Fast evolution of the retroprocessed mitochondrial rps3 gene in Conifer II and further evidence for the phylogeny of gymnosperms

The popular view that plant mitochondrial genome evolves slowly in sequence has been recently challenged by the extraordinarily high substitution rates of mtDNA documented mainly from several angiosperm genera, but high substitution rate acceleration accompanied with great length variation has been very rarely reported in plant mitochondrial genes. Here, we studied evolution of the mitochondrial rps3 gene that encodes the ribosomal small subunit protein 3 and found a dramatically high variation in both length and sequence of an exon region of it in Conifer II. A sequence comparison between cDNA and genomic DNA showed that there are no RNA editing sites in the Conifer II rps3 gene. Southern blotting analyses of the total DNA and mtDNA, together with the real-time PCR analysis, showed that rps3 exists as a single mitochondrial locus in gymnosperms. It is very likely that the Conifer II rps3 gene has experienced retroprocessing, i.e., the re-integration of its cDNA into the mitochondrial genome, followed by an evolutionary acceleration due to the intron loss. In addition, the phylogenetic analysis of rps3 supports the sister relationship between conifers and Gnetales. In particular, the monophyly of conifer II is strongly supported by the shared loss of two rps3 introns. Our results also indicate that the mitochondrial gene tree would be affected in topology when the “edited” paralogs are analyzed together with their genomic sequences.     

Regulation of Exocytosis and Fusion Pores by Synaptotagmin-Effector Interactions

Synaptotagmin (syt) serves as a Ca²⁺ sensor in the release of neurotransmitters and hormones. This function depends on the ability of syt to interact with other molecules. Syt binds to phosphatidylserine (PS)-containing lipid bilayers as well as to soluble N-ethylmaleimide sensitive factor receptors (SNAREs) and promotes SNARE assembly. All these interactions are regulated by Ca²⁺, but their specific roles in distinct kinetic steps of exocytosis are not well understood. To explore these questions we used amperometry recording from PC12 cells to investigate the kinetics of exocytosis. Syt isoforms and syt I mutants were overexpressed to perturb syt-PS and syt-SNARE interactions to varying degrees and evaluate the effects on fusion event frequency and the rates of fusion pore transitions. Syt I produced more rapid dilation of fusion pores than syt VII or syt IX, consistent with its role in synchronous synaptic release. Stronger syt-PS interactions were accompanied by a higher frequency of fusion events and more stable fusion pores. By contrast, syt-SNARE interactions and syt-induced SNARE assembly were uncorrelated with rates of exocytosis. This associates the syt-PS interaction with two distinct kinetic steps in Ca²⁺ triggered exocytosis and supports a role for the syt-PS interaction in stabilizing open fusion pores.     

Matched pairs of human prostate stromal cells display differential tropic effects on LNCaP prostate cancer cells

Prostate stromal cells may play binary roles in the process of prostate cancer development. As the first to be encountered by infiltrating prostate cancer cells, prostate stromal cells form the first defense line against prostate cancer progression and metastasis. However, interaction between prostate cancer and stromal cells may facilitate the formation of a tumor microenvironment favoring cancer cell growth and survival. To establish an experimental system for studying the interaction between cancer and stromal cells, we isolated three matched pairs of normal and cancer-associated human prostate stromal clones. In this report, we describe the morphologic and behavioral characteristics of these cells and their effect on LNCaP prostate cancer cells in co-culture. Unlike LNCaP prostate cancer cells, the isolated prostate stromal clones are large fibroblast-like cells with a slow proliferation rate. Growth and survival of these clones are not affected by androgens. The stromal cells display high resistance to serum starvation, while cancer-associated stromal clones have differentiated survival ability. In co-culture experiments, the stromal cells protected some LNCaP prostate cancer cells from death by serum starvation, and cancer-associated stromal clones showed more protection. This work thus established a panel of valuable human prostate stromal cell lines, which could be used in co-culture to study the interaction between prostate cancer and prostate stromal cells.     

Folding and membrane insertion of amyloid‐beta (25–35) peptide and its mutants: Implications for aggregation and neurotoxicity

The mechanisms of interfacial folding and membrane insertion of the Alzheimer's amyloid‐β fragment Aβ(25–35) and its less toxic mutant, N27A‐Aβ(25–35) and more toxic mutant, M35A‐Aβ(25–35), are investigated using replica–exchange molecular dynamics in an implicit water‐membrane environment. This study simulates the processes of interfacial folding and membrane insertion in a spontaneous fashion to identify their general mechanisms. Aβ(25–35) and N27A‐Aβ(25–35) peptides share similar mechanisms: the peptides are first located in the membrane hydrophilic region where their C‐terminal residues form helical structures. The peptides attempt to insert themselves into the membrane hydrophobic region using the C‐terminal or central hydrophobic residues. A small portion of peptides can successfully enter the membrane's hydrophobic core, led by their C‐terminal residues, through the formation of continuous helical structures. No detectable amount of M35A‐Aβ(25–35) peptides appeared to enter the membrane's hydrophobic core. The three studied peptides share a similar helical structure for their C‐terminal five residues, and these residues mainly buried within the membrane's hydrophobic region. In contrast, their N‐terminal properties are markedly different. With respect to the Aβ(25–35), the N27A‐Aβ(25–35) forms a more structured helix and is buried deeper within the membrane, which may result in a lower degree of aggregation and a lower neurotoxicity; in contrast, the less structured and more water‐exposed M35A‐Aβ(25–35) is prone to aggregation and has a higher neurotoxicity. Understanding the mechanisms of Aβ peptide interfacial folding and membrane insertion will provide new insights into the mechanisms of neurodegradation and may give structure‐based clues for rational drug design preventing amyloid associated diseases. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Value of human amniotic epithelial cells in tissue engineering for cornea

Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.     

Degradation of phenanthrene by the endophytic fungus <Emphasis Type="Italic">Ceratobasidum stevensii</Emphasis> found in <Emphasis Type="Italic">Bischofia polycarpa</Emphasis>

Some strains of white rot fungi, non-lignolytic fungi and litter-decomposing basidiomycetes have been recognized as PAH degraders. The purpose of our research was to enlarge the scope of PAH-degrading fungi and explore the huge endophytic microorganism resource for bioremediation of PAHs. In this study, phenanthrene was used as a model PAHs compound. Nine strains of endophytic fungi isolated from four kinds of plant from Eupharbiaceae were screened for degradation of phenanthrene. The endophytic fungus Ceratobasidum stevensii (strain B6) isolated from Bischofia polycarpam showed high degradation efficiency and was selected for further studies. Into the fungal culture, 100 mg l⁻¹ phenanthrene was added, and after 10 days of incubation, about 89.51% of the phenanthrene was removed by strain B6. Extracellular ligninolytic enzyme activities of strain B6 were tested. The results showed that manganese peroxidase [MnP] was the predominant ligninolytic enzyme and that its production was greatly induced by the presence of phenanthrene. To confirm the involvement of MnP in phenanthrene degradation, promotion and inhibition studies on MnP in different concentration level of Mn²⁺ and NaN₃ were performed. Additionally, fungal mycelium-free and resuspended experiments were carried out. The results showed no apparent correlation between MnP activity and phenanthrene degradation. The mycelium and fresh medium were the crucial factors affecting the degradation of phenanthrene. To date, this is the first report on PAH degradation by Ceratobasidum stevensii. This study suggests that endophytic fungi might be a novel and important resource for microorganisms that have PAH-degrading capabilities.     

Interference Between Tobacco necrosis virus and Turnip crinkle virus in Nicotiana benthamiana

Mixed infections of Nicotiana benthamiana plants by Tobacco necrosis virus (TNV) and Turnip crinkle virus (TCV) exhibited an interference interaction. Accumulation of TNV (+)RNA as well as capsid protein in mixed infection were considerably lower than that of singly infected plants. There were also a slight reduction in the levels of TCV (+)RNA and capsid protein in doubly infected plants, which displayed the concentration of both viruses decreased in dually infected plants. Tissue immunoblot analysis of systemic N. benthamiana leaves infected by TNV and TCV singly or doubly showed the interference between the two viruses in situ, which exhibited the decrease of both viruses in doubly infected leaves although the distribution of them did not change remarkably. These results were consistent with the hybridization analysis of viral genomic RNA and coat protein. Both cross‐protection test and mixed infection of the two viruses confirmed TCV had relatively stronger interference to the infection of TNV. Interference infection by TNV and TCV induced higher increase in the levels of cytochrome pathway respiration and alternative pathway respiration in host plants, especially the latter. Interference often occurred in different strains of one kind of virus or two different closely related viruses in one genus. Our results showed that interference could also occur in different viruses belonging to different genera.     

The β1-adrenergic receptor mediates extracellular signal-regulated kinase activation via Gαs

β-Adrenergic receptors can activate extracellular signal-regulated kinases (ERKs) via different mechanisms. In this study, we investigated the molecular mechanism of β1-adrenergic receptor (β1AR)-mediated ERK activation in African green monkey kidney COS-7 cells. Treatment of cells with isoproterenol (ISO), a β1AR selective agonist, induced phosphorylation of ERK1/2 in a dose-dependent manner. ISO-stimulated ERK phosphorylation was not influenced by the Gβγ inhibitor, βAR kinase carboxyl terminal (βARKct) or by the Gi inhibitor, pertussis toxin (PTX), but it was clearly abolished via inhibition of protein kinase A (PKA) with H89, or of mitogen-activated protein kinase kinase (MEK1) with PD98059, revealing that the Gαs subunit is involved in ERK regulation through the PKA/MEK1 pathway. We also tested the effect of the adenylate cyclase activator forskolin on ERK activation, and the result was identical to that of ISO stimulation. Moreover, pretreatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or with the Src tyrosine kinase inhibitor PP2 did not affect ERK activation. These observations suggest a mechanism of β1AR-mediated ERK activity that involves the Gαs subunit, but not EGFR or Src tyrosine kinase.