Cell-penetrating peptides derived from viral capsid proteins

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl(2) changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.     

Improving Detection Accuracy of Lung Cancer Serum Proteomic Profiling via Two-Stage Training Process

Background  

Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) is a frequently used technique for cancer biomarker research. The specificity of biomarkers detected by SELDI can be influenced by concomitant inflammation. This study aimed to increase detection accuracy using a two-stage analysis process.     

Metabolic capabilities and systems fluctuations in Haloarcula marismortui revealed by integrative genomics and proteomics analyses

The 1310 Haloarcula marismortui proteins identified from mid-log and late-log phase soluble and membrane proteomes were analyzed in metabolic and cellular process networks to predict the available systems and systems fluctuations upon environmental stresses. When the connected metabolic reactions with identified proteins were examined, the availability of a number of metabolic pathways and a highly connected amino acid metabolic network were revealed. Quantitative spectral count analyses suggested 300 or more proteins might have expression changes in late-log phase. Among these, integrative network analyses indicated approximately 106 were metabolic proteins that might have growth-phase dependent changes. Interestingly, a large proportion of proteins in affected biomodules had the same trend of changes in spectral counts. Disregard the magnitude of changes, we had successfully predicted and validated the expression changes of nine genes including the rimK, gltCP, rrnAC0132, and argC in lysine biosynthesis pathway which were downregulated in late-log phase. This study had not only revealed the expressed proteins but also the availability of biological systems in two growth phases, systems level changes in response to the stresses in late-log phase, cellular locations of identified proteins, and the likely regulated genes to facilitate further analyses in the postgenomic era.     

Proteome-wide Dysregulation by PRA1 Depletion Delineates a Role of PRA1 in Lipid Transport and Cell Migration

