Coordination of the recruitment of the FANCD2 and PALB2 Fanconi anemia proteins by an ubiquitin signaling network.

PubMed

Bick, Gregory; Zhang, Fan; Meetei, A Ruhikanta; Andreassen, Paul R

2017-06-01

Fanconi anemia (FA) is a chromosome instability syndrome and the 20 identified FA proteins are organized into two main arms which are thought to function at distinct steps in the repair of DNA interstrand crosslinks (ICLs). These two arms include the upstream FA pathway, which culminates in the monoubiquitination of FANCD2 and FANCI, and downstream breast cancer (BRCA)-associated proteins that interact in protein complexes. How, and whether, these two groups of FA proteins are integrated is unclear. Here, we show that FANCD2 and PALB2, as indicators of the upstream and downstream arms, respectively, colocalize independently of each other in response to DNA damage induced by mitomycin C (MMC). We also show that ubiquitin chains are induced by MMC and colocalize with both FANCD2 and PALB2. Our finding that the RNF8 E3 ligase has a role in recruiting FANCD2 and PALB2 also provides support for the hypothesis that the two branches of the FA-BRCA pathway are coordinated by ubiquitin signaling. Interestingly, we find that the RNF8 partner, MDC1, as well as the ubiquitin-binding protein, RAP80, specifically recruit PALB2, while a different ubiquitin-binding protein, FAAP20, functions only in the recruitment of FANCD2. Thus, FANCD2 and PALB2 are not recruited in a single linear pathway, rather we define how their localization is coordinated and integrated by a network of ubiquitin-related proteins. We propose that such regulation may enable upstream and downstream FA proteins to act at distinct steps in the repair of ICLs.

[Construction of FANCA mutant protein from Fanconi anemia patient and analysis of its function].

PubMed

Chen, Fei; Zhang, Ke-Jian; Zuo, Xue-Lan; Zeng, Xian-Chang

2007-11-01

To study FANCA protein expression in Fanconi anemia patient's (FA) cells and explore its function. FANCA protein expression was analyzed in 3 lymphoblast cell lines derived from 3 cases of type A FA (FA-A) patients using Western blot. Nucleus and cytoplasm localization of FANCA protein was analyzed in one case of FA-A which contained a truncated FANCA (exon 5 deletion). The FANCA mutant was constructed from the same patient and its interaction with FANCG was evaluated by mammalian two-hybrid (M2H) assay. FANCA protein was not detected in the 3 FA-A patients by rabbit anti-human MoAb, but a truncated FANCA protein was detected in 1 of them by mouse anti-human MoAb. The truncated FANCA could not transport from cytoplasm into nucleus. The disease-associated FANCA mutant was defective in binding to FANCG in M2H system. FANCA proteins are defective in the 3 FA-A patients. Disfunction of disease-associated FANCA mutant proved to be the pathogenic mutations in FANCA gene. Exon 5 of FANCA gene was involved in the interaction between FANCA and FANCG.

Identification of a Cullin5-ElonginB-ElonginC E3 complex in degradation of feline immunodeficiency virus Vif-mediated feline APOBEC3 proteins.

PubMed

Wang, Jiawen; Zhang, Wenyan; Lv, Mingyu; Zuo, Tao; Kong, Wei; Yu, Xianghui

2011-12-01

Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.

Middle region of FancM interacts with Mhf and Rmi1 in silkworms, a species lacking the Fanconi anaemia (FA) core complex.

PubMed

Sugahara, R; Mon, H; Lee, J M; Kusakabe, T

2014-04-01

The Fanconi anaemia (FA) pathway is responsible for interstrand crosslink (ICL) repair. Among the FA core complex components, FANCM is believed to act as a damage sensor for the ICL-blocked replication fork and also as a molecular platform for FA core complex assembly and interaction with Bloom's syndrome (BS) complex that is thought to play an important role in the processing of DNA structures such as stalled replication forks. In the present study, we found that in silkworms, Bombyx mori, a species lacking the major FA core complex components (FANCA, B, C, E, F, and G), FancM is required for FancD2 monoubiquitination and cell proliferation in the presence of mitomycin C (MMC). Silkworm FancM (BmFancM) was phosphorylated in the middle regions, and the modification was associated with its subcellular localization. In addition, BmFancM interacted with Mhf1, a histone-fold protein, and Rmi1, a subunit of the BS complex, in the different regions. The interaction region containing at least these two protein-binding domains played an essential role in FancM-dependent resistance to MMC. Our results suggest that BmFancM also acts as a platform for recruitment of both the FA protein and the BS protein, although the silkworm genome seems to lose FAAP24, a FancM-binding partner protein in mammals. © 2013 The Royal Entomological Society.

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters.

PubMed

Du, Wei; Rani, Reena; Sipple, Jared; Schick, Jonathan; Myers, Kasiani C; Mehta, Parinda; Andreassen, Paul R; Davies, Stella M; Pang, Qishen

2012-05-03

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Enterocyte fatty acid-binding proteins (FABPs): different functions of liver and intestinal FABPs in the intestine.

PubMed

Gajda, Angela M; Storch, Judith

2015-02-01

Fatty acid-binding proteins (FABP) are highly abundant cytosolic proteins that are expressed in most mammalian tissues. In the intestinal enterocyte, both liver- (LFABP; FABP1) and intestinal FABPs (IFABP; FABP2) are expressed. These proteins display high-affinity binding for long-chain fatty acids (FA) and other hydrophobic ligands; thus, they are believed to be involved with uptake and trafficking of lipids in the intestine. In vitro studies have identified differences in ligand-binding stoichiometry and specificity, and in mechanisms of FA transfer to membranes, and it has been hypothesized that LFABP and IFABP have different functions in the enterocyte. Studies directly comparing LFABP- and IFABP-null mice have revealed markedly different phenotypes, indicating that these proteins indeed have different functions in intestinal lipid metabolism and whole body energy homeostasis. In this review, we discuss the evolving knowledge of the functions of LFABP and IFABP in the intestinal enterocyte. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation

PubMed Central

Yamashita, Takayuki; Kupfer, Gary M.; Naf, Dieter; Suliman, Ahmed; Joenje, Hans; Asano, Shigetaka; D’Andrea, Alan D.

1998-01-01

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A–H). Two FA genes, corresponding to complementation groups A and C, have been cloned, but the function of the FAA and FAC proteins remains unknown. We have recently shown that the FAA and FAC proteins bind and form a nuclear complex. In the current study, we analyzed the FAA and FAC proteins in normal lymphoblasts and lymphoblasts from multiple FA complementation groups. In contrast to normal controls, FA cells derived from groups A, B, C, E, F, G, and H were defective in the formation of the FAA/FAC protein complex, the phosphorylation of the FAA protein, and the accumulation of the FAA/FAC protein complex in the nucleus. These biochemical events seem to define a signaling pathway required for the maintenance of genomic stability and normal hematopoiesis. Our results support the idea that multiple gene products cooperate in the FA Pathway. PMID:9789045

The Fanconi Anemia C Protein Binds to and Regulates Stathmin-1 Phosphorylation

PubMed Central

Magron, Audrey; Elowe, Sabine; Carreau, Madeleine

2015-01-01

The Fanconi anemia (FA) proteins are involved in a signaling network that assures the safeguard of chromosomes. To understand the function of FA proteins in cellular division events, we investigated the interaction between Stathmin-1 (STMN1) and the FA group C (FANCC) protein. STMN1 is a ubiquitous cytosolic protein that regulates microtubule dynamics. STMN1 activities are regulated through phosphorylation-dephosphorylation mechanisms that control assembly of the mitotic spindle, and dysregulation of STMN1 phosphorylation is associated with mitotic aberrancies leading to chromosome instability and cancer progression. Using different biochemical approaches, we showed that FANCC interacts and co-localizes with STMN1 at centrosomes during mitosis. We also showed that FANCC is required for STMN1 phosphorylation, as mutations in FANCC reduced serine 16- and 38-phosphorylated forms of STMN1. Phosphorylation of STMN1 at serine 16 is likely an event dependent on a functional FA pathway, as it is reduced in FANCA- and FANCD2-mutant cells. Furthermore, FA-mutant cells exhibited mitotic spindle anomalies such as supernumerary centrosomes and shorter mitotic spindles. These results suggest that FA proteins participate in the regulation of cellular division via the microtubule-associated protein STMN1. PMID:26466335

A systems biology approach for elucidating the interaction of curcumin with Fanconi anemia FANC G protein and the key disease targets of leukemia.

PubMed

Mahato, David; Samanta, Dipayan; Mukhopadhyay, Sudit S; Krishnaraj, R Navanietha

2017-06-01

Fanconi anemia (FA) is an autosomal recessive disorder with a high risk of malignancies including acute myeloid leukemia and squamous cell carcinoma. There is a constant search out of new potential therapeutic molecule to combat this disorder. In most cases, patients with FA develop haematological malignancies with acute myeloid leukemia and acute lymphoblastic leukemia. Identifying drugs which can efficiently block the pathways of both these disorders can be an ideal and novel strategy to treat FA. The curcumin, a natural compound obtained from turmeric is an interesting therapeutic molecule as it has been reported in the literature to combat both FA as well as leukemia. However, its complete mechanism is not elucidated. Herein, a systems biology approach for elucidating the therapeutic potential of curcumin against FA and leukemia is investigated by analyzing the computational molecular interactions of curcumin ligand with FANC G of FA and seven other key disease targets of leukemia. The proteins namely DOT1L, farnesyl transferase (FDPS), histone decetylase (EP3000), Polo-like kinase (PLK-2), aurora-like kinase (AUKRB), tyrosine kinase (ABL1), and retinoic acid receptor alpha (RARA) were chosen as disease targets for leukemia and modeled structure of FANC G protein as the disease target for FA. The docking investigations showed that curcumin had a very high binding affinity of -8.1 kcal/mol with FANC G protein. The key disease targets of leukemia namely tyrosine kinase (ABL1), aurora-like kinase (AUKRB), and polo-like kinase (PLK-2) showed that they had the comparable binding affinities of -9.7 k cal/mol, -8.7 k cal/mol, and -8.6 k cal/mol, respectively with curcumin. Further, the percentage similarity scores obtained from PAM50 using EMBOSS MATCHER was shown to provide a clue to understand the structural relationships to an extent and to predict the binding affinity. This investigation shows that curcumin effectively interacts with the disease targets of both FA and leukemia.

A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation.

PubMed

Chilton, Scott S; Falbel, Tanya G; Hromada, Susan; Burton, Briana M

2017-08-01

Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation. IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA. Copyright © 2017 American Society for Microbiology.

A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA.

PubMed

Yagasaki, H; Adachi, D; Oda, T; Garcia-Higuera, I; Tetteh, N; D'Andrea, A D; Futaki, M; Asano, S; Yamashita, T

2001-12-15

Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FA genes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA(-) cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.

Experimental Gonococcal Infection in Male Volunteers: Cumulative Experience with Neisseria Gonorrhoeae Strains FA1090 and MS11mkC

DTIC Science & Technology

2011-05-31

improved our understanding of the requirements for gonococcal LOS structures, pili, opacity proteins , IgA1 protease, and the ability of infecting...indicated by the horizontal dotted line) is 1.8× 103 cfu for MS11mkC and 1.0× 105 cfu for FA1090. contained predominantly piliated (P+), Opacity protein ...Gonococcal genetic island Absent Present Dillard and Seifert, (2001) Lactoferrin utilization (expression of lactoferrin-binding proteins B and A) Lf

Resistance to mitomycin C requires direct interaction between the Fanconi anemia proteins FANCA and FANCG in the nucleus through an arginine-rich domain.

PubMed

Kruyt, F A; Abou-Zahr, F; Mok, H; Youssoufian, H

1999-11-26

Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, birth defects, and chromosomal instability. Because FA cells are sensitive to mitomycin C (MMC), FA gene products could be involved in cellular defense mechanisms. The FANCA and FANCG proteins deficient in FA groups A and G interact directly with each other. We have localized the mutual interaction domains of these proteins to amino acids 18-29 of FANCA and to two noncontiguous carboxyl-terminal domains of FANCG encompassing amino acids 400-475 and 585-622. Site-directed mutagenesis of FANCA residues 18-29 revealed a novel arginine-rich interaction domain (RRRAWAELLAG). By alanine mutagenesis, Arg(1), Arg(2), and Leu(8) but not Arg(3), Trp(5), and Glu(7) appeared to be critical for binding to FANCG. Similar immunolocalization for FANCA and FANCG suggested that these proteins interact in vivo. Moreover, targeting of FANCA to the nucleus or the cytoplasm with nuclear localization and nuclear export signals, respectively, showed concordance between the localization patterns of FANCA and FANCG. The complementation function of FANCA was abolished by mutations in its FANCG-binding domain. Conversely, stable expression of FANCA mutants encoding intact FANCG interaction domains induced hypersensitivity to MMC in HeLa cells. These results demonstrate that FANCA-FANCG complexes are required for cellular resistance to MMC. Because the FANCC protein deficient in FA group C works within the cytoplasm, we suggest that FANCC and the FANCA-FANCG complexes suppress MMC cytotoxicity within distinct cellular compartments.

Modular Integration of Upconverting Nanocrystal-Dendrimer Composites for Folate Receptor-Specific NIR Imaging and Light-Triggered Drug Release.

PubMed

Wong, Pamela T; Chen, Dexin; Tang, Shengzhuang; Yanik, Sean; Payne, Michael; Mukherjee, Jhindan; Coulter, Alexa; Tang, Kenny; Tao, Ke; Sun, Kang; Baker, James R; Choi, Seok Ki

2015-12-02

Upconversion nanocrystals (UCNs) display near-infrared (NIR)-responsive photoluminescent properties for NIR imaging and drug delivery. The development of effective strategies for UCN integration with other complementary nanostructures for targeting and drug conjugation is highly desirable. This study reports on a core/shell-based theranostic system designed by UCN integration with a folate (FA)-conjugated dendrimer for tumor targeting and with photocaged doxorubicin as a cytotoxic agent. Two types of UCNs (NaYF4:Yb/Er (or Yb/Tm); diameter = ≈50 to 54 nm) are described, each displaying distinct emission properties upon NIR (980 nm) excitation. The UCNs are surface modified through covalent attachment of photocaged doxorubicin (ONB-Dox) and a multivalent FA-conjugated polyamidoamine (PAMAM) dendrimer G5(FA)6 to prepare UCN@(ONB-Dox)(G5FA). Surface plasmon resonance experiments performed with G5(FA)6 dendrimer alone show nanomolar binding avidity (KD = 5.9 × 10(-9) M) to the folate binding protein. This dendrimer binding corresponds with selective binding and uptake of UCN@(ONB-Dox)(G5FA) by FAR-positive KB carcinoma cells in vitro. Furthermore, UCN@(ONB-Dox)(G5FA) treatment of FAR(+) KB cells inhibits cell growth in a light dependent manner. These results validate the utility of modularly integrated UCN-dendrimer nanocomposites for cell type specific NIR imaging and light-controlled drug release, thus serving as a new theranostic system. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-resolution neutron and X-ray diffraction room-temperature studies of an H-FABP-oleic acid complex: study of the internal water cluster and ligand binding by a transferred multipolar electron-density distribution.

PubMed

Howard, E I; Guillot, B; Blakeley, M P; Haertlein, M; Moulin, M; Mitschler, A; Cousido-Siah, A; Fadel, F; Valsecchi, W M; Tomizaki, Takashi; Petrova, T; Claudot, J; Podjarny, A

2016-03-01

Crystal diffraction data of heart fatty acid binding protein (H-FABP) in complex with oleic acid were measured at room temperature with high-resolution X-ray and neutron protein crystallography (0.98 and 1.90 Å resolution, respectively). These data provided very detailed information about the cluster of water molecules and the bound oleic acid in the H-FABP large internal cavity. The jointly refined X-ray/neutron structure of H-FABP was complemented by a transferred multipolar electron-density distribution using the parameters of the ELMAMII library. The resulting electron density allowed a precise determination of the electrostatic potential in the fatty acid (FA) binding pocket. Bader's quantum theory of atoms in molecules was then used to study interactions involving the internal water molecules, the FA and the protein. This approach showed H⋯H contacts of the FA with highly conserved hydrophobic residues known to play a role in the stabilization of long-chain FAs in the binding cavity. The determination of water hydrogen (deuterium) positions allowed the analysis of the orientation and electrostatic properties of the water molecules in the very ordered cluster. As a result, a significant alignment of the permanent dipoles of the water molecules with the protein electrostatic field was observed. This can be related to the dielectric properties of hydration layers around proteins, where the shielding of electrostatic interactions depends directly on the rotational degrees of freedom of the water molecules in the interface.

Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

PubMed Central

Karttunen, Heidi; Savas, Jeffrey N.; McKinney, Caleb; Chen, Yu-Hung; Yates, John R.; Hukkanen, Veijo; Huang, Tony T.; Mohr, Ian

2015-01-01

SUMMARY DNA damage associated with viral DNA synthesis can result in double strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi Anemia (FA) genomic stability pathway is exploited by HSV1 to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV1-infected cells resulted in monoubiquitination of FA effector proteins, FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments and FANCI-D2 interacted with a multi-subunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, while HSV1 productive growth was impaired in monoubiquitination-defective FA patient cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for non-homologous end-joining (NHEJ). This identifies the FA-pathway as a new cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral lifecycle. PMID:24954902

Replication Protein A (RPA) deficiency activates the Fanconi anemia DNA repair pathway.

PubMed

Jang, Seok-Won; Jung, Jin Ki; Kim, Jung Min

2016-09-01

The Fanconi anemia (FA) pathway regulates DNA inter-strand crosslink (ICL) repair. Despite our greater understanding of the role of FA in ICL repair, its function in the preventing spontaneous genome instability is not well understood. Here, we show that depletion of replication protein A (RPA) activates the FA pathway. RPA1 deficiency increases chromatin recruitment of FA core complex, leading to FANCD2 monoubiquitination (FANCD2-Ub) and foci formation in the absence of DNA damaging agents. Importantly, ATR depletion, but not ATM, abolished RPA1 depletion-induced FANCD2-Ub, suggesting that ATR activation mediated FANCD2-Ub. Interestingly, we found that depletion of hSSB1/2-INTS3, a single-stranded DNA-binding protein complex, induces FANCD2-Ub, like RPA1 depletion. More interestingly, depletion of either RPA1 or INTS3 caused increased accumulation of DNA damage in FA pathway deficient cell lines. Taken together, these results indicate that RPA deficiency induces activation of the FA pathway in an ATR-dependent manner, which may play a role in the genome maintenance.

Folic acid inhibits COLO-205 colon cancer cell proliferation through activating the FRα/c-SRC/ERK1/2/NFκB/TP53 pathway: in vitro and in vivo studies

PubMed Central

Kuo, Chun-Ting; Chang, Chieh; Lee, Wen-Sen

2015-01-01

To investigate the molecular mechanism underlying folic acid (FA)-induced anti-colon caner activity, we showed that FA caused G0/G1 arrest in COLO-205. FA activated the proto-oncogene tyrosine-protein kinase Src (c-SRC)-mediated signaling pathway to enhance nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) nuclear translocation and binding onto the tumor protein p53 (TP53) gene promoter, and up-regulated expressions of TP53, cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 1B (CDKN1B). Knock-down of TP53 abolished FA-induced increases in the levels of CDKN1A and CDKN1B protein and G0/G1 arrest in COLO-205. Knock-down of folate receptor alpha (FRα) abolished FA-induced activations in the c-SRC-mediated pathway and increases in the levels of CDKN1A, CDKN1B and TP53 protein. These data suggest that FA inhibited COLO-205 proliferation through activating the FRα/c-SRC/mitogen-activated protein kinase 3/1 (ERK1/2)/NFκB/TP53 pathway-mediated up-regulations of CDKN1A and CDKN1B protein. In vivo studies demonstrated that daily i.p. injections of FA led to profound regression of the COLO-205 tumors and prolong the lifespan. In these tumors, the levels of CDKN1A, CDKN1B and TP53 protein were increased and von willebrand factor (VWF) protein levels were decreased. These findings suggest that FA inhibits COLO-205 colon cancer growth through anti-cancer cell proliferation and anti-angiogenesis. PMID:26056802

Fanconi Anemia and Laron Syndrome.

PubMed

Castilla-Cortazar, Inma; de Ita, Julieta Rodriguez; Aguirre, Gabriel Amador; Castorena-Torres, Fabiola; Ortiz-Urbina, Jesús; García-Magariño, Mariano; de la Garza, Rocío García; Diaz Olachea, Carlos; Elizondo Leal, Martha Irma

2017-05-01

Fanconi anemia (FA) is a condition characterized by genetic instability and short stature, which is due to growth hormone (GH) deficiency in most cases. However, no apparent relationships have been identified between FA complementation group genes and GH. In this study, we thereby considered an association between FA and Laron syndrome (LS) (insulin-like growth factor 1 [IGF-1] deficiency). A 21-year-old female Mexican patient with a genetic diagnosis of FA was referred to our research department for an evaluation of her short stature. Upon admission to our facility, her phenotype led to a suspicion of LS; accordingly, serum levels of IGF-1 and IGF binding protein 3 were analyzed and a GH stimulation test was performed. In addition, we used a next-generation sequencing approach for a molecular evaluation of FA disease-causing mutations and genes involved in the GH-IGF signaling pathway. Tests revealed low levels of IGF-1 and IGF binding protein 3 that remained within normal ranges, as well as a lack of response to GH stimulation. Sequencing confirmed a defect in the GH receptor signaling pathway. To the best of our knowledge, this study is the first to suggest an association between FA and LS. We propose that IGF-1 administration might improve some FA complications and functions based upon IGF-1 beneficial actions observed in animal, cell and indirect clinical models: erythropoiesis modulation, immune function improvement and metabolic regulation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

New insights into the operative network of FaEO, an enone oxidoreductase from Fragaria x ananassa Duch.

PubMed

Collu, Gabriella; Farci, Domenica; Esposito, Francesca; Pintus, Francesca; Kirkpatrick, Joanna; Piano, Dario

2017-05-01

The 2-methylene-furan-3-one reductase or Fragaria x ananassa Enone Oxidoreductase (FaEO) catalyses the last reductive step in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a major component in the characteristic flavour of strawberries. In the present work, we describe the association between FaEO and the vacuolar membrane of strawberry fruits. Even if FaEO lacks epitopes for stable or transient membrane-interactions, it contains a calmodulin-binding region, suggesting that in vivo FaEO may be associated with the membrane via a peripheral protein complex with calmodulin. Moreover, we also found that FaEO occurs in dimeric form in vivo and, as frequently observed for calmodulin-regulated proteins, it may be expressed in different isoforms by alternative gene splicing. Further mass spectrometry analysis confirmed that the isolated FaEO consists in the already known isoform and that it is the most characteristic during ripening. Finally, a characterization by absorption spectroscopy showed that FaEO has specific flavoprotein features. The relevance of these findings and their possible physiological implications are discussed.

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

PubMed

Narczyk, Marta; Bertoša, Branimir; Papa, Lucija; Vuković, Vedran; Leščić Ašler, Ivana; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Luić, Marija; Štefanić, Zoran

2018-04-01

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (P i ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes P i binding, while a three-site model is needed to characterize FA binding, irrespective of P i presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with P i and FA shows, however, that P i binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that P i moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463. © 2018 Federation of European Biochemical Societies.

Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

PubMed

Oka, Yasuyoshi; Bekker-Jensen, Simon; Mailand, Niels

2015-05-12

Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway. © 2015 The Authors.

Production of a Brassica napus low-molecular mass acyl-coenzyme A-binding protein in Arabidopsis alters the acyl-coenzyme A pool and acyl composition of oil in seeds

USDA-ARS?s Scientific Manuscript database

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expressio...

Fatty Acid Synthesis and Oxidation in Cumulus Cells Support Oocyte Maturation in Bovine

PubMed Central

Sanchez-Lazo, Laura; Brisard, Daphné; Elis, Sébastien; Maillard, Virginie; Uzbekov, Rustem; Labas, Valérie; Desmarchais, Alice; Papillier, Pascal; Monget, Philippe

2014-01-01

Oocyte meiotic maturation requires energy from various substrates including glucose, amino acids, and lipids. Mitochondrial fatty acid (FA) β-oxidation (FAO) in the oocyte is required for meiotic maturation, which is accompanied by differential expression of numerous genes involved in FAs metabolism in surrounding cumulus cells (CCs) in vivo. The objective was to elucidate components involved in FAs metabolism in CCs during oocyte maturation. Twenty-seven genes related to lipogenesis, lipolysis, FA transport, and FAO were chosen from comparative transcriptome analysis of bovine CCs before and after maturation in vivo. Using real-time PCR, 22 were significantly upregulated at different times of in vitro maturation (IVM) in relation to oocyte meiosis progression from germinal vesicle breakdown to metaphase-II. Proteins FA synthase, acetyl-coenzyme-A carboxylase, carnitine palmitoyltransferase, perilipin 2, and FA binding protein 3 were detected by Western blot and immunolocalized to CCs and oocyte cytoplasm, with FA binding protein 3 concentrated around oocyte chromatin. By mass spectrometry, CCs lipid profiling was shown to be different before and after IVM. FAO inhibitors etomoxir and mildronate dose-dependently decreased the oocyte maturation rate in vitro. In terms of viability, cumulus enclosed oocytes were more sensitive to etomoxir than denuded oocytes. In CCs, etomoxir (150μM) led to downregulation of lipogenesis genes and upregulated lipolysis and FAO genes. Moreover, the number of lipid droplets decreased, whereas several lipid species were more abundant compared with nontreated CCs after IVM. In conclusion, FAs metabolism in CCs is important to maintain metabolic homeostasis and may influence meiosis progression and survival of enclosed oocytes. PMID:25058602

Oleic Acid in the Ventral Tegmental Area Inhibits Feeding, Food Reward, and Dopamine Tone.

PubMed

Hryhorczuk, Cecile; Sheng, Zhenyu; Décarie-Spain, Léa; Giguère, Nicolas; Ducrot, Charles; Trudeau, Louis-Éric; Routh, Vanessa H; Alquier, Thierry; Fulton, Stephanie

2018-02-01

Long-chain fatty acids (FAs) act centrally to decrease food intake and hepatic glucose production and alter hypothalamic neuronal activity in a manner that depends on FA type and cellular transport proteins. However, it is not known whether FAs are sensed by ventral tegmental area (VTA) dopamine (DA) neurons to control food-motivated behavior and DA neurotransmission. We investigated the impact of the monounsaturated FA oleate in the VTA on feeding, locomotion, food reward, and DA neuronal activity and DA neuron expression of FA-handling proteins and FA uptake. A single intra-VTA injection of oleate, but not of the saturated FA palmitate, decreased food intake and increased locomotor activity. Furthermore, intra-VTA oleate blunted the rewarding effects of high-fat/sugar food in an operant task and inhibited DA neuronal firing. Using sorted DA neuron preparations from TH-eGFP mice we found that DA neurons express FA transporter and binding proteins, and are capable of intracellular transport of long-chain FA. Finally, we demonstrate that a transporter blocker attenuates FA uptake into DA neurons and blocks the effects of intra-VTA oleate to decrease food-seeking and DA neuronal activity. Together, these results suggest that DA neurons detect FA and that oleate has actions in the VTA to suppress DA neuronal activity and food seeking following cellular incorporation. These findings highlight the capacity of DA neurons to act as metabolic sensors by responding not only to hormones but also to FA nutrient signals to modulate food-directed behavior.

A new herbicidal site of action: Cinmethylin binds to acyl-ACP thioesterase and inhibits plant fatty acid biosynthesis.

PubMed

Campe, Ruth; Hollenbach, Eva; Kämmerer, Lara; Hendriks, Janneke; Höffken, Hans Wolfgang; Kraus, Helmut; Lerchl, Jens; Mietzner, Thomas; Tresch, Stefan; Witschel, Matthias; Hutzler, Johannes

2018-06-01

The prevalent occurrence of herbicide resistant weeds increases the necessity for new site of action herbicides for effective control as well as to relax selection pressure on the known sites of action. As a consequence, interest increased in the unexploited molecule cinmethylin as a new solution for the control of weedy grasses in cereals. Therefore, the mechanism of action of cinmethylin was reevaluated. We applied the chemoproteomic approach cellular Target Profiling™ from Evotec to identify the cinmethylin target in Lemna paucicostata protein extracts. We found three potential targets belonging to the same protein family of fatty acid thioesterases (FAT) to bind to cinmethylin with high affinity. Binding of cinmethylin to FAT proteins from Lemna and Arabidopsis was confirmed by fluorescence-based thermal shift assay. The plastid localized enzyme FAT plays a crucial role in plant lipid biosynthesis, by mediating the release of fatty acids (FA) from its acyl carrier protein (ACP) which is necessary for FA export to the endoplasmic reticulum. GC-MS analysis of free FA composition in Lemna extracts revealed strong reduction of unsaturated C18 as well as saturated C14, and C16 FAs upon treatment with cinmethylin, indicating that FA release for subsequent lipid biosynthesis is the primary target of cinmethylin. Lipid biosynthesis is a prominent target of different herbicide classes. To assess whether FAT inhibition constitutes a new mechanism of action within this complex pathway, we compared physiological effects of cinmethylin to different ACCase and VLCFA synthesis inhibitors and identified characteristic differences in plant symptomology and free FA composition upon treatment with the three herbicide classes. Also, principal component analysis of total metabolic profiling of treated Lemna plants showed strong differences in overall metabolic changes after cinmethylin, ACCase or VLCFA inhibitor treatments. Our results identified and confirmed FAT as the cinmethylin target and validate FAT inhibition as a new site of action different from other lipid biosynthesis inhibitor classes. Copyright © 2018 BASF SE. Published by Elsevier Inc. All rights reserved.

AMP-activated protein kinase is involved in the activation of the Fanconi anemia/BRCA pathway in response to DNA interstrand crosslinks.

PubMed

Chun, Min Jeong; Kim, Sunshin; Hwang, Soo Kyung; Kim, Bong Sub; Kim, Hyoun Geun; Choi, Hae In; Kim, Jong Heon; Goh, Sung Ho; Lee, Chang-Hun

2016-08-16

Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway.

Liver Fatty acid binding protein (L-Fabp) modulates murine stellate cell activation and diet induced nonalcoholic fatty liver disease

PubMed Central

Chen, Anping; Tang, Youcai; Davis, Victoria; Hsu, Fong-Fu; Kennedy, Susan M.; Song, Haowei; Turk, John; Brunt, Elizabeth M.; Newberry, Elizabeth P.; Davidson, Nicholas O.

2013-01-01

Activation of hepatic stellate cells (HSCs) is crucial to the development of fibrosis in nonalcoholic fatty liver disease. Quiescent HSCs contain lipid droplets (LDs), whose depletion upon activation induces a fibrogenic gene program. Here we show that liver fatty acid-binding protein (L-Fabp), an abundant cytosolic protein that modulates fatty acid (FA) metabolism in enterocytes and hepatocytes also modulates HSC FA utilization and in turn regulates the fibrogenic program. L-Fabp expression decreased 10-fold following HSC activation, concomitant with depletion of LDs. Primary HSCs isolated from L-FABP−/− mice contain fewer LDs than wild type (WT) HSCs, and exhibit upregulated expression of genes involved in HSC activation. Adenoviral L-Fabp transduction inhibited activation of passaged WT HSCs and increased both the expression of prolipogenic genes and also augmented intracellular lipid accumulation, including triglyceride and FA, predominantly palmitate. Freshly isolated HSCs from L-FABP−/− mice correspondingly exhibited decreased palmitate in the free FA pool. To investigate whether L-FABP deletion promotes HSC activation in vivo, we fed L-FABP−/− and WT mice a high fat diet supplemented with trans-fatty acids and fructose (TFF). TFF-fed L-FABP−/− mice exhibited reduced hepatic steatosis along with decreased LD abundance and size compared to WT mice. In addition, TFF-fed L-FABP−/− mice exhibited decreased hepatic fibrosis, with reduced expression of fibrogenic genes, compared to WT mice. Conclusion L-FABP deletion attenuates both diet-induced hepatic steatosis and fibrogenesis, despite the observation that L-Fabp paradoxically promotes FA and LD accumulation and inhibits HSC activation in vitro. These findings highlight the importance of cell-specific modulation of hepatic lipid metabolism in promoting fibrogenesis in nonalcoholic fatty liver disease. PMID:23401290

Insight into the heavy metal binding potential of dissolved organic matter in MSW leachate using EEM quenching combined with PARAFAC analysis.

PubMed

Wu, Jun; Zhang, Hua; He, Pin-Jing; Shao, Li-Ming

2011-02-01

Dissolved organic matter (DOM) plays an important role in heavy metal migration from municipal solid waste (MSW) to aquatic environments via the leachate pathway. In this study, fluorescence excitation-emission matrix (EEM) quenching combined with parallel factor (PARAFAC) analysis was adopted to characterize the binding properties of four heavy metals (Cu, Pb, Zn and Cd) and DOM in MSW leachate. Nine leachate samples were collected from various stages of MSW management, including collection, transportation, incineration, landfill and subsequent leachate treatment. Three humic-like components and one protein-like component were identified in the MSW-derived DOM by PARAFAC. Significant differences in quenching effects were observed between components and metal ions, and a relatively consistent trend in metal quenching curves was observed among various leachate samples. Among the four heavy metals, Cu(II) titration led to fluorescence quenching of all four PARAFAC-derived components. Additionally, strong quenching effects were only observed in protein-like and fulvic acid (FA)-like components with the addition of Pb(II), which suggested that these fractions are mainly responsible for Pb(II) binding in MSW-derived DOM. Moreover, the significant quenching effects of the FA-like component by the four heavy metals revealed that the FA-like fraction in MSW-derived DOM plays an important role in heavy metal speciation; therefore, it may be useful as an indicator to assess the potential ability of heavy metal binding and migration. © 2010 Elsevier Ltd. All rights reserved.

ACF7 regulates cytoskeletal-focal adhesion dynamics and migration and has ATPase activity.

PubMed

Wu, Xiaoyang; Kodama, Atsuko; Fuchs, Elaine

2008-10-03

Coordinated interactions between microtubule (MT) and actin cytoskeletons are involved in many polarized cellular processes. Spectraplakins are enormous (>500 kDa) proteins able to bind both MTs and actin filaments (F-actin) directly. To elucidate the physiological significance and functions of mammalian spectraplakin ACF7, we've conditionally targeted it in skin epidermis. Intriguingly, ACF7 deficiency compromises the targeting of microtubules along F-actin to focal adhesions (FAs), stabilizes FA-actin networks, and impairs epidermal migration. Exploring underlying mechanisms, we show that ACF7's binding domains for F-actin, MTs, and MT plus-end proteins are not sufficient to rescue the defects in FA-cytoskeletal dynamics and migration functions of ACF7 null keratinocytes. We've uncovered an intrinsic actin-regulated ATPase domain in ACF7 and demonstrate that it is both functional and essential for these roles. Our findings provide insight into the functions of this important cytoskeletal crosslinking protein in regulating dynamic interactions between MTs and F-actin to sustain directional cell movement.

Maternal BMI and gestational diabetes alter placental lipid transporters and fatty acid composition.

PubMed

Segura, Maria Teresa; Demmelmair, Hans; Krauss-Etschmann, Susanne; Nathan, Petra; Dehmel, Stefan; Padilla, Maria Carmen; Rueda, Ricardo; Koletzko, Berthold; Campoy, Cristina

2017-09-01

Placental fatty acid (FA) uptake and metabolism depend on maternal supply which may be altered when women have a high pre-pregnancy body mass index (BMI) or develop gestational diabetes (GDM). Consequently, an impaired FA transport to the fetus may negatively affect fetal development. While placental adaptation of maternal-fetal glucose transfer in mild GDM has been described, knowledge on placental FA acid metabolism and possible adaptations in response to maternal obesity or GDM is lacking. We aimed to analyze the FA composition and the expression of key genes involved in FA uptake and metabolism in placentas from women with pre-pregnancy normal weight (18.5 ≤ BMI<25 kg/m2), overweight (25 ≤ BMI<30 kg/m 2 ), obesity (BMI ≥ 30 kg/m 2 ), and lean pregnant women with GDM. Placental FA content was determined by gas liquid chromatography. Placental mRNA expression of FA transport proteins (FATP1, FATP4, FATP6), FA binding proteins (FABP3, FABP4, FABP7), FA translocase (FAT/CD36) and enzymes (Endothelial lipase (EL) and lipoprotein lipase (LPL)) were quantified by qRT-PCR. High pre-pregnancy BMI and GDM were associated with decreased placental FATP1, FATP4, EL and increased FAT/CD36 and FATP6 expressions. LPL mRNA levels and placental total FA content were similar among groups. Specific FA, including some long-chain polyunsaturated FA, were altered. Our results demonstrate that high pre-pregnancy BMI or GDM independently alter mRNA expression levels of genes involved in FA uptake and metabolism and the placental FA profile, which could affect fetal development and long-term health. Copyright © 2017. Published by Elsevier Ltd.

Ferulic Acid Administered at Various Time Points Protects against Cerebral Infarction by Activating p38 MAPK/p90RSK/CREB/Bcl-2 Anti-Apoptotic Signaling in the Subacute Phase of Cerebral Ischemia-Reperfusion Injury in Rats.

PubMed

Cheng, Chin-Yi; Tang, Nou-Ying; Kao, Shung-Te; Hsieh, Ching-Liang

2016-01-01

This study aimed to evaluate the effects of ferulic acid (FA) administered at various time points before or after 30 min of middle cerebral artery occlusion (MCAo) followed by 7 d of reperfusion and to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the cortical penumbra. FA was intravenously administered to rats at a dose of 100 mg/kg 24 h before ischemia (B-FA), 2 h before ischemia (P-FA), immediately after ischemic insult (I-FA), 2 h after reperfusion (R-FA), or 24 h after reperfusion (D-FA). Our study results indicated that P-FA, I-FA, and R-FA effectively reduced cerebral infarct areas and neurological deficits. P-FA, I-FA, and R-FA significantly downregulated glial fibrillary acidic protein (GFAP), mitochondrial Bax, cytochrome c, and cleaved caspase-3 expression, and effectively restored the phospho-p38 MAPK (p-p38 MAPK)/p38 MAPK ratio, phospho-90 kDa ribosomal S6 kinase (p-p90RSK) expression, phospho-Bad (p-Bad) expression, the phospho-cAMP response element-binding protein (p-CREB)/CREB ratio, the cytosolic and mitochondrial Bcl-2/Bax ratios, and the cytosolic Bcl-xL/Bax ratio in the cortical penumbra 7 d after reperfusion. SB203580, a specific inhibitor of p38 MAPK, administered 30 min prior to ischemia abrogated the downregulating effects of I-FA on cerebral infarction, and mitochondrial Bax and cleaved caspase-3 expression, and the upregulating effects of I-FA on the p-p38 MAPK/p38 MAPK ratio, p-p90RSK expression, p-Bad expression, and the p-CREB/CREB, and cytosolic and mitochondrial Bcl-2/Bax ratios. Our study results thus indicate that P-FA, I-FA, and R-FA effectively suppress reactive astrocytosis and exert neuroprotective effects against cerebral infarction by activating p38 MAPK signaling. The regulating effects of P-FA, I-FA, and R-FA on Bax-induced apoptosis result from activation of the p38 MAPK/p90RSK/CREB/Bcl-2 signaling pathway, and eventually contribute to inhibition of the cytochrome c-mediated caspase-3-dependent apoptotic pathway in the cortical penumbra 7 d after reperfusion.

Ferulic Acid Administered at Various Time Points Protects against Cerebral Infarction by Activating p38 MAPK/p90RSK/CREB/Bcl-2 Anti-Apoptotic Signaling in the Subacute Phase of Cerebral Ischemia-Reperfusion Injury in Rats

PubMed Central

Cheng, Chin-Yi; Tang, Nou-Ying; Kao, Shung-Te; Hsieh, Ching-Liang

2016-01-01

Objectives This study aimed to evaluate the effects of ferulic acid (FA) administered at various time points before or after 30 min of middle cerebral artery occlusion (MCAo) followed by 7 d of reperfusion and to examine the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in the cortical penumbra. Methods FA was intravenously administered to rats at a dose of 100 mg/kg 24 h before ischemia (B-FA), 2 h before ischemia (P-FA), immediately after ischemic insult (I-FA), 2 h after reperfusion (R-FA), or 24 h after reperfusion (D-FA). Results Our study results indicated that P-FA, I-FA, and R-FA effectively reduced cerebral infarct areas and neurological deficits. P-FA, I-FA, and R-FA significantly downregulated glial fibrillary acidic protein (GFAP), mitochondrial Bax, cytochrome c, and cleaved caspase-3 expression, and effectively restored the phospho-p38 MAPK (p-p38 MAPK)/p38 MAPK ratio, phospho-90 kDa ribosomal S6 kinase (p-p90RSK) expression, phospho-Bad (p-Bad) expression, the phospho-cAMP response element-binding protein (p-CREB)/CREB ratio, the cytosolic and mitochondrial Bcl-2/Bax ratios, and the cytosolic Bcl-xL/Bax ratio in the cortical penumbra 7 d after reperfusion. SB203580, a specific inhibitor of p38 MAPK, administered 30 min prior to ischemia abrogated the downregulating effects of I-FA on cerebral infarction, and mitochondrial Bax and cleaved caspase-3 expression, and the upregulating effects of I-FA on the p-p38 MAPK/p38 MAPK ratio, p-p90RSK expression, p-Bad expression, and the p-CREB/CREB, and cytosolic and mitochondrial Bcl-2/Bax ratios. Conclusions Our study results thus indicate that P-FA, I-FA, and R-FA effectively suppress reactive astrocytosis and exert neuroprotective effects against cerebral infarction by activating p38 MAPK signaling. The regulating effects of P-FA, I-FA, and R-FA on Bax-induced apoptosis result from activation of the p38 MAPK/p90RSK/CREB/Bcl-2 signaling pathway, and eventually contribute to inhibition of the cytochrome c-mediated caspase-3-dependent apoptotic pathway in the cortical penumbra 7 d after reperfusion. PMID:27187745

AMP-activated protein kinase is involved in the activation of the Fanconi anemia/BRCA pathway in response to DNA interstrand crosslinks

PubMed Central

Hwang, Soo Kyung; Kim, Bong Sub; Kim, Hyoun Geun; Choi, Hae In; Kim, Jong Heon; Goh, Sung Ho; Lee, Chang-Hun

2016-01-01

Fanconi anemia complementation group (FANC) proteins constitute the Fanconi Anemia (FA)/BRCA pathway that is activated in response to DNA interstrand crosslinks (ICLs). We previously performed yeast two-hybrid screening to identify novel FANC-interacting proteins and discovered that the alpha subunit of AMP-activated protein kinase (AMPKα1) was a candidate binding partner of the FANCG protein, which is a component of the FA nuclear core complex. We confirmed the interaction between AMPKα and both FANCG using co-immunoprecipitation experiments. Additionally, we showed that AMPKα interacted with FANCA, another component of the FA nuclear core complex. AMPKα knockdown in U2OS cells decreased FANCD2 monoubiquitination and nuclear foci formation upon mitomycin C-induced ICLs. Furthermore, AMPKα knockdown enhanced cellular sensitivity to MMC. MMC treatment resulted in an increase in AMPKα phosphorylation/activation, indicating AMPK is involved in the cellular response to ICLs. FANCA was phosphorylated by AMPK at S347 and phosphorylation increased with MMC treatment. MMC-induced FANCD2 monoubiquitination and nuclear foci formation were compromised in a U2OS cell line that stably overexpressed the S347A mutant form of FANCA compared to wild-type FANCA-overexpressing cells, indicating a requirement for FANCA phosphorylation at S347 for proper activation of the FA/BRCA pathway. Our data suggest AMPK is involved in the activation of the FA/BRCA pathway. PMID:27449087

FMRFamide-related peptides: anti-opiate transmitters acting in apoptosis.

