can be activated by any one of four different classes of receptors. The other mechanism is mediated by cyclic AMP and can be activated by either of two different classes of receptors. In addition to stimulating enzyme secretion, a secretagogue can cause potentiation of secretion, desensitization to the subsequent stimulation caused by the same or other secretagogues as well as residual stimulation of enzyme secretion. Although each class of secretagogue receptors can cause the same final effect, stimulation of enzyme secretion, the existence of multiple classes of receptors and the different mechanisms of action endow the acinar cell with a wide range of patterns of response depending on which of the several classes of receptors are activated.     

Anthramycin inhibition of restriction endonuclease cleavage and its use as a reversible blocking agent in DNA constructions.

Anthramycin can form a stable complex with DNA which does not dissociate upon repeated ethanol precipitations. The complex forms in less than one hour at pH 5.5. Bound anthramycin seems to be located in the minor groove of the DNA helix in the anthramycin DNA complex, since methylation of adenosine residues at N-3 by dimethylsulfate is reduced. The anthramycin-DNA complex is resistant to digestion by an excess of a number of restriction enzymes. Anthramycin can be removed from DNA by incubation at acid pH. The released DNA can then be cleaved by restriction enzymes. Anthramycin-DNA complexes can be acted upon by T4 polynucleotide ligase to form longer DNA molecules. The ability of anthramycin to form a stable but reversible complex which is not cleaved by restriction enzymes but can engage in joining reactions may allow a wider variety of DNA fragments to be more readily constructed in vitro.     

组织酸化参与外周痛觉传递的离子通道机制

组织酸化可以导致痛觉的产生.初级感觉神经元可以通过离子通道来感受外周的组织酸化.已鉴定了几个离子通道家族可能参与了外周组织酸化的感受：a.酸敏感离子通道(ASICs)是可以被酸直接门控的阳离子通道;b.辣椒素受体(VR1)可被酸敏化,同时可被pH＜6.0直接激活;c.P2X2和P2X2/3受体通道反应被酸上调;d.TwIK相关的酸感受钾通道（TASK）是被酸关闭的双孔内向整流钾通道.这些通道被酸所调控的共同结果就是提高了神经元的兴奋性.因此,它们在介导了组织酸化所诱导的痛觉感受和传递中具有重要作用.     

一种实时检测剪切力引起培养的大鼠动脉内皮细胞内一氧化氮含量的新方法

建立一个稳定和实时检测在不同剪切力作用下内皮细胞内一氧化氮含量的方法。利用流动小室建立内皮细胞剪切模型 ,在内皮细胞用DAF FM染色后 ,用Zeiss荧光共聚焦显微镜和ICCD摄象头检测细胞内的荧光强度。DAF FM的荧光强度可以反映一氧化氮的胞内含量。剪切力引起内皮细胞合成一氧化氮增加 ,并且这种作用是随着剪切力的增加而增加。剪切力的作用被一氧化氮合酶抑制剂L NAME全部抑制 ,被无Ca2 缓冲液部分抑制。这个方法可以实时反映一氧化氮含量的变化 ,可以用来研究剪切力引起一氧化氮变化的机制以及用来评价内皮细胞对剪切力的反应特性     

Effects of RNase and RNA on in vitro aster assembly

RNase alters the in vitro assembly of spindle asters in homogenates of meiotically dividing surf clam (Spisula solidissima) oocytes. Some effects of RNase, such as reduced astral fiber length, appear nonenzymatic and probably result from RNase binding to tubulin. However, RNase-induced changes in the microtubule organizing center are also observed. Since other polycations can mimic RNase effects, the existence of an RNA component of the spindle organizing center remains uncertain. Effects of RNase and other polycations on astral fiber length can be prevented and reversed by the RNase inhibitor, polyguanylic acid. Polyguanylic acid can also augment astral fiber length in the absence of added RNase or other polycations. Augmentation by polyguanylic acid is favored by high ionic strength, and can be duplicated by polyuridylic acid and, with less efficiency, by polyadenylic acid. Polucytidylic acid and unfractionated yeast RNA, however, are unable to augment aster assembly. Polyguanylic acid can also augment the length of astral fibers on complete spindles isolated under polymerizing condition. These results demonstrate that specfic polyribonucleotides can alter spindle assembly in vitro. The presence of an inhibitor of microtubule assembly in Spisula oocytes, which can be inactivated by specific RNAs, is suggested.     

