Requirement of rRNA methylation for 80S ribosome assembly on a cohort of cellular internal ribosome entry sites

Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.     

The A2B adenosine receptor promotes Th17 differentiation via stimulation of dendritic cell IL-6

Adenosine is an endogenous metabolite produced during hypoxia or inflammation. Previously implicated as an anti-inflammatory mediator in CD4(+) T cell regulation, we report that adenosine acts via dendritic cell (DC) A(2B) adenosine receptor (A(2B)AR) to promote the development of Th17 cells. Mouse naive CD4(+) T cells cocultured with DCs in the presence of adenosine or the stable adenosine mimetic 5'-(N-ethylcarboximado) adenosine resulted in the differentiation of IL-17- and IL-22-secreting cells and elevation of mRNA that encode signature Th17-associated molecules, such as IL-23R and RORγt. The observed response was similar when DCs were generated from bone marrow or isolated from small intestine lamina propria. Experiments using adenosine receptor antagonists and cells from A(2B)AR(-/-) or A(2A)AR(-/-)/A(2B)AR(-/-) mice indicated that the DC A(2B)AR promoted the effect. IL-6, stimulated in a cAMP-independent manner, is an important mediator in this pathway. Hence, in addition to previously noted direct effects of adenosine receptors on regulatory T cell development and function, these data indicated that adenosine also acts indirectly to modulate CD4(+) T cell differentiation and suggested a mechanism for putative proinflammatory effects of A(2B)AR.     

Antigen is required for maturation and activation of pathogenic anti-DNA antibodies and systemic inflammation

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies and systemic inflammation that results in part from dendritic cell activation by nucleic acid containing immune complexes. There are many mouse models of lupus, some spontaneous and some induced. We have been interested in an induced model in which estrogen is the trigger for development of a lupus-like serology. The R4A transgenic mouse expresses a transgene-encoded H chain of an anti-DNA Ab. This mouse maintains normal B cell tolerance with deletion of high-affinity DNA-reactive B cells and maturation to immunocompetence of B cells making nonglomerulotropic, low-affinity DNA-reactive Abs. When this mouse is given estradiol, normal tolerance mechanisms are altered; high-affinity DNA-reactive B cells mature to a marginal zone phenotype, and the mice are induced to make high titers of anti-DNA Abs. We now show that estradiol administration also leads to systemic inflammation with increased B cell-activating factor and IFN levels and induction of an IFN signature. DNA must be accessible to B cells for both the production of high-affinity anti-DNA Abs and the generation of the proinflammatory milieu. When DNase is delivered to the mice at the same time as estradiol, there is no evidence for an abrogation of tolerance, no increased B cell-activating factor and IFN, and no IFN signature. Thus, the presence of autoantigen is required for positive selection of autoreactive B cells and for the subsequent positive feedback loop that occurs secondary to dendritic cell activation by DNA-containing immune complexes.     

Variable developmental rate and survival of navel orangeworm (Lepidoptera: Pyralidae) on pistachio

A series of laboratory and field studies were conducted using two lines of navel orangeworm, reared on different stages of new crop and mummy pistachios, Pistacia vera L. This study demonstrated the potential importance of malformed pistachios (pea splits) to the population dynamics of navel orangeworm, because these nuts, which are available as early as two months before mature nuts, supported navel orangeworm development and survival. Overall, the developmental rate on new crop pistachios is fastest on mature nuts, 422.3 +/- 123 degree-days (DD, degrees C), but other factors such as exposure to insecticide residue also sped development, although survival decreased. Development took the longest on unharvested nuts (mummies) dried at 90 degrees C for 24 h, 2664.7 +/- 131.4 DD. In most trials development was variable and two generations could develop at the fastest rate before the slowest individual completed development, which in turn calls into question the concept of discrete generations. Generally, survival was highest on mature pistachios and other stages of new crop nut and lowest on mummies collected in May. Survival was also higher on the new varieties 'Lost Hills' and 'Golden Hills' (24.7 and 32.0%, respectively) than on the most extensively planted variety 'Kerman' (13.3%). In our trials, both the rate of development and survival were dependent on nut stage, age, variety, and quality, indicating that pistachios, like almonds, Prunus dulcis (Mill.) D. A. Webb, are a dynamic rather than a static nutrient source for navel orangeworm.     

Effects of rearing density, age, sex, and food deprivation on flight initiation of the red flour beetle (Coleoptera: Tenebrionidae)

Effects of rearing density, adult density and sex ratio in the flight chamber, adult age, sex, presence or absence of food, and duration of food deprivation on rate of and time to flight initiation of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), were studied in the laboratory. Rates of flight initiation were slightly lower at lower rearing densities, but they did not differ with age or sex of adults, presence or absence of food in the flight chamber, or duration of food deprivation. Focal adults were less likely to fly when individuals of the opposite sex were present in flight chambers. Presence of the same sex or mixed sexes and numbers of individuals in flight chambers did not affect tendency to fly of focal individuals. Mean time to flight of older beetles (7-20 d old) was less than that of younger beetles (1-4 d old). No young beetles flew during the first 24 h of flight tests. Mean time to flight did not differ with rearing density, sex, presence or absence of food, or duration of food deprivation. Our results indicate that this species is highly dispersive during the adult stage and that flight does not seem to be associated with just prereproductive or postreproductive dispersal phases, high rearing density, or short to medium periods of food deprivation. Therefore, T. castaneum level of flight activity does not seem to be associated with factors that have been shown in the literature to increase dispersal by walking for this species and to increase flight initiation in other stored-product species.     

DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes

The total contribution of the acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, to mammalian triacylglycerol (TG) synthesis has not been determined. Similarly, whether DGAT enzymes are required for lipid droplet (LD) formation is unknown. In this study, we examined the requirement for DGAT enzymes in TG synthesis and LDs in differentiated adipocytes with genetic deletions of DGAT1 and DGAT2. Adipocytes with a single deletion of either enzyme were capable of TG synthesis and LD formation. In contrast, adipocytes with deletions of both DGATs were severely lacking in TG and did not have LDs, indicating that DGAT1 and DGAT2 account for nearly all TG synthesis in adipocytes and appear to be required for LD formation during adipogenesis. DGAT enzymes were not absolutely required for LD formation in mammalian cells, however; macrophages deficient in both DGAT enzymes were able to form LDs when incubated with cholesterol-rich lipoproteins. Although adipocytes lacking both DGATs had no TG or LDs, they were fully differentiated by multiple criteria. Our findings show that DGAT1 and DGAT2 account for the vast majority of TG synthesis in mice, and DGAT function is required for LDs in adipocytes, but not in all cell types.     

Conformational transformation and selection of synthetic prion strains

Prion protein is capable of folding into multiple self-replicating prion strains that produce phenotypically distinct neurological disorders. Although prion strains often breed true upon passage, they can also transform or “mutate” despite being devoid of nucleic acids. To dissect the mechanism of prion strain transformation, we studied the physicochemical evolution of a mouse synthetic prion (MoSP) strain, MoSP1, after repeated passage in mice and cultured cells. We show that MoSP1 gradually adopted shorter incubation times and lower conformational stabilities. These changes were accompanied by structural transformation, as indicated by a shift in the molecular mass of the protease-resistant core of MoSP1 from approximately 19 kDa [MoSP1(2)] to 21 kDa [MoSP1(1)]. We show that MoSP1(1) and MoSP1(2) can breed with fidelity when cloned in cells; however, when present as a mixture, MoSP1(1) preferentially proliferated, leading to the disappearance of MoSP1(2). In culture, the rate of this transformation process can be influenced by the composition of the culture media and the presence of polyamidoamines. Our findings demonstrate that prions can exist as a conformationally diverse population of strains, each capable of replicating with high fidelity. Rare conformational conversion, followed by competitive selection among the resulting pool of conformers, provides a mechanism for the adaptation of the prion population to its host environment.     

RGK family G-domain:GTP analog complex structures and nucleotide-binding properties

The RGK family of small G-proteins, including Rad, Gem, Rem1, and Rem2, is inducibly expressed in various mammalian tissues and interacts with voltage-dependent calcium channels and Rho kinase. Many questions remain regarding their physiological roles and molecular mechanism. Previous crystallographic studies reported RGK G-domain:guanosine di-phosphate structures. To test whether RGK proteins undergo a nucleotide-induced conformational change, we determined the crystallographic structures of Rad:GppNHp and Rem2:GppNHp to 1.7 and 1.8 Å resolutions, respectively. Also, we characterized the nucleotide-binding properties and conformations for Gem, Rad, and several structure-based mutants using fluorescence spectroscopy. The results suggest that RGK G-proteins may not behave as Ras-like canonical nucleotide-induced molecular switches. Further, the RGK proteins have differing structures and nucleotide-binding properties, which may have implications for their varied action on effectors.     

Unstimulated primary CD4+ T cells from HIV-1-positive elite suppressors are fully susceptible to HIV-1 entry and productive infection

Elite controllers or suppressors (ES) are a group of HIV-1-infected individuals who maintain viral loads below the limit of detection of commercial assays for many years. The mechanisms responsible for this remarkable control are under intense study, with the hope of developing therapeutic vaccines effective against HIV-1. In this study, we addressed the question of the intrinsic susceptibility of ES CD4(+) T cells to infection. While we and others have previously shown that CD4(+) T cells from ES can be infected by HIV-1 isolates in vitro, these studies were confounded by exogenous activation and in vitro culture of CD4(+) T cells prior to infection. In order to avoid the changes in chemokine receptor expression that have been associated with such exogenous activation, we infected purified CD4(+) T cells directly after isolation from the peripheral blood of ES, viremic patients, and uninfected donors. We utilized a green fluorescent protein (GFP)-expressing proviral construct pseudotyped with CCR5-tropic or CXCR4-tropic envelope to compare viral entry using a fluorescence resonance energy transfer-based, single-round virus-cell fusion assay. The frequency of productive infection was also compared by assessing GFP expression. CD4(+) T cells from ES were as susceptible as or more susceptible than cells from viremic patients and uninfected donors to HIV-1 entry and productive infection. The results of this physiological study strongly suggest that differences in HIV-1 entry and infection of CD4(+) T cells alone cannot explain the elite control of viral replication.