Inhibitory Effects of Shikonin Dispersion, an Extract of Lithospermum erythrorhizon Encapsulated in ß-1,3-1,6 Glucan, on Streptococcus mutans and Non-Mutans Streptococci.

Shikonin is extracted from the roots of Lithospermum erythrorhizon, and shikonin extracts have been shown to have inhibitory effects on several bacteria. However, shikonin extracts are difficult to formulate because of their poor water solubility. In the present study, we prepared a shikonin dispersion, which was solubilized by the inclusion of ß-1,3-1,6 glucan, and analysed the inhibitory effects of this dispersion on Streptococcus mutans and non-mutans streptococci. The shikonin dispersion showed pronounced anti-S. mutans activity, and inhibited growth of and biofilm formation by this bacterium. The shikonin dispersion also showed antimicrobial and antiproliferative effects against non-mutans streptococci. In addition, a clinical trial was conducted in which 20 subjects were asked to brush their teeth for 1 week using either shikonin dispersion-containing or non-containing toothpaste, respectively. The shikonin-containing toothpaste decreased the number of S. mutans in the oral cavity, while no such effect was observed after the use of the shikonin-free toothpaste. These results suggest that shikonin dispersion has an inhibitory effect on S. mutans and non-mutans streptococci, and toothpaste containing shikonin dispersion may be effective in preventing dental caries.

Comparison of Evolutionary Relationships between Branchiostoma floridae, Ciona intestinalis, and Homo sapiens Globins Provide Evidence of Gene Co-Option and Convergent Evolution.

Globins have been studied as model proteins to elucidate the principles of protein evolution. This was achieved by understanding the relationship between amino acid sequence, three-dimensional structure, physicochemical properties, and physiological function. Previous molecular phylogenies of chordate globin genes revealed the monophyletic evolution of urochordate globins and suggested convergent evolution. However, to provide evidence of convergent evolution, it is necessary to determine the physicochemical and functional similarities between vertebrates and urochordate globins. In this study, we determined the expression patterns of Ciona globin genes using real-time RT-PCR. Two genes (Gb-1 and Gb-2) were predominantly expressed in the branchial sac, heart, and hemocytes and were induced under hypoxia. Combined with the sequence analysis, our findings suggest that Gb-1/-2 correspond to vertebrate hemoglobin-α/-ß. However, we did not find a robust similarity between Gb-3, Gb-4, and vertebrate globins. These results suggested that, even though Ciona globins obtained their unique functions differently from vertebrate globins, the two of them shared some physicochemical features and physiological functions. Our findings offer a good example for understanding the molecular mechanisms underlying gene co-option and convergence, which could lead to evolutionary innovations.

Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.

Inhibitory Effect of Adsorption of Streptococcus mutans onto Scallop-Derived Hydroxyapatite.

Hydroxyapatite adsorbs various substances, but little is known about the effects on oral bacteria of adsorption onto hydroxyapatite derived from scallop shells. In the present study, we analyzed the effects of adsorption of Streptococcus mutans onto scallop-derived hydroxyapatite. When scallop-derived hydroxyapatite was mixed with S. mutans, a high proportion of the bacterial cells adsorbed onto the hydroxyapatite in a time-dependent manner. An RNA sequencing analysis of S. mutans adsorbed onto hydroxyapatite showed that the upregulation of genes resulted in abnormalities in pathways involved in glycogen and histidine metabolism and biosynthesis compared with cells in the absence of hydroxyapatite. S. mutans adsorbed onto hydroxyapatite was not killed, but the growth of the bacteria was inhibited. Electron microscopy showed morphological changes in S. mutans cells adsorbed onto hydroxyapatite. Our results suggest that hydroxyapatite derived from scallop shells showed a high adsorption ability for S. mutans. This hydroxyapatite also caused changes in gene expression related to the metabolic and biosynthetic processes, including the glycogen and histidine of S. mutans, which may result in a morphological change in the surface layer and the inhibition of the growth of the bacteria.

Thixotropic ß-Chitin Nanofiber Hydrogel with a Living Body-Responsive Self-Hardening Property and Its Application as a Sprayable Antiadhesion Barrier.