Efficient PRNP gene targeting in bovine fibroblasts by adeno-associated virus vectors

Gene-targeted livestock can be created by combining ex vivo manipulation of cultured nuclear donor cells with cloning by nuclear transfer. However, this process can be limited by the low gene targeting frequencies obtained by transfection methods, and the limited ex vivo life span of the normal nuclear donor cells. We have developed an alternative gene targeting method based on the delivery of linear, single-stranded DNA molecules by adeno-associated virus (AAV) vectors, which can be used to introduce a variety of different mutations at single copy loci in normal human cells. Here we show that AAV vectors can efficiently target the PRNP gene encoding the prion protein PrP in bovine fetal fibroblasts, which can be used as nuclear donors to clone cattle. Cattle with both PRNP genes disrupted should be resistant to bovine spongiform encephalopathy.     

The role of iron in oxygen radical mediated lipid peroxidation

The role of iron in the peroxidation of polyunsaturated fatty acids is reviewed, especially with respect to the involvement of oxygen radicals. The hydroxyl radical can be generated by a superoxide-driven Haber-Weiss reaction or by Fenton's reaction; and the hydroxyl radical can initiate lipid peroxidation. However, lipid peroxidation is frequently insensitive to hydroxyl radical scavengers or superoxide dismutase. We propose that the hydroxyl radical may not be involved in the peroxidation of membrane lipids, but instead lipid peroxidation requires both Fe2+ and Fe3+. The inability of superoxide dismutase to affect lipid peroxidation can be explained by the fact that the direct reduction of iron can occur, exemplified by rat liver microsomal NADPH-dependent lipid peroxidation. Catalase can be stimulatory, inhibitory or without affect because H2O2 may oxidize some Fe2+ to form the required Fe3+, or, alternatively, excess H2O2 may inhibit by excessive oxidation of the Fe2+. In an analogous manner reductants can form the initiating complex by reduction of Fe3+, but complete reduction would inhibit lipid peroxidation. All of these redox reactions would be influenced by iron chelation.     

Percoll and Ficoll self-generated density gradients by low-speed osmocentrifugation

Percoll and Ficoll self-generated density gradients can be obtained by low-speed centrifugation of their solutions within dialysis cells. Useful Percoll density gradients can be obtained after 10-30 min centrifugation at 220-2010g, within dialysis cells. Ficoll density gradients, which are more difficult to self-generate, can be obtained by the same technique. Red cell band formation in a Percoll density gradient can be done in a single step by using dialysis cells as the centrifugation solution container.     

Partitioning additive genetic variance into genomic and remaining polygenic components for complex traits in dairy cattle

