Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent, but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells.
Flavin adenine dinucleotide (FAD); Intrinsic fluorescence; Silver island film (SIF); Plasmon resonance; Near-field interaction; Fluorescence cell imaging
Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus–cytoplasm transport of galectin-3, which is a β-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the β-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the β-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to β-catenin to inhibit transcriptional activity. Reduced expression of β-catenin target genes is consistent with the Nup98 reduction and the galectin-3–nucleus translocation rate. Overall, the results show Nup98’s involvement in nuclear–cytoplasm translocation of galectin-3 and β-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.
Nucleoporin; Nuclear pore complex; Galectin-3; Nuclear transport; Cancer progression
Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4+ T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4+ T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4+ T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4+ T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4+ T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.
Sphingosine 1-phosphate; RANKL; Rheumatoid arthritis
PIWI-interacting RNAs (piRNAs) are new class of small RNAs specifically expressed in male germ cells. It is known to bind to PIWI class of Argonaute proteins-Mili, Miwi, and Miwi-2. To decipher the mechanism of piRNA function, here we report a real-time PCR based multiplex assay for piRNA expression. Firstly, we showed that the assay specifically detects piRNA expression in adult testis, consistent with Northern blot result. Then we confirmed that we can simultaneously detect at least eight piRNAs expression using only 10pg total RNA –equivalent to RNA amount of a single cell. This is five to six orders more sensitive than corresponding Northern blot. Finally we use this assay to analyze eight piRNAs expression in primordial germ cells (PGCs) in genital ridges from E12.5, the time point piRNA-binding protein Mili starts to express in PGCs. This multiplex piRNA assay can be further expanded to assay a few hundred of piRNAs simultaneously from as little as total RNA of a single cell, that will help to understand the mechanism and function of piRNAs for germ cell development.
The polyether ionophoric antibiotics including monensin, salinomycin, and narasin, are widely used in veterinary medicine and as food additives and growth promoters in animal husbandry including poultry farming. Their effects on human health, however, are not fully understood. Recent studies showed that salinomycin is a cancer stem cell inhibitor. Since poultry consumption has risen sharply in the last three decades, we asked whether the consumption of meat tainted with growth promoting antibiotics might have effects on adipose cells. We showed in this report that the ionophoric antibiotics inhibit the differentiation of preadipocytes into adipocytes. The block of differentiation is not due to the induction of apoptosis nor the inhibition of cell proliferation. In addition, salinomycin also suppresses the transcriptional activity of the CCAAT/enhancer binding proteins and the peroxisome proliferator-activated receptor γ. These results suggest that the ionophoric antibiotics can be exploited as novel anti-obesity therapeutics and as pharmacological probes for the study of adipose biology. Further, the pharmacological effects of salinomycin could be a harbinger of its toxicity on the adipose tissue and other susceptible target cells in cancer therapy.
Adipogenesis; salinomycin; polyether antibiotic; ionophoric antibiotic; obesity
•Mutations in the ROC, COR and Kinase domain of LRRK2 alter the autophagic response to starvation.•LC3-I/II ratio following starvation is altered by mutations, as well as p62 and WIPI2 positive puncta.•This occurs independently of any alteration in downstream targets of mTORC1.
LRRK2 is one of the most important genetic contributors to Parkinson’s disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.