We have previously identified prenylated Rab acceptor 1 (PRA1) as a novel cellular interacting partner for Epstein-Barr virus-encoded oncoprotein, latent membrane protein 1 (LMP1). The intracellular trafficking and full signaling of LMP1 requires its interaction with PRA1. To further explore the role of PRA1 in Epstein-Barr virus-associated nasopharyngeal carcinoma (NPC) cells, we generated several PRA1-knockdown cell clones, which exhibited altered cell morphology and increased cell motility. We identified proteins differentially expressed in the knockdown clones by means of isobaric mass tags labeling coupled with multidimensional liquid chromatography-mass spectrometry. We validated a panel of proteins, which showed consistent up-regulation in PRA1-knockdown clones and participated in regulating lipid homeostasis and cell migration. Immunofluorescence staining further revealed altered localization of these proteins and accumulation of intracellular cholesterol in PRA1-knockdown clones. These effects were phenocopied by treatment with a cholesterol transport inhibitor, U18666A. Moreover, overexpressed PRA1 was able to alleviate the dysregulation of these affected proteins either from PRA1 knockdown or U18666A treatment, implying a role for PRA1 in regulating the levels of these affected proteins in response to altered cholesterol homeostasis. We further demonstrated that LMP1 expression caused PRA1 sequestration in NPC cells, leading to a consequence reminiscent of PRA1 knockdown. Finally, the immunohistochemistry showed a physiological relevance of the PRA1-associated proteome-wide changes in NPC biopsy tissues. In sum, our findings delineated novel roles of PRA1 in lipid transport and cell migration, and provided additional insights into the molecular basis of NPC morphogenesis, namely a consequence of LMP1-PRA1 interaction.Prenylated Rab acceptor 1 (PRA1)¹, which is a transmembrane protein of 21 kDa, is ubiquitously expressed in human tissues and localizes at the Golgi apparatus, post-Golgi vesicles, endosomes, and the plasma membrane (1, 2). As revealed by its name, PRA1 interacts with numerous Rab GTPases (2, 3), the latter of which function in a wide variety of biological processes such as endocytosis and exocytosis and have emerging roles in diseases (4–6). The PRA1-Rab interactions may assist in the packaging of Rabs into vesicles for transport to the destined compartments (2). Moreover, PRA1 also acts as a dual receptor for vesicle-associated membrane protein 2 (VAMP2) and GDP dissociation inhibitor 1 (GDI1) (7, 8). As a GDI displacement factor, PRA1 is able to catalytically dissociate endosomal Rabs (Rab9 and Rab5) from GDI-bound complexes and thereby escorts the liberated Rabs onto membranes (9). Given this relative lack of Rab specificity, PRA1-mediated regulation of Rab proteins is probably restricted by the cellular localization of PRA1, i.e. PRA1 regulates the Rabs present in the organelles with which PRA1 associates.Although its precise physical role remains to be better elucidated, PRA1 seems to function in the regulation of docking and fusion of transport vesicles both in the Golgi apparatus and at the plasma membrane, or alternately function as a sorting protein in the Golgi apparatus (10). PRA1 can form a complex with Rab3a and VAMP2, and the interaction of this complex can result in VAMP2 activation (7). Once activated, VAMP2 interacts with syntaxin, followed by the docking and fusion of transport vesicles with target membrane (11). Since syntaxin and VAMP2 are enriched in Golgi-derived lipid rafts (12), PRA1 is thought to associate with lipid rafts (13).As a platform for lipid-lipid and lipid-protein interactions, lipid rafts play critical roles in protein transport, sorting, targeting, signaling as well as membrane trafficking, and are essential for enveloped virus budding and assembly (14). In agreement with this notion, several viral proteins have been shown to interact with PRA1 to benefit the survival of viruses. For instance, the spike protein VP4 encoded by rotavirus and the envelope transmembrane protein gp41 encoded by retrovirus can interact with PRA1, and their interaction with PRA1 may in turn enhance the assembly of rotavirus and retrovirus particles, respectively (13, 15). In this regard, it is conceivable to speculate a role for PRA1 in promoting or stabilizing protein association with lipid rafts.In the previous study, we have identified PRA1 as a novel binding partner for the Epstein-Barr virus (EBV)-encoded oncoprotein, latent membrane protein 1 (LMP1) (16). EBV is closely associated with human diseases including nasopharyngeal carcinoma (NPC) (17), which is one of the common cancers in Taiwan and southern China, and LMP1 is shown to mainly contribute to these EBV-associated malignancies (18). By mimicking members of tumor necrosis factor receptor (TNFR) family, LMP1 can induce several signaling pathways in a constitutively-activated manner to exert its oncogenic potency (19–21). Importantly, the intracellular trafficking of LMP1 requires its interaction with PRA1, and this requirement is critical for full activation of LMP1-meditaed signaling (16). Accordingly, delineating the propensity of PRA would shed light on the nature of PRA1-LMP1 interaction and yield additional insights into the tumorigenesis of NPC.To further assess the role of PRA1 in NPC cells, in this study we generated several PRA1-knockdown NPC cell clones, which displayed altered cell morphology, and used these clones to analyze the effect of PRA1 on cell morphology and relevant biological processes. We discovered a panel of dysregulated proteins in PRA1-knockdown clones, which participate in lipid metabolism and transport and cell adhesion and migration, by using isobaric mass tags (iTRAQ) labeling approaches combined with multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). To determine the physiological relevancy of our findings, we investigated the functional consequence of PRA1 sequestration in LMP1-expressing cells. We confirmed the phenotype of LMP1-expressing cells, namely intracellular cholesterol accumulation, elevated expression levels of those PRA1-affected proteins, and increased cell motility, consistent with the effect of PRA1 knockdown. We also validated the PRA1-associated dysregulation of selected proteins in NPC tissues using immunohistochemistry.Taken together, our findings revealed a PRA1-involved modulation in lipid homeostasis and cell migration, and implied an unexpected association of the LMP1-PRA1 interaction with NPC morphogenesis.     

Gene targeting and expression analysis of mouse Tem1/endosialin using a lacZ reporter

TEM1 (endosialin) expression is increased in the stroma and tumor vasculature of several common human cancers. The exact physiological role of TEM1 is still unknown since Tem1-deficient mice are viable and show only a lower rate of abdominal site-specific tumor invasion in tumor transplantation experiments. Previous studies have reported Tem1 expression in mouse embryos and adults, but did not determine the timing or location of the earliest expression, and did not examine all organ systems. Using the highly sensitive Bluo-Gal staining method for detecting temporal and spatial Tem1-lacZ activity in lacZ knock-in (+/lacZ) mice, we found that Tem1 gene expression was initially detectable in the dorsal aortic wall, the heart, the umbilical vessels, the first branchial arch, and the cephalic mesenchyme at E9.5. From E10.5 to E14.5, Tem1 gene expression was additionally seen mainly in the genital tubercle, the mesonephros, the whisker follicles, the mesenchymal tissues around the eye, and the lung. Remarkably, the kidney expressed abundant Tem1-lacZ starting from E16.5. Postnatally, Tem1 expression decreased in most organs but elevated expression persisted in the renal glomerulus and the uterus, where the expression pattern varied at different estrous cycle stages. Co-localization studies indicated that most vimentin-positive cells co-expressed Tem1-lacZ, while a large portion of CD31- or desmin-positive cells were also positive for Tem1-lacZ. Taken together, our observations suggest that Tem1 is expressed throughout embryonic and adult development in several types of mesenchymal cells closely related to blood vessels.     

