Identification of differentially expressed salt-responsive proteins in roots of two perennial grass species contrasting in salinity tolerance

This study was designed to identify physiological responses and differential proteomic responses to salinity stress in roots of a salt-tolerant grass species, seashore paspalum (Paspalum vaginatum), and a salt-sensitive grass species, centipedegrass (Eremochloa ophiuroides). Plants of both species were exposed to salinity stress by watering the soil with 300 mM NaCl solution for 20 d in a growth chamber. The 2-DE analysis revealed that the abundance of 8 protein spots significantly increased and 14 significantly decreased in seashore paspalum, while 19 and 16 protein spots exhibited increase and decrease in abundance in centipedegrass, respectively. Eight protein spots that exhibited enhanced abundance in seashore paspalum under salinity stress were subjected to mass spectrometry analysis. Seven protein spots were successfully identified, they are peroxidase (POD, 2.36-fold), cytoplasmic malate dehydrogenase (cMDH, 5.84-fold), asorbate peroxidase (APX, 4.03-fold), two mitochondrial ATPSδ chain (2.26-fold and 4.78-fold), hypothetical protein LOC100274119 (5.01-fold) and flavoprotein wrbA (2.20-fold), respectively. Immunblotting analysis indicated that POD and ATPSδ chain were significantly up-regulated in seashore paspalum at 20 d of salinity treatment while almost no expression in both control and salt treatment of centipedegrass. These results indicated that the superior salinity tolerance in seashore paspalum, compared to centipedegrass, could be associated with a high abundance of proteins involved in ROS detoxification and energy metabolism.     

Effect of Angeli's salt on the glutamate/glutamine cycle activity and on glutamate excitotoxicity in the hamster retina

Glutamate is the main excitatory neurotransmitter in the retina, but it is toxic when present in excessive amounts. It is well known that NO is involved in glutamate excitotoxicity, but information regarding the possibility that NO-related species could reciprocally affect glutamate synaptic levels was not previously provided. The dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. The aim of the present work was to comparatively analyze the effect of nitroxyl and NO on the retinal glutamate/glutamine cycle in vitro activity. For this purpose, Angeli's salt (AS) and diethylamine NONOate (DEA/NO) were used as nitroxyl and NO donor, respectively. AS and DEA/NO significantly decreased retinal l-glutamate uptake and glutamine synthetase activity, but only AS decreased l-glutamine influx. Dithiothreitol prevented all the effects of AS and DEA/NO. The intravitreal injection of DEA/NO (but not AS) or a supraphysiological concentration of glutamate induced retinal histological alterations. Although AS could increase glutamate synaptic concentration in vitro, the histological alterations induced by glutamate were abrogated by AS. These results suggest that nitroxyl could regulate the hamster retinal glutamatergic pathway by acting through differential mechanisms at pre- and postsynaptic level.     

Molecular and expression characterization of a nanos1 homologue in Chinese sturgeon, Acipenser sinensis

The nanos gene family was essential for germ line development in diverse organisms. In the present study, the full-length cDNA of a nanos1 homologue in A. sinensis, Asnanos1, was isolated and characterized. The cDNA sequence of Asnanos1 was 1489 base pairs (bp) in length and encoded a peptide of 228 amino acid residues. Multiple sequence alignment showed that the zinc-finger motifs of Nanos1 were highly conserved in vertebrates. By RT-PCR analysis, Asnanos1 mRNAs were ubiquitously detected in all tissues examined except for the fat, including liver, spleen, heart, ovary, kidney, muscle, intestines, pituitary, hypothalamus, telencephalon, midbrain, cerebellum, and medulla oblongata. Moreover, a specific polyclonal antibody was prepared from the in vitro expressed partial AsNanos1 protein. Western blot analysis revealed that the tissue expression pattern of AsNanos1 was not completely coincided with that of its mRNAs, which was not found in fat, muscle and intestines. Additionally, by immunofluoresence localization, it was observed that AsNanos1 protein was in the cytoplasm of primary oocytes and spermatocytes. The presented results indicated that the expression pattern of Asnanos1 was differential conservation and divergence among diverse species.     

Kidney proteome responses in the teleost fish Paralichthys olivaceus indicate a putative immune response against Streptococcus parauberis

The proteomic response to bacterial infection in a teleost fish (Paralichthys olivaceus) infected with Streptococcus parauberis was analyzed using label-free protein quantitation coupled with LC-MS(E) tandem mass spectrometry. A total of 82 proteins from whole kidney, a major lymphoid organ in this fish, were found to be differentially expressed between healthy and diseased fish analyzed 6, 24, 72 and 120 h post-infection. Among the differentially expressed proteins, those involved in mediating immune responses (e.g., heat shock proteins, cathepsins, goose-type lysozyme and complement components) were most significantly up-regulated by infection. In addition, cell division cycle 48 (CDC48) and calreticulin, which are associated with cellular recovery and glycoprotein synthesis, were up-regulated in the universal protein group, whereas the other proteins in that group were down-regulated. There was continuous activation of expression of immune-associated proteins during infection, but there was also loss of expression of proteins not involved in immune function. We expect that our findings regarding immune response at the protein level would offer new insight into the systemic response to bacterial infection of a major immune organ in teleost fish.     

