Interleukin-7 inactivates the pro-apoptotic protein Bad promoting T cell survival

Interleukin-7 (IL-7) is a cytokine that is required for T cell development and survival. The anti-apoptotic function of IL-7 is partly through induction of Bcl-2 synthesis and cytosolic retention of Bax. Here we show that the Bcl-2 homology 3 domain-only protein, Bad, is involved in cell death following IL-7 withdrawal from D1 cells, an IL-7-dependent murine thymocyte cell line. IL-7 stimulation resulted in the inactivation of Bad by phosphorylation at Ser-112, -136, and -155. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated previously in Bad phosphorylation. In response to IL-7, the PI3K/Akt pathway induced phosphorylation at Ser-136 and -155, but Ser-112 was partly independent of the PI3K/Akt pathway, indicating an as yet unknown pathway in this response. Following IL-7 withdrawal, dephosphorylated Bad translocated from cytosol to mitochondria, bound to Bcl-2, and accelerated cell death. Thus, the inactivation of Bad contributes to the survival function of IL-7.     

Identification of GBF1 as a Cellular Factor Required for Hepatitis C Virus RNA Replication

In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.Hepatitis C virus (HCV) is an important human pathogen. It mainly infects human hepatocytes, and this often leads to chronic hepatitis, cirrhosis, or hepatocarcinoma. HCV studies have been hampered for many years by the difficulty in propagating this virus in vitro. Things have recently changed with the development of a cell culture model referred to as HCVcc (34, 60, 65), which allows the study of the HCV life cycle in cell culture and facilitates studies of the interactions between HCV and the host cell.HCV is an enveloped positive-strand RNA virus belonging to the family Flaviviridae (35). The viral genome contains a single open reading frame, which is flanked by two noncoding regions that are required for translation and replication. All viral proteins that are produced after proteolytic processing of the initially synthesized polyprotein are membrane associated (15, 43). This reflects the fact that virtually all steps of the viral life cycle occur in close association with cellular membranes.Interactions of HCV with cell membranes begin during entry. Several receptors, coreceptors, and other entry factors have been discovered over the years, which link HCV entry to specialized domains of the plasma membrane, such as tetraspanin-enriched microdomains and tight junctions (8, 16, 59). The internalization of the viral particle occurs by clathrin-mediated endocytosis (5, 40). The fusion of the viral envelope with the membrane of an acidic endosome likely mediates the transfer of the viral genome to the cytosol of the cell (5, 40, 57). However, little is known regarding the pre- and postfusion intracellular transport steps of entering viruses in the endocytic pathway.HCV RNA replication is also associated with cellular membranes. Replication begins with the translation of the genomic RNA of an incoming virus. This leads to the production of viral proteins, which in turn initiate the actual replication of the viral RNA. Mechanisms regulating the transition from the translation of the genomic RNA to its replication are not yet known. All viral proteins are not involved in RNA replication. Studies performed with subgenomic replicons demonstrated that proteins NS3-4A, NS4B, NS5A, and NS5B are necessary and sufficient for replication (6, 27, 37). RNA replication proceeds through the synthesis of a cRNA strand (negative strand), catalyzed by the RNA-dependent RNA polymerase activity of NS5B, which is then used as a template for the synthesis of new positive strands.Electron microscopy studies using a subgenomic replicon model suggested that replication takes place in membrane structures made of small vesicles, referred to as “membranous webs,” which are induced by the virus (26). Membranous webs are detectable not only in cells carrying subgenomic replicons but also in infected cells (50). They appear to be associated with the endoplasmic reticulum (ER) (26). In addition to the membranous webs, a second type of ER-associated replicase that is smaller and more mobile has recently been described (63). Cellular mechanisms leading to these membrane alterations are still poorly understood. In cells replicating and secreting infectious viruses effectively, the situation appears to be even more complex, since replicase components appear to be, at least in part, associated with cytoplasmic lipid droplets (41, 50, 56). This association depends on the capsid protein (41) and may reflect a coupling between replication and assembly. Indeed, HCV assembly and secretion show some similarities with very-low-density lipoprotein (VLDL) maturation and secretion (24, 64).Our knowledge of the cellular membrane mechanisms involved in the HCV life cycle is still limited. The expression of NS4B alone induces membrane alterations that are reminiscent of membranous webs (19). However, cellular factors that participate in this process are still unknown. On the other hand, several cellular proteins potentially involved in the HCV life cycle have been identified through their interactions with viral proteins. For some of these proteins, a functional role in infection was recently confirmed using RNA interference (48). It is very likely that other cellular factors critical to HCV infection have yet to be identified.To gain more insight into cellular mechanisms underlying HCV infection, we made use of brefeldin A (BFA), a macrocyclic lactone of fungal origin that exhibits a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways (30). BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARFs). ARFs are small GTP-binding proteins of the Ras superfamily. They function as regulators of vesicular traffic, actin remodeling, and phospholipid metabolism by recruiting effectors to membranes. BFA does not actually interfere directly with ARF GTPases but rather interferes with their activation by regulators known as guanine nucleotide exchange factors (GEFs) (14, 25). We now report the identification of an ARF GEF as a cellular BFA-sensitive factor that is required for HCV replication.     

