Studying fatty aldehyde metabolism in living cells with pyrene-labeled compounds

The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome (SLS) patient cells not only impairs the conversion of fatty aldehydes into their corresponding fatty acid but also has an effect on connected pathways. Alteration of the lipid profile in these cells is thought to be responsible for severe symptoms such as ichtyosis, mental retardation, and spasticity. Here we present a novel approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the culture medium of living cells using HPLC separation and fluorescence detection. Our results show that in fibroblasts from SLS patients, fatty aldehyde is not accumulating but is converted readily into fatty alcohol. In control cells, in contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed to induce toxicity in SLS patient and in control cells.     

Comprehensive proteome analysis in Cenococcum geophilum Fr. as a tool to discover drought-related proteins

Cenococcum geophilum is a widely distributed ectomycorrhizal fungus potentially playing a significant role in resistance and resilience mechanisms of its tree hosts exposed to drought stress. In this study, we performed a large scale protein analysis in pure cultures of C. geophilum in order to gain first global insights into the proteome assembly of this fungus. Using 1-D gel electrophoresis coupled with ESI-MS/MS, we indentified 638 unique proteins. Most of these proteins were related to the metabolic and cellular processes, and the transport machinery of cells. In a second step, we examined the influence of water deprivation on the proteome of C. geophilum pure cultures at three time points of gradually imposed drought. The results indicated that 12 proteins were differentially abundant in mycelia subjected to drought compared to controls. The induced responses in C. geophilum point towards regulation of osmotic stress, maintainance of cell integrity, and counteracting increased levels of reactive oxygen species formed during water deprivation.     

Synthesis and analytical investigation of C‐terminally modified peptide aldehydes and ketone: application to oxime ligation

C‐terminally modified peptides aldehyde (glycinal and alpha‐oxo aldehyde peptides) and ketone (pyruvic acid‐containing peptide) were synthesised to get new insights into the mechanism of acido‐catalysed oxime ligation. Their tetrahedral hydrated forms were investigated in solution and in the gas phase, using NMR and in‐source collision‐induced dissociation mass spectrometry, respectively, and the kinetics of the oximation reactions followed using analytical HPLC. The results obtained confirmed that the first step of the oximation reaction was the limiting step for the pyruvic acid‐containing peptides because of the steric effect and of the carbon angular strain of the ketone. The second step is the determining step for the aldehyde peptides because the basicity of the oxygen of the hydroxyl function of the tetrahedral form is greater for glycinal than for alpha‐oxo aldehyde. These data strongly suggest that the hydrated form of the aldehyde partner has to be considered when oxime reactions are performed in aqueous buffer. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Abnormal fatty alcohol metabolism in cultured keratinocytes from patients with Sjögren-Larsson syndrome

Sj?gren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder characterized by ichthyosis, mental retardation, spasticity, and deficient activity of fatty aldehyde dehydrogenase (FALDH). FALDH is an enzyme component of fatty alcohol:NAD oxidoreductase (FAO), which is necessary for fatty alcohol metabolism. To better understand the biochemical basis for the cutaneous symptoms in this disease, we investigated lipid metabolism in cultured keratinocytes from SLS patients. Enzyme activities of FALDH and FAO in SLS cells were <10% of normal. SLS keratinocytes accumulated 45-fold more fatty alcohol (hexadecanol, octadecanol, and octadecenol) than normal, whereas wax esters and 1-O-alkyl-2,3-diacylglycerols were increased by 5.6-fold and 7.5-fold, respectively. SLS keratinocytes showed a reduced incorporation of radioactive octadecanol into fatty acid (24% of normal) and triglyceride (13% of normal), but incorporation into wax esters and 1-O-alkyl-2,3-diacylglycerol was increased by 2.5-fold and 2.8-fold, respectively. Our results indicate that FALDH deficiency in SLS keratinocytes causes the accumulation and diversion of fatty alcohol into alternative biosynthetic pathways. The striking lipid abnormalities in cultured SLS keratinocytes are distinct from those seen in fibroblasts and may be related to the stratum corneum dysfunction and ichthyosis in SLS.     

