Steroidal saponins and ecdysterone from Asparagus filicinus and their cytotoxic activities

Two new spirostanoides, filiasparosides E (1) and F (2), one new furostanoside, filiasparoside G (3), and one new ecdysterone, stachysterone A-20, 22-acetonide (4), together with six known steroidal saponins, asparagusin A (5), filiasparoside A (6), filiasparoside B (7), aspafilioside A (8), aspafilioside B (9), and filiasparoside C (10) were isolated from the roots of Asparagus filicinus Buch.-Ham. Their structures were elucidated on the basis of spectroscopic and chemical evidence. Compounds 1-10 were investigated for their cytotoxicities against human breast adenocarcinoma MDA-MB-231 cell line and compounds 8-10 exhibited cytotoxic activities with IC₅₀ values ranging from 3.4 to 6.6 μM.     

New,Closely Related Haloarchaeal Viral Elements with Different Nucleic Acid Types

During the search for haloarchaeal viruses, we isolated and characterized a new pleomorphic lipid-containing virus, Haloarcula hispanica pleomorphic virus 1 (HHPV-1), that infects the halophilic archaeon Haloarcula hispanica. The virus contains a circular double-stranded DNA genome of 8,082 bp in size. The organization of the genome shows remarkable synteny and amino acid sequence similarity to the genome and predicted proteins of the halovirus HRPV-1, a pleomorphic single-stranded DNA virus that infects a halophilic archaeon Halorubrum sp. Analysis of the two halovirus sequences, as well as the entire nucleotide sequence of the 10.8-kb pHK2-plasmid and a 12.6-kb chromosomal region in Haloferax volcanii, allows us to suggest a new group of closely related viruses with genomes of either single-stranded or double-stranded DNA. Currently, closely related viruses are considered to have the same genome type. Our observation clearly contradicts this categorization and indicates that we should reconsider the way we classify viruses. Our results also provide a new example of related viruses where the viral structural proteins have not diverged as much as the proteins associated with genome replication. This result further strengthens the proposal for higher-order classification to be based on virion architecture rather than on genome type or replication mechanism.Metagenomic studies have increased the amount of information on the nucleotide sequence space in our environment. It has also increased our awareness of the viral abundance and diversity not recognized before (16, 24, 26). Along with this new information, we have learned to acknowledge the significance of viruses in the evolution and behavior of other organisms (55). To reveal the dynamics and molecular interactions in the interplay between a particular virus and its host, however, isolation of single viruses and their hosts is needed. Even though a number of viruses pathogenic to humans, domestic animals, and plants, as well as some bacteriophages, have been studied in great detail, much of the diversity of the archaeal viruses has remained unknown. By the year 2007 only 44 archaeal viruses had been described (2). That embraces less than 1% of all reported viruses. Although the diversity among these few isolated archaeal viruses is considerable, a head-and-tail morphology is prevalent among isolated viruses infecting euryarchaeal cells. In contrast, viruses of Crenarchaeota are diverse and often unusual with no viruses having a head-tail morphology (53).Archaeal haloviruses infect euryarchaeal hosts living in environments up to saturated salt. This makes them an interesting group of viruses that reside in a very restricted habitat. In samples taken from high salt environments, the Dead Sea and Spanish solar salterns, viral morphotypes most often observed were spindle-shaped, head-and-tail or tailless icosahedral particles (25, 31, 47). Isolated haloviruses, however, do not seem to reflect the proportions of different morphotypes found in the nature as nearly all of the isolates possess a head-and-tail morphology (2). Molecular level studies on only two spindle-shaped (10, 11) and one tailless icosahedral particle have been carried out (37, 51). Virus-like particles of other morphologies have also been observed in high-salt environments (47), but only one additional morphotype has been described in detail (50). This recently isolated lipid containing halovirus, HRPV-1, is the first archaeal virus containing a single-stranded DNA (ssDNA) genome (50). It infects Halorubrum sp. and has a pleomorphic appearance with glycosylated spike structures protruding from its external membrane (49, 50).The evolution of prokaryotic viral genome sequences is very fast (18), and the assessment of viral relationships using homology of the genome sequences applies only to closely related viruses (17, 19). Current higher-order classification of viruses is based on the host organism, the nature of the genome (RNA/DNA, single stranded versus double stranded) and the virion morphology. Recently, a higher-order clustering of virus families has been proposed based on common principles of virion architectures as well as on the fold of the major capsid protein (1, 6, 12, 13, 42). Consequently, major capsid proteins most probably belong to the vertically inherited viral “self” (4), whereas proteins involved in replication of the viral genome can be swapped by horizontal exchange (21, 63). The proposal is based on observations that structurally related viruses have been found to infect organisms that reside in all three domains of life.We have isolated a new pleomorphic virus infecting Haloarcula hispanica (Har. hispanica pleomorphic virus 1 [HHPV-1]). Here, we determine the molecular constituents of HHPV-1 and its genetic relatedness to other archaeal viruses and putative proviruses. Sequence homology and gene order (synteny) shows distinct genomic regions shared between four genetic elements separating replication, virus assembly, and integration functions. Surprisingly, in spite of the close relatedness of HRPV-1 and HHPV-1, the genome types of these two viruses differ (ssDNA and dsDNA, respectively).     

