亚热带地区阔叶林与杉木林土壤活性有机质比较

通过对亚热带3个地区地带性阔叶林和杉木林土壤活性有机质的比较,分析森林类型变化及杉木连栽对土壤有机碳和养分含量的影响.结果表明：地带性阔叶林转变为杉木林后,土壤总有机碳含量下降27.8%～52.1%、腐殖酸碳下降32.2%～52.8%、胡敏酸下降36.4%～59.0%、富里酸下降29.7%～50.0%;杉木连栽也使土壤总有机碳和腐殖质含量下降.森林类型改变和杉木连栽对土壤活性有机质的影响更明显.杉木林取代阔叶林后,土壤微生物生物量碳、微生物生物量氮、可溶性有机碳和可溶性有机氮含量的最大降幅分别为61.8%、38.2%、43.3%和69.0%;与第1代杉木林相比,第2代杉木林土壤微生物生物量碳、微生物生物量氮、可溶性有机碳和可溶性有机氮含量的最大降幅分别为34.7%、29.3%、30.4%、18.4%.经相关性分析,除冷水浸提有机氮外,土壤活性有机质与养分含量之间具有密切的相关关系.     

PKA modulates GSK-3beta- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3beta (GSK-3beta), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3beta, but the site-specific modulation of GSK-3beta-catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3beta-catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases.     

SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因

Ｈｉｓｔａｇ／ＮｉＮＴＡ系统是新发展起来的一个亲和纯化重组蛋白的有用工具,现常用于基因编码产物的特性研究中。ＳＤＳＰＡＧＥ是实验室测定蛋白质分子量通常采用的方法,而许多实验室用此方法检测Ｈｉｓｔａｇ融合蛋白时却常发现测得的分子量偏大,产生偏差的原因尚未阐明。为弄清这一问题,本实验室在研究一个Ｈｉｓｔａｇ融合蛋白Ｐ７３Ｈｉｓ时,首先用ＳＤＳＰＡＧＥ法测得其分子量确实比理论计算值大,然后对其进行Ｃ末端氨基酸顺序测定、电喷雾质谱分析,结果证实其实际分子量与理论值一致。酶切去除包括Ｈｉｓｔａｇ在内的部分肽段使ＳＤＳＰＡＧＥ法测量蛋白分子量的偏差大大降低,证实Ｈｉｓｔａｇ确实是造成偏差的原因之一。推测由于Ｈｉｓｔａｇ中的碱性氨基酸的作用造成蛋白在ＳＤＳＰＡＧＥ中迁移变慢,而导致偏差。这一现象值得引起有关研究者的注意。     

Peptide models of four possible insulin folding intermediates with two disulfides

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.     

紫稻细胞质雄性不育系叶片全蛋白双向电泳分析

通过对几种不育系叶片全蛋白双向电泳图谱分析证明;紫稻不育系具有不同于野败型,红莲型和马协型不育系的蛋白图谱,说明其可能是一种新的细胞质质源,同时,紫稻不育系与保持系蛋白图谱之间在三叶期和分蘖期时差异均不明显,但各材料本身蛋白图谱在两个不同时期之间差异很大,不育系与保持系图谱表现出的蛋白质（多肽）的差异,可能与不育系雄性不育有关。     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

Application of free-flow electrophoresis/2-dimentional gel electrophoresis for fractionation and characterization of native proteome of Pseudomonas putida KT2440

Free Flow Electrophoresis (FFE) is a liquid-based isoelectric focusing method. Unlike conventional in-gel fractionation of proteins, FFE can resolve proteins in their native forms and fractionation of subcellular compartments of the cell is also possible. To test the efficacy of the FFE method, the native cytosol proteome of a bacterium, Pseudomonas putida KT2440 was fractionated by FFE and the spectrum of protein elutes was characterized in association with 2-dimentional gel electrophoresis (2-DE). Major native proteins of P. putida KT2440 were eluted in the range of pH 4.8 approximately 6.0 in FFE, whereas the denatured proteome of P. putida KT2440 was widely distributed in the rage of pH 4 approximately 10 in the 2-DE analysis. In addition, one of the three FFE major fractions, which was eluted at pH 5.0, was further analyzed using 2-DE/MS-MS. Then, the pH range of identified proteins eluted in 2-DE/MS-MS was 4.72 approximately 5.89, indicating that observed pi values of native cytosolic proteomes in FFE were narrower than those of denatured cytosolic proteome. These results suggest that FFE fractionation and 2-DE/MS analysis may be useful tools for characterization of native proteomes of P. putida KT2440 and comparative analysis between denatured and native proteomes.     

Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase)

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene.     

A meta-analysis on pyrogenic organic matter induced priming effect

Pyrogenic organic matter (PyOM) is considered an important soil carbon (C) sink. However, there are evidences that its addition to soil may induce a priming effect (PE) thus influencing its C abatement potential. The direction, the size and the mechanisms responsible for PyOM induced PE are far from being understood. We collected approximately 650 data points from 18 studies to analyse the characteristics of the PE induced by PyOM. The database was divided between the PE induced on the native soil organic matter and on fresh organic matter. Most of the studies were short-term incubation therefore the projections of findings on the long term may be critical. Our findings indicate that over 1 year PyOM induces an average positive PE of 0.3 mg C g⁻¹ soil on native soil organic matter and a PE of approximately the same size but opposite direction on fresh organic matter. We studied the correlation of PE with several properties of soil, of the added PyOM, and time after PyOM addition. We found that PyOM primes positively the native soil organic matter in the first 20 days while negative PE appears in a later stage. Negative PE was correlated with the soil C content. PyOM characterized by a low C content induced a higher positive PE on native soil organic carbon. No correlation was found between the factors record in our database and the PE induced on the fresh organic matter. We reviewed the mechanisms proposed in literature to explain PE and discussed them based on findings from our meta-analysis. We believe that the presence of a labile fraction in PyOM may trigger the activity of soil microorganisms on the short term and therefore induce a positive PE, while on the long term PyOM may induce a negative PE by promoting physical protection mechanisms.