WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop

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Backbone and sidechain 1H, 13C and 15N resonance assignments of the RGS domain from human RGS14

We have assigned ¹H, ¹⁵N and ¹³C resonances of the RGS domain from the human RGS14 protein, a multi-domain member of the RGS (Regulators of G-protein signalling) family of proteins, important in the down-regulation of specific G-protein signalling pathways.     

Tick-borne Langat/mosquito-borne dengue flavivirus chimera, a candidate live attenuated vaccine for protection against disease caused by members of the tick-borne encephalitis virus complex: evaluation in rhesus monkeys and in mosquitoes

Langat virus (LGT), strain TP21, a naturally avirulent tick-borne flavivirus, was used to construct a chimeric candidate virus vaccine which contained LGT genes for premembrane (preM) and envelope (E) glycoprotein and all other sequences derived from dengue type 4 virus (DEN4). The live virus vaccine was developed to provide resistance to the highly virulent, closely related tick-borne flaviviruses that share protective E epitopes among themselves and with LGT. Toward that end the chimera, initially recovered in mosquito cells, was adapted to grow to high titer in qualified simian Vero cells. When inoculated intraperitoneally (i.p.), the Vero cell-adapted LGT TP21/DEN4 chimera remained completely attenuated for SCID mice. Significantly, the chimera protected immunocompetent mice against the most virulent tick-borne encephalitis virus (TBEV). Subsequently, rhesus monkeys were immunized in groups of 4 with 10(5) or 10(7) PFU of LGT strain TP21, with 10(5) PFU of DEN4, or with 10(3), 10(5), or 10(7) PFU of the chimera. Each of the monkeys inoculated with DEN4 or LGT TP21 became viremic, and the duration of viremia ranged from 1 to 5 days. In contrast, viremia was detected in only 1 of 12 monkeys inoculated with the LGT TP21/DEN4 chimera; in this instance the level of viremia was at the limit of detection. All monkeys immunized with the chimera or LGT TP21 virus developed a moderate to high level of neutralizing antibodies against LGT TP21 as well as TBEV and were completely protected against subsequent LGT TP21 challenge, whereas monkeys previously immunized with DEN4 virus became viremic when challenged with LGT TP21. These observations suggest that the chimera is attenuated, immunogenic, and able to induce a protective immune response. Furthermore, passive transfer of serum from monkeys immunized with chimera conferred significant protection to mice subsequently challenged with 100 i.p. 50% lethal doses of the highly virulent TBEV. The issue of transmissibility of the chimera by mosquitoes was addressed by inoculating a nonhematophagous mosquito, Toxorhynchites splendens, intrathoracically with the chimera or its DEN4 or LGT parent. Neither the LGT TP21/DEN4 vaccine candidate nor the wild-type LGT TP21 virus was able to infect this mosquito species, which is highly permissive for dengue viruses. Certain properties of the chimera, notably its attenuation for monkeys, its immunogenicity, and its failure to infect a highly permissive mosquito host, make it a promising vaccine candidate for use in immunization against severe disease caused by many tick-borne flaviviruses.     

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

Fluorescence and FT-IR spectroscopic studies of Suwannee River fulvic acid complexation with aluminum, terbium and calcium.

In this study fluorescence emission and IR spectroscopy have been used to investigate the interaction of the class A (oxygen seeking "hard acid") metal Al(3+), with Suwannee River fulvic acid. Addition of Al(3+) ion results in a significant enhancement in fulvic acid fluorescence emission (at lambda(em)=424 nm) and significant red shift of the excitation wavelength (from lambda(ex)=324 nm to lambda(ex)=344 nm) at low pH values (pH approximately 4.0-5.0). At pH 4.0 (0.1 M ionic strength), where the predominant aluminum ion species is the "free" (aquo) ion, the fulvic acid fluorescence reaches 142% of the value in the absence of added metal ion. Analysis of the pH 4.0 and pH 5.0 fluorescence enhancement data with the nonlinear (single site) model of Ryan and Weber indicated binding constants in the range of 4.67.10(4)-2.87.10(6) M(-1) and concentrations of ligand sites in the range of 18.6.10(-6)-24.0.10(-6) M, both consistent with previous studies performed on both aquatic and soil fulvic acids. Companion fluorescence experiments performed on two other class A metal ions, Ca(2+) and Tb(3+), indicated no significant enhancement or quenching with Ca(2+) and only slight quenching with Tb(3+). Comparison of FT-IR spectra collected on fulvic acid alone and fulvic acid in the presence of the three class A metals (Al(3+), Ca(2+) and Tb(3+)) provides strong evidence for the involvement of carboxyl carbonyl functions in the binding of all three metal ions, which is not unexpected. The spectra also reveal, however, a very pronounced difference in the 4000-2000 cm(-1) IR spectral region between the Al(3+) spectrum and the Ca(2+) and Tb(3+) spectra. The -OH stretch spectral region in the Al(3+) spectrum has a major component shifted to higher energy (compared to fulvic acid alone or to fulvic acid in the presence of Ca(2+) or Tb(3+)). Even more striking is the emergence of a pronounced IR band at 2407 cm(-1), which is present only in the Al(3+) spectrum. The results of fluorescence and IR experiments with the model compounds salicylic acid and phthalic acid further confirm that both salicylic acid-like sites and phthalic acid-like sites are likely complexation sites for Al(3+) in fulvic acid and are major contributors to the observed spectroscopic changes associated with Al(3+) ion complexation. From a comparison of both the fluorescence and IR spectral results for all three class A metals, differing most strongly in the value of their ionic index, it seems clear that major sources of the deviation in spectral properties between Al(3+) and Ca(2+)/Tb(3+) is the unusually high value of its charge density and relatively low propensity for involvement in covalent bonding interactions (very high ionic index and relatively low covalent index in the Nieboer and Richardson classification of environmental metals), as well as affinity for certain functional groups.     