Cloning and characterization of a new gene from the PAT protein family,in a marsupial,the stripe‐faced dunnart (Sminthopsis macroura)

Recent studies of PAT proteins in Drosophila and Xenopus have revealed significant roles for this family of proteins in the polarized transport of lipid droplets and maternal determinants during early embryogenesis. In mammals, PAT proteins are known to function mainly in lipid metabolism, yet research has yet to establish a role for PAT proteins in mammalian embryogenesis. Oocytes and early cleavage stages in Sminthopsis macroura show obvious polarized cytoplasmic distribution of organelles, somewhat similar to Drosophila and Xenopus, suggesting that a PAT protein may also be involved in S. macroura embryonic development. In the present study, we identified a new marsupial gene for PAT family proteins, DPAT, from S. macroura. Expression analyses by RT‐PCR and whole mount fluorescent in situ hybridization revealed that DPAT expression was specific to oocytes and cleavage stage conceptuses. Analysis of the localization of lipid droplets during S. macroura early embryonic development found a polarized distribution of lipid droplets at the two‐ and four‐cell stage, and an asymmetric enrichment in blastomeres on one side of conceptuses from two‐ to eight‐cell stage. Lipid droplets largely segregate to pluriblast cells at the 16‐cell stage, suggesting a role in pluriblast lineage allocation. Mol. Reprod. Dev. 77: 373–383, 2010. © 2010 Wiley‐Liss, Inc.     

Reduced gene dosage of histone H4 prevents CENP-A mislocalization and chromosomal instability in Saccharomyces cerevisiae

Mislocalization of the centromeric histone H3 variant (Cse4 in budding yeast, CID in flies, CENP-A in humans) to noncentromeric regions contributes to chromosomal instability (CIN) in yeast, fly, and human cells. Overexpression and mislocalization of CENP-A have been observed in cancers, however, the mechanisms that facilitate the mislocalization of overexpressed CENP-A have not been fully explored. Defects in proteolysis of overexpressed Cse4 (GALCSE4) lead to its mislocalization and synthetic dosage lethality (SDL) in mutants for E3 ubiquitin ligases (Psh1, Slx5, SCF^Met30, and SCF^Cdc4), Doa1, Hir2, and Cdc7. In contrast, defects in sumoylation of overexpressed cse4K215/216/A/R prevent its mislocalization and do not cause SDL in a psh1Δ strain. Here, we used a genome-wide screen to identify factors that facilitate the mislocalization of overexpressed Cse4 by characterizing suppressors of the psh1Δ GALCSE4 SDL. Deletions of histone H4 alleles (HHF1 or HHF2), which were among the most prominent suppressors, also suppress slx5Δ, cdc4-1, doa1Δ, hir2Δ, and cdc7-4 GALCSE4 SDL. Reduced dosage of H4 leads to defects in sumoylation and reduced mislocalization of overexpressed Cse4, which contributes to suppression of CIN when Cse4 is overexpressed. We determined that the hhf1-20, cse4-102, and cse4-111 mutants, which are defective in the Cse4-H4 interaction, also exhibit reduced sumoylation of Cse4 and do not display psh1Δ GALCSE4 SDL. In summary, we have identified genes that contribute to the mislocalization of overexpressed Cse4 and defined a role for the gene dosage of H4 in facilitating Cse4 sumoylation and mislocalization to noncentromeric regions, leading to CIN when Cse4 is overexpressed.     

Attenuation of Hind-Limb Ischemia in Mice with Endothelial-Like Cells Derived from Different Sources of Human Stem Cells

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however, their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC), human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC), and compared their in-vitro tube formation, migration and cytokine expression profiles, and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless, BM-EC, hESC-EC and hiPSC-EC exhibited typical cobblestone morphology, had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein, and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (P>0.05). While increased expression of major angiogenic factors including epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor, placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (P<0.05), the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (P<0.05). Compared with medium, transplanting BM-EC (n = 6), HUVEC (n = 6), hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion, functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases, and hESC-EC or iPSC-EC are readily available as “off-the-shelf” format for the treatment of tissue ischemia.     

