Microbial resource allocation for phosphatase synthesis reflects the availability of inorganic phosphorus across various soils

According to the resource allocation model for extracellular enzyme synthesis, microorganisms should preferentially allocate their resources to phosphorus (P)-acquiring enzyme synthesis when P availability is low in soils. However, the validity of this model across different soil types and soils differing in their microbial community composition has not been well demonstrated. Here we investigated whether the resource allocation model for phosphatase synthesis is applicable across different soil types (Andosols, Acrisols, Cambisols, and Fluvisols) and land uses (arable and forest), and we examined which soil test P and/or P fraction microorganisms responded to when investing their resources in phosphatase synthesis in the soils. The ratio of alkaline phosphatase (ALP) to β-d-glucosidase (BG) activities in the arable soils and the ratio of acid phosphatase (ACP) to BG activities in the forest soils were significantly negatively related with the available inorganic P concentration. We also observed significant effects of available inorganic P, pH, soil types, and land uses on the (ACP + ALP)/BG ratio when the data for the arable and forest soils were combined and used in a stepwise multiple regression analysis. These results suggest that microbial resource allocation for phosphatase synthesis is primarily controlled by available inorganic P concentration and soil pH, but the effects of soil types and land uses are also significant.     

Amyloidogenic Potential of Transthyretin Variants: INSIGHTS FROM STRUCTURAL AND COMPUTATIONAL ANALYSES*