PubMed

Rőszer, Tamás; Bánfalvi, Gáspár

2012-03-01

Members of the FMRFamide-related peptide (FaRP) family are neurotransmitters, hormone-like substances and tumor suppressor peptides. In mammals, FaRPs are considered as anti-opiate peptides due to their ability to inhibit opioid signaling. Some FaRPs are asserted to attenuate opiate tolerance. A recently developed chimeric FaRP (Met-enkephalin-FMRFa) mimics the analgesic effects of opiates without the development of opiate-dependence, displaying a future therapeutical potential in pain reduction. In this review we support the notion, that opiates and representative members of the FaRP family show overlapping effects on apoptosis. Binding of FaRPs to opioid receptors or to their own receptors (G-protein linked membrane receptors and acid-sensing ion channels) evokes or suppresses cell death, in a cell- and receptor-type manner. With the dramatically increasing incidence of opiate abuse and addiction, understanding of opioid-induced cell death, and in this context FaRPs will deserve growing attention. Copyright © 2011 Elsevier Inc. All rights reserved.

Plant-mediated silencing of the fatty acid- and retinoid-binding Pp-far-1 gene can reduce development of the root lesion nematode, Pratylenchus penetrans

USDA-ARS?s Scientific Manuscript database

Pratylenchus penetrans is one of the most important plant-parasitic nematodes and can act as a limiting factor of important agricultural, horticultural and industrial crops. Fatty acid- and retinoid- (FAR) binding proteins are unique to nematodes. The cDNA corresponding to a putative P. penetrans FA...

Fatty acid-binding protein 5 (FABP5) promotes lipolysis of lipid droplets, de novo fatty acid (FA) synthesis and activation of nuclear factor-kappa B (NF-κB) signaling in cancer cells.

PubMed

Senga, Shogo; Kobayashi, Narumi; Kawaguchi, Koichiro; Ando, Akira; Fujii, Hiroshi

2018-06-12

Fatty acid-binding proteins (FABPs) are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting them to the appropriate compartments in the cell. Epidermal fatty acid-binding protein (FABP5) is an intracellular lipid-binding protein that is abundantly expressed in adipocytes and macrophages. Previous studies have revealed that the FABP5 expression level is closely related to malignancy in various types of cancer. However, its precise functions in the metabolisms of cancer cells remain unclear. Here, we revealed that FABP5 knockdown significantly induced downregulation of the genes expression, such as hormone-sensitive lipase (HSL), monoacylglycerol lipase (MAGL), elongation of long-chain fatty acid member 6 (Elovl6), and acyl-CoA synthetase long-chain family member 1 (ACSL1), which are involved in altered lipid metabolism, lipolysis, and de novo FA synthesis in highly aggressive prostate and breast cancer cells. Moreover, we demonstrated that FABP5 induced inflammation and cytokine production through the nuclear factor-kappa B signaling pathway activated by reactive oxygen species and protein kinase C in PC-3 and MDA-MB-231 cells. Thus, FABP5 might regulate lipid quality and/or quantity to promote aggressiveness such as cell growth, invasiveness, survival, and inflammation in prostate and breast cancer cells. In the present study, we have revealed for the first time that high expression of FABP5 plays a critical role in alterations of lipid metabolism, leading to cancer development and metastasis in highly aggressive prostate and breast cancer cells. Copyright © 2018. Published by Elsevier B.V.

The potassium channel FaTPK1 plays a critical role in fruit quality formation in strawberry (Fragaria × ananassa).

PubMed

Wang, Shufang; Song, Miaoyu; Guo, Jiaxuan; Huang, Yun; Zhang, Fangfang; Xu, Cheng; Xiao, Yinghui; Zhang, Lusheng

2018-03-01

Potassium (K + ), an abundant cation in plant cells, is important in fruit development and plant resistance. However, how cellular K + is directed by potassium channels in fruit development and quality formation of strawberry (Fragaria × ananassa) is not yet fully clear. Here, a two-pore K + (TPK) channel gene in strawberry, FaTPK1, was cloned using reverse transcription-PCR. A green fluorescent protein subcellular localization analysis showed that FaTPK1 localized in the vacuole membrane. A transcription analysis indicated that the mRNA expression level of FaTPK1 increased rapidly and was maintained at a high level in ripened fruit, which was coupled with the fruit's red colour development, suggesting that FaTPK1 is related to fruit quality formation. The down- and up-regulation of the FaTPK1 mRNA expression levels using RNA interference and overexpression, respectively, inhibited and promoted fruit ripening, respectively, as demonstrated by consistent changes in firmness and the contents of soluble sugars, anthocyanin and abscisic acid, as well as the transcript levels of ripening-regulated genes PG1 (polygalacturonase), GAL6 (beta-galactosidase), XYL2 (D-xylulose reductase), SUT1 (sucrose transporter), CHS (chalcone synthase) and CHI (chalcone flavanone isomerase). Additionally, the regulatory changes influenced fruit resistance to Botrytis cinerea. An isothermal calorimetry analysis showed that the Escherichia coli-expressed FaTPK1 recombinant protein could bind K + with a binding constant of 2.1 × 10 -3  m -1 and a dissociation constant of 476 μm. Thus, the strawberry TPK1 is a ubiquitously expressed, tonoplast-localized two-pore potassium channel that plays important roles in fruit ripening and quality formation. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

A critical role of fatty acid binding protein 4 and 5 (FABP4/5) in the systemic response to fasting.

PubMed

Syamsunarno, Mas Rizky A A; Iso, Tatsuya; Hanaoka, Hirofumi; Yamaguchi, Aiko; Obokata, Masaru; Koitabashi, Norimichi; Goto, Kosaku; Hishiki, Takako; Nagahata, Yoshiko; Matsui, Hiroki; Sano, Motoaki; Kobayashi, Masaki; Kikuchi, Osamu; Sasaki, Tsutomu; Maeda, Kazuhisa; Murakami, Masami; Kitamura, Tadahiro; Suematsu, Makoto; Tsushima, Yoshito; Endo, Keigo; Hotamisligil, Gökhan S; Kurabayashi, Masahiko

2013-01-01

During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the (125)I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the modulation of energy homeostasis.

A Critical Role of Fatty Acid Binding Protein 4 and 5 (FABP4/5) in the Systemic Response to Fasting

PubMed Central

Syamsunarno, Mas Rizky A. A.; Iso, Tatsuya; Hanaoka, Hirofumi; Yamaguchi, Aiko; Obokata, Masaru; Koitabashi, Norimichi; Goto, Kosaku; Hishiki, Takako; Nagahata, Yoshiko; Matsui, Hiroki; Sano, Motoaki; Kobayashi, Masaki; Kikuchi, Osamu; Sasaki, Tsutomu; Maeda, Kazuhisa; Murakami, Masami; Kitamura, Tadahiro; Suematsu, Makoto; YoshitoTsushima; Endo, Keigo; Hotamisligil, Gökhan S.; Kurabayashi, Masahiko

2013-01-01

During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the 125I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the modulation of energy homeostasis. PMID:24244493

Structural and Binding Properties of Two Paralogous Fatty Acid Binding Proteins of Taenia solium Metacestode

PubMed Central

Yang, Hyun-Jong; Shin, Joo-Ho; Diaz-Camacho, Sylvia Paz; Nawa, Yukifumi; Kang, Insug; Kong, Yoon

2012-01-01

Background Fatty acid (FA) binding proteins (FABPs) of helminths are implicated in acquisition and utilization of host-derived hydrophobic substances, as well as in signaling and cellular interactions. We previously demonstrated that secretory hydrophobic ligand binding proteins (HLBPs) of Taenia solium metacestode (TsM), a causative agent of neurocysticercosis (NC), shuttle FAs in the surrounding host tissues and inwardly transport the FAs across the parasite syncytial membrane. However, the protein molecules responsible for the intracellular trafficking and assimilation of FAs have remained elusive. Methodology/Principal Findings We isolated two novel TsMFABP genes (TsMFABP1 and TsMFABP2), which encoded 133- and 136-amino acid polypeptides with predicted molecular masses of 14.3 and 14.8 kDa, respectively. They shared 45% sequence identity with each other and 15–95% with other related-members. Homology modeling demonstrated a characteristic β-barrel composed of 10 anti-parallel β-strands and two α-helices. TsMFABP2 harbored two additional loops between β-strands two and three, and β-strands six and seven, respectively. TsMFABP1 was secreted into cyst fluid and surrounding environments, whereas TsMFABP2 was intracellularly confined. Partially purified native proteins migrated to 15 kDa with different isoelectric points of 9.2 (TsMFABP1) and 8.4 (TsMFABP2). Both native and recombinant proteins bound to 11-([5-dimethylaminonaphthalene-1-sulfonyl]amino)undecannoic acid, dansyl-DL-α-amino-caprylic acid, cis-parinaric acid and retinol, which were competitively inhibited by oleic acid. TsMFABP1 exhibited high affinity toward FA analogs. TsMFABPs showed weak binding activity to retinol, but TsMFABP2 showed relatively high affinity. Isolation of two distinct genes from an individual genome strongly suggested their paralogous nature. Abundant expression of TsMFABP1 and TsMFABP2 in the canal region of worm matched well with the histological distributions of lipids and retinol. Conclusions/Significance The divergent biochemical properties, physiological roles and cellular distributions of the TsMFABPs might be one of the critical mechanisms compensating for inadequate de novo FA synthesis. These proteins might exert harmonized or independent roles on lipid assimilation and intracellular signaling. The specialized distribution of retinol in the canal region further implies that cells in this region might differentiate into diverse cell types during metamorphosis into an adult worm. Identification of bioactive systems pertinent to parasitic homeostasis may provide a valuable target for function-related drug design. PMID:23150743

Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase

PubMed Central

Sareen, Archana; Chaudhury, Indrajit; Adams, Nicole; Sobeck, Alexandra

2012-01-01

Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2–FANCI complex versus the monomeric proteins are. We show that the FANCD2–FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2–FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to—and independently of—FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase. PMID:22753026

Multiple TPR motifs characterize the Fanconi anemia FANCG protein.

PubMed

Blom, Eric; van de Vrugt, Henri J; de Vries, Yne; de Winter, Johan P; Arwert, Fré; Joenje, Hans

2004-01-05

The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.

Association of polymorphisms in solute carrier family 27, isoform A6 (SLC27A6) and fatty acid-binding protein-3 and fatty acid-binding protein-4 (FABP3 and FABP4) with fatty acid composition of bovine milk.

PubMed

Nafikov, R A; Schoonmaker, J P; Korn, K T; Noack, K; Garrick, D J; Koehler, K J; Minick-Bormann, J; Reecy, J M; Spurlock, D E; Beitz, D C

2013-09-01

The main goal of this study was to develop tools for genetic selection of animals producing milk with a lower concentration of saturated fatty acids (SFA) and a higher concentration of unsaturated fatty acids (UFA). The reasons for changing milk fatty acid (FA) composition were to improve milk technological properties, such as for production of more spreadable butter, and milk nutritional value with respect to the potentially adverse effects of SFA on human health. We hypothesized that genetic polymorphisms in solute carrier family 27, isoform A6 (SLC27A6) fatty acid transport protein gene and fatty acid binding protein (FABP)-3 and FABP-4 (FABP3 and FABP4) would affect the selectivity of FA uptake into, and FA redistribution inside, mammary epithelial cells, resulting in altered FA composition of bovine milk. The objectives of our study were to discover genetic polymorphisms in SLC27A6, FABP3, and FABP4, and to test those polymorphisms for associations with milk FA composition. The results showed that after pairwise comparisons between SLC27A6 haplotypes for significantly associated traits, haplotype H3 was significantly associated with 1.37 weight percentage (wt%) lower SFA concentration, 0.091 lower SFA:UFA ratio, and 0.17 wt% lower lauric acid (12:0) concentration, but 1.37 wt% higher UFA and 1.24 wt% higher monounsaturated fatty acid (MUFA) concentrations compared with haplotype H1 during the first 3 mo of lactation. Pairwise comparisons between FABP4 haplotypes for significantly associated traits showed that haplotype H3 was significantly associated with 1.04 wt% lower SFA concentration, 0.079 lower SFA:UFA ratio, 0.15 wt% lower lauric acid (12:0), and 0.27 wt% lower myristic acid (14:0) concentrations, but 1.04 wt% higher UFA and 0.91 wt% higher MUFA concentrations compared with haplotype H1 during the first 3 mo of lactation. Percentages of genetic variance explained by H3 versus H1 haplotype substitutions for SLC27A6 and FABP4 ranged from 2.50 to 4.86% and from 4.91 to 7.22%, respectively. Tag single nucleotide polymorphisms were identified to distinguish haplotypes H3 of SLC27A6 and FABP4 from others encompassing each gene. We found no significant associations between FABP3 haplotypes and milk FA composition. In conclusion, polymorphisms in FABP4 and SLC27A6 can be used to select for cattle producing milk with lower concentrations of SFA and higher concentrations of UFA. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Insulin enhances the peroxidase activity of heme by forming heme-insulin complex: Relevance to type 2 diabetes mellitus.

PubMed

Huang, Yi; Yang, Zhen; Xu, Huan; Zhang, Pengfei; Gao, Zhonghong; Li, Hailing

2017-09-01

Evidences have implicated the involvement of heme in the type 2 diabetes mellitus (T2Dm) pathogenesis, but possible mediators linking between heme and diabetes are still poorly understood. Here, we explored a potential mechanism that linked heme, insulin and diabetes. Our results demonstrated the formation of heme-insulin complex by two classical methods, i.e. UV-vis and capillary electrophoresis-frontal analysis (CE-FA). UV-vis results implied heme binding insulin via bis-histidine sites, and CE-FA further revealed that, when insulin uses two sites binding with heme, this interaction occurs at high affinity (K d =3.13×10 -6 M). Molecule docking supported that histidine-B5 of insulin binds with heme-Fe. In addition to that, tyrosine-B26, phenylalanine-B1 and valine-B2 are also contributed to binding heme. The binding amplified the peroxidase activity of heme itself. Under oxidative and nitrative stress, it affects pathogenesis of diabetes from two aspects: promoting insulin cross-linking that leads to permanent loss of insulin functionality on one hand, and enhancing protein tyrosine nitration that may result in inactivation of proteins associated with diabetes on the other hand. This study suggested that the enhanced peroxidase activity of heme through binding with insulin might be a previously unrecognized contributor to the pathogenesis of T2Dm in some heme-associated disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Long-chain fatty acid uptake by skeletal muscle is impaired in homozygous, but not heterozygous, heart-type-FABP null mice.

PubMed

Luiken, J J F P; Koonen, D P Y; Coumans, W A; Pelsers, M M A L; Binas, B; Bonen, A; Glatz, J F C

2003-04-01

Previous studies with cardiac myocytes from homozygous heart-type fatty acid (FA)-binding protein (H-FABP) -/- mice have indicated that this intracellular receptor protein for long-chain FA is involved in the cellular uptake of these substrates. Based on the knowledge that muscle FA uptake is a process highly sensitive to regulation by hormonal and mechanical stimuli, we studied whether H-FABP would play a role in this regulation. A suitable model system to answer this question is provided by H-FABP +/- mice, because in hindlimb muscles the content of H-FABP was measured to be 34% compared to wild-type mice. In these H-FABP +/- skeletal muscles, just as in H-FABP -/- muscles, contents of FA transporters, i.e., 43-kDa FABPpm and 88-kDa FAT/CD36, were similar compared to wild-type muscles, excluding possible compensatory mechanisms at the sarcolemmal level. Palmitate uptake rates were measured in giant vesicles prepared from hindlimb muscles of H-FABP -/-, H-FABP +/-, and H-FABP +/+ mice. For comparison, giant vesicles were isolated from liver, the tissue of which expresses a distinct type of FABP (i.e., L-FABP). Whereas in H-FABP -/- skeletal muscle FA uptake was reduced by 42-45%, FA uptake by H-FABP +/- skeletal muscle was not different from that in wild-type mice. In contrast, in liver from H-FABP -/- and from H-FABP +/- mice, FA uptake was not altered compared to wild-type animals, indicating that changes in FA uptake are restricted to H-FABP expressing tissues. It is concluded that H-FABP plays an important, yet merely permissive, role in FA uptake into muscle tissues.

Effects of antiglucocorticoid RU 486 on development of obesity in obese fa/fa Zucker rats.

PubMed

Langley, S C; York, D A

1990-09-01

The effects of RU 486 (mitepristone), an antagonist of type II glucocorticoid receptors (GR), on the development of obesity in young 5-wk-old obese fa/fa rats has been investigated. After 15 days of treatment, body composition of obese RU 486-treated rats was similar to that of lean-vehicle rats. Analysis of body composition changes showed that RU 486 effectively reversed the obesity. It stopped fat deposition in obese rats but increased protein deposition to the level of lean-vehicle rats. RU 486 prevented the development of hyperphagia and reduced gross energetic efficiency in the obese rats but had little effect on lean rats. Brown adipose tissue mitochondrial GDP binding was increased in obese rats but was reduced in lean rats by RU 486 treatment. RU 486 also reduced the elevated activity of hippocampal glycerophosphate dehydrogenase, a glucocorticoid-responsive enzyme, of obese rats to the level of lean rats. The evidence suggests that abnormal activity of glucocorticoid GR receptors or abnormal cellular responsiveness to corticosterone receptor complexes may be important in the development of obesity in the fa/fa rat.

DNA interstrand cross-link repair: understanding role of Fanconi anemia pathway and therapeutic implications.

PubMed

Shukla, Pallavi; Solanki, Avani; Ghosh, Kanjaksha; Vundinti, Babu Rao

2013-11-01

Interstrand cross-links (ICLs) are extremely toxic DNA lesions that prevent DNA double-helix separation due to the irreversible covalent linkage binding of some agents on DNA strands. Agents that induce these ICLs are thus widely used as chemotherapeutic drugs but may also lead to tumor growth. Fanconi anemia (FA) is a rare genetic disorder that leads to ICL sensitivity. This review provides update on current understanding of the role of FA proteins in repairing ICLs at various stages of cell cycle. We also discuss link between DNA cross-link genotoxicity caused by aldehydes in FA pathway. Besides this, we summarize various ICL agents that act as drugs to treat different types of tumors and highlight strategies for modulating ICL sensitivity for therapeutic interventions that may be helpful in controlling cancer and life-threatening disease, FA. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Experimental determination and modeling of arsenic complexation with humic and fulvic acids.

PubMed

Fakour, Hoda; Lin, Tsair-Fuh

2014-08-30

The complexation of humic acid (HA) and fulvic acid (FA) with arsenic (As) in water was studied. Experimental results indicate that arsenic may form complexes with HA and FA with a higher affinity for arsenate than for arsenite. With the presence of iron oxide based adsorbents, binding of arsenic to HA/FA in water was significantly suppressed, probably due to adsorption of As and HA/FA. A two-site ligand binding model, considering only strong and weak site types of binding affinity, was successfully developed to describe the complexation of arsenic on the two natural organic fractions. The model showed that the numbers of weak sites were more than 10 times those of strong sites on both HA and FA for both arsenic species studied. The numbers of both types of binding sites were found to be proportional to the HA concentrations, while the apparent stability constants, defined for describing binding affinity between arsenic and the sites, are independent of the HA concentrations. To the best of our knowledge, this is the first study to characterize the impact of HA concentrations on the applicability of the ligand binding model, and to extrapolate the model to FA. The obtained results may give insights on the complexation of arsenic in HA/FA laden groundwater and on the selection of more effective adsorption-based treatment methods for natural waters. Copyright © 2014 Elsevier B.V. All rights reserved.

Uncertainty analysis of the nonideal competitive adsorption-donnan model: effects of dissolved organic matter variability on predicted metal speciation in soil solution.

PubMed

Groenenberg, Jan E; Koopmans, Gerwin F; Comans, Rob N J

2010-02-15

Ion binding models such as the nonideal competitive adsorption-Donnan model (NICA-Donnan) and model VI successfully describe laboratory data of proton and metal binding to purified humic substances (HS). In this study model performance was tested in more complex natural systems. The speciation predicted with the NICA-Donnan model and the associated uncertainty were compared with independent measurements in soil solution extracts, including the free metal ion activity and fulvic (FA) and humic acid (HA) fractions of dissolved organic matter (DOM). Potentially important sources of uncertainty are the DOM composition and the variation in binding properties of HS. HS fractions of DOM in soil solution extracts varied between 14 and 63% and consisted mainly of FA. Moreover, binding parameters optimized for individual FA samples show substantial variation. Monte Carlo simulations show that uncertainties in predicted metal speciation, for metals with a high affinity for FA (Cu, Pb), are largely due to the natural variation in binding properties (i.e., the affinity) of FA. Predictions for metals with a lower affinity (Cd) are more prone to uncertainties in the fraction FA in DOM and the maximum site density (i.e., the capacity) of the FA. Based on these findings, suggestions are provided to reduce uncertainties in model predictions.

Architecture and DNA Recognition Elements of the Fanconi Anemia FANCM-FAAP24 Complex

PubMed Central

Coulthard, Rachel; Deans, Andrew J.; Swuec, Paolo; Bowles, Maureen; Costa, Alessandro; West, Stephen C.; McDonald, Neil Q.

2013-01-01

Summary Fanconi anemia (FA) is a disorder associated with a failure in DNA repair. FANCM (defective in FA complementation group M) and its partner FAAP24 target other FA proteins to sites of DNA damage. FANCM-FAAP24 is related to XPF/MUS81 endonucleases but lacks endonucleolytic activity. We report a structure of an FANCM C-terminal fragment (FANCMCTD) bound to FAAP24 and DNA. This S-shaped structure reveals the FANCM (HhH)2 domain is buried, whereas the FAAP24 (HhH)2 domain engages DNA. We identify a second DNA contact and a metal center within the FANCM pseudo-nuclease domain and demonstrate that mutations in either region impair double-stranded DNA binding in vitro and FANCM-FAAP24 function in vivo. We show the FANCM translocase domain lies in proximity to FANCMCTD by electron microscopy and that binding fork DNA structures stimulate its ATPase activity. This suggests a tracking model for FANCM-FAAP24 until an encounter with a stalled replication fork triggers ATPase-mediated fork remodeling. PMID:23932590

Increased Expression of Fatty-Acid and Calcium Metabolism Genes in Failing Human Heart

PubMed Central

Rodríguez-Penas, Diego; Feijóo-Bandín, Sandra; Noguera-Moreno, Teresa; Calaza, Manuel; Álvarez-Barredo, María; Mosquera-Leal, Ana; Parrington, John; Brugada, Josep; Portolés, Manuel; Rivera, Miguel; González-Juanatey, José Ramón; Lago, Francisca

2012-01-01

Background Heart failure (HF) involves alterations in metabolism, but little is known about cardiomyopathy-(CM)-specific or diabetes-independent alterations in gene expression of proteins involved in fatty-acid (FA) uptake and oxidation or in calcium-(Ca2+)-handling in the human heart. Methods RT-qPCR was used to quantify mRNA expression and immunoblotting to confirm protein expression in left-ventricular myocardium from patients with HF (n = 36) without diabetes mellitus of ischaemic (ICM, n = 16) or dilated (DCM, n = 20) cardiomyopathy aetiology, and non-diseased donors (CTL, n = 6). Results Significant increases in mRNA of genes regulating FA uptake (CD36) and intracellular transport (Heart-FA-Binding Protein (HFABP)) were observed in HF patients vs CTL. Significance was maintained in DCM and confirmed at protein level, but not in ICM. mRNA was higher in DCM than ICM for peroxisome-proliferator-activated-receptor-alpha (PPARA), PPAR-gamma coactivator-1-alpha (PGC1A) and CD36, and confirmed at the protein level for PPARA and CD36. Transcript and protein expression of Ca2+-handling genes (Two-Pore-Channel 1 (TPCN1), Two-Pore-Channel 2 (TPCN2), and Inositol 1,4,5-triphosphate Receptor type-1 (IP3R1)) increased in HF patients relative to CTL. Increases remained significant for TPCN2 in all groups but for TPCN1 only in DCM. There were correlations between FA metabolism and Ca2+-handling genes expression. In ICM there were six correlations, all distinct from those found in CTL. In DCM there were also six (all also different from those found in CTL): three were common to and three distinct from ICM. Conclusion DCM-specific increases were found in expression of several genes that regulate FA metabolism, which might help in the design of aetiology-specific metabolic therapies in HF. Ca2+-handling genes TPCN1 and TPCN2 also showed increased expression in HF, while HF- and CM-specific positive correlations were found among several FA and Ca2+-handling genes. PMID:22701570

Alterations in Oral [1-14C] 18:1n-9 Distribution in Lean Wild-Type and Genetically Obese (ob/ob) Mice

PubMed Central

Wang, Xinxia; Feng, Jie; Yu, Caihua; Shen, Qingwu W.; Wang, Yizhen

2015-01-01

Obesity may result from altered fatty acid (FA) disposal. Altered FA distribution in obese individuals is poorly understood. Lean wild-type C57BL/6J and obese C57BL/6Job/ob mice received an oral dose of [1-14C]18:1n-9 (oleic acid), and the radioactivity in tissues was evaluated at various time points. The 14C concentration decreased rapidly in gastrointestinal tract but gradually increased and peaked at 96 h in adipose tissue, muscle and skin in lean mice. The 14C concentration was constant in adipose tissue and muscle of obese mice from 4h to 168h. 14C-label content in adipose tissue was significantly affected by genotype, whereas muscle 14C-label content was affected by genotype, time and the interaction between genotype and time. There was higher total 14C retention (47.7%) in obese mice than in lean mice (9.0%) at 168 h (P<0.05). The 14C concentrations in the soleus and gastrocnemius muscle were higher in obese mice than in lean mice (P<0.05). Perirenal adipose tissue contained the highest 14C content in lean mice, whereas subcutaneous adipose tissue (SAT) had the highest 14C content and accounted for the largest proportion of total radioactivity among fat depots in obese mice. More lipid radioactivity was recovered as TAG in SAT from obese mice than from lean mice (P<0.05). Gene expression suggested acyl CoA binding protein and fatty acid binding protein are important for FA distribution in adipose tissue and muscle. The FA distribution in major tissues was altered in ob/ob mice, perhaps contributing to obesity. Understanding the disparity in FA disposal between lean and obese mice may reveal novel targets for the treatment and prevention of obesity. PMID:25826747

Low-ω3 Fatty Acid and Soy Protein Attenuate Alcohol-Induced Fatty Liver and Injury by Regulating the Opposing Lipid Oxidation and Lipogenic Signaling Pathways

PubMed Central

Reyes-Gordillo, Karina; Shah, Ruchi; Varatharajalu, Ravi; Garige, Mamatha; Leckey, Leslie C.

2016-01-01

Chronic ethanol-induced downregulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and upregulation of peroxisome proliferator-activated receptor gamma coactivator 1-beta (PGC1β) affect hepatic lipid oxidation and lipogenesis, respectively, leading to fatty liver injury. Low-ω3 fatty acid (Low-ω3FA) that primarily regulates PGC1α and soy protein (SP) that seems to have its major regulatory effect on PGC1β were evaluated for their protective effects against ethanol-induced hepatosteatosis in rats fed with Lieber-deCarli control or ethanol liquid diets with high or low ω3FA fish oil and soy protein. Low-ω3FA and SP opposed the actions of chronic ethanol by reducing serum and liver lipids with concomitant decreased fatty liver. They also prevented the downregulation of hepatic Sirtuin 1 (SIRT1) and PGC1α and their target fatty acid oxidation pathway genes and attenuated the upregulation of hepatic PGC1β and sterol regulatory element-binding protein 1c (SREBP1c) and their target lipogenic pathway genes via the phosphorylation of 5′ adenosine monophosphate-activated protein kinase (AMPK). Thus, these two novel modulators attenuate ethanol-induced hepatosteatosis and consequent liver injury potentially by regulating the two opposing lipid oxidation and lipogenic pathways. PMID:28074114

Design and Fabrication of Multifunctional Sericin Nanoparticles for Tumor Targeting and pH-Responsive Subcellular Delivery of Cancer Chemotherapy Drugs.

PubMed

Huang, Lei; Tao, Kaixiong; Liu, Jia; Qi, Chao; Xu, Luming; Chang, Panpan; Gao, Jinbo; Shuai, Xiaoming; Wang, Guobin; Wang, Zheng; Wang, Lin

2016-03-01

The severe cytotoxicity of cancer chemotherapy drugs limits their clinical applications. Various protein-based nanoparticles with good biocompatibility have been developed for chemotherapy drug delivery in hope of reducing drugs' side effects. Sericin, a natural protein from silk, has no immunogenicity and possesses diverse bioactivities that have prompted sericin's application studies. However, the potential of sericin as a multifunctional nanoscale vehicle for cancer therapy have not been fully explored. Here we report the successful fabrication and characterization of folate-conjugated sericin nanoparticles with cancer-targeting capability for pH-responsive release of doxorubicin (these nanoparticles are termed "FA-SND"). DOX is covalently linked to sericin through pH-sensitive hydrazone bonds that render a pH-triggered release property. The hydrophobicity of DOX and the hydrophilicity of sericin promote the self-assembly of sericin-DOX (SND) nanoconjugates. Folate (FA) is then covalently grafted to SND nanoconjugates as a binding unit for actively targeting cancer cells that overexpress folate receptors. Our characterization study shows that FA-SND nanoparticles exhibit negative surface charges that would reduce nonspecific clearance by circulation. These nanoparticles possess good cytotoxicity and hemocompatibiliy. Acidic environment (pH 5.0) triggers effective DOX release from FA-SND, 5-fold higher than does a neutral condition (pH 7.4). Further, FA-SND nanoparticles specifically target folate-receptor-rich KB cells, and endocytosed into lysosomes, an acidic organelle. The acidic microenvironment of lysosomes promotes a rapid release of DOX to nuclei, producing cancer specific chemo-cytotoxicity. Thus, FA-mediated cancer targeting and lysosomal-acidity promoting DOX release, two sequentially-occurring cellular events triggered by the designed components of FA-SND, form the basis for FA-SND to achieve its localized and intracellular chemo-cytotoxicity. Together, this study suggests that these FA-SND nanoparticles may be a potentially effective carrier particularly useful for delivering hydrophobic chemotherapeutic agents for treating cancers with high-level expression of folate receptors.

Identification of Novel Fusion Inhibitors of Influenza A Virus by Chemical Genetics

PubMed Central

Lai, Kin Kui; Cheung, Nam Nam; Yang, Fang; Dai, Jun; Liu, Li; Chen, Zhiwei; Sze, Kong Hung; Chen, Honglin

2015-01-01

ABSTRACT A previous screening of more than 50,000 compounds led to the identification of a pool of bioactive small molecules with inhibitory effect on the influenza A virus. One of these compounds, now widely known as nucleozin, is a small molecule that targets the influenza A virus nucleoprotein. Here we identify and characterize two structurally different novel fusion inhibitors of the influenza A virus group 1 hemagglutinin (HA), FA-583 and FA-617, with low nanomolar activities. Escape mutants that are highly resistant to each of these compounds were generated, and both were found to carry mutations localized in close proximity to the B-loop of the hemagglutinin 2 protein, which plays a crucial role in the virion-host cell fusion process. Recombinant virus, generated through reverse genetics, confirmed the resistance phenotype. In addition, the proposed binding pockets predicted by molecular docking studies are in accordance with the resistance-bearing mutation sites. We show through mechanistic studies that FA-583 and FA-617 act as fusion inhibitors by prohibiting the low-pH-induced conformational change of hemagglutinin. Our study has offered concrete biological and mechanistic explorations for the strategic development of novel fusion inhibitors of influenza A viruses. IMPORTANCE Here we report two structurally distinctive novel fusion inhibitors of influenza A virus that act by interfering with the structural change of HA at acidic pH, a process necessary for successful entry of the virus. Mutational and molecular docking studies have identified their binding pockets situated in close proximity to the B-loop region of hemagglutinin 2. The reduced sensitivity of FA-583- or FA-617-associated mutants to another compound suggests a close proximity and even partial overlap of their binding sites on hemagglutinin. Amino acid sequence alignments and crystal structure analyses of group 1 and group 2 hemagglutinins have shed light on the possible binding mode of these two compounds. This report offers new lead compounds for the design of fusion inhibitors for influenza A viruses and further shows that analysis by forward chemical genetics is a highly effective approach for the identification of novel compounds that can perturb the infectivity of viruses and to probe new druggable targets or druggable domains in various viruses. PMID:26676787

Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chi; Miller, Darcie J.; Guibao, Cristina D.

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by whichmore » the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ~4.2 μM) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.« less

Recruitment of Fanconi Anemia and Breast Cancer Proteins to DNA Damage Sites is differentially Governed by Replication

PubMed Central

Shen, Xi; Do, Huong; Li, Yongjian; Chung, Woo-Hyun; Tomasz, Maria; de Winter, Johan P.; Xia, Bing; Elledge, Stephen J.; Wang, Weidong; Li, Lei

2009-01-01

Summary Fanconi anemia (FA) is characterized by cellular hypersensivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remains unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Inter-dependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status. PMID:19748364

A High-Protein Diet Reduces Weight Gain, Decreases Food Intake, Decreases Liver Fat Deposition, and Improves Markers of Muscle Metabolism in Obese Zucker Rats

PubMed Central

French, William W.; Dridi, Sami; Shouse, Stephanie A.; Wu, Hexirui; Hawley, Aubree; Lee, Sun-Ok; Gu, Xuan; Baum, Jamie I.

2017-01-01

A primary factor in controlling and preventing obesity is through dietary manipulation. Diets higher in protein have been shown to improve body composition and metabolic health during weight loss. The objective of this study was to examine the effects of a high-protein diet versus a moderate-protein diet on muscle, liver and fat metabolism and glucose regulation using the obese Zucker rat. Twelve-week old, male, Zucker (fa/fa) and lean control (Fa/fa) rats were randomly assigned to either a high-protein (40% energy) or moderate-protein (20% energy) diet for 12 weeks, with a total of four groups: lean 20% protein (L20; n = 8), lean 40% protein (L40; n = 10), obese 20% protein (O20; n = 8), and obese 40% protein (O40; n = 10). At the end of 12 weeks, animals were fasted and euthanized. There was no difference in food intake between L20 and L40. O40 rats gained less weight and had lower food intake (p < 0.05) compared to O20. O40 rats had lower liver weight (p < 0.05) compared to O20. However, O40 rats had higher orexin (p < 0.05) levels compared to L20, L40 and O20. Rats in the L40 and O40 groups had less liver and muscle lipid deposition compared to L20 and L40 diet rats, respectively. O40 had decreased skeletal muscle mechanistic target of rapamycin complex 1 (mTORC1) phosphorylation and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression compared to O20 (p < 0.05), with no difference in 5′ AMP-activated protein kinase (AMPK), eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), protein kinase B (Akt) or p70 ribosomal S6 kinase (p70S6K) phosphorylation. The data suggest that high-protein diets have the potential to reduce weight gain and alter metabolism, possibly through regulation of an mTORC1-dependent pathway in skeletal muscle. PMID:28594375

A High-Protein Diet Reduces Weight Gain, Decreases Food Intake, Decreases Liver Fat Deposition, and Improves Markers of Muscle Metabolism in Obese Zucker Rats.

PubMed

French, William W; Dridi, Sami; Shouse, Stephanie A; Wu, Hexirui; Hawley, Aubree; Lee, Sun-Ok; Gu, Xuan; Baum, Jamie I

2017-06-08

A primary factor in controlling and preventing obesity is through dietary manipulation. Diets higher in protein have been shown to improve body composition and metabolic health during weight loss. The objective of this study was to examine the effects of a high-protein diet versus a moderate-protein diet on muscle, liver and fat metabolism and glucose regulation using the obese Zucker rat. Twelve-week old, male, Zucker (fa/fa) and lean control (Fa/fa) rats were randomly assigned to either a high-protein (40% energy) or moderate-protein (20% energy) diet for 12 weeks, with a total of four groups: lean 20% protein (L20; n = 8), lean 40% protein (L40; n = 10), obese 20% protein (O20; n = 8), and obese 40% protein (O40; n = 10). At the end of 12 weeks, animals were fasted and euthanized. There was no difference in food intake between L20 and L40. O40 rats gained less weight and had lower food intake ( p < 0.05) compared to O20. O40 rats had lower liver weight ( p < 0.05) compared to O20. However, O40 rats had higher orexin ( p < 0.05) levels compared to L20, L40 and O20. Rats in the L40 and O40 groups had less liver and muscle lipid deposition compared to L20 and L40 diet rats, respectively. O40 had decreased skeletal muscle mechanistic target of rapamycin complex 1 (mTORC1) phosphorylation and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression compared to O20 ( p < 0.05), with no difference in 5' AMP-activated protein kinase (AMPK), eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), protein kinase B (Akt) or p70 ribosomal S6 kinase (p70S6K) phosphorylation. The data suggest that high-protein diets have the potential to reduce weight gain and alter metabolism, possibly through regulation of an mTORC1-dependent pathway in skeletal muscle.

The effect of natural organic matter on the adsorption of mercury to bacterial cells

NASA Astrophysics Data System (ADS)

Dunham-Cheatham, Sarrah; Mishra, Bhoopesh; Myneni, Satish; Fein, Jeremy B.

2015-02-01

We investigated the ability of non-metabolizing Bacillus subtilis, Shewanella oneidensis MR-1, and Geobacter sulfurreducens bacterial species to adsorb mercury in the absence and presence of Suwanee River fulvic acid (FA). Bulk adsorption and X-ray absorption spectroscopy (XAS) experiments were conducted at three pH conditions, and the results indicate that the presence of FA decreases the extent of Hg adsorption to biomass under all of the pH conditions studied. Hg XAS results show that the presence of FA does not alter the binding environment of Hg adsorbed onto the biomass regardless of pH or FA concentration, indicating that ternary bacteria-Hg-FA complexes do not form to an appreciable extent under the experimental conditions, and that Hg binding on the bacteria is dominated by sulfhydryl binding. We used the experimental results to calculate apparent partition coefficients, Kd, for Hg under each experimental condition. The calculations yield similar coefficients for Hg onto each of the bacterial species studies, suggesting there is no significant difference in Hg partitioning between the three bacterial species. The calculations also indicate similar coefficients for Hg-bacteria and Hg-FA complexes. S XAS measurements confirm the presence of sulfhydryl sites on both the FA and bacterial cells, and demonstrate the presence of a wide range of S moieties on the FA in contrast to the bacterial biomass, whose S sites are dominated by thiols. Our results suggest that although FA can compete with bacterial binding sites for aqueous Hg, because of the relatively similar partition coefficients for the types of sorbents, the competition is not dominated by either bacteria or FA unless the concentration of one type of site greatly exceeds that of the other.

Identification of rare heterozygous missense mutations in FANCA in esophageal atresia patients using next-generation sequencing.

PubMed

Feng, Yu; Chen, Runsen; Da, Min; Qian, Bo; Mo, Xuming

2018-06-30

Esophageal atresia and tracheoesophageal fistula (EA/TEF) are relatively common malformations in newborns, but the etiology of EA/TEF remains unknown. Fanconi anemia (FA) complementation group A (FANCA) is a key component of the FA core complex and is essential for the activation of the DNA repair pathway. The middle region (amino acids 674-1208) of FANCA is required for its interaction with FAAP20. We performed targeted sequencing of this binding region of FANCA (exons 23-36) in 40 EA/TEF patients. We also investigated the effect of the p.A958V mutation on the protein-protein interaction between FANCA and FAAP20 using an in vitro binding assay and co-immunoprecipitation. Immunolocalization analysis was performed to investigate the subcellular localization of FANCA, and tissue sections and immunohistochemistry were used to explore the expression of FANCA. We identified four rare missense variants in the FANCA binding region. FANCA mutations were significantly overrepresented in EA/TEF patients compared with 4300 control subjects from the NHLBI-ESP project (Fisher's exact p = 2.17 × 10 -5 , odds ratio = 31.75). p.A958V, a novel de novo mutation in the FANCA gene, was identified in one patient with EA/TEF. We provide further evidence that the p.A958V mutation reduces the binding affinity of FANCA for FAAP20. Interestingly, the p.A958V mutation impaired the nuclear localization of the FANCA protein expressed in HeLa cells. We found that FANCA was more highly expressed in stratified squamous epithelium than in smooth muscle. In conclusion, mutations in the FANCA gene are associated with EA/TEF in humans. Copyright © 2018. Published by Elsevier B.V.

Identification of a New cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae

PubMed Central

Zhao, Can; Abdelgaffar, Heba M.; Pan, Hongyu; Song, Fuping

2015-01-01

Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with 125I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution. PMID:25795679

Identification of a New cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae.

