Significance of Basal Membrane Permeability of Epithelial Cells in Predicting Intestinal Drug Absorption.

Drug absorption from the gastrointestinal tract is often restricted by efflux transport by P-glycoprotein (P-gp) and metabolism by CYP3A4. Both localize in the epithelial cells, and thus, their activities are directly affected by the intracellular drug concentration, which should be regulated by the ratio of permeability between apical (A) and basal (B) membranes. In this study, using Caco-2 cells with forced expression of CYP3A4, we assessed the transcellular permeation of A-to-B and B-to-A directions and the efflux from the preloaded cells to both sides of 12 representative P-gp or CYP3A4 substrate drugs and obtained the parameters for permeabilities, transport, metabolism, and unbound fraction in the enterocytes (fent) using simultaneous and dynamic model analysis. The membrane permeability ratios for B to A (RBA) and fent varied by 8.8-fold and by more than 3000-fold, respectively, among the drugs. The RBA values for digoxin, repaglinide, fexofenadine, and atorvastatin were greater than 1.0 (3.44, 2.39, 2.27, and 1.90, respectively) in the presence of a P-gp inhibitor, thus suggesting the potential involvement of transporters in the B membrane. The Michaelis constant for quinidine for P-gp transport was 0.077 µM for the intracellular unbound concentration. These parameters were used to predict overall intestinal availability (FAFG) by applying an intestinal pharmacokinetic model, advanced translocation model (ATOM), in which permeability of A and B membranes accounted separately. The model predicted changes in the absorption location for P-gp substrates according to its inhibition, and FAFG values of 10 of 12 drugs, including quinidine at varying doses, were explained appropriately. SIGNIFICANCE STATEMENT: Pharmacokinetics has improved predictability by identifying the molecular entities of metabolism and transport and by using mathematical models to appropriately describe drug concentrations at the locations where they act. However, analyses of intestinal absorption so far have not been able to accurately consider the concentrations in the epithelial cells where P-glycoprotein and CYP3A4 exert effects. In this study, the limitation was removed by measuring the apical and basal membrane permeability separately and then analyzing these values using new appropriate models.

Phosphorylation of human phospholipase A1 DDHD1 at newly identified phosphosites affects its subcellular localization.

Phospholipase A1 (PLA1) hydrolyzes the fatty acids of glycerophospholipids, which are structural components of the cellular membrane. Genetic mutations in DDHD1, an intracellular PLA1, result in hereditary spastic paraplegia (HSP) in humans. However, the regulation of DDHD1 activity has not yet been elucidated in detail. In the present study, we examined the phosphorylation of DDHD1 and identified the responsible protein kinases. We performed MALDI-TOF MS/MS analysis and Phos-tag SDS-PAGE in alanine-substitution mutants in HEK293 cells and revealed multiple phosphorylation sites in human DDHD1, primarily Ser8, Ser11, Ser723, and Ser727. The treatment of cells with a protein phosphatase inhibitor induced the hyperphosphorylation of DDHD1, suggesting that multisite phosphorylation occurred not only at these major, but also at minor sites. Site-specific kinase-substrate prediction algorithms and in vitro kinase analyses indicated that cyclin-dependent kinase CDK1/cyclin A2 phosphorylated Ser8, Ser11, and Ser727 in DDHD1 with a preference for Ser11 and that CDK5/p35 also phosphorylated Ser11 and Ser727 with a preference for Ser11. In addition, casein kinase CK2α1 was found to phosphorylate Ser104, although this was not a major phosphorylation site in cultivated HEK293 cells. The evaluation of the effects of phosphorylation revealed that the phosphorylation mimic mutants S11/727E exhibit only 20% reduction in PLA1 activity. However, the phosphorylation mimics were mainly localized to focal adhesions, whereas the phosphorylation-resistant mutants S11/727A were not. This suggested that phosphorylation alters the subcellular localization of DDHD1 without greatly affecting its PLA1 activity.