Human transthyretin (TTR) is an amyloidogenic protein whose mild amyloidogenicity is enhanced by many point mutations affecting considerably the amyloid disease phenotype. To ascertain whether the high amyloidogenic potential of TTR variants may be explained on the basis of the conformational change hypothesis, an aim of this work was to determine structural alterations for five amyloidogenic TTR variants crystallized under native and/or destabilizing (moderately acidic pH) conditions. While at acidic pH structural changes may be more significant because of a higher local protein flexibility, only limited alterations, possibly representing early events associated with protein destabilization, are generally induced by mutations. This study was also aimed at establishing to what extent wild-type TTR and its amyloidogenic variants are intrinsically prone to β-aggregation. We report the results of a computational analysis predicting that wild-type TTR possesses a very high intrinsic β-aggregation propensity which is on average not enhanced by amyloidogenic mutations. However, when located in β-strands, most of these mutations are predicted to destabilize the native β-structure. The analysis also shows that rat and murine TTR have a lower intrinsic β-aggregation propensity and a similar native β-structure stability compared with human TTR. This result is consistent with the lack of in vitro amyloidogenicity found for both murine and rat TTR. Collectively, the results of this study support the notion that the high amyloidogenic potential of human pathogenic TTR variants is determined by the destabilization of their native structures, rather than by a higher intrinsic β-aggregation propensity.Protein misfolding and aggregation are involved in the pathogenesis of particularly relevant human deposition diseases, known as amyloidoses. In such diseases, normally soluble proteins undergo misfolding and become insoluble, causing the extracellular deposition of fibrillar aggregates (for reviews, see Ref. 1, 2). To date, more than 40 distinct human proteins have been associated with amyloidoses. For some of such proteins, including transthyretin (TTR),⁴ lysozyme, gelsolin, ApoAI, and ApoAII, fibrinogen A α-chain and cystatin C, the amyloidogenic potential is induced, or is enhanced as in the case of TTR (see below), by specific mutations. The most frequent hereditary amyloidoses are caused by the genetic variants of human TTR (2).TTR is a homotetramer of about 55 kDa involved in the transport of thyroxine in the extracellular fluids and in the co-transport of vitamin A, by forming a macromolecular complex with retinol-binding protein, the specific plasma carrier of retinol (3–5). Its three-dimensional structure is known at high resolution (6, 7). The structure is characterized by a large predominance of β-strands, and its four monomers are arranged according to a 222 symmetry, where one of the 2-fold symmetry axes of the molecule coincides with a long channel that transverses the entire tetramer and harbors two symmetrical binding sites for the thyroid hormone thyroxine. Each monomer contains eight β-strands (A-H), arranged in a β-sandwich of two four-stranded β-sheets, with a short α-helix connecting two of the eight β-strands. In the tetramer, the four monomers are organized as a dimer of dimers. Two monomers are held together, forming a stable dimer through a net of H-bond interactions involving the two external β-strands H and F. The two dimers associate back to back and form the tetramer, by interacting mostly through hydrophobic contacts between residues of the AB and GH loops.Normal TTR possesses an inherent potential, albeit low, to generate amyloid fibrils, giving rise to Senile Systemic Amyloidosis (SSA) in ∼25% of the population aged over 80 years (8). More than 100 point mutations are described for human TTR. Most of them are involved in the hereditary amyloidoses known as familial amyloidotic polyneuropathy (FAP) or cardiomyopathy (FAC) (9). Single point mutations enhance the amyloidogenicity of TTR, so that patients show an earlier age of onset and a faster disease progression compared with SSA patients. The observation that single point mutations can drastically influence the disease phenotype is particularly relevant. In fact, the study of pathogenic TTR variants may provide clues to the mechanism of their abnormal behavior leading to amyloid formation. Although amyloidogenic proteins in general may be structurally unrelated to each other, and lead to various pathological phenotypes in humans, the amyloid fibrils originating from different proteins share the common cross-β structure, consisting in continuous β-sheets lying parallel to the longitudinal axis of the fibril, with the constituent β-strands running perpendicular to this axis. Therefore, the amyloidogenic proteins have to undergo structural alterations to be able to generate the cross-β structure, i.e. new β-pairing interactions have to be established on the way to fibril formation. However, the molecular mechanisms underlying protein misfolding and aggregation into highly ordered fibrillar structures are not clarified definitely, although significant progress is recently been made toward their elucidation (1, 10, 11).Based on the seminal observation that the rates of aggregation into amyloid fibrils in vitro correlate with simple physico-chemical amino acid features (12), several algorithms were introduced in recent years to predict, with good success, the intrinsic β-aggregation propensities of protein and peptide sequences (for a review, see Ref. 13). The intrinsic β-aggregation propensity is a measure of the tendency polypeptide chains may have to aggregate into the amyloid structure, provided that aggregation proceeds from unstructured monomers. The prediction of intrinsic propensities to β-aggregation for amyloidogenic or non-amyloidogenic variants of the same sequence was used to explain in several instances their relative ability to speed up/slow down in vitro fibrillogenesis or the enhancement/reduction of their amyloidogenic potential in vivo (14). However, a high intrinsic aggregation propensity may not result in an actual aggregation, due to the protecting role of the ordered native structure (15, 16). Therefore, the amyloidogenic potential in the TTR variants may depend further on the change of stability in the native TTR tetramer induced by mutations. In particular, it remains to be clarified to what extent human TTR possesses an intrinsic propensity to β-aggregation, and whether amyloidogenic mutations enhance such a propensity, or only destabilize the TTR tetramer, thereby facilitating the misfolding and misassembly of a protein which is in itself prone to β-aggregation.With regard to the pathway from native to misfolded TTR and to amyloid aggregation, the results of a number of in vitro studies are consistent with the rate-limiting dissociation of the TTR tetramer, followed by misfolding of TTR monomers and their downhill polymerization to generate pathological aggregates (17–25). The crystal structures of amyloidogenic TTR variants are generally well conserved (26–30). Accordingly, the functional properties of the variants, such as the ability to interact with retinol-binding protein (5), are maintained, being consistent with the fact that large conformational changes are not induced by amyloidogenic mutations, at least under native-like conditions (11). In vitro studies have shown that a moderately acidic medium (pH 4–5) facilitates TTR fibrillogenesis (17) and that the extent of fibril formation is remarkably enhanced for amyloidogenic TTR variants in comparison to wild-type TTR (31). Recently, it has been shown by x-ray analysis that an acidic pH (4.6) causes a large local conformational change in an amyloidogenic TTR variant (I84S) affecting two subunits within the tetramer, which probably destabilizes the TTR tetramer (32). In contrast, no significant structural changes for wild-type TTR at pH 4.6 and for I84S TTR at neutral pH were found, suggesting that conformational changes associated with a destabilization of the TTR native state may be induced or enhanced in amyloidogenic TTR variants by partially denaturing conditions (32). Pursuing these observations, we extend here our investigation to include other amyloidogenic TTR variants in comparison to the wild-type protein, with the aim to unravel structural alterations that are possibly associated with an enhanced amyloidogenic potential, according to the conformational change hypothesis (11). In addition, we report the results of a computational analysis of the mutational effects on both the intrinsic propensity to β-aggregation and the stability of the native β-structure. The same analysis is performed on murine and rat TTRs, whose structural organizations are very similar to that of the human protein (33, 34).     

