Genomics and expression analysis of DHHC-cysteine-rich domain S-acyl transferase protein family in apple

S-acylation is one of a group of lipid modifications that occurs on eukaryotic proteins, mediated by DHHC-CRD-containing proteins, which plays an important role in regulating the membrane association, trafficking and function of target proteins. Although genome-wide identification of PAT family has been carried out in yeast, mice, humans and Arabidopsis, little is known about apple PAT genes. In this study, a total of 33 putative apple PAT proteins, containing DHHC-CRD by domain analysis, have been identified, and were classified into three groups according to the phylogenetic analysis of PAT proteins in apple and Arabidopsis. More complex TMDs in the most MdPATs revealed the PM location of the gene family. Gene structure, gene chromosomal location and paralogs analysis of MdPAT genes within the apple genome demonstrated that tandem and segmental duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of the PAT gene family in apple. According to the microarray and expressed sequence tag (ESTs) analysis, the different expression patterns indicate that they may play different roles during fruit development and rootstock-scion interactions process. Moreover, PATs were performed expression profile analyses in different tissues, indicating that the PATs are involved in various aspects of physiological and developmental processes of apple. To our knowledge, this is the first report of a genome-wide analysis of the apple PAT gene family, and this genomic analysis of apple DHHC-CRD PAT genes provides the first step towards a functional study of this gene family in apple.     

The unusual intersubunit ferroxidase center of Listeria innocua Dps is required for hydrogen peroxide detoxification but not for iron uptake. A study with site-specific mutants

The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.     

Clonal immune responses of Mycobacterium-specific γδ T cells in tuberculous and non-tuberculous tissues during M. tuberculosis infection

Background

We previously demonstrated that unvaccinated macaques infected with large-dose M.tuberculosis(Mtb) exhibited delays for pulmonary trafficking of Ag-specific αβ and γδ T effector cells, and developed severe lung tuberculosis(TB) and “secondary” Mtb infection in remote organs such as liver and kidney. Despite delays in lungs, local immunity in remote organs may accumulate since progressive immune activation after pulmonary Mtb infection may allow IFNγ-producing γδ T cells to adequately develop and traffic to lately-infected remote organs. As initial efforts to test this hypothesis, we comparatively examined TCR repertoire/clonality, tissue trafficking and effector function of Vγ2Vδ2 T cells in lung with severe TB and in liver/kidney without apparent TB.

Methodology/Principal Findings

We utilized conventional infection-immunity approaches in macaque TB model, and employed our decades-long expertise for TCR repertoire analyses. TCR repertoires in Vγ2Vδ2 T-cell subpopulation were broad during primary Mtb infection as most TCR clones found in lymphoid system, lung, kidney and liver were distinct. Polyclonally-expanded Vγ2Vδ2 T-cell clones from lymphoid tissues appeared to distribute and localize in lung TB granuloms at the endpoint after Mtb infection by aerosol. Interestingly, some TCR clones appeared to be more predominant than others in lymphocytes from liver or kidney without apparent TB lesions. TCR CDR3 spetratyping revealed such clonal dominance, and the clonal dominance of expanded Vγ2Vδ2 T cells in kidney/liver tissues was associated with undetectable or low-level TB burdens. Furthermore, Vγ2Vδ2 T cells from tissue compartments could mount effector function for producing anti-mycobacterium cytokine.

Conclusion

We were the first to demonstrate clonal immune responses of mycobacterium-specific Vγ2Vδ2 T cells in the lymphoid system, heavily-infected lungs and lately subtly-infected kidneys or livers during primary Mtb infection. While clonally-expanded Vγ2Vδ2 T cells accumulated in lately-infected kidneys/livers without apparent TB lesions, TB burdens or lesions appeared to impact TCR repertoires and tissue trafficking patterns of activated Vγ2Vδ2 T cells.     

