Processes of organic carbon in mangrove ecosystems

In addition to carbon accumulation in plants, processes of organic carbon in mangrove ecosystems include origins of sediment organic carbon, carbon fluxes between mangroves and their adjacent systems (coastal waters and atmosphere), and cycling processes. Sediment organic carbon originates from suspending solids in coastal waters, mangrove plants and benthic algae. In mangroves with low organic carbon content in sediments, tidal seawater is the main origin of sediment organic carbon, while in mangroves with high sediment organic carbon contents, sediment organic carbon mainly originates from mangrove plants. Due to tidal flush, there is large material exchange between mangrove ecosystems and their adjacent coastal waters. In China, exports of organic carbon in litter falls and dissolved organic carbon from mangroves to their adjacent coastal waters have not been documented. Processes of mangrove litter falls, including production, decomposition, export and animal consumption, determine linkages among organic carbon among mangrove plants, secondary production and coastal ocean. Consumers especially benthic animals may influence organic carbon in mangrove ecosystems, because (1) their consumption rates are high, and their selective feeding on some food sources will change the relative quantities of export, bury and mineralization of organic carbon from different origins; (2) their consumption is much more than assimilation, resulting in the changes in sizes, forms and qualities of non-assimilated organic matters, and then the changes in availability of export, consumption or mineralization of organic carbon. Respiration and sulfate reduction are important mineralization processes of organic carbon in mangrove sediments. Mineralization rates of organic carbon in mangrove sediments are influenced by quantities, activities and particle sizes of organic matters, and other factors such as forest ages, root activities and animal burrowing activities. Researches on processes of mangrove organic carbon should be based on open systems, and ecological processes of organic carbon should be coupled with vegetation restoration.     

人乳铁蛋白cDNA 基因乳腺表达载体的构建与鉴定

为了构建人乳铁蛋白基因 (hLF) 的乳腺表达载体并验证其在乳腺细胞中的表达情况,本载体以山羊β-casein基因上游包括启动子、外显子1、内含子1、部分外显子2作为5′端调控序列,下游包括部分外显子7、内含子7、外显子8、内含子8、外显子9及3′部分基因组片段作为3′端调控序列,长度分别为6.2 kb和7.1 kb,将hLF基因 (目的基因) 和Neo基因 (筛选标记) 分别插入到5′端调控序列和3′端调控序列的下游,构建成pBC1-hLF-Neo载体,其全长为25.348 kb。为了检测该载体的生物学     

Cytoplasmic linker protein-170 enhances spreading and phagocytosis in activated macrophages by stabilizing microtubules

Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-gamma and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated alpha-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-gamma/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-gamma/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.     

Molecular and functional dissection of a putative RNA-binding region in yeast mitochondrial leucyl-tRNA synthetase

Aminoacylation and editing by leucyl-tRNA synthetases (LeuRS) require migration of the tRNA acceptor stem end between the canonical aminoacylation core and a separate domain called CP1 that is responsible for amino acid editing. The LeuRS CP1 domain can also support group I intron RNA splicing in the yeast mitochondria, although splicing-sensitive sites have been identified on the main body. The RDW peptide, a highly conserved peptide within an RDW-containing motif, resides near one of the beta-strand linkers that connects the main body to the CP1 domain. We hypothesized that the RDW peptide was important for interactions of one or more of the LeuRS-RNA complexes. An assortment of X-ray crystallography structures suggests that the RDW peptide is dynamic and forms unique sets of interactions with the aminoacylation and editing complexes. Mutational analysis identified specific sites within the RDW peptide that failed to support protein synthesis activity in complementation experiments. In vitro enzymatic investigations of mutations at Trp445, Arg449, and Arg451 in yeast mitochondrial LeuRS suggested that these sites within the RDW peptide are critical to the aminoacylation complex, but impacted amino acid editing activity to a much less extent. We propose that these highly conserved sites primarily influence productive tRNA interactions in the aminoacylation complex.     

Necrotic death of neurons following an excitotoxic insult is prevented by a peptide inhibitor of c-jun N-terminal kinase

Peptide inhibitors of c-Jun N-terminal kinase (JNK) have been shown to potently protect against cerebral ischemia. The protective effect has been ascribed to prevention of apoptosis, but cell death following cerebral ischemia is a consequence of both apoptotic and necrotic cell death. We evaluated whether a peptide inhibitor (TAT-TIJIP) of JNK could prevent necrotic cell death in an in vitro model of excitotoxic neuronal death. We find that TAT-TIJIP effectively prevented cell death by interfering with several processes which have been identified as leading to cell death by necrosis. In particular, reactive oxygen species production was reduced, as indicated by an 88% decrease in the rate of dihydroethidium fluorescence in the presence of TAT-TIJIP. Furthermore, TAT-TIJIP attenuated the increase in cytosolic calcium following the excitotoxic insult. The potent neuroprotective properties of JNK peptide inhibitors likely reflects their abilities to prevent cell death by necrosis as well as apoptosis.     

