Cryptococcosis as an opportunistic infection in immunodeficiency secondary to paracoccidioidomycosis

We describe the case reports of two patients with immunodeficiency secondary to paracoccidioidomycosis (PCM) and opportunistic Cryptococcus neoformans infections. Secondary immunodeficiency likely occurred as a consequence of the intestinal loss of proteins and lymphocytes associated with malabsorption syndrome due to obstructed lymphatic drainage. Both patients had had severe abdominal involvement during the acute PCM disease. Immunological evaluation showed cellular and humoral immunity impairment. Cryptococcosis manifested as relatively well circumscribed lesions: osteolytic lesions of the skull in one patient, and pulmonary nodules in the other. The latter was treated surgically and with amphotericin B, whereas the other was treated with the combination amphotericin-B and flucytosine. Both patients had a good response to treatment with complete regression of the lesions. They have now 2 and 4 years of follow-up with maintenance therapy and no indication of reactivation of the infection. PCM also did not reactivate. The clinical and immunological characteristics of these patients are discussed and compared to the opportunistic C. neoformans infections of AIDS and transplant patients.     

Identification of peptide antagonists to glycoprotein Ibalpha that selectively inhibit von Willebrand factor dependent platelet aggregation

GPIbalpha is an integral membrane protein of the GPIb-IX-V complex found on the platelet surface that interacts with the A1 domain of von Willebrand factor (vWF-A1). The interaction of GPIbalpha with vWF-A1 under conditions of high shear stress is the first step in platelet-driven thrombus formation. Phage display was used to identify peptide antagonists of the GPIbalpha-vWF-A1 interaction. Two nine amino acid cysteine-constrained phage display libraries were screened against GPIbalpha revealing peptides that formed a consensus sequence. A peptide with sequence most representative of the consensus, designated PS-4, was used as the basis for an optimized library. The optimized selection identified additional GPIbalpha binding peptides with sequences nearly identical to the parent peptide. Surface plasmon resonance of the PS-4 parent and two optimized synthetic peptides, OS-1 and OS-2, determined their equilibrium dissociation GPIbalpha binding constants ( K Ds) of 64, 0.74, and 31 nM, respectively. Isothermal calorimetry corroborated the K D of peptide PS-4 with a resulting affinity value of 68 nM. An ELISA demonstrated that peptides PS-4, OS-1, and OS-2 competitively inhibited the interaction between the vWF-A1 domain and GPIbalpha-Fc in a concentration-dependent manner. All three peptides inhibited GPIbalpha-vWF-mediated platelet aggregation induced under high shear conditions using the platelet function analyzer (PFA-100) with full blockade observed at 150 nM for OS-1. In addition, OS-1 blocked ristocetin-induced platelet agglutination of human platelets in plasma with no influence on platelet aggregation induced by several agonists of alternative platelet aggregation pathways, demonstrating that this peptide specifically disrupted the GPIbalpha-vWF-A1 interaction.     

Influence of different mineral nitrogen sources (NO 3 − -N vs. NH 4 + -N) on arbuscular mycorrhiza development and N transfer in a Glomus intraradices–cowpea symbiosis

Labeled nitrogen (¹⁵?N) was applied to a soil-based substrate in order to study the uptake of N by Glomus intraradices extraradical mycelium (ERM) from different mineral N (NO ₃ ^? vs. NH ₄ ⁺ ) sources and the subsequent transfer to cowpea plants. Fungal compartments (FCs) were placed within the plant growth substrate to simulate soil patches containing root-inaccessible, but mycorrhiza-accessible, N. The fungus was able to take up both N-forms, NO ₃ ^? and NH ₄ ⁺ . However, the amount of N transferred from the FC to the plant was higher when NO ₃ ^? was applied to the FC. In contrast, analysis of ERM harvested from the FC showed a higher ¹⁵?N enrichment when the FC was supplied with ¹⁵NH ₄ ⁺ compared with ¹⁵NO ₃ ^? . The ¹⁵?N shoot/root ratio of plants supplied with ¹⁵NO ₃ ^? was much higher than that of plants supplied with ¹⁵NH ₄ ⁺ , indicative of a faster transfer of ¹⁵NO ₃ ^? from the root to the shoot and a higher accumulation of ¹⁵NH ₄ ⁺ in the root and/or intraradical mycelium. It is concluded that hyphae of the arbuscular mycorrhizal fungus may absorb NH ₄ ⁺ preferentially over NO ₃ ^? but that export of N from the hyphae to the root and shoot may be greater following NO ₃ ^? uptake. The need for NH ₄ ⁺ to be assimilated into organically bound N prior to transport into the plant is discussed.     

