Enzymatic Improvement of Food Flavor. V. Oxidation of Aldehydes in Soybean Extracts by an NAD+-Regenerating System Made up of Aldehyde Dehydrogenase and Diaphorase

Fourteen new and three known o-arylethenylbenzoic acids (10 ~ 18), required for testing as potential plant growth regulators, have been synthesized by the Wittig reaction. The cis- and trans-isomers were separated by fractional crystallization and identified by NMR spectroscopy. The preliminary assay results on cress seedlings are reported and show that both geometric isomers appear to have nearly identical anit-geotropic activity in this test system.     

Enzymatic Improvement of Food Flavor I. Purification and Characterization of Bovine Liver Mitochondrial Aldehyde Dehydrogenase

Bovine liver mitochondrial aldehyde dehydrogenase (aldehyde: NAD⁺ oxidoreductase, EC 1.2.1.3) has been purified to homogeneity by conventional purification procedures. The enzyme was found to have a molecular weight of 215,000 based on gel filtration. The protein is composed of polypeptides having the same molecular weight, 54,000 and thus it appears to consist of four subunits of equal size. The enzyme exhibited a broad aldehyde specificity, oxidizing irreversibly a wide variety of aliphatic and aromatic aldehydes to corresponding carboxylic acids. Km values for straight-chain saturated aldehydes were below 0.1 µm, and relatively constant independent of the carbon chain lengths of the aldehydes. The maximum velocities for saturated aldehydes also did not vary appreciably with their carbon chain lengths. Maximum activity was observed at pH 9.3 and 50°C. The enzyme activity was affected by some divalent cations. Ca²⁺ enhanced the activity, while Mg²⁺ inhibited it. The enzyme was quite stable at neutral pH, but was unstable above pH 9 or below pH 6. Bovine liver has three isozymes of aldehyde dehydrogenase which are located in the mitochondrial, cytosolic, and microsomal fractions. Comparison of enzymic properties among these isozymes and yeast enzyme indicates that the mitochondrial enzyme is very suitable for improving the objectionable flavor due to aldehydes in foods.     

Arginine 469 is a pivotal residue for the Hsc70-GlcNAc-binding property

The members of the 70 kDa-heat shock proteins (HSP70) family play numerous fundamental functions in the cell such as promoting the assembly of multimeric complexes or helping the correct folding of nascent proteins to take place. In numerous previous studies we demonstrated that Hsp70 and its constitutive isoform Hsc70 are endowed of a GlcNAc-binding activity. The molecular modeling of the substrate binding domain of Hsc70 and in silico docking experiments using Ser/Thr-O-GlcNAc motifs allowed to define the potential carbohydrate-recognition region and to point out the crucial position of Arg469 as an amino-acid directly interacting with the sugar moiety. We cloned a flagged Hsc70 in a pCMV.SPORT6 vector and we showed that the mutation R469A decreased the GlcNAc-binding property of the chaperone of around 70%. This is the first work reporting the localization of the GlcNAc-binding domain of a member of the HSP70 family.     

Possible involvement of MAP kinase pathways in acquired metal-tolerance induced by heat in plants

Cross tolerance is a phenomenon that occurs when a plant, in resisting one form of stress, develops a tolerance to another form. Pretreatment with nonlethal heat shock has been known to protect cells from metal stress. In this study, we found that the treatment of rice roots with more than 25 muM of Cu(2+) caused cell death. However, heat shock pretreatment attenuated Cu(2+)-induced cell death. The mechanisms of the cross tolerance phenomenon between heat shock and Cu(2+) stress were investigated by pretreated rice roots with the protein synthesis inhibitor cycloheximide (CHX). CHX effectively block heat shock protection, suggesting that protection of Cu(2+)-induced cell death by heat shock was dependent on de novo protein synthesis. In addition, heat pretreatment downregulated ROS production and mitogen-activated protein kinase (MAPK) activities, both of which can be greatly elicited by Cu(2+) stress in rice roots. Moreover, the addition of purified recombinant GST-OsHSP70 fusion proteins inhibited Cu(2+)-enhanced MAPK activities in an in vitro kinase assay. Furthermore, loss of heat shock protection was observed in Arabidopsis mkk2 and mpk6 but not in mpk3 mutants under Cu(2+) stress. Taken together, these results suggest that the interaction of OsHSP70 with MAPKs may contribute to the cellular protection in rice roots from excessive Cu(2+) toxicity.     

