Ionomycin ameliorates hypophosphatasia via rescuing alkaline phosphatase deficiency-mediated L-type Ca^(2+) channel internalization in mesenchymal stem cells

The loss-of-function mutations in the ALPL result in hypophosphatasia(HPP), an inborn metabolic disorder that causes skeletal mineralization defects. In adults, the main clinical features are early loss of primary or secondary teeth, osteoporosis, bone pain,chondrocalcinosis, and fractures. However, guidelines for the treatment of adults with HPP are not available. Here, we show that ALPL deficiency caused a reduction in intracellular Ca2+ influx, resulting in an osteoporotic phenotype due to downregulated osteogenic differentiation and upregulated adipogenic differentiation in both human and mouse bone marrow mesenchymal stem cells(BMSCs). Increasing the intracellular level of calcium in BMSCs by ionomycin treatment rescued the osteoporotic phenotype in alpl+/- mice and BMSC-specific(Prrx1-alpl-/-) conditional alpl knockout mice. Mechanistically, ALPL was found to be required for the maintenance of intracellular Ca2+ influx, which it achieves by regulating L-type Ca2+ channel trafficking via binding to the α2δsubunits to regulate the internalization of the L-type Ca2+ channel. Decreased Ca2+ flux inactivates the Akt/GSK3β/β-catenin signaling pathway, which regulates lineage differentiation of BMSCs. This study identifies a previously unknown role of the ectoenzyme ALPL in the maintenance of calcium channel trafficking to regulate stem cell lineage differentiation and bone homeostasis. Accelerating Ca2+ flux through L-type Ca2+ channels by ionomycin treatment may be a promising therapeutic approach for adult patients with HPP.

EXTERNALIZATION AND INTERNALIZATION OF CARDIAC ENDOTHELIN RECEPTORS DURING DIFFERENT PHASES OF SEPSIS IN RAT

To study the redistribution of endothelin- 1 (ET- 1) receptors in two subcellular organelles , the sarcolemmal membrane and the light vesicle, of rat heart during the progression of sepsis. Methods. Sepsis was induced by cecal ligation and puncture (CLP). ET1 receptor was assayed by using [125I]- ET1 binding. Marker enzyme activities, protein yield, and dry- to- wet weight ratio of cardiac membranes were measured. Results. Septic rat heart exhibited two distinct phases: an initial hyperdynamic phase( 9h after CLP; early stage of sepsis) followed by a hypodynamic (18h after CLP, late stage of sepsis) phase. [125I]- ET1 binding study showed that during early stage of sepsis, the Bmax of ET1 receptors was increased by 30％ in sarcolemma but decreased by 19％ in light vesicles, while during late stage of sepsis, the Bmax was decreased by 24％ in sarcolemma but increased by 38％ in light vesicles.The total binding of sarcolemma and light vesicles was increased by 25％ during early stage of sepsis but decreased by 17％ during late stage of sepsis. Conclusions. These data indicated that ET1 receptors in the rat heart were externalized from light vesicles to sarcolemmal membranes during early hyperdynamic phase while internalized from surface membranes to intracellular compartment during late hypodynamic phase of sepsis.

Intracellular distribution and internalization pathways of guanidinylated bioresponsive poly(amido amine)s in gene delivery

Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, weresynthesized by Michael-type addition reaction between guanidine hydrochloride(CAR) orchlorhexidine(CHL) and N,N-cystaminebisacrylamide(CBA). Previous studies have shownthat both polymers had high transfection efficiencies as gene delivery carriers. In this study,we investigated the nucleolus localization abilities and cellular internalization pathways ofthese two polymers in gene delivery. Each polymer condensed plasmid DNA(p DNA) andformed nanoparticle complexes, and then their transfection studies were performed inMCF-7 cells. Both complexes were found enriched in nucleolus after cellular transfection,and their transfection efficiencies were significantly improved when transfection was per-formed on MCF-7 cells arrested at M phase. The transfection efficiency of CAR-CBA-pDNAwas inhibited by chlorpromazine, and cell endosomes were disrupted after being exposedto CAR-CBA-pDNA. In regards to CHL-CBA-pDNA, its transfection efficiency was not affected by three types of endocytosis inhibitors used in the study, and CHL-CBA-pDNA showed no effect on endosomes. Cellular lactate dehydrogenase release and membrane morphology were changed after cells were transfected by the two complexes. The results indicated that both CAR-CBA and CHL-CBA polymers demonstrated good nucleolus localization abilities. It was beneficial for transfection when cells were arrested at M phase. CAR-CBA-pDNA cellular internalization was involved with clathrin-mediated endocytosis pathway, and escaping from endosomal entrapment, while the cellular uptake of CHL-CBA-pDNA occurs via clathrin-and caveolae-independent mechanism.