Considering recent advances in surgical techniques, sprayable antiadhesion barriers that are compatible with minimally invasive procedures are needed. However, the relatively low mechanical stiffness of the current thixotropic reversible sol-to-gel transition hydrogels has hindered their medical application. Herein, we show a thixotropic sprayable ß-chitin nanofiber hydrogel that spontaneously lost the thixotropic property in response to the environments within the living body. Furthermore, interactions between hydrogels and the biological environment result in a significant increase in mechanical stiffness. Due to these advantageous properties, ß-chitin nanofiber hydrogels administered by spray prevent postoperative abdominal adhesions and are thus promising sprayable antiadhesion barriers.

The iron chelator deferriferrichrysin induces paraptosis via extracellular signal-related kinase activation in cancer cells.

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.

Morphological Evidence for Novel Roles of Microtubules in Macrophage Phagocytosis.

Although the phagocytic activity of macrophages has long been studied, the involvement of microtubules in the process is not well understood. In this study, we improved the fixation protocol and revealed a dynamically rearranging microtubule network in macrophages, consisting of a basal meshwork, thick bundles at the cell edge, and astral microtubules. Some astral microtubules extended beneath the cell cortex and continued to form bundles at the cell edge. These microtubule assemblies were mutually exclusive of actin accumulation during membrane ruffling. Although the stabilization of microtubules with paclitaxel did not affect the resting stage of the macrophages, it reduced the phagocytic activity and membrane ruffling of macrophages activated with serum-MAF, which induced rapid phagocytosis. In contrast, the destabilization of microtubules with nocodazole enhanced membrane ruffling and the internalization of phagocytic targets suggesting an inhibitory effect of the microtubule network on the remodeling of the actin network. Meanwhile, the microtubule network was necessary for phagosome maturation. Our detailed analyses of cytoskeletal filaments suggest a phagocytosis control system involving Ca2+ influx, the destabilization of microtubules, and activation of actin network remodeling, followed by the translocation and acidification of phagosomes on the microtubule bundles.

Actin Filament in the First Cell Cycle Contributes to the Determination of the Anteroposterior Axis in Ascidian Development.

In many animal species, the body axis is determined by the relocalization of maternal determinants, organelles, or unique cell populations in a cytoskeleton-dependent manner. In the ascidian first cell cycle, the myoplasm, including mitochondria, endoplasmic reticulum (ER), and maternal mRNAs, move to the future posterior side concomitantly (called ooplasmic segregation or cytoplasmic and cortical reorganization). This translocation consists of first and second phases depending on the actin and microtubule, respectively. However, the transition from first to second phase, that is, translocation of myoplasmic components from microfilaments to microtubules, has been poorly investigated. In this study, we analyzed the relationship between these cytoskeletons and myoplasmic components during the first cell cycle and their role in morphogenesis by inhibitor experiments. Owing to our improved visualization techniques, there was unexpected F-actin accumulation at the vegetal pole during this transition period. When this F-actin was depolymerized, the microtubule structure was strongly affected, the myoplasmic components, including maternal mRNA, were mislocalized, and the anteroposterior axis formation was disordered. These results suggested the importance of F-actin during the first cell cycle and the existence of interactions between microfilaments and microtubules, implying the enigmatic mechanism of ooplasmic segregation. Solving this mystery leads us to an improved understanding of ascidian early development.

Dynamic changes in the association between maternal mRNAs and endoplasmic reticulum during ascidian early embryogenesis.

Axis formation is one of the most important events occurring at the beginning of animal development. In the ascidian egg, the antero-posterior axis is established at this time owing to a dynamic cytoplasmic movement called cytoplasmic and cortical reorganisation. During this movement, mitochondria, endoplasmic reticulum (ER), and maternal mRNAs (postplasmic/PEM RNAs) are translocated to the future posterior side. Although accumulating evidence indicates the crucial roles played by the asymmetrical localisation of these organelles and the translational regulation of postplasmic/PEM RNAs, the organisation of ER has not been described in sufficient detail to date owing to technical difficulties. In this study, we developed three different multiple staining protocols for visualising the ER in combination with mitochondria, microtubules, or mRNAs in whole-mount specimens. We defined the internally expanded "dense ER" using these protocols and described cisterna-like structures of the dense ER using focused ion beam-scanning electron microscopy. Most importantly, we described the dynamic changes in the colocalisation of postplasmic/PEM mRNAs and dense ER; for example, macho-1 mRNA was detached and excluded from the dense ER during the second phase of ooplasmic movements. These detailed descriptions of the association between maternal mRNA and ER can provide clues for understanding the translational regulation mechanisms underlying axis determination during ascidian early embryogenesis.