PubMed

Zhao, Can; Jurat-Fuentes, Juan Luis; Abdelgaffar, Heba M; Pan, Hongyu; Song, Fuping; Zhang, Jie

2015-06-01

Pyramiding of diverse cry toxin genes from Bacillus thuringiensis with different modes of action is a desirable strategy to delay the evolution of resistance in the European corn borer (Ostrinia nubilalis). Considering the dependency of susceptibility to Cry toxins on toxin binding to receptors in the midgut of target pests, a diverse mode of action is commonly defined as recognition of unique binding sites in the target insect. In this study, we present a novel cry1Ie toxin gene (cry1Ie2) as a candidate for pyramiding with Cry1Ab or Cry1Fa in corn to control Ostrinia species larvae. The new toxin gene encodes an 81-kDa protein that is processed to a protease-resistant core form of approximately 55 kDa by trypsin digestion. The purified protoxin displayed high toxicity to Ostrinia furnacalis and O. nubilalis larvae but low to no activity against Spodoptera or heliothine species or the coleopteran Tenebrio molitor. Results of binding assays with (125)I-labeled Cry1Ab toxin and brush border membrane vesicles from O. nubilalis larvae demonstrated that Cry1Ie2 does not recognize the Cry1Ab binding sites in that insect. Reciprocal competition binding assays with biotin-labeled Cry1Ie2 confirmed the lack of shared sites with Cry1Ab or Cry1Fa in O. nubilalis brush border membrane vesicles. These data support Cry1Ie2 as a good candidate for pyramiding with Cry1Ab or Cry1Fa in corn to increase the control of O. nubilalis and reduce the risk of resistance evolution. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cloning and expressing a highly functional and substrate specific farnesoic acid o-methyltransferase from the Asian citrus psyllid (Diaphorina citri Kuwayama).

PubMed

Van Ekert, Evelien; Shatters, Robert G; Rougé, Pierre; Powell, Charles A; Smagghe, Guy; Borovsky, Dov

2015-01-01

The Asian citrus psyllid, Diaphorina citri, transmits a phloem-limited bacterium, Candidatus 'Liberibacter' asiaticus that causes citrus greening disease. Because juvenile hormone (JH) plays an important role in adult and nymphal development, we studied the final steps in JH biosynthesis in D. citri. A putative JH acid methyltransferase ortholog gene (jmtD) and its cognate cDNA were identified by searching D. citri genome database. Expression analysis shows expression in all life stages. In adults, it is expressed in the head-thorax, (containing the corpora allata), and the abdomen (containing ovaries and male accessory glands). A 3D protein model identified the catalytic groove with catalytically active amino acids and the S-adenosyl methionine (SAM)-binding loop. The cDNA was expressed in Escherichia coli cells and the purified enzyme showed high preference for farnesoic acid (FA) and homoFA (kcat of 0.752 × 10(-3) and 0.217 × 10(-3) s(-1), respectively) as compared to JH acid I (JHA I) (cis/trans/cis; 2Z, 6E, 10cis), JHA III (2E, 6E, 10cis), and JHA I (trans/cis/cis; 2E, 2Z, 10cis) (kcat of 0.081 × 10(-3), 0.013 × 10(-3), and 0.003 × 10(-3) s(-1), respectively). This suggests that this ortholog is a DcFA-o-methyl transferase gene (fmtD), not a jmtD, and that JH biosynthesis in D. citri proceeds from FA to JH III through methyl farnesoate (MF). DcFA-o-MT does not require Ca(2+), Mg(2+) or Zn(2+), however, Zn(2+) (1 mM) completely inhibits the enzyme probably by binding H115 at the active groove. This represents the first purified FA-o-MT from Hemiptera with preferred biological activity for FA and not JHA.

Structural analysis of binding functionality of folic acid-PEG dendrimers against folate receptor.

PubMed

Sampogna-Mireles, Diana; Araya-Durán, Ingrid D; Márquez-Miranda, Valeria; Valencia-Gallegos, Jesús A; González-Nilo, Fernando D

2017-03-01

Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystal Structures of Penicillin-Binding Protein 2 From Penicillin-Susceptible And -Resistant Strains of Neisseria Gonorrhoeae Reveal An Unexpectedly Subtle Mechanism for Antibiotic Resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Powell, A.J.; Tomberg, J.; Deacon, A.M.

Penicillin-binding protein 2 (PBP2) from N. gonorrhoeae is the major molecular target for {beta}-lactam antibiotics used to treat gonococcal infections. PBP2 from penicillin-resistant strains of N. gonorrhoeae harbors an aspartate insertion after position 345 (Asp-345a) and 4-8 additional mutations, but how these alter the architecture of the protein is unknown. We have determined the crystal structure of PBP2 derived from the penicillin-susceptible strain FA19, which shows that the likely effect of Asp-345a is to alter a hydrogen-bonding network involving Asp-346 and the SXN triad at the active site. We have also solved the crystal structure of PBP2 derived from themore » penicillin-resistant strain FA6140 that contains four mutations near the C terminus of the protein. Although these mutations lower the second order rate of acylation for penicillin by 5-fold relative to wild type, comparison of the two structures shows only minor structural differences, with the positions of the conserved residues in the active site essentially the same in both. Kinetic analyses indicate that two mutations, P551S and F504L, are mainly responsible for the decrease in acylation rate. Melting curves show that the four mutations lower the thermal stability of the enzyme. Overall, these data suggest that the molecular mechanism underlying antibiotic resistance contributed by the four mutations is subtle and involves a small but measurable disordering of residues in the active site region that either restricts the binding of antibiotic or impedes conformational changes that are required for acylation by {beta}-lactam antibiotics.« less

Structural insights into the functions of the FANCM-FAAP24 complex in DNA repair

PubMed Central

Yang, Hui; Zhang, Tianlong; Tao, Ye; Wang, Fang; Tong, Liang; Ding, Jianping

2013-01-01

Fanconi anemia (FA) is a genetically heterogeneous disorder associated with deficiencies in the FA complementation group network. FA complementation group M (FANCM) and FA-associated protein 24 kDa (FAAP24) form a stable complex to anchor the FA core complex to chromatin in repairing DNA interstrand crosslinks. Here, we report the first crystal structure of the C-terminal segment of FANCM in complex with FAAP24. The C-terminal segment of FANCM and FAAP24 both consist of a nuclease domain at the N-terminus and a tandem helix-hairpin-helix (HhH)2 domain at the C-terminus. The FANCM-FAAP24 complex exhibits a similar architecture as that of ApXPF. However, the variations of several key residues and the electrostatic property at the active-site region render a catalytically inactive nuclease domain of FANCM, accounting for the lack of nuclease activity. We also show that the first HhH motif of FAAP24 is a potential binding site for DNA, which plays a critical role in targeting FANCM-FAAP24 to chromatin. These results reveal the mechanistic insights into the functions of FANCM-FAAP24 in DNA repair. PMID:24003026

Investigating noncovalent squarylium dye-protein interactions by capillary electrophoresis-frontal analysis.

PubMed

Yan, Weiying; Colyer, Christa L

2006-11-24

Noncovalent interactions between fluorescent probe molecules and protein analyte molecules, which typically occur with great speed and minimal sample handling, form the basis of many high sensitivity analytical techniques. Understanding the nature of these interactions and the composition of the resulting complexes represents an important area of study that can be facilitated by capillary electrophoresis (CE). Specifically, we will present how frontal analysis (FA) and Hummel-Dreyer (HD) methods can be implemented with CE to determine association constants and stoichiometries of noncovalent complexes of the red luminescent squarylium dye Red-1c with bovine serum albumin (BSA) and beta-lactoglobulin A. By adjusting solution conditions, such as pH or ionic strength, it is possible to selectively modify the binding process. As such, conditions for optimal selectivity for labeling reactions can be established by capillary electrophoresis-frontal analysis (CE-FA) investigations.

Analysis of TP53 gene expression and p53 level of human hypopharyngeal FaDu (HTB-43) head and neck cancer cell line after microRNA-181a inhibition.

PubMed

Cheah, Y K; Cheng, R W; Yeap, S K; Khoo, C H; See, H S

2014-03-17

The identification of new biomarkers for early detection of highly recurrent head and neck cancer is urgently needed. MicroRNAs (miRNAs) are small and non-coding RNAs that regulate cancer-related gene expression, such as tumor protein 53 (TP53) gene expression. This study was carried out to analyze TP53 gene expression using real-time PCR and to determine changes in intracellular p53 level by flow cytometry after downregulation of miRNA-181a miRNA inhibitor in the FaDu cell line. TP53 gene expression showed a 3-fold increment and the p53 protein level was also increased in the miRNA-181a-treated cells. In conclusion, miRNA-181a binds to the TP53 gene and inhibits its expression, decreasing the synthesis of p53.

Production of a Brassica napus Low-Molecular Mass Acyl-Coenzyme A-Binding Protein in Arabidopsis Alters the Acyl-Coenzyme A Pool and Acyl Composition of Oil in Seeds.

PubMed

Yurchenko, Olga; Singer, Stacy D; Nykiforuk, Cory L; Gidda, Satinder; Mullen, Robert T; Moloney, Maurice M; Weselake, Randall J

2014-06-01

Low-molecular mass (10 kD) cytosolic acyl-coenzyme A-binding protein (ACBP) has a substantial influence over fatty acid (FA) composition in oilseeds, possibly via an effect on the partitioning of acyl groups between elongation and desaturation pathways. Previously, we demonstrated that the expression of a Brassica napus ACBP (BnACBP) complementary DNA in the developing seeds of Arabidopsis (Arabidopsis thaliana) resulted in increased levels of polyunsaturated FAs at the expense of eicosenoic acid (20:1 cisΔ11 ) and saturated FAs in seed oil. In this study, we investigated whether alterations in the FA composition of seed oil at maturity were correlated with changes in the acyl-coenzyme A (CoA) pool in developing seeds of transgenic Arabidopsis expressing BnACBP. Our results indicated that both the acyl-CoA pool and seed oil of transgenic Arabidopsis lines expressing cytosolic BnACBP exhibited relative increases in linoleic acid (18:2 cisΔ9,12 ; 17.9%-44.4% and 7%-13.2%, respectively) and decreases in 20:1 cisΔ11 (38.7%-60.7% and 13.8%-16.3%, respectively). However, alterations in the FA composition of the acyl-CoA pool did not always correlate with those seen in the seed oil. In addition, we found that targeting of BnACBP to the endoplasmic reticulum resulted in FA compositional changes that were similar to those seen in lines expressing cytosolic BnACBP, with the most prominent exception being a relative reduction in α-linolenic acid (18:3 cisΔ9,12,15 ) in both the acyl-CoA pool and seed oil of the former (48.4%-48.9% and 5.3%-10.4%, respectively). Overall, these data support the role of ACBP in acyl trafficking in developing seeds and validate its use as a biotechnological tool for modifying the FA composition of seed oil. © 2014 American Society of Plant Biologists. All Rights Reserved.

Fanconi anemia complementation group A (FANCA) localizes to centrosomes and functions in the maintenance of centrosome integrity.

PubMed

Kim, Sunshin; Hwang, Soo Kyung; Lee, Mihee; Kwak, Heejin; Son, Kook; Yang, Jiha; Kim, Sung Hak; Lee, Chang-Hun

2013-09-01

Fanconi anemia (FA) proteins are known to play roles in the cellular response to DNA interstrand cross-linking lesions; however, several reports have suggested that FA proteins play additional roles. To elucidate novel functions of FA proteins, we used yeast two-hybrid screening to identify binding partners of the Fanconi anemia complementation group A (FANCA) protein. The candidate proteins included never-in-mitosis-gene A (NIMA)-related kinase 2 (Nek2), which functions in the maintenance of centrosome integrity. The interaction of FANCA and Nek2 was confirmed in human embryonic kidney (HEK) 293T cells. Furthermore, FANCA interacted with γ-tubulin and localized to centrosomes, most notably during the mitotic phase, confirming that FANCA is a centrosomal protein. Knockdown of FANCA increased the frequency of centrosomal abnormalities and enhanced the sensitivity of U2OS osteosarcoma cells to nocodazole, a microtubule-interfering agent. In vitro kinase assays indicated that Nek2 can phosphorylate FANCA at threonine-351 (T351), and analysis with a phospho-specific antibody confirmed that this phosphorylation occurred in response to nocodazole treatment. Furthermore, U2OS cells overexpressing the phosphorylation-defective T351A FANCA mutant showed numerical centrosomal abnormalities, aberrant mitotic arrest, and enhanced nocodazole sensitivity, implying that the Nek2-mediated T351 phosphorylation of FANCA is important for the maintenance of centrosomal integrity. Taken together, this study revealed that FANCA localizes to centrosomes and is required for the maintenance of centrosome integrity, possibly through its phosphorylation at T351 by Nek2. Copyright © 2013 Elsevier Ltd. All rights reserved.

A novel, visible light-induced, rapidly cross-linkable gelatin scaffold for osteochondral tissue engineering

PubMed Central

Mazaki, Tetsuro; Shiozaki, Yasuyuki; Yamane, Kentaro; Yoshida, Aki; Nakamura, Mariko; Yoshida, Yasuhiro; Zhou, Di; Kitajima, Takashi; Tanaka, Masato; Ito, Yoshihiro; Ozaki, Toshifumi; Matsukawa, Akihiro

2014-01-01

Osteochondral injuries remain difficult to repair. We developed a novel photo-cross-linkable furfurylamine-conjugated gelatin (gelatin-FA). Gelatin-FA was rapidly cross-linked by visible light with Rose Bengal, a light sensitizer, and was kept gelled for 3 weeks submerged in saline at 37°C. When bone marrow-derived stromal cells (BMSCs) were suspended in gelatin-FA with 0.05% Rose Bengal, approximately 87% of the cells were viable in the hydrogel at 24 h after photo-cross-linking, and the chondrogenic differentiation of BMSCs was maintained for up to 3 weeks. BMP4 fusion protein with a collagen binding domain (CBD) was retained in the hydrogels at higher levels than unmodified BMP4. Gelatin-FA was subsequently employed as a scaffold for BMSCs and CBD-BMP4 in a rabbit osteochondral defect model. In both cases, the defect was repaired with articular cartilage-like tissue and regenerated subchondral bone. This novel, photo-cross-linkable gelatin appears to be a promising scaffold for the treatment of osteochondral injury. PMID:24662725

Dysfunction of protein kinase FA/GSK-3 alpha in lymphocytes of patients with schizophrenic disorder.

PubMed

Yang, S D; Yu, J S; Lee, T T; Yang, C C; Ni, M H; Yang, Y Y

1995-09-01

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP.Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA/glycogen synthase kinase-3 alpha (kinase FA/GSK-3 alpha) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA/GSK-3 alpha. Immunoblotting and kinase activity analysis in an anti-kinase FA/GSK-3 alpha immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA/GSK-3 alpha in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA/GSK-3 alpha activity in the high levels of 14.8 +/- 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA/GSK-3 alpha activity in the low levels of 2.8 +/- 1.6 units/mg, indicating that the different levels of kinase FA/GSK-3 alpha activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide initial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA/GSK-3 alpha, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes.

Rac1 Recruitment to the Archipelago Structure of the Focal Adhesion through the Fluid Membrane as Revealed by Single-Molecule Analysis

PubMed Central

Shibata, Akihiro C E; Chen, Limin H; Nagai, Rie; Ishidate, Fumiyoshi; Chadda, Rahul; Miwa, Yoshihiro; Naruse, Keiji; Shirai, Yuki M; Fujiwara, Takahiro K; Kusumi, Akihiro

2013-01-01

The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress-fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter-island channels in the FA zone rather freely, and the integrins in the FA-protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G-protein regulating FA formation, and its activators αPIX and βPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively-active Rac1 mutant exhibited temporary and all-time immobilizations in the FA zone, suggesting that upon PIX-induced Rac1 activation at the FA-protein islands, Rac1 tends to be immobilized at the FA-protein islands. © 2013 Wiley Periodicals, Inc PMID:23341328

Fanconi Anemia Proteins, DNA Interstrand Crosslink Repair Pathways, and Cancer Therapy

PubMed Central

Andreassen, Paul R.; Ren, Keqin

2016-01-01

DNA interstrand crosslinkers, a chemically diverse group of compounds which also induce alkylation of bases and DNA intrastrand crosslinks, are extensively utilized for cancer therapy. Understanding the cellular response to DNA damage induced by these agents is critical for more effective utilization of these compounds and for the identification of novel therapeutic targets. Importantly, the repair of DNA interstrand crosslinks (ICLs) involves many distinct DNA repair pathways, including nucleotide excision repair, translesion synthesis (TLS), and homologous recombination (HR). Additionally, proteins implicated in the pathophysiology of the multigenic disease Fanconi anemia (FA) have a role in the repair of ICLs that is not well understood. Cells from FA patients are hypersensitive to agents that induce ICLs, therefore FA proteins are potentially novel therapeutic targets. Here we will review current research directed at identifying FA genes and understanding the function of FA proteins in DNA damage responses. We will also examine interactions of FA proteins with other repair proteins and pathways, including signaling networks, which are potentially involved in ICL repair. Potential approaches to the modulation of FA protein function to enhance therapeutic outcome will be discussed. Also, mutation of many genes that encode proteins involved in ICL repair, including FA genes, increases susceptibility to cancer. A better understanding of these pathways is therefore critical for the design of individualized therapies tailored to the genetic profile of a particular malignancy. For this purpose, we will also review evidence for the association of mutation of FA genes with cancer in non-FA patients. PMID:19200054

The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG.

PubMed

de Winter, J P; van der Weel, L; de Groot, J; Stone, S; Waisfisz, Q; Arwert, F; Scheper, R J; Kruyt, F A; Hoatlin, M E; Joenje, H

2000-11-01

Fanconi anemia (FA) is a chromosomal instability syndrome associated with a strong predisposition to cancer, particularly acute myeloid leukemia and squamous cell carcinoma. At the cellular level, FA is characterized by spontaneous chromosomal breakage and a unique hypersensitivity to DNA cross-linking agents. Complementation analysis has indicated that at least seven distinct genes are involved in the pathogenesis of FA. Despite the identification of four of these genes (FANCA, FANCC, FANCF and FANCG), the nature of the 'FA pathway' has remained enigmatic, as the FA proteins lack sequence homologies or motifs that could point to a molecular function. To further define this pathway, we studied the subcellular localizations and mutual interactions of the FA proteins, including the recently identified FANCF protein, in human lymphoblasts. FANCF was found predominantly in the nucleus, where it complexes with FANCA, FANCC and FANCG. These interactions were detected in wild-type and FA-D lymphoblasts, but not in lymphoblasts of other FA complementation groups. This implies that each of the FA proteins, except FANCD, is required for these complexes to form. Similarly, we show that the interaction between FANCA and FANCC is restricted to wild-type and FA-D cells. Furthermore, we document the subcellular localization of FANCA and the FANCA/FANCG complex in all FA complementation groups. Our results, along with published data, culminate in a model in which a multi-protein FA complex serves a nuclear function to maintain genomic integrity.

FMISO accumulation in tumor is dependent on glutathione conjugation capacity in addition to hypoxic state.

PubMed

Masaki, Yukiko; Shimizu, Yoichi; Yoshioka, Takeshi; Nishijima, Ken-Ichi; Zhao, Songji; Higashino, Kenichi; Numata, Yoshito; Tamaki, Nagara; Kuge, Yuji

2017-10-01

18 F-fluoromisonidazole (FMISO), a well-known PET imaging probe for diagnosis of hypoxia, is believed to accumulate in hypoxic cells via covalent binding with macromolecules after reduction of the nitro group. Previously, we showed the majority of 18 F-FMISO was incorporated into low-molecular-weight metabolites in hypoxic tumors, and the glutathione conjugate of reduced FMISO (amino-FMISO-GS) distributed in the tumor hypoxic regions as revealed by imaging mass spectrometry (IMS). The present study was conducted to clarify whether FMISO is metabolized to amino-FMISO-GS within tumor cells and how amino-FMISO-GS contributes to FMISO accumulation in hypoxic cells. We also evaluated the relationship between FMISO accumulation and the glutathione conjugation-related factors in the cells. Tumor cells (FaDu, LOVO, and T24) were treated with 18 F-FMISO and incubated under normoxic or hypoxic conditions for 4 h. The FMISO metabolites were analyzed with LC-ESI-MS. Several glutathione conjugation-related factors of tumor cells were evaluated in vitro. FaDu tumor-bearing mice were intravenously injected with 18 F-FMISO and the tumors were excised at 4 h post-injection. Autoradiography, IMS and histologic studies were performed. Amino-FMISO-GS was the main contributor to FMISO incorporated in hypoxic FaDu cells in vitro and in vivo. Total FMISO uptake levels and amino-FMISO-GS levels were highest in FaDu, followed by LOVO, and then T24 (total uptake: 0.851 ± 0.009 (FaDu), 0.617 ± 0.021 (LOVO) and 0.167 ± 0.006 (T24) % dose/mg protein; amino-FMISO-GS: 0.502 ± 0.035 (FaDu), 0.158 ± 0.013 (LOVO), and 0.007 ± 0.001 (T24) % dose/mg protein). The glutathione level of FaDu was significantly higher than those of LOVO and T24. The enzyme activity of glutathione-S-transferase catalyzing the glutathione conjugation reaction in FaDu was similar levels to that in LOVO, and was higher than that in T24. Quantitative RT-PCR analysis revealed that the expression levels of efflux transporters of the glutathione conjugate (multidrug resistance-associated protein 1) were lowest in FaDu, followed by LOVO, and then T24. FMISO accumulates in hypoxic cells through reductive metabolism followed by glutathione conjugation. We illustrated the possibility that increased production and decreased excretion of amino-FMISO-GS contribute to FMISO accumulation in tumor cells under hypoxic conditions.

Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-induced Macrophage Cell Death through Enhancing Ceramide Production

PubMed Central

Zhang, Yuwen; Rao, Enyu; Zeng, Jun; Hao, Jiaqing; Sun, Yanwen; Liu, Shujun; Sauter, Edward R.; Bernlohr, David A.; Cleary, Margot P.; Suttles, Jill; Li, Bing

2016-01-01

Macrophages play a critical role in obesity-associated chronic inflammation and disorders. However, the molecular mechanisms underlying the response of macrophages to elevated fatty acids (FAs) and their contribution to metabolic inflammation in obesity remain to be fully elucidated. Here, we report a new mechanism by which dietary FAs, in particular saturated FAs, are able to directly trigger macrophage cell death. We demonstrated that excess saturated FAs, but not unsaturated FAs, induced the production of cytotoxic ceramides in macrophage cell lines. Most importantly, expression of adipose fatty acid binding protein (A-FABP) in macrophages facilitated metabolism of excess saturated FAs for ceramide synthesis. Inhibition or deficiency of A-FABP in macrophage cell lines decreased saturated FA-induced ceramide production, thereby resulting in reduced cell death. Furthermore, we validated the role of A-FABP in promoting saturated FA-induced macrophage cell death with primary bone-marrow derived macrophages and high-fat diet-induced obese mice. Altogether, our data reveal that excess dietary saturated FAs may serve as direct triggers in induction of ceramide production and macrophage cell death through elevated expression of A-FABP, thus establishing A-FABP as a new molecular sensor in triggering macrophage-associated sterile inflammation in obesity. PMID:27920274

Mobility-based correction for accurate determination of binding constants by capillary electrophoresis-frontal analysis.

PubMed

Qian, Cheng; Kovalchik, Kevin A; MacLennan, Matthew S; Huang, Xiaohua; Chen, David D Y

2017-06-01

Capillary electrophoresis frontal analysis (CE-FA) can be used to determine binding affinity of molecular interactions. However, its current data processing method mandate specific requirement on the mobilities of the binding pair in order to obtain accurate binding constants. This work shows that significant errors are resulted when the mobilities of the interacting species do not meet these requirements. Therefore, the applicability of CE-FA in many real word applications becomes questionable. An electrophoretic mobility-based correction method is developed in this work based on the flux of each species. A simulation program and a pair of model compounds are used to verify the new equations and evaluate the effectiveness of this method. Ibuprofen and hydroxypropyl-β-cyclodextrinare used to demonstrate the differences in the obtained binding constant by CE-FA when different calculation methods are used, and the results are compared with those obtained by affinity capillary electrophoresis (ACE). The results suggest that CE-FA, with the mobility-based correction method, can be a generally applicable method for a much wider range of applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[FANCA gene mutation analysis in Fanconi anemia patients].

PubMed

Chen, Fei; Peng, Guang-Jie; Zhang, Kejian; Hu, Qun; Zhang, Liu-Qing; Liu, Ai-Guo

2005-10-01

To screen the FANCA gene mutation and explore the FANCA protein function in Fanconi anemia (FA) patients. FANCA protein expression and its interaction with FANCF were analyzed using Western blot and immunoprecipitation in 3 cases of FA-A. Genomic DNA was used for MLPA analysis followed by sequencing. FANCA protein was undetectable and FANCA and FANCF protein interaction was impaired in these 3 cases of FA-A. Each case of FA-A contained biallelic pathogenic mutations in FANCA gene. No functional FANCA protein was found in these 3 cases of FA-A, and intragenic deletion, frame shift and splice site mutation were the major pathogenic mutations found in FANCA gene.

Shikonin, a constituent of Lithospermum erythrorhizon exhibits anti-allergic effects by suppressing orphan nuclear receptor Nr4a family gene expression as a new prototype of calcineurin inhibitors in mast cells.

PubMed

Wang, Xiaoyu; Hayashi, Shusaku; Umezaki, Masahito; Yamamoto, Takeshi; Kageyama-Yahara, Natsuko; Kondo, Takashi; Kadowaki, Makoto

2014-12-05

Over the last few decades, food allergy (FA) has become a common disease in infants in advanced countries. However, anti-allergic medicines available in the market have no effect on FA, and consequently effective drug therapies for FA are not yet available. We have already demonstrated that mucosal mast cells play an essential role in the development of FA in a murine model. Thus, we screened many constituents from medicinal herbs for the ability to inhibit rat basophilic leukemia-2H3 mast-like cell degranulation, and found that shikonin, a naphthoquinone dye from Lithospermum erythrorhizon, exhibited the most potent inhibitory effect among them. Furthermore, shikonin extremely inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of tumor necrosis factor (TNF)-α mRNA expression in mucosal-type bone marrow-derived mast cells (mBMMCs). Global gene expression analysis confirmed by real-time PCR revealed that shikonin drastically inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of mRNA expression of the nuclear orphan receptor 4a family (Nr4a1, Nr4a2 and Nr4a3) in mBMMCs, and knockdown of Nr4a1 or Nr4a2 suppressed the IgE/antigen-induced upregulation of TNF-α mRNA expression. Computational docking simulation of a small molecule for a target protein is a useful technique to elucidate the molecular mechanisms underlying the effects of drugs. Therefore, the simulation revealed that the predicted binding sites of shikonin to immunophilins (cyclophilin A and FK506 binding protein (FKBP) 12) were almost the same as the binding sites of immunosuppressants (cyclosporin A and FK506) to immunophilins. Indeed, shikonin inhibited the calcineurin activity to a similar extent as cyclosporin A that markedly suppressed the IgE/antigen-enhanced mRNA expression of TNF-α and the Nr4a family in mBMMCs. These findings suggest that shikonin suppresses mucosal mast cell activation by reducing Nr4a family gene expression through the inhibition of calcineurin activity. Therefore, shikonin has therapeutic potential for the treatment of allergic diseases as a new calcineurin inhibitor. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

PubMed Central

James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

2015-01-01

Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

Interactions between stepwise-eluted sub-fractions of fulvic acids and protons revealed by fluorescence titration combined with EEM-PARAFAC.

PubMed

Song, Fanhao; Wu, Fengchang; Guo, Fei; Wang, Hao; Feng, Weiying; Zhou, Min; Deng, Yanghui; Bai, Yingchen; Xing, Baoshan; Giesy, John P

2017-12-15

In aquatic environments, pH can control environmental behaviors of fulvic acid (FA) via regulating hydrolysis of functional groups. Sub-fractions of FA, eluted using pyrophosphate buffers with initial pHs of 3.0 (FA 3 ), 5.0 (FA 5 ), 7.0 (FA 7 ), 9.0 (FA 9 ) and 13.0 (FA 13 ), were used to explore interactions between the various, operationally defined, FA fractions and protons, by use of EEM-PARAFAC analysis. Splitting of peaks (FA 3 and FA 13 ), merging of peaks (FA 7 ), disappearance of peaks (FA 9 and FA 13 ), and red/blue-shifting of peaks were observed during fluorescence titration. Fulvic-like components were identified from FA 3 -FA 13 , and protein-like components were observed in fractions FA 9 and FA 13 . There primary compounds (carboxylic-like, phenolic-like, and protein-like chromophores) in PARAFAC components were distinguished based on acid-base properties. Dissociation constants (pK a ) for fulvic-like components with proton ranged from 2.43 to 4.13 in an acidic pH and from 9.95 to 11.27 at basic pH. These results might be due to protonation of di-carboxylate and phenolic functional groups. At basic pH, pK a values of protein-like components (9.77-10.13) were similar to those of amino acids. However, at acidic pH, pK a values of protein-like components, which ranged from 3.33 to 4.22, were 1-2units greater than those of amino acids. Results presented here, will benefit understanding of environmental behaviors of FA, as well as interactions of FA with environmental contaminants. Copyright © 2017 Elsevier B.V. All rights reserved.

Placental fatty acid transport in maternal obesity.

PubMed

Cetin, I; Parisi, F; Berti, C; Mandò, C; Desoye, G

2012-12-01

Pregestational obesity is a significant risk factor for adverse pregnancy outcomes. Maternal obesity is associated with a specific proinflammatory, endocrine and metabolic phenotype that may lead to higher supply of nutrients to the feto-placental unit and to excessive fetal fat accumulation. In particular, obesity may influence placental fatty acid (FA) transport in several ways, leading to increased diffusion driving force across the placenta, and to altered placental development, size and exchange surface area. Animal models show that maternal obesity is associated with increased expression of specific FA carriers and inflammatory signaling molecules in placental cotyledonary tissue, resulting in enhanced lipid transfer across the placenta, dislipidemia, fat accumulation and possibly altered development in fetuses. Cell culture experiments confirmed that inflammatory molecules, adipokines and FA, all significantly altered in obesity, are important regulators of placental lipid exchange. Expression studies in placentas of obese-diabetic women found a significant increase in FA binding protein-4 expression and in cellular triglyceride content, resulting in increased triglyceride cord blood concentrations. The expression and activity of carriers involved in placental lipid transport are influenced by the endocrine, inflammatory and metabolic milieu of obesity, and further studies are needed to elucidate the strong association between maternal obesity and fetal overgrowth.

Alpha-fetoprotein and Fanconi Anemia: Relevance to DNA Repair and Breast Cancer Susceptibility.

PubMed

Lakhi, Nisha A; Mizejewski, Gerald J

2017-02-01

Elevations of serum alpha-fetoprotein (sAFP) have been reported in fetal and infant states of anemia. Fanconi anemia (FA) belongs to a family of genetic instability disorders which lack the capability to repair DNA breaks. The lesion occurs at a checkpoint regulatory step of the G2 to mitotic transition, allowing FA cells to override cell-cycle arrest. FA DNA repair pathways contain complementation groups known as FANC proteins. FANC proteins form multi-protein complexes with BRCA proteins and are involved in homologous DNA repair. An impaired cascade in these events imparts an increased breast cancer susceptibility to female FA patients. Elevations of sAFP have availed this fetal protein to serve as a biomarker for FA disease. However, the origin of the synthesis of sAFA has not been determined in FA patients. We hypothesize that hematopoietic multipotent progenitor stem cells in the bone marrow are the source of sAFP production in FA patients.

Omega-3 fatty acid supplement prevents development of intracranial atherosclerosis.

PubMed

Shen, Jiamei; Hafeez, Adam; Stevenson, James; Yang, Jianjie; Yin, Changbin; Li, Fengwu; Wang, Sainan; Du, Huishan; Ji, Xunming; Rafols, Jose A; Geng, Xiaokun; Ding, Yuchuan

2016-10-15

Intracranial atherosclerotic stenosis (ICAS) is one of the most common causes of stroke worldwide and, in particular, has been implicated as a leading cause of recurrent ischemic stroke. We adapted a rat model of atherosclerosis to study brain intracranial atherosclerosis, and further investigated the effect of omega-3 fatty acids (O3FA) in attenuating development of ICAS. Adult male Sprague-Dawley rats were divided into control normal-cholesterol or high-cholesterol diet groups with or without O3FA for up to 6weeks. During the first 2weeks, NG-nitro-l-arginine methyl ester (l-NAME, 3mg/mL) was added to the drinking water of the high-cholesterol groups. The rats received supplementation with O3FA (5mg/kg/day) by gavages. Blood lipids including low density lipoprotein (LDL), cholesterol (CHO), triglycerides (TG) and high density lipoprotein (HDL) were measured at 3 and 6weeks. The lumen of middle cerebral artery (MCA) and the thickness of the vessel wall were assessed. Inflammatory molecular markers were assessed by Western blot. A high-cholesterol diet exhibited a significant increase in the classic blood markers (LDL, CHO, and TG) for atherosclerosis, as well as a decrease in HDL. These markers were found to be progressively more severe with time. Lumen stenosis and intimal thickening were increased in MCA. O3FA showed attenuation of blood lipids with an absence of morphological changes. O3FA significantly reduced the inflammatory marker CD68 in MCA and prevented monocyte chemotactic protein (MCP-1) and interferon-γ (IFN-γ) expression in the brain. O3FA similarly decreased inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6), markers affiliated with monocyte activity in atherosclerosis. Furthermore, O3FA significantly inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1), a marker for endothelial activation. Lastly, O3FA increased ATP-binding cassette transporter A1 (ABCA1) protein expression via silent information regulator 1 (SIRT1) activation, thus increasing cholesterol efflux from macrophages to HDL. Long-term O3FA dietary supplementation prevents the development of intracranial atherosclerosis. This O3FA effect appears to be mediated by its prevention of macrophage infiltration into the vessel wall, therefore reducing inflammation and intimal thickening. While similar effects in humans need to be determined, O3FA dietary supplement shows promising results in the prevention of ICAS. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Emerging functions of the Fanconi anemia pathway at a glance.

PubMed

Sumpter, Rhea; Levine, Beth

2017-08-15

Fanconi anemia (FA) is a rare disease, in which homozygous or compound heterozygous inactivating mutations in any of 21 genes lead to genomic instability, early-onset bone marrow failure and increased cancer risk. The FA pathway is essential for DNA damage response (DDR) to DNA interstrand crosslinks. However, proteins of the FA pathway have additional cytoprotective functions that may be independent of DDR. We have shown that many FA proteins participate in the selective autophagy pathway that is required for the destruction of unwanted intracellular constituents. In this Cell Science at a Glance and the accompanying poster, we briefly review the role of the FA pathway in DDR and recent findings that link proteins of the FA pathway to selective autophagy of viruses and mitochondria. Finally, we discuss how perturbations in FA protein-mediated selective autophagy may contribute to inflammatory as well as genotoxic stress. © 2017. Published by The Company of Biologists Ltd.

Fluorocoxib A Loaded Nanoparticles Enable Targeted Visualization of Cyclooxygenase-2 in Inflammation and Cancer

PubMed Central

Uddin, Md. Jashim; Werfel, Thomas A.; Crews, Brenda C.; Gupta, Mukesh K.; Kavanaugh, Taylor E.; Kingsley, Philip J.; Boyd, Kelli; Marnett, Lawrence J.; Duvall, Craig L.

2016-01-01

Cyclooxygenase-2 (COX-2) is expressed in virtually all solid tumors and its overexpression is a hallmark of inflammation. Thus, it is a potentially powerful biomarker for the early clinical detection of inflammatory disease and human cancers. We report a reactive oxygen species (ROS) responsive micellar nanoparticle, PPS-b-POEGA, that solubilizes the first fluorescent COX-2-selective inhibitor fluorocoxib A (FA) for COX-2 visualization in vivo. Pharmacokinetics and biodistribution of FA-PPS-b-POEGA nanoparticles (FA-NPs) were assessed after a fully-aqueous intravenous (i.v.) administration in wild-type mice and revealed 4 – 8 h post-injection as an optimal fluorescent imaging window. Carrageenan-induced inflammation in the rat and mouse footpads and 1483 HNSCC tumor xenografts were successfully visualized by FA-NPs with fluorescence up to 10-fold higher than that of normal tissues. The targeted binding of the FA cargo was blocked by pretreatment with the COX-2 inhibitor indomethacin, confirming COX-2-specific binding and local retention of FA at pathological sites. Our collective data indicate that FA-NPs are the first i.v.-ready FA formulation, provide high signal-to-noise in inflamed, premalignant, and malignant tissues, and will uniquely enable clinical translation of the poorly water-soluble FA compound. PMID:27043768

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei[S

PubMed Central

Esteves, Adriana; Knoll-Gellida, Anja; Canclini, Lucia; Silvarrey, Maria Cecilia; André, Michèle; Babin, Patrick J.

2016-01-01

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC12 but not BODIPY-FLC5 to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC12 to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC12 was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity. PMID:26658423

Fatty acid binding proteins have the potential to channel dietary fatty acids into enterocyte nuclei.

PubMed

Esteves, Adriana; Knoll-Gellida, Anja; Canclini, Lucia; Silvarrey, Maria Cecilia; André, Michèle; Babin, Patrick J

2016-02-01

Intracellular lipid binding proteins, including fatty acid binding proteins (FABPs) 1 and 2, are highly expressed in tissues involved in the active lipid metabolism. A zebrafish model was used to demonstrate differential expression levels of fabp1b.1, fabp1b.2, and fabp2 transcripts in liver, anterior intestine, and brain. Transcription levels of fabp1b.1 and fabp2 in the anterior intestine were upregulated after feeding and modulated according to diet formulation. Immunofluorescence and electron microscopy immunodetection with gold particles localized these FABPs in the microvilli, cytosol, and nuclei of most enterocytes in the anterior intestinal mucosa. Nuclear localization was mostly in the interchromatin space outside the condensed chromatin clusters. Native PAGE binding assay of BODIPY-FL-labeled FAs demonstrated binding of BODIPY-FLC(12) but not BODIPY-FLC(5) to recombinant Fabp1b.1 and Fabp2. The binding of BODIPY-FLC(12) to Fabp1b.1 was fully displaced by oleic acid. In vivo experiments demonstrated, for the first time, that intestinal absorption of dietary BODIPY-FLC(12) was followed by colocalization of the labeled FA with Fabp1b and Fabp2 in the nuclei. These data suggest that dietary FAs complexed with FABPs are able to reach the enterocyte nucleus with the potential to modulate nuclear activity. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

FANCJ Helicase Operates in the Fanconi Anemia DNA Repair Pathway and the Response to Replicational Stress

PubMed Central

Wu, Yuliang; Brosh, Robert M.

2009-01-01

Fanconi anemia (FA) is an autosomal recessive disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. Cells from FA patients exhibit spontaneous chromosomal instability and hypersensitivity to DNA interstrand cross-linking (ICL) agents. Although the precise mechanistic details of the FA/BRCA pathway of ICL-repair are not well understood, progress has been made in the identification of the FA proteins that are required for the pathway. Among the 13 FA complementation groups from which all the FA genes have been cloned, only a few of the FA proteins are predicted to have direct roles in DNA metabolism. One of the more recently identified FA proteins, shown to be responsible for complementation of the FA complementation group J, is the BRCA1 Associated C-terminal Helicase (BACH1, designated FANCJ), originally identified as a protein associated with breast cancer. FANCJ has been proposed to function downstream of FANCD2 monoubiquitination, a critical event in the FA pathway. Evidence supports a role for FANCJ in a homologous recombination (HR) pathway of double strand break (DSB) repair. In this review, we will summarize the current knowledge in terms of FANCJ functions through its enzymatic activities and protein interactions. The molecular roles of FANCJ in DNA repair and the response to replicational stress will be discussed. PMID:19519404

Free Fatty Acid Storage in Human Visceral and Subcutaneous Adipose Tissue

PubMed Central

Ali, Asem H.; Koutsari, Christina; Mundi, Manpreet; Stegall, Mark D.; Heimbach, Julie K.; Taler, Sandra J.; Nygren, Jonas; Thorell, Anders; Bogachus, Lindsey D.; Turcotte, Lorraine P.; Bernlohr, David; Jensen, Michael D.

2011-01-01

OBJECTIVE Because direct adipose tissue free fatty acid (FFA) storage may contribute to body fat distribution, we measured FFA (palmitate) storage rates and fatty acid (FA) storage enzymes/proteins in omental and abdominal subcutaneous fat. RESEARCH DESIGN AND METHODS Elective surgery patients received a bolus of [1-14C]palmitate followed by omental and abdominal subcutaneous fat biopsies to measure direct FFA storage. Long chain acyl-CoA synthetase (ACS) and diacylglycerol acyltransferase activities, CD36, fatty acid-binding protein, and fatty acid transport protein 1 were measured. RESULTS Palmitate tracer storage (dpm/g adipose lipid) and calculated palmitate storage rates were greater in omental than abdominal subcutaneous fat in women (1.2 ± 0.8 vs. 0.7 ± 0.4 μmol ⋅ kg adipose lipid−1 ⋅ min−1, P = 0.005) and men (0.7 ± 0.2 vs. 0.2 ± 0.1, P < 0.001), and both were greater in women than men (P < 0.0001). Abdominal subcutaneous adipose tissue palmitate storage rates correlated with ACS activity (women: r = 0.66, P = 0.001; men: r = 0.70, P = 0.007); in men, CD36 was also independently related to palmitate storage rates. The content/activity of FA storage enzymes/proteins in omental fat was dramatically lower in those with more visceral fat. In women, only omental palmitate storage rates were correlated (r = 0.54, P = 0.03) with ACS activity. CONCLUSIONS Some adipocyte FA storage factors correlate with direct FFA storage, but sex differences in this process in visceral fat do not account for sex differences in visceral fatness. The reduced storage proteins in those with greater visceral fat suggest that the storage factors we measured are not a predominant cause of visceral adipose tissue accumulation. PMID:21810594

Association of protein kinase FA/GSK-3alpha (a proline-directed kinase and a regulator of protooncogenes) with human cervical carcinoma dedifferentiation/progression.

PubMed

Yang, S D; Yu, J S; Lee, T T; Ni, M H; Yang, C C; Ho, Y S; Tsen, T Z

1995-10-01

Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in poorly differentiated cervical carcinoma (82.8 +/- 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 +/- 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 +/- 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 +/- 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3alpha in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3alpha may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3alpha may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3alpha may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression.

