Thermodynamic and Kinetic Characterization of the Protein Z-dependent Protease Inhibitor (ZPI)-Protein Z Interaction Reveals an Unexpected Role for ZPI Lys-239

The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), circulates in blood as a tight complex with its cofactor, protein Z (PZ), enabling it to function as a rapid inhibitor of membrane-associated factor Xa. Here, we show that N,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-3-oxa-1,3-diazol-4-yl)ethylenediamine (NBD)-fluorophore-labeled K239C ZPI is a sensitive, moderately perturbing reporter of the ZPI-PZ interaction and utilize the labeled ZPI to characterize in-depth the thermodynamics and kinetics of wild-type and variant ZPI-PZ interactions. NBD-labeled K239C ZPI bound PZ with ∼3 nm K_D and ∼400% fluorescence enhancement at physiologic pH and ionic strength. The NBD-ZPI-PZ interaction was markedly sensitive to ionic strength and pH but minimally affected by temperature, consistent with the importance of charged interactions. NBD-ZPI-PZ affinity was reduced ∼5-fold by physiologic calcium levels to resemble NBD-ZPI affinity for γ-carboxyglutamic acid/EGF1-domainless PZ. Competitive binding studies with ZPI variants revealed that in addition to previously identified Asp-293 and Tyr-240 hot spot residues, Met-71, Asp-74, and Asp-238 made significant contributions to PZ binding, whereas Lys-239 antagonized binding. Rapid kinetic studies indicated a multistep binding mechanism with diffusion-limited association and slow complex dissociation. ZPI complexation with factor Xa or cleavage decreased ZPI-PZ affinity 2–7-fold by increasing the rate of PZ dissociation. A catalytic role for PZ was supported by the correlation between a decreased rate of PZ dissociation from the K239A ZPI-PZ complex and an impaired ability of PZ to catalyze the K239A ZPI-factor Xa reaction. Together, these results reveal the energetic basis of the ZPI-PZ interaction and suggest an important role for ZPI Lys-239 in PZ catalytic action.     

In vitro plant regeneration from organogenic callus of <Emphasis Type="Italic">Curcuma kwangsiensis</Emphasis> Lindl. (Zingiberaceae)

A novel protocol for callus-mediated shoot regeneration was established for an important medicinal and ornamental plant native to South China, Curcuma kwangsiensis, using shoot base sections excised from seedlings in vitro as explant sources. The frequency of callus formation reached 91% for explants cultured on MS medium containing 1.4 μM TDZ, 4.4 μM BA and 2.3 μM 2,4-D. 8.2 shoots per callus was achieved on MS medium supplemented with 1.4 μM TDZ, 17.8 μM BA and 2.7 μM NAA. Single shoots transferred into MS medium free of plant growth regulator rooted well. Regenerated plants acclimatized ex vitro at 100%, and grew vigorously under shaded greenhouse conditions.     

Comparative Growth,Biomass Production and Fuel Properties Among Different Perennial Plants,Bamboo and <Emphasis Type="Italic">Miscanthus</Emphasis>

Bamboo and Miscanthus species are perennial low-input plants that are excellent candidates for bioenergy feedstock production. Biological characteristics, dry matter yields and fuel properties of the bamboo and Miscanthus have been studied. Genotype growth characteristics were determined by measurements of plant height, tillering, tuft diameter, and shoot diameter. To date, comparisons of biomass yields of bamboo and Miscanthus have not been previously reported in the literature. Bamboo and Miscanthus species were collected and previous articles describing the productivity of bamboo and Miscanthus were examined. Genotypes differed in plant height, tillering, tuft diameter, and shoot diameter. Nitrogen, temperature, water and plant density have effects on mature stands biomass production, which ranged from 5.9 to 49.5 tonnes/ha/yr for bamboo and 3.2 to 49.0 tonnes/ha/yr for Miscanthus. With such biomass yields, bamboo and Miscanthus should be considered as two very promising plants for biomass production in Zhejiang, China in the near future.     

