Development of an Ultra-Rapid Diagnostic Method Based on Heart-Type Fatty Acid Binding Protein Levels in the CSF of CJD Patients

Creutzfeldt-Jakob disease (CJD) is a transmissible, fatal, neurodegenerative disease in humans. Recently, various drugs have been reported to be useful in the treatment of CJD; however, for such treatments to be useful it is essential to rapidly and accurately diagnose CJD. 124 CJD patients and 87 with other diseases causing rapid progressive dementia were examined. Cerebral spinal fluid (CSF) from CJD patients was analyzed by 2D-PAGE and the protein expression pattern was compared with that from healthy subjects. One of three CJD-specific spots was found to be fatty acid binding protein (FABP), and heart-type FABP (H-FABP) was analyzed as a new biochemical marker for CJD. H-FABP ELISA results were compared between CJD patients and patients with other diseases (n = 211). Visual readout accuracy of the Rapicheck^® H-FABP test panel for CSF was analyzed using an independent measure of CSF H-FABP concentration. The distribution of H-FABP in the brains of CJD patients was examined by immunohistochemistry. ELISA sensitivity and specificity were 90.3% and 92.9%, respectively, and Rapicheck^® H-FABP sensitivity and specificity were 87.9% and 96.0%, respectively. ELISA and Rapicheck^® H-FABP assays provided comparable results for 14-3-3 protein and total tau protein. Elevated H-FABP levels were associated with an accumulation of abnormal prion protein, astrocytic gliosis, and neuronal loss in the cerebral cortices of CJD patients. In conclusion, Rapicheck^® H-FABP of CSF specimens enabled quick and frequent diagnosis of CJD. H-FABP represents a new biomarker for CJD distinct from 14-3-3 protein and total tau protein.     

Clonal spread of antimicrobial-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates among pups in two kennels

Although the dog breeding industry is common in many countries, the presence of antimicrobial resistant bacteria among pups in kennels has been infrequently investigated. This study was conducted to better understand the epidemiology of antimicrobial-resistant Escherichia coli isolates from kennel pups not treated with antimicrobials. We investigated susceptibilities to 11 antimicrobials, and prevalence of extended-spectrum β-lactamase (ESBL) in 86 faecal E. coli isolates from 43 pups in two kennels. Genetic relatedness among all isolates was assessed using pulsed-field gel electrophoresis (PFGE). Susceptibility tests revealed that 76% of the isolates were resistant to one or more of tested antimicrobials, with resistance to dihydrostreptomycin most frequently encountered (66.3%) followed by ampicillin (60.5%), trimethoprim-sulfamethoxazole (41.9%), oxytetracycline (26.7%), and chloramphenicol (26.7%). Multidrug resistance, defined as resistance against two or more classes of antimicrobials, was observed in 52 (60.5%) isolates. Three pups in one kennel harboured SHV-12 ESBL-producing isolates. A comparison between the two kennels showed that frequencies of resistance against seven antimicrobials and the variation in resistant phenotypes differed significantly. Analysis by PFGE revealed that clone sharing rates among pups of the same litters were not significantly different in both kennels (64.0% vs. 88.9%), whereas the rates among pups from different litters were significantly different between the two kennels (72.0% vs. 33.3%, P < 0.05). The pups in the two kennels had antimicrobial-resistant E. coli clones, including multidrug-resistant and ESBL-producing clones. It is likely that resistant and susceptible bacteria can clonally spread among the same and/or different litters thus affecting the resistance prevalence.     

Shigella effector IpaH9.8 binds to a splicing factor U2AF(35) to modulate host immune responses

Shigella effectors injected into the host cell via the type III secretion system are involved in various aspects of infection. Here, we show that one of the effectors, IpaH9.8, plays a role in modulating inflammatory responses to Shigella infection. In murine lung infection model, DeltaipaH9.8 mutant caused more severe inflammatory responses with increased pro-inflammatory cytokine production levels than did wild-type Shigella, which resulted in a 30-fold decrease in bacterial colonization. Binding assays revealed that IpaH9.8 has a specific affinity to U2AF(35), a mammalian splicing factor, which interferes with U2AF(35)-dependent splicing as assayed for IgM pre-mRNA. Reducing the U2AF(35) level in HeLa cells and infecting HeLa cells with wild-type caused a decrease in the expression of the il-8, RANTES, GM-CSF, and il-1beta genes as examined by RT-PCR. The results indicate that IpaH9.8 plays a role in Shigella infection to optimize the host inflammatory responses, thus facilitating bacterial colonization within the host epithelial cells.     

Activation of Protein Kinase C Suppresses the γ-Aminobutyric AcidB Receptor-Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine

Modulation of the gamma-aminobutyric acidB (GABAB) receptor-mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation-evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the high K(+)-evoked Ca2+-dependent release of NA and ACh, but not the ouabain-evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic-vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K(+)-evoked release of NA and ACh, but not the ouabain-evoked Ca(2+)-independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor-mediated inhibition of high K(+)-evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation-evoked Ca(2+)-dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor-mediated response.     

Stereospecific Reduction of Ethyl 2′ -Ketopantothenate to Ethyl D-(+)-Pantothenate with Microbial Cells as a Catalyst

A novel enzymatic process for the synthesis of D-(+)-pantothenic acid through the asymmetric reduction of the 2′ -ketopantothenate ester is described. Candida macedoniensis AKU 4588 was found to convert ethyl 2′ -ketopantothenate (80 mg/ml) almost specifically to ethyl D-(+)-pantothenate (>98% enantiomeric excess), with a molar yield of 97.2%.     

Polarization-based brightness discrimination in the foraging butterfly, Papilio xuthus

The human eye is insensitive to the angular direction of the light e-vector, but several animal species have the ability to discriminate differently polarized lights. How the polarization is detected is often unclear, however. Egg-laying Papilio butterflies have been shown to see false colours when presented with differently polarized lights. Here we asked whether this also holds in foraging butterflies. After training individuals to feed on nectar in front of an unpolarized spectral light, we carried out three dual-choice tests, where the discrimination of (i) the spectral content, (ii) the light intensity, and (iii) the e-vector orientation were investigated. In the first test, the butterflies selected the trained spectrum irrespective of its intensity, and in the second test they chose the light with the higher intensity. The result of the e-vector discrimination test was very similar to that of the second test, suggesting that foraging butterflies discriminate differently polarized lights as differing in brightness rather than as differing in colour. Papilio butterflies are clearly able to use at least two modes of polarization vision depending on the behavioural context.     

Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

Background

Neural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain.

Methodology/Principal Findings

In this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner.

Conclusions

This study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of a potential value in stem cell therapies in various brain diseases.