Infection of B cell-deficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lung pathology

Long-term survival of mice infected with Mycobacterium tuberculosis is dependent upon IFN-gamma and T cells, but events in early phases of the immune response are not well understood. In this study, we describe a role for B cells during early immune responses to infection with a clinical isolate of M. tuberculosis (CDC 1551). Following a low-dose infection with M. tuberculosis CDC 1551, similar numbers of bacteria were detected in the lungs of both B cell knockout (IgH 6-, BKO) and C57BL/6J (wild-type) mice. However, despite comparable bacterial loads in the lungs, less severe pulmonary granuloma formation and delayed dissemination of bacteria from lungs to peripheral organs were observed in BKO mice. BKO mice reconstituted with naive B cells, but not those given M. tuberculosis-specific Abs, before infection developed pulmonary granulomas and dissemination patterns similar to wild-type animals. Further analysis of lung cell populations revealed greater numbers of lymphocytes, especially CD8+ T cells, macrophages, and neutrophils in wild-type and reconstituted mice than in BKO mice. Thus, less severe lesion formation and delayed dissemination of bacteria found in BKO mice were dependent on B cells, not Abs, and were associated with altered cellular infiltrate to the lungs. These observations demonstrate an important, previously unappreciated, role for B cells during early immune responses to M. tuberculosis infections.     

Wide range of viral load in healthy african green monkeys naturally infected with simian immunodeficiency virus

The distribution and levels of simian immunodeficiency virus (SIV) in tissues and plasma were assessed in naturally infected African green monkeys (AGM) of the vervet subspecies (Chlorocebus pygerythrus) by limiting-dilution coculture, quantitative PCR for viral DNA and RNA, and in situ hybridization for SIV expression in tissues. A wide range of SIV RNA levels in plasma was observed among these animals (<1,000 to 800,000 copies per ml), and the levels appeared to be stable over long periods of time. The relative numbers of SIV-expressing cells in tissues of two monkeys correlated with the extent of plasma viremia. SIV expression was observed in lymphoid tissues and was not associated with immunopathology. Virus-expressing cells were observed in the lamina propria and lymphoid tissue of the gastrointestinal tract, as well as within alveolar macrophages in the lung tissue of one AGM. The range of plasma viremia in naturally infected AGM was greater than that reported in naturally infected sooty mangabeys. However, the degree of viremia in some AGM was similar to that observed during progression to AIDS in human immunodeficiency virus-infected individuals. Therefore, containment of viremia is an unlikely explanation for the lack of pathogenicity of SIVagm in its natural host species, AGM.     

Survival of secondary lethal systemic Francisella LVS challenge depends largely on interferon gamma

Although survival of primary infection with the live vaccine strain (LVS) of Francisella tularensis depends on interferon gamma (IFN-γ), the relative importance of IFN-γ to secondary protective immunity in vivo has not been clearly established. Here we examine the role of IFN-γ in T cell priming and expression of vaccine-induced protection against lethal intraperitoneal challenge of mice. Large amounts of IFN-γ were detected between days 3 and 7 in the sera of LVS-immunized mice, while relatively small amounts were found transiently after secondary LVS challenge. Consistent with the production of this cytokine, mice lacking IFN-γ (gamma interferon knockout, GKO, mice) could not be successfully vaccinated with LVS or an attenuated mglA mutant of F. novicida to withstand secondary Francisella LVS challenge. Further, splenocytes from such primed mice did not adoptively transfer protection to naive GKO recipient mice in vivo, nor control the intramacrophage growth of LVS in vitro. Finally, LVS-immune WT mice depleted of IFN-γ prior to intraperitoneal challenge survived only the lowest doses of challenge. Thus successful priming of protective LVS-immune T cells, as well as complete expression of protection against Francisella during secondary challenge, depends heavily on IFN-γ.     

Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1

Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson''s disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser⁶⁵) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser¹¹¹) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser¹¹¹ of each of the Rabs, we demonstrate that Rab Ser¹¹¹ phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser¹¹¹ phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser¹¹¹ phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser⁶⁵. We further show mechanistically that phosphorylation at Ser¹¹¹ significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser¹¹¹ may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson''s disease.