Kinectin participates in microtubule-dependent hormone secretion in pancreatic islet beta-cells

Kinectin (KNT) is a candidate membrane receptor for kinesin in the movement of intracellular organelles along microtubules. Isoforms of KNT exist containing different combinations of six small (residues 23-33) variable domains (vd) vd1-6 within the C-terminus. Here we investigate a role for KNT and its isoform KNTvd4(-) in the transport of amylin and insulin-containing secretory vesicles in the pancreatic islet beta-cell line RINm5F. KNTvd4(-) lacks vd4 that forms the kinesin-binding domain, and hence its role in the cell is an enigma. We report that amylin-containing vesicles also contained insulin, and exhibited microtubule, and small G-protein-dependent secretion. Knockdown of KNT by small interference RNA (siRNA) inhibited amylin expression and secretion. In contrast, recombinant KNTvd4(-) overexpressed in RINm5F cells associated with amylin-containing vesicles and inhibited amylin secretion, but had no discernible affect on amylin expression. The data suggests that both KNT and KNTvd4(-) participate in microtubule-dependent secretion of amylin in islet beta-cells.     

Airway smooth muscle relaxation results from a reduction in the frequency of Ca<Superscript>2+</Superscript> oscillations induced by a cAMP-mediated inhibition of the IP<Subscript>3</Subscript> receptor

Background

It has been shown that the contractile state of airway smooth muscle cells (SMCs) in response to agonists is determined by the frequency of Ca²⁺ oscillations occurring within the SMCs. Therefore, we hypothesized that the relaxation of airway SMCs induced by agents that increase cAMP results from the down-regulation or slowing of the frequency of the Ca²⁺ oscillations.

Methods

The effects of isoproterenol (ISO), forskolin (FSK) and 8-bromo-cAMP on the relaxation and Ca²⁺ signaling of airway SMCs contracted with methacholine (MCh) was investigated in murine lung slices with phase-contrast and laser scanning microscopy.

Results

All three cAMP-elevating agents simultaneously induced a reduction in the frequency of Ca²⁺ oscillations within the SMCs and the relaxation of contracted airways. The decrease in the Ca²⁺ oscillation frequency correlated with the extent of airway relaxation and was concentration-dependent. The mechanism by which cAMP reduced the frequency of the Ca²⁺ oscillations was investigated. Elevated cAMP did not affect the re-filling rate of the internal Ca²⁺ stores after emptying by repetitive exposure to 20 mM caffeine. Neither did elevated cAMP limit the Ca²⁺ available to stimulate contraction because an elevation of intracellular Ca²⁺ concentration induced by exposure to a Ca²⁺ ionophore (ionomycin) or by photolysis of caged-Ca²⁺ did not reverse the effect of cAMP. Similar results were obtained with iberiotoxin, a blocker of Ca²⁺-activated K⁺ channels, which would be expected to increase Ca²⁺ influx and contraction. By contrast, the photolysis of caged-IP₃ in the presence of agonist, to further elevate the intracellular IP₃ concentration, reversed the slowing of the frequency of the Ca²⁺ oscillations and relaxation of the airway induced by FSK. This result implied that the sensitivity of the IP₃R to IP₃ was reduced by FSK and this was supported by the reduced ability of IP₃ to release Ca²⁺ in SMCs in the presence of FSK.

Conclusion

These results indicate that the relaxant effect of cAMP-elevating agents on airway SMCs is achieved by decreasing the Ca²⁺ oscillation frequency by reducing internal Ca²⁺ release through IP₃ receptors.

     

A conserved TGFβ1/HuR feedback circuit regulates the fibrogenic response in fibroblasts

Persistent fibroblast activation in wound repair is believed to be the key reason for fibrosis and transforming growth factor (TGF)β is considered as one of the key mediators for the fibrogenic response, with the detailed mechanism largely unknown. Here we found that TGFβ1 treatment could induce a significant increase of endogenous TGFβ1 expression by enhancing the mRNA stability in cardiac fibroblasts. Further study revealed that TGFβ1 treatment translocated the nuclear HuR into cytoplasm, which in turn bound the ARE in the 3'UTR of TGFβ1 and increased the mRNA stability as seen from the RNA-IP and reporter assay. Knockdown of HuR decreased the endogenous expression of TGFβ1 under exogenous TGFβ1 treatment, simultaneously with the decrease of Col1a, Col3a and fibronectin expression. Our study here established a TGFβ1/HuR feedback circuit regulating the fibrogenic response in fibroblasts, and targeting this feedback loop is of great potential to control fibrosis.     