ABSTRACT: BACKGROUND: Low cost genotyping of individuals using high density genomic markers were recently introduced as genomic selection in genetic improvement programs in dairy cattle. Most implementations of genomic selection only use marker information, in the models used for prediction of genetic merit. However, in other species it has been shown that only a fraction of the total genetic variance can be explained by markers. Using 5217 bulls in the Nordic Holstein population that were genotyped and had genetic evaluations based on progeny, we partitioned the total additive genetic variance into a genomic component explained by markers and a remaining component explained by familial relationships. The traits analyzed were production and fitness related traits in dairy cattle. Furthermore, we estimated the genomic variance that can be attributed to individual chromosomes and we illustrate methods that can predict the amount of additive genetic variance that can be explained by sets of markers with different density. RESULTS: The amount of additive genetic variance that can be explained by markers was estimated by an analysis of the matrix of genomic relationships. For the traits in the analysis, most of the additive genetic variance can be explained by 44 K informative SNP markers. The same amount of variance can be attributed to individual chromosomes but surprisingly the relation between chromosomal variance and chromosome length was weak. In models including both genomic (marker) and familial (pedigree) effects most (on average 77.2%) of total additive genetic variance was explained by genomic effects while the remaining was explained by familial relationships. CONCLUSIONS: Most of the additive genetic variance for the traits in the Nordic Holstein population can be explained using 44 K informative SNP markers. By analyzing the genomic relationship matrix it is possible to predict the amount of additive genetic variance that can be explained by a reduced (or increased) set of markers. For the population analyzed the improvement of genomic prediction by increasing marker density beyond 44 K is limited.     

表面活性剂对土壤中多环芳烃生物有效性影响的研究进展

表面活性剂能够改变多环节烃(Polycyclic aromatic hydrocarbons,PAHs）在土壤中的溶解度、吸附/解吸平衡和与土壤微生物的相互作用,从而改变PAHs的生物有效性,表面活性剂主要通过降低土壤-水之间的界面张力,增加PAHs的溶解度、促进PAHs的运输等方式来加强PAHs的生物有效性,但由于表面活性剂本身对微生物的毒害作用或无毒的表面活性剂优先作为微生物的生长基质,可能会对PAHs的生物有效性起到抑制作用,另外,表面活性剂对土壤中不同形态的PAHs生物有效性的影响不同,表面活性剂、PAHs和土壤微生物的类型浓度以及土壤的物理化学条件等都对PAHs的生物有效性有影响。     

A new procedure for enzymatic semisynthesis of human insulin by hydrolysis of single-chain des-(b-30)-lnsulin precursor with lysyl endopeptidase

It has been shown that the single-chain des-(B-30)-insulin precursor (SCI) can be converted into human insulin ester by transpeptidation using trypsin in the presence of a threonine derivative. The present study demonstrates that Achromobacter lyticus protease 1 (lysyl endopeptidase) can catalyze the transpeptidation reaction more efficiently than can trypsin. It is also shown that des-(B-30)-insulin (DAI) can be produced by hydrolysis of SCI with the lysyl endopeptidase. Since it is well known that SCI can be produced by gene technology, the following method is recommended for industrial production of human insulin ester: hydrolysis of SCI with lysyl endopeptidase followed by coupling of the resulting DAI with a threonine derivative using trypsin or lysyl endopeptidase.     

噬菌体表面呈现技术研究进展

噬菌体表面呈现技术是1985年建立的一种将外源基因表达呈现在噬菌体颗粒表面的方法,可用于建立随机多肽文库、抗体文库等。经特定配基的筛选,可获得与其特异结合的配体分子。通过改构,还可将cDNA产物表达于噬菌体颗粒的尾部构建cDNA文库。SIP技术通过将配体和配基分别与基因Ⅲ蛋白的C末端和N末端融合表达,基因Ⅲ的C-末端参与噬菌体颗粒的组装,配基与配体的结合能够重建基因Ⅲ蛋白的功能,才能形成有感染能力的噬菌体,这样就大大提高了筛选效率。     

铜绿假单胞菌产酰化高丝氨酸内酯信号分子抑制剂的生产及分离

目的：通过自体诱导信号分子抑制剂的生产获得部分分离纯化的酰化高丝氨酸内酯（AHL）抑制剂。方法：病原菌铜绿假单胞菌经摇床培养后获得AHL抑制剂,采用溶解度差异性和树脂进行分离纯化。结果：铜绿假单胞菌PAO1不仅分泌自体诱导信号分子,而且在生长的后期还合成一种信号分子抑制剂,该信号分子抑制剂对群体感应中的AHL类信号分子有明显的抑制作用;该抑制剂具有醇溶性和水溶性,采用乙醇溶解可以除去糖类和无机小分子等不溶于醇的物质;大孔吸附树脂不具有吸附抑制剂的能力,但可以除去醇溶性糖类物质;阴离子交换树脂能够吸附信号分子抑制剂,具有较好的分离效率。结论：获得了除去大部分杂质,得到部分分离纯化的AHL抑制剂。     

