Cytochalasin D can improve heterologous protein productivity in adherent Chinese hamster ovary cells

We generated a series of adherent gene-amplified CHO clones expressing human secreted alkaline phosphatase (SEAP) as a model for heterologous protein production. Clones demonstrate a 26- to 52-fold increase in productivity compared to controls after dhfr/methotrexate-mediated gene amplification and clone selection. SEAP is stably expressed in these clones over at least a 6-week period without significant productivity loss. Two-dimensional protein electrophoresis identified 21 proteins that exhibited altered expression in clones of increasing SEAP productivity. Based on MALDI TOF/TOF mass spectrometry of relevant protein spots, changes in translation, energy pathways, chaperones, regulatory proteins, and cytoskeletal proteins were observed, including a 4-fold expression increase in actin capping protein. We hypothesized that an alteration of the actin cytoskeleton using cytochalasin D as a mimic for actin-capping protein could have a beneficial effect on heterologous protein secretion. Treatment with 0.5 mug/mL cytochalasin D increased SEAP productivity 2- to 3-fold compared to an amplified control which resulted in an increase in productivity from 52- to 150-fold compared to a nonamplified parent.     

Expression profiling of murine double-negative regulatory T cells suggest mechanisms for prolonged cardiac allograft survival

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 +/- 11.1 days) compared with untreated controls (22.8 +/- 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including Fc epsilon RI gamma subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival.     

Applications of affinity chromatography in proteomics

Affinity chromatography is a powerful protein separation method that is based on the specific interaction between immobilized ligands and target proteins. Peptides can also be separated effectively by affinity chromatography through the use of peptide-specific ligands. Both two-dimensional electrophoresis (2-DE)- and non-2-DE-based proteomic approaches benefit from the application of affinity chromatography. Before protein separation by 2-DE, affinity separation is used primarily for preconcentration and pretreatment of samples. Those applications entail the removal of one protein or a class of proteins that might interfere with 2-DE resolution, the concentration of low-abundance proteins to enable them to be visualized in the gel, and the classification of total protein into two or more groups for further separation by gel electrophoresis. Non-2-DE-based approaches have extensively employed affinity chromatography to reduce the complexity of protein and peptide mixtures. Prior to mass spectrometry (MS), preconcentration and capture of specific proteins or peptides to enhance sensitivity can be accomplished by using affinity adsorption. Affinity purification of protein complexes followed by identification of proteins by MS serves as a powerful tool for generating a map of protein-protein interactions and cellular locations of complexes. Affinity chromatography of peptide mixtures, coupled with mass spectrometry, provides a tool for the study of protein posttranslational modification (PTM) sites and quantitative proteomics. Quantitation of proteomes is possible via the use of isotope-coded affinity tags and isolation of proteolytic peptides by affinity chromatography. An emerging area of proteomics technology development is miniaturization. Affinity chromatography is becoming more widely used for exploring PTM and protein-protein interactions, especially with a view toward developing new general tag systems and strategies of chemical derivatization on peptides for affinity selection. More applications of affinity-based purification can be expected, including increasing the resolution in 2-DE, improving the sensitivity of MS quantification, and incorporating purification as part of multidimensional liquid chromatography experiments.     

Gene knockdown by intro-thoracic injection of double-stranded RNA in the brown planthopper,Nilaparvata lugens

RNA interference (RNAi) is a powerful strategy for gene function study in insects. Here, we described the development of a RNAi technique by microinjection of double-stranded RNA (dsRNA) in the brown planthopper Nilaparvata lugens. Based on the mortality and RNAi efficiency criteria, the conjunctive between prothorax and mesothorax was selected as the injection site and 50 nl as injection volume. Three genes with different expression patterns were selected to evaluate the RNAi efficiency. A comparable 40% decrease of gene expression was observed at the 4th day after injection for the ubiquitously expressed calreticulin and the gut specific cathepsin-B genes, but only 25% decrease at the 5th day for the central nervous system specific Nlβ2 gene. Double injection could increase the RNAi efficiency, such as from 25% to 53% for Nlβ2 gene. The gene knockdown technique developed in this study will be an essential post-genomic tool for further investigations in N. lugens.     

