Plasmodium ookinetes coopt mammalian plasminogen to invade the mosquito midgut

Ookinete invasion of the mosquito midgut is an essential step for the development of the malaria parasite in the mosquito. Invasion involves recognition between a presumed mosquito midgut receptor and an ookinete ligand. Here, we show that enolase lines the ookinete surface. An antienolase antibody inhibits oocyst development of both Plasmodium berghei and Plasmodium falciparum, suggesting that enolase may act as an invasion ligand. Importantly, we demonstrate that surface enolase captures plasminogen from the mammalian blood meal via its lysine motif (DKSLVK) and that this interaction is essential for midgut invasion, because plasminogen depletion leads to a strong inhibition of oocyst formation. Although addition of recombinant WT plasminogen to depleted serum rescues oocyst formation, recombinant inactive plasminogen does not, thus emphasizing the importance of plasmin proteolytic activity for ookinete invasion. The results support the hypothesis that enolase on the surface of Plasmodium ookinetes plays a dual role in midgut invasion: by acting as a ligand that interacts with the midgut epithelium and, further, by capturing plasminogen, whose conversion to active plasmin promotes the invasion process.     

Opposite effects of pioglitazone and rosiglitazone on mitochondrial respiration in skeletal muscle of patients with type 2 diabetes

Aim: Skeletal muscle insulin resistance has been linked to mitochondrial dysfunction. We examined how improvements in muscular insulin sensitivity following rosiglitazone (ROSI) or pioglitazone (PIO) treatment would affect muscle mitochondrial function in patients with type 2 diabetes mellitus (T2DM). Methods: Muscle biopsies were obtained from 21 patients with T2DM before and after 12 weeks on either ROSI (4 mg once daily) [n = 12; age, 59.2 ± 2.2 years; body mass index (BMI), 29.6 ± 0.7 kg/m²] or PIO (30 mg once daily) (n = 9; age, 56.3 ± 2.4 years; BMI, 29.5 ± 1.5 kg/m²). An age‐ and BMI‐matched control group was also included (n = 8; age, 61.8 ± 2.3 years; BMI, 28.4 ± 0.6 kg/m²). Insulin sensitivity, citrate synthase‐ and β‐hydroxyacyl‐CoA‐dehydrogenase (HAD) activity, intramuscular triglyceride (IMTG) and protein content of complexes I–IV were measured, while mitochondrial respiration per milligram muscle was measured in saponin‐treated skinned muscle fibres using high‐resolution respirometry. Results: Mitochondrial respiration per milligram muscle was lower in T2DM compared to controls at baseline and decreased during ROSI treatment but increased during PIO treatment. Citrate synthase activity and average protein content of complexes I–IV were unchanged in the ROSI group, but protein content of complexes II and III increased during PIO treatment. Insulin sensitivity improved in all patients, but IMTG levels were unchanged. Conclusions: We show opposite effects of ROSI and PIO on mitochondrial respiration, and also show that insulin sensitivity can be improved independently of changes in mitochondrial respiration. We confirm that mitochondrial respiration is reduced in T2DM compared to age‐ and BMI‐matched control subjects.     

Co-localisation of the Kir6.2/SUR1 channel complex with glucagon-like peptide-1 and glucose-dependent insulinotrophic polypeptide expression in human ileal cells and implications for glycaemic control in new onset type 1 diabetes

OBJECTIVE: The ATP-dependent K+-channel (K(ATP)) is critical for glucose sensing and normal glucagon and insulin secretion from pancreatic endocrine alpha- and beta-cells. Gastrointestinal endocrine L- and K-cells are also glucose-sensing cells secreting glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotrophic polypeptide (GIP) respectively. The aims of this study were to 1) investigate the expression and co-localisation of the K(ATP) channel subunits, Kir6.2 and SUR1, in human L- and K-cells and 2) investigate if a common hyperactive variant of the Kir6.2 subunit, Glu23Lys, exerts a functional impact on glucose-sensing tissues in vivo that may affect the overall glycaemic control in children with new-onset type 1 diabetes. DESIGN AND METHODS: Western blot and immunohistochemical analyses were performed for expression and co-localisation studies. Meal-stimulated C-peptide test was carried out in 257 children at 1, 6 and 12 months after diagnosis. Genotyping for the Glu23Lys variant was by PCR-restriction fragment length polymorphism. RESULTS: Kir6.2 and SUR1 co-localise with GLP-1 in L-cells and with GIP in K-cells in human ileum tissue. Children with type 1 diabetes carrying the hyperactive Glu23Lys variant had higher HbA1C at diagnosis (coefficient = 0.61%, P = 0.02) and 1 month after initial insulin therapy (coefficient = 0.30%, P = 0.05), but later disappeared. However, when adjusting HbA1C for the given dose of exogenous insulin, the dose-adjusted HbA1C remained higher throughout the 12 month study period (coefficient = 0.42%, P = 0.03). CONCLUSIONS: Kir6.2 and SUR1 co-localise in the gastrointestinal endocrine L- and K-cells. The hyperactive Glu23Lys variant of the K(ATP) channel subunit Kir6.2 may cause defective glucose sensing in several tissues and impaired glycaemic control in children with type 1 diabetes.     