LRRK2, leucine rich repeat kinase 2; ROC, ras of complex proteins; COR, C-terminal of ROC; PD, Parkinson’s disease; ICC, Immunocytochemistry; LRRK2; Parkinson’s disease; Autophagy; Lysosomes; Signaling pathways
km23-1 was originally identified as a TGFβ receptor-interacting protein that plays an important role in TGFβ signaling. Moreover, km23-1 is actually part of an ancient superfamily of NTPase-regulatory proteins, widely represented in archaea and bacteria. To further elucidate the function of km23-1, we identified novel protein interacting partners for km23-1 by using tandem affinity purification (TAP) and tandem mass spectrometry (MS). Here we show that km23-1 interacted with a class of proteins involved in actin-based cell motility and modulation of the actin cytoskeleton. We further showed that km23-1 modulates the formation of a highly organized stress fiber network. More significantly, we demonstrated that knockdown (KD) of km23-1 decreased RhoA activation in Mv1Lu epithelial cells. Finally, our results demonstrated for the first time that depletion of km23-1 inhibited cell migration of human colon carcinoma cells (HCCCs) in wound-healing assays. Overall, our findings demonstrate that km23-1 regulates RhoA and motility-associated actin modulating proteins, suggesting that km23-1 may represent a novel target for anti-metastatic therapy.
km23-1; cell migration; actin; Rho; dynein
Dendritic cell (DC)-based vaccine strategies have gained increasing popularity in recent years. Methods for ex vivo generation of immunocompetent mature DCs still require optimization. DCs have been shown to phenotypically mature under elevated pressure. We compared the effects of pressure on DC maturation with LPS- and cytokine-stimulation. Human monocyte-derived immature or LPS- and cytokine-matured DCs were exposed to ambient or 40mmHg increased pressure for 12-hrs., then assessed for expression of CD80, CD86, CD40, MHC-I/II, and inflammatory cytokine production. DCs were also evaluated for capacity to stimulate T-cell proliferation by co-culture with allogeneic lymphocytes. Pressure significantly increased cytokine production and expression of all surface molecules on immature DC other than MHC-I and CD40. Pressure/LPS-treated DCs displayed further upregulation of MHC-I, CD40, and IL-12p70. Cytokine-matured DCs appeared less responsive to pressure. T-cell proliferation correlated with MHC expression. Results suggest mechanical stimulation of DCs may provide a useful adjuvant to TLR-agonist maturation strategies.
dendritic cell; maturation; vaccine; pressure; mechanotransduction
Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7 Å resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.
Streptococcus pneumoniae; Pseudomonas aeruginosa; neuraminidase; crystal structure; pneumonia
We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterixnull embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblastic cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TNW suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.
Tenascin-W ; osteogenesis; Bmp; preosteoblast
Acetaminophen (APAP) overdose is widely regarded as a major cause of acute liver failure in the United States. Intentional or accidental overdose of APAP in man or rodent elicits direct hepatocellular injury that is accompanied by hepatic depletion of the antioxidant, glutathione (GSH). In recent years, the innate immune response has also been shown to promote the development of APAP hepatotoxicity via indirect liver damage. In the present study, we demonstrate that Jα18−/− mice, which are selectively deficient in the innate immune T cell, Vα14iNKT cells, were resistant to APAP hepatotoxicity relative to WT mice as reflected by biochemical and histological liver injury markers. In parallel, improvement in the biochemical and histological parameters of liver injury in Jα18−/− mice was associated with a significant increase in hepatic levels of GSH, which detoxified APAP metabolites to attenuate hepatic oxidative stress, liver injury and necrosis. Notably, the protective effect of hepatic GSH during Vα14iNKT cells deficiency was demonstrated by its depletion in Jα18−/− mice using DL-buthionine-[S,R]-sulfoximine which exacerbated hepatic oxidative and nitrosative stress as well as liver necrosis and caused mice mortality. Extraordinarily, APAP metabolism in Jα18−/− mice was altered in favor of hepatic GSH conjugates and decreased glucuronide conjugates. In summary, we reveal a novel finding establishing a unique association between hepatic innate immunity and GSH levels in altering APAP metabolism to suppress liver injury and necrosis during Vα14iNKT cells deficiency in Jα18−/− mice.