N-(4-Hydroxyphenyl) Retinamide Suppresses SARS-CoV-2 Spike Protein-Mediated Cell-Cell Fusion by a Dihydroceramide Δ4-Desaturase 1-Independent Mechanism.

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

Star-Shaped Peptide-Polymer Hybrids as Fast pH-Responsive Supramolecular Hydrogels.

Significant challenges have gone into the design of smart hydrogels, with numerous potential applications in the industrial, cosmetic, and biomedical fields. Herein, we report the synthesis of novel 4-arm self-assembling peptide-polyethylene glycol (PEG) hybrid star-shaped polymers and their comprehensive hydrogel properties. ß-sheet-forming oligopeptides with alternating hydrophobic Leu/ionizable Glu repeats and Cys residues were successfully conjugated to 4-arm PEG via a thiol-maleimide click reaction. The hybrid star-shaped polymers demonstrated good cytocompatibility and reversible ß-sheet (lightly acidic pH)-to-random coil (neutral and basic pH) transition in dilute aqueous solutions. At increasing polymer concentrations up to 0.5 wt %, the star-shaped polymers formed transparent hydrogels with shear-thinning and self-healing behaviors via ß-sheet self-assembly, as well as a conformation-dependent gel-sol transition. Interestingly, the star-shaped polymers responded rapidly to pH changes, causing gelation to occur rapidly within a few seconds from the change in pH. Hydrogel characteristics could be modulated by manipulating the length and net charge of the peptide blocks. Furthermore, these star-shaped polymers served as satisfactory network scaffolds that could respond to dynamic environmental changes in the pH-oscillation system, owing to their excellent gelation capability and pH sensitivity. As such, they are highly favorable for diverse applications, such as pH-responsive controlled release.

Lysophosphatidylinositol Induced Morphological Changes and Stress Fiber Formation through the GPR55-RhoA-ROCK Pathway.

We previously reported that lysophosphatidylinositol (LPI) functions as an endogenous agonist of GPR55, a novel cannabinoid receptor. However, the physiological roles of LPI-GPR55 have not yet been elucidated in detail. In the present study, we found that LPI induced morphological changes in GPR55-expressing HEK293 cells. LPI induced the cell rounding of GPR55-expressing HEK293 cells but not of empty-vector-transfected cells. LPI also induced the activation of small GTP-binding protein RhoA and increased stress fiber formation in GPR55-expressing HEK293 cells. The inhibition of RhoA and Rho kinase ROCK by the C3 exoenzyme and the ROCK inhibitor reduced LPI-induced cell rounding and stress fiber formation. These results clearly indicated that the LPI-induced morphological changes and the assembly of the cytoskeletons were mediated through the GPR55-RhoA-ROCK pathway.

Antitumor effects of novel mAbs against cationic amino acid transporter 1 (CAT1) on human CRC with amplified CAT1 gene.

Copy number alterations detected by comparative genomic hybridization (CGH) can lead to the identification of novel cancer-related genes. We analyzed chromosomal aberrations in a set of 100 human primary colorectal cancers (CRCs) using CGH and found a solute carrier (SLC) 7A1 gene, which encodes cationic amino acid transporter 1 (CAT1) with 14 putative transmembrane domains, in a chromosome region (13q12.3) with a high frequency of gene amplifications. SLC7A1/CAT1 is a transporter responsible for the uptake of cationic amino acids (arginine, lysine, and ornithine) essential for cellular growth. Microarray and PCR analyses have revealed that mRNA transcribed from CAT1 is overexpressed in more than 70% of human CRC samples, and RNA interference-mediated knockdown of CAT1 inhibited the cell growth of CRCs. Rats were immunized with rat hepatoma cells expressing CAT1 tagged with green fluorescent protein (GFP), and rat splenocytes were fused with mouse myeloma cells. Five rat monoclonal antibodies (mAbs) (CA1 ~ CA5) reacting with HEK293 cells expressing CAT1-GFP in a GFP expression-dependent manner were selected from established hybridoma clones. Novel anti-CAT1 mAbs selectively reacted with human CRC tumor tissues compared with adjacent normal tissues according to immuno-histochemical staining and bound strongly to numerous human cancer cell lines by flow cytometry. Anti-CAT1 mAbs exhibited internalization activity, antibody-dependent cellular cytotoxicity, and migration inhibition activity against CRC cell lines. Furthermore, CA2 inhibited the in vivo growth of human HT29 and SW-C4 CRC tumors in nude mice. This study suggested CAT1 to be a promising target for mAb therapy against CRCs.