Sorghum bicolor SbHSP110 has an elongated shape and is able of protecting against aggregation and replacing human HSPH1/HSP110 in refolding and disaggregation assays

Perturbations in the native structure, often caused by stressing cellular conditions, not only impair protein function but also lead to the formation of aggregates, which can accumulate in the cell leading to harmful effects. Some organisms, such as plants, express the molecular chaperone HSP100 (homologous to HSP104 from yeast), which has the remarkable capacity to disaggregate and reactivate proteins. Recently, studies with animal cells, which lack a canonical HSP100, have identified the involvement of a distinct system composed of HSP70/HSP40 that needs the assistance of HSP110 to efficiently perform protein breakdown. As sessile plants experience stressful conditions more severe than those experienced by animals, we asked whether a plant HSP110 could also play a role in collaborating with HSP70/HSP40 in a system that increases the efficiency of disaggregation. Thus, the gene for a putative HSP110 from the cereal Sorghum bicolor was cloned and the protein, named SbHSP110, purified. For comparison purposes, human HsHSP110 (HSPH1/HSP105) was also purified and investigated in parallel. First, a combination of spectroscopic and hydrodynamic techniques was used for the characterization of the conformation and stability of recombinant SbHSP110, which was produced folded. Second, small-angle X-ray scattering and combined predictors of protein structure indicated that SbHSP110 and HsHSP110 have similar conformations. Then, the chaperone activities, which included protection against aggregation, refolding, and reactivation, were investigated, showing that SbHSP110 and HsHSP110 have similar functional activities. Altogether, the results add to the structure/function relationship study of HSP110s and support the hypothesis that plants have multiple strategies to act upon the reactivation of protein aggregates.     

Induction of barotolerance by heat shock treatment in yeast

In Saccharomyces cerevisiae, heat shock treatment provides protection against subsequent hydrostatic pressure damage. Such an induced hydrostatic pressure resistance (barotolerance) closely resembles the thermotolerance similarly induced by heat shock treatment. The parallel induction of barotolerance and thermotolerance by heat shock suggests that hydrostatic pressure and high temperature effects in yeast may be tightly linked physiologically.     

热休克反应中小鼠心脏HSP70表达的免疫组织化学研究

用免疫组织化学方法研究了小鼠心脏在不同温度（40、41、44、46℃）热休克处理后,各恢复期（2、4、8、12、24小时）HSP70的表达。结果表明,（1）44、46℃热处理能诱导心肌细胞合成HSP70,以46℃为多（P＜0．01）,且于恢复期4－8小时为合成高峰（P＜0．01）。（2）阳性免疫反应定位于心肌细胞质中,核呈阴性反应。提示了心脏有较强的耐热能力。     

HSP70 kinetics study by continuous observation of HSP-GFP fusion protein expression on a perfusion heating stage

The direct correlation between levels of heat shock protein expression and efficiency of its tissue protection function motivates this study of how thermal doses can be used for an optimal stress protocol design. Heat shock protein 70 (HSP70) expression kinetics were visualized continuously in cultured bovine aortic endothelial cells (BAECs) on a microscope heating stage using green fluorescent protein (GFP) as a reporter. BAECs were transfected with a DNA vector, HSP(p)-HSP70-GFP which expresses an HSP70-GFP fusion protein under control of the HSP70 promoter. Expression levels were validated by western blot analysis. Transfected cells were heated on a controlled temperature microscope stage at 42 degrees C for a defined period, then shifted to 37 degrees C for varied post-heating times. The expression of HSP70-GFP and its sub-cellular localization were visualized via fluorescence microscopy. The progressive expression kinetics were measured by quantitative analysis of serial fluorescence images captured during heating protocols from 1 to 2 h and post-heating times from 0 to 20 h. The results show two sequential peaks in HSP70 expression at approximately 3 and 12 h post-heat shock. A progressive translocation of HSP70 from the cytoplasm to the nucleus was observed from 6 to 16 h. We conclude that we have successfully combined molecular cloning and optical imaging to study HSP70 expression kinetics. The kinetic profile for HSP70-GFP fusion protein is consistent with the endogenous HSP70. Furthermore, information on dynamic intracellular translocation of HSP70 was extracted from the same experimental data.     

