Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement

The effects of monopalmitoylphosphatidylcholine (MPPC or lysophosphatidylcholine) and a series of short-chain primary alcohols (ethanol, 1-butanol and 1-hexanol) on cell shape, hemolysis, viscoelastic properties and membrane lipid packing of human red blood cells (RBCs) were studied. For MPPC, the effective membrane concentration to induce the formation of stage 3 echinocytes (8 x 10(6) molecules per cell) was one order of magnitude lower than that needed to induce 50% hemolysis (7 x 10(7) molecules per cell). In contrast, short-chain alcohols induced both shape changes and hemolysis within close concentration range (2.5 x 10(8) to 3.5 x 10(8) molecules per cell). Viscoelastic properties of the RBCs were studied by micropipette aspiration and correlated with shape change. Ethanol-treated RBCs showed a decrease in membrane elastic modulus and an increase in membrane viscosity in the recovery phase at the early stage of shape change. MPPC-treated cells showed the same type of viscoelastic changes, but these were not observed until the formation of stage 2 echinocytes. High-resolution solid-state 13C nuclear magnetic resonance technique was applied to study membrane lipid packing in the ghost membrane by following the chemical shift of hydrocarbon chains. Both MPPC and ethanol caused the 13C-NMR chemical shift to move upfield, indicating that membrane lipids were expanded due to the intercalation of these exogenous molecules. Using data obtained from model compounds, we convert values of chemical shift into a lipid packing parameter, i.e., number of gauche bonds for fatty acyl hydrocarbon chains. Approximately 10(8) interacting molecules per cell are required to induce a detectable change of lipid packing by both MPPC and ethanol. The results indicate that homolysis occurs at a smaller surface area for MPPC- than ethanol-treated RBCs. Our findings suggest that progressive changes in the molecular packing in the membrane lead eventually to hemolysis, but the mode responsible for shape transformation varies with these amphipaths.     

Nuclear Receptor Nur77 Facilitates Melanoma Cell Survival under Metabolic Stress by Protecting Fatty Acid Oxidation

1. Download high-res image (276KB)
2. Download full-size image

     