[The Strength of the Sense of Shame of the Aged in Care Scenes: Comparison Between Whether or not There is Anyone Else Around Them].

Medical staff in a hospital or nursing facility should take care of aged individuals with dignity and respect. We conducted a survey on aged individuals to derive under what care circumstances they had a sense of shame, using 12 illustrations, drawn by ourselves, which were common daily care scenes where nurses and patients meet. This survey was conducted at 4 care facilities in A prefecture, Japan. The number of surveyed persons was 43, with the following exclusion criteria: over 60 years old, more than third level of care needed, and non suspected of having dementia. We got the following results from the answers of 41 persons: 1. When elder persons are surrounded by people other than the care staff, they feel more of a sense of shame than when alone; 2. They feel more sense of shame when they use a wheelchair than when they use crutches; 3. They do not feel much shame when they get a bed-bath, even if other persons are there; and 4. Male patients feel more shame than females when they meet their family. These results suggest that elderly patients feel a stronger sense of shame when they are seen by others than when they are seen by care staff. The result 2 suggests that the use of a wheelchair exposes their physical weakness to others. Males feel a stronger sense of shame when they show a weakness in their gender role. We conclude that the sense of shame of aged individuals in daily life scenes in a care facility depends on their gender and whether or not they are surrounded by other persons.

Complex formation of sphingomyelin synthase 1 with glucosylceramide synthase increases sphingomyelin and decreases glucosylceramide levels.

Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in cis-Golgi, and SM synthesis is thought to occur in medial/trans-Golgi; however, SMS1 and GCS are known to partially co-localize in cisternae, especially in medial/trans-Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile α-motif of SMS1 reduced the stability of the SMS1-GCS complex, resulting in a significant reduction in SM synthesis in vivo In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis in vivo These results suggest that formation of the SMS1-GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1-GCS complex was confirmed by CRISPR/Cas9-mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.

Autonomous Movement System Induced by Synergy between pH Oscillation and a pH-Responsive Oil Droplet.

A sustainable droplet motion that is driven by pH oscillation was obtained. The pH oscillation is only of a single pulse in a batch reactor. However, it shows continuous oscillation around the moving droplet, as the motion itself controls the diffusion flux in an asymmetric manner. Various types of motions that are spontaneous in nature may be obtained by a single-pulse oscillation coupled with mass transport.

Carboxyl-terminal Tail-mediated Homodimerizations of Sphingomyelin Synthases Are Responsible for Efficient Export from the Endoplasmic Reticulum.

Sphingomyelin synthase (SMS) is the key enzyme for cross-talk between bioactive sphingolipids and glycerolipids. In mammals, SMS consists of two isoforms: SMS1 is localized in the Golgi apparatus, whereas SMS2 is localized in both the Golgi and plasma membranes. SMS2 seems to exert cellular functions through protein-protein interactions; however, the existence and functions of quaternary structures of SMS1 and SMS2 remain unclear. Here we demonstrate that both SMS1 and SMS2 form homodimers. The SMSs have six membrane-spanning domains, and the N and C termini of both proteins face the cytosolic side of the Golgi apparatus. Chemical cross-linking and bimolecular fluorescence complementation revealed that the N- and/or C-terminal tails of the SMSs were in close proximity to those of the other SMS in the homodimer. Homodimer formation was significantly decreased by C-terminal truncations, SMS1-ΔC22 and SMS2-ΔC30, indicating that the C-terminal tails of the SMSs are primarily responsible for homodimer formation. Moreover, immunoprecipitation using deletion mutants revealed that the C-terminal tail of SMS2 mainly interacted with the C-terminal tail of its homodimer partner, whereas the C-terminal tail of SMS1 mainly interacted with a site other than the C-terminal tail of its homodimer partner. Interestingly, homodimer formation occurred in the endoplasmic reticulum (ER) membrane before trafficking to the Golgi apparatus. Reduced homodimerization caused by C-terminal truncations of SMSs significantly reduced ER-to-Golgi transport. Our findings suggest that the C-terminal tails of SMSs are involved in homodimer formation, which is required for efficient transport from the ER.