Neural Coding with Graded Membrane Potential Changes and Spikes

The neural encoding of sensory stimuli is usually investigated for spike responses, although many neurons are known to convey information by graded membrane potential changes. We compare by model simulations how well different dynamical stimuli can be discriminated on the basis of spiking or graded responses. Although a continuously varying membrane potential contains more information than binary spike trains, we find situations where different stimuli can be better discriminated on the basis of spike responses than on the basis of graded responses. Spikes can be superior to graded membrane potential fluctuations if spikes sharpen the temporal structure of neuronal responses by amplifying fast transients of the membrane potential. Such fast membrane potential changes can be induced deterministically by the stimulus or can be due to membrane potential noise that is influenced in its statistical properties by the stimulus. The graded response mode is superior for discrimination between stimuli on a fine time scale.     

A simple and novel DNA combing methodology for Fiber-FISH and optical mapping

Single molecule analysis can help us study genomics efficiently. It involves studying single DNA molecules for genomic studies. DNA combing is one of such techniques which allowed us to study single DNA molecules for multiple uses. DNA combing technology can be used to perform Fiber-FISH and optical mapping. Physical mapping of genomes can be studied by restriction digestion of combed DNA on glass slides. Restriction fragments can be arranged into optical maps by gathering fluorescent intensity data by CCD camera and image analysis by softwares. Physical mapping and DNA segment rearrangements can be studied by Fiber-FISH which involves application of probes on genomic DNA combed over glass slides. We developed a novel methodology involving combing solution optimization, denatured combed DNA and performed restriction digestion of combed DNA. Thus we provided an efficient and robust combing platform for its application in Fiber-FISH and optical mapping.     

Multidimensional curve fitting program for biological data

A program written for use with the IBM-PC can be used to find least squares solutions to linearized multidimensional equations. The program is 'user-friendly' by requiring little from the user except to make decisions; most responses can be entered by a single keystroke. Once data are entered by the user, they can be repeatedly manipulated, graphed, and correlated. Many models relating data variables can be tried relatively easily, and best fit results found. Examples using respiratory mechanical data illustrate the ease of model comparisons.     

Use of bioluminescence in nucleic acid hybridization reactions

Luminescence reactions can be used to detect specific nucleic acid sequences hybridized with a nucleic probe. Different labels such as cytidine sulphone, fluorescein, and biotin can be incorporated into DNA or oligonucleotide molecules and detected by antibody or avidin conjugates coupled to glucose-6P dehydrogenase. On supports such as nitrocellulose filters, sensitivity is not greatly increased using luminescence, but detection is rapid and easy to perform using polaroid film. Moreover, hybridization can be performed with different labelled probes on the same sample. In solution, luminescence can be used to monitor sandwich reactions. The method is less sensitive than detection on filters but can easily be automated. The performance of these assays can be increased considerably by enzymatic amplification of the target catalysed by a thermostable polymerase.     

Synthesis and rapid purification of UDP-[6-3H]galactose

UDP[6-3H]galactose of high specificity can be obtained by oxidation of the C-6 hydroxymethyl group of UDP-galactose by galactose oxidase and subsequent reduction by sodium borotritide. One-step purification of the nucleotide sugar involves anion-exchange chromatography on a Pharmacia Mono Q column. Radiolabeled UDP-N-acetylgalactosamine can also be synthesized and purified by this procedure. Both nucleotide sugars can be used for sugar incorporation studies using the appropriate glycosyltransferase.