Direct eIF2-eIF3 contact in the multifactor complex is important for translation initiation in vivo

Translation initiation factor 3 (eIF3) of Saccharo myces cerevisiae forms a multifactor complex (MFC) with eIFs 1, 2, 5 and Met-tRNA(i)(Met). We previously constructed a subunit interaction model for the MFC. Here we incorporated affinity tags into the three largest eIF3 subunits (eIF3a/TIF32, eIF3b/PRT1 and eIF3c/NIP1) and deleted predicted binding domains in each tagged protein. By characterizing the mutant subcomplexes, we confirmed all key predictions of our model and uncovered new interactions of NIP1 with PRT1 and of TIF32 with eIF1. In addition to the contact between eIF2 and the N-terminal domain (NTD) of NIP1 bridged by eIF5, the C-terminal domain (CTD) of TIF32 binds eIF2 directly and is required for eIF2-eIF3 association in vivo. Overexpressing a CTD-less form of TIF32 exacerbated the initiation defect of an eIF5 mutation that weakens the NIP1-eIF5-eIF2 connection. Thus, the two independent eIF2-eIF3 contacts have additive effects on translation in vivo. Overexpressing the NIP1-NTD sequestered eIF1-eIF5-eIF2 in a defective subcomplex that derepressed GCN4 translation, providing the first in vivo evidence that association with eIF3 promotes binding of eIF2 and Met-tRNA(i)(Met) to 40S ribosomes.     

Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.     

Colonization models and initial genetic diversity in the Americas

The mode and tempo of colonization of the Americas established the initial pattern of continental genetic diversity. Despite a long history of study, the process of settlement remains controversial in terms of date, rate, and pattern. While there is agreement that Asia was the source population, several different models have been proposed for the colonization process. A classic model postulates a rapid spread of population ("blitzkrieg") from a small band of hunters entering through the corridor between the continental ice sheets circa 11,000 years B.P. Colonization occurred as a wave of expansion across the land masses of North and South America. An alternative model envisions the original colonists initially limiting settlement to the coastline, using boats, and entering the Americas at an earlier date, circa 13,500 B.P. Range expansion along this linear habitat from North to South America could be rapid without requiring population saturation of entire continental regions. These models have markedly different implications for genetic variation among Native Americans. The blitzkrieg colonization process would have generated multiple founder effects leading to extreme loss of genetic variation. Computer simulation of this model shows nearly complete fixation in 30 generations. Simulation of the coastal model, on the other hand, requires less extreme demographic assumptions and maintains substantial genetic variability after 100 generations. Although with the coastal model continental interiors are occupied less rapidly than with the blitzkrieg model, the coastal model allows earlier entry and rapid expansion to the southern limits of the hemisphere.     

Segregation analysis of blood pressure and body mass index in a rural US community

To assess evidence for a gene with large effect on systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI), we conducted segregation analyses on 261 nuclear families collected from a rural Caucasian community in Michigan. The families were ascertained through a hypertensive proband. Each phenotype was adjusted for significant covariate effects (e.g., gender and age). We used class D regressive models to conduct the segregation analyses. Our analysis results support the segregation of a major gene for BMI, but not for SBP or DBP. A recessive locus effect provided the best explanation for BMI where approximately 43% of the variance of BMI was due to this gene.     

Nitrite reductase of Nitrosomonas europaea is not essential for production of gaseous nitrogen oxides and confers tolerance to nitrite

A gene that encodes a periplasmic copper-type nitrite reductase (NirK) was identified in Nitrosomonas europaea. Disruption of this gene resulted in the disappearance of Nir activity in cell extracts. The nitrite tolerance of NirK-deficient cells was lower than that of wild-type cells. Unexpectedly, NirK-deficient cells still produced nitric oxide (NO) and nitrous oxide (N(2)O), the latter in greater amounts than that of wild-type cells. This demonstrates that NirK is not essential for the production of NO and N(2)O by N. europaea. Inactivation of the putative fnr gene showed that Fnr is not essential for the expression of nirK.     

The insulin receptor catalyzes the tyrosine phosphorylation of caveolin-1

Our previous studies revealed that insulin stimulates the tyrosine phosphorylation of caveolin in 3T3L1 adipocytes. To explore the mechanisms involved in this event, we evaluated the association of the insulin receptor with caveolin. The receptor was detected in a Triton-insoluble low density fraction, co-sedimenting with caveolin and flotillin on sucrose density gradients. We also detected the receptor in caveolin-enriched rosette structures by immunohistochemical analysis of plasma membrane sheets from 3T3L1 adipocytes. Insulin stimulated the phosphorylation of caveolin-1 on Tyr(14). This effect of the hormone was not blocked by overexpression of mutant forms of the Cbl-associated protein that block the translocation of phospho-Cbl to the caveolin-enriched, lipid raft microdomains. Moreover, this phosphorylation event was also unaffected by inhibitors of the MAPK and phosphatidylinositol 3-kinase pathways. Although previous studies demonstrated that the Src family kinase Fyn was highly enriched in caveolae, an inhibitor of this kinase had no effect on insulin-stimulated caveolin phosphorylation. Interestingly, overexpression of a mutant form of caveolin that failed to interact with the insulin receptor did not undergo phosphorylation. Taken together, these data indicate that the insulin receptor directly catalyzes the tyrosine phosphorylation of caveolin.     

Characterization of thermodynamic diversity between transmissible spongiform encephalopathy agent strains and its theoretical implications.

Some transmissible spongiform encephalopathy (TSE) (or "prion") strains, notably those derived from bovine spongiform encephalopathy, are highly resistant to total inactivation by heat. When three TSE strains derived from sheep with scrapie were heated, little inactivation took place at low temperatures, but at higher temperatures, considerable inactivation occurred. The temperature at which substantial inactivation first occurred varied according to TSE strain, and it was calculated to be 70 degrees C for the 22C strain, 84 degrees C for ME7, and 97 degrees C for 22A by fitting the data to a model based on competition between a destructive and a protective reaction. However, PrP(Sc) from mice infected with a range of TSE strains retained similar resistance to proteinase K digestion after heating to below or above these temperatures, showing that the properties of PrP(Sc) responsible for proteinase resistance do not correlate with those conferring thermostability on the TSE agent. The simplest explanation of these data is that the causal agent contains a macromolecular component that is structurally independent of the host, that it varies covalently between TSE strains, and that it is protected by other macromolecular components. The model is in accord with the virino hypothesis, which proposes a host-independent informational molecule protected by the host protein PrP.     

Microbial recognition via Toll-like receptor-dependent and -independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.