Nursing Education Trial Using a Virtual Nightingale Ward.

Nursing department students are expected to correctly grasp the entire concept of nursing through their education. The authors created a movie of a Nightingale ward (virtual ward, hereafter) with an architectural computer design software for education. The students' reaction to the virtual ward was categorized into three viewpoints: that of nurses, of patients, and of nurses and patients in common. Most of the reactions in each viewpoint were: "easy to observe patients" in the nurses' viewpoint; "no privacy" in the patients' viewpoint; and "wide room" in the common viewpoint, respectively. These reactions show the effectiveness of using a virtual ward in nursing education. Because these reactions are characteristics of a Nightingale ward, and even students, who have generally less experiences, recognized these characteristics from the both viewpoints of nurses and patients.

Insulin-like growth factor-II regulates bone sialoprotein gene transcription.

Insulin-like growth factor-I and -II (IGF-I and IGF-II) have been found in bone extracts of several different species, and IGF-II is the most abundant growth factor stored in bone. Bone sialoprotein (BSP) is a noncollagenous extracellular matrix glycoprotein associated with mineralized connective tissues. In this study, we have investigated the regulation of BSP transcription by IGF-II in rat osteoblast-like ROS17/2.8 cells. IGF-II (50 ng/ml) increased BSP mRNA and protein levels after 6-h stimulation, and enhanced luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Effects of IGF-II were inhibited by tyrosine kinase, extracellular signal-regulated kinase1/2 and phosphatidylinositol 3-kinase inhibitors, and abrogated by 2-bp mutations in cAMP response element (CRE), FGF2 response element (FRE) and homeodomain protein-binding site (HOX). The results of gel shift assays showed that nuclear proteins binding to CRE, FRE and HOX sites were increased by IGF-II (50 ng/ml) at 3 and 6 h. CREB1, phospho-CREB1, c-Fos and c-Jun antibodies disrupted the formation of the CRE-protein complexes. Dlx5 and Runx2 antibodies disrupted the FRE- and HOX-protein complex formations. These studies therefore demonstrated that IGF-II increased BSP transcription by targeting CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB1, c-Fos, c-Jun, Dlx5 and Runx2 transcription factors appear to be key regulators of IGF-II effects on BSP transcription.

Correlation Between Body Movements and Salivary Secretion During Sedation.

During dental sedation, control of the cough reflex is crucial for a safe and smooth procedure. Accumulated saliva is one of the predisposing factors for coughing. Body movements during dental sedation appear to enhance salivation. Therefore, the aim of this study was to investigate the difference in salivary secretion between the with-movements state and the without-movements state during sedation. Salivary weight for 1 min was measured 3 times in 27 patients with intellectual disability during dental treatment under deep sedation with midazolam and propofol. The observed variables were body movements, bispectral index (BIS), and predicted propofol effect-site concentration. A total of 81 measurements were classified into the with-movements state (n = 39; ie, measurements during which body movements were observed) or the without-movements state (n = 42; ie, measurements during which no body movements were observed). The median salivary weight was significantly smaller in the without-movements state compared with the with-movements state (0.03 vs 0.11 g, P < .0001). The BIS was significantly lower in the without-movements state. There was no significant difference in the predicted propofol effect-site concentration between the 2 states. Significant correlation was observed between salivary weight and BIS in the with-movements state (r = 0.44, P = .004). The findings indicate that salivary secretion decreased according to deep sedation. Furthermore, immobility also reduced salivary secretion. We concluded that one reason that immobility is beneficial is because of the resulting decreased salivary secretion during dental treatment under deep sedation.

