Survival and growth of two heterotrophic hydrothermal vent archaea, <Emphasis Type="Italic">Pyrococcus</Emphasis> strain GB-D and <Emphasis Type="Italic">Thermococcus fumicolans</Emphasis>, under low pH and high sulfide concentrations in combination with high temperature and pressure regimes

Growth and survival of hyperthermophilic archaea in their extreme hydrothermal vent and subsurface environments are controlled by chemical and physical key parameters. This study examined the effects of elevated sulfide concentrations, temperature, and acidic pH on growth and survival of two hydrothermal vent archaea (Pyrococcus strain GB-D and Thermococcus fumicolans) under high temperature and pressure regimes. These two strains are members of the Thermococcales, a family of hyperthermophilic, heterotrophic, sulfur-reducing archaea that occur in high densities at vent sites. As actively growing cells, these two strains tolerated regimes of pH, pressure, and temperature that were in most cases not tolerated under severe substrate limitation. A moderate pH of 5.5–7 extends their survival and growth range over a wider range of sulfide concentrations, temperature and pressure, relative to lower pH conditions. T. fumicolans and Pyrococcus strain GB-D grew under very high pressures that exceeded in-situ pressures typical of hydrothermal vent depths, and included deep subsurface pressures. However, under the same conditions, but in the absence of carbon substrates and electron acceptors, survival was generally lower, and decreased rapidly when low pH stress was combined with high pressure and high temperature. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Targeting GRB7/ERK/FOXM1 Signaling Pathway Impairs Aggressiveness of Ovarian Cancer Cells

Ovarian cancer is a highly lethal disease with poor prognosis and especially in high-grade tumor. Emerging evidence has reported that aberrant upregulation and activation of GRB7, ERK as well as FOXM1 are closely associated with aggresivenesss of human cancers. However, the interplay between these factors in the pathogenesis of human cancers still remains unclear. In this study, we found that GRB7 (P<0.0001), ERK phosphorylation (P<0.0001) and FOXM1 (P = 0.001) were frequently increased and associated with high-grade tumors, as well as a high tendency in association with advanced stage ovarian cancer by immunohistochemical analysis. Intriguingly, the expressions of GRB7 (P<0.0001), ERK phosphorylation (P<0.001) and FOXM1 (P<0.001) showed a significant stepwise increase pattern along Grade 1 to Grade 3 ovarian cancers. Biochemical studies using western blot analysis demonstrated that enforced expression or knockdown of GRB7 showed GRB7 could elevate the levels of ERK phosphorylation and FOXM1, whereas enforced expression of FOXM1 could not alter levels of GRB7 and ERK phosphorylation. But inhibition of ERK signaling by U0126 or PD98059 could reduce the level of FOXM1 in GRB7-overexpressing ovarian cancer cells, suggesting that GRB7, ERK and FOXM1 are regulated orderly. Moreover, inhibition of ERK activity by U0126 or PD98059, or decreased FOXM1 expression by Thiostrepton significantly inhibited cell migration/invasion, tumor growth in vitro and in vivo. Collectively, our findings confer that targeting GRB7/ERK/FOXM1 signaling cascade may be a promising molecular therapeutic choice in combating ovarian cancer.     

TMEM41B and VMP1 are scramblases and regulate the distribution of cholesterol and phosphatidylserine

TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.     

Opposing roles for TRAF1 in the alternative versus classical NF-κB pathway in T cells

T cells lacking TRAF1 hyperproliferate in response to T cell receptor signaling but have impaired signaling downstream of specific TNFR family members such as 4-1BB. Here we resolve this paradox by showing that while TRAF1 is required for maximal activation of the classical NF-κB pathway downstream of 4-1BB in primary T cells, TRAF1 also restricts the constitutive activation of NIK in anti-CD3-activated T cells. Activation of the alternative NF-κB pathway is restricted in unstimulated cells by a cIAP1/2:TRAF2:TRAF3:NIK complex. Using knockdown of NIK by siRNA we show that in activated CD8 T cells TRAF1 is also involved in this process and that constitutive activation of the alternative NF-κB pathway is responsible for costimulation independent hyperproliferation and excess cytokine production in TRAF1-deficient CD8 T cells compared with WT CD8 T cells. The T cell costimulatory molecule 4-1BB critically regulates the survival of activated and memory CD8 T cells. We demonstrate that stimulation through 4-1BB induces cIAP1-dependent TRAF3 degradation and activation of the alternative NF-κB pathway. We also show that while both TRAF1 and cIAP1 have non-redundant roles in suppressing the alternative NF-κB pathway in T cells activated in the absence of costimulation, activation of the classical NF-κB pathway downstream of 4-1BB requires TRAF1, whereas cIAP1 plays a redundant role with cIAP2. Collectively these results demonstrate that TRAF1 plays a critical role in regulating T cell activation both through restricting the costimulation independent activation of NIK in activated T cells and by promoting the 4-1BB-induced classical NF-κB pathway.     