The Years of the Monkey

The fact that mammals are diploid sets a barrier to rapidly understand the function of non-coding and coding genes in the genome. Recently, Yang et al. reported successful derivation of monkey haploid embryonic stem cells from parthenotes, which provide an effective platform for studying mammalian gene function and enable reverse genetic screening of genes for recessive phenotypes in monkeys.According to the Zodiac in the Chinese Calendar, the next year of the monkey is not slated until February 2016, but a recent paper in this month''s Cell Research suggests that it may have arrived early for the field of stem cell biology. In a stunning technical “Tour de Force”, Jinsong Li and his colleagues report for the first time the generation of several independent haploid monkey embryonic stem (ES) cell lines¹, building on the previous work from their lab and others that described the generation of murine haploid ES cell lines^2,3,4,5 (Figure 1). They first activated metaphase II monkey oocytes with ionomycin followed by cycloheximide treatment. These activated oocytes could develop into blastocysts in vitro and haploid ES cells (haESCs) can be derived by culturing the inner cell mass in a standard monkey ES cell culture system and using Hoechst FACS technique. Remarkably, one of the cell lines remained stable during long term passage, obviating the need for FACS sorting for the haploid cell lines during subsequent propagation. The cell lines can be genetically manipulated by insertional mutagenesis or by PiggyBac transposon technology, suggesting the possibility of genome-wide screening strategies. In this regard, a series of parallel scientific advances suggest that this technology platform may be particularly timely as the field of stem cell biology moves towards regenerative medicine and therapeutics.Open in a separate windowFigure 1The scheme of parthenogenetic (PG) and androgenetic (AG) haploid embryonic stem cells (haESCs) derivation. (A) For the generation of PG-haESCs, metaphase II oocytes were activated with either strontium chloride (SrCl₂) for mice or ionomycin/cycloheximide (CHX) for monkeys and further cultivated to the blastocyst stage. With the help of Hoechst FACS technique, PG-haESCs can be derived. (B) For the generation of AG-haESCs, metaphase II oocytes were enucleated followed by sperm injection. In addition, the reconstructed oocytes were activated with SrCl₂ for mice and further developed to the blastocyst stage in vitro. AG-haESCs can be derived by several rounds of Hoechst FACS based on DNA contents. The derivation of non-human primate AG-haESCs has not been reported yet.For many years, it has proven quite difficult to engineer site-specific mutations, knock-ins, and knock-outs in human ES or induced pluripotent stem (iPS) cells, and only a handful of genetically engineered lines have been created by conventional homologous recombination strategies⁶. However, recent advances in RNA-guided nuclease technology has led to a marked improvement in the efficiency of the knockout of genes in human pluripotent stem cells⁷, suggesting that it may be possible to create knock-out haploid non-human primate (NHP) ES cell lines that harbor specific disease genes and surrogate reporter readouts, and then to look for genetic complementation that could identify critical genes that could be potential drug targets. A library of individual NHP haploid ES cell lines that harbor a loss-of-function mutation across the entire NHP genome could find multiple uses in quickly identifying signaling pathways in differentiated cell types. Given recent advances in screening in human ES and iPS cell lines⁸, direct drug screening on the haploid monkey ES cell lines should also be possible. In addition, it will likely be possible to set up genome-wide screening to systematically identify entire network of genes that drive specific differentiation events, and early steps of primate organogenesis. If androgenetic NHP haploid cell lines can be developed (see Figure 1), a leap in the efficiency of the generation of monkey KO animal models could be envisioned over the long term. In this regard, the recent generation of chimeric monkeys⁹, as well as future technical advances related to this achievement, could become of significant interest.At the same time, the study indirectly raises the query as to the need for monkey model systems when the technology for genetic manipulation in the mouse is without peer, and human ES and iPS cell lines can now be easily generated and genetically manipulated. The recent pronouncement of the termination of NIH support for primate research (http://news.sciencemag.org/people-events/2013/06/nih-will-retire-most-research-chimps-end-many-projects), along with the growing awareness of the need to re-examine the need for NHP models, suggests that there must be very solid scientific grounds for pursuing NHP model systems in the future.In this regard, a growing body of evidence is now pointing to the lack of fidelity of mouse models of human disease to the in vivo human setting, a problem that has plagued cancer therapeutics for decades. Recently, the lack of predictability of human responses from models of murine sepsis has been cogently made¹⁰, and the divergence in the physiology of mice and humans, particularly in terms of metabolism and cardiovascular, are enormous. The complexity and scalability of primate versus murine organogenesis also may be an issue. For example, the human heart is 10 000 times larger than the murine, has a much larger diversity of cell types, and a level of tertiary morphology that is not found in the murine heart (for review see¹¹). Murine cardiogenesis is largely completed with 48 h, while human cardiogenesis occurs over months, and recent studies that suggest a much larger diversity and markedly extended period of proliferation of the family of heart progenitors in the human fetal versus murine heart¹². To date, there are no approved drugs that have come from genetically engineered murine models of cardiovascular (CV) disease, and the biggest CV drugs have actually been discovered based on human genetics (statins, PCSK9, etc.). The increased importance of CV side effects for new drugs in the diabetes space, as well as for other chronic diseases, points to the importance of their study in more sophisticated primate systems, as all these drugs (Avandia, Vioxx, etc.) had cleared conventional screening in rodent model systems. Given the above, we may have to put the Chinese Calendar on auto-repeat mode, as we enter the “Years of the Monkey” in this decade and the next.     

