Oxidative stress in fungal fermentation processes: the roles of alternative respiration

Filamentous fungi are arguably the most industrially important group of microorganisms. Production processes involving these simple eukaryotes are often highly aerobic in nature, which implies these cultures are routinely subject to oxidative stress. Despite this, little is known about how filamentous fungi cope with high levels of oxidative stress as experienced in fermenter systems. More surprisingly, much of our knowledge of oxidative stress responses in fungi comes from environmental or medical studies. Here, the current understanding of oxidative stress effects and cellular responses in filamentous fungi is critically discussed. In particular the role of alternative respiration is evaluated, and the contributions of the alternative oxidase and alternative dehydrogenases in defence against oxidative stress, and their profound influence on fungal metabolism is critically examined. Finally, the importance of further research which would underpin a less empirical approach to optimising fungal strains for the fermenter environment is emphasised.     

An efficient and rapid method for cDNA cloning from difficult templates using codon optimization and SOE-PCR: with human RANK and TIMP2 gene as examples

As gene cloning from difficult templates with regionalized high GC content is a long recognized problem, we have developed a novel and reliable method to clone such genes. Firstly, the high GC content region of the target cDNA was synthesized directly after codon optimization and the remaining cDNA fragment without high GC content was generated by routine RT-PCR. Then the entire redesigned coding sequence of the target gene was obtained by fusing the above available two cDNA fragments with SOE-PCR (splicing by overlapping extension-PCR). We have cloned the human RANK gene (ten exons; CDS 1851 bp) using this strategy. The redesigned cDNA was transfected into an eukaryotic expression system (A459 cells) to verify its expression. RT-PCR and western blotting confirmed this. To validate our method, we also successfully cloned human TIMP2 gene (five exons; CDS 660 bp) also having a regionalized high GC content. Our strategy for combining codon optimization and SOE-PCR to clone difficult genes is thus feasible and potentially universally applicable.     

Identification of Two Species of Yeast-like Symbiotes in the Brown Planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis>

To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed spacer (ITS)–5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that the two different 18S and ITS–5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and ITS–5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS–5.8S rDNA, showed the high similarity to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes.     

正常人长时数字记忆信息提取的神经基础的功能磁共振研究

本文旨在通过功能磁共振成像(functional magnetic resonance imaging,fMRI)技术研究正常人进行长时数字记忆信息提取的神经基础。选取22名右利手志愿者进行长时数字记忆任务实验,采用组块设计,记忆任务与对照任务交替进行,同时利用Siemens 1.5T超导型磁共振仪进行fMRI成像,采用SPM99软件进行数据分析,脑功能区定位在Talairach坐标中显示。结果显示被试者在进行长时数字记忆提取任务时,激活最显著的皮层是左侧额中回(Brodmann分区9区,BA9区),另外左额叶内侧回、左额下回、右额下回、扣带回、左顶下小叶、左顶上小叶、右顶上小叶、右颞中回、左枕舌回、左枕中回、右中脑、小脑、右尾状核尾部等结构也有激活,各大脑皮层的激活均呈现明显的左侧半球优势。根据上述结果推论,长时数字记忆由以左侧大脑半球为优势的各脑区共同参与完成,其中左侧额叶外侧面可能是信息提取的重要结构,而其它脑叶及其之间的广泛联系可能在数字信息的加工、处理和存储中起重要作用。     

Limnonectins: a new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis)

Amphibian skin secretions are rich sources of biologically-active peptides with antimicrobial peptides predominating in many species. Several studies involving molecular cloning of biosynthetic precursor-encoding cDNAs from skin or skin secretions have revealed that these exhibit highly-conserved domain architectures with an unusually high degree of conserved nucleotide and resultant amino acid sequences within the signal peptides. This high degree of nucleotide sequence conservation has permitted the design of primers complementary to such sites facilitating “shotgun” cloning of skin or skin secretion-derived cDNA libraries from hitherto unstudied species. Here we have used such an approach using a skin secretion-derived cDNA library from an unstudied species of Chinese frog - the Fujian large-headed frog, Limnonectes fujianensis - and have discovered two 16-mer peptides of novel primary structures, named limnonectin-1Fa (SFPFFPPGICKRLKRC) and limnonectin-1Fb (SFHVFPPWMCKSLKKC), that represent the prototypes of a new class of amphibian skin antimicrobial peptide. Unusually these limnonectins display activity only against a Gram-negative bacterium (MICs of 35 and 70 μM) and are devoid of haemolytic activity at concentrations up to 160 μM. Thus the “shotgun” cloning approach described can exploit the unusually high degree of nucleotide conservation in signal peptide-encoding domains of amphibian defensive skin secretion peptide precursor-encoding cDNAs to rapidly expedite the discovery of novel and functional defensive peptides in a manner that circumvents specimen sacrifice without compromising robustness of data.     