2018年中国植物科学若干领域重要研究进展

2018年中国植物科学继续呈现快速发展的态势, 我国科学家在国际植物科学主流学术刊物发表论文数量大幅增加, 取得了多项具有重要影响的成果。调控植物生长-代谢平衡实现可持续农业发展入选2018年度中国科学十大进展; 中国被子植物区系进化历史研究入选2018年度中国生命科学十大进展。以水稻为代表的农作物和果蔬等经济作物研究在国际上已呈现出明显的优势, 若干领域已从“追赶”状态跨越到“领跑”地位。该文对2018年中国科学家在植物科学若干领域取得的重要研究成果进行了概括性评述, 旨在全面追踪和报道当前中国植物科学领域的发展前沿和热点, 展示中国科学家所取得的杰出成就。     

IL-18基因转染对大鼠C6胶质瘤细胞生长特性的影响

探讨IL-18基因转染对大鼠C6胶质瘤细胞生长特性的影响。用MTT法和流式细胞术检测C6／IL-18细胞和C6细胞的增殖特性和细胞周期分布。免疫细胞化学检测C6／IL-18细胞和C6细胞的增殖细胞核抗原(PCNA)、波形蛋白表达。结果显示,与C6细胞相比C6／IL-18细胞的增殖能力降低,G0/G1期细胞增多而G2／M期细胞减少;PCNA、波形蛋白表达降低。研究表明,IL-18基因具有抑制C6胶质瘤细胞增殖、降低其恶性程度的作用。     

Dimers initiate and propagate serine protease inhibitor polymerisation

The serine protease inhibitor (serpin) family can readily form long-chain polymers by a process that underlies a variety of diseases. We show here that monomers of plasma serpins α₁-antitrypsin and antithrombin are stable on incubation with the rate-limiting step in their polymerisation being the formation of the initial dimer. Once formed, the dimers readily interlink to form tetramers and can bind monomers to form trimers and longer oligomers. Cleavage of the only exposed reactive loop, in unit I of the dimers, prevents their interlinkage, but these cleaved dimers can still link to monomers. The rapid binding by the cleaved dimers of a peptide specific to the lower half of β-sheet A of the molecule indicates the ready opening of this β-sheet in unit II of the dimers. The failure of the cleaved dimers to bind peptide-complexed monomers, together with the relative inaccessibility of the P14 hinge residue in the oligomers, is evidence that partial insertion of the reactive loop into its own A-sheet is required for polymer formation. We propose that serpin dimers initiate and propagate polymerisation by having one exposed loop with an optimal conformation as a β-strand donor and a readily opened β-sheet as an acceptor. The sequential reformation of these activated β-interfaces as the oligomer extends, molecule by molecule, provides a model for the fibril and amyloid formation of conformational diseases in general as well as for the infectivity of prion encephalopathies.     

The subcellular localization of an unusual rice calmodulin isoform,OsCaM61, depends on its prenylation status

Calmodulin (CaM) is a small Ca²⁺-binding protein highly conserved in eukaryotes. We have reported previously a novel rice CaM-like protein (OsCaM61) which contains an N-terminal CaM domain and a C-terminal extension with a potential prenylation site. Here we report in vitro activity assays confirm OsCaM61 as a functional CaM. Using the green fluorescent protein (GFP) as a visual marker, we further studied the subcellular localization of OsCaM61 in stably transformed tobacco cells. The GFP-OsCaM61 fusion protein was membrane-associated whereas OsCaM61-GFP was mainly detected in the nucleoplasm. GFP-OsCaM61 was transported into the nucleoplasm upon a block in isoprenoid biosynthesis by mevinolin treatment of cells. These results indicate that the prenylated OsCaM61 molecules are mainly membrane-associated whereas its unprenylated counterparts are transported into the nucleoplasm. Thus, OsCaM61 may play functions in co-ordinating Ca²⁺ signaling with isoprenoid metabolism.     

The Arabidopsis anaphase-promoting complex or cyclosome: molecular and genetic characterization of the APC2 subunit

In yeast and animals, the anaphase-promoting complex or cyclosome (APC/C) is an essential ubiquitin protein ligase that regulates mitotic progression and exit by controlling the stability of cell cycle regulatory proteins, such as securin and the mitotic cyclins. In plants, the function, regulation, and substrates of the APC/C are poorly understood. To gain more insight into the roles of the plant APC/C, we characterized at the molecular level one of its subunits, APC2, which is encoded by a single-copy gene in Arabidopsis. We show that the Arabidopsis gene is able to partially complement a budding yeast apc2 ts mutant. By yeast two-hybrid assays, we demonstrate an interaction of APC2 with two other APC/C subunits: APC11 and APC8/CDC23. A reverse-genetic approach identified Arabidopsis plants carrying T-DNA insertions in the APC2 gene. apc2 null mutants are impaired in female megagametogenesis and accumulate a cyclin-beta-glucuronidase reporter protein but do not display metaphase arrest, as observed in other systems. The APC2 gene is expressed in various plant organs and does not seem to be cell cycle regulated. Finally, we report intriguing differences in APC2 protein subcellular localization compared with that in other systems. Our observations support a conserved function of the APC/C in plants but a different mode of regulation.