Follicle-stimulating Hormone(FSH) Induced Internalization of Porcine FSH Receptor in Cultured Porcine Granulosa Cells and Chinese Hamster Ovary Cells Transfected with Recombinant Porcine FSH Receptor cDNA

In order to study the fate of human follicle-stimulating hormone (FSH) when hormone binds to its receptor, a quick biochemical method that can differentiate between the surface-bound and internalized hormone was used to determine the internalization induced by FSH in cultured both porcine granulosa cells and Chinese hamster ovary (CHO) cells expressing recombinant porcine FSH receptor. The results showed that FSH was slowly internalized, and the internalized radioactivity (acid resistant) reached a peak 10-12 h after addition of 125 I-hFSH. It was suggested that FSHR do not get internalized rapidly under physiological circumstances precisely because the appropriate sequences are absent.

GPCR/endocytosis/ERK signaling/S2R is involved in the regulation of the internalization,mitochondria-targeting and-activating properties of human salivary histatin 1

Human salivary histatin 1(Hst1)exhibits a series of cell-activating properties,such as promoting cell spreading,migration,and metabolic activity.We recently have shown that fluorescently labeled Hst1(F-Hst1)targets and activates mitochondria,presenting an important molecular mechanism.However,its regulating signaling pathways remain to be elucidated.We investigated the influence of specific inhibitors of G protein-coupled receptors(GPCR),endocytosis pathways,extracellular signal-regulated kinases1/2(ERK1/2)signaling,p38 signaling,mitochondrial respiration and Na+/K+-ATPase activity on the uptake,mitochondria-targeting and-activating properties of F-Hst1.We performed a si RNA knockdown(KD)to assess the effect of Sigma-2 receptor(S2R)/Transmembrane Protein 97(TMEM97)—a recently identified target protein of Hst1.We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1.Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1.The inhibitors of GPCR,ERK1/2,phagocytosis,and clathrin-mediated endocytosis(CME)as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake,which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity.Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1.We further showed the intracellular trafficking and targeting process of F-Hst1,in which early endosome plays an important role.Overall,phagocytosis,CME,GPCR,ERK signaling,and S2R/TMEM97 are involved in the internalization of Hst1,while only CME and S2R/TMEM97 are critical for its subcellular targeting.The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.

Body-focused Anxiety in Women: Associations with Internalization of the Thin-ideal, Dieting Frequency, Body Mass Index and Media Effects

Exposure to media that portrays thin women as ideal and attractive can lead to women internalizing the thin ideal, which results in incorporating societal standards of thinness into belief systems. Internalization of the thin-ideal is associated with numerous detrimental effects on women, including decreased levels of self-esteem and increased levels of body-focused anxiety, negative emotions and disordered eating. The present study utilized a sample of women (N = 208) aged between 18 and 67 years (M = 29.44, SD = 13.08) to examine the relationship between internalization of the thin- ideal, body-focused anxiety, body mass index (BMI), and dieting frequency. Correlational, regression and mediation analyses conducted on the data showed that internalization of the thin-ideal, BMI and dieting frequency significantly contributed to body-focused anxiety in women. In addition, body-focused anxiety fully mediated the relationship between internalization of the thin-ideal and dieting frequency among women. BMI did not moderate the relationship between internalization of the thin-ideal and body-focused, indicating that women who internalize the thin-ideal are less vulnerable to dieting unless experiencing body-focused anxiety. The results of the current study enhance our understanding of the relationship between internalization of the thin-ideal, body-focused anxiety, BMI, and dieting frequency among women. Clinical implications will be discussed.