Fanconi Anemia Proteins and Their Interacting Partners: A Molecular Puzzle

PubMed Central

Kaddar, Tagrid; Carreau, Madeleine

2012-01-01

In recent years, Fanconi anemia (FA) has been the subject of intense investigations, primarily in the DNA repair research field. Many discoveries have led to the notion of a canonical pathway, termed the FA pathway, where all FA proteins function sequentially in different protein complexes to repair DNA cross-link damages. Although a detailed architecture of this DNA cross-link repair pathway is emerging, the question of how a defective DNA cross-link repair process translates into the disease phenotype is unresolved. Other areas of research including oxidative metabolism, cell cycle progression, apoptosis, and transcriptional regulation have been studied in the context of FA, and some of these areas were investigated before the fervent enthusiasm in the DNA repair field. These other molecular mechanisms may also play an important role in the pathogenesis of this disease. In addition, several FA-interacting proteins have been identified with roles in these “other” nonrepair molecular functions. Thus, the goal of this paper is to revisit old ideas and to discuss protein-protein interactions related to other FA-related molecular functions to try to give the reader a wider perspective of the FA molecular puzzle. PMID:22737580

Fanconi Anemia Core Complex Gene Promoters Harbor Conserved Transcription Regulatory Elements

PubMed Central

Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5′ region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3′ regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters. PMID:21826217

Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

PubMed

Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

Small-molecule inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity

PubMed Central

Lan, Hong; Cheng, Cliff C.; Kowalski, Timothy J.; Pang, Ling; Shan, Lixin; Chuang, Cheng-Chi; Jackson, James; Rojas-Triana, Alberto; Bober, Loretta; Liu, Li; Voigt, Johannes; Orth, Peter; Yang, Xianshu; Shipps, Gerald W.; Hedrick, Joseph A.

2011-01-01

Fatty acid binding protein-4 (FABP4) and FABP5 are two closely related FA binding proteins expressed primarily in adipose tissue and/or macrophages. The small-molecule FABP4 inhibitor BMS309403 was previously reported to improve insulin sensitivity in leptin-deficient Lepob/Lepob (ob/ob) mice. However, this compound was not extensively characterized in the more physiologically relevant animal model of mice with diet-induced obesity (DIO). Here, we report the discovery and characterization of a novel series of FABP4/5 dual inhibitors represented by Compounds 1–3. Compared with BMS309403, the compounds had significant in vitro potency toward both FABP4 and FABP5. In cell-based assays, Compounds 2 and 3 were more potent than BMS309403 to inhibit lipolysis in 3T3-L1 adipocytes and in primary human adipocytes. They also inhibited MCP-1 release from THP-1 macrophages as well as from primary human macrophages. When chronically administered to DIO mice, BMS309403 and Compound 3 reduced plasma triglyceride and free FA levels. Compound 3 reduced plasma free FAs at a lower dose level than BMS309403. However, no significant change was observed in insulin, glucose, or glucose tolerance. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia but not insulin resistance in DIO mice. PMID:21296956

Small-molecule inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity.

PubMed

Lan, Hong; Cheng, Cliff C; Kowalski, Timothy J; Pang, Ling; Shan, Lixin; Chuang, Cheng-Chi; Jackson, James; Rojas-Triana, Alberto; Bober, Loretta; Liu, Li; Voigt, Johannes; Orth, Peter; Yang, Xianshu; Shipps, Gerald W; Hedrick, Joseph A

2011-04-01

Fatty acid binding protein-4 (FABP4) and FABP5 are two closely related FA binding proteins expressed primarily in adipose tissue and/or macrophages. The small-molecule FABP4 inhibitor BMS309403 was previously reported to improve insulin sensitivity in leptin-deficient Lep(ob)/Lep(ob) (ob/ob) mice. However, this compound was not extensively characterized in the more physiologically relevant animal model of mice with diet-induced obesity (DIO). Here, we report the discovery and characterization of a novel series of FABP4/5 dual inhibitors represented by Compounds 1-3. Compared with BMS309403, the compounds had significant in vitro potency toward both FABP4 and FABP5. In cell-based assays, Compounds 2 and 3 were more potent than BMS309403 to inhibit lipolysis in 3T3-L1 adipocytes and in primary human adipocytes. They also inhibited MCP-1 release from THP-1 macrophages as well as from primary human macrophages. When chronically administered to DIO mice, BMS309403 and Compound 3 reduced plasma triglyceride and free FA levels. Compound 3 reduced plasma free FAs at a lower dose level than BMS309403. However, no significant change was observed in insulin, glucose, or glucose tolerance. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia but not insulin resistance in DIO mice.

The Fanconi anemia protein interaction network: casting a wide net.

PubMed

Rego, Meghan A; Kolling, Frederick W; Howlett, Niall G

2009-07-31

It has long been hypothesized that a defect in the repair of damaged DNA is central to the etiology of Fanconi anemia (FA). Indeed, an increased sensitivity of FA patient-derived cells to the lethal effects of various forms of DNA damaging agents was described over three decades ago [A.J. Fornace, Jr., J.B. Little, R.R. Weichselbaum, DNA repair in a Fanconi's anemia fibroblast cell strain, Biochim. Biophys. Acta 561 (1979) 99-109; Y. Fujiwara, M. Tatsumi, Repair of mitomycin C damage to DNA in mammalian cells and its impairment in Fanconi's anemia cells, Biochem. Biophys. Res. Commun. 66 (1975) 592-598; A.J. Rainbow, M. Howes, Defective repair of ultraviolet- and gamma-ray-damaged DNA in Fanconi's anaemia, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 31 (1977) 191-195]. Furthermore, the cytological hallmark of FA, the DNA crosslink-induced radial chromosome formation, exemplifies an innate impairment in the repair of these particularly cytotoxic DNA lesions [A.D. Auerbach, Fanconi anemia diagnosis and the diepoxybutane (DEB) test, Exp. Hematol. 21 (1993) 731-733]. Precisely defining the collective role of the FA proteins in DNA repair, however, continues to be one of the most enigmatic and challenging questions in the FA field. The first six identified FA proteins (A, C, E, F, G, and D2) harbored no recognizable enzymatic features, precluding association with a specific metabolic process. Consequently, our knowledge of the role of the FA proteins in the DNA damage response has been gleaned primarily through biochemical association studies with non-FA proteins. Here, we provide a chronological discourse of the major FA protein interaction network discoveries, with particular emphasis on the DNA damage response, that have defined our current understanding of the molecular basis of FA.

Fluocinolone acetonide partially restores the mineralization of LPS-stimulated dental pulp cells through inhibition of NF-κB pathway and activation of AP-1 pathway

PubMed Central

Liu, Zhongning; Jiang, Ting; Wang, Xinzhi; Wang, Yixiang

2013-01-01

BACKGROUND AND PURPOSE Fluocinolone acetonide (FA) is commonly used as a steroidal anti-inflammatory drug. We recently found that in dental pulp cells (DPCs) FA has osteo-/odonto-inductive as well as anti-inflammatory effects. However, the mechanism by which FA induces these effects in DPCs is poorly understood. EXPERIMENTAL APPROACH The effect of FA on the mineralization of DPCs during inflammatory conditions and the underlying mechanism were investigated by real-time PCR, Western blot, EMSA, histochemical staining, immunostaining and pathway blockade assays. KEY RESULTS FA significantly inhibited the inflammatory response in LPS-treated DPCs not only by down-regulating the expression of pro–inflammation-related genes, but also by up-regulating the expression of the anti-inflammatory gene PPAR-γ and mineralization-related genes. Moreover, histochemical staining and immunostaining showed that FA could partially restore the expressions of alkaline phosphatase, osteocalcin and dentin sialophosphoprotein (DSPP) and mineralization in LPS-stimulated DPCs. Real-time PCR and Western blot analysis revealed that FA up-regulated DSPP and runt-related transcription factor 2 expression by inhibiting the expression of phosphorylated-NF-κB P65 and activating activator protein-1 (AP-1) (p-c-Jun and Fra-1). These results were further confirmed through EMSA, by detection of NF-κB DNA-binding activity and pathway blockade assays using a NF-κB pathway inhibitor, AP-1 pathway inhibitor and glucocorticoid receptor antagonist. CONCLUSIONS AND IMPLICATIONS Inflammation induced by LPS suppresses the mineralization process in DPCs. FA partially restored this osteo-/odonto-genesis process in LPS-treated DPCs and had an anti-inflammatory effect through inhibition of the NF-κB pathway and activation of the AP-1 pathway. Hence, FA is a potential new treatment for inflammation-associated bone/teeth diseases. PMID:24024985

Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence

PubMed Central

Zhang, Huan; Pan, Yue; Wu, Yao; Tian, Xiu-Qi; Wang, Fang-Fang; Wang, Li

2017-01-01

As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication. PMID:28369120

Inhibitory effects of marine-derived DNA-binding anti-tumour tetrahydroisoquinolines on the Fanconi anaemia pathway.

PubMed

Martínez, Sandra; Pérez, Laura; Galmarini, Carlos M; Aracil, Miguel; Tercero, Juan C; Gago, Federico; Albella, Beatriz; Bueren, Juan A

2013-10-01

We have previously shown that cells with a defective Fanconi anaemia (FA) pathway are hypersensitive to trabectedin, a DNA-binding anti-cancer tetrahydroisoquinoline (DBAT) whose adducts functionally mimic a DNA inter-strand cross link (ICL). Here we expand these observations to new DBATs and investigate whether our findings in primary untransformed cells can be reproduced in human cancer cells. Initially, the sensitivity of transformed and untransformed cells, deficient or not in one component of the FA pathway, to mitomycin C (MMC) and three DBATs, trabectedin, Zalypsis and PM01183, was assessed. Then, the functional interaction of these drugs with the FA pathway was comparatively investigated. While untransformed FA-deficient haematopoietic cells were hypersensitive to both MMC and DBATs, the response of FA-deficient squamous cell carcinoma (SCC) cells to DBATs was similar to that of their respective FA-competent counterparts, even though these FA-deficient SCC cells were hypersensitive to MMC. Furthermore, while MMC always activated the FA pathway, the DBATs inhibited the FA pathway in the cancer cell lines tested and this enhanced their response to MMC. Our data show that although DBATs functionally interact with DNA as do agents that generate classical ICL, these drugs should be considered as FA pathway inhibitors rather than activators. Moreover, this effect was most significant in a variety of cancer cells. These inhibitory effects of DBATs on the FA pathway could be exploited clinically with the aim of 'fanconizing' cancer cells in order to make them more sensitive to other anti-tumour drugs. © 2013 The British Pharmacological Society.

Inhibitory effects of marine-derived DNA-binding anti-tumour tetrahydroisoquinolines on the Fanconi anaemia pathway

PubMed Central

Martínez, Sandra; Pérez, Laura; Galmarini, Carlos M; Aracil, Miguel; Tercero, Juan C; Gago, Federico; Albella, Beatriz; Bueren, Juan A

2013-01-01

BACKGROUND AND PURPOSE We have previously shown that cells with a defective Fanconi anaemia (FA) pathway are hypersensitive to trabectedin, a DNA-binding anti-cancer tetrahydroisoquinoline (DBAT) whose adducts functionally mimic a DNA inter-strand cross link (ICL). Here we expand these observations to new DBATs and investigate whether our findings in primary untransformed cells can be reproduced in human cancer cells. EXPERIMENTAL APPROACH Initially, the sensitivity of transformed and untransformed cells, deficient or not in one component of the FA pathway, to mitomycin C (MMC) and three DBATs, trabectedin, Zalypsis and PM01183, was assessed. Then, the functional interaction of these drugs with the FA pathway was comparatively investigated. KEY RESULTS While untransformed FA-deficient haematopoietic cells were hypersensitive to both MMC and DBATs, the response of FA-deficient squamous cell carcinoma (SCC) cells to DBATs was similar to that of their respective FA-competent counterparts, even though these FA-deficient SCC cells were hypersensitive to MMC. Furthermore, while MMC always activated the FA pathway, the DBATs inhibited the FA pathway in the cancer cell lines tested and this enhanced their response to MMC. CONCLUSIONS AND IMPLICATIONS Our data show that although DBATs functionally interact with DNA as do agents that generate classical ICL, these drugs should be considered as FA pathway inhibitors rather than activators. Moreover, this effect was most significant in a variety of cancer cells. These inhibitory effects of DBATs on the FA pathway could be exploited clinically with the aim of ‘fanconizing’ cancer cells in order to make them more sensitive to other anti-tumour drugs. PMID:23937566

Cell Communication during Aggregation and Development of the Cellular Slime Mould Distyostelium discoideum.

DTIC Science & Technology

1985-01-01

of actin protein xg relative centrifugal force glorin N-propionyl- Y -L-glutawyl-L-ornithine- S- lactam ethyl ester [3 H]FA [7,9,3’,5 ’-3H]folic acid...solubilize the pellet and radioactivity was measured on a LKB Rack Beta scintillation counter. cAMP Binding to Whole Cells. This assay followed the well...inserts, pre-filled with 4ml of Unisolve I scintillant, and radioactivity measured on a LKB Rack Beta scintillation counter. Controls included: a) no

De Novo Chromosome Copy Number Variation in Fanconi Anemia-Associated Hematopoietic Defects

DTIC Science & Technology

2014-08-01

protein, and not FANCA , is required for the suppression of spontaneous de novo CNV. These findings support a model whereby the FANCD2 protein, possibly...cancer susceptibility gene products BRCA1 and BRCA2 function cooperatively in the FA-BRCA pathway to repair damaged DNA. Recent studies have...the FA-BRCA pathway in the suppression of spontaneous de novo CNVs Task 1. Correction of FA-A, FA-C, and FA-D2 hTERT cells with pLenti6.2/V5- FANCA

Epidermal fatty acid-binding protein protects nerve growth factor-differentiated PC12 cells from lipotoxic injury

PubMed Central

Liu, Jo-Wen; Montero, Manuel; Bu, Liming; De Leon, Marino

2015-01-01

Epidermal fatty acid-binding protein (E-FABP/FABP5/DA11) binds and transport long-chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E-FABP protects nerve growth factor-differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM-induced lipotoxicity (PAM-LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E-FABP. Antioxidants MCI-186 and N-acetyl cysteine prevented E-FABP's induction in expression by PAM-LTx, while tert-butyl hydroperoxide increased ROS and E-FABP expression. Non-metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E-FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE-FABP showed reduced E-FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E-FABP cellular levels by pre-loading the cells with recombinant E-FABP diminished the PAM-induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E-FABP expression and enhanced the resistance of NGFDPC12 cells to PAM-LTx. We conclude that E-FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS. Epidermal fatty acid-binding protein (E-FABP) may protect nerve cells from the damaging exposure to high levels of free fatty acids (FA). We show that E-FABP can neutralize the effects of reactive oxygen species (ROS) generated by the high levels of FA in the cell and protect PC12 cells from lipotoxic injuries common in Type 2 diabetes neuropathy. Potentially, E-FABP gene up-regulation may be mediated through the NFkB pathway and future studies are needed to further evaluate this proposition. PMID:25147052

Direct fluorescence anisotropy assay for cocaine using tetramethylrhodamine-labeled aptamer.

PubMed

Liu, Yingxiong; Zhao, Qiang

2017-06-01

Development of simple, sensitive, and rapid method for cocaine detection is important in medicine and drug abuse monitoring. Taking advantage of fluorescence anisotropy and aptamer, this study reports a direct fluorescence anisotropy (FA) assay for cocaine by employing an aptamer probe with tetramethylrhodamine (TMR) labeled on a specific position. The binding of cocaine and the aptamer causes a structure change of the TMR-labeled aptamer, leading to changes of the interaction between labeled TMR and adjacent G bases in aptamer sequence, so FA of TMR varies with increasing of cocaine. After screening different labeling positions of the aptamer, including thymine (T) bases and terminals of the aptamer, we obtained a favorable aptamer probe with TMR labeled on the 25th base T in the sequence, which exhibited sensitive and significant FA-decreasing responses upon cocaine. Under optimized assay conditions, this TMR-labeled aptamer allowed for direct FA detection of cocaine as low as 5 μM. The maximum FA change reached about 0.086. This FA method also enabled the detection of cocaine spiked in diluted serum and urine samples, showing potential for applications. Graphical Abstract The binding of cocaine to the TMR-labeled aptamer causes conformation change and alteration of the intramolecular interaction between TMR and bases of aptamer, leading to variance of fluorescence anisotropy (FA) of TMR, so direct FA analyis of cocaine is achieved.

Evaluation of New Diagnostic Biomarkers in Pediatric Sepsis: Matrix Metalloproteinase-9, Tissue Inhibitor of Metalloproteinase-1, Mid-Regional Pro-Atrial Natriuretic Peptide, and Adipocyte Fatty-Acid Binding Protein

PubMed Central

Alqahtani, Mashael F.; Smith, Craig M.; Weiss, Scott L.; Dawson, Susan; Ralay Ranaivo, Hantamalala; Wainwright, Mark S.

2016-01-01

Elevated plasma concentrations of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), mid-regional pro-atrial natriuretic peptide (mrProANP), and adipocyte fatty-acid-binding proteins (A-FaBPs) have been investigated as biomarkers for sepsis or detection of acute neurological injuries in adults, but not children. We carried out a single-center, prospective observational study to determine if these measures could serve as biomarkers to identify children with sepsis. A secondary aim was to determine if these biomarkers could identify children with neurologic complications of sepsis. A total of 90 patients ≤ 18 years-old were included in this study. 30 with severe sepsis or septic shock were compared to 30 age-matched febrile and 30 age-matched healthy controls. Serial measurements of each biomarker were obtained, beginning on day 1 of ICU admission. In septic patients, MMP9-/TIMP-1 ratios (Median, IQR, n) were reduced on day 1 (0.024, 0.004–0.174, 13), day 2 (0.020, 0.002–0.109, 10), and day 3 (0.018, 0.003–0.058, 23) compared with febrile (0.705, 0.187–1.778, 22) and healthy (0.7, 0.4–1.2, 29) (p< 0.05) controls. A-FaBP and mrProANP (Median, IQR ng/mL, n) were elevated in septic patients compared to control groups on first 2 days after admission to the PICU (p <0.05). The area under the curve (AUC) for MMP-9/TIMP-1 ratio, mrProANP, and A-FaBP to distinguish septic patients from healthy controls were 0.96, 0.99, and 0.76, respectively. MMP-9/TIMP-1 ratio was inversely and mrProANP was directly related to PIM-2, PELOD, and ICU and hospital LOS (p<0.05). A-FaBP level was associated with PELOD, hospital and ICU length of stay (p<0.05). MMP-9/TIMP-1 ratio associated with poor Glasgow Outcome Score (p<0.05). A-FaBP levels in septic patients with neurological dysfunction (29.3, 17.2–54.6, 7) were significantly increased compared to septic patients without neurological dysfunction (14.6, 13.3–20.6, 11). MMP-9/TIMP-1 ratios were significantly lower, while A-FaBP and mrProANP were higher in septic patients compared to the control groups. Each biomarker was associated with hospital morbidity and length of stay. These results suggest that these biomarkers merit further prospective study for the early identification of children with sepsis. PMID:27089280

Evaluation of New Diagnostic Biomarkers in Pediatric Sepsis: Matrix Metalloproteinase-9, Tissue Inhibitor of Metalloproteinase-1, Mid-Regional Pro-Atrial Natriuretic Peptide, and Adipocyte Fatty-Acid Binding Protein.

PubMed

Alqahtani, Mashael F; Smith, Craig M; Weiss, Scott L; Dawson, Susan; Ralay Ranaivo, Hantamalala; Wainwright, Mark S

2016-01-01

Elevated plasma concentrations of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), mid-regional pro-atrial natriuretic peptide (mrProANP), and adipocyte fatty-acid-binding proteins (A-FaBPs) have been investigated as biomarkers for sepsis or detection of acute neurological injuries in adults, but not children. We carried out a single-center, prospective observational study to determine if these measures could serve as biomarkers to identify children with sepsis. A secondary aim was to determine if these biomarkers could identify children with neurologic complications of sepsis. A total of 90 patients ≤ 18 years-old were included in this study. 30 with severe sepsis or septic shock were compared to 30 age-matched febrile and 30 age-matched healthy controls. Serial measurements of each biomarker were obtained, beginning on day 1 of ICU admission. In septic patients, MMP9-/TIMP-1 ratios (Median, IQR, n) were reduced on day 1 (0.024, 0.004-0.174, 13), day 2 (0.020, 0.002-0.109, 10), and day 3 (0.018, 0.003-0.058, 23) compared with febrile (0.705, 0.187-1.778, 22) and healthy (0.7, 0.4-1.2, 29) (p< 0.05) controls. A-FaBP and mrProANP (Median, IQR ng/mL, n) were elevated in septic patients compared to control groups on first 2 days after admission to the PICU (p <0.05). The area under the curve (AUC) for MMP-9/TIMP-1 ratio, mrProANP, and A-FaBP to distinguish septic patients from healthy controls were 0.96, 0.99, and 0.76, respectively. MMP-9/TIMP-1 ratio was inversely and mrProANP was directly related to PIM-2, PELOD, and ICU and hospital LOS (p<0.05). A-FaBP level was associated with PELOD, hospital and ICU length of stay (p<0.05). MMP-9/TIMP-1 ratio associated with poor Glasgow Outcome Score (p<0.05). A-FaBP levels in septic patients with neurological dysfunction (29.3, 17.2-54.6, 7) were significantly increased compared to septic patients without neurological dysfunction (14.6, 13.3-20.6, 11). MMP-9/TIMP-1 ratios were significantly lower, while A-FaBP and mrProANP were higher in septic patients compared to the control groups. Each biomarker was associated with hospital morbidity and length of stay. These results suggest that these biomarkers merit further prospective study for the early identification of children with sepsis.

The Fanconi anemia ID2 complex: dueling saxes at the crossroads.

PubMed

Boisvert, Rebecca A; Howlett, Niall G

2014-01-01

Fanconi anemia (FA) is a rare recessive genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility in early adulthood. FA is caused by biallelic germ-line mutation of any one of 16 genes. While several functions for the FA proteins have been ascribed, the prevailing hypothesis is that the FA proteins function cooperatively in the FA-BRCA pathway to repair damaged DNA. A pivotal step in the activation of the FA-BRCA pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Despite their importance for DNA repair, the domain structure, regulation, and function of FANCD2 and FANCI remain poorly understood. In this review, we provide an overview of our current understanding of FANCD2 and FANCI, with an emphasis on their posttranslational modification and common and unique functions.

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

PubMed Central

Boisvert, Rebecca A; Howlett, Niall G

2014-01-01

Fanconi anemia (FA) is a rare recessive genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility in early adulthood. FA is caused by biallelic germ-line mutation of any one of 16 genes. While several functions for the FA proteins have been ascribed, the prevailing hypothesis is that the FA proteins function cooperatively in the FA-BRCA pathway to repair damaged DNA. A pivotal step in the activation of the FA-BRCA pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Despite their importance for DNA repair, the domain structure, regulation, and function of FANCD2 and FANCI remain poorly understood. In this review, we provide an overview of our current understanding of FANCD2 and FANCI, with an emphasis on their posttranslational modification and common and unique functions. PMID:25486561

Autotoxicity mechanism of Oryza sativa: transcriptome response in rice roots exposed to ferulic acid

PubMed Central

2013-01-01

Background Autotoxicity plays an important role in regulating crop yield and quality. To help characterize the autotoxicity mechanism of rice, we performed a large-scale, transcriptomic analysis of the rice root response to ferulic acid, an autotoxin from rice straw. Results Root growth rate was decreased and reactive oxygen species, calcium content and lipoxygenase activity were increased with increasing ferulic acid concentration in roots. Transcriptome analysis revealed more transcripts responsive to short ferulic-acid exposure (1- and 3-h treatments, 1,204 genes) than long exposure (24 h, 176 genes). Induced genes were involved in cell wall formation, chemical detoxification, secondary metabolism, signal transduction, and abiotic stress response. Genes associated with signaling and biosynthesis for ethylene and jasmonic acid were upregulated with ferulic acid. Ferulic acid upregulated ATP-binding cassette and amino acid/auxin permease transporters as well as genes encoding signaling components such as leucine-rich repeat VIII and receptor-like cytoplasmic kinases VII protein kinases, APETALA2/ethylene response factor, WRKY, MYB and Zinc-finger protein expressed in inflorescence meristem transcription factors. Conclusions The results of a transcriptome analysis suggest the molecular mechanisms of plants in response to FA, including toxicity, detoxicification and signaling machinery. FA may have a significant effect on inhibiting rice root elongation through modulating ET and JA hormone homeostasis. FA-induced gene expression of AAAP transporters may contribute to detoxicification of the autotoxin. Moreover, the WRKY and Myb TFs and LRR-VIII and SD-2b kinases might regulate downstream genes under FA stress but not general allelochemical stress. This comprehensive description of gene expression information could greatly facilitate our understanding of the mechanisms of autotoxicity in plants. PMID:23705659

A Role for the Juxtamembrane Cytoplasm in the Molecular Dynamics of Focal Adhesions

PubMed Central

Wolfenson, Haguy; Lubelski, Ariel; Regev, Tamar; Klafter, Joseph; Henis, Yoav I.; Geiger, Benjamin

2009-01-01

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico simulations and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplasmic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentration and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxtamembrane domain acts as an intermediary layer, enabling an efficient regulation of FA formation and reorganization. PMID:19172999

Fanconi anemia protein, FANCG, is a phosphoprotein and is upregulated with FANCA after TNF-alpha treatment.

PubMed

Futaki, M; Watanabe, S; Kajigaya, S; Liu, J M

2001-02-23

Fanconi anemia (FA) is a genetic syndrome characterized by bone marrow failure, birth defects, and a predisposition to malignancy. At this time, six FA genes have been identified, and several gene products have been found to interact in a protein complex. FA cells appear to overexpress the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). We therefore examined the effects of TNF-alpha on the regulation of FA complementation group proteins, FANCG and FANCA. We found that treatment with TNF-alpha induced FANCG protein expression. FANCA was induced concurrently with FANCG, and the FANCA/FANCG complex was increased in the nucleus following TNF-alpha treatment. Inactivation of inhibitory kappa B kinase-2 modulated the expression of FANCG. We also found that both nuclear and cytoplasmic FANCG fractions were phosphorylated. These results show that FANCG is a phosphoprotein and suggest that the cellular accumulation of FA proteins is subject to regulation by TNF-alpha signaling.

Cholinergic Receptor Binding in Alzheimer Disease and Healthy Aging: Assessment In Vivo with Positron Emission Tomography Imaging.

PubMed

Sultzer, David L; Melrose, Rebecca J; Riskin-Jones, Hannah; Narvaez, Theresa A; Veliz, Joseph; Ando, Timothy K; Juarez, Kevin O; Harwood, Dylan G; Brody, Arthur L; Mandelkern, Mark A

2017-04-01

To compare regional nicotinic cholinergic receptor binding in older adults with Alzheimer disease (AD) and healthy older adults in vivo and to assess relationships between receptor binding and clinical symptoms. Using cross-sectional positron emission tomography (PET) neuroimaging and structured clinical assessment, outpatients with mild to moderate AD (N = 24) and healthy older adults without cognitive complaints (C group; N = 22) were studied. PET imaging of α4β2* nicotinic cholinergic receptor binding using 2-[ 18 F]fluoro-3-(2(S)azetidinylmethoxy)pyridine (2FA) and clinical measures of global cognition, attention/processing speed, verbal memory, visuospatial memory, and neuropsychiatric symptoms were used. 2FA binding was lower in the AD group compared with the C group in the medial thalamus, medial temporal cortex, anterior cingulate, insula/opercula, inferior caudate, and brainstem (p < 0.05, corrected cluster), but binding was not associated with cognition. The C group had significant inverse correlations between 2FA binding in the thalamus (left: r s  = -0.55, p = 0.008; right: r s  = -0.50, p = 0.02; N = 22) and hippocampus (left: r s  = -0.65, p = 0.001; right: r s  = -0.55, p = 0.009; N = 22) and the Trails A score. The AD group had inverse correlation between 2FA binding in anterior cingulate (left: r s  = -0.50, p = 0.01; right: r s  = -0.50, p = 0.01; N = 24) and Neurobehavioral Rating Scale agitation/disinhibition factor score. Cholinergic receptor binding is reduced in specific brain regions in mild to moderate AD and is related to neuropsychiatric symptoms. Among healthy older adults, lower receptor binding may be associated with slower processing speed. Cholinergic receptor binding in vivo may reveal links to other key brain changes associated with aging and AD and may provide a potential molecular treatment target. Published by Elsevier Inc.

Stress and DNA repair biology of the Fanconi anemia pathway

PubMed Central

Longerich, Simonne; Li, Jian; Xiong, Yong; Sung, Patrick

2014-01-01

Fanconi anemia (FA) represents a paradigm of rare genetic diseases, where the quest for cause and cure has led to seminal discoveries in cancer biology. Although a total of 16 FA genes have been identified thus far, the biochemical function of many of the FA proteins remains to be elucidated. FA is rare, yet the fact that 5 FA genes are in fact familial breast cancer genes and FA gene mutations are found frequently in sporadic cancers suggest wider applicability in hematopoiesis and oncology. Establishing the interaction network involving the FA proteins and their associated partners has revealed an intersection of FA with several DNA repair pathways, including homologous recombination, DNA mismatch repair, nucleotide excision repair, and translesion DNA synthesis. Importantly, recent studies have shown a major involvement of the FA pathway in the tolerance of reactive aldehydes. Moreover, despite improved outcomes in stem cell transplantation in the treatment of FA, many challenges remain in patient care. PMID:25237197

Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

PubMed

Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

2017-03-15

Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2.

PubMed

Hussain, Shobbir; Wilson, James B; Blom, Eric; Thompson, Larry H; Sung, Patrick; Gordon, Susan M; Kupfer, Gary M; Joenje, Hans; Mathew, Christopher G; Jones, Nigel J

2006-05-10

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.

Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP.

PubMed

Chen, Ying-Hui; Wang, Gao-Yuan; Hao, Hao-Chao; Chao, Chun-Jiang; Wang, Yamei; Jin, Quan-Wen

2017-03-01

GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe , we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale. © 2017. Published by The Company of Biologists Ltd.

Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake.

PubMed

Cheng, Qing; Wållberg, Helena; Grafström, Jonas; Lu, Li; Thorell, Jan-Olov; Hägg Olofsson, Maria; Linder, Stig; Johansson, Katarina; Tegnebratt, Tetyana; Arnér, Elias S J; Stone-Elander, Sharon; Ahlzén, Hanna-Stina Martinsson; Ståhl, Stefan

2016-12-01

Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors. The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377-ST and ZTaq:3638-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with (11)C for in vivo PET studies. Kinetic scans with the (11)C-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions. Flow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZEGFR:2377-ST-DyLight488. [Methyl-(11)C]-labeled ZEGFR:2377-ST-CH3 and ZTaq:3638-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-(11)C]-ZEGFR:2377-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since ZEGFR:2377 binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels. Same-day in vivo dual tracer imaging enabled by the Sel-tag technology and (11)C-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand.

Mechanism of the development of nonalcoholic steatohepatitis after pancreaticoduodenectomy.

PubMed

Nagaya, Tadanobu; Tanaka, Naoki; Kimura, Takefumi; Kitabatake, Hiroyuki; Fujimori, Naoyuki; Komatsu, Michiharu; Horiuchi, Akira; Yamaura, Takahiro; Umemura, Takeji; Sano, Kenji; Gonzalez, Frank J; Aoyama, Toshifumi; Tanaka, Eiji

2015-06-01

It is recognized that nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), may develop after pancreaticoduodenectomy (PD). However, the mechanism of NASH development remains unclear. This study aimed to examine the changes in gene expression associated with NASH occurrence following PD. The expression of genes related to fatty acid/triglyceride (FA/TG) metabolism and inflammatory signaling was examined using liver samples obtained from 7 post-PD NASH patients and compared with 6 healthy individuals and 32 conventional NASH patients. The livers of post-PD NASH patients demonstrated significant up-regulation of the genes encoding CD36, FA-binding proteins 1 and 4, acetyl-coenzyme A carboxylase α, diacylglycerol acyltransferase 2, and peroxisome proliferator-activated receptor (PPAR) γ compared with normal and conventional NASH livers. Although serum apolipoprotein B (ApoB) and TG were decreased in post-PD NASH patients, the mRNAs of ApoB and microsomal TG transfer protein were robustly increased, indicating impaired TG export from the liver as very-low-density lipoprotein (VLDL). Additionally, elevated mRNA levels of myeloid differentiation primary response 88 and superoxide dismutases in post-PD NASH livers suggested significant activation of innate immune response and augmentation of oxidative stress generation. Enhanced FA uptake into hepatocytes and lipogenesis, up-regulation of PPARγ, and disruption of VLDL excretion into the circulation are possible mechanisms of steatogenesis after PD. These results provide a basis for understanding the pathogenesis of NAFLD/NASH following PD.

Ubiquitylation and the Fanconi Anemia Pathway

PubMed Central

Garner, Elizabeth; Smogorzewska, Agata

2012-01-01

The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic stability. PMID:21605559

A defined role for multiple Fanconi anemia gene products in DNA-damage-associated ubiquitination.

PubMed

Tan, Winnie; Deans, Andrew J

2017-06-01

Fanconi anemia (FA) is an inherited blood disorder that causes bone marrow failure and high predisposition to cancers. The FA pathway guards the cell's genome stability by orchestrating the repair of interstrand cross-linking during the S phase of the cell cycle, preventing the chromosomal instability that is a key event in bone marrow failure syndrome. Central to the FA pathway is loss of monoubiquitinated forms of the Fanconi proteins FANCI and FANCD2, a process that is normally mediated by a "core complex" of seven other Fanconi proteins. Each protein, when mutated, can cause FA. The FA core-complex-catalyzed reaction is critical for signaling DNA cross-link damage such as that induced by chemotherapies. Here, we present a perspective on the current understanding of FANCI and FANCD2 monoubiquitination-mediated DNA repair. Our recent biochemical reconstitution of the monoubiquitination (and deubiquitination) reactions creates a paradigm for understanding FA. Further biochemical analysis will create new opportunities to address the leukemic phenotype of FA patients. Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Increased mitochondrial matrix directed superoxide production by fatty acid hydroperoxides in skeletal muscle mitochondria

PubMed Central

Bhattacharya, Arunabh; Lustgarten, Michael; Shi, Yun; Liu, Yuhong; Jang, Youngmok C; Pulliam, Daniel; Jernigan, Amanda L; Van Remmen, Holly

2013-01-01

Previous studies have shown that muscle atrophy is associated with mitochondrial dysfunction and an increased rate of mitochondrial reactive oxygen species production. We recently demonstrated that fatty acid hydroperoxides (FA-OOH) are significantly elevated in mitochondria isolated from atrophied muscles. The purpose of the current study is to determine whether FA-OOH can alter skeletal muscle mitochondrial function. We found that FA-OOH (at low micromolar concentrations) induces mitochondrial dysfunction assessed by decrease in the rate of ATP production, oxygen consumption and activity of respiratory chain complexes I and III. Using methods to distinguish superoxide release towards the matrix and inter-membrane space, we demonstrate that FA-OOH significantly elevates oxidative stress in the mitochondrial matrix (and not the inter-membrane space) with complex I as the major site of superoxide production (most likely from a site upstream of the ubiquinone binding site but downstream from the flavin binding site-the iron sulfur clusters). Our results are the first to indicate that FA-OOH’s are important modulators of mitochondrial function and oxidative stress in skeletal muscle mitochondria and may play an important role in muscle atrophies that are associated with increased generation of FA-OOH’s, e.g., denervation-induced muscle atrophy. PMID:21172427

The FANCJ/MutLα interaction is required for correction of the cross-link response in FA-J cells

PubMed Central

Peng, Min; Litman, Rachel; Xie, Jenny; Sharma, Sudha; Brosh, Robert M; Cantor, Sharon B

2007-01-01

FANCJ also called BACH1/BRIP1 was first linked to hereditary breast cancer through its direct interaction with BRCA1. FANCJ was also recently identified as a Fanconi anemia (FA) gene product, establishing FANCJ as an essential tumor suppressor. Similar to other FA cells, FANCJ-null (FA-J) cells accumulate 4N DNA content in response to DNA interstrand crosslinks (ICLs). This accumulation is corrected by reintroduction of wild-type FANCJ. Here, we show that FANCJ interacts with the mismatch repair complex MutLα, composed of PMS2 and MLH1. Specifically, FANCJ directly interacts with MLH1 independent of BRCA1, through its helicase domain. Genetic studies reveal that FANCJ helicase activity and MLH1 binding, but not BRCA1 binding, are essential to correct the FA-J cells' ICL-induced 4N DNA accumulation and sensitivity to ICLs. These results suggest that the FANCJ/MutLα interaction, but not FANCJ/BRCA1 interaction, is essential for establishment of a normal ICL-induced response. The functional role of the FANCJ/MutLα complex demonstrates a novel link between FA and MMR, and predicts a broader role for FANCJ in DNA damage signaling independent of BRCA1. PMID:17581638

FANCD2 monoubiquitination and activation by hexavalent chromium [Cr(VI)] exposure

PubMed Central

Vilcheck, Susan K.; Ceryak, Susan; O’Brien, Travis J.; Patierno, Steven R.

2007-01-01

Fanconi anemia (FA) is a rare autosomal recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. FA cells are hypersensitive to DNA crosslinking agents. FA is a genetically heterogeneous disease with at least 11 complementation groups. The eight cloned FA proteins interact in a common pathway with established DNA-damage-response proteins, including BRCA1 and ATM. Six FA proteins (A, C, E, F, G, and L) regulate the monoubiquitination of FANCD2 after DNA damage by crosslinking agents, which targets FANCD2 to BRCA1 nuclear foci containing BRCA2 (FANCD1) and RAD51. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including DNA interstrand crosslinks. We have shown that FA-A fibroblasts are hypersensitive to both Cr(VI)-induced apoptosis and clonogenic lethality. Here we show that Cr(VI) treatment induced monoubiquitination of FANCD2 in normal human fibroblasts, providing the first molecular evidence of Cr(VI)-induced activation of the FA pathway. FA-A fibroblasts demonstrated no FANCD2 monoubiquitination, in keeping with the requirement of FA-A for this modification. We also found that Cr(VI) treatment induced significantly more S-phase-dependent DNA double strand breaks (DSBs), as measured by γ-H2AX expression, in FA-A fibroblasts compared to normal cells. However, and notably, DSBs were repaired equally in both normal and FA-A fibroblasts during recovery from Cr(VI) treatment. While previous research on FA has defined the genetic causes of this disease, it is critical in terms of individual risk assessment to address how cells from FA patients respond to genotoxic insult. PMID:16893675

Chitosan-coated doxorubicin nano-particles drug delivery system inhibits cell growth of liver cancer via p53/PRC1 pathway.

PubMed

Ye, Bai-Liang; Zheng, Ru; Ruan, Xiao-Jiao; Zheng, Zhi-Hai; Cai, Hua-Jie

2018-01-01

Nano-particles have been widely used in target-specific drug delivery system and showed advantages in cancers treatment. This study aims to evaluate the effect of chitosan coated doxorubicin nano-particles drug delivery system in liver cancer. The chitosan nano-particles were prepared by using the ionic gelation method. The characterizations of the nano-particles were determined by transmission electron microscopy. The cytotoxicity was detected by MTT assay, and the endocytosis, cell apoptosis and cell cycle were examined by flow cytometry. The protein level was analyzed with western blot. The dual luciferase reporter assay was performed to assess the interaction between p53 and the promoter of PRC1, and chromatin immune-precipitation was used to verify the binding between them. The FA-CS-DOX nano-particles were irregular and spherical particles around 30-40 nm, with uniform size and no adhesion. No significant difference was noted in doxorubicin release rate between CS-DOX and FA-CS-DOX. FA-CS-DOX nano-particles showed stronger cytotoxicity than CS-DOX. FA-CS-DOX nano-particles promoted the apoptosis and arrested cell cycle at G2/M phase, and they up-regulated p53. FA-CS-DOX nano-particles inhibited cell survival through p53/PRC1 pathway. Chitosan-coated doxorubicin nano-particles drug delivery system inhibits cell growth of liver cancer by promoting apoptosis and arresting cell cycle at G2/M phase through p53/PRC1 pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

N-3 fatty acids reduced trans fatty acids retention and increased docosahexaenoic acid levels in the brain.

PubMed

Lavandera, Jimena Verónica; Saín, Juliana; Fariña, Ana Clara; Bernal, Claudio Adrián; González, Marcela Aída

2017-09-01

The levels of docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) are critical for the normal structure and function of the brain. Trans fatty acids (TFA) and the source of the dietary fatty acids (FA) interfere with long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. The aim of this study was to investigate the effect of TFA supplementation in diets containing different proportions of n-9, n-6, and n-3 FA on the brain FA profile, including the retention of TFA, LC-PUFA levels, and n-6/n-3 PUFA ratios. These parameters were also investigated in the liver, considering that LC-PUFA are mainly bioconverted from their dietary precursors in this tissue and transported by serum to the brain. Also, stearoyl-CoA desaturase-1 (SCD1) and sterol regulatory element-binding protein-1c (SREBP-1c) gene expressions were evaluated. Male CF1 mice were fed (16 weeks) diets containing different oils (olive, corn, and rapeseed) with distinct proportions of n-9, n-6, and n-3 FA (55.2/17.2/0.7, 32.0/51.3/0.9, and 61.1/18.4/8.6), respectively, substituted or not with 0.75% of TFA. FA composition of the brain, liver, and serum was assessed by gas chromatography. TFA were incorporated into, and therefore retained in the brain, liver, and serum. However, the magnitude of retention was dependent on the tissue and type of isomer. In the brain, total TFA retention was lower than 1% in all diets. Dietary n-3 PUFA decreased TFA retention and increased DHA accretion in the brain. The results underscore the importance of the type of dietary FA on the retention of TFA in the brain and also on the changes of the FA profile.

Overexpression of protein kinase FA/GSK-3 alpha (a proline-directed protein kinase) correlates with human hepatoma dedifferentiation/progression.