Sphingomyelin synthase 2, but not sphingomyelin synthase 1, is involved in HIV-1 envelope-mediated membrane fusion.

Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was ∼5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor·co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.

Postoperative intravenous patient-controlled analgesia improves pain management after subcutaneous implantable defibrillator implantation.

Objective: Postoperative pain is a major issue with subcutaneous implantable cardioverter defibrillators (S-ICD). In 2020, we introduced intravenous patient-controlled analgesia (IV-PCA) in addition to the conventional, request-based analgesia for postoperative pain control in S-ICD. To determine the effect and safety, we quantitatively assessed the effect of IV-PCA after S-ICD surgery over conventional methods. Methods: During the study period, a total of 113 consecutive patients (age, 50.1 ± 15.5 years: males, 101) underwent a de novo S-ICD implantation under general anesthesia. While the postoperative pain was addressed with either request-based analgesia (by nonsteroid anti-inflammatory drugs, N = 68, dubbed as "PCA absent") or fentanyl-based IV-PCA in addition to the standard care (N = 45, dubbed as "PCA present"). The degree of postoperative pain from immediately after surgery to 1 week were retrospectively investigated by the numerical rating scale (NRS) divided into four groups at rest and during activity (0: no pain, 1-3: mild pain, 4-6: moderate pain, 7-10: severe pain). Results: Although IV-PCA was removed on Day 1, it was associated with continued better pain control compared to PCA absent group. At rest, the proportion of patients expressing pain (mild or more) was significantly lower in the PCA present group from Day 0 to Day 4. In contrast to at rest, a better pain control continued through the entire study period of 7 days. No serious adverse events were observed. A few patients experienced nausea in both groups and the inter-group difference was not found significant. Conclusion: IV-PCA suppresses postoperative pain in S-ICD without major safety concerns.

The actions and metabolism of lysophosphatidylinositol, an endogenous agonist for GPR55.

Lysophosphatidylinositol (LPI) is a subspecies of lysophospholipid and is assumed to be not only a degradation product of phosphatidylinositol (PI), but also a bioactive lysophospholipid mediator. However, not much attention has been directed toward LPI compared to lysophosphatidic acid (LPA), since the receptor for LPI has not been identified. During screening for an agonist for the orphan G protein coupled receptor GPR55, we identified LPI, 2-arachidonoyl LPI in particular, as an agonist for GPR55. Our efforts to identify an LPI receptor facilitated research on LPI as a lipid messenger. In addition, we also found that DDHD1, previously identified as phosphatidic acid-preferring phospholipase A1, was one of the synthesizing enzymes of 2-arachidonoyl LPI. Here, we summarized the background for discovering the LPI receptor, and the actions/metabolism of LPI. We also referred to the biosynthesis of PI, a 1-stearoyl-2-arachidonoyl species, since the molecule is the precursor of 2-arachidonoyl LPI. Furthermore, we discussed physiological and/or pathophysiological processes involving LPI and GPR55, including the relevance of LPI-GPR55 and cannabinoids, since GPR55 was previously postulated to be another cannabinoid receptor. Although there is no doubt that GPR55 is the LPI receptor, we should re-consider whether or not GPR55 is in fact another cannabinoid receptor.

STP4: spatio temporal path planning based on pedestrian trajectory prediction in dense crowds.

This article proposes a means of autonomous mobile robot navigation in dense crowds based on predicting pedestrians' future trajectories. The method includes a pedestrian trajectory prediction for a running mobile robot and spatiotemporal path planning for when the path crosses with pedestrians. The predicted trajectories are converted into a time series of cost maps, and the robot achieves smooth navigation without dodging to the right or left in crowds; the path planner does not require a long-term prediction. The results of an evaluation implementing this method in a real robot in a science museum show that the trajectory prediction works. Moreover, the proposed planning's arrival times is 26.4% faster than conventional 2D path planning's arrival time in a simulation of navigation in a crowd of 50 people.

Aggregatibacter actinomycetemcomitans lipopolysaccharide regulates bone sialoprotein gene transcription.

Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is believed to be associated with aggressive periodontitis characterized by a rapid bone loss. A. actinomycetemcomitans lipopolysaccharide (LPS) has a similar structure to Escherichia coli LPS, and they are Toll-like receptor 4 agonists. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of A. actinomycetemcomitans LPS on bone formation, we targeted BSP as a marker for osteogenic differentiation and bone formation. BSP mRNA levels were decreased by 0.1 µg/ml and increased by 0.01 µg/ml A. actinomycetemcomitans LPS at 6 h in osteoblast-like ROS17/2.8 cells. In transient transfection analyses, 0.1 µg/ml decreased and 0.01 µg/ml A. actinomycetemcomitans LPS increased luciferase activities of the construct (-116 to +60). Introduction of 2 bp mutations to the constructs showed that the effects of A. actinomycetemcomitans LPS were mediated by a cAMP response element (CRE), a FGF2 response element (FRE), and a homeodomain protein-binding site (HOX). Tyrosine kinase, ERK1/2, and PI3-kinase/Akt participated in the effects of both 0.1 and 0.01 µg/ml A. actinomycetemcomitans LPS. The results of gel shift showed that 0.1 µg/ml decreased while 0.01 µg/ml A. actinomycetemcomitans LPS increased CRE-, FRE-, and HOX-binding protein complexes formation at 6 h, and revealed that 0.01 µg/ml A. actinomycetemcomitans LPS induced BSP transcription through CREB1, JunD, Fra2, c-Fos, Runx2, Dlx5, and Smad1 targeting those response elements. These studies therefore indicated that 0.1 µg/ml suppressed and 0.01 µg/ml A. actinomycetemcomitans LPS increased BSP gene transcription mediated through CRE, FRE, and HOX elements in the rat BSP gene promoter.

Further evidence for involvement of a noncanonical function of uracil DNA glycosylase in class switch recombination.

Activation-induced cytidine deaminase (AID) introduces DNA cleavage in the Ig gene locus to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. The DNA deamination model assumes that AID deaminates cytidine (C) on DNA and generates uridine (U), resulting in DNA cleavage after removal of U by uracil DNA glycosylase (UNG). Although UNG deficiency reduces CSR efficiency to one tenth, we reported that catalytically inactive mutants of UNG were fully proficient in CSR and that several mutants at noncatalytic sites lost CSR activity, indicating that enzymatic activity of UNG is not required for CSR. In this report we show that CSR activity by many UNG mutants critically depends on its N-terminal domain, irrespective of their enzymatic activities. Dissociation of the catalytic and CSR activity was also found in another UNG family member, SMUG1, and its mutants. We also show that Ugi, a specific peptide inhibitor of UNG, inhibits CSR without reducing DNA cleavage of the S (switch) region, confirming dispensability of UNG in DNA cleavage in CSR. It is therefore likely that UNG is involved in a repair step after DNA cleavage in CSR. Furthermore, requirement of the N terminus but not enzymatic activity of UNG mutants for CSR indicates that the UNG protein structure is critical. The present findings support our earlier proposal that CSR depends on a noncanonical function of the UNG protein (e.g., as a scaffold for repair enzymes) that might be required for the recombination reaction after DNA cleavage.

Phosphorylation and subcellular localization of human phospholipase A1, DDHD1/PA-PLA1.

Protein phosphorylation is the most common post-translational modification of proteins and functions as a molecular switch for their regulation. This modification is reversibly regulated by protein kinases and phosphatases. In most cases, the phosphorylation of enzymes positively or negatively regulates enzyme activity. However, we found that the phosphorylation of DDHD1 phospholipase A1 (PLA1) did not affect PLA1 activity. Integrated analyses, including phospho-proteomics, Phos-tag SDS-PAGE, PLA1 enzyme assays, and immunofluorescent microscopy, revealed the subcellular localization of DDHD1 without greatly affecting its PLA1 activity. Our findings may contribute to understanding rare clinical cases that concern the implications of protein phosphorylation.