Glutathione; APAP; Vα14iNKT cells; ROS; Liver
Vertebrate TLR5 directly binds bacterial flagellin proteins and activates innate immune responses against pathogenic flagellated bacteria. Structural and biochemical studies on the TLR5/flagellin interaction have been challenging due to the technical difficulty in obtaining active recombinant proteins of TLR5 ectodomain (TLR5-ECD). We recently succeeded in production of the N-terminal leucine rich repeats (LRRs) of Danio rerio (dr) TLR5-ECD in a hybrid with another LRR protein, hagfish variable lymphocyte receptor (VLR), and determined the crystal structure of its complex with flagellin D1–D2–D3 domains. Although the structure provides valuable information about the interaction, it remains to be revealed how the C-terminal region of TLR5-ECD contributes to the interaction. Here, we present two methods to obtain recombinant TLR5 proteins that contain the C-terminal region in a baculovirus expression system. First, production of biologically active full-length drTLR5-ECD was substantially enhanced by supplementation of expression culture with purified flagellin proteins. Second, we designed TLR5-VLR hybrids using an LRR hybrid technology by single and double LRR fusions and were able to express diverse regions of drTLR5-ECD, allowing us to detect a previously unidentified TLR5/flagellin interaction. The drTLR5-VLR hybrid technique was also successfully applied to human TLR5-ECD whose expression has been highly problematic. These alternative TLR5 expression strategies provide an opportunity to obtain a complete view of the TLR5/flagellin interaction and can be applied to other LRR proteins.
Toll-like receptor 5; Flagellin; Leucine-rich repeat; LRR hybrid; Variable lymphocyte receptor; Innate immunity
Hemagglutinin (HA) of influenza A has been reported as the key protein in viral infection. Therefore, the density and the dynamic pattern of this protein in viral envelope will affect the virus to infect target cells. We used a lentiviral system to study the influenza A H1N1 viral infection. Herein we demonstrate that the influenza non-structural proteins (NS) significantly promote viral infection. By substituting NS gene segment from an H1N1 genome set of A/WSN/1933 with the NS segment isolated from another H1N1 substrain genome set, China246, we found that viral infection tropism was significantly altered. The reassortant H1N1 shows almost identical infectivity compared with its parental virus, A/WSN/1933, for the human epithelial cell line HOT, but shows only 1/100 infectivity of its parental virus when infecting the Madin-Darby canine kidney (MDCK) cell line. These results suggest that not only is NS important in the infectivity of human influenza virus, but that it may play a critical role in viral tropism, allowing the virus to mutate and spread to other species.
Influenza A; H1N1; Lentivirus; influenza non-structural proteins; NS; Infection tropism
The myogenic transcription factor Pax3, a member of the paired class homeodomain family of transcription factors, plays an essential role in early skeletal muscle development. We previously demonstrated that Pax3 is phosphorylated at three specific residues (Ser201, Ser205, and Ser209) and that the pattern of phosphorylation at these sites changes throughout early myogenesis. Further, we demonstrated that the protein kinase CK2 phosphorylates Pax3 at Ser205 and that this phosphorylation event is required for the subsequent phosphorylation of Ser201 by GSK3β. However, the kinase that phosphorylates Pax3 at Ser209 has yet to be identified. In the present work we use standard purification methods and in vitro biochemical analyses to provide solid evidence identifying the protein kinase CK2 as phosphorylating Pax3 at Ser209. Further, we qualitatively demonstrate that the phosphorylation of Pax3 at Ser209 by CK2 is enhanced when Ser205 is previously phosphorylated. Taken together, our results allow us to propose a mechanism to describe the ordered phosphorylation of Pax3 throughout early myogenesis.
Pax3; Myogenesis; CK2; Phosphorylation
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the major carcinogens in tobacco. NNK has been associated with various cancers in tobacco users, especially lung cancer. However, the effects of NNK on cytotoxic T lymphocytes (CTLs), the cells responsible for destrcution of maligant and pathogen-infected cells, has not been elucidated. Using transgenic CTLs in vitro and in vivo, we show that NNK can directly affect CTL activation. NNK can enhance the expression of adhesion molecule CD62L in CTLs during their activation in vitro, but has no effects on their expansion and production of effector molecules such as IFN and granzyme B. After transferred into recipient mice, however, the NNK pretreated CTLs suffer an early loss in expansion. The percentage of memory precursors is higher in NNK pretreated CTLs, but the total amount of memory precursors is similar to controls. The final memory CTL population from NNK pretreated CTLs is reduced, but sustains a more central memory phenotype. In conclusion, NNK can affect CTL activation by modulating adhension molecule expression and reducing memory programming.
