RNA binding properties of the AU-rich element-binding recombinant Nup475/TIS11/tristetraprolin protein

Regulation of messenger RNA stability by AU-rich elements is an important means of regulating genes induced by growth factors and cytokines. Nup475 (also known as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding Cys(3)His motif proteins required for proper regulation of tumor necrosis factor mRNA stability in macrophages. We developed an Escherichia coli expression system to produce soluble Nup475 protein in quantity to study its RNA binding properties. Nup475 protein bound a tumor necrosis factor AU-rich element over a broad range of pH and salt concentrations by RNA gel shift. This binding was inhibited by excess zinc metal, providing a potential mechanism for previous reports of zinc stabilization of AU-rich element (ARE) containing messenger RNAs. Immobilized Nup475 protein was used to select its optimal binding site by RNA SELEX and revealed a strong preference for the extended sequence UUAUUUAUU, rather than a simple AUUUA motif. These findings were confirmed by site-directed mutagenesis of the tumor necrosis factor ARE and RNA gel shifts on c-fos, interferon-gamma, and interferon-beta ARE fragments. A weaker binding activity toward adenine-rich sites, such as a poly(A) tail RNA fragment, can partially disrupt the Nup475-tumor necrosis factor AU-rich element complex.     

Molding of the regenerate in mandibular distraction: clinical experience

Initial clinical experience with distraction osteogenesis has demonstrated the risk of developing postdistraction malocclusion that requires secondary orthodontic correction. In addition, optimal mandibular form is not always achieved. Both animal studies and preliminary clinical investigations have suggested that the regenerate can be successfully "molded" during active mandibular distraction. The authors have applied this concept clinically to obtain a more desirable occlusal relationship in a group of mandibular distraction patients. Eleven patients are described in whom angulation of the distraction device or intermaxillary/interdental elastics were employed to mold the regenerate. Two representative case studies are provided to illustrate the principles. When using elastic traction to close an anterior open bite, care must be taken that extrusion of individual teeth is minimized by distributing the force over the entire dental arch, especially the basilar portions of the jaws. The authors demonstrate that molding of the regenerate can be successfully accomplished not only during device activation but also early in the consolidation period. The outer limit of the time window in which molding is effective remains to be defined.     

Mesd encodes an LRP5/6 chaperone essential for specification of mouse embryonic polarity

Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. Here, we demonstrate that mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3(-)(/)(-) embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling.     

Stress and the developing hippocampus: a double-edged sword?

The mechanisms that regulate neuronal function are a sum of genetically determined programs and experience. The effect of experience on neuronal function is particularly important during development, because early-life positive and adverse experience (stress) may influence the still “plastic” nervous system long-term. Specifically, for hippocampal-mediated learning and memory processes, acute stress may enhance synaptic efficacy and overall learning ability, and conversely, chronic or severe stress has been shown to be detrimental. The mechanisms that enable stress to act as this “double-edged sword” are unclear. Here, we discuss the molecular mediators of the stress response in the hippocampus with an emphasis on novel findings regarding the role of the neuropeptide known as corticotropin-releasing hormone (CRH). We highlight the physiological and pathological roles of this peptide in the developing hippocampus, and their relevance to the long-term effects of early-life experience on cognitive function during adulthood.     

Caenorhabditis elegans SOS-1 is necessary for multiple RAS-mediated developmental signals

Vulval induction in Caenorhabditis elegans has helped define an evolutionarily conserved signal transduction pathway from receptor tyrosine kinases (RTKs) through the adaptor protein SEM-5 to RAS. One component present in other organisms, a guanine nucleotide exchange factor for Ras, has been missing in C.ELEGANS: To understand the regulation of this pathway it is crucial to have all positive-acting components in hand. Here we describe the identification, cloning and genetic characterization of C.ELEGANS: SOS-1, a putative guanine nucleotide exchanger for LET-60 RAS. RNA interference experiments suggest that SOS-1 participates in RAS-dependent signaling events downstream of LET-23 EGFR, EGL-15 FGFR and an unknown RTK. We demonstrate that the previously identified let-341 gene encodes SOS-1. Analyzing vulval development in a let-341 null mutant, we find an SOS-1-independent pathway involved in the activation of RAS signaling. This SOS-1-independent signaling is not inhibited by SLI-1/Cbl and is not mediated by PTP-2/SHP, raising the possibility that there could be another RasGEF.     