Conservation value of degraded habitats for forest birds in southern Peninsular Malaysia

Clearance of tropical forest for agricultural purposes is generally assumed to seriously threaten the survival of forest species. In this study, we quantified the conservation value, for forest bird species, of three degraded habitat types in Peninsular Malaysia, namely rubber tree plantations, oil palm plantations, and open areas. We surveyed these degraded habitats using point counts to estimate their forest bird species richness and abundance. We assessed whether richness, abundance, and activities of different avian dietary groups (i.e. insectivores and frugivores) varied among the habitats. We identified the critical habitat elements that accounted for the distribution of forest avifauna in these degraded habitats. Our results showed that these habitats harboured a moderate fraction of forest avifauna (approximately 46–76 species) and their functions were complementary (i.e. rubber tree plantations for moving; open habitats for perching; shrubs in oil palm plantations for foraging). In terms of species richness and abundance, rubber tree plantations were more important than oil palm plantations and open habitats. The relatively high species richness of this agricultural landscape was partly due to the contiguity of our study areas with extensive forest areas. Forecasts of forest-species presence under various canopy cover scenarios suggest that leaving isolated trees among non-arboreal crops could greatly attract relatively tolerant species that require tree canopy. The conservation value of degraded habitats in agricultural landscapes seems to depend on factors such as the type of crops planted and distance to primary forest remnants.     

Tuberin: a stimulus-regulated tumor suppressor protein controlled by a diverse array of receptor tyrosine kinases and G protein-coupled receptors

Tuberin, a tumor suppressor protein, is involved in various cellular functions including survival, proliferation, and growth. It has emerged as an important effector regulated by receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Regulation of tuberin by RTKs and GPCRs is highly complex and dependent on the type of receptors and their associated signaling molecules. Apart from Akt, the first kinase recognized to phosphorylate and inactivate tuberin upon growth factor stimulation, an increasing number of kinases upstream of tuberin have been identified. Furthermore, recruitment of different scaffolding adaptor components to the activated receptors appears to play an important role in the regulation of tuberin activity. More recently, the differential regulation of tuberin by various G protein family members have also been intensively studied, it appears that G proteins can both facilitate (e.g., G(i/o)) as well as inhibit (e.g., G(q)) tuberin phosphorylation. In the present review, we attempt to summarize our emerging understandings of the roles of RTKs, GPCRs, and their cross-talk on the regulation of tuberin.     

Taxonomical and morphological notes on two species of mudskippers, Periophthalmus walailakae and Periophthalmodon schlosseri (Teleostei: Gobiidae) from Singapore

The mudskipper Periophthalmus walailakae is recorded from Singapore, where it was previously misidentified as Periophthalmodon schlosseri, with which it is syntopic. Periophthalmus walailakae is distinguished from its congeners by the following combination of characters: pelvic fins completely united and shaped like a disk, and first dorsal fin dark brown or black, with a rounded posterior edge and a white distal margin. This species most closely resembles Pn. schlosseri but has one row of teeth on the upper jaw, scales on the isthmus, and a different upper lip and jaw morphology. Contrary to an earlier report, scales are present on the snout, interorbital, and isthmus of Ps. walailakae. The two species can also be distinguished by size, external morphology, and body color patterns.     

Fgf8 expression in the Tbx1 domain causes skeletal abnormalities and modifies the aortic arch but not the outflow tract phenotype of Tbx1 mutants

Fgf8 and Tbx1 have been shown to interact in patterning the aortic arch, and both genes are required in formation and growth of the outflow tract of the heart. However, the nature of the interaction of the two genes is unclear. We have utilized a novel Tbx1(Fgf8) allele which drives Fgf8 expression in Tbx1-positive cells and an inducible Cre-LoxP recombination system to address the role of Fgf8 in Tbx1 positive cells in modulating cardiovascular development. Results support a requirement of Fgf8 in Tbx1 expressing cells to finely control patterning of the aortic arch and great arteries specifically during the pharyngeal arch artery remodeling process and indicate that the endoderm is the most likely site of this interaction. Furthermore, our data suggest that Fgf8 and Tbx1 play independent roles in regulating outflow tract development. This finding is clinically relevant since TBX1 is the candidate for DGS/VCFS, characterized clinically by variable expressivity and reduced penetrance of cardiovascular defects; Fgf8 gene variants may provide molecular clues to this variability.     

A simple fluorescence labeling method for studies of protein oxidation, protein modification, and proteolysis

Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve (3)H or (14)C methylation, which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid precipitation. Alternative labeling methods have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, (3)H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well suited to studying increased proteolytic susceptibility after protein modification, because the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite and is stable over time and to extremes of pH, temperature (even boiling), freeze-thaw, mercaptoethanol, and methanol.