Expression of C4.4A in precursor lesions of pulmonary adenocarcinoma and squamous cell carcinoma

The protein C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor, is a potential new biomarker in non-small cell lung cancer, with high levels of expression recently shown to correlate to poor survival of adenocarcinoma patients. In this study, C4.4A immunoreactivity in precursor lesions of lung squamous cell carcinoma and adenocarcinoma was investigated by stainings with a specific anti-C4.4A antibody. In the transformation from normal bronchial epithelium to squamous cell carcinoma, C4.4A was weakly expressed in basal cell hyperplasia but dramatically increased in squamous metaplasia. This was confined to the cell membrane and sustained in dysplasia, carcinoma in situ, and the invasive carcinoma. The induction of C4.4A already at the stage of hyperplasia could indicate that it is a marker of very early squamous differentiation, which aligns well with our earlier finding that C4.4A expression levels do not provide prognostic information on the survival of squamous cell carcinoma patients. In the progression from normal alveolar epithelium to peripheral adenocarcinoma, we observed an unexpected, distinct cytoplasmic staining for C4.4A in a fraction of atypical adenomatous hyperplasias, while most bronchioloalveolar carcinomas were negative. Likewise, only a fraction of the invasive adenocarcinomas was positive for C4.4A. With a view to the prognostic impact of C4.4A in adenocarcinoma patients, this finding might suggest that C4.4A could be an early biomarker for a possibly more malignant subtype of this disease.     

Imaging of urokinase-type plasminogen activator receptor expression using a 64Cu-labeled linear peptide antagonist by microPET.

PURPOSE: Malignant tumors are capable of degrading the surrounding extracellular matrix, resulting in local invasion or metastasis. Urokinase-type plasminogen activator (uPA) and its cell surface receptor (uPAR) are central molecules in one of the major protease systems involved in extracellular matrix degradation. Noninvasive imaging of this receptor in vivo with radiolabeled peptides that specifically target uPAR may therefore be useful to decipher the potential invasiveness of malignant lesions. EXPERIMENTAL DESIGN: In this study, we developed a (64)Cu-labeled uPAR-binding peptide for positron emission tomography (PET) imaging. A linear, high-affinity uPAR-binding peptide antagonist AE105 was conjugated with 1,4,7,10-tetraazadodecane-N,N',N',N'-tetraacetic acid (DOTA) and labeled with (64)Cu for microPET imaging of mice bearing U87MG human glioblastoma (uPAR positive) and MDA-MB-435 human breast cancer (uPAR negative). RESULTS: Surface plasmon resonance measurements show that AE105 with DOTA conjugated at the alpha-amino group (DOTA-AE105) has high affinity toward uPAR. microPET imaging reveals a rapid and high accumulation of (64)Cu-DOTA-AE105 in uPAR-positive U87MG tumors (10.8 +/- 1.5%ID/g at 4.5 hours, n = 3) but not in uPAR-negative MDA-MB-435 tumors (1.2 +/- 0.6%ID/g at 4.5 hours, n = 3). Specificity of this peptide-based imaging of uPAR was validated by further control experiments. First, a nonbinding variant of AE105 carrying a single amino acid replacement (Trp-->Glu) does not target U87MG tumors in vivo. Second, targeting of U87MG tumors by (64)Cu-DOTA-AE105 is specifically inhibited by a nonlabeled antagonist. CONCLUSION: The successful demonstration of the ability of a (64)Cu labeled uPAR-specific probe to visualize uPAR expression in vivo may allow clinical translation of this class of radiopharmaceuticals for uPAR-positive cancer detection and patient stratification for uPA/uPAR system-based cancer therapy.     

39A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor

The urokinase receptor, uPAR, is a GPI-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear if the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements, or if lateral interactions between uPAR and other membrane proteins can induce the same response. In this paper, we show that both of these triggering mechanisms can be operative and that uPAR dependent modulation of cell morphology can indeed occur independently of vitronectin binding. Expression of wildtype uPAR on HEK-293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements whereas a mutant uPAR, uPAR_W32A with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR_W32A with the protease ligand, pro-uPA or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored even though this did not rescue the vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR_Y57A, in the presence of a synthetic uPAR-binding peptide, as well as with wildtype uPAR which underwent cytoskeletal rearrangements even when cultivated in uPA deficient serum.     

Decay of house-dust mite allergen Der f 1 at indoor climatic conditions.

BACKGROUND: The decay of house-dust mite allergens is important for the outcome of avoidance measures for house-dust mite-allergic patients. OBJECTIVE: To quantify the stability of Der f 1 from mattress dust when exposed to domestic conditions. METHODS: Three samples of mattress dust were individually homogenized and divided into 64 subsamples. Mites were killed by freezing for 48 hours at -30 degrees C. The subsamples were exposed in eight homes, three storerooms, and one greenhouse, where temperature and relative humidity were recorded. Der f 1 was determined in extracts of subsamples (enzyme-linked immunoadsorbent assay) at 0, 3, 12, and 24 months. RESULTS: In the three samples of mattress dust, the initial concentrations of Der f 1 (mean +/- standard deviation; STD) were: 169 (12), 3.9 (0.4), and 31 (2.6) microg/g, respectively. Median half-life of Der f 1 in the mattress dust samples was 10 years in the exposure homes, 18 years in the store rooms, and 1.0 year in the greenhouse. No correlations among preserved Der f 1 and temperature, relative humidity, and absolute humidity in homes were found (Spearman rank correlation test). CONCLUSION: Natural decay of Der f 1, with an estimated half-life of 10 years at housing conditions, has no practical consequence in reducing allergen exposure. Therefore, avoidance measures should include an active removal of the allergens.