Connexin 32 down-regulates the fibrinolytic factors in metastatic renal cell carcinoma cells

Fibrinolytic factors have an important role in tumor progression through the degradation of extracellular matrix. The increased levels of urokinase-type plasminogen activator (uPA), uPA-receptor (uPAR) and type-1 PA inhibitor (PAI-1) are reported in human renal cell carcinoma (RCC). Connexin (Cx) gene, a member of gap junction, is known to act as a tumor suppressor gene. We have reported that Cx32 improves malignant phenotypes of metastatic RCC cells via the inhibition of Src-dependent signaling. In this study, we examined the effect of expression of Cx32 gene on the production of uPA, uPAR and PAI-1, and on the induction of PAI-1 stimulated by hypoxia in a human metastatic RCC cell line, Caki-1 cells. Cx32 expression decreased both mRNA level and production of PAI-1, uPA and uPAR in Caki-1 cells. Cx32 also decreased hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha mRNA level. PP1, a Src inhibitor, significantly decreased PAI-1, uPA, uPAR and HIF-alpha mRNA levels in Caki-1 cells. Furthermore, Cx32 suppressed the induction of HIF-2alpha protein in Caki-1 cells under hypoxia. PAI-1 mRNA level in Cx32-transfected Caki-1 cells was lower than that of mock transfectant under hypoxic conditions. These results suggest that Cx32 might reduce PAI-1, uPA and uPAR production in metastatic RCC cells via the inhibition of Src-dependent induction of HIF-1alpha and HIF-2alpha gene expression and that Cx32 might suppress hypoxia-inducible gene expression under hypoxic conditions.     

PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation

Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin–proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles.     

Multivariable Logistic Regression Model: A Novel Mathematical Model that Predicts Visual Field Sensitivity from Macular Ganglion Cell Complex Thickness in Glaucoma

Purpose

To design a mathematical model that can predict the relationship between the ganglion cell complex (GCC) thickness and visual field sensitivity (VFS) in glaucoma patients.

Design

Retrospective cross-sectional case series.

Method

Within 3 months from VFS measurements by the Humphrey field analyzer 10-2 program, 83 eyes underwent macular GCC thickness measurements by spectral-domain optical coherence tomography (SD-OCT). Data were used to construct a multiple logistic model that depicted the relationship between the explanatory variables (GCC thickness, age, sex, and spherical equivalent of refractive errors) determined by a regression analysis and the mean VFS corresponding to the SD-OCT scanned area. Analyses were performed in half or 8 segmented local areas as well as in whole scanned areas. A simple logistic model that included GCC thickness as the single explanatory variable was also constructed. The ability of the logistic models to depict the real GCC thickness/VFS in SAP distribution was analyzed by the χ² test of goodness-of-fit. The significance of the model effect was analyzed by analysis of variance (ANOVA).

Results

Scatter plots between the GCC thickness and the mean VFS showed sigmoid curves. The χ² test of goodness-of-fit revealed that the multiple logistic models showed a good fit for the real GCC thickness/VFS distribution in all areas except the nasal-inferior-outer area. ANOVA revealed that all of the multiple logistic models significantly predicted the VFS based on the explanatory variables. Although simple logistic models also exhibited significant VFS predictability based on the GCC thickness, the model effect was less than that observed for the multiple logistic models.

Conclusions

The currently proposed logistic models are useful methods for depicting relationships between the explanatory variables, including the GCC thickness, and the mean VFS in glaucoma patients.     

Knock Out of S1P3 Receptor Signaling Attenuates Inflammation and Fibrosis in Bleomycin-Induced Lung Injury Mice Model

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein–coupled receptors (S1P1–5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor β1 (TGF-β1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis.     