Suppression of PACT-Induced Type I Interferon Production by Herpes Simplex Virus 1 Us11 Protein

Herpes simplex virus 1 (HSV-1) Us11 protein is a double-stranded RNA-binding protein that suppresses type I interferon production through the inhibition of the cytoplasmic RNA sensor RIG-I. Whether additional cellular mediators are involved in this suppression remains to be determined. In this study, we report on the requirement of cellular double-stranded RNA-binding protein PACT for Us11-mediated perturbation of type I interferon production. Us11 associates with PACT tightly to prevent it from binding with and activating RIG-I. The Us11-deficient HSV-1 was indistinguishable from the Us11-proficient virus in the suppression of interferon production when PACT was compromised. More importantly, HSV-1-induced activation of interferon production was abrogated in PACT knockout murine embryonic fibroblasts. Our findings suggest a new mechanism for viral evasion of innate immunity through which a viral double-stranded RNA-binding protein interacts with PACT to circumvent type I interferon production. This mechanism might also be used by other PACT-binding viral interferon-antagonizing proteins such as Ebola virus VP35 and influenza A virus NS1.     

Whole-body protein turnover reveals the cost of detoxification of secondary metabolites in a vertebrate browser

The detoxification limitation hypothesis predicts that the metabolism and biotransformation of plant secondary metabolites (PSMs) elicit a cost to herbivores. There have been many attempts to estimate these costs to mammalian herbivores in terms of energy, but this ignores what may be a more important cost—increases in protein turnover and concomitant losses of amino acids. We measured the effect of varying dietary protein concentrations on the ingestion of two PSMs (1,8 cineole—a monoterpene, and benzoic acid—an aromatic carboxylic acid) by common brushtail possums (Trichosurus vulpecula). The dietary protein concentration had a small effect on how much cineole possums ingested. In contrast, protein had a large effect on how much benzoate they ingested, especially at high dietary concentrations of benzoate. This prompted us to measure the effects of dietary protein and benzoate on whole-body protein turnover using the end-product method following an oral dose of [¹⁵N] glycine. Increasing the concentration of dietary protein in diets without PSMs improved N balance but did not influence whole-body protein turnover. In contrast, feeding benzoate in a low-protein diet pushed animals into negative N balance. The concomitant increases in the rates of whole-body protein turnover in possums eating diets with more benzoate were indicative of a protein cost of detoxification. This was about 30 % of the dietary N intake and highlights the significant effects that PSMs can have on nutrient metabolism and retention.     

Degradation of Newly Synthesized Polypeptides by Ribosome-Associated RACK1/c-Jun N-Terminal Kinase/Eukaryotic Elongation Factor 1A2 Complex

Folding of newly synthesized polypeptides (NSPs) into functional proteins is a highly regulated process. Rigorous quality control ensures that NSPs attain their native fold during or shortly after completion of translation. Nonetheless, signaling pathways that govern the degradation of NSPs in mammals remain elusive. We demonstrate that the stress-induced c-Jun N-terminal kinase (JNK) is recruited to ribosomes by the receptor for activated protein C kinase 1 (RACK1). RACK1 is an integral component of the 40S ribosome and an adaptor for protein kinases. Ribosome-associated JNK phosphorylates the eukaryotic translation elongation factor 1A isoform 2 (eEF1A2) on serines 205 and 358 to promote degradation of NSPs by the proteasome. These findings establish a role for a RACK1/JNK/eEF1A2 complex in the quality control of NSPs in response to stress.     

Structural basis of FliG–FliM interaction in Helicobacter pylori

FliG and FliM are switch proteins that regulate the rotation and switching of the flagellar motor. Several assembly models for FliG and FliM have recently been proposed; however, it remains unclear whether the assembly of the switch proteins is conserved among different bacterial species. We applied a combination of pull‐down, thermodynamic and structural analyses to characterize the FliM–FliG association from the mesophilic bacterium Helicobacter pylori. FliM binds to FliG with micromolar binding affinity, and their interaction is mediated through the middle domain of FliG (FliG_M), which contains the EHPQR motif. Crystal structures of the middle domain of H. pylori FliM (FliM_M) and FliG_M–FliM_M complex revealed that FliG binding triggered a conformational change of the FliM α3‐α1′ loop, especially Asp130 and Arg144. We furthermore showed that various highly conserved residues in this region are required for FliM–FliG complex formation. Although the FliM–FliG complex structure displayed a conserved binding mode when compared with Thermotoga maritima, variable residues were identified that may contribute to differential binding affinities across bacterial species. Comparison of the thermodynamic parameters of FliG–FliM interactions between H. pylori and Escherichia coli suggests that molecular basis and binding properties of FliM to FliG is likely different between these two species.