pVHL and PTEN tumour suppressor proteins cooperatively suppress kidney cyst formation

In patients with von Hippel-Lindau (VHL) disease, renal cysts and clear cell renal cell carcinoma (ccRCC) arise from renal tubular epithelial cells containing biallelic inactivation of the VHL tumour suppressor gene. However, it is presumed that formation of renal cysts and their conversion to ccRCC involve additional genetic changes at other loci. Here, we show that cystic lesions in the kidneys of patients with VHL disease also demonstrate activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Strikingly, combined conditional inactivation of Vhlh and the Pten tumour suppressor gene, which normally antagonises PI3K signalling, in the mouse kidney, elicits cyst formation after short latency, whereas inactivation of either tumour suppressor gene alone failed to produce such a phenotype. Interestingly, cells lining these cysts frequently lack a primary cilium, a microtubule-based cellular antenna important for suppression of uncontrolled kidney epithelial cell proliferation and cyst formation. Our results support a model in which the PTEN tumour suppressor protein cooperates with pVHL to suppress cyst development in the kidney.     

Novel route for elimination of brain oxysterols across the blood-brain barrier: conversion into 7alpha-hydroxy-3-oxo-4-cholestenoic acid

Recently, we demonstrated a net blood-to-brain passage of the oxysterol 27-hydroxycholesterol corresponding to 4-5 mg/day. As the steady-state levels of this sterol are only 1-2 mug/g brain tissue, we hypothesized that it is metabolized and subsequently eliminated from the brain. To explore this concept, we first measured the capacity of in vitro systems representing the major cell populations found in the brain to metabolize 27-hydroxycholesterol. We show here that 27-hydroxycholesterol is metabolized into the known C(27) steroidal acid 7alpha-hydroxy-3-oxo-4-cholestenoic acid by neuronal cell models only. Using an in vitro model of the blood-brain barrier, we demonstrate that 7alpha-hydroxy-3-oxo-4-cholestenoic acid is efficiently transferred across monolayers of primary brain microvascular endothelial cells. Finally, we measured the concentration of 7alpha-hydroxy-3-oxo-4-cholestenoic acid in plasma from the internal jugular vein and brachial artery of healthy volunteers. Calculation of the arteriovenous concentration difference revealed a significant in vivo flux of this steroid from the brain into the circulation in human. Together, these studies identify a novel metabolic route for the elimination of 27-hydroxylated sterols from the brain. Given the emerging connections between cholesterol and neurodegeneration, this pathway may be of importance for the development of these conditions.     

Delayed-type hypersensitivity response in an isogenic murine model of paracoccidioidomycosis

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265)Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses againstP. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.Abbreviations DTH delayed-type hypersensitivity - DNFB dinitrofluorobenzene - FN18 Fava Netto's antigen obtained from isolate Pb18 - FN265 Fava Netto's antigen obtained from isolate Pb265 - SRBC sheep red blood cells     

Net uptake of atmospheric CO2 by coastal submerged aquatic vegetation

‘Blue Carbon’, which is carbon captured by marine living organisms, has recently been highlighted as a new option for climate change mitigation initiatives. In particular, coastal ecosystems have been recognized as significant carbon stocks because of their high burial rates and long‐term sequestration of carbon. However, the direct contribution of Blue Carbon to the uptake of atmospheric CO₂ through air‐sea gas exchange remains unclear. We performed in situ measurements of carbon flows, including air‐sea CO₂ fluxes, dissolved inorganic carbon changes, net ecosystem production, and carbon burial rates in the boreal (Furen), temperate (Kurihama), and subtropical (Fukido) seagrass meadows of Japan from 2010 to 2013. In particular, the air‐sea CO₂ flux was measured using three methods: the bulk formula method, the floating chamber method, and the eddy covariance method. Our empirical results show that submerged autotrophic vegetation in shallow coastal waters can be functionally a sink for atmospheric CO_2. This finding is contrary to the conventional perception that most near‐shore ecosystems are sources of atmospheric CO₂. The key factor determining whether or not coastal ecosystems directly decrease the concentration of atmospheric CO₂ may be net ecosystem production. This study thus identifies a new ecosystem function of coastal vegetated systems; they are direct sinks of atmospheric CO₂.     