PubMed

Yang, S D; Yu, J S; Yang, C C; Lee, S C; Lee, T T; Ni, M H; Kuan, C Y; Chen, H C

1996-05-01

Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3 alpha (kinase F(A)/GSK-3 alpha) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P < 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 +/- 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 +/- 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 +/- 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3 alpha is due to overexpression of the protein. Elevated kinase FA/GSK-3 alpha expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be fivefold overexpressed in well differentiated hepatoma and 13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3 alpha is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3 alpha is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression.

Cloning and characterization of murine fanconi anemia group A gene: Fanca protein is expressed in lymphoid tissues, testis, and ovary.

PubMed

van de Vrugt, H J; Cheng, N C; de Vries, Y; Rooimans, M A; de Groot, J; Scheper, R J; Zhi, Y; Hoatlin, M E; Joenje, H; Arwert, F

2000-04-01

Fanconi anemia (FA) is an autosomal recessive disorder in humans characterized by bone marrow failure, cancer predisposition, and cellular hypersensitivity to cross-linking agents such as mitomycin C and diepoxybutane. FA genes display a caretaker function essential for maintenance of genomic integrity. We have cloned the murine homolog of FANCA, the gene mutated in the major FA complementation group (FA-A). The full-length mouse Fanca cDNA consists of 4503 bp and encodes a protein with a predicted molecular weight of 161 kDa. The deduced Fanca mouse protein shares 81% amino acid sequence similarity and 66% identity with the human protein. The nuclear localization signal and partial leucine zipper consensus motifs found in the human FANCA protein were also present in the murine homolog. In spite of the species difference, the murine Fanca cDNA was capable of correcting the cross-linker sensitive phenotype of human FA-A cells, suggesting functional conservation. Based on Northern as well as Western blots, Fanca was mainly expressed in lymphoid tissues, testis, and ovary. This expression pattern correlates with some of the clinical symptoms observed in FA patients. The availability of the murine Fanca cDNA now allows the gene to be studied in experimental mouse models.

Folic acid-decorated polyamidoamine dendrimer mediates selective uptake and high expression of genes in head and neck cancer cells.

PubMed

Xu, Leyuan; Kittrell, Shannon; Yeudall, W Andrew; Yang, Hu

2016-11-01

Folic acid (FA)-decorated polyamidoamine dendrimer G4 (G4-FA) was synthesized and studied for targeted delivery of genes to head and neck cancer cells expressing high levels of folate receptors (FRs). Cellular uptake, targeting specificity, cytocompatibility and transfection efficiency were evaluated. G4-FA competes with free FA for the same binding site. G4-FA facilitates the cellular uptake of DNA plasmids in a FR-dependent manner and selectively delivers plasmids to FR-high cells, leading to enhanced gene expression. G4-FA is a suitable vector to deliver genes selectively to head and neck cancer cells. The fundamental understandings of G4-FA as a vector and its encouraging transfection results for head and neck cancer cells provided support for its further testing in vivo.

Protein source in a high-protein diet modulates reductions in insulin resistance and hepatic steatosis in fa/fa Zucker rats.

PubMed

Wojcik, Jennifer L; Devassy, Jessay G; Wu, Yinghong; Zahradka, Peter; Taylor, Carla G; Aukema, Harold M

2016-01-01

High-protein diets are being promoted to reduce insulin resistance and hepatic steatosis in metabolic syndrome. Therefore, the effect of protein source in high-protein diets on reducing insulin resistance and hepatic steatosis was examined. Fa/fa Zucker rats were provided normal-protein (15% of energy) casein, high-protein (35% of energy) casein, high-protein soy, or high-protein mixed diets with animal and plant proteins. The high-protein mixed diet reduced area under the curve for insulin during glucose tolerance testing, fasting serum insulin and free fatty acid concentrations, homeostatic model assessment index, insulin to glucose ratio, and pancreatic islet cell area. The high-protein mixed and the high-protein soy diets reduced hepatic lipid concentrations, liver to body weight ratio, and hepatic steatosis rating. These improvements were observed despite no differences in body weight, feed intake, or adiposity among high-protein diet groups. The high-protein casein diet had minimal benefits. A high-protein mixed diet was the most effective for modulating reductions in insulin resistance and hepatic steatosis independent of weight loss, indicating that the source of protein within a high-protein diet is critical for the management of these metabolic syndrome parameters. © 2015 The Obesity Society.

Rescue of replication failure by Fanconi anaemia proteins.

PubMed

Constantinou, Angelos

2012-02-01

Chromosomal aberrations are often associated with incomplete genome duplication, for instance at common fragile sites, or as a consequence of chemical alterations in the DNA template that block replication forks. Studies of the cancer-prone disease Fanconi anaemia (FA) have provided important insights into the resolution of replication problems. The repair of interstrand DNA crosslinks induced by chemotherapy drugs is coupled with DNA replication and controlled by FA proteins. We discuss here the recent discovery of new FA-associated proteins and the development of new tractable repair systems that have dramatically improved our understanding of crosslink repair. We focus also on how FA proteins protect against replication failure in the context of fragile sites and on the identification of reactive metabolites that account for the development of Fanconi anaemia symptoms.

Snm1B/Apollo functions in the Fanconi anemia pathway in response to DNA interstrand crosslinks.

PubMed

Mason, Jennifer M; Sekiguchi, JoAnn M

2011-07-01

Fanconi anemia (FA) is an inherited chromosomal instability disorder characterized by childhood aplastic anemia, developmental abnormalities and cancer predisposition. One of the hallmark phenotypes of FA is cellular hypersensitivity to agents that induce DNA interstrand crosslinks (ICLs), such as mitomycin C (MMC). FA is caused by mutation in at least 14 genes which function in the resolution of ICLs during replication. The FA proteins act within the context of a protein network in coordination with multiple repair factors that function in distinct pathways. SNM1B/Apollo is a member of metallo-β-lactamase/βCASP family of nucleases and has been demonstrated to function in ICL repair. However, the relationship between SNM1B and the FA protein network is not known. In the current study, we establish that SNM1B functions epistatically to the central FA factor, FANCD2, in cellular survival after ICL damage and homology-directed repair of DNA double-strand breaks. We also demonstrate that MMC-induced chromosomal anomalies are increased in SNM1B-depleted cells, and this phenotype is not further exacerbated upon depletion of either FANCD2 or another key FA protein, FANCI. Furthermore, we find that SNM1B is required for proper localization of critical repair factors, including FANCD2, BRCA1 and RAD51, to MMC-induced subnuclear foci. Our findings demonstrate that SNM1B functions within the FA pathway during the repair of ICL damage.

A leu-rich repeat receptor-like protein kinase, FaRIPK1, interacts with the ABA receptor, FaABAR, to regulate fruit ripening in strawberry.

PubMed

Hou, Bing-Zhu; Xu, Cheng; Shen, Yuan-Yue

2018-03-24

Strawberry (Fragaria×ananassa) is a model plant for studying non-climacteric fruit ripening regulated by abscisic acid (ABA); however, its exact molecular mechanisms are yet not fully understood. In this study, a predicted leu-rich repeat (LRR) receptor-like kinase in strawberry, red-initial protein kinase 1 (FaRIPK1), was screened and, using a yeast two-hybrid assay, was shown to interact with a putative ABA receptor, FaABAR. This association was confirmed by bimolecular fluorescence complementation and co-immunoprecipitation assays, and shown to occur in the nucleus. Expression analysis by real-time PCR showed that FaRIPK1 is expressed in roots, stems, leaves, flowers, and fruit, with a particularly high expression in white fruit at the onset of coloration. Down-regulation of FaRIPK1 expression in strawberry fruit, using Tobacco rattle virus-induced gene silencing, inhibited ripening, as evidenced by suppression of ripening-related physiological changes and reduced expression of several genes involved in softening, sugar content, pigmentation, and ABA biosynthesis and signaling. The yeast-expressed LRR and STK (serine/threonine protein kinase) domains of FaRIPK1 bound ABA and showed kinase activity, respectively. A fruit disc-incubation test revealed that FaRIPK1 expression was induced by ABA and ethylene. The synergistic action of FaRIPK1 with FaABAR in regulation of strawberry fruit ripening is discussed.

Immunofluorescence detection of nitrogenase proteins in whole cells.

PubMed

Rennie, R J

1976-12-01

Fluorescent antibodies (FA) prepared against the Mo-Fe and Fe proteins of nitrogenase from Klebsiella pneumoniae M5aI were used to detect these protein components in toluene-treated whole cells that were actively reducing acetylene. The FA were highly specific, staining only nitrogenase component proteins originating from Klebsiella. Cross-reactions between the FA and purified nitrogenase proteins from other dinitrogen-fixing micro-organisms did not occur, except in the case of Bacillus polymyxa. The tests rapidly and accurately assayed the component proteins in Klebsiella mutants and derivatives to which Klebsiella nif genes had been transferred either by plasmid or by other means. Cross-reactions also indicated the degree of relatedness between nitrogenase proteins from dinitrogen-fixing micro-organisms of various origins.

Recent discoveries in the molecular pathogenesis of the inherited bone marrow failure syndrome Fanconi anemia.

PubMed

Mamrak, Nicholas E; Shimamura, Akiko; Howlett, Niall G

2017-05-01

Fanconi anemia (FA) is a rare autosomal and X-linked genetic disease characterized by congenital abnormalities, progressive bone marrow failure (BMF), and increased cancer risk during early adulthood. The median lifespan for FA patients is approximately 33years. The proteins encoded by the FA genes function together in the FA-BRCA pathway to repair DNA damage and to maintain genome stability. Within the past two years, five new FA genes have been identified-RAD51/FANCR, BRCA1/FANCS, UBE2T/FANCT, XRCC2/FANCU, and REV7/FANCV-bringing the total number of disease-causing genes to 21. This review summarizes the discovery of these new FA genes and describes how these proteins integrate into the FA-BRCA pathway to maintain genome stability and critically prevent early-onset BMF and cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Szkudlarek-Haśnik, A.; Zubik-Skupień, I.; Góra, A.; Dubas, M.; Korzonek-Szlacheta, I.; Wielkoszyński, T.; Żurawiński, W.; Sosada, K.

2012-04-01

Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

2015-07-15

Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effectsmore » of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in normal human cells. • Protective role of proteasomes is linked to repair of DNA–protein crosslinks.« less

Sterol regulatory element-binding protein-1 participates in the regulation of fatty acid synthase expression in colorectal neoplasia.

PubMed

Li, J N; Mahmoud, M A; Han, W F; Ripple, M; Pizer, E S

2000-11-25

Endogenous fatty acid synthesis has been observed in certain rapidly proliferating normal and neoplastic tissues. Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the expression of lipogenic genes including fatty acid synthase (FAS), the major biosynthetic enzyme for fatty acid synthesis. We have previously shown that SREBP-1, FAS, and Ki-67, a proliferation marker, colocalized in the crypts of the fetal gastrointestinal tract epithelium. This study sought to determine whether SREBP-1 participates in the regulation of proliferation-associated fatty acid synthesis in colorectal neoplasia. An immunohistochemical analysis of SREBP-1, FAS, and Ki-67 expression in 25 primary human colorectal carcinoma specimens showed colocalization in 22 of these. To elucidate a functional linkage between SREBP-1 activation and proliferation-associated FA synthesis, SREBP-1 and FAS content were assayed during the adaptive response of cultured HCT116 colon carcinoma cells to pharmacological inhibition of FA synthesis. Cerulenin and TOFA each inhibited the endogenous synthesis of fatty acids in a dose-dependent manner and each induced increases in both precursor and mature forms of SREBP-1. Subsequently, both the transcriptional activity of the FAS promoter in a luciferase reporter gene construct and the FAS expression increased. These results demonstrate that tumor cells recognize and respond to a deficiency in endogenous fatty acid synthesis by upregulating both SREBP-1 and FAS expression and support the model that SREBP-1 participates in the transcriptional regulation of lipogenic genes in colorectal neoplasia. Copyright 2000 Academic Press.

Cleavable DNA-protein hybrid molecular beacon: A novel efficient signal translator for sensitive fluorescence anisotropy bioassay.

PubMed

Hu, Pan; Yang, Bin

2016-01-15

Due to its unique features such as high sensitivity, homogeneous format, and independence on fluorescent intensity, fluorescence anisotropy (FA) assay has become a hotspot of study in oligonucleotide-based bioassays. However, until now most FA probes require carefully customized structure designs, and thus are neither generalizable for different sensing systems nor effective to obtain sufficient signal response. To address this issue, a cleavable DNA-protein hybrid molecular beacon was successfully engineered for signal amplified FA bioassay, via combining the unique stable structure of molecular beacon and the large molecular mass of streptavidin. Compared with single DNA strand probe or conventional molecular beacon, the DNA-protein hybrid molecular beacon exhibited a much higher FA value, which was potential to obtain high signal-background ratio in sensing process. As proof-of-principle, this novel DNA-protein hybrid molecular beacon was further applied for FA bioassay using DNAzyme-Pb(2+) as a model sensing system. This FA assay approach could selectively detect as low as 0.5nM Pb(2+) in buffer solution, and also be successful for real samples analysis with good recovery values. Compatible with most of oligonucleotide probes' designs and enzyme-based signal amplification strategies, the molecular beacon can serve as a novel signal translator to expand the application prospect of FA technology in various bioassays. Copyright © 2015 Elsevier B.V. All rights reserved.

Towards understanding the E. coli PNP binding mechanism and FRET absence between E. coli PNP and formycin A.

PubMed

Prokopowicz, Małgorzata; Greń, Bartosz; Cieśla, Joanna; Kierdaszuk, Borys

2017-11-01

The aim of this study is threefold: (1) augmentation of the knowledge of the E. coli PNP binding mechanism; (2) explanation of the previously observed 'lack of FRET' phenomenon and (3) an introduction of the correction (modified method) for FRET efficiency calculation in the PNP-FA complexes. We present fluorescence studies of the two E. coli PNP mutants (F159Y and F159A) with formycin A (FA), that indicate that the aromatic amino acid is indispensable in the nucleotide binding, additional hydroxyl group at position 159 probably enhances the strength of binding and that the amino acids pair 159-160 has a great impact on the spectroscopic properties of the enzyme. The experiments were carried out in hepes and phosphate buffers, at pH7 and 8.3. Two methods, a conventional and a modified one, that utilizes the dissociation constant, for calculations of the energy transfer efficiency (E) and the acceptor-to-donor distance (r) between FA and the Tyr (energy donor) were employed. Total difference spectra were calculated for emission spectra (λ ex 280nm, 295nm, 305nm and 313nm) for all studied systems. Time-resolved techniques allowed to conclude the existence of a specific structure formed by amino acids at positions 159 and 160. The results showed an unexpected pattern change of FRET in the mutants, when compared to the wild type enzyme and a probable presence of a structure created between 159 and 160 residue, that might influence the binding efficiency. Additionally, we confirmed the indispensable role of the modification of the FRET efficiency (E) calculation on the fraction of enzyme saturation in PNP-FA systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation by fluorescence, STD-NMR, docking and semi-empirical calculations of the o-NBA photo-acid interaction with BSA

NASA Astrophysics Data System (ADS)

Chaves, Otávio A.; Jesus, Catarina S. H.; Cruz, Pedro F.; Sant'Anna, Carlos M. R.; Brito, Rui M. M.; Serpa, Carlos

2016-12-01

Serum albumins present reversible pH dependent conformational transitions. A sudden laser induced pH-jump is a methodology that can provide new insights on localized protein (un)folding processes that occur within the nanosecond to microsecond time scale. To generate the fast pH jump needed to fast-trigger a protein conformational event, a photo-triggered acid generator as o-nitrobenzaldehyde (o-NBA) can be conveniently used. In order to detect potential specific or nonspecific interactions between o-NBA and BSA, we have performed ligand-binding studies using fluorescence spectroscopy, saturation transfer difference (STD) NMR, molecular docking and semi-empirical calculations. Fluorescence quenching indicates the formation of a non-fluorescent complex in the ground-state between the fluorophore and the quencher, but o-NBA does not bind much effectively to the protein (Ka 4.34 × 103 M- 1) and thus can be considered a relatively weak binder. The corresponding thermodynamic parameters: ΔG°, ΔS° and ΔH° showed that the binding process is spontaneous and entropy driven. Results of 1H STD-NMR confirm that the photo-acid and BSA interact, and the relative intensities of the signals in the STD spectra show that all o-NBA protons are equally involved in the binding process, which should correspond to a nonspecific interaction. Molecular docking and semi-empirical calculations suggest that the o-NBA binds preferentially to the Trp-212-containing site of BSA (FA7), interacting via hydrogen bonds with Arg-217 and Tyr-149 residues.

Evaluation by fluorescence, STD-NMR, docking and semi-empirical calculations of the o-NBA photo-acid interaction with BSA.

PubMed

Chaves, Otávio A; Jesus, Catarina S H; Cruz, Pedro F; Sant'Anna, Carlos M R; Brito, Rui M M; Serpa, Carlos

2016-12-05

Serum albumins present reversible pH dependent conformational transitions. A sudden laser induced pH-jump is a methodology that can provide new insights on localized protein (un)folding processes that occur within the nanosecond to microsecond time scale. To generate the fast pH jump needed to fast-trigger a protein conformational event, a photo-triggered acid generator as o-nitrobenzaldehyde (o-NBA) can be conveniently used. In order to detect potential specific or nonspecific interactions between o-NBA and BSA, we have performed ligand-binding studies using fluorescence spectroscopy, saturation transfer difference (STD) NMR, molecular docking and semi-empirical calculations. Fluorescence quenching indicates the formation of a non-fluorescent complex in the ground-state between the fluorophore and the quencher, but o-NBA does not bind much effectively to the protein (Ka~4.34×10(3)M(-1)) and thus can be considered a relatively weak binder. The corresponding thermodynamic parameters: ΔG°, ΔS° and ΔH° showed that the binding process is spontaneous and entropy driven. Results of (1)H STD-NMR confirm that the photo-acid and BSA interact, and the relative intensities of the signals in the STD spectra show that all o-NBA protons are equally involved in the binding process, which should correspond to a nonspecific interaction. Molecular docking and semi-empirical calculations suggest that the o-NBA binds preferentially to the Trp-212-containing site of BSA (FA7), interacting via hydrogen bonds with Arg-217 and Tyr-149 residues. Copyright © 2016 Elsevier B.V. All rights reserved.

De Novo Chromosome Copy Number Variation in Fanconi Anemia-Associated Hematopoietic Defects

DTIC Science & Technology

2012-04-01

Appendix 1. Expansion of monoclonal populations of FA-A hTERT and FA-A + FANCA hTERT cells Appendix 2. Expansion of monoclonal populations of FA...marrow failure (BMF) and pronounced cancer susceptibility. The FA proteins and the major breast cancer susceptibility gene products BRCA1 and BRCA2...Correction of FA-A, FA-C, and FA-D2 hTERT cells with pLenti6.2/V5- FANCA , -FANCC, and FANCD2, respectively. Sub-task 1. Selection and expansion of clonal

Fanconi Anemia complementation group C protein in metabolic disorders.

PubMed

Nepal, Manoj; Ma, Chi; Xie, Guoxiang; Jia, Wei; Fei, Peiwen

2018-06-21

Given importance of 22-Fanconi Anemia (FA) proteins together to act in a signaling pathway in preventing deleterious clinical symptoms, e.g. severe bone marrow failure, congenital defects, an early onset of aging and cancer, studies on each FA protein become increasingly attractive. However, an unbiased and systematic investigation of cellular effects resulting from each FA protein is missing. Here, we report roles of FA complementation C group protein (FANCC) in the protection from metabolic disorders. This study was prompted by the diabetes-prone feature displayed in FANCC knockout mice, which is not typically shown in patients with FA. We found that in cells expressing FANCC at different levels, there are representative alterations in metabolites associated with aging (glycine, citrulline, ornithine, L-asparagine, L-tyrosine, L-arginine, L-glutamine, L-leucine, L-isoleucine, L-valine, L-proline and L-alanine), Diabetes Mellitus (DM) (carbon monoxide, collagens, fatty acids, D-glucose, fumaric acid, 2-oxoglutaric acid, C3), inflammation (inosine, L-arginine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, hypoxanthine, L-methionine), and cancer ( L-methionine, sphingomyelin, acetyl-L-carnitine, L-aspartic acid, L-glutamic acid, niacinamide, phospho-rylethanolamine). We also found that FANCC can act in an FA-pathway-independent manner in tumor suppression. Collectively, featured-metabolic alterations are readouts of functional mechanisms underlying reduced tumorigenicity driven by FANCC, demonstrating close links among cancer, aging, inflammation and DM.

Body distributioin of RGD-mediated liposome in brain-targeting drug delivery.

PubMed

Qin, Jing; Chen, DaWei; Hu, Haiyang; Qiao, MingXi; Zhao, XiuLi; Chen, Baoyu

2007-09-01

RGD conjugation liposomes (RGD-liposomes) were evaluated for brain-targeting drug delivery. The flow cytometric in vitro study demonstrated that RGD-liposomes could bind to monocytes and neutrophils effectively. Ferulic acid (4-hydroxy-3-methoxycinnamic, FA) was loaded into liposomes. Rats were subjected to intrastriatal microinjections of 100 units of human recombinant IL-1beta to produce brain inflammation and caudal vein injection of three formulations (FA solution, FA liposome and RGD-coated FA liposome). Animals were sacrificed 15, 30, 60 and 120 min after administration to study the body distribution of the FA in the three formulations. HPLC was used to determine the concentration of FA in vivo with salicylic acid as internal standard. The results of body distribution indicated that RGD-coated liposomes could be mediated into the brain with a 6-fold FA concentration compared to FA solution and 3-fold in comparison to uncoated liposome. Brain targeted delivery was achieved and a reduction in dosage might be allowed.

Folic acid-modified soy protein nanoparticles for enhanced targeting and inhibitory.

PubMed

Cheng, Xu; Wang, Xin; Cao, Zhipeng; Yao, Weijing; Wang, Jun; Tang, Rupei

2017-02-01

Soy protein isolate (SPI) was hydrolyzed by compound enzymes to give water soluble low molecular soy protein (SP). SP and folic acid (FA) modified SP was polymerized with N-3- acrylamidophenylboronic acid (APBA) monomer in aqueous solution to give SP nanoparticles (SP NPs) and FA modified nanoparticles (FA-SP NPs), respectively. These NPs display excellent stability in different conditions, and have a uniform spherical shape with a diameter around of 200nm. Doxorubicin (DOX) was then successfully loaded into SP and FA-SP NPs with a desirable loading content of 13.33% and 16.01%, respectively. The cellular uptake and cytotoxicity of DOX-loaded SP NPs and FA-SP NPs were investigated using the two-dimensional (2D) monolayer cell model and three-dimensional (3D) multicellular spheroids (MCs). In vivo, tumor accumulation and growth inhibitory were then examined using H22 tumor-bearing mice. All these results demonstrated that conjugation of FA can efficiently enhance SP-based NPs' tumor accumulation and antitumor effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Self-reported dietary intake of omega-3 fatty acids and association with bone and lower extremity function.

PubMed

Rousseau, James H; Kleppinger, Alison; Kenny, Anne M

2009-10-01

To assess the relationship between self-reported omega-3 fatty acid (O3FA) intake and bone mineral density (BMD) and lower extremity function in older adults. Cross-sectional analysis of baseline information from three separate ongoing studies of older adults, pooled for this analysis. Academic health center. Two hundred forty-seven men (n=118) and women (n=129) residing in the community or an assisted living facility. Self-reported dietary intake (O3FA, omega-6 fatty acids (O6FA), protein, and total calorie); BMD of the hip or heel; and lower extremity function including leg strength, chair rise time, walking speed, Timed Up and Go, and frailty. The mean reported intake of O3FA was 1.27 g/day. Correlation coefficients (r) between O3FA and T-scores from total femur (n=167) were 0.210 and 0.147 for combined femur and heel T scores. Similar correlations were found for leg strength (r=0.205) and chair rise time (r=-0.178), but the significance was lost when corrected for protein intake. Subjects with lower reported O3FA intake (<1.27 g/day) had lower BMD than those with higher reported O3FA intake. In a multiple regression analysis with femoral neck BMD as the dependent variable and reported intake of O3FA, O6FA, protein, and vitamin D as independent variables, reported O3FA intake was the only significant variable, accounting for 6% of the variance in BMD. Older adults had low reported intakes of O3FA. There was an association between greater reported O3FA intake and higher BMD. There was no independent association between reported O3FA intake and lower extremity function. Results from this preliminary report are promising and suggest further investigation.

Differential Cationization of Fatty Acids with Monovalent Cations Studied by ESI-MS/MS and Computational Approach.

PubMed

Sudarshana Reddy, B; Pavankumar, P; Sridhar, L; Saha, Soumen; Narahari Sastry, G; Prabhakar, S

2018-04-24

The intercellular and intracellular transport of charged species (Na + /K + ) entail interaction of the ions with neutral organic molecules and formation of adduct ions. The rate of transport of the ions across the cell membrane(s) may depend on the stability of the adduct ions, which in turn rely on structural aspects of the organic molecules that interact with the ions. Positive ion ESI mass spectra were recorded for the solutions containing fatty acids (FAs) and monovalent cations (X=Li + , Na + , K + , Rb + and Cs + ). Product ion spectra of the [FA+X] + ions were recorded at different collision energies. Theoretical studies were exploited under both gas phase and solvent phase to investigate the structural effects of the fatty acids during cationization. Stability of [FA+X] + adduct ions were further estimated by means of AIM topological analyses and interaction energy (IE) values. Positive ion ESI-MS analyses of the solution of FAs and X + ions showed preferential binding of the K + ions to FAs. The K + ion binding increased with the increase in number of double bonds of FAs, while decreased with increase in the number of carbons of FAs. Dissociation curves of [FA+X] + ions indicated the relative stability order of the [FA+X] + ions and it was in line with the observed trends in ESI-MS. The solvent phase computational studies divulged the mode of binding and the binding efficiencies of different FAs with monovalent cations. Among the studied monovalent cations, the cationization of FAs follow the order K + >Na + >Li + >Rb + >Cs + . The docosahexaenoic acid showed high efficiency in binding with K + ion. The K + ion binding efficiency of FAs depends on the number of double bonds in unsaturated FAs and the carbon chain length in saturated FAs. The cationization trends of FAs obtained from the ESI-MS, ESI-MS/MS analyses were in good agreement with solvent phase computational studies. This article is protected by copyright. All rights reserved.

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin*

PubMed Central

Kwak, Tae Kyoung; Lee, Mi-Sook; Ryu, Jihye; Choi, Yoon-Ju; Kang, Minkyung; Jeong, Doyoung; Lee, Jung Weon

2012-01-01

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration. PMID:22761432

Fatty acid elongase-5 (Elovl5) regulates hepatic triglyceride catabolism in obese C57BL/6J mice[S

PubMed Central

Tripathy, Sasmita; Lytle, Kelli A.; Stevens, Robert D.; Bain, James R.; Newgard, Christopher B.; Greenberg, Andrew S.; Huang, Li-Shin; Jump, Donald B.

2014-01-01

Nonalcoholic fatty liver disease is a major public health concern in the obese and type 2 diabetic populations. The high-fat lard diet induces obesity and fatty liver in C57BL/6J mice and suppresses expression of the PPAR-target gene, FA elongase 5 (Elovl5). Elovl5 plays a key role in MUFA and PUFA synthesis. Increasing hepatic Elovl5 activity in obese mice lowered hepatic TGs and endoplasmic reticulum stress markers (X-box binding protein 1 and cAMP-dependent transcription factor 6α) and increased TG catabolism and fatty acyl carnitines. Increased hepatic Elovl5 activity did not increase hepatic capacity for β-oxidation. Elovl5 effects on hepatic TG catabolism were linked to increased protein levels of adipocyte TG lipase (ATGL) and comparative gene identification 58 (CGI58). Elevated hepatic Elovl5 activity also induced the expression of some (pyruvate dehydrogenase kinase 4 and fibroblast growth factor 21), but not other cytochrome P450 4A10 (CYP4A10), PPAR-target genes. FA products of Elovl5 activity increased ATGL, but not CGI58, mRNA through PPARβ-dependent mechanisms in human HepG2 cells. Treatment of mouse AML12 hepatocytes with the PPARβ agonist (GW0742) decreased 14C-18:2,n-6 in TGs but did not affect β-oxidation. These studies establish that Elovl5 activity regulates hepatic levels of FAs controlling PPARβ activity, ATGL expression, and TG catabolism, but not FA oxidation. PMID:24814977

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice*

PubMed Central

Simhadri, Srilatha; Peterson, Shaun; Patel, Dharm S.; Huo, Yanying; Cai, Hong; Bowman-Colin, Christian; Miller, Shoreh; Ludwig, Thomas; Ganesan, Shridar; Bhaumik, Mantu; Bunting, Samuel F.; Jasin, Maria; Xia, Bing

2014-01-01

PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis. PMID:25016020

Protective Effects of Ferulic Acid on High Glucose-Induced Protein Glycation, Lipid Peroxidation, and Membrane Ion Pump Activity in Human Erythrocytes

PubMed Central

Sompong, Weerachat; Cheng, Henrique; Adisakwattana, Sirichai

2015-01-01

Ferulic acid (FA) is the ubiquitous phytochemical phenolic derivative of cinnamic acid. Experimental studies in diabetic models demonstrate that FA possesses multiple mechanisms of action associated with anti-hyperglycemic activity. The mechanism by which FA prevents diabetes-associated vascular damages remains unknown. The aim of study was to investigate the protective effects of FA on protein glycation, lipid peroxidation, membrane ion pump activity, and phosphatidylserine exposure in high glucose-exposed human erythrocytes. Our results demonstrated that FA (10-100 μM) significantly reduced the levels of glycated hemoglobin (HbA1c) whereas 0.1-100 μM concentrations inhibited lipid peroxidation in erythrocytes exposed to 45 mM glucose. This was associated with increased glucose consumption. High glucose treatment also caused a significant reduction in Na+/K+-ATPase activity in the erythrocyte plasma membrane which could be reversed by FA. Furthermore, we found that FA (0.1-100 μM) prevented high glucose-induced phosphatidylserine exposure. These findings provide insights into a novel mechanism of FA for the prevention of vascular dysfunction associated with diabetes. PMID:26053739

BRCA1 interacts directly with the Fanconi anemia protein FANCA.

PubMed

Folias, Alexandra; Matkovic, Mara; Bruun, Donald; Reid, Sonja; Hejna, James; Grompe, Markus; D'Andrea, Alan; Moses, Robb

2002-10-01

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by skeletal defects, anemia, chromosomal instability and increased risk of leukemia. At the cellular level FA is characterized by increased sensitivity to agents forming interstrand crosslinks (ICL) in DNA. Six FA genes have been cloned and interactions among individual FANC proteins have been found. The FANCD2 protein co-localizes in nuclear foci with the BRCA1 protein following DNA damage and during S-phase, requiring the FANCA, C, E and G proteins to do so. This finding may reflect a direct role for the BRCA1 protein in double strand break (DSB) repair and interaction with the FANC proteins. Therefore interactions between BRCA1 and the FANC proteins were investigated. Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740-1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.

Evidence of Field-Evolved Resistance of Spodoptera frugiperda to Bt Corn Expressing Cry1F in Brazil That Is Still Sensitive to Modified Bt Toxins

PubMed Central

Monnerat, Rose; Martins, Erica; Macedo, Cristina; Queiroz, Paulo; Praça, Lilian; Soares, Carlos Marcelo; Moreira, Helio; Grisi, Isabella; Silva, Joseane; Soberon, Mario; Bravo, Alejandra

2015-01-01

Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda. PMID:25830928

Evidence of field-evolved resistance of Spodoptera frugiperda to Bt corn expressing Cry1F in Brazil that is still sensitive to modified Bt toxins.

PubMed

Monnerat, Rose; Martins, Erica; Macedo, Cristina; Queiroz, Paulo; Praça, Lilian; Soares, Carlos Marcelo; Moreira, Helio; Grisi, Isabella; Silva, Joseane; Soberon, Mario; Bravo, Alejandra

2015-01-01

Brazil ranked second only to the United States in hectares planted to genetically modified crops in 2013. Recently corn producers in the Cerrado region reported that the control of Spodoptera frugiperda with Bt corn expressing Cry1Fa has decreased, forcing them to use chemicals to reduce the damage caused by this insect pest. A colony of S. frugiperda was established from individuals collected in 2013 from Cry1Fa corn plants (SfBt) in Brazil and shown to have at least more than ten-fold higher resistance levels compared with a susceptible colony (Sflab). Laboratory assays on corn leaves showed that in contrast to SfLab population, the SfBt larvae were able to survive by feeding on Cry1Fa corn leaves. The SfBt population was maintained without selection for eight generations and shown to maintain high levels of resistance to Cry1Fa toxin. SfBt showed higher cross-resistance to Cry1Aa than to Cry1Ab or Cry1Ac toxins. As previously reported, Cry1A toxins competed the binding of Cry1Fa to brush border membrane vesicles (BBMV) from SfLab insects, explaining cross-resistance to Cry1A toxins. In contrast Cry2A toxins did not compete Cry1Fa binding to SfLab-BBMV and no cross-resistance to Cry2A was observed, although Cry2A toxins show low toxicity to S. frugiperda. Bioassays with Cry1AbMod and Cry1AcMod show that they are highly active against both the SfLab and the SfBt populations. The bioassay data reported here show that insects collected from Cry1Fa corn in the Cerrado region were resistant to Cry1Fa suggesting that resistance contributed to field failures of Cry1Fa corn to control S. frugiperda.

Effect of different intestinal conditions on the intermolecular interaction between insulin and cell-penetrating peptide penetratin and on its contribution to stimulation of permeation through intestinal epithelium.

PubMed

Kamei, Noriyasu; Aoyama, Yukina; Khafagy, El-Sayed; Henmi, Mao; Takeda-Morishita, Mariko

2015-08-01

Our recent studies have shown that the coadministration of cell-penetrating peptides (CPPs) is a potential strategy for oral delivery of peptide- and protein-based biopharmaceuticals. The intermolecular interaction between drug and CPP is an essential factor in the effective delivery of these drugs, but the characteristics of the interaction under the conditions of the intestinal lumen remain unknown. In this study, therefore, we examined the characteristics of binding of the amphipathic CPP penetratin to insulin and the efficiency of its enhancement of epithelial insulin transport at different pH and in simulated intestinal fluids (SIFs). The binding between insulin and penetratin was pH dependent and particularly decreased at pH 5.0. In addition, we clarified that the sodium taurocholate (NaTC) present in two types of SIF (fasted-state SIF [FaSSIF] and fed-state SIF [FeSSIF]) affected binding efficiency. However, the permeation of insulin through a Caco-2 cell monolayer was significantly facilitated by coincubation with l- or d-penetratin at various pH values. Moreover, the permeation-stimulating effect of l-penetratin was observed in FaSSIF containing NaTC and lecithin, but not in 3mM NaTC solution, suggesting that the presence of lecithin was the key factor in maintaining the ability of penetratin to enhance the intestinal absorption of biopharmaceuticals. This report describes the essential considerations for in vivo use and clinical application of a CPP-based oral delivery strategy. Copyright © 2015 Elsevier B.V. All rights reserved.

Diets containing salmon fillet delay development of high blood pressure and hyperfusion damage in kidneys in obese Zucker fa/fa rats.

PubMed

Vikøren, Linn A; Drotningsvik, Aslaug; Mwakimonga, Angela; Leh, Sabine; Mellgren, Gunnar; Gudbrandsen, Oddrun A

2018-04-01

Hypertension is the leading risk factor for cardiovascular and chronic renal diseases, affecting more than 1 billion people. Fish intake is inversely correlated with the prevalence of hypertension in several, but not all, studies, and intake of fish oil and fish proteins has shown promising potential to delay development of high blood pressure in rats. The effects of baked and raw salmon fillet intake on blood pressure and renal function were investigated in obese Zucker fa/fa rats, which spontaneously develop hypertension with proteinuria and renal failure. Rats were fed diets containing baked or raw salmon fillet in an amount corresponding to 25% of total protein from salmon and 75% of protein from casein, or casein as the sole protein source (control group) for 4 weeks. Results show lower blood pressure and lower urine concentrations of albumin and cystatin C (relative to creatinine) in salmon diet groups when compared to control group. Morphological examinations revealed less prominent hyperfusion damage in podocytes from rats fed diets containing baked or raw salmon when compared to control rats. In conclusion, diets containing baked or raw salmon fillet delayed the development of hypertension and protected against podocyte damage in obese Zucker fa/fa rats. Copyright © 2018 American Heart Association. Published by Elsevier Inc. All rights reserved.

Development of LSPR and SPR sensor for the detection of an anti-cancer drug for chemotherapy

NASA Astrophysics Data System (ADS)

Zhao, Sandy Shuo; Bolduc, Olivier R.; Colin, Damien Y.; Pelletier, Joelle N.; Masson, Jean-François

2012-03-01

The anti-cancer drug, methotrexate (MTX) as a strong inhibitor of human dihydrofolate reductase (hDHFR) has been studied in localized surface plasmon resonance (LSPR) and surface plasmon resonance (SPR) competitive binding assays with folic acid stabilized gold nanoparticles (FA AuNP). The latter with a diameter of 15 nm were prepared in a simple step with sequential characterization using UV-Vis, FTIR, and Raman. A LSPR competitive binding assay between different concentrations of MTX and FA AuNP for hDHFR in solution was designed to quantify MTX by using UV-Vis spectroscopy. Sensitivity of the assay was optimized with respect to both concentrations of the enzyme and FA. The detection and quantification of spiked MTX was demonstrated in phosphate buffer saline and in fetal bovine serum accompanied by solid-phase extraction treatment of the serum. In addition, this assay could also provide as a screening tool for potential inhibitors of hDHFR. In another perspective, MTX was measured in a competitive binding assay with FA AuNP for histidine-tagged hDHFR immobilized on a SPR sensitive surface. In this case, FA AuNP offer a secondary amplification of the analytical response which is indirectly proportional to the concentration of MTX. This alternative approach could contribute to the realization of direct detection of MTX in complex biological fluids. A comparison of characteristics and analytical parameters such as sensitivity, dynamic range and limit of detection between the LSPR and SPR sensing platforms will also be presented. Both assays offer potential in tackling real biological samples for the purpose of monitoring and validating anti-cancer drug levels in human serum during chemotherapy.

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress.

PubMed

Li, Yibo; Amarachintha, Surya; Wilson, Andrew F; Li, Xue; Du, Wei

2017-06-18

Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G 2 /M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress.

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress

PubMed Central

Wilson, Andrew F.; Li, Xue

2017-01-01

ABSTRACT Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G2/M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress. PMID:28475398

HSP90 Shapes the Consequences of Human Genetic Variation.

PubMed

Karras, Georgios I; Yi, Song; Sahni, Nidhi; Fischer, Máté; Xie, Jenny; Vidal, Marc; D'Andrea, Alan D; Whitesell, Luke; Lindquist, Susan

2017-02-23

HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90 ≥ HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Mixed compared with single-source proteins in high-protein diets affect kidney structure and function differentially in obese fa/fa Zucker rats.

PubMed

Devassy, Jessay G; Wojcik, Jennifer L; Ibrahim, Naser H M; Zahradka, Peter; Taylor, Carla G; Aukema, Harold M

2017-02-01

Questions remain regarding the potential negative effects of dietary high protein (HP) on kidney health, particularly in the context of obesity in which the risk for renal disease is already increased. To examine whether some of the variability in HP effects on kidney health may be due to source of protein, obese fa/fa Zucker rats were given HP (35% of energy from protein) diets containing either casein, soy protein, or a mixed source of animal and plant proteins for 12 weeks. Control lean and obese rats were given diets containing casein at normal protein (15% of energy from protein) levels. Body weight and blood pressure were measured, and markers of renal structural changes, damage, and function were assessed. Obesity alone resulted in mild renal changes, as evidenced by higher kidney weights, proteinuria, and glomerular volumes. In obese rats, increasing the protein level using the single, but not mixed, protein sources resulted in higher renal fibrosis compared with the lean rats. The mixed-protein HP group also had lower levels of serum monocyte chemoattractant protein-1, even though this diet further increased kidney and glomerular size. Soy and mixed-protein HP diets also resulted in a small number of damaged glomeruli, while soy compared with mixed-protein HP diet delayed the increase in blood pressure over time. Since obesity itself confers added risk of renal disease, an HP diet from mixed-protein sources that enables weight loss but has fewer risks to renal health may be advantageous.

Cloning and expression analysis of FaPR-1 gene in strawberry

NASA Astrophysics Data System (ADS)

Mo, Fan; Luo, Ya; Ge, Cong; Mo, Qin; Ling, Yajie; Luo, Shu; Tang, Haoru

2018-04-01

The FaPR-1 gene was cloned by RT-PCR from `Benihoppe' strawberry and its bioinformatics analysis was conducted. The results showed that the open reading frame was 483 bp encoding encoding l60 amino acids which protein molecular weight and theoretical isoelectricity were 17854.17 and 8.72 respectively. Subcellular localization prediction shows that this gene is located extracellularly. By comparing strawberry FaPR-l and other plant Pathogenesis-related protein, homology and phylogenetic tree construction showed that the homology with grapes, peach is relatively close. In the treatments of ABA, sucrose and the mixture of the two, the expression of FaPR-1 in strawberry fruit were significantly increased.

STAT3-activated CD36 facilitates fatty acid uptake in chronic lymphocytic leukemia cells

PubMed Central

Rozovski, Uri; Harris, David M.; Li, Ping; Liu, Zhiming; Jain, Preetesh; Ferrajoli, Alessandra; Burger, Jan; Thompson, Phillip; Jain, Nitin; Wierda, William; Keating, Michael J.; Estrov, Zeev

2018-01-01

Although several studies established that unlike normal B cells chronic lymphocytic leukemia (CLL) cells metabolize fatty acids (FA), how CLL cells internalize FA is poorly understood. Because in various cell types CD36 facilitates FA uptake, we wondered whether a similar mechanism is operative CLL. We found that CD36 levels are higher in CLL cells than in normal B cells, and that small interfering RNA, CD36 neutralizing antibodies or sulfosuccinimidyl oleate (SSO) that inhibits CD36 significantly reduced the oxygen consumption of CLL cells incubated with FA. Because CD36 is oeverexpressed and STAT3 is constitutively activated in CLL cells, we wondered whether STAT3 induces CD36 expression. Sequence analysis identified putative STAT3 binding sites in the CD36 gene promoter. Chromatin immunoprecipitation and an electrophoretic mobility shift assay revealed that STAT3 binds to the CD36 gene promoter. A luciferase assay and STAT3-small hairpin RNA, that significantly decreased the levels of CD36 in CLL cells, established that STAT3 activates the transcription of the CD36 gene. Furthermore, SSO induced a dose-dependent apoptosis of CLL cells. Taken together, our data suggest that STAT3 activates CD36 and that CD36 facilitates FA uptake in CLL cells. Whether CD36 inhibition would provide clinical benefits in CLL remains to be determined. PMID:29765537

A novel Trojan-horse targeting strategy to reduce the non-specific uptake of nanocarriers by non-cancerous cells.