A primer-based approach to genome walking

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.     

Morphogenesis of the marine ciliate, Pseudoamphisiella alveolata (Kahl, 1932) Song & Warren, 2000 (Ciliophora, Stichotrichia, Urostylida) during binary fission

Morphogenesis during the binary fission of the stichotrich ciliate Pseudoamphisiella alveolata, isolated from Jiaozhou Bay near Qingdao, China, was investigated using protargol silver impregnation. The process is characterized as follows: (1) in the proter, only the posterior part of the parental adoral zone of membranelles is renewed, where the membranelles dedifferentiate and then rebuild the UM-anlage and the missing membranelles, (2) the oral primordium in the opisthe and the FVT-anlagen in both dividers are formed de novo on the cell surface, (3) an "extra" anlage, which is generated on the right of the right marginal anlage, develops into three or four "extra" marginal cirri that connect the caudal cirri with the marginal rows, (4) the right marginal anlage is formed within the old structure, (5) the FVT-cirri develop in a primary mode, and (6) unlike most stichotrichs, the right marginal anlagen in both dividers generate closely together. As an additional contribution, the diversity of morphogenetic patterns within the genus Pseudoamphisiella is discussed. Based on both morphogenetic and SS rRNA gene sequencing data, the systematic position of the genus Pseudoamphisiella as well as the family Pseudoamphisiellidae Song et al. 1997 is briefly analyzed. The results indicate that they should very possibly represent a higher evolved group in the order Urostylida.     

CD9 is expressed on human male germ cells that have a long-term repopulation potential after transplantation into mouse testes

Human spermatogonial stem cells (SSCs) play critical roles in lifelong maintenance of male fertility and regeneration of spermatogenesis. These cells are expected to provide an important resource for male fertility preservation and restoration. A basic strategy has been proposed that would involve harvesting testis biopsy specimens from a cancer patient prior to cancer therapies, and transplanting them back to the patient at a later time; then, SSCs included in the specimens would regenerate spermatogenesis. To clinically apply this strategy, isolating live human SSCs is important. In this study, we investigated whether CD9, a known rodent SSC marker, is expressed on human male germ cells that can repopulate recipient mouse testes upon transplantation. Testicular tissues were obtained from men with obstructive azoospermia. Using immunohistochemistry, we found that CD9 was expressed in human male germ cells in the basal compartment of the seminiferous epithelium. Following immunomagnetic cell sorting, CD9-positive cells were enriched for germ cells expressing MAGEA4, which is expressed by spermatogonia and some early spermatocytes, compared with unsorted cells. We then transplanted CD9-positive cells into nude mouse testes and detected an approximately 3- to 4-fold enrichment of human germ cells that repopulated mouse testes for at least 4 mo after transplantation, compared with unsorted cells. We also observed that some cell turnover occurred in human germ cell colonies in recipient testes. These results demonstrate that CD9 identifies human male germ cells with capability of long-term survival and cell turnover in the xenogeneic testis environment.     