Expression and purification of chicken beta interferon and its antivirus immunological activity

Interferon beta (IFN-β) belongs to a class of natural proteins that can inhibit virus replication. In this paper, nucleic acid sequence encoding chicken interferon beta (chIFN-β) mature protein was cloned and highly expressed in Escherichia coli (E. coli) by 1mM IPTG induction. The expressed chIFN-β was about 20% of total bacterial protein. The recombinant protein in form of inclusion body was then solubilised, refolded and purified to a purity of greater than 95% by immobilized metal ion affinity chromatography (IMAC). This recombinant chIFN-β could inhibit vesicular stomatitis Indiana virus (VSV) and infectious bursal disease virus (IBDV) replication in vitro. Animal experiments showed that the recombinant chIFN-β could decrease pathological lesions caused by the IBDV in bursa of Fabricius. These results will provide useful information for further investigations on veterinary clinical applications and fundamental research.     

福建沿海地区的景观生态建设途径探讨

景观生态学研究景观和区域尺度的资源、环境经营与管理问题 ,具有综合整体性和宏观区域性特色 ,并以中尺度的景观结构与生态过程关系研究见长[8] 。自 2 0世纪 80年代后期 ,景观生态学逐渐成为资源、环境生态方面研究的一个热点。福建沿海地区是指北从福鼎县南至诏安县的广大沿海县、市 ,行政上包括 33个县市行政单位。该区域是福建省经济最为活跃 ,发展速度最快的地区 ,近 2 0年来经济年均增长率达 18% ,随着经济的发展 ,带来了一系列的环境问题 :城市生活污水、生活垃圾、工业废料和农业化肥等都成为福建省沿海地区严重的污染源。如何协调…     

瘤足蕨科及其近缘类群植物叶表皮微形态扫描电镜观察

瘤足蕨科是一个自然的群,但由于各类群之间的形态差异及种内变异比较大,其分类和系统位置一直有争议。该研究利用扫描电子显微镜对瘤足蕨科10种及其近缘类群桫椤科2种、蚌壳蕨科1种共13种植物,即瘤足蕨(Plagiogyria adnata)、耳形瘤足蕨(P.stenoptera)、镰羽瘤足蕨(P.falcata)、峨嵋瘤足蕨(P.assurgens)、密羽瘤足蕨(P.pycnophylla)、灰背瘤足蕨(P.glauca)、华东瘤足蕨(P.japonica)、华中瘤足蕨(P.euphlebia)、日本瘤足蕨(P.matsumureana)、美洲瘤足蕨(P.pectinata)、桫椤(Alsophila spinulosa)、西亚黑桫椤(A.khasyana)和金毛狗(Cibotium barometz)的叶表皮微形态进行了观察和比较,探讨其分类学意义,并据此编制了供试10个种类的分类检索表。结果表明:13种瘤足蕨科及其近缘类群植物气孔均仅分布在叶下表皮,气孔微下陷,气孔器椭圆形至圆形;叶上下表皮角质层纹饰复杂,类型多样,少数类群叶下表皮具毛状物;多数类群气孔外拱盖凹陷;气孔外拱盖内缘浅波状或齿状;保卫细胞两极大多数有明显的"T"型加厚。不同种间叶表皮扫描电镜特征表现出一定差异,对种的划分有一定的分类学鉴定意义。     

影响被子植物花粉管萌发的因素--关于"影响被子植物花粉管萌发因素的探究"一文的补充

进行创新教育,培养学生创造性思维,并引导学生进行研究性学习是目前时代的要求。这也是目前高中新教材增加了许多研究性实验的原因。“观察被子植物的花粉管”便是其中的1个。虽然,花粉管的发现距今已有180年的历史,但观察花粉管的萌发及生殖细胞（或精子）在其中的活动,仍然是认识和理解被子植物有性生殖过程的一个重要环节。这应该是实验设计者的最初用意。研究性实验给我们的教和学提供了很大的拓展空间．如“影响被子植物花粉管萌发因素的探究”（见《生物学通报》2003年第7期,以下简称“探究”）一文在引导学生观察被子植物花粉管基础上,继而对影响花粉萌发的因素做了探究。     

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 μM) and capsaicin (1 μM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 μM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 μM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.     

Mitochondrial dysfunction and increased reactive oxygen species impair insulin secretion in sphingomyelin synthase 1-null mice

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic β-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic β-cells.