Structural and biochemical characterization of a novel aldehyde dehydrogenase encoded by the benzoate oxidation pathway in Burkholderia xenovorans LB400

The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDH_C) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD ⁺ and NADP ⁺). Our data definitively show that ALDH_C is more active in the presence of NADP ⁺ and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-Å crystal structure of ALDH_C in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2′-phosphoryl moiety of NADP ⁺ and a threonine/lysine pair on ALDH_C. The catalytic preference of ALDH_C for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDH_C induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDH_C in benzoate degradation.     

Critical role of acrolein in secondary injury following ex vivo spinal cord trauma

The pathophysiology of spinal cord injury (SCI) is characterized by the initial, primary injury followed by secondary injury processes in which oxidative stress is a critical component. Secondary injury processes not only exacerbate pathology at the site of primary injury, but also result in spreading of injuries to the adjacent, otherwise healthy tissue. The lipid peroxidation byproduct acrolein has been implicated as one potential mediator of secondary injury. To further and rigorously elucidate the role of acrolein in secondary injury, a unique ex vivo model is utilized to isolate the detrimental effects of mechanical injury from toxins such as acrolein that are produced endogenously following SCI. We demonstrate that (i) acrolein-Lys adducts are capable of diffusing from compressed tissue to adjacent, otherwise uninjured tissue; (ii) secondary injury by itself produces significant membrane damage and increased superoxide production; and (iii) these injuries are significantly attenuated by the acrolein scavenger hydralazine. Furthermore, hydralazine treatment results in significantly less membrane damage 2 h following compression injury, but not immediately after. These findings support our hypothesis that, following SCI, acrolein is increased to pathologic concentrations, contributes significantly to secondary injury, and thus represents a novel target for scavenging to promote improved recovery.     

Variations in the Volatile Organic Compound Emission Potential of Plant Functional Groups in the Temperate Grassland Vegetation of Inner Mongolia, China

The biogenic volatile organic compounds (VOC) emitted by the vegetation of a terrestrial ecosystem play a key role in both regional air quality and tropospheric chemistry. To describe the general emission properties of VOC of different plant functional groups (PFG) in a typical temperate grassland in Inner Mongolia, China, we randomly selected 175 plant species and measured the quantities of isoprene and monoterpene in situ. Results showed that most plants had low VOC emission potential at the species level,especially for some dominant plants, such as Leymus chinensis Tzvel., Stipa grandis Smirn., and Agropyron cristatum Gaertn. At the PFG level, the lowest VOC emission potential was found for perennial rhizome grasses, a major PFG in a typical temperate grassland ecosystem. The effects of overgrazing and subsequent vegetation succession on the emission of VOC by different plant life form functional groups (PLFG)were also discussed.     

介质表面修饰对蛋白质芯片固定率和反应性的影响

评价最常用的二种玻璃表面修饰方法对蛋白质芯片质量的影响．选择蛋白质的固定效率、反应性作为检测指标,对戊二醛修饰法和多聚赖氨酸修饰法进行比较,由机械手将探针蛋白质分别固定在两种玻片上,靶蛋白用荧光染料Cy3标记,两种修饰方法的芯片均可使蛋白质保持较好的固定效率和反应活性．由共价键偶联的醛基修饰玻片制备的蛋白质芯片不仅有更高的反应活性,而且图象佳,但背景偏高、用醛基修饰的玻片制备蛋白质芯片是较理想的选择、     

通过农杆菌介导法获得耐盐转甜菜碱醛脱氢酶基因白三叶草

通过农杆菌介导法将耐盐植物山菠菜甜菜碱醛脱氢酶(Betaine Aldehyde Dehydrogenase,BADH)基因成功地转化了白三叶草。转基因植株在经过48h 1％NaCl胁迫后相对电导率为20％左右,而非转基因植株高达40％,表明转基因植株细胞膜在盐胁迫下受到的伤害较非转基因的轻,并且转基因植株能够在含有0．5％NaCl的水培养中正常生长两周以上,而非转基因植株则呈现不正常生长。