Neutralization of human papillomavirus with monoclonal antibodies reveals different mechanisms of inhibition

The mechanisms of human papillomavirus (HPV) neutralization by antibodies are incompletely understood. We have used HPV16 pseudovirus infection of HaCaT cells to analyze how several neutralizing monoclonal antibodies (MAbs) generated against HPV16 L1 interfere with the process of keratinocyte infection. HPV16 capsids normally bind to both the cell surface and extracellular matrix (ECM) of HaCaT cells. Surprisingly, two strongly neutralizing MAbs, V5 and E70, did not prevent attachment of capsids to the cell surface. However, they did block association with the ECM and prevented internalization of cell surface-bound capsids. In contrast, MAb U4 prevented binding to the cell surface but not to the ECM. The epitope recognized by U4 was inaccessible when virions were bound to the cell surface but became accessible after endocytosis, presumably coinciding with receptor detachment. Treatment of capsids with heparin, which is known to interfere with binding to cell surface heparan sulfate proteoglycans (HSPGs), also resulted in HPV16 localization to the ECM. These results suggest that the U4 epitope on the intercapsomeric C-terminal arm is likely to encompass the critical HSPG interaction residues for HPV16, while the V5 and E70 epitopes at the apex of the capsomer overlap the ECM-binding sites. We conclude that neutralizing antibodies can inhibit HPV infection by multiple distinct mechanisms, and understanding these mechanisms can add insight to the HPV entry processes.     

Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha subunits

AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic alpha subunit and two regulatory subunits, beta and gamma. It had been reported that the mammalian AMPK alpha subunit contained an autoinhibitory domain (alpha1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the alpha subunit (alpha1-(313-335)), which includes a predicted alpha-helix, is responsible for alpha subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for alpha1 autoinhibition. An autoinhibitory structural model of human AMPK alpha1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 A and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK alpha1-(1-394) with deletion of residues 313-335 (Deltaalpha394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with Deltaalpha394, V298G, or L328Q mutants, which indicated that these AMPK alpha1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK alpha autoinhibitory function.     

Plasma membrane targeting is essential for Rem-mediated Ca2+ channel inhibition

The small GTPase Rem is a potent negative regulator of high voltage-activated Ca(2+) channels and a known interacting partner for Ca(2+) channel accessory beta subunits. The mechanism for Rem-mediated channel inhibition remains controversial, although it has been proposed that Ca(V)beta association is required. Previous work has shown that a C-terminal truncation of Rem (Rem-(1-265)) displays reduced in vivo binding to membrane-localized beta 2a and lacks channel regulatory function. In this paper, we describe a role for the Rem C terminus in plasma membrane localization through association with phosphatidylinositol lipids. Moreover, Rem-(1-265) can associate with beta 2a in vitro and beta 1b in vivo, suggesting that the C terminus does not directly participate in Ca(V)beta association. Despite demonstrated beta 1b binding, Rem-(1-265) was not capable of regulating a Ca(V)1.2-beta 1b channel complex, indicating that beta subunit binding is not sufficient for channel regulation. However, fusion of the CAAX domain from K-Ras4B or H-Ras to the Rem-(1-265) C terminus restored membrane localization and Ca(2+) channel regulation, suggesting that beta binding and membrane localization are independent events required for channel inhibition.     

Gap junction-mediated intercellular communication in the immune system

Immune cells are usually considered non-attached blood cells, which would exclude the formation of gap junctions. This is a misconception since many immune cells express connexin 43 (Cx43) and other connexins and are often residing in tissue. The role of gap junctions is largely ignored by immunologists as is the immune system in the field of gap junction research. Here, the current knowledge of the distribution of connexins and the function of gap junctions in the immune system is discussed. Gap junctions appear to play many roles in antibody productions and specific immune responses and may be important in sensing danger in tissue by the immune system. Gap junctions not only transfer electrical and metabolical but also immunological information in the form of peptides for a process called cross-presentation. This is essential for proper immune responses to viruses and possibly tumours. Until now only 40 research papers on gap junctions in the immune system appeared and this will almost certainly expand with the increased mutual interest between the fields of immunology and gap junction research.     

A genome-wide gain-of-function analysis of rice genes using the FOX-hunting system

The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.