Identification and characterization of the core region of protein phosphatase-1

PP1, PP2A and PP2B all belong to PPP family of serine/threonine protein phosphatases. Their primary structures are highly conserved, particularly in the catalytic domain. In order to obtain correlative information about this conserved region, we constructed N-, C-deletion and N/C double-deletion mutants. We found that the N- and Csingle-deletion mutants exhibited higher enzymatic activities, while specific activity of N/C double-deletion mutant PP1 (9-306) did not notably change. The results of kinetics analysis showed that k_cat and k_cat/K_m increased about 16-fold in the single-deletion mutants; while the two parameters of the double-deletion were lower than the single-deletions. We further explored stability of all mutants in existing denaturant guanidine hydrochloride (GdnHCl). It was noticeable that stability of PP1-(9-306) in all mutants was the highest. We speculated that PP1-(9-306) maybe retains a compact spherical structure, thus accordingly affected molecular catalysis. On the other hand the structures of single-deletion mutants were relatively relaxed, which were able to bind substrate easily, so activities of single-deletion mutants were higher than that of double-deletion mutant. We therefore deduced that PP1-(9-306) may be close to core region of PP1 molecule. In order to further solidify this idea, we used fluorescence spectra method to explore changes of space conformation. We found that emission peaks of all single-deletions were blue shifted in different degree in the absence of denaturant, while emission peak of N/C double-deletion mutant did not change obviously compared with that of the wild-type PP1. Conformation change of N/C double-deletion mutant was significantly less than those of single-deletion mutants in different GdnHCl concentration.     

Increasing serum half-life and extending cholesterol lowering in vivo by engineering antibody with pH-sensitive binding to PCSK9

Target-mediated clearance and high antigen load can hamper the efficacy and dosage of many antibodies. We show for the first time that the mouse, cynomolgus, and human cross-reactive, antagonistic anti-proprotein convertase substilisin kexin type 9 (PCSK9) antibodies J10 and the affinity-matured and humanized J16 exhibit target-mediated clearance, resulting in dose-dependent pharmacokinetic profiles. These antibodies prevent the degradation of low density lipoprotein receptor, thus lowering serum levels of LDL-cholesterol and potently reducing serum cholesterol in mice, and selectively reduce LDL-cholesterol in cynomolgus monkeys. In order to increase the pharmacokinetic and efficacy of this promising therapeutic for hypercholesterolemia, we engineered pH-sensitive binding to mouse, cynomolgus, and human PCSK9 into J16, resulting in J17. This antibody shows prolonged half-life and increased duration of cholesterol lowering in two species in vivo by binding to endogenous PCSK9 in mice and cynomolgus monkeys, respectively. The proposed mechanism of this pH-sensitive antibody is that it binds with high affinity to PCSK9 in the plasma at pH 7.4, whereas the antibody-antigen complex dissociates at the endosomal pH of 5.5-6.0 in order to escape from target-mediated degradation. Additionally, this enables the antibody to bind to another PCSK9 and therefore increase the antigen-binding cycles. Furthermore, we show that this effect is dependent on the neonatal Fc receptor, which rescues the dissociated antibody in the endosome from degradation. Engineered pH-sensitive antibodies may enable less frequent or lower dosing of antibodies hampered by target-mediated clearance and high antigen load.     

The dietary phytochemical 3,3'-diindolylmethane induces G2/M arrest and apoptosis in oral squamous cell carcinoma by modulating Akt-NF-κB, MAPK, and p53 signaling

In light of the growing incidence of oral cancer in Taiwan, this study is aimed at investigating the antitumor activity of 3,3'-diindolylmethane (DIM), an active metabolite of the phytochemical indole-3-carbinol (I3C), in oral squamous cell carcinoma (OSCC). DIM exhibited substantially higher antiproliferative potency than I3C in three OSCC cell lines with IC(50) values in SCC2095, SCC9, and SCC15 cells, respectively, of 22 versus 168μM, 25 versus 176μM, and 29versus 300μM. Flow cytometric analysis and Comet assay indicated that DIM suppressed the viability of SCC2095 cells by inducing apoptosis and G2/M arrest. Western blot analysis of various signaling markers revealed the ability of DIM to target pathways mediated by Akt, mitogen-activated protein (MAP) kinases, nuclear factor (NF)-κB, and p53, of which the concerted action underlined its antitumor efficacy. The concomitant inactivation of Akt and MAP kinases in response to DIM facilitated the dephosphorylation of the proapoptotic protein Bad at Ser-136 and Ser-112, respectively. Through endoplasmic reticulum (ER) stress, DIM stimulated the activation of p53 via Ser-15 phosphorylation, leading to increased expression of the BH3-only proapoptotic Bcl-2 members Puma and Noxa. Together, these changes decreased the mitochondrial threshold for apoptosis. G2/M arrest might be attributable to the suppressive effect of DIM on the expression of cyclin B1 and cdc25c. As many downstream effectors of the Akt-NF-κB pathway, including glycogen synthase kinase 3β, IκB kinase α, and cyclooxygenase-2, have been shown to promote oral tumorigenesis, the ability of DIM to inhibit this signaling axis underscores its chemopreventive potential in oral cancer.     