Genetic architecture and adaptive significance of the selfing syndrome in Capsella

The transition from outcrossing to predominant self-fertilization is one of the most common evolutionary transitions in flowering plants. This shift is often accompanied by a suite of changes in floral and reproductive characters termed the selfing syndrome. Here, we characterize the genetic architecture and evolutionary forces underlying evolution of the selfing syndrome in Capsella rubella following its recent divergence from the outcrossing ancestor C. grandiflora. We conduct genotyping by multiplexed shotgun sequencing and map floral and reproductive traits in a large (N= 550) F2 population. Our results suggest that in contrast to previous studies of the selfing syndrome, changes at a few loci, some with major effects, have shaped the evolution of the selfing syndrome in Capsella. The directionality of QTL effects, as well as population genetic patterns of polymorphism and divergence at 318 loci, is consistent with a history of directional selection on the selfing syndrome. Our study is an important step toward characterizing the genetic basis and evolutionary forces underlying the evolution of the selfing syndrome in a genetically accessible model system.     

Characterization and expression profile analysis of a sucrose synthase gene from common bean (Phaseolus vulgaris L.) during seed development

A full-length cDNA encoding common bean (Phaseolus vulgaris L.) sucrose synthase (designated as Pv_BAT93 Sus), which catalyses the synthesis and cleavage of sucrose, was isolated from seeds at 15 days after pollination (DAP) by rapid amplification of cDNA ends (RACE). The full-length cDNA of Pv_BAT93 Sus had a 2,418 bp open reading frame (ORF) encoding a protein of 806 amino acid residues. Sequence comparison analysis showed that Pv_BAT93 Sus was very similar to several members of the sucrose synthase family of other plant species. Tissue expression pattern analysis showed that Pv_BAT93 Sus was expressed in leaves, flowers, stems, roots, cotyledons, and particularly during seed development. Expression studies using in situ hybridization revealed altered spatial and temporal patterns of Sus expression in the EMS mutant relative to wild-type and confirmed Sus expression in common bean developing seeds. The expression and accumulation of Sus mRNA was clearly shown in several tissues, such as the suspensor and embryo, but also in the transfer cells and endothelium. The results highlight the diverse roles that Sus might play during seed development in common bean.     

Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor

Measles vaccination remains the most efficient way to control the spread of the virus. This work focuses on the production of a measles vaccine using stirred conditions as an advanced option for process scale up. Non-porous Cytodex 1 microcarriers were used to support MRC-5 cell growth in suspension cultures. Virus replication was first optimized in spinner flasks, and the effects of various operational parameters were investigated. Cell infection with AIK-C measles strain at an MOI (multiplicity of infection) of 0.005, without glucose regulation and in M199 medium, resulted in a virus titer of 10^6.25 TCID₅₀ (median tissue culture infective dose)/ml. To optimize the production process in a 7-l bioreactor, we carried out various perfused cultures using minimum essential medium (MEM) + 5% FCS diluted with phosphate-buffered saline (PBS). We achieved a high cell density level (4.1 × 10⁶ cells/ml) with an efficient use of the medium when MEM + 5% FCS diluted with PBS at 25% was used during the cell amplification step. Optimization of measles production in MRC-5 cells grown on Cytodex 1 beads in a 7-l bioreactor showed that perfusion was the most efficient when compared to repeated-batch culture. Perfusion at a rate of 0.25 V (reactor volume)/day showed the highest specific productivity (1.6 IVP [infectious virus particle] cell⁻¹ day⁻¹). Testing of several stabilizers containing pharmaceutically improved components such as sugars, amino acids, and charged ions showed that the formulation composed of sucrose and MgCl₂, led to the maintenance of the infectivity of the AIK-C measles virus strain to a significant level, when stored at +28 °C, +4 °C and −60 °C.