Carcinogen; CTLs; NNK; CD62L; Cytokines; Memory; Activation
IRE1α (Inositol-requiring enzyme 1 α), an endoplasmic reticulum (ER)-resident sensor for mammalian unfolded protein response, is a type I transmembrane protein which has a bifunctional enzyme containing kinase and RNase domains. Although the luminal domain and cytosolic domain of IRE1α are thought to play crucial roles in regulating the protein activity, no functional and structural studies of the transmembrane domain exist thus far. Herein, using CD spectroscopy, we report that the transmembrane domain of the IRE1α is alpha helical in a membrane-like environment. In addition, SDS-PAGE and FRET analyses support that the transmembrane domain forms oligomers in SDS micelles. Thus, the study would provide insights into how the transmembrane domain plays a role in regulating the IRE1α protein activity.
endoplasmic reticulum; transmembrane domain; dimerization; IRE1α
The major constituent of the eukaryotic ER protein-translocation channel (Sec61p in yeast, Sec61 in higher eukaryotes) shows a high degree of evolutionary conservation from yeast to humans. The vast majority of eukaryotic species have a conserved di-tyrosine in the 4th ER luminal loop. Previously, we discovered through a screen of ethylnitrosourea- (ENU-) mutagenized mice that substitution of the latter of these tyrosines with histidine (Y344H) of the murine Sec61 protein results in diabetes and hepatic steatosis in mice that is a result of ER stress. To further characterize the mechanism behind ER stress in these mice we made the homologous mutation in yeast Sec61p (Y345H). We found that this mutation increased sensitivity of yeast to ER stressing agents and to reduction of Inositol Requiring Enzyme 1 (IRE1) activity. Furthermore, we found that, while this mutation did not affect translocation, it did delay degradation of the model ER-associated degradation (ERAD) substrate CPY*. Replacing both ER luminal tyrosines with alanines resulted in a destabilization of the Sec61 protein that was rescued by over expression of Sss1p. This double mutant still lacked a noticeable translocation defect after stabilization by Sss1p, but exhibited a similar defect in CPY* degradation.
Sec61; ER stress; ER associated degradation
Elevated cyclooygenase-2 (COX-2) expression is frequently observed in human non–small cell lung cancer (NSCLC) and associated with poor prognosis, indicating critical involvement of the inflammatory pathway in lung carcinogenesis. Recently, we found that green tea extract (GTE) induced annexin-1 (ANX1) in the lung adenocarcinoma A549 cells. ANX1 is a glucocorticoid-inducible 37 kDa protein involved in a wide range biological function and is an important anti-inflammatory mediator. The present study further examines the interplay between the expressions and production of ANX1, COX-2, phospholipase A2 (cPLA2) and prostaglandin E2 (PGE2) following the treatment of NSCLC cell lines with GTE. We found that GTE induced ANX1 and inhibited COX-2 expression in lung cancer A549, H157 and H460 cell lines. Addition of pro-inflammatory cytokine IL-1β diminished GTE-induced ANX1. Silence of ANX1 in cells abrogates the inhibitory activity on COX-2, indicating that the anti-inflammatory activity of GTE is mediated at least partially by the up-regulation of ANX1. However, differential pattern of inhibitory effects of ANX1 on cPLA2 expression was observed among various cell types, suggesting that the anti-inflammatory activity mediated by ANX1 is cell type specific. Our study may provide a new mechanism of GTE on the prevention of lung cancer and other diseases related to inflammation.