PubMed

Shen, Zheyu; Wu, Hao; Yang, Sugeun; Ma, Xuehua; Li, Zihou; Tan, Mingqian; Wu, Aiguo

2015-11-01

One big challenge with active targeting of nanocarriers is non-specific binding between targeting molecules and non-target moieties expressed on non-cancerous cells, which leads to non-specific uptake of nanocarriers by non-cancerous cells. Here, we propose a novel Trojan-horse targeting strategy to hide or expose the targeting molecules of nanocarriers on-demand. The non-specific uptake by non-cancerous cells can be reduced because the targeting molecules are hidden in hydrophilic polymers. The nanocarriers are still actively targetable to cancer cells because the targeting molecules can be exposed on-demand at tumor regions. Typically, Fe3O4 nanocrystals (FN) as magnetic resonance imaging (MRI) contrast agents were encapsulated into albumin nanoparticles (AN), and then folic acid (FA) and pH-sensitive polymers (PP) were grafted onto the surface of AN-FN to construct PP-FA-AN-FN nanoparticles. Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), transmission electron microscope (TEM) and gel permeation chromatography (GPC) results confirm successful construction of PP-FA-AN-FN. According to difference of nanoparticle-cellular uptake between pH 7.4 and 5.5, the weight ratio of conjugated PP to nanoparticle FA-AN-FN (i.e. graft density) and the molecular weight of PP (i.e. graft length) are optimized to be 1.32 and 5.7 kDa, respectively. In vitro studies confirm that the PP can hide ligand FA to prevent it from binding to cells with FRα at pH 7.4 and shrink to expose FA at pH 5.5. In vivo studies demonstrate that our Trojan-horse targeting strategy can reduce the non-specific uptake of the PP-FA-AN-FN by non-cancerous cells. Therefore, our PP-FA-AN-FN might be used as an accurately targeted MRI contrast agent. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hypolipidemic mechanism of oryzanol components- ferulic acid and phytosterols.

PubMed

Bhaskaragoud, G; Rajath, S; Mahendra, V P; Kumar, G Sunil; Gopala Krishna, A G; Kumar, G Suresh

2016-07-22

The effect of oryzanol (well known hypolipidemic component in rice bran oil) and its chemical constituents- ferulic acid (FA) and phytosterols on hypolipidemia were investigated. Docking (in silico) studies showed that FA had a better binding ability with lipase while sterols bound well with HMG-CoA reductase. Further in vivo studies of feeding high fat (30%) to rats increased body weights, serum TC, TG, non-HDL-C and reduced HDL-C were observed, compared to normal diet fed group (ND). ORZ treated groups alleviated the lipid profile. Furthermore, increased organ weights, higher intestinal lipase activity, and liver lipid peroxidation was observed in the high-fat group (HF). These effects were ameliorated in oryzanol concentrate fed groups (ORZ). Higher fecal fat was found in ORZ groups, analysis of fecal matter by mass spectroscopy revealed the presence of FA. In vitro, a bile acid binding study supported the strong affinity of sterol towards bile acids. In conclusion, oryzanol in the intestine is cleaved into FA and sterol by intestinal lipase enzymes both lipase and HMG-CoA reductase activities were inhibited, respectively. These hydrolysates eliminated the bile acids, thus lowering lipid profiles. Copyright © 2016 Elsevier Inc. All rights reserved.

Development and application of nanoparticles synthesized with folic acid-conjugated soy protein

USDA-ARS?s Scientific Manuscript database

In this study, soy protein isolate (SPI) was conjugated with folic acid (FA) to prepare nanoparticles for target-specific drug delivery. Successful conjugation was evidenced by UV spectrophotometry and primary amino group analysis. An increase in count rate by at least 142% was observed in FA-conjug...

Fluorophore Absorption Size Exclusion Chromatography (FA-SEC): An Alternative Method for High-Throughput Detergent Screening of Membrane Proteins.

PubMed

Lin, Sung-Yao; Sun, Xing-Han; Hsiao, Yu-Hsuan; Chang, Shao-En; Li, Guan-Syun; Hu, Nien-Jen

2016-01-01

Membrane proteins play key roles in many fundamental functions in cells including ATP synthesis, ion and molecule transporter, cell signalling and enzymatic reactions, accounting for ~30% genes of whole genomes. However, the hydrophobic nature of membrane proteins frequently hampers the progress of structure determination. Detergent screening is the critical step in obtaining stable detergent-solubilized membrane proteins and well-diffracting protein crystals. Fluorescence Detection Size Exclusion Chromatography (FSEC) has been developed to monitor the extraction efficiency and monodispersity of membrane proteins in detergent micelles. By tracing the FSEC profiles of GFP-fused membrane proteins, this method significantly enhances the throughput of detergent screening. However, current methods to acquire FSEC profiles require either an in-line fluorescence detector with the SEC equipment or an off-line spectrofluorometer microplate reader. Here, we introduce an alternative method detecting the absorption of GFP (FA-SEC) at 485 nm, thus making this methodology possible on conventional SEC equipment through the in-line absorbance spectrometer. The results demonstrate that absorption is in great correlation with fluorescence of GFP. The comparably weaker absorption signal can be improved by using a longer path-length flow cell. The FA-SEC profiles were congruent with the ones plotted by FSEC, suggesting FA-SEC could be a comparable and economical setup for detergent screening of membrane proteins.

Identification of Heat Shock Transcription Factor Genes Involved in Thermotolerance of Octoploid Cultivated Strawberry

PubMed Central

Liao, Wan-Yu; Lin, Lee-Fong; Jheng, Jing-Lian; Wang, Chun-Chung; Yang, Jui-Hung; Chou, Ming-Lun

2016-01-01

Heat shock transcription factors (HSFs) are mainly involved in the activation of genes in response to heat stress as well as other abiotic and biotic stresses. The growth, development, reproduction, and yield of strawberry are strongly limited by extreme temperatures and droughts. In this study, we used Illumina sequencing and obtained transcriptome data set from Fragaria × ananassa Duchessne cv. Toyonoka. Six contigs and three unigenes were confirmed to encode HSF proteins (FaTHSFs). Subsequently, we characterized the biological functions of two particularly selected unigenes, FaTHSFA2a and FaTHSFB1a, which were classified into class A2 and B HSFs, respectively. Expression assays revealed that FaTHSFA2a and FaTHSFB1a expression was induced by heat shock and correlated well with elevated ambient temperatures. Overexpression of FaTHSFA2a and FaTHSFB1a resulted in the activation of their downstream stress-associated genes, and notably enhanced the thermotolerance of transgenic Arabidopsis plants. Besides, both FaTHSFA2a and FaTHSFB1a fusion proteins localized in the nucleus, indicating their similar subcellular distributions as transcription factors. Our yeast one-hybrid assay suggested that FaTHSFA2a has trans-activation activity, whereas FaTHSFB1a expresses trans-repression function. Altogether, our annotated transcriptome sequences provide a beneficial resource for identifying most genes expressed in octoploid strawberry. Furthermore, HSF studies revealed the possible insights into the molecular mechanisms of thermotolerance, thus rendering valuable molecular breeding to improve the tolerance of strawberry in response to high-temperature stress. PMID:27999304

Identification of Heat Shock Transcription Factor Genes Involved in Thermotolerance of Octoploid Cultivated Strawberry.

PubMed

Liao, Wan-Yu; Lin, Lee-Fong; Jheng, Jing-Lian; Wang, Chun-Chung; Yang, Jui-Hung; Chou, Ming-Lun

2016-12-17

Heat shock transcription factors (HSFs) are mainly involved in the activation of genes in response to heat stress as well as other abiotic and biotic stresses. The growth, development, reproduction, and yield of strawberry are strongly limited by extreme temperatures and droughts. In this study, we used Illumina sequencing and obtained transcriptome data set from Fragaria × ananassa Duchessne cv. Toyonoka. Six contigs and three unigenes were confirmed to encode HSF proteins (FaTHSFs). Subsequently, we characterized the biological functions of two particularly selected unigenes, FaTHSFA2a and FaTHSFB1a , which were classified into class A2 and B HSFs, respectively. Expression assays revealed that FaTHSFA2a and FaTHSFB1a expression was induced by heat shock and correlated well with elevated ambient temperatures. Overexpression of FaTHSFA2a and FaTHSFB1a resulted in the activation of their downstream stress-associated genes, and notably enhanced the thermotolerance of transgenic Arabidopsis plants. Besides, both FaTHSFA2a and FaTHSFB1a fusion proteins localized in the nucleus, indicating their similar subcellular distributions as transcription factors. Our yeast one-hybrid assay suggested that FaTHSFA2a has trans-activation activity, whereas FaTHSFB1a expresses trans-repression function. Altogether, our annotated transcriptome sequences provide a beneficial resource for identifying most genes expressed in octoploid strawberry. Furthermore, HSF studies revealed the possible insights into the molecular mechanisms of thermotolerance, thus rendering valuable molecular breeding to improve the tolerance of strawberry in response to high-temperature stress.

Different Fatty Acids Compete with Arachidonic Acid for Binding to the Allosteric or Catalytic Subunits of Cyclooxygenases to Regulate Prostanoid Synthesis*

PubMed Central

Dong, Liang; Zou, Hechang; Yuan, Chong; Hong, Yu H.; Kuklev, Dmitry V.; Smith, William L.

2016-01-01

Prostaglandin endoperoxide H synthases (PGHSs), also called cyclooxygenases (COXs), convert arachidonic acid (AA) to PGH2. PGHS-1 and PGHS-2 are conformational heterodimers, each composed of an (Eallo) and a catalytic (Ecat) monomer. Previous studies suggested that the binding to Eallo of saturated or monounsaturated fatty acids (FAs) that are not COX substrates differentially regulate PGHS-1 versus PGHS-2. Here, we substantiate and expand this concept to include polyunsaturated FAs known to modulate COX activities. Non-substrate FAs like palmitic acid bind Eallo of PGHSs stimulating human (hu) PGHS-2 but inhibiting huPGHS-1. We find the maximal effects of non-substrate FAs on both huPGHSs occurring at the same physiologically relevant FA/AA ratio of ∼20. This inverse allosteric regulation likely underlies the ability of PGHS-2 to operate at low AA concentrations, when PGHS-1 is effectively latent. Unlike FAs tested previously, we observe that C-22 FAs, including ω-3 fish oil FAs, have higher affinities for Ecat than Eallo subunits of PGHSs. Curiously, C-20 ω-3 eicosapentaenoate preferentially binds Ecat of huPGHS-1 but Eallo of huPGHS-2. PGE2 production decreases 50% when fish oil consumption produces tissue EPA/AA ratios of ≥0.2. However, 50% inhibition of huPGHS-1 itself is only seen with ω-3 FA/AA ratios of ≥5.0. This suggests that fish oil-enriched diets disfavor AA oxygenation by altering the composition of the FA pool in which PGHS-1 functions. The distinctive binding specificities of PGHS subunits permit different combinations of non-esterified FAs, which can be manipulated dietarily, to regulate AA binding to Eallo and/or Ecat thereby controlling COX activities. PMID:26703471

Relationships among the serum omega fatty acid levels, serum C-reactive protein levels and arterial stiffness/wave reflection in Japanese men.

PubMed

Tomiyama, Hirofumi; Matsumoto, Chisa; Odaira, Mari; Yamada, Jiko; Yoshida, Masanobu; Shiina, Kazuki; Nagata, Mikio; Yamashina, Akira

2011-08-01

We examined the relationship among the serum omega-3 and omega-6 fatty acid (O3FA and O6FA) levels, serum C-reactive protein (CRP) levels, and arterial stiffness/wave reflection (AS/WR) in healthy Japanese men. In 2206 Japanese healthy men, parameters related to the AS/WR (i.e., brachial-ankle pulse wave velocity and radial arterial pulse wave analysis) were measured. No significant inverse relationships were observed between the serum O3FA levels and the AS/WR-related parameters. Adjusted values of the AS/WR-related parameters and serum CRP levels were higher in the subjects with serum O6FA levels in the highest tertile than in those with serum O6FA levels in the lowest tertile. In healthy Japanese men with known high dietary intakes of O3FAs, the serum O3FA levels may not reflect the pathophysiological abnormalities related to AS/WR. Increased serum O6FA levels appeared to be independently associated with the unfavorable conditions related to AS/WR and inflammation. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Fanconi anemia: causes and consequences of genetic instability.

PubMed

Kalb, R; Neveling, K; Nanda, I; Schindler, D; Hoehn, H

2006-01-01

Fanconi anemia (FA) is a rare recessive disease that reflects the cellular and phenotypic consequences of genetic instability: growth retardation, congenital malformations, bone marrow failure, high risk of neoplasia, and premature aging. At the cellular level, manifestations of genetic instability include chromosomal breakage, cell cycle disturbance, and increased somatic mutation rates. FA cells are exquisitely sensitive towards oxygen and alkylating drugs such as mitomycin C or diepoxybutane, pointing to a function of FA genes in the defense against reactive oxygen species and other DNA damaging agents. FA is caused by biallelic mutations in at least 12 different genes which appear to function in the maintenance of genomic stability. Eight of the FA proteins form a nuclear core complex with a catalytic function involving ubiquitination of the central FANCD2 protein. The posttranslational modification of FANCD2 promotes its accumulation in nuclear foci, together with known DNA maintenance proteins such as BRCA1, BRCA2, and the RAD51 recombinase. Biallelic mutations in BRCA2 cause a severe FA-like phenotype, as do biallelic mutations in FANCD2. In fact, only leaky or hypomorphic mutations in this central group of FA genes appear to be compatible with life birth and survival. The newly discovered FANCJ (= BRIP1) and FANCM (= Hef ) genes correspond to known DNA-maintenance genes (helicase resp. helicase-associated endonuclease for fork-structured DNA). These genes provide the most convincing evidence to date of a direct involvement of FA genes in DNA repair functions associated with the resolution of DNA crosslinks and stalled replication forks. Even though genetic instability caused by mutational inactivation of the FANC genes has detrimental effects for the majority of FA patients, around 20% of patients appear to benefit from genetic instability since genetic instability also increases the chance of somatic reversion of their constitutional mutations. Intragenic crossover, gene conversion, back mutation and compensating mutations in cis have all been observed in revertant, and, consequently, mosaic FA-patients, leading to improved bone marrow function. There probably is no other experiment of nature in our species in which causes and consequences of genetic instability, including the role of reactive oxygen species, can be better documented and explored than in FA.

Shaping up the protein folding funnel by local interaction: lesson from a structure prediction study.

PubMed

Chikenji, George; Fujitsuka, Yoshimi; Takada, Shoji

2006-02-28

Predicting protein tertiary structure by folding-like simulations is one of the most stringent tests of how much we understand the principle of protein folding. Currently, the most successful method for folding-based structure prediction is the fragment assembly (FA) method. Here, we address why the FA method is so successful and its lesson for the folding problem. To do so, using the FA method, we designed a structure prediction test of "chimera proteins." In the chimera proteins, local structural preference is specific to the target sequences, whereas nonlocal interactions are only sequence-independent compaction forces. We find that these chimera proteins can find the native folds of the intact sequences with high probability indicating dominant roles of the local interactions. We further explore roles of local structural preference by exact calculation of the HP lattice model of proteins. From these results, we suggest principles of protein folding: For small proteins, compact structures that are fully compatible with local structural preference are few, one of which is the native fold. These local biases shape up the funnel-like energy landscape.

Shaping up the protein folding funnel by local interaction: Lesson from a structure prediction study

PubMed Central

Chikenji, George; Fujitsuka, Yoshimi; Takada, Shoji

2006-01-01

Predicting protein tertiary structure by folding-like simulations is one of the most stringent tests of how much we understand the principle of protein folding. Currently, the most successful method for folding-based structure prediction is the fragment assembly (FA) method. Here, we address why the FA method is so successful and its lesson for the folding problem. To do so, using the FA method, we designed a structure prediction test of “chimera proteins.” In the chimera proteins, local structural preference is specific to the target sequences, whereas nonlocal interactions are only sequence-independent compaction forces. We find that these chimera proteins can find the native folds of the intact sequences with high probability indicating dominant roles of the local interactions. We further explore roles of local structural preference by exact calculation of the HP lattice model of proteins. From these results, we suggest principles of protein folding: For small proteins, compact structures that are fully compatible with local structural preference are few, one of which is the native fold. These local biases shape up the funnel-like energy landscape. PMID:16488978

Postpartum changes in maternal and infant erythrocyte fatty acids are likely to be driven by restoring insulin sensitivity and DHA status.

PubMed

Kuipers, Remko S; Luxwolda, Martine F; Sango, Wicklif S; Kwesigabo, Gideon; Velzing-Aarts, Francien V; Dijck-Brouwer, D A Janneke; Muskiet, Frits A J

2011-06-01

Perinatal changes in maternal glucose and lipid fluxes and de novo lipogenesis (DNL) are driven by hormones and nutrients. Docosahexaenoic acid (DHA) reduces, whereas insulin augments, nuclear abundance of sterol-regulatory-element-binding-protein-1 (SREBP-1), which promotes DNL, stearoyl-CoA-desaturase (SCD, also Δ9-desaturase), fatty acid-(FA)-elongation (Elovl) and FA-desaturation (FADS). Decreasing maternal insulin sensitivity with advancing gestation and compensatory hyperinsulinemia cause augmented postprandial glucose levels, adipose tissue lipolysis and hepatic glucose- and VLDL-production. Hepatic VLDL is composed of dietary, body store and DNL derived FA. Decreasing insulin sensitivity increases the contribution of FA from hepatic-DNL in VLDL-triacylglycerols, and consequently saturated-FA and monounsaturated-FA (MUFA) in maternal serum lipids increase during pregnancy. Although other authors described changes in maternal serum and RBC essential-FA (EFA) after delivery, none went into detail about the changes in non-EFA and the mechanisms behind -and/or functions of- the observed changes. Postpartum FA-changes result from changing enzymatic activities that are influenced by the changing hormonal milieu after delivery and DHA-status. We studied FA-profiles and FA-ratios (as indices for enzymatic activities) of maternal and infant RBC at delivery and after 3 months exclusive breastfeeding in three populations with increasing freshwater-fish intakes. DNL-, SCD- and FADS2-activities decreased after delivery. Elongation-6 (Elovl-6)- and FADS1-activities increased. The most pronounced postpartum changes for mothers were increases in 18:0, linoleic (LA), arachidonic acid (AA) and decreases in 16:0, 18:1ω9 and DHA; and for infants increases in 18:1ω9, 22:5ω3, LA and decreases in 16:0 and AA. Changes were in line with the literature. Postpartum increases in 18:0, and decreases in 16:0 and 18:1ω9, might derive from reduced insulin-promoted DNL-activity, with more reduced SCD- than Elovl-activity that leaves more 16:0 to be converted to 18:0 (Elovl-activity) than to MUFA (SCD-activity). Postpartum changes in ΣDNL, saturated-FA and MUFA related negatively to RBC-DHA. This concurs with suppression of both SCD- and Elovl-6 activities by DHA, through its influence on SREBP. Infant MUFA and LA increased at expense of their mothers. Sustained transport might be important for myelination (MUFA) and skin barrier development (LA). Maternal postpartum decreases in FADS2-, and apparent increases in FADS1-activity, together with increases in LA, AA, and 22:5ω3, but decrease in DHA, confirm that FADS2 is rate limiting in EFA-desaturation. Maternal LA and AA increases might be the result of rerouting from transplacental transfer to the incorporation into milk lipids and discontinued placental AA-utilization. Perinatal changes in maternal and infant FA status may be strongly driven by changing insulin sensitivity and DHA status. Copyright © 2011 Elsevier Ltd. All rights reserved.

Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

PubMed

Zhao, Hongyu; Yao, Zhenyu; Chen, Xiangbin; Wang, Xinquan; Chen, Guo-Qiang

2017-11-01

Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate (PHA) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of PhaP A h from Aeromonas hydrophila has been solved to date. Based on the structure of PhaP A h , homology models of PhaP A z from Azotobacter sp. FA-8 and PhaP TD from Halomonas bluephagenesis TD were successfully established, allowing rational mutagenesis to be conducted to enhance the stability and surfactant properties of these proteins. PhaP A z mutants, including PhaP A z Q38L and PhaP A z Q78L, as well as PhaP TD mutants, including PhaP TD Q38M and PhaP TD Q72M, showed better emulsification properties and improved thermostability (6-10°C higher melting temperatures) compared with their wild-type homologues under the same conditions. Importantly, the established PhaP homology-modelling approach, based on the high-resolution structure of PhaP A h , can be generalized to facilitate the study of other PhaP members. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

ATR-dependent phosphorylation of FANCA on serine 1449 after DNA damage is important for FA pathway function

PubMed Central

Collins, Natalie B.; Wilson, James B.; Bush, Thomas; Thomashevski, Andrei; Roberts, Kate J.; Jones, Nigel J.

2009-01-01

Previous work has shown several proteins defective in Fanconi anemia (FA) are phosphorylated in a functionally critical manner. FANCA is phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation are unknown. Mass spectrometry of FANCA revealed one phosphopeptide, phosphorylated on serine 1449. Serine 1449 phosphorylation was induced after DNA damage but not during S phase, in contrast to other posttranslational modifications of FA proteins. Furthermore, the S1449A mutant failed to completely correct a variety of FA-associated phenotypes. The DNA damage response is coordinated by phosphorylation events initiated by apical kinases ATM (ataxia telangectasia mutated) and ATR (ATM and Rad3-related), and ATR is essential for proper FA pathway function. Serine 1449 is in a consensus ATM/ATR site, phosphorylation in vivo is dependent on ATR, and ATR phosphorylated FANCA on serine 1449 in vitro. Phosphorylation of FANCA on serine 1449 is a DNA damage–specific event that is downstream of ATR and is functionally important in the FA pathway. PMID:19109555

Fatty Acids Regulate Germline Sex Determination through ACS-4-Dependent Myristoylation.

PubMed

Tang, Hongyun; Han, Min

2017-04-20

Fat metabolism has been linked to fertility and reproductive adaptation in animals and humans, and environmental sex determination potentially plays a role in the process. To investigate the impact of fatty acids (FA) on sex determination and reproductive development, we examined and observed an impact of FA synthesis and mobilization by lipolysis in somatic tissues on oocyte fate in Caenorhabditis elegans. The subsequent genetic analysis identified ACS-4, an acyl-CoA synthetase and its FA-CoA product, as key germline factors that mediate the role of FA in promoting oocyte fate through protein myristoylation. Further tests indicated that ACS-4-dependent protein myristoylation perceives and translates the FA level into regulatory cues that modulate the activities of MPK-1/MAPK and key factors in the germline sex-determination pathway. These findings, including a similar role of ACS-4 in a male/female species, uncover a likely conserved mechanism by which FA, an environmental factor, regulates sex determination and reproductive development. Copyright © 2017 Elsevier Inc. All rights reserved.

Update of the human and mouse Fanconi anemia genes.

PubMed

Dong, Hongbin; Nebert, Daniel W; Bruford, Elspeth A; Thompson, David C; Joenje, Hans; Vasiliou, Vasilis

2015-11-24

Fanconi anemia (FA) is a recessively inherited disease manifesting developmental abnormalities, bone marrow failure, and increased risk of malignancies. Whereas FA has been studied for nearly 90 years, only in the last 20 years have increasing numbers of genes been implicated in the pathogenesis associated with this genetic disease. To date, 19 genes have been identified that encode Fanconi anemia complementation group proteins, all of which are named or aliased, using the root symbol "FANC." Fanconi anemia subtype (FANC) proteins function in a common DNA repair pathway called "the FA pathway," which is essential for maintaining genomic integrity. The various FANC mutant proteins contribute to distinct steps associated with FA pathogenesis. Herein, we provide a review update of the 19 human FANC and their mouse orthologs, an evolutionary perspective on the FANC genes, and the functional significance of the FA DNA repair pathway in association with clinical disorders. This is an example of a set of genes--known to exist in vertebrates, invertebrates, plants, and yeast--that are grouped together on the basis of shared biochemical and physiological functions, rather than evolutionary phylogeny, and have been named on this basis by the HUGO Gene Nomenclature Committee (HGNC).

Mutations in the Paxillin-binding Site of Integrin-linked Kinase (ILK) Destabilize the Pseudokinase Domain and Cause Embryonic Lethality in Mice*

PubMed Central

Moik, Daniel; Böttcher, Anika; Makhina, Tatiana; Grashoff, Carsten; Bulus, Nada; Zent, Roy; Fässler, Reinhard

2013-01-01

Integrin-linked kinase (ILK) localizes to focal adhesions (FAs) where it regulates cell spreading, migration, and growth factor receptor signaling. Previous reports showed that overexpressed ILK in which Val386 and Thr387 were substituted with glycine residues (ILK-VT/GG) could neither interact with paxillin nor localize to FA in cells expressing endogenous wild-type ILK, implying that paxillin binding to ILK is required for its localization to FAs. Here, we show that introducing this mutation into the germ line of mice (ILK-VT/GG) caused vasculogenesis defects, resulting in a general developmental delay and death at around embryonic day 12.5. Fibroblasts isolated from ILK-VT/GG mice contained mutant ILK in FAs, showed normal adhesion to and spreading on extracellular matrix substrates but displayed impaired migration. Biochemical analysis revealed that VT/GG substitutions decreased ILK protein stability leading to decreased ILK levels and reduced binding to paxillin and α-parvin. Because paxillin depletion did not affect ILK localization to FAs, the embryonic lethality and the in vitro migration defects are likely due to the reduced levels of ILK-VT/GG and diminished binding to parvins. PMID:23658024

SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE2.6, an Ortholog of OPEN STOMATA1, Is a Negative Regulator of Strawberry Fruit Development and Ripening1[OPEN

PubMed Central

Dang, Ruihong; Li, Jinxi; Jiang, Jinzhu; Zhang, Ning; Jia, Meiru; Wei, Lingzhi; Li, Ziqiang; Li, Bingbing; Jia, Wensuo

2015-01-01

Whereas the regulatory mechanisms that direct fruit ripening have been studied extensively, little is known about the signaling mechanisms underlying this process, especially for nonclimacteric fruits. In this study, we demonstrated that a SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE2, designated as FaSnRK2.6, is a negative regulator of fruit development and ripening in the nonclimacteric fruit strawberry (Fragaria × ananassa) and can also mediate temperature-modulated strawberry fruit ripening. FaSnRK2.6 was identified as an ortholog of OPEN STOMATA1. Levels of FaSnRK2.6 transcript rapidly decreased during strawberry fruit development and ripening. FaSnRK2.6 was found to be capable of physically interacting with strawberry ABSCISIC ACID INSENSITIVE1, a negative regulator in strawberry fruit ripening. RNA interference-induced silencing of FaSnRK2.6 significantly promoted fruit ripening. By contrast, overexpression of FaSnRK2.6 arrested fruit ripening. Strawberry fruit ripening is highly sensitive to temperature, with high temperatures promoting ripening and low temperatures delaying it. As the temperature increased, the level of FaSnRK2.6 expression declined. Furthermore, manipulating the level of FaSnRK2.6 expression altered the expression of a variety of temperature-responsive genes. Taken together, this study demonstrates that FaSnRK2.6 is a negative regulator of strawberry fruit development and ripening and, furthermore, that FaSnRK2.6 mediates temperature-modulated strawberry fruit ripening. PMID:25609556

Role of Fanconi Anemia FANCG in Preventing Double-Strand Breakage and Chromosomal Rearrangement during DNA Replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tebbs, R S; Hinz, J M; Yamada, N A

The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are unknown. By constructing and characterizing a null fancg mutant of hamster CHO cells, we present several new insights for FA. The fancg cells show a broad sensitivity to genotoxic agents, not supporting the conventional concept of sensitivity to only DNA crosslinking agents. The aprt mutation rate is normal, but hprt mutations are reduced, which we ascribe to the lethality of large deletions. CAD and dhfr gene amplification rates are increased, implying excess chromosomal breakage during DNA replication, andmore » suggesting amplification as a contributing factor to cancer-proneness in FA patients. In S-phase cells, both spontaneous and mutagen-induced Rad51 nuclear foci are elevated. These results support a model in which FancG protein helps to prevent collapse of replication forks by allowing translesion synthesis or lesion bypass through homologous recombination.« less

Regulation of Fanconi anemia protein FANCD2 monoubiquitination by miR-302

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suresh, Bharathi; College of Medicine, Hanyang University, Seoul; Kumar, A. Madhan

2015-10-16

Fanconi anemia (FA) is a recessively inherited multigene disease characterized by congenital defects, progressive bone marrow failure, and heightened cancer susceptibility. Monoubiquitination of the FA pathway member FANCD2 contributes to the repair of replication stalling DNA lesions. However, cellular regulation of FANCD2 monoubiquitination remains poorly understood. In the present study, we identified the miR-302 cluster as a potential regulator of FANCD2 by bioinformatics analysis. MicroRNAs (miRNAs) are the major posttranscriptional regulators of a wide variety of biological processes, and have been implicated in a number of diseases. Expression of the exogenous miR-302 cluster (without miR-367) reduced FANCD2 monoubiquitination and nuclearmore » foci formation. Furthermore, miR-302 cells showed extensive chromosomal breakage upon MMC treatment when compared to mock control cells. Taken together, our results suggest that overexpression of miR-302 plays a critical role in the regulation of FANCD2 monoubiquitination, resulting in characteristic defects in DNA repair within cells. - Highlights: • miR-302 binds to the 3′UTR promoter of the FANCD2 gene to regulate gene expression. • miR-302 cluster down-regulates FANCD2 protein expression. • miR-302 cluster reduces FANCD2 monoubiquitination and nuclear foci formation. • miR-302 exhibits the characteristic defects in DNA repair in cells.« less

Role of Fatty Acid Kinase in Cellular Lipid Homeostasis and SaeRS-Dependent Virulence Factor Expression in Staphylococcus aureus

PubMed Central

Ericson, Megan E.; Subramanian, Chitra; Frank, Matthew W.

2017-01-01

ABSTRACT The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus, but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nonesterified FA pool in S. aureus that is elevated in strains lacking FA kinase activity. SaeRS-mediated transcription is restored in FA kinase-negative strains when the intracellular FA pool is reduced either by growth with FA-depleted bovine serum albumin to extract the FA into the medium or by the heterologous expression of Neisseria gonorrhoeae acyl-acyl carrier protein synthetase to activate FA for phospholipid synthesis. These data show that FAs act as negative regulators of SaeRS signaling, and FA kinase activates SaeRS-dependent virulence factor production by lowering inhibitory FA levels. Thus, FA kinase plays a role in cellular lipid homeostasis by activating FA for incorporation into phospholipid, and it indirectly regulates SaeRS signaling by maintaining a low intracellular FA pool. PMID:28765222

Acid-base properties of humic substances from composted and thermally-dried sewage sludges and amended soils as determined by potentiometric titration and the NICA-Donnan model.

PubMed

Fernández, José M; Plaza, César; Senesi, Nicola; Polo, Alfredo

2007-09-01

The acid-base properties of humic acids (HAs) and fulvic acids (FAs) isolated from composted sewage sludge (CS), thermally-dried sewage sludge (TS), soils amended with either CS or TS at a rate of 80 t ha(-1)y(-1) for 3y and the corresponding unamended soil were investigated by use of potentiometric titrations. The non-ideal competitive adsorption (NICA)-Donnan model for a bimodal distribution of proton binding sites was fitted to titration data by use of a least-squares minimization method. The main fitting parameters of the NICA-Donnan model obtained for each HA and FA sample included site densities, median affinity constants and widths of affinity distributions for proton binding to low and high affinity sites, which were assumed to be, respectively, carboxylic- and phenolic-type groups. With respect to unamended soil HA and FA, the HAs and FAs from CS, and especially TS, were characterized by smaller acidic functional group contents, larger proton binding affinities of both carboxylic- and phenolic-type groups, and smaller heterogeneity of carboxylic and phenolic-type groups. Amendment with CS or TS led to a decrease of acidic functional group contents and a slight increase of proton binding affinities of carboxylic- and phenolic-type groups of soil HAs and FAs. These effects were more evident in the HA and FA fractions from CS-amended soil than in those from TS-amended soil.

[Molecular basis of Fanconi's anemia].

PubMed

Digweed, M

1999-01-01

Fanconi anaemia (FA) is an autosomal recessive genetic disorder characterised clinically by progressive bone marrow failure, skeletal deformities and a predisposition to neoplasia. Patient cells manifest an extreme chromosomal instability and hypersensitivity to polyfunctional alkylating agents. It is assumed that the basic defect is related to the repair of DNA damage, in particular that of so-called DNA crosslinks. Currently there are eight complementation groups in FA (FA-A-FA-H) which indicates that at least eight independent genes can lead to FA. Three of these genes have been identified: FANCA, FANCC and FANCG. In this review, the molecular biology and genetics of FA are presented and possible functions of the FANC proteins are discussed.

ATM and KAT5 safeguard replicating chromatin against formaldehyde damage

PubMed Central

Ortega-Atienza, Sara; Wong, Victor C.; DeLoughery, Zachary; Luczak, Michal W.; Zhitkovich, Anatoly

2016-01-01

Many carcinogens damage both DNA and protein constituents of chromatin, and it is unclear how cells respond to this compound injury. We examined activation of the main DNA damage-responsive kinase ATM and formation of DNA double-strand breaks (DSB) by formaldehyde (FA) that forms histone adducts and replication-blocking DNA-protein crosslinks (DPC). We found that low FA doses caused a strong and rapid activation of ATM signaling in human cells, which was ATR-independent and restricted to S-phase. High FA doses inactivated ATM via its covalent dimerization and formation of larger crosslinks. FA-induced ATM signaling showed higher CHK2 phosphorylation but much lower phospho-KAP1 relative to DSB inducers. Replication blockage by DPC did not produce damaged forks or detectable amounts of DSB during the main wave of ATM activation, which did not require MRE11. Chromatin-monitoring KAT5 (Tip60) acetyltransferase was responsible for acetylation and activation of ATM by FA. KAT5 and ATM were equally important for triggering of intra-S-phase checkpoint and ATM signaling promoted recovery of normal human cells after low-dose FA. Our results revealed a major role of the KAT5-ATM axis in protection of replicating chromatin against damage by the endogenous carcinogen FA. PMID:26420831

Biallelic inactivation of REV7 is associated with Fanconi anemia.

PubMed

Bluteau, Dominique; Masliah-Planchon, Julien; Clairmont, Connor; Rousseau, Alix; Ceccaldi, Raphael; Dubois d'Enghien, Catherine; Bluteau, Olivier; Cuccuini, Wendy; Gachet, Stéphanie; Peffault de Latour, Régis; Leblanc, Thierry; Socié, Gérard; Baruchel, André; Stoppa-Lyonnet, Dominique; D'Andrea, Alan D; Soulier, Jean

2016-09-01

Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient's cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV.

Biallelic inactivation of REV7 is associated with Fanconi anemia

PubMed Central

Masliah-Planchon, Julien; Clairmont, Connor; Rousseau, Alix; Ceccaldi, Raphael; Dubois d’Enghien, Catherine; Bluteau, Olivier; Cuccuini, Wendy; Gachet, Stéphanie; Peffault de Latour, Régis; Leblanc, Thierry; Socié, Gérard; Baruchel, André; Stoppa-Lyonnet, Dominique; D’Andrea, Alan D.

2016-01-01

Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, γH2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient’s cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV. PMID:27500492

Low-protein, high-carbohydrate diet increases glucose uptake and fatty acid synthesis in brown adipose tissue of rats.

PubMed

Aparecida de França, Suélem; Pavani Dos Santos, Maísa; Nunes Queiroz da Costa, Roger Vinícius; Froelich, Mendalli; Buzelle, Samyra Lopes; Chaves, Valéria Ernestânia; Giordani, Morenna Alana; Pereira, Mayara Peron; Colodel, Edson Moleta; Marlise Balbinotti Andrade, Cláudia; Kawashita, Nair Honda

2014-04-01

The aim of this study was to evaluate glucose uptake and the contribution of glucose to fatty acid (FA) synthesis and the glycerol-3-phosphate (G3P) of triacylglycerol synthesis by interscapular brown adipose tissue (IBAT) of low-protein, high-carbohydrate (LPHC) diet-fed rats. LPHC (6% protein; 74% carbohydrate) or control (17% protein; 63% carbohydrate) diets were administered to rats (∼ 100 g) for 15 d. Total FA and G3P synthesis and the synthesis of FA and G3P from glucose were evaluated in vivo by (3)H2O and (14)C-glucose. Sympathetic neural contribution for FA synthesis was evaluated by comparing the synthesis in denervated (7 d before) IBAT with that of the contralateral innervated side. The insulin signaling and β3 adrenergic receptor (β3-AR) contents, as well as others, were determined by Western blot (Student's t test or analysis of variance; P ≤ 0.05). Total FA synthesis in IBAT was 133% higher in the LPHC group and was reduced 85% and 70% by denervation for the LPHC and control groups, respectively. Glucose uptake was 3.5-fold higher in the IBAT of LPHC rats than in that of the control rats, and the contribution of glucose to the total FA synthesis increased by 12% in control rats compared with 18% in LPHC rats. The LPHC diet increased the G3P generation from glucose by 270% and the insulin receptor content and the p-AKT insulin stimulation in IBAT by 120% and reduced the β3-AR content by 50%. The LPHC diet stimulated glucose uptake, both the total rates and the rates derived from glucose-dependent FA and G3P synthesis, by increasing the insulin sensitivity and the sympathetic flux, despite a reduction in the β3-AR content. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of folic acid-PAMAM conjugates: drug loading efficacy and dendrimer morphology.

PubMed

Chanphai, P; Tajmir-Riahi, H A

2018-05-01

We report the loading efficacy of folic acid (FA) by polyamidoamine (PAMAM-G3 and PAMAM-G4) nanoparticles in aqueous solution at physiological pH. Thermodynamic parameters ΔH = -47.57 (kJ Mol -1 ), ΔS = -122.78 (J Mol -1 , K -1 ) and ΔG = -10.96 (kJ Mol -1 ) showed FA-PAMAM bindings occur via H-bonding and van der Waals contacts. The stability of acid-PAMAM conjugate increased as polymer size increased. The acid loading efficacy was 40 to 50%. TEM images exhibited major polymer morphological changes upon acid encapsulation. PAMAM dendrimers are capable of FA delivery in vitro.

Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

PubMed

Shynlova, Oksana; Chow, Michelle; Lye, Stephen J

2009-10-01

The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions.

Application of Ferulic Acid for Alzheimer’s Disease: Combination of Text Mining and Experimental Validation

PubMed Central

Meng, Guilin; Meng, Xiulin; Ma, Xiaoye; Zhang, Gengping; Hu, Xiaolin; Jin, Aiping; Liu, Xueyuan

2018-01-01

Alzheimer’s disease (AD) is an increasing concern in human health. Despite significant research, highly effective drugs to treat AD are lacking. The present study describes the text mining process to identify drug candidates from a traditional Chinese medicine (TCM) database, along with associated protein target mechanisms. We carried out text mining to identify literatures that referenced both AD and TCM and focused on identifying compounds and protein targets of interest. After targeting one potential TCM candidate, corresponding protein-protein interaction (PPI) networks were assembled in STRING to decipher the most possible mechanism of action. This was followed by validation using Western blot and co-immunoprecipitation in an AD cell model. The text mining strategy using a vast amount of AD-related literature and the TCM database identified curcumin, whose major component was ferulic acid (FA). This was used as a key candidate compound for further study. Using the top calculated interaction score in STRING, BACE1 and MMP2 were implicated in the activity of FA in AD. Exposure of SHSY5Y-APP cells to FA resulted in the decrease in expression levels of BACE-1 and APP, while the expression of MMP-2 and MMP-9 increased in a dose-dependent manner. This suggests that FA induced BACE1 and MMP2 pathways maybe novel potential mechanisms involved in AD. The text mining of literature and TCM database related to AD suggested FA as a promising TCM ingredient for the treatment of AD. Potential mechanisms interconnected and integrated with Aβ aggregation inhibition and extracellular matrix remodeling underlying the activity of FA were identified using in vitro studies. PMID:29896095

Application of Ferulic Acid for Alzheimer's Disease: Combination of Text Mining and Experimental Validation.

PubMed

Meng, Guilin; Meng, Xiulin; Ma, Xiaoye; Zhang, Gengping; Hu, Xiaolin; Jin, Aiping; Zhao, Yanxin; Liu, Xueyuan

2018-01-01

Alzheimer's disease (AD) is an increasing concern in human health. Despite significant research, highly effective drugs to treat AD are lacking. The present study describes the text mining process to identify drug candidates from a traditional Chinese medicine (TCM) database, along with associated protein target mechanisms. We carried out text mining to identify literatures that referenced both AD and TCM and focused on identifying compounds and protein targets of interest. After targeting one potential TCM candidate, corresponding protein-protein interaction (PPI) networks were assembled in STRING to decipher the most possible mechanism of action. This was followed by validation using Western blot and co-immunoprecipitation in an AD cell model. The text mining strategy using a vast amount of AD-related literature and the TCM database identified curcumin, whose major component was ferulic acid (FA). This was used as a key candidate compound for further study. Using the top calculated interaction score in STRING, BACE1 and MMP2 were implicated in the activity of FA in AD. Exposure of SHSY5Y-APP cells to FA resulted in the decrease in expression levels of BACE-1 and APP, while the expression of MMP-2 and MMP-9 increased in a dose-dependent manner. This suggests that FA induced BACE1 and MMP2 pathways maybe novel potential mechanisms involved in AD. The text mining of literature and TCM database related to AD suggested FA as a promising TCM ingredient for the treatment of AD. Potential mechanisms interconnected and integrated with Aβ aggregation inhibition and extracellular matrix remodeling underlying the activity of FA were identified using in vitro studies.