＂Half-a-Day＂ Syndrome： A Mode of Internalization of Vocational and Technical Education by the Youths in the 20th Century Nigeria

This research focused on ＂half-a-day＂ as a mode of internalizing vocational and technical education by the youths in Nigeria. It traced the origin of ＂half-a-day＂ linking it with the ingenuity of the youths to make the formal education that they have acquired operational. It also discussed the traditional education with its apprenticeship system as being modified through the ＂half-a-day＂ syndrome. The study is historical and descriptive therefore, social survey method was employed using unscheduled interview and consulting relevant literature. It was discovered that, apart from the failure of the educational system due to lack of proper infrastructure, the youths, especially those from the lower class, have reasoned that the salaries from the white-collar jobs may not be enough to sustain them. It is, therefore, recommended, among others, that： Government can institutionalise the ＂half-a-day＂ as a form of summer school and certify both the centres of learning and learners.

The Observation of FSH’s Cellular Internalization

Follicle stimulating hormone (FSH) is a kind of glycoprotein gonadotropin, and plays an important role in the diagnosis and treatment of infertility. Follicle stimulating hormone receptor (FSHR) is a kind of G protein coupled receptor (GPCR), found in the ovary and testes, and its activation is required for the hormonal operation during the breeding period. In this study, an experimental model of FSHR mediated FSH into cell membrane, which exhibited a phenomenon of fluorescent localized on cell surfaces internalized into cell interior, was established to verify biological activity of FSH.

On the necessity and countermeasures of the ＂internalization＂ construction of the China-characterized public diplomacy under the new situations

In the era of the globalization of the economy, the network of the information, and the socialization of the citizens, there is the tendency that a country＇s domestic problems will be internationalized, while the international issues will be domestic. Combined with the background of China to realize the ＂China Dreams＂, in the field of the public diplomacy, we should pay sufficient attention to the ＂internalization＂ construction in the aspects of the value ideas, the mode of the systems, the political propositions, and the cultural influence and so on, which is the benign interaction involving the governments, the citizens and the social organizations. In this paper, the author casts a brick to attract jade. Through the discussion of the necessity of the ＂internalization＂ construction of the public diplomacy, the author carries out the discussion from the social and the cultural dimensions, in order to arouse the attention to the ＂internalization＂ construction of the public diplomacy, so that our country will be more influential in the politics, and more approachable in the images, and more appealing in the morality.

Research for Competitiveness Enhancement Problem of Regional Industry Based on Brand Internalization Theory

Along with the quick development of social economy, market competition has already entered into a new stage, namely brand competition era. This paper is to put forward scientifically rational countermeasures and suggestions through taking brand internalization theory as basis and starting from regional industry competitiveness. Thus it would increase regional industry competitiveness of brand.

The role repositioning of teacher and student in the process of knowledge internalization under flipped classroom

Flipped classroom model led to the transformation of university teachers＇ teaching role. From the perspective of the sociology of knowledge, the teacher professional development is a process of transformation of the role of teachers, and role transformation and the way of actors involved in the knowledge are associated. Participating in knowledge in different ways, role types are also different. Thus, changing the way ofparticipating in knowledge is the basic way to participate in the transformation of the role. Classification of roles is not the classification ofhumans.A social may might play many roles of many types in his life andmay have the function of a variety of roles at the same time. Meanwhile, it can be transferred between characters, especially in the case of depending on the same knowledge system.As long as the ways of involvement are different,the functions and types of roles are also different. University teachers are those who play a variety of roles of knowledge and society, some being the teaching teachers of organization and dissemination of knowledge, some being scientific and teaching teachers who focus on organizing and disseminating knowledge as well as knowledge creation, and knowledge creation and heavy teacher teaching and research, while others are knowledge creation based research teachers. In the field of the sociology of knowledge, university teachers should be scientific teaching roles depending on a deep knowledge and participating in knowledge in a variety of ways. From the perspective of the sociology of knowledge, according to Eph · Znaniecki＇ s role classification, university teachers participate in knowledge in three ways： teaching, learning and researching. Teaching, learning and researching are relative but different ways of participating in knowledge. State they are relative because itshows teaching can promote learning, learning can promote researching, researching can also promote learning and teaching. The homochromous interaction of teaching, leaming and researching, knowledge is in constant state of development and generation.

