Resilience of Rhodobacter sphaeroides cytochrome bc1 to heme c1 ligation changes

Typically, c hemes are bound to the protein through two thioether bonds to cysteines and two axial ligands to the heme iron. In high-potential class I c-type cytochromes, these axial ligands are commonly His-Met. A change in this methionine axial ligand is often correlated with a dramatic drop in the heme redox potential and loss of function. Here we describe a bacterial cytochrome c with an unusual tolerance to the alternations in the heme ligation pattern. Substitution of the heme ligating methionine (M185) in cytochrome c1 of the Rhodobacter sphaeroides cytochrome bc1 complex with Lys and Leu lowers the redox midpoint potential but not enough to prevent physiologically competent electron transfer in these fully functional variants. Only when Met-185 is replaced with His is the drop in the redox potential sufficiently large to cause cytochrome bc1 electron transfer chain failure. Functional mutants preserve the structural integrity of the heme crevice: only the nonfunctional His variant allows carbon monoxide to bind to reduced heme, indicating a significant opening of the heme environment. This range of cytochrome c1 ligand mutants exposes both the relative resilience to sixth axial ligand change and the ultimate thermodynamic limits of operation of the cofactor chains in cytochrome bc1.     

An integrated BAC and genome sequence physical map of Phytophthora sojae

Phytophthora spp. are serious pathogens that threaten numerous cultivated crops, trees, and natural vegetation worldwide. The soybean pathogen P. sojae has been developed as a model oomycete. Here, we report a bacterial artificial chromosome (BAC)-based, integrated physical map of the P. sojae genome. We constructed two BAC libraries, digested 8,681 BACs with seven restriction enzymes, end labeled the digested fragments with four dyes, and analyzed them with capillary electrophoresis. Fifteen data sets were constructed from the fingerprints, using individual dyes and all possible combinations, and were evaluated for contig assembly. In all, 257 contigs were assembled from the XhoI data set, collectively spanning approximately 132 Mb in physical length. The BAC contigs were integrated with the draft genome sequence of P. sojae by end sequencing a total of 1,440 BACs that formed a minimal tiling path. This enabled the 257 contigs of the BAC map to be merged with 207 sequence scaffolds to form an integrated map consisting of 79 superscaffolds. The map represents the first genome-wide physical map of a Phytophthora sp. and provides a valuable resource for genomics and molecular biology research in P. sojae and other Phytophthora spp. In one illustration of this value, we have placed the 350 members of a superfamily of putative pathogenicity effector genes onto the map, revealing extensive clustering of these genes.     

Molecular characterization of geminivirus-derived small RNAs in different plant species

DNA geminiviruses are thought to be targets of RNA silencing. Here, we characterize small interfering (si) RNAs—the hallmarks of silencing—associated with Cabbage leaf curl begomovirus in Arabidopsis and African cassava mosaic begomovirus in Nicotiana benthamiana and cassava. We detected 21, 22 and 24 nt siRNAs of both polarities, derived from both the coding and the intergenic regions of these geminiviruses. Genetic evidence showed that all the 24 nt and a substantial fraction of the 22 nt viral siRNAs are generated by the dicer-like proteins DCL3 and DCL2, respectively. The viral siRNAs were 5′ end phosphorylated, as shown by phosphatase treatments, and methylated at the 3′-nucleotide, as shown by HEN1 miRNA methylase-dependent resistance to β-elimination. Similar modifications were found in all types of endogenous and transgene-derived siRNAs tested, but not in a major fraction of siRNAs from a cytoplasmic RNA tobamovirus. We conclude that several distinct silencing pathways are involved in DNA virus-plant interactions.     

Effects of elevated atmospheric CO2 on soil enzyme activities at different nitrogen application treatments

It has been predicted that elevated atmospheric CO₂ will increase enzyme activity as a result of CO₂-induced carbon entering the soil. The objective of this study was to investigate the effects of elevated atmospheric CO₂ on soil enzyme activities under a rice/wheat rotation. This experiment was conducted in Wuxi, Jiangsu, China as part of the China FACE (Free Air Carbon Dioxide Enrichment) Project. Two atmospheric CO₂ concentrations (580±60) and (380±40) μmol·mol^-1) and three N application treatments (low-150, normal-250 and high-350 kg N·hm^-2) were included. Soil samples (0-10 cm) were collected for analysis of β-glucosidase, invertase, urease, acid phosphates and β-glucosaminidase activities. The results revealed that with elevated atmospheric CO₂ β-glucosidase activity significantly decreased (P < 0.05) at low N application rates; had no significant effect with a normal N application rate; and significantly increased (P < 0.05) with a high N application rate. For urease activity, at low and normal N application rates (but not high N application rate), elevated atmospheric CO₂ significantly increased (P < 0.05) it. With acid phosphatase elevated atmospheric CO₂ only had significant higher effects (P < 0.05) at high N application rates. Under different CO₂ concentration, effects of N fertilization are also different. Soil β-glucosidase activity at ambient CO₂ concentration decreased with N fertilization, while it increased at elevated CO₂ concentration. In addition, invertase and acid phosphatase activities at elevated CO₂ concentration, significantly increased (P < 0.05) with N treatments, but there was no effect with the ambient CO₂ concentration. For urease activity, at ambient CO₂ concentration, N fertilization increased it significantly (P < 0.05), whereas at elevated CO₂ concentration it was not significant. Additionally, with β-glucosaminidase activity, there were no significant effects from N application. In general, then, elevated atmospheric CO₂ increased soil enzyme activity, which may be attributed to the following two factors: (1) elevated atmospheric CO₂ led to more plant biomass in the soil, which in turn stimulated soil microbial biomass and activity; and (2) elevated atmospheric CO₂ increased plant photosynthesis, thereby increasing plant-derived soil enzymes.     

Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules

BACKGROUND: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general. METHODS: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes. RESULTS: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later. CONCLUSIONS: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.     

Preparation of yuanhuacine and relative daphne diterpene esters from Daphne genkwa and structure-activity relationship of potent inhibitory activity against DNA topoisomerase I

Two new daphne diterpene esters Yuanhuajine (2) and Yuanhuagine (4), together with three known daphne diterpene esters yuanhuacine (1), yuanhuadine (3), and yuanhuapine (5), were isolated and identified from Daphne genkwa, a traditional Chinese medicine. Their structures were elucidated by a combination of UV, IR, MS and NMR ((1)H NMR, (13)C NMR, HSQC, and HMBC) spectra. In order to explore the structure-activity relationship, three compounds 6, 7, and 8 were prepared as three derivatives of 1. Inhibitory activities against DNA topoisomerase I (topo I) were assessed for the compounds 1-8. These compounds, except for 8, exhibited potent inhibitory activities against DNA topo I at IC(50) levels of 11.1-53.4 microM and they are new type of topo I inhibitors bearing different structures compared with the known topo I inhibitors. The agarose-gel electrophoresis experiments showed that the orthoester group of daphne diterpene esters was necessary for the inhibitory activity against DNA topo I, and the inhibition against DNA topo I is probably one of the anti-tumor mechanisms of daphne diterpene esters.     

Proteome analysis of human lung squamous carcinoma

Few lung cancer-specific molecular markers have been established in regard of "early-stage" diagnosis and prognosis. In this study the proteome analysis of human lung squamous carcinoma (hLSC) was carried out using two strategies to explore the carcinogenic mechanisms and identify its molecular markers more directly and comprehensively. Comparative proteome analysis on 20 hLSC tissues and paired normal bronchial epithelial tissues revealed 76 differential proteins, among which 68 proteins were identified by PMF. The identified proteins fell into three categories: oncoproteins, cell cycle regulators and signaling molecules. To validate the identified differential proteins, the expressions levels of three differential proteins mdm2, c-jun and EGFR were determined by immunohistochemical staining and immunoblots. The results verified proteome analysis results. Serological proteome analysis (SERPA) of ten hLSC tissues was performed to identify the tumor-associated antigens. The results revealed 36 +/- 8 differential proteins reactive with patients' autologous sera, of which 14 proteins were identified. Six of the 14 proteins, alpha enolase, pre-B cell-enhancing factor precursor, triosephosphate isomerase, phosphoglycerate mutase 1, fructose-bisphosphate aldolase A, and guanine nucleotide-binding protein beta subunit-like protein, were also up-regulated in hLSCs in the comparative proteomic study, which suggests potential application of these 6 hLSC-associated antigens in diagnosis and therapy of hLSC.     

Solution structure and dynamics of human metallothionein-3 (MT-3)

Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the alpha-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (beta-domain) and one in the C-terminus (alpha-domain). The overall fold of the alpha-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the beta-domain exhibits similar internal motion to the alpha-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.     

A 1-D model to explore the effects of tissue loading and tissue concentration gradients in the revised Starling principle

The recent experiments in Hu et al. (Am J Physiol Heart Circ Physiol 279: H1724-H1736, 2000) and Adamson et al. (J Physiol 557: 889-907, 2004) in frog and rat mesentery microvessels have provided strong evidence supporting the Michel-Weinbaum hypothesis for a revised asymmetric Starling principle in which the Starling force balance is applied locally across the endothelial glycocalyx layer rather than between lumen and tissue. These experiments were interpreted by a three-dimensional (3-D) mathematical model (Hu et al.; Microvasc Res 58: 281-304, 1999) to describe the coupled water and albumin fluxes in the glycocalyx layer, the cleft with its tight junction strand, and the surrounding tissue. This numerical 3-D model converges if the tissue is at uniform concentration or has significant tissue gradients due to tissue loading. However, for most physiological conditions, tissue gradients are two to three orders of magnitude smaller than the albumin gradients in the cleft, and the numerical model does not converge. A simpler multilayer one-dimensional (1-D) analytical model has been developed to describe these conditions. This model is used to extend Michel and Phillips's original 1-D analysis of the matrix layer (J Physiol 388: 421-435, 1987) to include a cleft with a tight junction strand, to explain the observation of Levick (Exp Physiol 76: 825-857, 1991) that most tissues have an equilibrium tissue concentration that is close to 0.4 lumen concentration, and to explore the role of vesicular transport in achieving this equilibrium. The model predicts the surprising finding that one can have steady-state reabsorption at low pressures, in contrast to the experiments in Michel and Phillips, if a backward-standing gradient is established in the cleft that prevents the concentration from rising behind the glycocalyx.