Substrate inhibition and allosteric regulation by heparan sulfate of Trypanosoma brucei cathepsin L

The cysteine protease brucipain is an important drug target in the protozoan Trypanosoma brucei, the causative agent of both Human African trypanosomiasis and Animal African trypanosomiasis. Brucipain is closely related to mammalian cathepsin L and currently used as a framework for the development of inhibitors that display anti-parasitic activity. We show that recombinant brucipain lacking the C-terminal extension undergoes inhibition by the substrate benzyloxycarbonyl-FR-7-amino-4-methylcoumarin at concentrations above the K_m, but not by benzyloxycarbonyl-VLR-7-amino-4-methylcoumarin. The allosteric modulation exerted by the substrate is controlled by temperature, being apparent at 25 °C but concealed at 37 °C. The behavior of the enzyme in vitro can be explained by discrete conformational changes caused by the shifts in temperature that render it less susceptible to substrate inhibition. Enzyme inhibition by the di-peptydyl substrate impaired the degradation of human fibrinogen at 25 °C, but not at 37 °C. We also found that heparan sulfate acts as a natural allosteric modulator of the enzyme through interactions that prevent substrate inhibition. We propose that brucipain shifts between an active and an inactive form as a result of temperature-dependent allosteric regulation.     

蛋白质O-GlcNAc修饰的检测技术

O-连接的N-乙酰葡糖胺（O-GlcNAc）修饰是位于细胞浆和细胞核蛋白质的丝氨酸或苏氨酸上的一种翻译后修饰,在高等真核生物细胞中广泛存在.越来越多的研究表明,O-GlcNAc修饰在代谢调控、压力应激、细胞周期、凋亡、糖尿病、心血管疾病和癌症等多种生理和病理过程中发挥重要作用,因此, O-GlcNAc修饰已受到众多生命科学领域研究人员的关注.然而,由于O-GlcNAc修饰与传统的N聚糖和O聚糖修饰有所不同,常规糖基化修饰的检测方法并不适用于O-GlcNAc.本文对O-GlcNAc修饰的检测及其修饰位点的确定方法进行了综述,并分析了各种方法的优缺点.     

Specific heteromeric association of four transmembrane peptides derived from platelet glycoprotein Ib-IX complex

As the receptor on the platelet surface for von Willebrand factor, glycoprotein (GP) Ib-IX complex is critically involved in hemostasis and thrombosis. How the complex is assembled from GP Ibα, GP Ibβ and GP IX subunits, all of which are type I transmembrane proteins, is not entirely clear. Genetic and mutational analyses have identified the transmembrane (TM) domains of these subunits as active participants in assembly of the complex. In this study, peptides containing the transmembrane domain of each subunit have been produced and their interaction with one another characterized. Only the Ibβ TM sequence, but not the Ibα and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ibβ-Ibα-Ibβ peptide complex (αβ₂) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibα and Ibβ TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native αβ₂ complex, reproducing the same effect by the IX subunit in cells expressing the GP Ib-IX complex. Specific fluorescence resonance energy transfer was observed between donor-labeled αβ₂ peptide complex and acceptor-conjugated IX TM peptide in micelles. Finally, the mutation D135K in the IX TM peptide could hamper both the formation of the αβ₂ complex and the energy transfer, consistent with its reported effect in the full-length complex. Overall, our results have demonstrated directly the native-like heteromeric interaction among the isolated Ibα, Ibβ and IX TM peptides, which provides support for the four-helix bundle model of the TM domains in the GP Ib-IX complex and paves the way for further structural analysis. The methods developed in this study may be applicable to other studies of heteromeric interaction among multiple TM helices.     

Study on structure-property-reactivity-function relationship of human neuronal growth inhibitory factor (hGIF)

Human metallothionein-3 (hMT3), also named as human neuronal growth inhibitory factor (hGIF), can inhibit the outgrowth of embryonic cortical neurons in the presence of brain extracts. In order to systematically study the structure-property-reactivity-function relationship of hGIF, our laboratory designed a series of mutants and studied their structure, property, reactivity and functions by a series of chemical and biological tools including UV spectroscopy, CD spectroscopy, NMR, chemical reaction and primary neuronal culture assays. In summary, we concluded that the bioactivity of hGIF was regulated by multiple factors, including the ⁶CPCP⁹ motif, an additional threonine insert at sequence position 5, domain-domain interactions, the structure and stability of the metal-thiolate cluster and the linker. Our studies provide more and more evidences which revealed that the bioactivity of hGIF is mainly related to the essential metal release and its characteristic conformation.     

Entamoeba histolytica: response of the parasite to metronidazole challenge on the levels of mRNA and protein expression

Entamoeba histolytica remains a cause of significant morbidity and mortality in many countries. Although metronidazole has been used for the treatment of amoebiasis for several decades, little is known on how the amoebae react to this challenge on the levels of mRNA and protein expression. In this study, we examined their response using a focused microarray, quantitative RT-PCR, and two-dimensional gel electrophoresis (2DE). The amoebae modestly increased the levels of mRNA coding for superoxide dismutase, peroxiredoxin, ferredoxin, thioredoxin reductase, and the galactose/N-acetylgalactosamine specific lectin light and heavy chains. The mRNAs encoding actin and the 70 kDa and 101 kDa heat shock proteins were decreased. All the changes occured within 1 h of exposition, with very little further changes. In addition, the proteome revealed only very few changes. Taken together, E. histolytica appears to make only modest mRNA and protein expression changes when confronted with an unknown chemical stress.