Cloning and Characterization of a Unique Cytotoxic Protein Parasporin-5 Produced by Bacillus thuringiensis A1100 Strain

Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which was designated parasporin-5 (PS5). PS5 was purified from B. thuringiensis serovar tohokuensis strain A1100 based on its cytocidal activity against human leukemic T cells (MOLT-4). The 50% effective concentration (EC₅₀) of PS5 to MOLT-4 cells was approximately 0.075 μg/mL. PS5 was expressed as a 33.8-kDa inactive precursor protein and exhibited cytocidal activity only when degraded by protease at the C-terminal into smaller molecules of 29.8 kDa. Although PS5 showed no significant homology with other known parasporins, a Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) search revealed that the protein showed slight homology to, not only some B. thuringiensis Cry toxins, but also to aerolysin-type β-pore-forming toxins (β-PFTs). The recombinant PS5 protein could be obtained as an active protein only when it was expressed in a precursor followed by processing with proteinase K. The cytotoxic activities of the protein against various mammalian cell lines were evaluated. PS5 showed strong cytocidal activity to seven of 18 mammalian cell lines tested, and low to no cytotoxicity to the others.     

Factors associated with development and distribution of granular/fuzzy astrocytes in neurodegenerative diseases

Granular/fuzzy astrocytes (GFAs), a subtype of “aging‐related tau astrogliopathy,” are noted in cases bearing various neurodegenerative diseases. However, the pathogenic significance of GFAs remains unclear. We immunohistochemically examined the frontal cortex, caudate nucleus, putamen and amygdala in 105 cases composed of argyrophilic grain disease cases (AGD, N = 26), and progressive supranuclear palsy (PSP, N = 10), Alzheimer’s disease (AD, N = 20) and primary age‐related tauopathy cases (PART, N = 18) lacking AGD, as well as 31 cases bearing other various neurodegenerative diseases to clarify (i) the distribution patterns of GFAs in AGD, and PSP, AD and PART lacking AGD, (ii) the impacts of major pathological factors and age on GFA formation and (iii) immunohistochemical features useful to understand the formation process of GFAs. In AGD cases, GFAs consistently occurred in the amygdala (100%), followed by the putamen (69.2%) and caudate nucleus and frontal cortex (57.7%, respectively). In PSP cases without AGD, GFAs were almost consistently noted in all regions examined (90–100%). In AD cases without AGD, GFAs were less frequent, developing preferably in the putamen (35.0%) and caudate nucleus (30.0%). PART cases without AGD had GFAs most frequently in the amygdala (35.3%), being more similar to AGD than to AD cases. Ordered logistic regression analyses using all cases demonstrated that the strongest independent factor of GFA formation in the frontal cortex and striatum was the diagnosis of PSP, while that in the amygdala was AGD. The age was not significantly associated with GFA formation in any region. In GFAs in AGD cases, phosphorylation and conformational change of tau, Gallyas‐positive glial threads indistinguishable from those in tufted astrocytes, and the activation of autophagy occurred sequentially. Given these findings, AGD, PSP, AD and PART cases may show distinct distributions of GFAs, which may provide clues to predict the underlying processes of primary tauopathies.     

Histamine and histidine decarboxylase: Immunomodulatory functions and regulatory mechanisms

Histamine is a bioactive monoamine that is synthesized by the enzymatic activity of histidine decarboxylase (HDC) in basophils, mast cells, gastric enterochromaffin‐like (ECL) cells and histaminergic neuronal cells. Upon a series of cellular stimuli, these cells release stored histamine, which elicits allergies, inflammation, and gastric acid secretion and regulates neuronal activity. Recent studies have shown that certain other types of myeloid lineage cells also produce histamine with HDC induction under various pathogenic stimuli. Histamine has been shown to play a series of pathophysiological roles by modulating immune and inflammatory responses in a number of disease conditions, whereas the mechanistic aspects underlying induced HDC expression remain elusive. In the present review, we summarize the current understanding of the regulatory mechanism of Hdc gene expression and the roles played by histamine in physiological contexts as well as pathogenic processes. We also introduce a newly developed histaminergic cell‐monitoring transgenic mouse line (Hdc‐BAC‐GFP) that serves as a valuable experimental tool to identify the source of histamine and dissect upstream regulatory signals.     

A simple and practical workflow for genotyping of CRISPR–Cas9‐based knockout phenotypes using multiplexed amplicon sequencing

Founder animals carrying high proportions of somatic mutation induced by CRISPR–Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next‐generation sequencing is the most suitable approach for large‐scale management of multiple samples and accurate evaluation of the efficiency of Cas9‐induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR–Cas9‐based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single‐indexed library on the Illumina MiSeq platform. Additionally, a user‐friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype–phenotype correlations in CRISPR–Cas9‐based loss‐of‐function screening of numerous target genes in various organisms.     

OPN4 belongs to the photosensitive system of the human skin

The human skin has previously been described to be affected by light; however, the underlying mechanism remains unknown. OPN4 (melanopsin) expression was first identified in the skin of amphibians; however, whether it is also expressed and functioned in the human skin has not yet been identified. Here, we show that OPN4 was expressed in the human skin tissue and cultures of isolated keratinocytes, melanocytes and fibroblasts. Additionally, Ca²⁺ influx in vitro and ex vivo and phosphorylation of extracellular signal‐regulated kinases 1/2 in human fibroblasts were observed by stimulation of blue light irradiation. Notably, our findings showed that this Ca²⁺ influx and phosphorylation of extracellular signal‐regulated kinases 1/2 are promoted in an intensity‐dependent manner, indicating that the light signal is converted to an intracellular signal via OPN4 in the human skin. Overall, in this study we showed that the human skin functions as a photoreceptor by demonstrating that in human skin, the photoreceptive protein was expressed, and photoreception was conducted via photoreceptive protein.     

Immunohistochemical and Western blot analyses of collar enamel in the jaw teeth of gars,Lepisosteus oculatus,an actinopterygian fish

Although most fish have no enamel layer in their teeth, those belonging to Lepisosteus (gars), an extant actinopterygian fish genus, do and so can be used to study amelogenesis. In order to examine the collar enamel matrix in gar teeth, we subjected gar teeth to light and electron microscopic immunohistochemical examinations using an antibody against bovine amelogenin (27?kDa) and antiserum against porcine amelogenin (25?kDa), as well as region-specific antibodies and antiserum against the C-terminus and middle region, and N-terminus of porcine amelogenin, respectively. The enamel matrix exhibited intense immunoreactivity to the anti-bovine amelogenin antibody and the anti-porcine amelogenin antiserum in addition to the C-terminal and middle region-specific antibodies, but not to the N-terminal-specific antiserum. These results suggest that the collar enamel matrix of gar teeth contains amelogenin-like proteins and that these proteins possess domains that closely resemble the C-terminal and middle regions of porcine amelogenin. Western blot analyses of the tooth germs of Lepisosteus were also performed. As a result, protein bands with molecular weights of 78?kDa and 65?kDa were clearly stained by the anti-bovine amelogenin antibody as well as the antiserum against porcine amelogenin and the middle-region-specific antibody. It is likely that the amelogenin-like proteins present in Lepisosteus do not correspond to the amelogenins found in mammals, although they do possess domains that are shared with mammalian amelogenins.