Green tea; annexin-1; cycolooxygenase-2; anti-inflammatory; lung cancer
Aquaporin-4 (AQP4) is the predominant water channel in the central nervous system, where it has been reported to be involved in many pathophysiological roles including water transport. In this paper, the AQP4 tetramer was modeled from its PDB structure file, embedded in a palmitoyl-oleoyl-phosphatidyl-choline (POPC) lipid bilayer, solvated in water, then minimized and equilibrated by means of molecular dynamics simulations. Analysis of the equilibrated structure showed that the central pore along the fourfold axis of the tetramers is formed with hydrophobic amino acid residues. In particular, Phe-195, Leu-191 and Leu-75, form the narrowest part of the pore. Therefore water molecules are not expected to transport through the central pore, which was confirmed by MD simulations. Each monomer of the AQP4 tetramers forms a channel whose walls consist mostly of hydrophilic residues. There are eight water molecules in single file observed in each of the four channels, transporting through the selectivity filter containing Arg-216, His-201, Phe-77, Ala-210, and the two conserved Asn-Pro-Ala (NPA) motifs containing Asn-213 and Asn-97. By using Brownian dynamics fluctuation–dissipation-theorem (BD-FDT), the overall free-energy profile was obtained for water transporting through AQP4 for the first time, which gives a complete map of the entire channel of water permeation.
Aquaporin-4; Molecular dynamics simulations; Asn-Pro-Ala motifs; Central fourfold axis; Brownian dynamics fluctuation–dissipation-theorem (BD-FDT); Free energy computation
Since the endoplasmic reticulum (ER) plays a vital role in hepatocyte function, it is not surprising that a variety of liver-related diseases are associated with ER stress. As in other tissues, ER stress in the liver leads to generation of the unfolded-protein response resulting in activation of a transcriptional program that promotes restoration of homeostasis within the lumen of the ER. Previous studies using cells in culture demonstrated that ER stress induces expression of REDD1 (regulated in development and DNA damage responses), a potent repressor of signaling through the protein kinase referred to as the mechanistic target of rapamycin in complex 1 (mTORC1). In the present study, the results from the cell culture experiments were extended to show that tunicamycin-mediated ER stress in the liver in vivo also induces REDD1 gene expression. Moreover, the induction of REDD1 gene expression was shown to require the protein kinase PERK and enhanced phosphorylation of its substrate, the α-subunit of eukaryotic initiation factor 2.
REDD1; Ddit4; eIF2; ER stress; unfolded protein response; PERK
Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic β cells. In the present study, we have identified the expression of TGR5 in pancreatic β cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated Gαs and caused an increase in intracellular cAMP and Ca2+. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective Gαs inhibitor) or U73122 (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, U73122 or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on Gs/cAMP/Ca2+ pathway. 8-pCPT-2′-O-Me-cAMP, a cAMP analogue, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic β cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.
Bile salts; Oleanolic acid; TGR5 receptor; G-protein coupled receptor; insulin resistance; incretins
The podocyte is a highly specialized kidney glomerular epithelial cell that plays an essential role in glomerular filtration and is believed to be the target of numerous glomerular diseases leading to proteinuria. Despite the leaps in our understanding of podocyte biology, new methodologies are needed to facilitate research into the cell. Multiphoton microscopy (MPM) was used to image the nephrin knockout/green fluorescent protein (GFP) knock-in heterozygote (Nphs1tm1Rkl/J) mouse. The nephrin promoter restricts GFP expression to the podocytes that fluoresce green under excitation. From the exterior of an intact kidney, MPM can peer into the renal parenchyma and visualize the podocytes that outline the globular shape of the glomeruli. Details as fine as the podocyte’s secondary processes can be resolved. In contrast, podocytes exhibit no fluorescence in the wildtype mouse and are invisible to MPM. Phenotypically, there are no significant differences between wildtype and Nphs1tm1Rkl/J mice in body weight, urinary albumin excretion, creatinine clearance, or glomerular depth. Interestingly, the glomeruli are closer to the kidney capsule in female mice, making the gender the preferred choice for MPM. For the first time, green fluorescent podocytes in a mouse model free of confounding phenotypes can be visualized unequivocally and in the “positive” by MPM, facilitating intravital studies of the podocyte.
green fluorescent protein; 2-photon microscopy; podocyte processes; renal corpuscle
Ion channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca2+-activated Cl− channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16Ainh-A01 as a specific inhibitor of Ano1.
