Oxysterol regulation of estrogen receptor alpha-mediated gene expression in a transcriptional activation assay system using HeLa cells

In order to test the estrogenic activity of sterol oxidation products from cholesterol and phytosterols, an estrogen-dependent gene expression assay was performed in estrogen receptor alpha-stably transformed HeLa cells. The ranking of the estrogenic potency of these compounds was different: 17beta-estradiol > genistein > beta-epoxycholesterol = daidzein = cholestanetriol = 22(R)-hydroxycholesterol = 20(S)-hydroxycholesterol = sitostanetriol > campestanetriol = beta-epoxysitosterol = 7beta-hydroxycholesterol. These compounds were not estrogenic in estrogen receptor-negative HeLa cells.     

Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4

Presenilin (PS) genes linked to early-onset familial Alzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of gamma-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Overexpression of CALP did not affect the metabolism or stability of PS complex, and gamma-cleavage of betaAPP or Notch site 3 cleavage was not altered. However, co-expression of CALP and a voltage-gated potassium channel subunit Kv4.2 reconstituted the features of A-type K(+) currents and CALP directly bound Kv4.2, indicating that CALP functions as KChIPs that are known as components of native Kv4 channel complex. Taken together, CALP/KChIP4 is a novel EF-hand protein interacting with PS as well as with Kv4 that may modulate functions of a subset of membrane proteins in brain.     

Effects of acute vs. chronic phorbol ester exposure on transepithelial permeability and epithelial morphology.

In previous experiments we have shown that acute (30 minutes) exposure to phorbol esters or other protein kinase C activators causes increased transepithelial permeability, specifically by the increased paracellular permeability through tight junctions. However, the role of protein kinase C activators in carcinogenesis is predicted upon a chronic exposure of an effective dose at frequent intervals for a prolonged period of time. We therefore sought to determine the effect of chronic phorbol ester exposure on transepithelial permeability by exposing cells of the polar renal epithelial cell line, LLC-PK1, to phorbol esters for time periods as long as 16 weeks. The following changes ensued: (1) after the initial drop in transepithelial resistance due to phorbol ester exposure, i.e., an increase in transepithelial permeability (in the acute phase of exposure), an adaptive response occurs as transepithelial resistances in chronically exposed cultures recover to approximately 50% of control values, (2) the cell sheets in chronically exposed cultures lose their acute responsiveness of transepithelial permeability to phorbol ester exposure, (3) cell sheet architecture changes as cells occasionally multilayer and actual polyp-like cell masses appear at high frequency, and (4) cytosolic protein kinase C activity decreases to 50% of control level with acute exposure and then is further decreased to less than 1% of control level in chronically treated cells; membrane-associated PKC activity is not as sharply decreased. The possible role of transepithelial permeability in carcinogenesis and the value of chronically treated epithelial cell cultures as a model for two-stage carcinogenesis are discussed.     

Early-life atomic-bomb irradiation accelerates immunological aging and elevates immune-related intracellular reactive oxygen species

Reactive oxygen species (ROS) play an important role in immune responses; however, their excessive production and accumulation increases the risk of inflammation-related diseases. Although irradiation is known to accelerate immunological aging, the underlying mechanism is still unclear. To determine the possible involvement of ROS in this mechanism, we examined 10,023 samples obtained from 3752 atomic-bomb survivors in Hiroshima and Nagasaki, who participated in repeated biennial examinations from 2008 to 2016, for the effects of aging and radiation exposure on intracellular ROS (H₂O₂ and O₂^•−) levels, percentages of T-cell subsets, and the effects of radiation exposure on the relationship between cell percentages and intracellular ROS levels in T-cell subsets. The cell percentages and intracellular ROS levels in T-cell subsets were measured using flow cytometry, with both fluorescently labeled antibodies and the fluorescent reagents, carboxy-DCFDA and hydroethidine. The percentages of naïve CD4⁺ and CD8⁺ T cells decreased with increasing age and radiation dose, while the intracellular O₂^•− levels in central and effector memory CD8⁺ T cells increased. Additionally, when divided into three groups based on the percentages of naïve CD4⁺ T cells, intracellular O₂^•− levels of central and effector memory CD8⁺ T cells were significantly elevated with the lowest radiation dose group in the naïve CD4⁺ T cells. Thus, the radiation exposure-induced decrease in the naïve CD4⁺ T cell pool size may reflect decreased immune function, resulting in increased intracellular ROS levels in central and effector memory CD8⁺ T cells, and increased intracellular oxidative stress.     