Dissection of the insulin-sensitizing effect of liver X receptor ligands

The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Treatment of insulin-resistant mice with synthetic LXR ligands enhances glucose tolerance, inducing changes in gene expression expected to decrease hepatic gluconeogenesis (via indirect suppression of gluconeogenic enzymes) and increase peripheral glucose disposal (via direct up-regulation of glut4 in fat). To evaluate the relative contribution of each of these effects on whole-body insulin sensitivity, we performed hyperinsulinemic-euglycemic clamps in high-fat-fed insulin-resistant rats treated with an LXR agonist or a peroxisome proliferator-activated receptor gamma ligand. Both groups showed significant improvement in insulin action. Interestingly, rats treated with LXR ligand had lower body weight and smaller fat cells than controls. Insulin-stimulated suppression of the rate of glucose appearance (Ra) was pronounced in LXR-treated rats, but treatment failed to enhance peripheral glucose uptake (R'g), despite increased expression of glut4 in epididymal fat. To ascertain whether LXR ligands suppress hepatic gluconeogenesis directly, mice lacking LXRalpha (the primary isotype in liver) were treated with LXR ligand, and gluconeogenic gene expression was assessed. LXR activation decreased expression of gluconeogenic genes in wild-type and LXRbeta null mice, but failed to do so in animals lacking LXRalpha. Our observations indicate that despite inducing suggestive gene expression changes in adipose tissue in this model of diet-induced insulin resistance, the antidiabetic effect of LXR ligands is primarily due to effects in the liver that appear to require LXRalpha. These findings have important implications for clinical development of LXR agonists as insulin sensitizers.     

Maize Ribosome-Inactivating Protein Uses Lys158–Lys161 to Interact with Ribosomal Protein P2 and the Strength of Interaction Is Correlated to the Biological Activities

Ribosome-inactivating proteins (RIPs) inactivate prokaryotic or eukaryotic ribosomes by removing a single adenine in the large ribosomal RNA. Here we show maize RIP (MOD), an atypical RIP with an internal inactivation loop, interacts with the ribosomal stalk protein P2 via Lys158–Lys161, which is located in the N-terminal domain and at the base of its internal loop. Due to subtle differences in the structure of maize RIP, hydrophobic interaction with the ‘FGLFD’ motif of P2 is not as evidenced in MOD-P2 interaction. As a result, interaction of P2 with MOD was weaker than those with trichosanthin and shiga toxin A as reflected by the dissociation constants (K_D) of their interaction, which are 1037.50±65.75 µM, 611.70±28.13 µM and 194.84±9.47 µM respectively.Despite MOD and TCS target at the same ribosomal protein P2, MOD was found 48 and 10 folds less potent than trichosanthin in ribosome depurination and cytotoxicity to 293T cells respectively, implicating the strength of interaction between RIPs and ribosomal proteins is important for the biological activity of RIPs. Our work illustrates the flexibility on the docking of RIPs on ribosomal proteins for targeting the sarcin-ricin loop and the importance of protein-protein interaction for ribosome-inactivating activity.     

Parasite aquaporins: Current developments in drug facilitation and resistance

Background

Although being situated in a niche, research on parasite aquaporins is a lively field that has provided new insight into basic aquaporin structure–function relationships and physiological roles of water and solute transport. Moreover, it bears the potential to find novel approaches to antiparasitic chemotherapy.

Scope of review

Here, we summarize the current knowledge about the structure and substrate selectivity of aquaporins from protozoan and helminth parasites, review the current views on their physiological roles, and discuss their potency for chemotherapy.

Major conclusions

Parasite aquaporins fulfill highly diverse tasks in the physiology of the various organisms, yet their general protein structure is well conserved. Aquaporins are directly (antimonials) and indirectly (melarsoprol, pentamidine) linked to the uptake of antiparasitic drugs. Unfortunately, drug-like aquaporin inhibitors are still missing.

General significance

Aquaporins expression levels determine the degree of parasite resistance against certain drugs. Further studies on parasite aquaporins may provide data about overcoming drug resistance mechanisms or even spark novel treatments. This article is part of a Special Issue entitled Aquaporins.     

Survivin is essential for fertile egg production and female fertility in mice

Survivin is the smallest member of the inhibitor of apoptosis protein (IAP) family and acts as a bifunctional protein involved in mitosis regulation and apoptosis inhibition. To identify the physiological role of Survivin in female reproduction, we selectively disrupted Survivin expression in oocytes and granulosa cells (GCs), two major cell types in the ovary, by two different Cre-Loxp conditional knockout systems, and found that both led to defective female fertility. Survivin deletion in oocytes did not affect oocyte growth, viability and ovulation, but caused tetraploid egg production and thus female infertility. Further exploration revealed that Survivin was essential for regulating proper meiotic spindle organization, spindle assembly checkpoint activity, timely metaphase-to-anaphase transition and cytokinesis. Mutant mice with Survivin depleted in GCs showed reduced ovulation and subfertility, caused by defective follicular growth, increased follicular atresia and impaired luteinization. These findings suggest that Survivin has an important role in regulating folliculogenesis and oogenesis in the adult mouse ovary.     

Adenovirus type 12-specific RNA sequences during productive infection of KB cells.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.