Spectrum of FANCA mutations in Italian Fanconi anemia patients: identification of six novel alleles and phenotypic characterization of the S858R variant.

PubMed

Savino, Maria; Borriello, Adriana; D'Apolito, Maria; Criscuolo, Maria; Del Vecchio, Maria; Bianco, Anna Monica; Di Perna, Michele; Calzone, Rita; Nobili, Bruno; Zatterale, Adriana; Zelante, Leopoldo; Joenje, Hans; Della Ragione, Fulvio; Savoia, Anna

2003-10-01

Fanconi anemia (FA) is an autosomal recessive disorder characterized by genomic instability, bone marrow failure, congenital malformations, and cancer predisposition. FA is a genetically heterogeneous disease with at least seven genes so far identified. The role of FA proteins is unknown although they interact in a common functional pathway. Here, we report six novel FANCA sequence changes and review all the mutations identified in Italy. Except for two missense substitutions, all are expected to cause a premature termination of the FANCA protein at various sites throughout the molecule. The premature terminations are due to nonsense and splice site mutations, as well as small insertions and deletions, and large genomic rearrangements. The expected truncated proteins were not detectable on Western blot analyses. The FANCA-S858R variant is instead expressed at lower level than that seen in normal cell lines and is associated with a non-ubiquinated FANCD2 protein, strongly suggesting that the amino acid substitution is a disease-causing mutation. The spectrum of FA mutations is widely in agreement with the heterogeneous ethnic origin of the Italian population. Copyright 2003 Wiley-Liss, Inc.

Phenotypic and genetic variability of production traits and milk fatty acid contents across days in milk for Walloon Holstein first-parity cows.

PubMed

Bastin, C; Gengler, N; Soyeurt, H

2011-08-01

The objective of this study was to assess the phenotypic and genetic variability of production traits and milk fatty acid (FA) contents throughout lactation. Genetic parameters for milk, fat, and protein yields, fat and protein contents, and 19 groups and individual FA contents in milk were estimated for first-parity Holstein cows in the Walloon Region of Belgium using single-trait, test-day animal models and random regressions. Data included 130,285 records from 26,166 cows in 531 herds. Heritabilities indicated that de novo synthesized FA were under stronger genetic control than FA originating from the diet and from body fat mobilization. Estimates for saturated short- and medium-chain individual FA ranged from 0.35 for C4:0 to 0.44 for C8:0, whereas those for monounsaturated long-chain individual FA were lower (around 0.18). Moreover, de novo synthesized FA were more heritable in mid to late lactation. Approximate daily genetic correlations among traits were calculated as correlations between daily breeding values for days in milk between 5 and 305. Averaged daily genetic correlations between milk yield and FA contents did not vary strongly among FA (around -0.35) but they varied strongly across days in milk, especially in early lactation. Results indicate that cows selected for high milk yield in early lactation would have lower de novo synthesized FA contents in milk but a slightly higher content of C18:1 cis-9, indicating that such cows might mobilize body fat reserves. Genetic correlations among FA emphasized the combination of FA according to their origin: contents in milk of de novo FA were highly correlated with each other (from 0.64 to 0.99). Results also showed that genetic correlations between C18:1 cis-9 and other FA varied strongly during the first 100 d in milk and reinforced the statement that the release of long-chain FA inhibits FA synthesis in the mammary gland while the cow is in negative energy balance. Finally, results showed that the FA profile in milk changed during the lactation phenotypically and genetically, emphasizing the relationship between the physiological status of cow and milk composition. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Hydrogen Sulfide Inhibits Formaldehyde-Induced Endoplasmic Reticulum Stress in PC12 Cells by Upregulation of SIRT-1

PubMed Central

Zhang, Ping; Chen, Li-Xun; Wang, Li; Xie, Ming; Wang, Chun-Yan; Tang, Xiao-Qing

2014-01-01

Background Formaldehyde (FA), a well-known environmental pollutant, has been classified as a neurotoxic molecule. Our recent data demonstrate that hydrogen sulfide (H2S), the third gaseous transmitter, has a protective effect on the neurotoxicity of FA. However, the exact mechanisms underlying this protection remain largely unknown. Endoplasmic reticulum (ER) stress has been implicated in the neurotoxicity of FA. Silent mating type information regulator 2 homolog 1 (SIRT-1), a histone deacetylases, has various biological activities, including the extension of lifespan, the modulation of ER stress, and the neuroprotective action. Objective We hypothesize that the protection of H2S against FA-induced neurotoxicity involves in inhibiting ER stress by upregulation of SIRT-1. The present study attempted to investigate the protective effect of H2S on FA-induced ER stress in PC12 cells and the contribution of SIRT-1 to the protection of H2S against FA-induced injuries, including ER stress, cytotoxicity and apoptosis. Principal Findings We found that exogenous application of sodium hydrosulfide (NaHS; an H2S donor) significantly attenuated FA-induced ER stress responses, including the upregulated levels of glucose-regulated protein 78, C/EBP homologous protein, and cleaved caspase-12 expression. We showed that NaHS upregulates the expression of SIRT-1 in PC12 cells. Moreover, the protective effects of H2S on FA-elicited ER stress, cytotoxicity and apoptosis were reversed by Sirtinol, a specific inhibitor of SIRT-1. Conclusion/Significance These data indicate that H2S exerts its protection against the neurotoxicity of FA through overcoming ER stress via upregulation of SIRT-1. Our findings provide novel insights into the protective mechanisms of H2S against FA-induced neurotoxicity. PMID:24587076

Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Cytoplasm-dislocalized Leukemic Nucleophosmin Protein (NPMc)*

PubMed Central

Du, Wei; Li, Jie; Sipple, Jared; Chen, Jianjun; Pang, Qishen

2010-01-01

Eight of the Fanconi anemia (FA) proteins form a core complex that activates the FA pathway. Some core complex components also form subcomplexes for yet-to-be-elucidated functions. Here, we have analyzed the interaction between a cytoplasmic FA subcomplex and the leukemic nucleophosmin (NPMc). Exogenous NPMc was degraded rapidly in FA acute myeloid leukemia bone marrow cells. Knockdown of FANCA or FANCC in leukemic OCI/AML3 cells induced rapid degradation of endogenous NPMc. NPMc degradation was mediated by the ubiquitin-proteasome pathway involving the IBR-type RING-finger E3 ubiquitin ligase IBRDC2, and genetic correction of FA-A or FA-C lymphoblasts prevented NPMc ubiquitination. Moreover, cytoplasmic FANCA and FANCC formed a cytoplasmic complex and interacted with NPMc. Using a patient-derived FANCC mutant and a nuclearized FANCC, we demonstrated that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability. Finally, depletion of FANCA and FANCC in NPMc-positive leukemic cells significantly increased inflammation and chemoresistance through NF-κB activation. Our findings not only reveal the molecular mechanism involving cytoplasmic retention of NPMc but also suggest cytoplasmic function of FANCA and FANCC in NPMc-related leukemogenesis. PMID:20864535

Implications of obesity for tendon structure, ultrastructure and biochemistry: a study on Zucker rats.

PubMed

Biancalana, Adriano; Velloso, Lício Augusto; Taboga, Sebastião Roberto; Gomes, Laurecir

2012-02-01

The extracellular matrix consists of collagen, proteoglycans and non-collagen proteins. The incidence of obesity and associated diseases is currently increasing in developed countries. Obesity is considered to be a disease of modern times, and genes predisposing to the disease have been identified in humans and animals. The objective of the present study was to compare the morphological and biochemical aspects of the deep digital flexor tendon of lean (Fa/Fa or Fa/fa) and genetically obese (fa/fa) Zucker rats. Ultrastructural analysis showed the presence of lipid droplets in both groups, whereas disorganized collagen fibril bundles were observed in obese animals. Lean animals presented a larger amount of non-collagen proteins and glycosaminoglycans than obese rats. We propose that the overweight and lesser physical activity in obese animals may have provoked the alterations in the composition and organization of extracellular matrix components but a genetic mechanism cannot be excluded. These alterations might be related to organizational and structural modifications in the collagen bundles that influence the mechanical properties of tendons and the progression to a pathological state. Copyright © 2011 Elsevier Ltd. All rights reserved.

High incidence of HPV-associated head and neck cancers in FA deficient mice is associated with E7's induction of DNA damage through its inactivation of pocket proteins.

PubMed

Park, Jung Wook; Shin, Myeong-Kyun; Pitot, Henry C; Lambert, Paul F

2013-01-01

Fanconi anemia (FA) patients are highly susceptible to solid tumors at multiple anatomical sites including head and neck region. A subset of head and neck cancers (HNCs) is associated with 'high-risk' HPVs, particularly HPV16. However, the correlation between HPV oncogenes and cancers in FA patients is still unclear. We previously learned that FA deficiency in mice predisposes HPV16 E7 transgenic mice to HNCs. To address HPV16 E6's oncogenic potential under FA deficiency in HNCs, we utilized HPV16 E6-transgenic mice (K14E6) and HPV16 E6/E7-bi-transgenic mice (K14E6E7) on genetic backgrounds sufficient or deficient for one of the fanc genes, fancD2 and monitored their susceptibility to HNCs. K14E6 mice failed to develop tumor. However, E6 and fancD2-deficiency accelerated E7-driven tumor development in K14E6E7 mice. The increased tumor incidence was more correlated with E7-driven DNA damage than proliferation. We also found that deficiency of pocket proteins, pRb, p107, and p130 that are well-established targets of E7, could recapitulate E7's induction of DNA damage. Our findings support the hypothesis that E7 induces HPV-associated HNCs by promoting DNA damage through the inactivation of pocket proteins, which explains why a deficiency in DNA damage repair would increase susceptibility to E7-driven cancer. Our results further demonstrate the unexpected finding that FA deficiency does not predispose E6 transgenic mice to HNCs, indicating a specificity in the synergy between FA deficiency and HPV oncogenes in causing HNCs.

The fanconi anemia pathway limits human papillomavirus replication.

PubMed

Hoskins, Elizabeth E; Morreale, Richard J; Werner, Stephen P; Higginbotham, Jennifer M; Laimins, Laimonis A; Lambert, Paul F; Brown, Darron R; Gillison, Maura L; Nuovo, Gerard J; Witte, David P; Kim, Mi-Ok; Davies, Stella M; Mehta, Parinda A; Butsch Kovacic, Melinda; Wikenheiser-Brokamp, Kathryn A; Wells, Susanne I

2012-08-01

High-risk human papillomaviruses (HPVs) deregulate epidermal differentiation and cause anogenital and head and neck squamous cell carcinomas (SCCs). The E7 gene is considered the predominant viral oncogene and drives proliferation and genome instability. While the implementation of routine screens has greatly reduced the incidence of cervical cancers which are almost exclusively HPV positive, the proportion of HPV-positive head and neck SCCs is on the rise. High levels of HPV oncogene expression and genome load are linked to disease progression, but genetic risk factors that regulate oncogene abundance and/or genome amplification remain poorly understood. Fanconi anemia (FA) is a genome instability syndrome characterized at least in part by extreme susceptibility to SCCs. FA results from mutations in one of 15 genes in the FA pathway, whose protein products assemble in the nucleus and play important roles in DNA damage repair. We report here that loss of FA pathway components FANCA and FANCD2 stimulates E7 protein accumulation in human keratinocytes and causes increased epithelial proliferation and basal cell layer expansion in the HPV-positive epidermis. Additionally, FANCD2 loss stimulates HPV genome amplification in differentiating cells, demonstrating that the intact FA pathway functions to restrict the HPV life cycle. These findings raise the possibility that FA genes suppress HPV infection and disease and suggest possible mechanism(s) for reported associations of HPV with an FA cohort in Brazil and for allelic variation of FA genes with HPV persistence in the general population.

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5' flap DNA: basis of interstrand cross-link repair by FAN1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gwon, Gwang Hyeon; Kim, Youngran; Liu, Yaqi

2014-10-15

Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5' flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domainmore » playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5' flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.« less

Reversal of multidrug resistance in MCF-7/Adr cells by codelivery of doxorubicin and BCL2 siRNA using a folic acid-conjugated polyethylenimine hydroxypropyl-β-cyclodextrin nanocarrier

PubMed Central

Li, Jin-Ming; Zhang, Wei; Su, Hua; Wang, Yuan-Yuan; Tan, Cai-Ping; Ji, Liang-Nian; Mao, Zong-Wan

2015-01-01

Systemic administration of chemotherapy for cancer often faces drug resistance, limiting its applications in cancer therapy. In this study, we developed a simple multifunctional nanocarrier based on polyethylenimine (PEI) to codeliver doxorubicin (DOX) and BCL2 small interfering RNA (siRNA) for overcoming multidrug resistance (MDR) and enhancing apoptosis in MCF-7/Adr cancer cells by combining chemotherapy and RNA interference (RNAi) therapy. The low-molecular-weight branch PEI was used to conjugate hydroxypropyl-β-cyclodextrin (HP-β-CD) and folic acid (FA), forming the codelivery nanocarrier (FA-HP-β-CD-PEI) to encapsulate DOX with the cavity HP-β-CD and bind siRNA with the positive charge of PEI for tumor-targeting codelivering drugs. The drug-loaded nanocomplexes (FA-HP-β-CD-PEI/DOX/siRNA) showed uniform size distribution, high cellular uptake, and significant gene suppression of BCL2, displaying the potential of overcoming MDR for enhancing the effect of anticancer drugs. Furthermore, the nanocomplexes achieved significant cell apoptosis through a mechanism of downregulating the antiapoptotic protein BCL2, resulted in improving therapeutic efficacy of the coadministered DOX by tumor targeting and RNA interference. Our study indicated that combined RNAi therapy and chemotherapy using our functional codelivery nanocarrier could overcome MDR and enhance apoptosis in MDR cancer cells for a potential application in treating MDR cancers. PMID:25960653

Management, nutrition, and lactation performance are related to bulk tank milk de novo fatty acid concentration on northeastern US dairy farms.

PubMed

Woolpert, M E; Dann, H M; Cotanch, K W; Melilli, C; Chase, L E; Grant, R J; Barbano, D M

2016-10-01

This study investigated the relationship of management practices, dietary characteristics, milk composition, and lactation performance with de novo fatty acid (FA) concentration in bulk tank milk from commercial dairy farms with Holstein, Jersey, and mixed-breed cows. It was hypothesized that farms with higher de novo milk FA concentrations would more commonly use management and nutrition practices known to optimize ruminal conditions that enhance de novo synthesis of milk FA. Farms (n=44) located in Vermont and northeastern New York were selected based on a history of high de novo (HDN; 26.18±0.94g/100g of FA; mean ± standard deviation) or low de novo (LDN; 24.19±1.22g/100g of FA) FA in bulk tank milk. Management practices were assessed during one visit to each farm in March or April, 2014. Total mixed ration samples were collected and analyzed for chemical composition using near infrared spectroscopy. We found no differences in days in milk at the farm level. Yield of milk fat, true protein, and de novo FA per cow per day were higher for HDN versus LDN farms. The HDN farms had lower freestall stocking density (cows/stall) than LDN farms. Additionally, tiestall feeding frequency was higher for HDN than LDN farms. No differences between HDN and LDN farms were detected for dietary dry matter, crude protein, neutral detergent fiber, starch, or percentage of forage in the diet. However, dietary ether extract was lower for HDN than LDN farms. This research indicates that overcrowded freestalls, reduced feeding frequency, and greater dietary ether extract content are associated with lower de novo FA synthesis and reduced milk fat and true protein yields on commercial dairy farms. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Defective homing is associated with altered Cdc42 activity in cells from patients with Fanconi anemia group A

PubMed Central

Zhang, Xiaoling; Shang, Xun; Guo, Fukun; Murphy, Kim; Kirby, Michelle; Kelly, Patrick; Reeves, Lilith; Smith, Franklin O.; Williams, David A.

2008-01-01

Previous studies showed that Fanconi anemia (FA) murine stem cells have defective reconstitution after bone marrow (BM) transplantation. The mechanism underlying this defect is not known. Here, we report defective homing of FA patient BM progenitors transplanted into mouse models. Using cells from patients carrying mutations in FA complementation group A (FA-A), we show that when transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) recipient mice, FA-A BM cells exhibited impaired homing activity. FA-A cells also showed defects in both cell-cell and cell-matrix adhesion. Complementation of FA-A deficiency by reexpression of FANCA readily restored adhesion of FA-A cells. A significant decrease in the activity of the Rho GTPase Cdc42 was found associated with these defective functions in patient-derived cells, and expression of a constitutively active Cdc42 mutant was able to rescue the adhesion defect of FA-A cells. These results provide the first evidence that FA proteins influence human BM progenitor homing and adhesion via the small GTPase Cdc42-regulated signaling pathway. PMID:18565850

Dietary fish protein hydrolysates containing bioactive motifs affect serum and adipose tissue fatty acid compositions, serum lipids, postprandial glucose regulation and growth in obese Zucker fa/fa rats.

PubMed

Drotningsvik, Aslaug; Mjøs, Svein A; Pampanin, Daniela M; Slizyte, Rasa; Carvajal, Ana; Remman, Tore; Høgøy, Ingmar; Gudbrandsen, Oddrun A

2016-10-01

The world's fisheries and aquaculture industries produce vast amounts of protein-containing by-products that can be enzymatically hydrolysed to smaller peptides and possibly be used as additives to functional foods and nutraceuticals targeted for patients with obesity-related metabolic disorders. To investigate the effects of fish protein hydrolysates on markers of metabolic disorders, obese Zucker fa/fa rats consumed diets with 75 % of protein from casein/whey (CAS) and 25 % from herring (HER) or salmon (SAL) protein hydrolysate from rest raw material, or 100 % protein from CAS for 4 weeks. The fatty acid compositions were similar in the experimental diets, and none of them contained any long-chain n-3 PUFA. Ratios of lysine:arginine and methionine:glycine were lower in HER and SAL diets when compared with CAS, and taurine was detected only in fish protein hydrolysate diets. Motifs with reported hypocholesterolemic or antidiabetic activities were identified in both fish protein hydrolysates. Rats fed HER diet had lower serum HDL-cholesterol and LDL-cholesterol, and higher serum TAG, MUFA and n-3:n-6 PUFA ratio compared with CAS-fed rats. SAL rats gained more weight and had better postprandial glucose regulation compared with CAS rats. Serum lipids and fatty acids were only marginally affected by SAL, but adipose tissue contained less total SFA and more total n-3 PUFA when compared with CAS. To conclude, diets containing hydrolysed rest raw material from herring or salmon proteins may affect growth, lipid metabolism, postprandial glucose regulation and fatty acid composition in serum and adipose tissue in obese Zucker rats.

Fusaric Acid Induces DNA Damage and Post-Translational Modifications of p53 in Human Hepatocellular Carcinoma (HepG2 ) Cells.

PubMed

Ghazi, Terisha; Nagiah, Savania; Tiloke, Charlette; Sheik Abdul, Naeem; Chuturgoon, Anil A

2017-11-01

Fusaric acid (FA), a common fungal contaminant of maize, is known to mediate toxicity in plants and animals; however, its mechanism of action is unclear. p53 is a tumor suppressor protein that is activated in response to cellular stress. The function of p53 is regulated by post-translational modifications-ubiquitination, phosphorylation, and acetylation. This study investigated a possible mechanism of FA induced toxicity in the human hepatocellular carcinoma (HepG 2 ) cell line. The effect of FA on DNA integrity and post-translational modifications of p53 were investigated. Methods included: (a) culture and treatment of HepG 2 cells with FA (IC 50 : 580.32 μM, 24 h); (b) comet assay (DNA damage); (c) Western blots (protein expression of p53, MDM2, p-Ser-15-p53, a-K382-p53, a-CBP (K1535)/p300 (K1499), HDAC1 and p-Ser-47-Sirt1); and (d) Hoechst 33342 assay (apoptosis analysis). FA caused DNA damage in HepG 2 cells relative to the control (P < 0.0001). FA decreased the protein expression of p53 (0.24-fold, P = 0.0004) and increased the expression of p-Ser-15-p53 (12.74-fold, P = 0.0126) and a-K382-p53 (2.24-fold, P = 0.0096). This occurred despite the significant decrease in the histone acetyltransferase, a-CBP (K1535)/p300 (K1499) (0.42-fold, P = 0.0023) and increase in the histone deacetylase, p-Ser-47-Sirt1 (1.22-fold, P = 0.0020). The expression of MDM2, a negative regulator of p53, was elevated in the FA treatment compared to the control (1.83-fold, P < 0.0001). FA also inhibited cell proliferation and induced apoptosis in HepG 2 cells as evidenced by the Hoechst assay. Together, these results indicate that FA is genotoxic and post-translationally modified p53 leading to HepG 2 cell death. J. Cell. Biochem. 118: 3866-3874, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ni, Qian; Department of Pediatrics, Lanzhou University Second Hospital, Lanzhou; Shao, Yuan

Highlights: • Acute exposure to high altitude (HA) increased hepatic fatty acid (FA) β-oxidation. • Acute exposure of rats to HA increased hepatic FA synthesis. • PPARα and AMPK can regulate the FA metabolism. • FA may be a key energy fuel and a compensation for CHO during acute exposure to HA. • The acute changes of FA metabolism may be a mechanism of acclimatization. - Abstract: High altitude (HA) affects energy metabolism. The impact of acute and chronic HA acclimatization on the major metabolic pathways is still controversial. In this study, we aimed to unveil the impact of HAmore » on the key enzymes involved in the fatty acid (FA) metabolism in liver. Rats were exposed to an altitude of 4300 m for 30 days and the expressions of two key proteins involved in FA β-oxidation (carnitine palmitoyl transferase I, CPT-I; and peroxisome proliferator-activated receptor alpha, PPARα), two proteins involved in FA synthesis (acetyl CoA carboxylase-1, ACC-1; and AMP-activated protein kinase, AMPK), as well as the total ketone body in the liver and the plasma FFAs were examined. Rats without HA exposure were used as controls. We observed that the acute exposure of rats to HA (3 days) led to a significant increase in the expressions of CPT-I and PPARα and in the total hepatic ketone body. Longer exposure (15 days) caused a marked decrease in the expression of CPT-I and PPARα. By 30 days after HA exposure, the expression levels of CPT-I and PPARα returned to the control level. The hepatic ACC-1 level showed a significant increase in rats exposed to HA for 1 and 3 days. In contrast, the hepatic level of AMPK showed a significant reduction throughout the experimental period. Plasma FFA concentrations did not show any significant changes following HA exposure. Thus, increased hepatic FA oxidation and synthesis in the early phase of HA exposure may be among the important mechanisms for the rats to respond to the hypoxic stress in order to acclimatize themselves to the stressful environments.« less

Fetal asphyctic preconditioning modulates the acute cytokine response thereby protecting against perinatal asphyxia in neonatal rats.

PubMed

Vlassaks, Evi; Strackx, Eveline; Vles, Johan Sh; Nikiforou, Maria; Martinez-Martinez, Pilar; Kramer, Boris W; Gavilanes, Antonio Wd

2013-01-26

Perinatal asphyxia (PA) is a major cause of brain damage and neurodevelopmental impairment in infants. Recent investigations have shown that experimental sublethal fetal asphyxia (FA preconditioning) protects against a subsequent more severe asphyctic insult at birth. The molecular mechanisms of this protection have, however, not been elucidated. Evidence implicates that inflammatory cytokines play a protective role in the induction of ischemic tolerance in the adult brain. Accordingly, we hypothesize that FA preconditioning leads to changes in the fetal cytokine response, thereby protecting the newborn against a subsequent asphyctic insult. In rats, FA preconditioning was induced at embryonic day 17 by clamping the uterine vasculature for 30 min. At term birth, global PA was induced by placing the uterine horns, containing the pups, in a saline bath for 19 min. We assessed, at different time points after FA and PA, mRNA and protein expression of several cytokines and related receptor mRNA levels in total hemispheres of fetal and neonatal brains. Additionally, we measured pSTAT3/STAT3 levels to investigate cellular responses to these cytokines. Prenatally, FA induced acute downregulation in IL-1β, TNF-α and IL-10 mRNA levels. At 96 h post FA, IL-6 mRNA and IL-10 protein expression were increased in FA brains compared with controls. Two hours after birth, all proinflammatory cytokines and pSTAT3/STAT3 levels decreased in pups that experienced FA and/or PA. Interestingly, IL-10 and IL-6 mRNA levels increased after PA. When pups were FA preconditioned, however, IL-10 and IL-6 mRNA levels were comparable to those in controls. FA leads to prenatal changes in the neuroinflammatory response. This modulation of the cytokine response probably results in the protective inflammatory phenotype seen when combining FA and PA and may have significant implications for preventing post-asphyctic perinatal encephalopathy.

Fetal asphyctic preconditioning modulates the acute cytokine response thereby protecting against perinatal asphyxia in neonatal rats

PubMed Central

2013-01-01

Background Perinatal asphyxia (PA) is a major cause of brain damage and neurodevelopmental impairment in infants. Recent investigations have shown that experimental sublethal fetal asphyxia (FA preconditioning) protects against a subsequent more severe asphyctic insult at birth. The molecular mechanisms of this protection have, however, not been elucidated. Evidence implicates that inflammatory cytokines play a protective role in the induction of ischemic tolerance in the adult brain. Accordingly, we hypothesize that FA preconditioning leads to changes in the fetal cytokine response, thereby protecting the newborn against a subsequent asphyctic insult. Methods In rats, FA preconditioning was induced at embryonic day 17 by clamping the uterine vasculature for 30 min. At term birth, global PA was induced by placing the uterine horns, containing the pups, in a saline bath for 19 min. We assessed, at different time points after FA and PA, mRNA and protein expression of several cytokines and related receptor mRNA levels in total hemispheres of fetal and neonatal brains. Additionally, we measured pSTAT3/STAT3 levels to investigate cellular responses to these cytokines. Results Prenatally, FA induced acute downregulation in IL-1β, TNF-α and IL-10 mRNA levels. At 96 h post FA, IL-6 mRNA and IL-10 protein expression were increased in FA brains compared with controls. Two hours after birth, all proinflammatory cytokines and pSTAT3/STAT3 levels decreased in pups that experienced FA and/or PA. Interestingly, IL-10 and IL-6 mRNA levels increased after PA. When pups were FA preconditioned, however, IL-10 and IL-6 mRNA levels were comparable to those in controls. Conclusions FA leads to prenatal changes in the neuroinflammatory response. This modulation of the cytokine response probably results in the protective inflammatory phenotype seen when combining FA and PA and may have significant implications for preventing post-asphyctic perinatal encephalopathy. PMID:23351591

Fatty acid modulated human serum albumin binding of the β-carboline alkaloids norharmane and harmane.

PubMed

Domonkos, Celesztina; Fitos, Ilona; Visy, Júlia; Zsila, Ferenc

2013-12-02

Harmane and norharmane are representative members of the large group of natural β-carboline alkaloids featured with diverse pharmacological activities. In blood, these agents are transported by human serum albumin (HSA) which has a profound impact on the pharmacokinetic and pharmacodynamic properties of many therapeutic drugs and xenobiotics. By combination of various spectroscopic methods, the present contribution is aimed to elucidate how nonesterified fatty acids (FAs), the primary endogenous ligands of HSA, affect the binding properties of harmane and norharmane. Analysis of induced circular dichroism (CD) and fluorescence spectroscopic data indicates the inclusion of the neutral form of both molecules into the binding pocket of subdomain IIIA, which hosts two FA binding sites, too. The induced CD and UV absorption spectra of harmane and norharmane exhibit peculiar changes upon addition of FAs, suggesting the formation of ternary complexes in which the lipid ligands significantly alter the binding mode of the alkaloids via cooperative allosteric mechanism. To our knowledge, it is the first instance of the demonstration of drug-FA cobinding at site IIIA. In line with these results, molecular docking calculations showed two distinct binding positions of norharmane within subdomain IIIA. The profound increase in the affinity constants of β-carbolines estimated in the presence of FAs predicts that the unbound, pharmacologically active serum fraction of these compounds strongly depends on the actual lipid binding profile of HSA.

Towards a Molecular Understanding of the Fanconi Anemia Core Complex

PubMed Central

Hodson, Charlotte; Walden, Helen

2012-01-01

Fanconi Anemia (FA) is a genetic disorder characterized by the inability of patient cells to repair DNA damage caused by interstrand crosslinking agents. There are currently 14 verified FA genes, where mutation of any single gene prevents repair of DNA interstrand crosslinks (ICLs). The accumulation of ICL damage results in genome instability and patients having a high predisposition to cancers. The key event of the FA pathway is dependent on an eight-protein core complex (CC), required for the monoubiquitination of each member of the FANCD2-FANCI complex. Interestingly, the majority of patient mutations reside in the CC. The molecular mechanisms underlying the requirement for such a large complex to carry out a monoubiquitination event remain a mystery. This paper documents the extensive efforts of researchers so far to understand the molecular roles of the CC proteins with regard to its main function in the FA pathway, the monoubiquitination of FANCD2 and FANCI. PMID:22675617

Damaged mitochondria in Fanconi anemia - an isolated event or a general phenomenon?

PubMed

Pagano, Giovanni; Shyamsunder, Pavithra; Verma, Rama S; Lyakhovich, Alex

2014-01-01

Fanconi anemia (FA) is known as an inherited bone marrow failure syndrome associated with cancer predisposition and susceptibility to a number of DNA damaging stimuli, along with a number of clinical features such as upper limb malformations, increased diabetes incidence and typical anomalies in skin pigmentation. The proteins encoded by FA-defective genes (FANC proteins) display well-established roles in DNA damage and repair pathways. Moreover, some independent studies have revealed that mitochondrial dysfunction (MDF) is also involved in FA phenotype. Unconfined to FA, we have shown that other syndromes featuring DNA damage and repair (such as ataxia-telangiectasia, AT, and Werner syndrome, WS) display MDF-related phenotypes, along with oxidative stress (OS) that, altogether, may play major roles in these diseases. Experimental and clinical studies are warranted in the prospect of future therapies to be focused on compounds scavenging reactive oxygen species (ROS) as well as protecting mitochondrial functions.

Omega-3 fatty acid deficiency disrupts endocytosis, neuritogenesis, and mitochondrial protein pathways in the mouse hippocampus

PubMed Central

English, Jane A.; Harauma, Akiko; Föcking, Melanie; Wynne, Kieran; Scaife, Caitriona; Cagney, Gerard; Moriguchi, Toru; Cotter, David R.

2013-01-01

Omega-3 fatty acid (n-3 FA) deficiency is an environmental risk factor for schizophrenia, yet characterization of the consequences of deficiency at the protein level in the brain is limited. We aimed to identify the protein pathways disrupted as a consequence of chronic n-3 deficiency in the hippocampus of mice. Fatty acid analysis of the hippocampus following chronic dietary deficiency revealed a 3-fold decrease (p < 0.001) in n-3 FA levels. Label free LC-MS/MS analysis identified and profiled 1008 proteins, of which 114 were observed to be differentially expressed between n-3 deficient and control groups (n = 8 per group). The cellular processes that were most implicated were neuritogenesis, endocytosis, and exocytosis, while specific protein pathways that were most significantly dysregulated were mitochondrial dysfunction and clathrin mediated endocytosis (CME). In order to characterize whether these processes and pathways are ones influenced by antipsychotic medication, we used LC-MS/MS to test the differential expression of these 114 proteins in the hippocampus of mice chronically treated with the antipsychotic agent haloperidol. We observed 23 of the 114 proteins to be differentially expressed, 17 of which were altered in the opposite direction to that observed following n-3 deficiency. Overall, our findings point to disturbed synaptic function, neuritogenesis, and mitochondrial function as a consequence of dietary deficiency in n-3 FA. This study greatly aids our understanding of the molecular mechanism by which n-3 deficiency impairs normal brain function, and provides clues as to how n-3 FA exert their therapeutic effect in early psychosis. PMID:24194745

Digestion of fatty acids in ruminants: a meta-analysis of flows and variation factors. 1. Total fatty acids.

PubMed

Schmidely, P; Glasser, F; Doreau, M; Sauvant, D

2008-05-01

A database built from 95 experiments with 303 treatments was used to quantify the ruminal biohydrogenation (BH) of fatty acids (FA), efficiency of microbial protein synthesis (EMPS), duodenal flow and intestinal absorption of total FA and of FA with 12 to 18 C units, in response to variations in dietary FA content, source or technological treatment of fat supplement. Flows of FA were expressed relative to dry matter intake (DMI) to compile data from bovine and ovine species. BH tended to increase curvilinearly with FA intake, whereas dietary FA did not affect EMPS. A linear relationship between FA intake and duodenal flow of total FA was obtained, with a coefficient of 0.75 ± 0.06 g duodenal FA/kg DMI for each g FA intake/kg DMI. Between experiments, positive balances of total FA (intake - duodenum) were related to low EMPS. Relationships between duodenal flows of FA with 12 to 18 C units and their respective intakes were linear, with a coefficient that increased with the number of C units. Duodenal flow of bacterial FA was linearly related to FA intake (coefficient 0.33 ± 0.13), whereas contribution of bacterial lipid to duodenal flow decreased as FA intake increased. For each FA with 12 to 16 C units, prediction of FA absorption from its respective duodenal flow was linear. For total FA and FA with 18 C units, apparent absorption levelled off at high duodenal flows. All these relationships were discussed according to current knowledge on microbial metabolism in the rumen and on the intestinal digestibility of FA in the intestine.

Towards a deeper understanding of fatty acid bioaccessibility and its dependence on culinary treatment and lipid class: a case study of gilthead seabream (Sparus aurata).

PubMed

Costa, Sara; Afonso, Cláudia; Cardoso, Carlos; Oliveira, Rui; Alves, Francisca; Nunes, Maria L; Bandarra, Narcisa M

2016-11-08

The bioaccessibility of total lipids and fatty acids (FA) in raw and grilled gilthead seabream (Sparus aurata) was determined using an in vitro digestion model. The particular impact of grilling on the FA profile of seabream was also studied. In addition, the influence of lipid class on the bioaccessibility of each FA was analysed. Grilling did not change the relative FA profile, and only the absolute values were altered. However, the relative FA profile varied across lipid classes, being more dissimilar between TAG and phospholipids. Long-chain SFA and PUFA seemed to be less bioaccessible. Moreover, grilling reduced bioaccessibility of protein, fat and many FA, with the highest reductions found in PUFA such as the DHA. Strong evidence supporting a predominantly regioselective action of lipase during in vitro digestion was found, and the impact of this phenomenon on FA bioaccessibility was assessed.

Capacity of humic substances to complex with iron at different salinities in the Yangtze River estuary and East China Sea.

PubMed

Yang, Rujun; Su, Han; Qu, Shenglu; Wang, Xuchen

2017-05-03

The iron binding capacities (IBC) of fulvic acid (FA) and humic acid (HA) were determined in the salinity range from 5 to 40. The results indicated that IBC decreased while salinity increased. In addition, dissolved iron (dFe), FA and HA were also determined along the Yangtze River estuary's increasing salinity gradient from 0.14 to 33. The loss rates of dFe, FA and HA in the Yangtze River estuary were up to 96%, 74%, and 67%, respectively. The decreases in dFe, FA and HA, as well as the change in IBC of humic substances (HS) along the salinity gradient in the Yangtze River estuary were all well described by a first-order exponential attenuation model: y(dFe/FA/HA, S) = a 0 × exp(kS) + y 0 . These results indicate that flocculation of FA and HA along the salinity gradient resulted in removal of dFe. Furthermore, the exponential attenuation model described in this paper can be applied in the major estuaries of the world where most of the removal of dFe and HS occurs where freshwater and seawater mix.

Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage.

PubMed

Fu, Dechen; Dudimah, Fred Duafalia; Zhang, Jun; Pickering, Anna; Paneerselvam, Jayabal; Palrasu, Manikandan; Wang, Hong; Fei, Peiwen

2013-03-01

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair.

Recruitment of DNA polymerase eta by FANCD2 in the early response to DNA damage

PubMed Central

Fu, Dechen; Dudimah, Fred Duafalia; Zhang, Jun; Pickering, Anna; Paneerselvam, Jayabal; Palrasu, Manikandan; Wang, Hong; Fei, Peiwen

2013-01-01

How Fanconi anemia (FA) protein D2 (FANCD2) performs DNA damage repair remains largely elusive. We report here that translesion synthesis DNA polymerase (pol) eta is a novel mediator of FANCD2 function. We found that wild type (wt) FANCD2, not K561R (mt) FANCD2, can interact with pol eta. Upon DNA damage, the interaction of pol eta with FANCD2 occurs earlier than that with PCNA, which is in concert with our finding that FANCD2 monoubiquitination peaks at an earlier time point than that of PCNA monoubiquitination. FANCD2-null FA patient cells (PD20) carrying histone H2B-fused pol eta and wtFANCD2, respectively, show a similar tendency of low Mitomycin C (MMC) sensitivity, while cells transfected with empty vector control or pol eta alone demonstrate a similar high level of MMC sensitivity. It therefore appears that FANCD2 monoubiquitination plays a similar anchor role as histone to bind DNA in regulating pol eta. Collectively, our study indicates that, in the early phase of DNA damage response, FANCD2 plays crucial roles in recruiting pol eta to the sites of DNA damage for repair. PMID:23388460

Hepatic ATGL mediates PPAR-α signaling and fatty acid channeling through an L-FABP independent mechanism

PubMed Central

Ong, Kuok Teong; Mashek, Mara T.; Davidson, Nicholas O.; Mashek, Douglas G.

2014-01-01

Adipose TG lipase (ATGL) catalyzes the rate-limiting step in TG hydrolysis in most tissues. We have shown that hepatic ATGL preferentially channels hydrolyzed FAs to β-oxidation and induces PPAR-α signaling. Previous studies have suggested that liver FA binding protein (L-FABP) transports FAs from lipid droplets to the nucleus for ligand delivery and to the mitochondria for β-oxidation. To determine if L-FABP is involved in ATGL-mediated FA channeling, we used adenovirus-mediated suppression or overexpression of hepatic ATGL in either WT or L-FABP KO mice. Hepatic ATGL knockdown increased liver weight and TG content of overnight fasted mice regardless of genotype. L-FABP deletion did not impair the effects of ATGL overexpression on the oxidation of hydrolyzed FAs in primary hepatocyte cultures or on serum β-hydroxybutyrate concentrations in vivo. Moreover, L-FABP deletion did not influence the effects of ATGL knockdown or overexpression on PPAR-α target gene expression. Taken together, we conclude that L-FABP is not required to channel ATGL-hydrolyzed FAs to mitochondria for β-oxidation or the nucleus for PPAR-α regulation. PMID:24610891

An investigation about the structures, thermodynamics and kinetics of the formic acid involved molecular clusters

NASA Astrophysics Data System (ADS)

Zhang, Rui; Jiang, Shuai; Liu, Yi-Rong; Wen, Hui; Feng, Ya-Juan; Huang, Teng; Huang, Wei

2018-05-01

Despite the very important role of atmospheric aerosol nucleation in climate change and air quality, the detailed aerosol nucleation mechanism is still unclear. Here we investigated the formic acid (FA) involved multicomponent nucleation molecular clusters including sulfuric acid (SA), dimethylamine (DMA) and water (W) through a quantum chemical method. The thermodynamics and kinetics analysis was based on the global minima given by Basin-Hopping (BH) algorithm coupled with Density Functional Theory (DFT) and subsequent benchmarked calculations. Then the interaction analysis based on ElectroStatic Potential (ESP), Topological and Atomic Charges analysis was made to characterize the binding features of the clusters. The results show that FA binds weakly with the other molecules in the cluster while W binds more weakly. Further kinetic analysis about the time evolution of the clusters show that even though the formic acid's weak interaction with other nucleation precursors, its effect on sulfuric acid dimer steady state concentration cannot be neglected due to its high concentration in the atmosphere.

Perilipin 5 Regulates Islet Lipid Metabolism and Insulin Secretion in a cAMP-Dependent Manner: Implication of Its Role in the Postprandial Insulin Secretion

PubMed Central

Trevino, Michelle B.; Machida, Yui; Hallinger, Daniel R.; Garcia, Eden; Christensen, Aaron; Dutta, Sucharita; Peake, David A.; Ikeda, Yasuhiro

2015-01-01

Elevation of circulating fatty acids (FA) during fasting supports postprandial (PP) insulin secretion that is critical for glucose homeostasis and is impaired in diabetes. We tested our hypothesis that lipid droplet (LD) protein perilipin 5 (PLIN5) in β-cells aids PP insulin secretion by regulating intracellular lipid metabolism. We demonstrated that PLIN5 serves as an LD protein in human islets. In vivo, Plin5 and triglycerides were increased by fasting in mouse islets. MIN6 cells expressing PLIN5 (adenovirus [Ad]-PLIN5) and those expressing perilipin 2 (PLIN2) (Ad-PLIN2) had higher [3H]FA incorporation into triglycerides than Ad-GFP control, which support their roles as LD proteins. However, Ad-PLIN5 cells had higher lipolysis than Ad-PLIN2 cells, which increased further by 8-Br-cAMP, indicating that PLIN5 facilitates FA mobilization upon cAMP stimulation as seen postprandially. Ad-PLIN5 in islets enhanced the augmentation of glucose-stimulated insulin secretion by FA and 8-Br-cAMP in G-protein–coupled receptor 40 (GPR40)- and cAMP-activated protein kinase–dependent manners, respectively. When PLIN5 was increased in mouse β-cells in vivo, glucose tolerance after an acute exenatide challenge was improved. Therefore, the elevation of islet PLIN5 during fasting allows partitioning of FA into LD that is released upon refeeding to support PP insulin secretion in cAMP- and GPR40-dependent manners. PMID:25392244

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

PubMed Central

Johnson, Darryl; Boyes, Barry; Orlando, Ron

2013-01-01

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage. PMID:24294112

The use of ammonium formate as a mobile-phase modifier for LC-MS/MS analysis of tryptic digests.

PubMed

Johnson, Darryl; Boyes, Barry; Orlando, Ron

2013-12-01

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.

Mutational disruption of the ABCC2 gene in fall armyworm, Spodoptera frugiperda, confers resistance to the Cry1Fa and Cry1A.105 insecticidal proteins.