FUSE binding protein 1 interacts with untranslated regions of Japanese encephalitis virus RNA and negatively regulates viral replication

The untranslated regions (UTRs) located at the 5' and 3' ends of the Japanese encephalitis virus (JEV) genome, a positive-sense RNA, are involved in viral translation, the initiation of RNA synthesis, and the packaging of nascent virions. The cellular and viral proteins that participate in these processes are expected to interact with the UTRs. In this study, we used biotinylated RNA-protein pulldown and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses to identify that the far upstream element (FUSE) binding protein 1 (FBP1) binds with JEV 5' and 3' UTRs. The impact of FBP1 on JEV infection was determined in cells with altered FBP1 expression. JEV replication was enhanced by knockdown and reduced by the overexpression of FBP1, indicating a negative role for FBP1 in JEV infection. FBP1, a nuclear protein, was redistributed to the perinuclear region and appeared as cytoplasmic foci that partially colocalized with JEV RNA in the early stage of JEV infection. By using a JEV replicon reporter assay, FBP1 appeared to suppress JEV protein expression mediated by the 5' and 3' UTRs. Thus, we suggest that FBP1 binds with the JEV UTR RNA and functions as a host anti-JEV defense molecule by repressing viral protein expression.     

Human papillomavirus infection in oral papillary and verrucous lesions is a prognostic indicator of malignant transformation

Background: Recent studies have demonstrated an increase in the incidence of HPV-associated oral squamous cell carcinoma. The aim of this study was to investigate the presentation of HPV in verrucous and papillary lesions of the oral mucosa and the relationship with the prognosis of the patients. Methods: Fifty-three biopsy specimens from 31 patients were investigated by polymerase chain reaction using a consensus primer directed to the HPV L1 gene; this was followed by a confirmatory in situ hybridization to identify the HPV types. Result: Fifteen tumor biopsies (28.3%) were positive for the HPV L1 gene, but only 8 specimens (15.1%) were found to be positive using in situ hybridization. The positive rates of HPV L1 gene were 58.8% and 13.9% in malignant and benign verrucous lesions, respectively. HPV infection is independently associated with malignant transformation and disease-specific survival. Conclusion: The presence of HPV infection is relatively low; however, the clinical outcome of patients with HPV-positive papillary and verrucous lesions was poor.     

Mice carrying a conditional Serca2 allele for the generation of Ca handling-deficient mouse models

Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca²⁺ into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2^null mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2^flox allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner. The model was tested by mating Serca2^flox mice with MLC-2v^wt/Cre mice and with αMHC-Cre transgenic mice. In heterozygous Serca2^wt/floxMLC-2v^wt/Cre mice, the expression of SERCA2a and SERCA2b proteins were reduced in the heart and slow skeletal muscle, in accordance with the expression pattern of the MLC-2v gene. In Serca2^flox/flox Tg(αMHC-Cre) embryos with early homozygous cardiac Serca2 disruption, normal embryonic development and yolk sac circulation was maintained up to at least embryonic stage E10.5. The Serca2^flox mouse is the first murine conditional gene disruption model for the SERCA family of Ca²⁺ ATPases, and should be a powerful tool for investigating specific physiological roles of SERCA2 function in a range of tissues and organs in vivo both in adult and embryonic stages.     

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.     

Clathrin-Mediated Post-Golgi Membrane Trafficking in the Morphogenesis of Hepatitis Delta Virus

