Gene expression profiling identifies a set of transcripts that are up-regulated inhuman testicular seminoma.

Seminoma constitutes one subtype of human testicular germ cell tumors and is uniformly composed of cells that are morphologically similar to the primordial germ cells and/or the cells in the carcinoma in situ. We performed a genome-wide exploration of the genes that are specifically up-regulated in seminoma by oligonucleotide-based microarray analysis. This revealed 106 genes that are significantly and consistently up-regulated in the seminomas compared to the adjacent normal tissues of the testes. The microarray data were validated by semi-quantitative RT-PCR analysis. Of the 106 genes, 42 mapped to a small number of specific chromosomal regions, namely, 1q21, 2p23, 6p21-22, 7p14-15, 12pll, 12p13, 12q13-14 and 22q12-13. This list of up-regulated genes may be useful in identifying the causative oncogene(s) and/or the origin of seminoma. Furthermore, immunohistochemical analysis revealed that the seminoma cells specifically expressed the six gene products that were selected randomly from the list. These proteins include CCND2 and DNMT3A and may be useful as molecular pathological markers of seminoma.     

Osteoclasts adapt to physioxia perturbation through DNA demethylation

Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two‐photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia‐inducible factor activity. We observe that hypoxia decreases ten‐eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen‐dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.     

Design and synthesis of novel δ opioid receptor agonists with an azatricyclodecane skeleton for improving blood-brain barrier penetration

We designed and synthesized novel δ opioid receptor (DOR) agonists 3a–i with an azatricyclodecane skeleton, which was a novel structural class of DOR agonists. Among them, 3b exhibited high values of binding affinity and potent agonistic activity for the DOR that were approximately equivalent to those of 2 which bore an oxazatricyclodecane skeleton. In vitro assays using the blood-brain barrier (BBB) permeability test kit supported the idea that 3b achieved an excellent BBB permeability by converting an oxygen atom of 2 to a carbon atom (methylene group) in the core skeleton. As a result, 3b showed potent antinociceptive effects.     

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.     

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.     

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.     

Parkin stabilizes PINK1 through direct interaction

Parkinson disease (PD) is the most common movement disorder and is characterized by dopaminergic dysfunction. The majority of PD cases are sporadic; however, the discovery of genes linked to rare familial forms of the disease has provided crucial insight into the molecular mechanisms of disease pathogenesis. Multiple genes mediating familial forms of Parkinson’s disease (PD) have been identified, such as parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1: PINK1 (PARK6). Here, we showed that Parkin directly interacts with PINK1, but did not bind to pathogenic PINK1 mutants. Parkin, but not its pathogenic mutants, stabilizes PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway. In addition, the interaction between Parkin and PINK1 resulted in reciprocal reduction of their solubility. Our results indicate that Parkin regulates PINK1 stabilization via direct interaction with PINK1, and operates through a common pathway with PINK1 in the pathogenesis of early-onset PD.