Optimizing collagen antigen unmasking in paraffin-embedded tissues

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin--biotin-- peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 m phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml−1 pepsin in 0.01 n hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37°C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation     

The diversity of receptor recognition in cholesterol‐dependent cytolysins

Cholesterol‐dependent cytolysins (CDCs) are bacterial pore‐forming toxins secreted mainly by pathogenic Gram‐positive bacteria. CDCs generally recognize and bind to membrane cholesterol to create pores and lyse target cells. However, in contrast to typical CDCs such as streptolysin O, several atypical CDCs have been reported. The first of these was intermedilysin, which is secreted by Streptococcus intermedius and has human cell‐specificity, human CD59 (huCD59) being its receptor. In the study reported here, the diversity of receptor recognition among CDCs was investigated and multi‐receptor recognition characteristics were identified within this toxin family. Streptococcus mitis‐derived human platelet aggregation factor (Sm‐hPAF) secreted by S. mitis strain Nm‐65 isolated from a patient with Kawasaki disease was previously shown to hemolyze erythrocytes in a species‐dependent manner, its maximum activity being in human cells. In the present study, it was found that Sm‐hPAF recognizes both membrane cholesterol and huCD59 as receptors for triggering pore‐formation. Moreover, vaginolysin (VLY) of Gardnerella vaginalis showed similar characteristics to Sm‐hPAF regarding receptor recognition. On the basis of the results presented here, the mode of receptor recognition of CDCs can be categorized into the following three groups: (i) Group I, comprising typical CDCs with high affinity to cholesterol and no or very little affinity to huCD59; (ii) Group II, including atypical CDCs such as ILY, with no or very little affinity to cholesterol and high affinity to huCD59; and (iii) Group III, which contains atypical CDCs such as Sm‐hPAF and VLY with affinity to both cholesterol and huCD59.     

Climate and topography drives macroscale biodiversity through land‐use change in a human‐dominated world

Drivers of biodiversity at macroscales have long been of interest in ecology, and climate and topography are now considered to be major drivers. Because humans have transformed most of the Earth's land surface, land use may play a significant role as a driver of biodiversity at a macroscale. Here we disentangle the relationships among climate, topography, land use, available energy (measured by the normalized difference vegetation index [NDVI]), and species richness of Japanese forest birds. Species richness was better explained at 40‐ and 80‐km resolutions than at 5‐, 10‐ and 20‐km resolutions; it was explained by climate, topography, and land use, and the effects of land use were fully incorporated into those of climate and topography. As temperature increased and elevation decreased, natural forest area decreased, and this decrease intensified in warm lowland areas. With the loss of natural forest, species richness decreased below a certain threshold. As temperature increased and elevation decreased, species richness and NDVI increased slightly or were unchanged in cool highland areas and decreased in warm lowland areas. Species richness increased linearly with the increase in NDVI. Most effects of climate/topography on species richness in warm lowland areas were shared by those of land use, suggesting that the decrease in species richness in warm lowland areas has been caused by loss of natural forest. Therefore, it is suggested that climate and topography determined land use intensity, which in turn, drove species richness through the depletion of available energy. Increasing temperature and decreasing elevation leads to both benefits (increase in potential available energy) and costs (depletion of energy by human land‐use change) for forest birds. These costs seem to override benefits in warm lowland areas.     

Characterization of an Alteromonas long-type ulvan lyase involved in the degradation of ulvan extracted from Ulva ohnoi

Ulvan is a sulfated polysaccharide found in the cell wall of the green algae Ulva. We first isolated several ulvan-utilizing Alteromonas sp. from the feces of small marine animals. The strain with the highest ulvan-degrading activity, KUL17, was analyzed further. We identified a 55-kDa ulvan-degrading protein secreted by this strain and cloned the gene encoding for it. The deduced amino acid sequence indicated that the enzyme belongs to polysaccharide lyase family 24 and thus the protein was named ulvan lyase. The predicted molecular mass of this enzyme is 110 kDa, which is different from that of the identified protein. By deletion analysis, the catalytic domain was proven to be located on the N-terminal half of the protein. KUL17 contains two ulvan lyases, one long and one short, but the secreted and cleaved long ulvan lyase was demonstrated to be the major enzyme for ulvan degradation.     