Differential Requirement for CD70 and CD80/CD86 in Dendritic Cell-Mediated Activation of Tumor-Tolerized CD8 T Cells

Activated human blood γδ T cells have also been previously demonstrated to behave as professional APCs, although the processes that control APC function have not been characterized. n this study, we show that the acquisition of potent APC function by human blood γδ T cells is achieved after physical interaction with an Ab-coated target cell, a process that we refer to as licensing. In cancer models, licensing of γδ T cells by tumor-reactive mAbs promotes the uptake of tumor Ags and professional presentation to tumor-reactive αβ T cells. We propose that licensing by Ab is a mechanism whereby the adaptive properties of γδ T cells are induced by their innate functions in a spatially and temporally controlled manner.     

Antibody-dependent cellular cytotoxicity against primary HIV-infected CD4+ T cells is directly associated with the magnitude of surface IgG binding

Antibody (Ab)-dependent cellular cytotoxicity (ADCC) is thought to potentially play a role in vaccine-induced protection from HIV-1. The characteristics of such antibodies remain incompletely understood. Furthermore, correlates between ADCC and HIV-1 immune status are not clearly defined. We screened the sera of 20 HIV-1-positive (HIV-1(+)) patients for ADCC. Normal human peripheral blood mononuclear cells were used to derive HIV-infected CD4(+) T cell targets and autologous, freshly isolated, natural killer (NK) cells in a novel assay that measures granzyme B (GrB) and HIV-1-infected CD4(+) T cell elimination (ICE) by flow cytometry. We observed that complex sera mediated greater levels of ADCC than anti-HIV-1 envelope glycoprotein (Env)-specific monoclonal antibodies and serum-mediated ADCC correlated with the amount of IgG and IgG1 bound to HIV-1-infected CD4(+) T cells. No correlation between ADCC and viral load, CD4(+) T cell count, or neutralization of HIV-1(SF162) or other primary viral isolates was detected. Sera pooled from clade B HIV-1(+) individuals exhibited breadth in killing targets infected with HIV-1 from clades A/E, B, and C. Taken together, these data suggest that the total amount of IgG bound to an HIV-1-infected cell is an important determinant of ADCC and that polyvalent antigen-specific Abs are required for a robust ADCC response. In addition, Abs elicited by a vaccine formulated with immunogens from a single clade may generate a protective ADCC response in vivo against a variety of HIV-1 species. Increased understanding of the parameters that dictate ADCC against HIV-1-infected cells will inform efforts to stimulate ADCC activity and improve its potency in vaccinees.     

Follicle characteristics and follicle developmental related Wnt6 polymorphism in Chinese indigenous Wanxi-white goose

In birds, downy feather quantity mainly affected by the follicles. Wnt6, a secreted cysteine-rich protein, plays a key role in follicular development as an intercellular signaling molecule. The present study was to investigate the follicle development and Wnt6 polymorphism in Wanxi-white geese, a Chinese indigenous breed. In total, 300 fertilized eggs were hatched. At embryonic stage and on early birth goslings, the diameter and density of follicles from different sites were examined after sectioning and staining. The results showed that the diameter of primary feather follicles in thorax, venter, dorsum and flank had no difference at embryonic stage. In contrast, after birth, thorax and ventral feather follicles had greater diameter than those on dorsum and flank. Similarly, the primary feather follicle density was higher in thorax and venter than in dorsum and flank at embryonic stage. The secondary feather follicle diameter in flank was greater than that in other sites examined. The secondary follicle showed lush growth in E27 with thickest in ventral and thorax. Overall, follicle formed consistently in dorsal and flank, and follicle in thorax and ventral formed in another consistent way. The polymorphism study showed 2 single nucleotide polymorphisms of Wnt6 and 3 genotypes identified. Sequencing revealed two nucleotide transitions, T451C and A466G, which were synonymous mutations causing codons for aspartate and lysine to change from GAU to GAC and from AAA to AAG, respectively. This information about follicle development and Wnt6 polymorphisms would provide potential utilization in marker-assisted selection program for down feather selection.     

Ferroportin 1 and hephaestin expression in BeWo cell line with different iron treatment

The process of placental iron transfer is an important physiological process during pregnancy. However, the molecular mechanism of placental iron transport has not been completely elucidated until now. Ferroportin 1 (FPN1) and hephaestin (Heph) have been identified as the important molecules involved in duodenal iron export. However, whether they participate in the placental iron efflux has been undefined until now. In this study, the BeWo cells were treated with desferrioxamine and Holo-transferrin human in different concentrations and harvested at 48 and 72 h. The mRNA expression of FPN1 and Heph was detected with quantitative real-time polymerase chain reaction, and the protein expression was detected with western blots. The results showed an up-regulated FPN1 expression with desferrioxamine treatment and down-regulated expression with Holo-transferrin human supplementation. However, the change of FPN1 expression at protein level was limited. Heph expression enhanced when cells were treated with desferrioxamine although the quantity of Heph expression was low. Heph expression showed no significant change with Holo-transferrin human supplementation. It indicates that FPN1 may participate in placental iron transport, and placental FPN1 expression is obviously not dependent on the iron regular element/iron regular protein regulation. An alternatively spliced FPN1 isoform that lacks an iron regular element may be the predominant expression in BeWo cells. It also demonstrates that Heph is active in placenta but may not play a key role in placental iron transport because it is not the main part of placental copper oxidase.