CREG inhibits VSMCs proliferation by modulating the internalization of IGFII

Background To study the molecular mechanisms of CREG(the cellular repressor of E1A-stimulated gene) on proliferation of VSMCs in vitro.Methods The pRc/CMV-CREG plasmid or the pSM2-siCREG plasmid was transferred into human vascular smooth muscle cells(hVSMCs) to produce the cell clone that over-expression or down-expression of CREG respectively.BrdU assay and FACS cell cycle analysis were used to detect the proliferation of cells.Western blotting and immunocytochemistry show the expression and localization of IGF2R in hVSMCs.RT-PCR and ELISA assay determined the expression and secretion of IGFII factor. Alex488-labeled rhIGFII was used to investigate the endocysis of cells.And the blockade of IGFII internalization by treatment both the neutralized antibody of anti-IGF2R and rsIGF2R detected the effect of IGFII on VSMCs growth. Furthermore,Western blotting and signal pathway inhibitor were used to analysis the activation of PI3K/AKT and ERK on VSMCs proliferation.Results Western blotting identified that the expression of CREG in hVSMCs-CREG cells increased compared to control cell,and the decreased obviously in hVSMCs-siCREG cells.Meanwhile,the overexpression of CREG in cells was detected to inhibit the proliferation of VSMCs and to enhance the distribution of IGF2R in cellular membrane.Furthermore,overexpression of CREG also accelerated the endocysis of IGFII in hVSMCs-CREG,and attenuate the secretion of IGFII into cell medium by ELISA analysis and Alex488 labeled IGFII analysis.Blockade experiments both neutralized antibody of IGF2R and rhIGF2R fragment determined that enhancement of IGFII secretion promoted the VSMCs proliferation,and PI3K/AKT and ERK signal pathway mediated the effect of IGFII on VSMCs.Conclusions Altogether, these data indicate that CREG inhibits the proliferation of hVSMCs through interfering into the internalization pathway of IGF2R-IGFII.

Study on the Promoting Mechanism of Teachers'Ethics Internalization in Universities

On the basis of clarifying the teachers'Ethics internalization theory,this paper analyzes the typical dilemma in teachers'Ethics internalization.To resolve these problems,this paper construct an overall framework,including security mechanism,normative mechanism,education mechanism,evaluation mechanism and supervision mechanism,which the external conditions are used to constantly strengthen the moral needs of the subject,then enhance the consciousness of moral internalization,improve the effectiveness of teachers'moral construction.

Erratum to“Tumor Microenvironment-Specific Chemical Internalization for Enhanced Gene Therapy of Metastatic Breast Cancer”

In the Research Article “Tumor Microenvironment-Specific Chemical Internalization for Enhanced Gene Therapy of Metastatic Breast Cancer,” the authors made an error in Fig. 6A. In Fig. 6A, the image of “pre” in the “Free Cy5-siRNA” group was misused. When incorporating the final images in Fig. 6A, another image of “24 h” in the “Free Cy5-siRNA” group was inadvertently chosen as the “Pre” image, so they appear to be remarkably similar. After the error was discovered, the authors repeated the experiments that concluded that this error did not affect the results, discussion, or conclusion of this paper. Figure 6A has now been corrected in the PDF and HTML (full text).

Exploration and insights into the cellular internalization and intracellular fate of amphiphilic polymeric nanocarriers

The benefcial or deleterious effects of nanomedicines emerge from their complex interactions with intracellular pathways and their subcellular fate.Moreover,the dynamic nature of plasma membrane accounts for the movement of these nanocarriers within the cell towards different organelles thereby not only infuencing their pharmacokinetic and pharmacodynamic properties but also bioavailability,therapeutic effcacy and toxicity.Therefore,an in-depth understanding of underlying parameters controlling nanocarrier endocytosis and intracellular fate is essential.In order to direct nanoparticles towards specifc sub-cellular organelles the physicochemical attributes of nanocarriers can be manipulated.These include particle size,shape and surface charge/chemistry.Restricting the particle size of nanocarriers below 200 nm contributes to internalization via clathrin and caveolae mediated pathways.Similarly,a moderate negative surface potential confers endolysosomal escape and targeting towards mitochondria,endoplasmic reticulum(ER)and Golgi.This review aims to provide an insight into these physicochemical attributes of nanocarriers fabricated using amphiphilic graft copolymers affecting cellular internalization.Fundamental principles understood from experimental studies have been extrapolated to draw a general conclusion for the designing of optimized nanoparticulate drug delivery systems and enhanced intracellular uptake via specifc endocytic pathway.

HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis

Deletion of HAX1 in mice causes a severe reduction in the numbers of lymphocytes in the bone marrow and in the spleen. Additionally, B220＋ B progenitor cells in the bone marrow are reduced, suggesting an important function of HAX1 in B cell development. HAX1 is thought to play a protective role in apoptotic processes; therefore, we investigated the role of HAX1 in bone marrow B progenitor cells and splenic B cells. We did not observe an effect on the survival of Hax1-/- bone marrow cells but detected enhanced survival of splenic Hax1-/- B cells upon in vitro starvation/ growth-factor withdrawal. To explain this apparent inconsistency with previous reports of HAX1 function, we also studied the B cell receptor （BCR）-induced apoptosis of IgM-stimulated splenic naive B cells and found that apoptosis decreased in these cells. We further found impaired internalization of the BCR from Hax1-/- splenic B cells after IgM crosslinking; this impaired internalization may result in decreased BCR signaling and, consequently, decreased BCR-mediated apoptosis. We measured HAX1 binding to the cytoplasmic domains of different Ig subtypes and identified KVKWI（V）F as the putative binding motif for HAX1 within the cytoplasmic domains. Because this motif can be found in almost all Ig subtypes, it is likely that HAX1 plays a general role in BCR-mediated internalization events and BCR-mediated apoptosis.

INTERNALIZATION OF GAP JUNCTIONS BETWEEN NEURON AND GLIAL CELL AND FORMATION OF ANNULAR LAMELLAR BODIES IN CHINESE LEECH Whitmania pigra

This paper reveals the formation of annular lamellar body (ALB) in the ganglion of leech by means of in situ fixation and the lanthanum nitrate tracer technique. This formation involves both wrapping and internalization of the gap junctions between glial processes themselves, as well as between neuron and glial process. The results indicate that there is probably an active process of internalization of membrane structures involving gap junctions between neuron and glial cell in the central nervous system in leech. The functions of ALB are discussed.

Effects of the physicochemical properties of gold nanostructures on cellular internalization

Unique physicochemical properties of Au nanomaterials make them potential star materials in biomedicalapplications. However, we still know a little about the basic problem of what really mattersin fabrication of Au nanomaterials which can get into biological systems, especially cells, with highefficiency. An understanding of how the physicochemical properties of Au nanomaterials affecttheir cell internalization is of significant interest. Studies devoted to clarify the functions of variousproperties of Au nanostructures such as size, shape and kinds of surface characteristics in cell internalizationare under way. These fundamental investigations will give us a foundation for constructingAu nanomaterial-based biomedical devices in the future. In this review, we present the current advancesand rationales in study of the relationship between the physicochemical properties of Aunanomaterials and cell uptake. We also provide a perspective on the Au nanomaterial-cell interactionresearch.

pH-responsive Polymersome Based on PMCP-b-PDPA as a Drug Delivery System to Enhance Cellular Internalization and Intracellular Drug Release

Choline phosphate （CP） as a novel zwitterion possesses specific and excellent properties compared with phosphorylcholine （PC）, as well as its polymer, such as poly（2-（methacryloyloxy）ethyl choline phosphate） （PMCP）, has been studied extensively due to its unique characteristics of rapid cellular internalization via the special quadrupole interactions with the cell membrane. Recently, we reported a novel PMCP-based drug delivery system to enhance the cellular internalization where the drug was conjugated to the polymer via reversible acylhydrazone bond. Herein, to make full use of this feature of PMCP, we synthesized the diblock copolymer poly（2-（methacryloyloxy）ethyl choline phosphate）-b-poly（2- （diisopropylamino）ethyl methacrylate） （PMCP-b-PDPA）, which could self-assemble into polymersomes with hydrophilic PMCP corona and hydrophobic membrane wall in mild conditions when the pH value is 〉 6.4. It has been found that these polymersomes can be successfully used to load anticancer drug Dox with the loading content of about 11.30 wt%. After the polymersome is rapidly internalized by the cell with the aid of PMCP, the loaded drug can be burst-released in endosomes since PDPA segment is protonated at low pH environment, which renders PDPA to transfer from hydrophobic to hydrophilic, and the subsequent polymersomes collapse thoroughly. Ultimately, the ＂proton sponge＂ effect of PDPA chain can further accelerate the Dox to escape from endosome to cytoplasm to exert cytostatic effects. Meanwhile, the cell viability assays showed that the Dox-loaded polymersomes exhibited significant inhibitory effect on tumor cells, indicating its great potential as a targeted intracellular delivery system with high efficiency.