Optimizing collagen antigen unmasking in paraffin-embedded tissues

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin--biotin-- peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 m phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml−1 pepsin in 0.01 n hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37°C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation     

A Simple Method for Transportation of Mouse Embryos Using Microtubes and a Warm Box

Generally, transportation of preimplantation embryos without freezing requires incubators that can maintain an optimal culture environment with a suitable gas phase, temperature, and humidity. Such incubators are expensive to transport. We reported previously that normal offspring were obtained when the gas phase and temperature could be maintained during transportation. However, that system used plastic dishes for embryo culture and is unsuitable for long-distance transport of live embryos. Here, we developed a simple low-cost embryo transportation system. Instead of plastic dishes, several types of microtubes—usually used for molecular analysis—were tested for embryo culture. When they were washed and attached to a gas-permeable film, the rate of embryo development from the 1-cell to blastocyst stage was more than 90%. The quality of these blastocysts and the rate of full-term development after embryo transfer to recipient female mice were similar to those of a dish-cultured control group. Next, we developed a small warm box powered by a battery instead of mains power, which could maintain an optimal temperature for embryo development during transport. When 1-cell embryos derived from BDF1, C57BL/6, C3H/He and ICR mouse strains were transported by a parcel-delivery service over 3 days using microtubes and the box, they developed to blastocysts with rates similar to controls. After the embryos had been transferred into recipient female mice, healthy offspring were obtained without any losses except for the C3H/He strain. Thus, transport of mouse embryos is possible using this very simple method, which might prove useful in the field of reproductive medicine.     

Alterations of Regional Cerebral Blood Flow in Tinnitus Patients as Assessed Using Single-Photon Emission Computed Tomography

Tinnitus is the perception of phantom sound without an external auditory stimulus. Using neuroimaging techniques, such as positron emission tomography, electroencephalography, magnetoencephalography, and functional magnetic resonance imaging (fMRI), many studies have demonstrated that abnormal functions of the central nervous system are closely associated with tinnitus. In our previous research, we reported using resting-state fMRI that several brain regions, including the rectus gyrus, cingulate gyrus, thalamus, hippocampus, caudate, inferior temporal gyrus, cerebellar hemisphere, and medial superior frontal gyrus, were associated with tinnitus distress and loudness. To reconfirm these results and probe target regions for repetitive transcranial magnetic stimulation (rTMS), we investigated the regional cerebral blood flow (rCBF) between younger tinnitus patients (<60 years old) and the age-matched controls using single-photon emission computed tomography and easy Z-score imaging system. Compared with that of controls, the rCBF of tinnitus patients was significantly lower in the bilateral medial superior frontal gyri, left middle occipital gyrus and significantly higher in the bilateral cerebellar hemispheres and vermis, bilateral middle temporal gyri, right fusiform gyrus. No clear differences were observed between tinnitus patients with normal and impaired hearing. Regardless of the assessment modality, similar brain regions were identified as characteristic in tinnitus patients. These regions are potentially involved in the pathophysiology of chronic subjective tinnitus.     

Synergistic Proinflammatory Responses by IL-17A and Toll-Like Receptor 3 in Human Airway Epithelial Cells

Viral respiratory infections activate the innate immune response in the airway epithelium through Toll-like receptors (TLRs) and induce airway inflammation, which causes acute exacerbation of asthma. Although increases in IL-17A expression were observed in the airway of severe asthma patients, the interaction between IL-17A and TLR activation in airway epithelium remains poorly understood. In this study, we demonstrated that IL-17A and polyI:C, the ligand of TLR3, synergistically induced the expression of proinflammatory cytokines and chemokines (G-CSF, IL-8, CXCL1, CXCL5, IL-1F9), but not type I interferon (IFN-α1, -β) in primary culture of normal human bronchial epithelial cells. Synergistic induction after co-stimulation with IL-17A and polyI:C was observed from 2 to 24 hours after stimulation. Treatment with cycloheximide or actinomycin D had no effect, suggesting that the synergistic induction occurred without de novo protein synthesis or mRNA stabilization. Inhibition of the TLR3, TLR/TIR-domain-containing adaptor-inducing interferon β (TRIF), NF-κB, and IRF3 pathways decreased the polyI:C- and IL-17A/polyI:C-induced G-CSF and IL-8 mRNA expression. Comparing the levels of mRNA induction between co-treatment with IL-17A/polyI:C and treatment with polyI:C alone, blocking the of NF-κB pathway significantly attenuated the observed synergism. In western blotting analysis, activation of both NF-κB and IRF3 was observed in treatment with polyI:C and co-treatment with IL-17A/polyI:C; moreover, co-treatment with IL-17A/polyI:C augmented IκB-α phosphorylation as compared to polyI:C treatment alone. Collectively, these findings indicate that IL-17A and TLR3 activation cooperate to induce proinflammatory responses in the airway epithelium via TLR3/TRIF-mediated NF-κB/IRF3 activation, and that enhanced activation of the NF-κB pathway plays an essential role in synergistic induction after co-treatment with IL-17A and polyI:C in vitro.