Amelioration of Cold Injury-Induced Cortical Brain Edema Formation by Selective Endothelin ETB Receptor Antagonists in Mice

Brain edema is a potentially fatal pathological condition that often occurs in stroke and head trauma. Following brain insults, endothelins (ETs) are increased and promote several pathophysiological responses. This study examined the effects of ET_B antagonists on brain edema formation and disruption of the blood-brain barrier in a mouse cold injury model (Five- to six-week-old male ddY mice). Cold injury increased the water content of the injured cerebrum, and promoted extravasation of both Evans blue and endogenous albumin. In the injury area, expression of prepro-ET-1 mRNA and ET-1 peptide increased. Intracerebroventricular (ICV) administration of BQ788 (ET_B antagonist), IRL-2500 (ET_B antagonist), or {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 (ET_A antagonist) prior to cold injury significantly attenuated the increase in brain water content. Bolus administration of BQ788, IRL-2500, or {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 also inhibited the cold injury-induced extravasation of Evans blue and albumin. Repeated administration of BQ788 and IRL-2500 beginning at 24 h after cold injury attenuated both the increase in brain water content and extravasation of markers. In contrast, {"type":"entrez-nucleotide","attrs":{"text":"FR139317","term_id":"258103156","term_text":"FR139317"}}FR139317 had no effect on edema formation when administrated after cold injury. Cold injury stimulated induction of glial fibrillary acidic protein-positive reactive astrocytes in the injured cerebrum. Induction of reactive astrocytes after cold injury was attenuated by ICV administration of BQ788 or IRL-2500. These results suggest that ET_B receptor antagonists may be an effective approach to ameliorate brain edema formation following brain insults.     

Presence of Neutrophil Extracellular Traps and Citrullinated Histone H3 in the Bloodstream of Critically Ill Patients

Neutrophil extracellular traps (NETs), a newly identified immune mechanism, are induced by inflammatory stimuli. Modification by citrullination of histone H3 is thought to be involved in the in vitro formation of NETs. The purposes of this study were to evaluate whether NETs and citrullinated histone H3 (Cit-H3) are present in the bloodstream of critically ill patients and to identify correlations with clinical and biological parameters. Blood samples were collected from intubated patients at the time of ICU admission from April to June 2011. To identify NETs, DNA and histone H3 were visualized simultaneously by immunofluorescence in blood smears. Cit-H3 was detected using a specific antibody. We assessed relationships of the presence of NETs and Cit-H3 with the existence of bacteria in tracheal aspirate, SIRS, diagnosis, WBC count, and concentrations of IL-8, TNF-α, cf-DNA, lactate, and HMGB1. Forty-nine patients were included. The median of age was 66.0 (IQR: 52.5–76.0) years. The diagnoses included trauma (7, 14.3%), infection (14, 28.6%), resuscitation from cardiopulmonary arrest (8, 16.3%), acute poisoning (4, 8.1%), heart disease (4, 8.1%), brain stroke (8, 16.3%), heat stroke (2, 4.1%), and others (2, 4.1%). We identified NETs in 5 patients and Cit-H3 in 11 patients. NETs and/or Cit-H3 were observed more frequently in “the presence of bacteria in tracheal aspirate” group (11/22, 50.0%) than in “the absence of bacteria in tracheal aspirate” group (4/27, 14.8%) (p<.01). Multiple logistic regression analysis showed that only the presence of bacteria in tracheal aspirate was significantly associated with the presence of NETs and/or Cit-H3. The presence of bacteria in tracheal aspirate may be one important factor associated with NET formation. NETs may play a pivotal role in the biological defense against the dissemination of pathogens from the respiratory tract to the bloodstream in potentially infected patients.     

The anticoagulant properties of mast cell product, chondroitin sulphate E

The anticoagulant potency in vitro of chondroitin sulphate E has been found to be similar to that of the heparinoids. In purified systems chondroitin sulphate E was shown to be principally an activator of heparin cofactor II. Maximum acceleration of heparin cofactor II:thrombin interaction was 185-fold (9.3 X 10(7) M-1 min-1), antithrombin III:thrombin interaction was 11-fold (4.16 X 10(6) M-1 min-1) and antithrombin III:factor Xa was 146-fold (3.86 X 10(6) M-1 min-1). Chondroitin sulphate E was observed to prolong the thrombin clotting time of fibrinogen in the absence of antithrombin III and heparin cofactor II. The effect appeared to be related to interference in thrombin:fibrinogen interaction rather than in fibrin monomer polymerization.