Intramolecular G-quadruplex-hairpin loop structure competition of a GC-rich exon region in the TMPRSS2 gene.

We identified cytosine-rich regions adjacent to guanine-rich regions in protease genes. A typical GC-rich sequence derived from the TMPRSS2 gene showed structural competition between a G-quadruplex and a hairpin loop, and this competition significantly affected transcription efficiency. These results suggest an impact of neighboring sequences on the gene expression of guanine-rich sequences.

Involvement of Annexin A2 in Serum-MAF Dependent Phagocytic Activation of Macrophages.

BACKGROUND/AIM: Serum-derived macrophage activating factor (serum-MAF) is expected to have adjuvant effects through rapid phagocytic activation, which depends on F-actin accumulation in multi-layered membrane ruffles induced within 5 min after serum-MAF addition. This study aimed to elucidate the importance of annexin A2, which is a multifunctional Ca2+-binding protein related to cytoskeletal membrane dynamics, in serum-MAF signalling. MATERIALS AND METHODS: Annexin A2 and F-actin localizations were analyzed via immunostaining and confocal microscopy. Using EGTA as chelator, the role of Ca2+ in serum-MAF signalling was examined. RESULTS: Annexin A2 was found to translocate from the cytosol to the cell cortex within 30 s of serum-MAF stimulation. Ca2+ chelation inhibited the translocation of annexin A2, frill-like structure formation, and phagocytic activation by serum-MAF. CONCLUSION: Annexin A2 and Ca2+ were responsible for the rapid phagocytic activation by serum-MAF. This study provides an understanding of phagocytic activation in macrophages, which could be beneficial for cancer immunotherapy.

Dermal Fibroblasts Internalize Phosphatidylserine-Exposed Secretory Melanosome Clusters and Apoptotic Melanocytes.

Pigmentation in the dermis is known to be caused by melanophages, defined as melanosome-laden macrophages. In this study, we show that dermal fibroblasts also have an ability to uptake melanosomes and apoptotic melanocytes. We have previously demonstrated that normal human melanocytes constantly secrete melanosome clusters from various sites of their dendrites. After adding secreted melanosome clusters collected from the culture medium of melanocytes, time-lapse imaging showed that fibroblasts actively attached to the secreted melanosome clusters and incorporated them. Annexin V staining revealed that phosphatidylserine (PtdSer), which is known as an 'eat-me' signal that triggers the internalization of apoptotic cells by macrophages, is exposed on the surface of secreted melanosome clusters. Dermal fibroblasts were able to uptake secreted melanosome clusters as did macrophages, and those fibroblasts express TIM4, a receptor for PtdSer-mediated endocytosis. Further, co-cultures of fibroblasts and melanocytes demonstrated that dermal fibroblasts internalize PtdSer-exposed apoptotic melanocytes. These results suggest that not only macrophages, but also dermal fibroblasts contribute to the collection of potentially toxic substances in the dermis, such as secreted melanosome clusters and apoptotic melanocytes, that have been occasionally observed to drop down into the dermis from the epidermis.

Comparison of Macrophage Activation Using γ-Globulin and Serum-derived Macrophage Activating Factor.

BACKGROUND/AIM: Serum-derived macrophage activating factor (serum-MAF) can rapidly activate macrophage phagocytic activity by inducing characteristic membrane ruffles designated as Frill-like structures. Serum-MAF contains γ-globulin, an activator of phagocytosis. This study examined whether serum-MAF and γ-globulin activate macrophages similarly. MATERIALS AND METHODS: Morphological changes in macrophages were observed by time-lapse imaging and the efficiency of engulfment was analysed quantitatively. Immunological staining of talin-1 and a calpain inhibitor were performed. RESULTS: The engulfment efficiency of serum-MAF- and γ-globulin-activated macrophages was significantly different. Talin-1 showed weak co-localisation with the Frill-like structures. Treatment with a calpain inhibitor similarly down-regulated phagocytosis irrespective of the activation factor. CONCLUSION: There was a difference between macrophage activation mechanisms by γ-globulin and serum-MAF. Talin may slightly contribute to serum-MAF activation. It is possible to distinguish between the calpain-dependent fundamental 'mechanism of phagocytosis' and the activating factor-dependent rapid 'activation mechanism'.