Interaction of antimicrobial peptides with bacterial polysaccharides from lung pathogens

The interaction of two cathelicidin antimicrobial peptides, LL-37 and SMAP-29, with three bacterial polysaccharides, respectively, produced by Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae, was investigated to identify possible mechanisms adopted by lung pathogens to escape the action of innate immunity effectors. In vitro assays indicated that the antibacterial activity of both peptides was inhibited to a variable extent by the three polysaccharides. Circular dichroism experiments showed that these induced an alpha-helical conformation in the two peptides, with the polysaccharides from K. pneumoniae and B. cepacia showing, respectively, the highest and the lowest effect. Fluorescence measurements also indicated the presence of peptide-polysaccharide interactions. A model is proposed in which the binding of peptides to the polysaccharide molecules induces, at low polysaccharide to peptide ratios, a higher order of aggregation, due to peptide-peptide interactions. Overall, these results suggest that binding of the peptides by the polysaccharides produced by lung pathogens can contribute to the impairment of peptide-based innate defenses of airway surface.     

Heat-Induced Proteasomic Degradation of HSF1 in Serum-Starved Human Fibroblasts Aging in Vitro

The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.     

Single Ryanodine Receptor Channel Basis of Caffeine's Action on Ca Sparks

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca²⁺ release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca²⁺ sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca²⁺ level on both sides of the channel. Cytosolic Ca²⁺ enhanced RyR2 caffeine affinity, whereas luminal Ca²⁺ essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC₅₀ of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca²⁺ spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca²⁺ sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.     

Unconventional endocytosis and trafficking of transferrin receptor induced by iron

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH₄Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.     

Changes in paracrine interleukin-2 requirement, CCR7 expression, frequency, and cytokine secretion of human immunodeficiency virus-specific CD4+ T cells are a consequence of antigen load

Virus-specific CD4+ T-cell responses are thought to be required for the induction and maintenance of many effective CD8+ T-cell and B-cell immune responses in experimental animals and humans. Although the presence of human immunodeficiency virus (HIV)-specific CD4+ T cells has been documented in patients at all stages of HIV infection, many fundamental questions regarding their frequency and function remain. A 10-color, 12-parameter flow cytometric panel was utilized to examine the frequency, memory phenotype (CD27, CCR7, and CD45RA), and cytokine production (interleukin-2 [IL-2], gamma interferon, and tumor necrosis factor alpha) of CD4+ T cells specific for HIV antigens as well as for adenovirus, Epstein-Barr virus (EBV), influenza H1N1 virus, influenza H3N2 virus, cytomegalovirus, varicella-zoster virus (VZV), and tetanus toxoid in normal controls, long-term nonprogressors (LTNP), and HIV-infected patients with progressive disease on or off therapy. The HIV-specific CD4+ T-cell responses in LTNP and patients on therapy were similar in frequency, phenotype, and cytokine production to responses directed against adenovirus, EBV, influenza virus, and VZV. HIV-specific CD4+ T cells from patients off antiretroviral therapy demonstrated a shift towards a CCR7(-) CD45RA(-) phenotype and a reduced percentage of IL-2-producing cells. The alterations in cytokine production during HIV viremia were found to be intrinsic to the HIV-specific CD4+ T cells and caused a requirement for IL-2 supplied exogenously for proliferation to occur. These observations suggest that many previously described changes in HIV-specific CD4+ T-cell function and phenotype are a consequence of high levels of antigen in viremic patients. In addition, defects in function and phenotype of HIV-specific CD4+ T cells are not readily discernible in the context of antiretroviral therapy but rather are similar to responses to other viruses.