To investigate the effect of the T16Ainh-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1.
Inhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16Ainh-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively.
T16Ainh-A01 inhibited Ca2+- activated Cl− currents by 60% at 10 µM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16Ainh-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16Ainh-A01. In organotypic cultures of smooth muscle strips from mouse jejunum, the proliferation of ICC was reduced but the total number of proliferating cells/confocal stack was not affected, suggesting that the inhibitory effect was specific for ICC.
The selective Ano1 inhibitor T16Ainh-A01 inhibited Ca2+-activated Cl− currents, reduced the number of proliferating ICC in culture and inhibited proliferation in the pancreatic cancer cell line CFPAC-1. These data support the notion that chloride channels in general and Ano1 in particular are involved in the regulation of proliferation.
Interstitial cells of Cajal; ICC; TMEM16A; GIST; cancer; intestine
The family C G protein-coupled receptor (GPCR) T1R2 and T1R3 heterodimer functions as a broadly acting sweet taste receptor. Perception of sweet taste is a species-dependent physiological process. It has been widely reported that New World monkeys and rodents can not perceive some of the artificial sweeteners and sweet-tasting proteins that can be perceived by humans, apes, and Old World monkeys. Until now, only the sweet receptors of humans, mice and rats have been functionally characterized. Here we report characterization of the sweet taste receptor (T1R2/T1R3) from a species of New World squirrel monkey. Our results show that the heterodimeric receptor of squirrel monkey does not respond to artificial sweeteners aspartame, neotame, cyclamate, saccharin and sweet-tasting protein monellin, but surprisingly, it does respond to thaumatin at high concentrations (>18 μM). This is the first report that New World monkey species can perceive some specific sweet-tasting proteins. Furthermore, the receptor responses to the sweeteners cannot be inhibited by the sweet inhibitor lactisole. We compared the response differences of the squirrel monkey and human receptors and found that the residues in T1R2 determine species-dependent sweet taste toward saccharin, while the residues in either T1R2 or T1R3 are responsible for the sweet taste difference between humans and squirrel monkeys toward monellin. Molecular models indicated that electrostatic properties of the receptors probably mediate the species-dependent response to sweet-tasting proteins.
New World monkeys; squirrel monkey; sweet taste receptors; sweet-tasting proteins; G protein-coupled receptor; molecular modeling
Estrogen receptors are localized in mitochondria, but their functions in this organelle remain unclear. We previously found that ERα interacted with mitochondrial protein HADHB and affected the thiolytic cleavage activity of HADHB in β-oxidation. It is known that ERβ binds to ERα. In addition, ERβ is predominately located in mitochondria. These facts led us to speculate that ERβ may also be associated with HADHB in mitochondria. In order to test this hypothesis, we performed co-immunoprecipitation and confocal microscopy analyses with human breast cancer MCF7 cells. The results demonstrated that ERβ was indeed associated and colocalized with HADHB within mitochondria. Interestingly, in contrast to the stimulatory effect of ERα on HADHB enzyme activity observed in the previous study, silencing of ERβ enhanced the enzyme activity of HADHB in the present study, suggesting that ERβ plays an inhibitory role in HADHB enzyme activity in the breast cancer cells. Our results imply that ERα and ERβ may differentially affect cellular oxidative stress through influencing the rate of β-oxidation of fatty acids in breast cancer cells.
Breast cancer; estrogen receptor beta; hydroxyacyl-CoA dehydrogenase/trifunctional protein; beta subunit (HADHB); mitochondria