DMPK is a New Candidate Mediator of Tumor Suppressor p53-Dependent Cell Death.

Tumor suppressor p53 plays an integral role in DNA-damage induced apoptosis, a biological process that protects against tumor progression. Cell shape dramatically changes when cells undergo apoptosis, which is associated with actomyosin contraction; however, it remains entirely elusive how p53 regulates actomyosin contraction in response to DNA-damaging agents. To identify a novel p53 regulating gene encoding the modulator of myosin, we conducted DNA microarray analysis. We found that, in response to DNA-damaging agent doxorubicin, expression of myotonic dystrophy protein kinase (DMPK), which is known to upregulate actomyosin contraction, was increased in a p53-dependent manner. The promoter region of DMPK gene contained potential p53-binding sequences and its promoter activity was increased by overexpression of the p53 family protein p73, but, unexpectedly, not of p53. Furthermore, we found that doxorubicin treatment induced p73 expression, which was significantly attenuated by downregulation of p53. These data suggest that p53 induces expression of DMPK through upregulating p73 expression. Overexpression of DMPK promotes contraction of the actomyosin cortex, which leads to formation of membrane blebs, loss of cell adhesion, and concomitant caspase activation. Taken together, our results suggest the existence of p53-p73-DMPK axis which mediates DNA-damage induced actomyosin contraction at the cortex and concomitant cell death.

Characteristic Morphological Changes and Rapid Actin Accumulation in Serum-MAF-treated Macrophages.

BACKGROUND/AIM: Serum-derived macrophage activating factor, serum-MAF, is known to increase the phagocytic activity of macrophages by enhancing the engulfment efficiency. To elucidate the mechanisms underlying phagocytic activation, morphological changes were observed and analyzed. MATERIALS AND METHODS: Morphological changes in macrophages were observed and quantitatively analyzed using scanning electron microscope (SEM) and confocal microscope. RESULTS: SEM and confocal microscopy images revealed frill-like structures and active actin accumulations, respectively, in serum-MAF treated macrophages. Actin accumulation was induced within 5 min following serum-MAF treatment. CONCLUSION: Serum-MAF induced a rapid rearrangement of cytoskeletal actin and enhanced phagocytic activity. Findings of the current study may contribute to the development of techniques that facilitate activation of the human immune system, which in turn may be beneficial for cancer immunotherapy.

Non-centrosomal microtubule structures regulated by egg activation signaling contribute to cytoplasmic and cortical reorganization in the ascidian egg.

In the first ascidian cell cycle, cytoplasmic and cortical reorganization is required for distributing maternal factors to their appropriate positions, resulting in the formation of the embryonic axis. This cytoplasmic reorganization is considered to depend on the cortical microfilament network in the first phase and on the sperm astral microtubule (MT) in the second phase. Recently, we described three novel MT structures: a deeply extended MT meshwork (DEM) in the entire subcortical region of the unfertilized egg, transiently accumulated MT fragments (TAF) in the vegetal pole, and a cortical MT array in the posterior vegetal cortex (CAMP). Particularly, our previous study showed CAMP to contribute to the movement of myoplasm. In addition, it is very similar to the parallel MT array, which appears during cortical rotation in Xenopus eggs. However, how these MT structures are organized is still unclear. Here, we investigated the relationship between the egg activation pathway and MT structures during the first ascidian cell cycle. First, we carefully analyzed cell cycle progression through meiosis I and II and the first mitosis, and successfully established a standard time table of cell cycle events. Using this time table as a reference, we precisely described the behavior of novel MT structures and revealed that it was closely correlated with cell cycle events. Moreover, pharmacological experiments supported the relationship between these MT structures and the signal transduction mechanisms that begin after fertilization, including Ca2+ signaling, MPF signaling, and MEK/MAPK signaling. Especially, CAMP formation was directed by activities of cyclin-dependent kinases. As these MT structures are conserved, at least, within chordate group, we emphasize the importance of understanding the controlling mechanisms of MT dynamics, which is important for embryonic axis determination in the ascidian egg.