Metabolic consequences of NDUFS4 gene deletion in immortalized mouse embryonic fibroblasts

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.     

Effects of fumonisins on liver and kidney sphinganine and the sphinganine to sphingosine ratio during chronic exposure in ducks

Sa and the Sa/So ratio are very sensitive biomarkers of exposure to fumonisins in several species. We previously demonstrated that increases in Sa and in the Sa/So ratio in serum were less pronounced when ducks ingested fumonisins for more than 7 weeks than when animals were exposed for only 1-2 weeks [S.T. Tran, D. Tardieu, A. Auvergne, J.D. Bailly, R. Babilé, S. Durand, G. Benard, P. Guerre, Serum sphinganine and the sphinganine to sphingosine ratio as biomarker of dietary fumonisins during chronic exposure in ducks, Chem. Biol. Interact., in press]. The aim of this study was to investigate the kinetics of Sa and of the Sa/So in both liver and kidney of ducks that have been previously tested for Sa and the Sa/So ratio in serum. Analysis were performed on treatment days 0, 7, 14, 28 and 77 in five groups of ducks fed fumonisins obtained from an extract of Fusarium verticillioides culture material by daily gavage to obtain an exposure equal to 0, 2, 8, 32 and 128 mg FB1/kg feed. Sa and the Sa/So ratio in tissues were then correlated with Sa and the Sa/So ratio previously obtained in serum. The amounts on sphinganine 1-phosphate (Sa1P) and sphingosine1-phosphate (So1P) in the liver were also investigated. On day 7 of treatment, 2mg/kg FB1 in the feed were sufficient to increase Sa and the Sa/So ratio in liver (by 165 and 148%, respectively) and kidney (by 193 and 104%, respectively). At a rate of 128 mg/kg FB1 in the feed, a very high increase in Sa concentration was observed in both liver and kidney without mortality and/or signs of necrosis (respective increase of 2034 and 3768%). Although the precise mechanism of the resistance of ducks to fumonisin-induced hepatotoxicity is still uncertain, it might be linked to the rate at which the sphingoid bases sphinganine and sphingosine are converted to their 1-phosphate or other metabolite and eliminated from target tissues.     

Mitochondrial fusion and division: Regulation and role in cell viability

Discovery of various molecular components regulating dynamics and organization of the mitochondria in cells, together with novel insights into the role of mitochondrial fusion and division in the maintenance of cellular homeostasis, have provided some of the most exciting breakthroughs in the last decade of mitochondrial research. The focus of this review is on the regulation of mitochondrial fusion and division machineries. The newly identified factors associated with mitofusin/OPA1-dependent mitochondrial fusion, and Drp1-dependent mitochondrial division are discussed. Furthermore, the most recent findings on the role of mitochondrial fusion and division in the maintenance of cell function are also reviewed here in some detail.     

Inhibition of 5' to 3' mRNA degradation under stress conditions in Saccharomyces cerevisiae: from GCN4 to MET16

After deadenylation, most cytoplasmic mRNAs are decapped and digested by 5' to 3' exonucleases in Saccharomyces cerevisiae. Capped and deadenylated mRNAs are degraded to a lesser extent by 3' to 5' exonucleases. We have used a method, based on the electroporation of in vitro synthetised mRNAs, to study the relative importance of these two exonucleolytic pathways under stress conditions. We show that derepression of GCN4 upon amino acid starvation specifically limits the 5'-to-3'-degradation pathway. Because adenosine 3'-5' biphosphate (pAp), which is produced by Met16p, inhibits this degradation pathway to a comparable extent, we were prompted to analyse the role of Met16p in this phenomenon. We show that the inhibitory effects of amino acid limitation on 5' to 3' mRNA degradation are absent in a met16 mutant. We therefore conclude that the GCN4 dependence of MET16 expression is responsible for the decrease in 5' to 3' digestion under stress conditions and that cells use pAp as a signal to limit 5' to 3' RNA degradation under stress conditions. Because 3' to 5' mRNA degradation is unaffected, the relative importance of this pathway in the decay of certain RNAs may be increased under stress conditions.