The closest lineage of Archaeplastida is revealed by phylogenomics analyses that include Microheliella maris

By clarifying the phylogenetic positions of ‘orphan’ protists (unicellular micro-eukaryotes with no affinity to extant lineages), we may uncover the novel affiliation between two (or more) major lineages in eukaryotes. Microheliella maris was an orphan protist, which failed to be placed within the previously described lineages by pioneering phylogenetic analyses. In this study, we analysed a 319-gene alignment and demonstrated that M. maris represents a basal lineage of one of the major eukaryotic lineages, Cryptista. We here propose a new clade name ‘Pancryptista’ for Cryptista plus M. maris. The 319-gene analyses also indicated that M. maris is a key taxon to recover the monophyly of Archaeplastida and the sister relationship between Archaeplastida and Pancryptista, which is collectively called ‘CAM clade’ here. Significantly, Cryptophyceae tend to be attracted to Rhodophyta depending on the taxon sampling (ex., in the absence of M. maris and Rhodelphidia) and the particular phylogenetic ‘signal’ most likely hindered the stable recovery of the monophyly of Archaeplastida in previous studies.     

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.     

Involvement of ryanodine receptors in pacemaker Ca2+ oscillation in murine gastric ICC

Using a cell cluster preparation from the stomach smooth muscle tissue of mice, we measured intracellular Ca(2+) oscillations in interstitial cells of Cajal (ICCs) in the presence of nifedipine. Pacemaker [Ca(2+)](i) activity in ICCs was significantly suppressed by caffeine application and restored after washout. Application of either ryanodine or FK-506 terminated the pacemaker [Ca(2+)](i) activity irreversibly. Immunostaining of smooth muscle tissue showed that c-Kit-immunopositive cells (that form network-like structure cells in the myenteric plexus, equivalent to ICCs) clearly express ryanodine receptors (RyR). RT-PCR revealed that ICCs (identified with c-Kit-immunoreactivity) predominantly express type 3 RyR (RyR3). Furthermore, the FK-binding proteins 12 and 12.6, both of which would interact with RyR3, were detected. In conclusion, we provide first evidence for the essential contribution of RyR to generating pacemaker activity in gastric motility. Similar mechanisms might account for spontaneous rhythmicity seen in smooth muscle tissues distributed in the autonomic nervous system.     

Survival rate of microbes after freeze-drying and long-term storage

The survival rates of 10 species of microorganisms were investigated after freeze-drying and preserving in a vacuum at 5 degrees C. The survival rates varied with species. The survival rates immediately after freeze-drying were different among yeast, gram-positive bacteria, and gram-negative bacteria, and the change in the 10-year survival rate was species-specific. The survival rate of yeast, Saccharomyces cerevisiae, was about 10% immediately after drying, and the rate did not decrease significantly during the 10-year storage period. Survival rates after the drying of gram-positive bacteria, i.e., Brevibacterium flavum, B. lactofermentum, Corynebacterium acetoacidophilum, C. gultamicum, and Streptococcus mutans, were around 80%. The survival rate of Brevibacterium and Corynebacterium did not decrease greatly during the storage period, whereas the rate of S. mutans decreased to about 20% after 10 years. Survival rates after the drying of gram-negative bacteria, i.e., Escherichia coli, Pseudomonas putida, Serratia marcescens, and Alcaligenes faecalis, were around 50%. The survival rate decreased for the first 5 years and then stabilized to around 10% thereafter.