Clathrin is involved in the endocytosis and exocytosis of cellular proteins and the process of virus infection. We have previously demonstrated that large hepatitis delta antigen (HDAg-L) functions as a clathrin adaptor, but the detailed mechanisms of clathrin involvement in the morphogenesis of hepatitis delta virus (HDV) are not clear. In this study, we found that clathrin heavy chain (CHC) is a key determinant in the morphogenesis of HDV. HDAg-L with a single amino acid substitution at the clathrin box retained nuclear export activity but failed to interact with CHC and to assemble into virus-like particles. Downregulation of CHC function by a dominant-negative mutant or by short hairpin RNA reduced the efficiency of HDV assembly, but not the secretion of hepatitis B virus subviral particles. In addition, the coexistence of a cell-permeable peptide derived from the C terminus of HDAg-L significantly interfered with the intracellular transport of HDAg-L. HDAg-L, small HBsAg, and CHC were found to colocalize with the trans-Golgi network and were highly enriched on clathrin-coated vesicles. Furthermore, genotype II HDV, which assembles less efficiently than genotype I HDV does, has a putative clathrin box in its HDAg-L but interacted only weakly with CHC. The assembly efficiency of the various HDV genotypes correlates well with the CHC-binding activity of their HDAg-Ls and coincides with the severity of disease outcome. Thus, the clathrin box and the nuclear export signal at the C terminus of HDAg-L are potential new molecular targets for HDV therapy.Pathogens often take advantage of intracellular pathways involved in the trafficking of cellular macromolecules in order to carry out their life cycle, which consists of virus entry, translation, genome replication, assembly, and release. The clathrin-mediated endocytic route is a pathway commonly used for virus entry (29). Following clathrin-mediated endocytosis, incoming viruses are transported together with their receptors from the plasma membrane into early and late endosomes. Several links between clathrin adaptor complexes and viral biogenesis, including those of influenza virus (37), reovirus (13), and vesicular stomatitis virus (33), have been demonstrated.Clathrin and its adaptor proteins (APs), which constitute the major components of clathrin-coated vesicles (CCVs), are often the carriers of proteins and lipids that are transported from the trans-Golgi network (TGN) to the endosome (20, 35). Clathrin-mediated exocytosis has been found to participate in viral multiplication. The envelope protein of vesicular stomatitis virus, glycoprotein 1, recruits clathrin adaptor complex adaptor protein 1 (AP1) onto Golgi membranes and possibly leaves the TGN in CCVs for subsequent transport to endosomes (1). It is also known that interaction of AP1 with the matrix domain of human immunodeficiency virus type 1 Gag protein promotes viral release (5). In addition, Vpu inhibits the endosomal accumulation of the human immunodeficiency virus type 1 structural proteins Env and Gag, which is known to enhance viral assembly and release at the plasma membrane (39). Furthermore, large hepatitis delta antigen (HDAg-L) encoded by the hepatitis delta virus (HDV) has recently been identified as a novel clathrin adaptor-like protein (18). HDAg-L specifically interacts with clathrin heavy chain (CHC) at the TGN and inhibits clathrin-mediated protein transport. However, the role of CHC in the life cycle of HDV remains unclear.HDV is a highly pathogenic virus. The virion is coated with the envelope proteins of hepatitis B virus (HBV), the hepatitis B virus surface antigens (HBsAgs) (24). Superinfection or coinfection with HBV may result in fulminant hepatitis and progressive chronic liver cirrhosis (3, 36). The small HDAg (HDAg-S) lacks the unique C-terminal 19-amino-acid sequence of HDAg-L (6, 41, 43) and functions as a transactivator of HDV genome replication in the nucleus (23, 24). Both HDAg-S and HDAg-L possess nuclear localization signals (NLSs) spanning amino acid residues 35 to 88 and are mainly localized in the nuclei of transfected cells in the absence of HBsAg (7, 8). However, HDAg-L has been demonstrated to be a nucleocytoplasmic shuttling protein with a nuclear export signal (NES) at its unique C terminus, and this is important for HDV assembly (27). In the presence of HBsAg, HDAg-L relocalizes to the cytoplasm (29). In addition, a NES-interacting protein of HDAg-L, NESI, has been identified to be essential for the HDAg-L-mediated nuclear export of HDV RNA (42). Furthermore, the proline-rich motif within the unique 19-amino-acid extension together with isoprenylation of the CXXX motif (15) are essential for HDAg-L to form delta virus-like particles (VLPs) with HBsAg (19, 22). Taken together, these results imply that an intracellular association between HDAg-L and HBsAg in the cytoplasm is the driving force of HDV assembly. The interaction of HDAg-L with HBsAg facilitates the assembly and secretion of HDV particles. Nevertheless, the cellular proteins and pathways involved in the transport, packaging, and secretion of HDV are poorly understood.In this study, the involvement of clathrin-mediated trafficking in the propagation of HDV is biochemically characterized. Downregulation of functional CHC significantly reduced the efficiency of the CCV-mediated HDV assembly. However, CHC is not essential for the assembly of HBV subviral particles (SVPs). These results indicate that, although HBV and HDV share common surface antigens, different mechanisms are involved in their viral assembly and release. In addition, the assembly efficiency of the various HDV genotypes correlates well with the ability of HDAg-L to interact with CHC. This may reflect the fact that there is lower pathogenicity among patients infected with HDV genotype II than among those infected with genotype I.