酿酒酵母耐热元器件的筛选

以提高酿酒酵母耐热性、降低乙醇发酵过程控温能耗成本为目的,通过分析嗜热栖热菌(Thermus thermophiles)HB8热激蛋白基因,设计并构建了5个热激蛋白元器件,并导入酿酒酵母。通过梯度升温培养筛选出性能较好的耐热元器件FBA1p-groes-SLM5t,并利用恒定高温培养进一步验证了含有该元器件的酿酒酵母工程菌S.c-Gro ES具有良好的耐热性,研究表明在42℃培养48h的存活率是对照的3倍。此外,FBA1p-groes-SLM5t还能提高酵母的抗氧化性,42℃下菌株S.c-Gro ES的ROS水平比对照低37.6%,H2O2处理1 h后存活率是对照的1.62倍,说明耐热元器件在缓解热胁迫的同时对细胞的抗氧化性也有帮助。耐热工程酿酒酵母S.c-Gro ES,其40℃发酵乙醇产量相对于30℃对照和40℃对照分别提高了25%和13.8%。嗜热菌热激蛋白的引入可以明显提高酿酒酵母的耐热性及其乙醇合成效率。     

Improvement of the transformation efficiency of Sacchaaromyces cerevisiae by altering carbon sources in pre-culture

We show here that the transformation efficiency of Saccharomyces cerevisiae is improved by altering carbon sources in media for pre-culturing cells prior to the transformation reactions. The transformation efficiency was increased up to sixfold by combination with existing transformation protocols. This method is widely applicable for yeast research since efficient transformation can be performed easily without changing any of the other procedures in the transformation.     

核定位信号在热休克蛋白 70 抑制氧化应激所致 细胞核仁分离中的作用

为探讨核定位信号在热休克蛋白 70 (HSP70) 抑制 H²O² 所致核仁损伤中的作用,采用分子克隆技术分别构建了 4 个真核表达载体, pcDNA3.1(-)-HSP70^WT (HSP70 野生型), pcDNA3.1(-)-HSP70^ΔNLS (核定位信号缺失突变体 ), pEGFP-N1-HSP70^WT, pEGFP-N1- HSP70^ΔNLS. 向传代培养的 C²C¹²肌源细胞培养液中加入终浓度为 1.0 mmol/L 的 H²O² 模拟体外氧化应激 . 甲苯胺蓝染色细胞核仁发现,正常细胞仅有一个位于中央的、浓染致密的核仁颗粒 . 过氧化氢处理后 3 h ,可见明显的核仁分离 . 热休克反应处理的细胞及转 pcDNA3.1( － )-HSP70WT 细胞则能明显抑制氧化应激所致的核仁分离 . 荧光蛋白示踪及核仁蛋白质免疫印迹分析显示, H²O²处理后 1 h , HSP70WT 由正常时的细胞浆定位转为细胞核及核仁定位,而 HSP70ΔNLS 在 H²O²处理后仍定位于细胞浆,同时丧失了抑制核仁分离的作用 . 上述结果提示,野生型 HSP70 能显著抑制氧化应激所致细胞核仁分离,核定位信号通过介导 HSP70 向细胞核及核仁移位而决定 HSP70 对核仁损伤的保护作用 .     

HSP70 interacting protein prevents the accumulation of inclusions in polyglutamine disease1

Heat shock proteins (HSPs) are associated with the proteinaceous inclusions that characterise many neurodegenerative diseases. This suggests they may be associated with disease aetiology and/or represents an attempt to remove abnormal protein aggregates. In this study the adenoviral mediated over‐expression of HSP70 interacting protein (HIP) alone was shown to significantly reduce inclusion formation in both an in vitro model of Spinal Bulbar Muscular Atrophy and a primary neuronal model of polyglutamine disease. Experiments to determine the mechanism of action showed that: denatured luciferase activity (a measure of protein refolding) was not increased in the presence of HIP alone but was increased when HIP was co‐expressed with HSP70 or Heat Shock cognate protein 70 (HSC70); the expression of polyglutamine inclusions in cortical neurons mediated an increase in the levels of HSC70 but not HSP70. Our data suggest that HIP may prevent inclusion formation by facilitating the constitutive HSC70 refolding cycle and possibly by preventing aggregation. HIP expression is not increased following stress and its over‐expression may therefore reduce toxic polyglutamine aggregation events and contribute to an effective therapeutic strategy.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