ARK-1 inhibits EGFR signaling in C. elegans

A screen for synthetic enhancers of sli-1 identified ark-1 (forAck-related tyrosine kinase), a novel inhibitor of let-23 EGFR signaling in C. elegans. An ark-1 mutation synergizes with mutations in other negative regulators of let-23, resulting in increased RAS signaling. Genetic analysis suggests that ARK-1 acts upstream of RAS and is dependent upon SEM-5. ARK-1 inhibits LET-23-mediated ovulation, a RAS-independent function. ARK-1 physically interacts with SEM-5 in the yeast two-hybrid assay. We find that sem-5 also has a negative function in let-23-mediated ovulation and suggest that this negative function is mediated by the recruitment of inhibitors such as ARK-1.     

Splicing before import - an intein in a mitochondrially targeted preprotein folds and is catalytically active in the cytoplasm in vivo

Nuclear-encoded mitochondrial proteins are cytoplasmically synthesized and imported into the organelle. The intein-containing RecA protein of Mycobacterium tuberculosis, with or without the CoxIVp mitochondrial targeting signal (MTS), was used to determine where a protein targeted to mitochondria folds and becomes catalytically active. Analysis of fractions from Saccharomyces cerevisiae cells expressing RecA without the MTS revealed that RecA and intein proteins remained cytoplasmic. With the MTS, most of RecA was directed to mitochondria, while most of the intein remained in the cytoplasm. The intein therefore folds into a catalytically active state in the cytoplasm prior to RecA import into mitochondria.     

The Anti-Inflammatory Activity of Curcumin Protects the Genital Mucosal Epithelial Barrier from Disruption and Blocks Replication of HIV-1 and HSV-2

Inflammation is a known mechanism that facilitates HIV acquisition and the spread of infection. In this study, we evaluated whether curcumin, a potent and safe anti-inflammatory compound, could be used to abrogate inflammatory processes that facilitate HIV-1 acquisition in the female genital tract (FGT) and contribute to HIV amplification. Primary, human genital epithelial cells (GECs) were pretreated with curcumin and exposed to HIV-1 or HIV glycoprotein 120 (gp120), both of which have been shown to disrupt epithelial tight junction proteins, including ZO-1 and occludin. Pre-treatment with curcumin prevented disruption of the mucosal barrier by maintaining ZO-1 and occludin expression and maintained trans-epithelial electric resistance across the genital epithelium. Curcumin pre-treatment also abrogated the gp120-mediated upregulation of the proinflammatory cytokines tumor necrosis factor-α and interleukin (IL)-6, which mediate barrier disruption, as well as the chemokines IL-8, RANTES and interferon gamma-induced protein-10 (IP-10), which are capable of recruiting HIV target cells to the FGT. GECs treated with curcumin and exposed to the sexually transmitted co-infecting microbes HSV-1, HSV-2 and Neisseria gonorrhoeae were unable to elicit innate inflammatory responses that indirectly induced activation of the HIV promoter and curcumin blocked Toll-like receptor (TLR)-mediated induction of HIV replication in chronically infected T-cells. Finally, curcumin treatment resulted in significantly decreased HIV-1 and HSV-2 replication in chronically infected T-cells and primary GECs, respectively. All together, our results suggest that the use of anti-inflammatory compounds such as curcumin may offer a viable alternative for the prevention and/or control of HIV replication in the FGT.     

A Phase I Double Blind,Placebo-Controlled,Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults

Background

Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01) plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN) may lead to a unique immune profile, inducing both strong T-cell and antibody responses.

Methods

In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01_E or F4/AS01_B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01_B; or three co-administrations of Ad35-GRIN and F4/AS01_B. T cell and antibody responses were measured.

Results

The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration.

Conclusion

Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses.

Trial Registration

ClinicalTrials.gov NCT01264445