Differential amplification of antifreeze protein genes in the pleuronectinae

Summary The organization of antifreeze protein (AFP) genes in the yellowtail flounder was investigated by Southern blotting and the characterization of clones from a genomic library. This flounder, like the closely related winter flounder, has a set of 10–12 linked but irregularly spaced AFP genes. However, it lacks the tandemly amplified set of 20 such genes that are present in the winter flounder. DNA sequence analysis of a tandemly repeated gene from winter flounder showed that it can code for one of the two most abundant AFP components in the serum. Consistent with this higher AFP gene dosage, the peak serum AFP level in midwinter was 9 mg/ml in the winter flounder and only 4 mg/ml in the yellowtail flounder. A recent amplification of the AFP gene in the winter flounder lineage might be responsible for the higher serum AFP levels in this fish. This increase in gene dosage might have helped the winter flounder colonize the ice-laden, shallow-water niche that it currently occupies along the east coast of North America. Genomic Southern blotting of two other righteye flounders, the smooth flounder and the American plaice, illustrates another example of a differential amplification of AFP genes that correlates with a species' exposure to ice.     

Interactions between visitors and Formosan macaques (Macaca cyclopis) at Shou‐Shan Nature Park,Taiwan

Ecotourism involving feeding wildlife has raised public attention and is a controversial issue, especially concerning nonhuman primates. Between July 2002 and April 2005, the behavior of monkeys and tourists was collected through scan samplings, focal samplings and behavior samplings at the Shou‐Shan Nature Park located in Taiwan's second largest city—Kaohsiung. In addition, the number of tourists and monkeys was counted in different hours and places within the park. Four hundred visitors were interviewed using a questionnaire to gather data on sex, age, purpose and frequency of visit to the park. The number of tourists was significantly higher during weekends than in weekdays in all locations. Humans dominated in the initiation of interspecies interactions—the overall ratio of human‐initiated and monkey‐initiated interactions was 2.44:1. Human–monkey conflicts accounted for only 16.4% of the total interactions (n=2,166), and adult human males and adult male macaques participated in higher rates than other age/sex groups in these conflicts. Visitors showed more affiliative behavior (15.9%) than agonistic behavior (8%) toward the macaques. In response to visitors' threat or attack, the Formosan macaques mostly showed submissive behavior with bared teeth, squealed or ran away to avoid confrontation (69.1%)—only few responded with counteraggression (18.7%). This study for the first time provided evidence that food provisioning increased both the frequency and duration of aggression among Formosan macaques (P<0.001). During food provisioning, the average frequency and the duration of agonistic events of macaques were more than 4 times higher compared with those without food provisioning. The average frequency of food provision by tourists was 0.73 times/hr—more than twice the incident that monkeys grabbed the food from tourists (0.34 times/hr). If people refrain from feeding monkeys and destroying the city park's natural vegetation, monkeys can be used to educate public about nature conservation in an urban setting. Am. J. Primatol. 71:214–222, 2009. © 2008 Wiley‐Liss, Inc.     

Apo-E genotypes and cardiovascular diseases: a sensitivity study using cross-validatory criteria

The Apolipoprotein-E (Apo-E) gene, a gene that produces proteins which help to regulate lipid levels in the bloodstream, is of interest in the study of cardiovascular diseases. An approach to making inferences about the genetic effects of the Apo-E gene has been developed by Glickman and Gagnon (2002). The framework describes the role of genetic and risk factors on the onset ages of multiple diseases, and accounts for the possibility that an individual was censored for reasons related to the diseases of interest. The framework also allows for missing genetic information, so that subjects censored prior to genetic sampling, and therefore missing such information, may still be included in the analysis. We apply an extension to this framework to the original cohort of the Framingham Heart Study for measuring the effects of different Apo-E genotypes on the onset age of various cardiovascular disease events. In particular, we compare the fit of univariate versus multivariate onset age components to the model, whether to incorporate health covariates measured at baseline or at a point later in the study, and whether to assume a heritability model for Apo-E genotype frequencies. The results of the best fitting model are presented.