Characterization of the <Emphasis Type="Italic">GHR</Emphasis> gene genetic variation in Chinese indigenous goat breeds

The aim of the present work was to investigate single nucleotide polymorphism (SNP) of growth hormone receptor (GHR) gene exon 10, characterize the genetic variation in three Chinese indigenous goat breeds, and search for its potential association with cashmere traits. In this study, a polymerase chain reaction-single strand conformation polymorphism (PCR–SSCP) protocol has been developed for rapid genotyping of the GHR gene in goats. One hundred seventy-eight goats from Liaoning Cashmere (96), Inner Mongolia White Cashmere (40), and Chengdu Grey (42) breeds in China were genotyped at GHR locus using the protocol developed. In all goat breeds investigated, a SNP in exon 10 of GHR gene has been identified by analyzing genomic DNA. The polymorphism consists of a single nucleotide substitution A → G, resulting in two alleles named, respectively, A and G based on the nucleotide at the position. The allele A was found to be more common in the animals investigated, and seems to be more consistent with cattle and zebu at this polymorphic site found in goats. The Hardy–Weinberg equilibrium of genotype distributions of GHR locus was verified in Liaoning Cashmere, and Inner Mongolia White Cashmere breeds. According to the classification of polymorphism information content (PIC), Chengdu Grey was less polymorphic than Liaoning Cashmere and Inner Mongolia White Cashmere breeds at this locus. The phylogenetic tree of different species based on the nucleotide sequences of GHR gene exon 10 is generally in agreement with the known species relationship. No significant association was found between the polymorphism revealed and the cashmere traits analyzed in present work.     

Endotoxin-induced cardiovascular dysfunction in mice: effect of simvastatin

Lung infections are associated with acute lung injury (ALI), systemic inflammation, and vascular events. Clinical studies suggest that statins improve health outcomes of patients with pneumonia and ALI. The mechanisms by which this occurs are unknown. The aim of this study was to determine whether statins attenuate systemic inflammation and cardiovascular dysfunction related to ALI in mice. Simvastatin (SS; 20 mg/kg) or vehicle solution was instilled intraperitoneally into mice 24 h before and again just prior to intratracheal LPS instillation (1 μg/g). These mice were then anesthetized with 2.0% isoflurane and underwent a short surgical procedure to instill LPS. Four hours later, IL-6 levels in bronchoalveolar lavage fluid and in arterial and venous serum were measured. Cardiac function was evaluated using 2-D echocardiography, and endothelial function was determined using wire myography on aortic sections. LPS induced a significant increase in serum IL-6 levels. SS reduced venous (P = 0.040) but not arterial concentrations of IL-6 (P = 0.112). SS improved the maximal vasodilatory response of the aorta to ACh (P = 0.004) but not to sodium nitroprusside (P = 1.000). SS also improved cardiac output (P = 0.023). Vasodilatory response to ACh was impaired when aorta from untreated mice was incubated with ex vivo IL-6 (P = 0.004), whereas in the aorta from mice pretreated with SS, the vasodilatory response did not change with IL-6 incubation (P = 0.387). SS significantly improved LPS-induced cardiovascular dysfunction possibly by reducing systemic expression of IL-6 and its downstream signaling pathways. These findings may explain how statins improve health outcomes in patients with ALI.     

Cinnamic acid pretreatment mitigates chilling stress of cucumber leaves through altering antioxidant enzyme activity

To elucidate the physiological mechanism of chilling stress mitigated by cinnamic acid (CA) pretreatment, a cucumber variety (Cucumis sativus cv. Jinchun no. 4) was pretreated with 50 μM CA for 2 d and was then cultivated at two temperatures (15/8 and 25/18 °C) for 1 d. We investigated whether exogenous CA could protect cucumber plantlets from chilling stress (15/8 °C) and examined whether the protective effect was associated with the regulation of antioxidant enzymes and lipid peroxidation. At 2 d, exogenous CA did not influence plant growth, but induced the activities of some antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), glutathione peroxidase (GSH-Px, EC 1.6.4.2) and ascorbate peroxidase (APX, EC 1.11.1.11) in cucumber leaves, and it also elevated the contents of reduced glutathione (GSH) and ascorbate (AsA). When CA was rinsed and the CA-pretreated seedlings were exposed to different temperatures, the antioxidant activities in leaves at 3 d had undergone additional change. Chilling increased the activities of CAT, GSH-PX, APX, GSH and AsA in leaves, but the combination of CA pretreatment and chilling enhanced the antioxidant activities even more. Moreover, chilling inhibited plant growth and increased the contents of malonaldehyde (MDA), superoxide radical (O₂⁻) and hydrogen peroxide (H₂O₂) in cucumber leaves, and the stress resulted in 87.5% of the second leaves being withered. When CA pretreatment was combined with the chilling stress, we observed alleviated growth inhibition and decreased contents of MDA, H₂O₂ and O₂⁻ in comparison to non-pretreated stressed plants, and found that the withered leaves occurred at a rate of 25.0%. We propose that CA pretreatment increases antioxidant enzyme activities in chilling-stressed leaves and decreases lipid peroxidation to some extent, enhancing the tolerance of cucumber leaves to chilling stress.     

哺乳动物中的DNA主动去甲基化

DNA甲基化是一种相对稳定且可遗传的表观遗传标记,在植物和动物细胞中均发现有DNA主动去甲基化现象,其机制在植物中已基本得到阐释,但在哺乳动物中尚未鉴定出一种有效的DNA去甲基化酶,并且DNA主动去甲基化途径也存在争议。文章综合分析了近期的文献资料,阐述了哺乳动物中发生DNA主动去甲基化的时空特异性,并从细胞和组织特异性角度介绍DNA主动去甲基化的可能通路和机制,即5-甲基胞嘧啶的氧化作用、5-甲基胞嘧啶脱氨基以及DNA修复等,旨在为破译表观遗传重编程过程提供理论依据。