Massive cytoplasmic transport and microtubule organization in fertilized chordate eggs.

Eggs have developed their own strategies for early development. Amphibian, teleost fish, and ascidian eggs show cortical rotation and an accompanying structure, a cortical parallel microtubule (MT) array, during the one-cell embryonic stage. Cortical rotation is thought to relocate maternal deposits to a certain compartment of the egg and to polarize the embryo. The common features and differences among chordate eggs as well as localized maternal proteins and mRNAs that are related to the organization of MT structures are described in this review. Furthermore, recent studies report progress in elucidating the molecular nature and functions of the noncentrosomal MT organizing center (ncMTOC). The parallel array of MT bundles is presumably organized by ncMTOCs; therefore, the mechanism of ncMTOC control is likely inevitable for these species. Thus, the molecules related to the ncMTOC provide clues for understanding the mechanisms of early developmental systems, which ultimately determine the embryonic axis.

Phagocytic Activation of Macrophages with Serum MAF Depends on Engulfment Efficiency and Not Migratory Activity.

BACKGROUND/AIM: Serum-derived macrophage activating factor (serum MAF) is known to increase the phagocytic activity of macrophages and potentially plays a role in activating cancer immunity. In order to reveal the contributing factors for phagocytic activation, the migratory activity and the efficiency of engulfment was analyzed. MATERIALS AND METHODS: THP-1 macrophages were induced by 12-O-tetradecanoyl-13-acetate (TPA). The migratory activity and efficiency of engulfment were analyzed by time-lapse imaging and suspension assay, respectively. RESULTS: While the distance of migration did not change before and after activation with serum MAF, the efficiency of beads internalisation was significantly increased. CONCLUSION: Phagocytic activation of serum-MAF-treated macrophages was caused by increasing the efficiency of engulfment. This study contributes to the knowledge about the activation of the immune system through phagocytic activation of macrophages.

Nanocomposite injectable gels capable of self-replenishing regenerative extracellular microenvironments for in vivo tissue engineering.

Injectable hydrogels are biomaterials that have the potential to provide scaffolds to cells for in situ tissue regeneration with a minimally invasive implantation procedure. The success of in vivo tissue engineering utilizing injectable gels depends on providing cells with appropriate scaffolds that present an instructive extracellular microenvironment, which strongly influences the survival, proliferation, organization, and function of cells encapsulated within gels. One of the most important abilities of injectable gels to achieve this function is to adsorb and retain a wide variety of requisite bioactive molecules including nutrients, extracellular matrices, and growth/differentiation factors within gels. Previously, we developed nanocomposite injectable gels fabricated by simple combination of common biodegradable copolymers, poly(lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PLGA-PEG-PLGA), and synthetic clay nanoparticles (LAPONITE®). We revealed that the nanocomposite injectable gels strongly adsorb ECM molecules including collagen and heparin within gels and retain them due to the ability of LAPONITE® in synchronization with the degradation of PLGA-PEG-PLGA and subsequent release of the degradation products. Human dermal fibroblast cells cultured on the nanocomposite gels showed enough high cell viability and proliferation for at least a week. Moreover, various kinds of human cells encapsulated within the nanocomposite gels exhibited significantly higher survival, proliferation, and three-dimensional organization in comparison with the PLGA-PEG-PLGA gel, LAPONITE® gel, and Matrigel. Furthermore, transplantation of mouse myoblast cells with the nanocomposite gels in model mice of skeletal muscle injury dramatically enhanced tissue regeneration and functional recovery, whereas cell transplantation with the PLGA-PEG-PLGA gel did not. Thus, the nanocomposite injectable gels possess unique abilities to self-replenish the regenerative extracellular microenvironment within the gels in the body, demonstrating the potential utility of the nanocomposite injectable gels for in vivo tissue engineering.