Inactivation of the mitochondrial heat shock protein zim17 leads to aggregation of matrix hsp70s followed by pleiotropic effects on morphology and protein biogenesis

The biogenesis of mitochondrial matrix proteins involves the translocase of the outer membrane, the presequence translocase of the inner membrane and the presequence translocase-associated motor. The mitochondrial heat shock protein 70 (mtHsp70) forms the central core of the motor. Recent studies led to the identification of Zim17, a mitochondrial zinc finger motif protein that interacts with mtHsp70. Different views have been reported on the localization of Zim17 in the mitochondrial inner membrane or matrix. Depletion of Zim17 impairs several critical mitochondrial processes, leading to inhibition of protein import, defects of Fe/S protein biogenesis and aggregation of Hsp70s in the matrix. Additionally, we found that inactivation of Zim17 altered the morphology of mitochondria. These pleiotropic effects raise the question of the specific function of Zim17 in mitochondria. Here, we report that Zim17 is a heat shock protein of the mitochondrial matrix that is loosely associated with the inner membrane. To address the function of Zim17 in organello, we generated a temperature-sensitive mutant allele of the ZIM17 gene in yeast. Upon a short-term shift of the yeast mutant cells to a non-permissive temperature, matrix Hsp70s aggregated while protein import, Fe/S protein activity and mitochondrial morphology were not, or only mildly, affected. Only after a long-term shift to non-permissive temperature, were strong defects in protein import, Fe/S protein activity and mitochondrial morphology observed. These findings suggest that the heat shock protein Zim17 plays a specific role in preventing protein aggregation in the mitochondrial matrix, and that aggregation of Hsp70s causes pleiotropic effects on protein biogenesis and mitochondrial morphology.     

谢氏宽漠王HSP70基因cDNA片段的克隆及热激条件下的表达

谢氏宽漠王Mantichorula semenowi Reitter是一种沙漠指示性甲虫。本研究由该种甲虫体内克隆到两种不同的HSP70基因片段,分别为MsHSP70和MsHSC70。同源性发现表明这两个基因片段与已报道的其他昆虫的热休克蛋白核苷酸序列高度同源。半定量RT-PCR分析显示：经42℃热激1 h 后立即诱导MsHSP70表达至最高峰;在恢复到室温的1～4 h 内MsHSP70表达量逐渐降低,但仍然高于未热激对照组。而MsHSC70在42℃热激1 h后表达受到抑制,但在恢复2 h和4 h时有少量的表达,分别仅为未热激对照组的0.25和0.28倍。结果提示MsHSP70和MsHSC70在保护细胞方面具有不同的作用。本实验结果为谢氏宽漠王在极端的沙漠环境胁迫下的抗逆适应性研究提供了理论基础。     

Heat shock factor 1 is a key regulator of the stress response in Chlamydomonas

We report here on the characterization of heat shock factor 1 (HSF1), encoded by one of two HSF genes identified in the genome of Chlamydomonas reinhardtii. Chlamydomonas HSF1 shares features characteristic of class A HSFs of higher plants. HSF1 is weakly expressed under non-stress conditions and rapidly induced by heat shock. Heat shock also resulted in hyperphosphorylation of HSF1, and the extent of phosphorylation correlated with the degree of induction of heat shock genes, suggesting a role for phosphorylation in HSF1 activation. HSF1, like HSFs in yeasts, forms high-molecular-weight complexes, presumably trimers, under non-stress, stress and recovery conditions. Immunoprecipitation of HSF1 under these conditions led to the identification of cytosolic HSP70A as a protein constitutively interacting with HSF1. Strains in which HSF1 was strongly under-expressed by RNAi were highly sensitive to heat stress. 14C-labelling of nuclear-encoded proteins under heat stress revealed that synthesis of members of the HSP100, HSP90, HSP70, HSP60 and small HSP families in the HSF1-RNAi strains was dramatically reduced or completely abolished. This correlated with a complete loss of HSP gene induction at the RNA level. These data suggest that HSF1 is a key regulator of the stress response in Chlamydomonas.