PubMed

Flagel, Lex; Lee, Young Wha; Wanjugi, Humphrey; Swarup, Shilpa; Brown, Alana; Wang, Jinling; Kraft, Edward; Greenplate, John; Simmons, Jeni; Adams, Nancy; Wang, Yanfei; Martinelli, Samuel; Haas, Jeffrey A; Gowda, Anilkumar; Head, Graham

2018-05-08

The use of Bt proteins in crops has revolutionized insect pest management by offering effective season-long control. However, field-evolved resistance to Bt proteins threatens their utility and durability. A recent example is field-evolved resistance to Cry1Fa and Cry1A.105 in fall armyworm (Spodoptera frugiperda). This resistance has been detected in Puerto Rico, mainland USA, and Brazil. A S. frugiperda population with suspected resistance to Cry1Fa was sampled from a maize field in Puerto Rico and used to develop a resistant lab colony. The colony demonstrated resistance to Cry1Fa and partial cross-resistance to Cry1A.105 in diet bioassays. Using genetic crosses and proteomics, we show that this resistance is due to loss-of-function mutations in the ABCC2 gene. We characterize two novel mutant alleles from Puerto Rico. We also find that these alleles are absent in a broad screen of partially resistant Brazilian populations. These findings confirm that ABCC2 is a receptor for Cry1Fa and Cry1A.105 in S. frugiperda, and lay the groundwork for genetically enabled resistance management in this species, with the caution that there may be several distinct ABCC2 resistances alleles in nature.

Fatty Acid-Mediated Inhibition of Metal Binding to the Multi-Metal Site on Serum Albumin: Implications for Cardiovascular Disease.

PubMed

Blindauer, Claudia A; Khazaipoul, Siavash; Yu, Ruitao; Stewart, Alan J

2016-01-01

Human serum albumin (HSA) is the major protein in blood plasma and is responsible for circulatory transport of a range of small molecules including fatty acids, metal ions and drugs. We previously identified the major plasma Zn2+ transport site on HSA and revealed that fatty-acid binding (at a distinct site called the FA2 site) and Zn2+ binding are interdependent via an allosteric mechanism. Since binding affinities of long-chain fatty acids exceed those of plasma Zn2+, this means that under certain circumstances the binding of fatty acid molecules to HSA is likely to diminish HSA Zn2+-binding, and hence affects the control of circulatory and cellular Zn2+ dynamics. This relationship between circulatory fatty acid and Zn2+ dynamics is likely to have important physiological and pathological implications, especially since it has been recognised that Zn2+ acts as a signalling agent in many cell types. Fatty acid levels in the blood are dynamic, but most importantly, chronic elevation of plasma fatty acid levels is associated with some metabolic disorders and disease states - including myocardial infarction and other cardiovascular diseases. In this article, we briefly review the metal-binding properties of albumin and highlight the importance of their interplay with fatty acid binding. We also consider the impact of this dynamic link upon levels and speciation of plasma Zn2+, its effect upon cellular Zn2+ homeostasis and its relevance to cardiovascular and circulatory processes in health and disease.

Interplay between ChREBP and SREBP-1c coordinates postprandial glycolysis and lipogenesis in livers of mice[S

PubMed Central

Li, Shili; Choi, Hwa Y.; Fang, Fei; Fukasawa, Masashi; Uyeda, Kosaku; Hammer, Robert E.; Horton, Jay D.; Engelking, Luke J.; Liang, Guosheng

2018-01-01

Lipogenesis in liver is highest in the postprandial state; insulin activates SREBP-1c, which transcriptionally activates genes involved in FA synthesis, whereas glucose activates carbohydrate-responsive element-binding protein (ChREBP), which activates both glycolysis and FA synthesis. Whether SREBP-1c and ChREBP act independently of one another is unknown. Here, we characterized mice with liver-specific deletion of ChREBP (L-Chrebp−/− mice). Hepatic ChREBP deficiency resulted in reduced mRNA levels of glycolytic and lipogenic enzymes, particularly in response to sucrose refeeding following fasting, a dietary regimen that elicits maximal lipogenesis. mRNA and protein levels of SREBP-1c, a master transcriptional regulator of lipogenesis, were also reduced in L-Chrebp−/− livers. Adeno-associated virus-mediated restoration of nuclear SREBP-1c in L-Chrebp−/− mice normalized expression of a subset of lipogenic genes, while not affecting glycolytic genes. Conversely, ChREBP overexpression alone failed to support expression of lipogenic genes in the livers of mice lacking active SREBPs as a result of Scap deficiency. Together, these data show that SREBP-1c and ChREBP are both required for coordinated induction of glycolytic and lipogenic mRNAs. Whereas SREBP-1c mediates insulin’s induction of lipogenic genes, ChREBP mediates glucose’s induction of both glycolytic and lipogenic genes. These overlapping, but distinct, actions ensure that the liver synthesizes FAs only when insulin and carbohydrates are both present. PMID:29335275

Glial activation colocalizes with structural abnormalities in amyotrophic lateral sclerosis.

PubMed

Alshikho, Mohamad J; Zürcher, Nicole R; Loggia, Marco L; Cernasov, Paul; Chonde, Daniel B; Izquierdo Garcia, David; Yasek, Julia E; Akeju, Oluwaseun; Catana, Ciprian; Rosen, Bruce R; Cudkowicz, Merit E; Hooker, Jacob M; Atassi, Nazem

2016-12-13

In this cross-sectional study, we aimed to evaluate brain structural abnormalities in relation to glial activation in the same cohort of participants. Ten individuals with amyotrophic lateral sclerosis (ALS) and 10 matched healthy controls underwent brain imaging using integrated MR/PET and the radioligand [ 11 C]-PBR28. Diagnosis history and clinical assessments including Upper Motor Neuron Burden Scale (UMNB) were obtained from patients with ALS. Diffusion tensor imaging (DTI) analyses including tract-based spatial statistics and tractography were applied. DTI metrics including fractional anisotropy (FA) and diffusivities (mean, axial, and radial) were measured in regions of interest. Cortical thickness was assessed using surface-based analysis. The locations of structural changes, measured by DTI and the areas of cortical thinning, were compared to regional glial activation measured by relative [ 11 C]-PBR28 uptake. In this cohort of individuals with ALS, reduced FA and cortical thinning colocalized with regions demonstrating higher radioligand binding. [ 11 C]-PBR28 binding in the left motor cortex was correlated with FA (r = -0.68, p < 0.05) and cortical thickness (r = -0.75, p < 0.05). UMNB was correlated with glial activation (r = +0.75, p < 0.05), FA (r = -0.77, p < 0.05), and cortical thickness (r = -0.75, p < 0.05) in the motor cortex. Increased uptake of the glial marker [ 11 C]-PBR28 colocalizes with changes in FA and cortical thinning. This suggests a link between disease mechanisms (gliosis and inflammation) and structural changes (cortical thinning and white and gray matter changes). In this multimodal neuroimaging work, we provide an in vivo model to investigate the pathogenesis of ALS. © 2016 American Academy of Neurology.

Glial activation colocalizes with structural abnormalities in amyotrophic lateral sclerosis

PubMed Central

Alshikho, Mohamad J.; Zürcher, Nicole R.; Loggia, Marco L.; Cernasov, Paul; Chonde, Daniel B.; Izquierdo Garcia, David; Yasek, Julia E.; Akeju, Oluwaseun; Catana, Ciprian; Rosen, Bruce R.; Cudkowicz, Merit E.

2016-01-01

Objective: In this cross-sectional study, we aimed to evaluate brain structural abnormalities in relation to glial activation in the same cohort of participants. Methods: Ten individuals with amyotrophic lateral sclerosis (ALS) and 10 matched healthy controls underwent brain imaging using integrated MR/PET and the radioligand [11C]-PBR28. Diagnosis history and clinical assessments including Upper Motor Neuron Burden Scale (UMNB) were obtained from patients with ALS. Diffusion tensor imaging (DTI) analyses including tract-based spatial statistics and tractography were applied. DTI metrics including fractional anisotropy (FA) and diffusivities (mean, axial, and radial) were measured in regions of interest. Cortical thickness was assessed using surface-based analysis. The locations of structural changes, measured by DTI and the areas of cortical thinning, were compared to regional glial activation measured by relative [11C]-PBR28 uptake. Results: In this cohort of individuals with ALS, reduced FA and cortical thinning colocalized with regions demonstrating higher radioligand binding. [11C]-PBR28 binding in the left motor cortex was correlated with FA (r = −0.68, p < 0.05) and cortical thickness (r = −0.75, p < 0.05). UMNB was correlated with glial activation (r = +0.75, p < 0.05), FA (r = −0.77, p < 0.05), and cortical thickness (r = −0.75, p < 0.05) in the motor cortex. Conclusions: Increased uptake of the glial marker [11C]-PBR28 colocalizes with changes in FA and cortical thinning. This suggests a link between disease mechanisms (gliosis and inflammation) and structural changes (cortical thinning and white and gray matter changes). In this multimodal neuroimaging work, we provide an in vivo model to investigate the pathogenesis of ALS. PMID:27837005

Comparative Genomics of Regulation of Fatty Acid and Branched-chain Amino Acid Utilization in Proteobacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kazakov, Alexey E.; Rodionov, Dmitry A.; Arkin, Adam Paul

2008-10-31

Bacteria can use branched-chain amino acids (ILV, i.e. isoleucine, leucine, valine) and fatty acids (FA) as sole carbon and energy sources convering ILV into acetyl-CoA, propanoyl-CoA and propionyl-CoA, respectively. In this work, we used the comparative genomic approach to identify candidate transcriptional factors and DNA motifs that control ILV and FA utilization pathways in proteobacteria. The metabolic regulons were characterized based on the identification and comparison of candidate transcription factor binding sites in groups of phylogenetically related genomes. The reconstructed ILV/FA regulatory network demonstrates considerable variability and involves six transcriptional factors from the MerR, TetR and GntR families binding tomore » eleven distinct DNA motifs. The ILV degradation genes in gamma- and beta-proteobacteria are mainly regulated by anovel regulator from the MerR family (e.g., LiuR in Pseudomonas aeruginosa) (40 species), in addition, the TetR-type regulator LiuQ was identified in some beta-proteobacteria (8 species). Besides the core set of ILV utilization genes, the LiuR regulon in some lineages is expanded to include genes from other metabolic pathways, such as the glyoxylate shunt and glutamate synthase in the Shewanella species. The FA degradation genes are controlled by four regulators including FadR in gamma-proteobacteria (34 species), PsrA in gamma- and beta-proteobacteria (45 species), FadP in beta-proteobacteria (14 species), and LiuR orthologs in alpha-proteobacteria (22 species). The remarkable variability of the regulatory systems associated with the FA degradation pathway is discussed from the functional and evolutionary points of view.« less

Changes in UCP expression in tissues of Zucker rats fed diets with different protein content.

PubMed

Masanés, R M; Yubero, P; Rafecas, I; Remesar, X

2002-09-01

The effect of dietary protein content on the uncoupling proteins (UCP) 1, 2 and 3 expression in a number of tissues of Zucker lean and obese rats was studied. Thirty-day-old male Zucker lean (Fa/?) and obese (fa/fa) rats were fed on hyperproteic (HP, 30% protein), standard (RD, 17% protein) or hypoproteic (LP, 9% protein) diets ad libitum for 30 days. Although dietary protein intake affected the weights of individual muscles in lean and obese animals, these weights were similar. In contrast, huge differences were observed in brown adipose tissue (BAT) and liver weights. Lean rats fed on the LP diet generally increased UCP expression, whereas the HP group had lower values. Obese animals, HP and LP groups showed higher UCP expression in muscles, with slight differences in BAT and lower values for UCP3 in subcutaneous adipose tissue. The mean values of UCP expression in BAT of obese rats were lower than in their lean counterpart, whereas the expression in skeletal muscle was increased. Thus, expression of UCPs can be modified by dietary protein content, in lean and obese rats. A possible thermogenic function of UCP3 in muscle and WAT in obese rats must be taken into account.

Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides.

PubMed

Zhao, Qiang; Lv, Qin; Wang, Hailin

2015-08-15

We previously reported a fluorescence anisotropy (FA) approach for small molecules using tetramethylrhodamine (TMR) labeled aptamer. It relies on target-binding induced change of intramolecular interaction between TMR and guanine (G) base. TMR-labeling sites are crucial for this approach. Only terminal ends and thymine (T) bases could be tested for TMR labeling in our previous work, possibly causing limitation in analysis of different targets with this FA strategy. Here, taking the analysis of adenosine triphosphate (ATP) as an example, we demonstrated a success of conjugating TMR on other bases of aptamer adenine (A) or cytosine (C) bases and an achievement of full mapping various labeling sites of aptamers. We successfully constructed aptamer fluorescence anisotropy (FA) sensors for adenosine triphosphate (ATP). We conjugated single TMR on adenine (A), cytosine (C), or thymine (T) bases or terminals of a 25-mer aptamer against ATP and tested FA responses of 14 TMR-labeled aptamer to ATP. The aptamers having TMR labeled on the 16th base C or 23rd base A were screened out and exhibited significant FA-decreasing or FA-increasing responses upon ATP, respectively. These two favorable TMR-labeled aptamers enabled direct FA sensing ATP with a detection limit of 1 µM and the analysis of ATP in diluted serum. The comprehensive screening various TMR labeling sites of aptamers facilitates the successful construction of FA sensors using TMR-labeled aptamers. It will expand application of TMR-G interaction based aptamer FA strategy to a variety of targets. Copyright © 2015 Elsevier B.V. All rights reserved.

The Fanconi anemia pathway limits the severity of mutagenesis.

PubMed

Hinz, John M; Nham, Peter B; Salazar, Edmund P; Thompson, Larry H

2006-08-13

Fanconi anemia (FA) is a developmental and cancer predisposition disorder in which key, yet unknown, physiological events promoting chromosome stability are compromised. FA cells exhibit excess metaphase chromatid breaks and are universally hypersensitive to DNA interstrand crosslinking agents. Published mutagenesis data from single-gene mutation assays show both increased and decreased mutation frequencies in FA cells. In this review we discuss the data from the literature and from our isogenic fancg knockout hamster CHO cells, and interpret these data within the framework of a molecular model that accommodates these seemingly divergent observations. In FA cells, reduced rates of recovery of viable X-linked hypoxanthine phosphoribosyltransferase (hprt) mutants are characteristically observed for diverse mutagenic agents, but also in untreated cultures, indicating the relevance of the FA pathway for processing assorted DNA lesions. We ascribe these reductions to: (1) impaired mutagenic translesion synthesis within hprt during DNA replication and (2) lethality of mutant cells following replication fork breakage on the X chromosome, caused by unrepaired double-strand breaks or large deletions/translocations encompassing essential genes flanking hprt. These findings, along with studies showing increased spontaneous mutability of FA cells at two autosomal loci, support a model in which FA proteins promote both translesion synthesis at replication-blocking lesions and repair of broken replication forks by homologous recombination and DNA end joining. The essence of this model is that the FANC protein pathway serves to restrict the severity of mutational outcome by favoring base substitutions and small deletions over larger deletions and chromosomal rearrangements.

Peroxisome proliferator-activated receptor-α expression induces alterations in cardiac myofilaments in a pressure-overload model of hypertrophy

PubMed Central

Karam, Chehade N.; Warren, Chad M.; Henze, Marcus; Banke, Natasha H.; Lewandowski, E. Douglas

2017-01-01

Although alterations in fatty acid (FA) metabolism have been shown to have a negative impact on contractility of the hypertrophied heart, the targets of action remain elusive. In this study we compared the function of skinned fiber bundles from transgenic (Tg) mice that overexpress a relatively low level of the peroxisome proliferator-activated receptor α (PPARα), and nontransgenic (NTg) littermates. The mice (NTg-T and Tg-T) were stressed by transverse aortic constriction (TAC) and compared with shams (NTg-S and Tg-S). There was an approximate 4-fold increase in PPARα expression in Tg-S compared with NTg-S, but Tg-T hearts showed the same PPARα expression as NTg-T. Expression of PPARα did not alter the hypertrophic response to TAC but did reduce ejection fraction (EF) in Tg-T hearts compared with other groups. The rate of actomyosin ATP hydrolysis was significantly higher in Tg-S skinned fiber bundles compared with all other groups. Tg-T hearts showed an increase in phosphorylation of specific sites on cardiac myosin binding protein-C (cMyBP-C) and β-myosin heavy chain isoform. These results advance our understanding of potential signaling to the myofilaments induced by altered FA metabolism under normal and pathological states. We demonstrate that chronic and transient PPARα activation during pathological stress alters myofilament response to Ca2+ through a mechanism that is possibly mediated by MyBP-C phosphorylation and myosin heavy chain isoforms. NEW & NOTEWORTHY Data presented here demonstrate novel signaling to sarcomeric proteins by chronic alterations in fatty acid metabolism induced by PPARα. The mechanism involves modifications of key myofilament regulatory proteins modifying cross-bridge dynamics with differential effects in controls and hearts stressed by pressure overload. PMID:28130336

Fatty acid composition and desaturase gene expression in flax (Linum usitatissimum L.).

PubMed

Thambugala, Dinushika; Cloutier, Sylvie

2014-11-01

Little is known about the relationship between expression levels of fatty acid desaturase genes during seed development and fatty acid (FA) composition in flax. In the present study, we looked at promoter structural variations of six FA desaturase genes and their relative expression throughout seed development. Computational analysis of the nucleotide sequences of the sad1, sad2, fad2a, fad2b, fad3a and fad3b promoters showed several basic transcriptional elements including CAAT and TATA boxes, and several putative target-binding sites for transcription factors, which have been reported to be involved in the regulation of lipid metabolism. Using semi-quantitative reverse transcriptase PCR, the expression patterns throughout seed development of the six FA desaturase genes were measured in six flax genotypes that differed for FA composition but that carried the same desaturase isoforms. FA composition data were determined by phenotyping the field grown genotypes over four years in two environments. All six genes displayed a bell-shaped pattern of expression peaking at 20 or 24 days after anthesis. Sad2 was the most highly expressed. The expression of all six desaturase genes did not differ significantly between genotypes (P = 0.1400), hence there were no correlations between FA desaturase gene expression and variations in FA composition in relatively low, intermediate and high linolenic acid genotypes expressing identical isoforms for all six desaturases. These results provide further clues towards understanding the genetic factors responsible for FA composition in flax.

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis1[OPEN

PubMed Central

Hsiao, An-Shan; Xue, Yan

2017-01-01

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1. Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 (GL2), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1, smo1-1, and ACBP1+/−smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1+/−smo1-1. Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. PMID:28500265

Differential p53 engagement in response to oxidative and oncogenic stresses in Fanconi anemia mice.

PubMed

Rani, Reena; Li, Jie; Pang, Qishen

2008-12-01

Members of the Fanconi anemia (FA) protein family are involved in repair of genetic damage caused by DNA cross-linkers. It is not clear whether the FA proteins function in oxidative DNA damage and oncogenic stress response. Here, we report that deficiency in the Fanca gene in mice elicits a p53-dependent growth arrest and DNA damage response to oxidative DNA damage and oncogenic stress. Using a Fanca-/-Trp53-/- double knockout model and a functionally switchable p53 retrovirus, we define the kinetics, dependence, and persistence of p53-mediated response to oxidative and oncogenic stresses in Fanca-/- cells. Notably, oxidative stress induces persistent p53 response in Fanca-/- cells, likely due to accumulation of unrepaired DNA damage. On the other hand, whereas wild-type cells exhibit prolonged response to oncogene activation, the p53-activating signals induced by oncogenic ras are short-lived in Fanca-/- cells, suggesting that Fanca may be required for the cell to engage p53 during constitutive ras activation. We propose that the FA proteins protect cells from stress-induced proliferative arrest and tumor evolution by acting as a modulator of the signaling pathways that link FA to p53.

Differential p53 engagement in response to oxidative and oncogenic stresses in Fanconi anemia mice

PubMed Central

Rani, Reena; Li, Jie; Pang, Qishen

2008-01-01

Members of the Fanconi anemia (FA) protein family are involved in repair of genetic damage caused by DNA cross-linkers. It is not clear whether the FA proteins function in oxidative DNA damage and oncogenic stress response. Here we report that deficiency in the Fanca gene in mice elicits a p53-dependent growth arrest and DNA damage response to oxidative DNA damage and oncogenic stress. Using a Fanca-/- Trp53-/- double knockout model and a functionally switchable p53 retrovirus, we define the kinetics, dependence, and persistence of p53-mediated response to oxidative and oncogenic stresses in Fanca-/- cells. Notably, oxidative stress induces persistent p53 response in Fanca-/- cells, likely due to accumulation of unrepaired DNA damage. On the other hand, whereas WT cells exhibit prolonged response to oncogene activation, the p53-activating signals induced by oncogenic ras are short-lived in Fanca-/- cells, suggesting that Fanca may be required for the cell to engage p53 during constitutive ras activation. We propose that the FA proteins protect cells from stress-induced proliferative arrest and tumor evolution by acting as a modulator of the signaling pathways that link FA to p53. PMID:19047147

Folic acid conjugated mPEG-PEI600 as an efficient non-viral vector for targeted nucleic acid delivery.

PubMed

Xu, Zhenhua; Jin, Jiefu; Siu, Leo K S; Yao, Hong; Sze, Johnny; Sun, Hongzhe; Kung, Hsiang-Fu; Poon, Wai Sang; Ng, Samuel S M; Lin, Marie C

2012-04-15

In this study we describe a novel polymer, mPPS-FA, synthesized as a potential gene transfer vector. To complete mPPS-FA, folic acid was conjugated to a backbone (named mPPS) consisting of a copolymer of methyl PEG-2000, PEI-600, and sebacoyl chloride. (1)H NMR, FT-IR, and UV spectroscopy were used to characterize the structure of mPPS-FA. It was revealed that mPPS-FA holds the ability to bind plasmid DNA yielding positively charged particles (polyplexes). Dynamic light scattering (DLS) and TEM techniques were used to study the size and morphology of the formed mPPS-FA/DNA nanocomplexes. The mPPS-FA/DNA nanoparticles exhibited low cytotoxicity as transfection of B16-F0, U87MG, CHO-1, and Ho-8910 cells produced >80% viability indicating low cytotoxicity of the polymer. The ability of mPPS-FA to deliver EGFP plasmid to melanoma B16-F0, U87, CHO-1, Ho-8910, and A549 cells was investigated in vitro as compared to the lipid-based transfection agent Lipofectamine2000 and Linear PEI 22 kDa (L-PEI 22 kDa). We found that mPPS-FA/DNA complexes yielded the highest GFP transfection efficiency in B16-F0, U87, CHO-1, and Ho-8910 cells, which all highly express folate receptors (FR), at an mPPS-FA/DNA ratio (w/w) of 15. Furthermore, the transfection of mPPS-FA/DNA complexes in CHO-1 cells could be competitively blocked by free folic acid molecules. In contrast, in low FR expressing A549 cells, mPPS-FA showed similar low transfection efficiency as mPPS. Taken together, mPPS-FA showed the highest efficiency in vitro and the potential to be developed as a nonviral gene carrier. Copyright © 2012 Elsevier B.V. All rights reserved.

Nerolidol production in agroinfiltrated tobacco: Impact of protein stability and membrane targeting of strawberry (Fragraria ananassa) NEROLIDOL SYNTHASE1.

PubMed

Andrade, Paola; Manzano, David; Ramirez-Estrada, Karla; Caudepon, Daniel; Arro, Montserrat; Ferrer, Albert; Phillips, Michael A

2018-02-01

The sesquiterpene alcohol nerolidol, synthesized from farnesyl diphosphate (FDP), mediates plant-insect interactions across multiple trophic levels with major implications for pest management in agriculture. We compared nerolidol engineering strategies in tobacco using agroinfiltration to transiently express strawberry (Fragraria ananassa) linalool/nerolidol synthase (FaNES1) either at the endoplasmic reticulum (ER) or in the cytosol as a soluble protein. Using solid phase microextraction and gas chromatography-mass spectrometry (SPME-GCMS), we have determined that FaNES1 directed to the ER via fusion to the transmembrane domain of squalene synthase or hydroxymethylglutaryl - CoA reductase displayed significant improvements in terms of transcript levels, protein accumulation, and volatile production when compared to its cytosolic form. However, the highest levels of nerolidol production were observed when FaNES1 was fused to GFP and expressed in the cytosol. This SPME-GCMS method afforded a limit of detection and quantification of 1.54 and 5.13 pg, respectively. Nerolidol production levels, which ranged from 0.5 to 3.0 μg/g F.W., correlated more strongly to the accumulation of recombinant protein than transcript level, the former being highest in FaNES-GFP transfected plants. These results indicate that while the ER may represent an enriched source of FDP that can be exploited in metabolic engineering, protein accumulation is a better predictor of sesquiterpene production. Copyright © 2017 Elsevier B.V. All rights reserved.

A casein diet added isoflavone-enriched soy protein favorably affects biomarkers of steatohepatitis in obese Zucker rats.

PubMed

Gudbrandsen, Oddrun Anita; Wergedahl, Hege; Berge, Rolf Kristian

2009-05-01

Dietary supplementation of a soy protein enriched with isoflavones (HDI) has been shown to improve fatty liver in obese rats. The main objective of this study was to investigate whether HDI would influence the inflammatory status in livers of obese rats with fatty liver. Male obese Zucker fa/fa rats were fed casein (controls) or casein supplemented with HDI (containing 4.00 g of genistein and 4.50 g of daidzein per kilogram of diet) for 6 wk. The HDI-fed rats had a markedly lower hepatic concentration of triacylglycerol when compared with controls. The decreased aspartate transaminase/alanine transaminase ratio in plasma, together with lower circulating levels of alkaline phosphatase and bile acids after HDI feeding, implied an improved hepatitis. This was supported by decreased plasma and hepatic mRNA levels of tumor necrosis factor-alpha, lower plasma levels of interleukin-1beta and monocyte chemoattractant protein-1, and an increased anti-inflammatory fatty acid index in plasma. HDI also seemed to protect the rats from oxidative damage, because the level of lipid peroxides in triacylglycerol-rich lipoproteins after in vitro copper oxidation was lower for HDI-fed rats when compared with controls. These results show that isoflavone-enriched soy protein favorably affects biomarkers of hepatic inflammation in obese Zucker fa/fa rats with fatty liver. Thus, dietary soy proteins enriched in isoflavones may be a promising agent to improve steatohepatitis in patients.

Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair

PubMed Central

Nakanishi, Koji; Yang, Yun-Gui; Pierce, Andrew J.; Taniguchi, Toshiyasu; Digweed, Martin; D'Andrea, Alan D.; Wang, Zhao-Qi; Jasin, Maria

2005-01-01

Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2/FANCD1, proteins involved in homology-directed DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca–/– cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice. PMID:15650050

Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair.

PubMed

Nakanishi, Koji; Yang, Yun-Gui; Pierce, Andrew J; Taniguchi, Toshiyasu; Digweed, Martin; D'Andrea, Alan D; Wang, Zhao-Qi; Jasin, Maria

2005-01-25

Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. Cells from FA patients are hypersensitive to agents that produce DNA crosslinks and, after treatment with these agents, have pronounced chromosome breakage and other cytogenetic abnormalities. Eight FANC genes have been cloned, and the encoded proteins interact in a common cellular pathway. DNA-damaging agents activate the monoubiquitination of FANCD2, resulting in its targeting to nuclear foci that also contain BRCA1 and BRCA2/FANCD1, proteins involved in homology-directed DNA repair. Given the interaction of the FANC proteins with BRCA1 and BRCA2, we tested whether cells from FA patients (groups A, G, and D2) and mouse Fanca-/- cells with a targeted mutation are impaired for this repair pathway. We find that both the upstream (FANCA and FANCG) and downstream (FANCD2) FA pathway components promote homology-directed repair of chromosomal double-strand breaks (DSBs). The FANCD2 monoubiquitination site is critical for normal levels of repair, whereas the ATM phosphorylation site is not. The defect in these cells, however, is mild, differentiating them from BRCA1 and BRCA2 mutant cells. Surprisingly, we provide evidence that these proteins, like BRCA1 but unlike BRCA2, promote a second DSB repair pathway involving homology, i.e., single-strand annealing. These results suggest an early role for the FANC proteins in homologous DSB repair pathway choice.

FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3.

PubMed

Wilson, J B; Yamamoto, K; Marriott, A S; Hussain, S; Sung, P; Hoatlin, M E; Mathew, C G; Takata, M; Thompson, L H; Kupfer, G M; Jones, N J

2008-06-12

Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2, which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2-FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2-FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.

High Incidence of HPV-Associated Head and Neck Cancers in FA Deficient Mice Is Associated with E7’s Induction of DNA Damage through Its Inactivation of Pocket Proteins

PubMed Central

Park, Jung Wook; Shin, Myeong-Kyun; Pitot, Henry C.; Lambert, Paul F.

2013-01-01

Fanconi anemia (FA) patients are highly susceptible to solid tumors at multiple anatomical sites including head and neck region. A subset of head and neck cancers (HNCs) is associated with ‘high-risk’ HPVs, particularly HPV16. However, the correlation between HPV oncogenes and cancers in FA patients is still unclear. We previously learned that FA deficiency in mice predisposes HPV16 E7 transgenic mice to HNCs. To address HPV16 E6’s oncogenic potential under FA deficiency in HNCs, we utilized HPV16 E6-transgenic mice (K14E6) and HPV16 E6/E7-bi-transgenic mice (K14E6E7) on genetic backgrounds sufficient or deficient for one of the fanc genes, fancD2 and monitored their susceptibility to HNCs. K14E6 mice failed to develop tumor. However, E6 and fancD2-deficiency accelerated E7-driven tumor development in K14E6E7 mice. The increased tumor incidence was more correlated with E7-driven DNA damage than proliferation. We also found that deficiency of pocket proteins, pRb, p107, and p130 that are well-established targets of E7, could recapitulate E7’s induction of DNA damage. Our findings support the hypothesis that E7 induces HPV-associated HNCs by promoting DNA damage through the inactivation of pocket proteins, which explains why a deficiency in DNA damage repair would increase susceptibility to E7-driven cancer. Our results further demonstrate the unexpected finding that FA deficiency does not predispose E6 transgenic mice to HNCs, indicating a specificity in the synergy between FA deficiency and HPV oncogenes in causing HNCs. PMID:24086435

Calcite growth-rate inhibition by fulvic acid and magnesium ion—Possible influence on biogenic calcite formation

NASA Astrophysics Data System (ADS)

Reddy, Michael M.

2012-08-01

Increases in ocean surface water dissolved carbon dioxide (CO2) concentrations retard biocalcification by reducing calcite supersaturation (Ωc). Reduced calcification rates may influence growth-rate dependent magnesium ion (Mg) incorporation into biogenic calcite modifying the use of calcifying organisms as paleoclimate proxies. Fulvic acid (FA) at biocalcification sites may further reduce calcification rates. Calcite growth-rate inhibition by FA and Mg, two common constituents of seawater and soil water involved in the formation of biogenic calcite, was measured separately and in combination under identical, highly reproducible experimental conditions. Calcite growth rates (pH=8.5 and Ωc=4.5) are reduced by FA (0.5 mg/L) to 47% and by Mg (10-4 M) to 38%, compared to control experiments containing no added growth-rate inhibitor. Humic acid (HA) is twice as effective a calcite growth-rate inhibitor as FA. Calcite growth rate in the presence of both FA (0.5 mg/L) and Mg (10-4 M) is reduced to 5% of the control rate. Mg inhibits calcite growth rates by substitution for calcium ion at the growth site. In contrast, FA inhibits calcite growth rates by binding multiple carboxylate groups on the calcite surface. FA and Mg together have an increased affinity for the calcite growth sites reducing calcite growth rates.

Calcite growth-rate inhibition by fulvic acid and magnesium ion—Possible influence on biogenic calcite formation

USGS Publications Warehouse

Reddy, Michael M.

2012-01-01

Increases in ocean surface water dissolved carbon dioxide (CO2) concentrations retard biocalcification by reducing calcite supersaturation (Ωc). Reduced calcification rates may influence growth-rate dependent magnesium ion (Mg) incorporation into biogenic calcite modifying the use of calcifying organisms as paleoclimate proxies. Fulvic acid (FA) at biocalcification sites may further reduce calcification rates. Calcite growth-rate inhibition by FA and Mg, two common constituents of seawater and soil water involved in the formation of biogenic calcite, was measured separately and in combination under identical, highly reproducible experimental conditions. Calcite growth rates (pH=8.5 and Ωc=4.5) are reduced by FA (0.5 mg/L) to 47% and by Mg (10−4 M) to 38%, compared to control experiments containing no added growth-rate inhibitor. Humic acid (HA) is twice as effective a calcite growth-rate inhibitor as FA. Calcite growth rate in the presence of both FA (0.5 mg/L) and Mg (10−4 M) is reduced to 5% of the control rate. Mg inhibits calcite growth rates by substitution for calcium ion at the growth site. In contrast, FA inhibits calcite growth rates by binding multiple carboxylate groups on the calcite surface. FA and Mg together have an increased affinity for the calcite growth sites reducing calcite growth rates.

The impact of FANCD2 deficiency on formaldehyde-induced toxicity in human lymphoblastoid cell lines

PubMed Central

Ren, Xuefeng; Ji, Zhiying; McHale, Cliona M.; Yuh, Jessica; Bersonda, Jessica; Tang, Maycky; Smith, Martyn T.; Zhang, Luoping

2015-01-01

Formaldehyde (FA), a major industrial chemical and ubiquitous environmental pollutant, has recently been classified by the International Agency for Research on Cancer as a human leukemogen. The major mode of action of FA is thought to be the formation of DNA-protein crosslinks (DPCs). Repair of DPCs may be mediated by the Fanconi anemia pathway; however, data supporting the involvement of this pathway is limited, particularly in human hematopoietic cells. Therefore, we assessed the role of FANCD2, a critical component of the Fanconi anemia pathway, in FA-induced toxicity in human lymphoblast cell models of FANCD2-deficiency (PD20 cells) and FANCD2-sufficiency (PD20-D2 cells). After treatment of the cells with 0-150 μM FA for 24 hours, DPCs were increased in a dose-dependent manner in both cell lines, with greater increases in FANCD2-deficient PD20 cells. FA also induced cytotoxicity, micronuclei, chromosome aberrations, and apoptosis in a dose-dependent manner in both cell lines, with greater increases in cytotoxicity and apoptosis in PD20 cells. Increased levels of γ-ATR and γ-H2AX in both cell lines suggested the recognition of FA-induced DNA damage; however, the induction of BRCA2 was compromised in FANCD2-deficient PD20 cells, potentially reducing the capacity to repair DPCs. Together, these findings suggest that FANCD2 protein and the Fanconi anemia pathway are essential to protect human lymphoblastoid cells against FA toxicity. Future studies are needed to delineate the role of this pathway in mitigating FA-induced toxicity, particularly in hematopoietic stem cells, the target cells in leukemia. PMID:22872141

Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lasry, Inbal; Berman, Bluma; Glaser, Fabian

2009-08-28

The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structuresmore » of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.« less

Whey protein gel composites in the diet of goats increased the omega-3 and omega-6 content of milk fat.

PubMed

Weinstein, J A; Taylor, S J; Rosenberg, M; DePeters, E J

2016-08-01

Previously, feeding whey protein gels containing polyunsaturated fatty acids (PUFA) reduced their rumen biohydrogenation and increased their concentration in milk fat of Holstein cows. Our objective was to test the efficacy of whey protein isolate (WPI) gels produced in a steam tunnel as a method to alter the fatty acid (FA) composition of the milk lipids. Four primiparous Lamancha goats in midlactation were fed three diets in a 3 × 4 Latin square design. The WPI gels were added to a basal concentrate mix that contained one of three lipid sources: (i) 100% soya bean oil (S) to create (WPI/S), (ii) a 1:1 (wt/wt) mixture of S and linseed (L) oil to create (WPI/SL), or (iii) 100% L to create (WPI/L). Periods were 22 days with the first 10 days used as an adjustment phase followed by a 12-day experimental phase. During the adjustment phase, all goats received a rumen available source of lipid, yellow grease, to provide a baseline for milk FA composition. During the experimental phase, each goat received its assigned WPI. Milk FA concentration of C18:2 n-6 and C18:3 n-3 reached 9.3 and 1.64 g/100 g FA, respectively, when goats were fed WPI/S. Feeding WPI/SL increased the C18:2 n-6 and C18:3 n-3 concentration to 6.22 and 4.36 g/100 g FA, and WPI/L increased C18:2 n-6 and C18:3 n-3 to 3.96 and 6.13 g/100 g FA respectively. The adjusted transfer efficiency (%) of C18:3 n-3 to milk FA decreased significantly as dietary C18:3 n-3 intake increased. Adjusted transfer efficiency for C18:2 n-6 did not change with increasing intake of C18:2 n-6. The WPI gels were effective at reducing rumen biohydrogenation of PUFA; however, we observed a change in the proportion increase of C18:3 n-3 in milk FA suggesting possible regulation of n-3 FA to the lactating caprine mammary gland. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

Deficiency of UBE2T, the E2 Ubiquitin Ligase Necessary for FANCD2 and FANCI Ubiquitination, Causes FA-T Subtype of Fanconi Anemia.

PubMed

Rickman, Kimberly A; Lach, Francis P; Abhyankar, Avinash; Donovan, Frank X; Sanborn, Erica M; Kennedy, Jennifer A; Sougnez, Carrie; Gabriel, Stacey B; Elemento, Olivier; Chandrasekharappa, Settara C; Schindler, Detlev; Auerbach, Arleen D; Smogorzewska, Agata

2015-07-07

Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Palmitoleic acid (16:1n7) increases oxygen consumption, fatty acid oxidation and ATP content in white adipocytes.

PubMed

Cruz, Maysa M; Lopes, Andressa B; Crisma, Amanda R; de Sá, Roberta C C; Kuwabara, Wilson M T; Curi, Rui; de Andrade, Paula B M; Alonso-Vale, Maria I C

2018-03-20

We have recently demonstrated that palmitoleic acid (16:1n7) increases lipolysis, glucose uptake and glucose utilization for energy production in white adipose cells. In the present study, we tested the hypothesis that palmitoleic acid modulates bioenergetic activity in white adipocytes. For this, 3 T3-L1 pre-adipocytes were differentiated into mature adipocytes in the presence (or absence) of palmitic (16:0) or palmitoleic (16:1n7) acid at 100 or 200 μM. The following parameters were evaluated: lipolysis, lipogenesis, fatty acid (FA) oxidation, ATP content, oxygen consumption, mitochondrial mass, citrate synthase activity and protein content of mitochondrial oxidative phosphorylation (OXPHOS) complexes. Treatment with 16:1n7 during 9 days raised basal and isoproterenol-stimulated lipolysis, FA incorporation into triacylglycerol (TAG), FA oxidation, oxygen consumption, protein expression of subunits representing OXPHOS complex II, III, and V and intracellular ATP content. These effects were not observed in adipocytes treated with 16:0. Palmitoleic acid, by concerted action on lipolysis, FA esterification, mitochondrial FA oxidation, oxygen consumption and ATP content, does enhance white adipocyte energy expenditure and may act as local hormone.

Effects of baked and raw salmon fillet on lipids and n-3 PUFAs in serum and tissues in Zucker fa/fa rats​​​​​​​​​​​​​​​​​​​​.

PubMed

Vikøren, Linn A; Drotningsvik, Aslaug; Bergseth, Marthe T; Mjøs, Svein A; Mola, Nazanin; Leh, Sabine; Mellgren, Gunnar; Gudbrandsen, Oddrun A

2017-01-01

Knowledge of the health impact of consuming heat-treated versus raw fish fillet is limited. To investigate effects of baked or raw salmon fillet intake on lipids and n-3 PUFAs in serum and tissues, obese Zucker fa/fa rats were fed diets containing 25% of protein from baked or raw salmon fillet and 75% of protein from casein, or casein as the sole protein source (control group) for four weeks. Salmon diets had similar composition of amino and fatty acids. Growth and energy intake were similar in all groups. Amounts of lipids and n-3 PUFAs in serum, liver and skeletal muscle were similar between rats fed baked or raw salmon fillet. When compared to the control group, rats fed baked salmon had lower serum total and LDL cholesterol and higher serum triacylglycerol levels. Both raw and baked salmon groups had lower HDL cholesterol level when compared to control rats. In conclusion, baking as a preparation method does not alter protein and fat qualities of salmon fillets, and intake of baked and raw salmon fillets gave similar effects on lipids and n-3 PUFAs in serum and tissues from rats.

Effects of baked and raw salmon fillet on lipids and n-3 PUFAs in serum and tissues in Zucker fa/fa rats​​​​​​​​​​​​​​​​​​​​

PubMed Central

Vikøren, Linn A.; Drotningsvik, Aslaug; Bergseth, Marthe T.; Mjøs, Svein A.; Mola, Nazanin; Leh, Sabine; Mellgren, Gunnar; Gudbrandsen, Oddrun A.

2017-01-01

ABSTRACT Knowledge of the health impact of consuming heat-treated versus raw fish fillet is limited. To investigate effects of baked or raw salmon fillet intake on lipids and n-3 PUFAs in serum and tissues, obese Zucker fa/fa rats were fed diets containing 25% of protein from baked or raw salmon fillet and 75% of protein from casein, or casein as the sole protein source (control group) for four weeks. Salmon diets had similar composition of amino and fatty acids. Growth and energy intake were similar in all groups. Amounts of lipids and n-3 PUFAs in serum, liver and skeletal muscle were similar between rats fed baked or raw salmon fillet. When compared to the control group, rats fed baked salmon had lower serum total and LDL cholesterol and higher serum triacylglycerol levels. Both raw and baked salmon groups had lower HDL cholesterol level when compared to control rats. In conclusion, baking as a preparation method does not alter protein and fat qualities of salmon fillets, and intake of baked and raw salmon fillets gave similar effects on lipids and n-3 PUFAs in serum and tissues from rats. PMID:28659746

Mechanism of Ubiquitination and Deubiquitination in the Fanconi Anemia Pathway.

PubMed

van Twest, Sylvie; Murphy, Vincent J; Hodson, Charlotte; Tan, Winnie; Swuec, Paolo; O'Rourke, Julienne J; Heierhorst, Jörg; Crismani, Wayne; Deans, Andrew J

2017-01-19

Monoubiquitination and deubiquitination of FANCD2:FANCI heterodimer is central to DNA repair in a pathway that is defective in the cancer predisposition syndrome Fanconi anemia (FA). The "FA core complex" contains the RING-E3 ligase FANCL and seven other essential proteins that are mutated in various FA subtypes. Here, we purified recombinant FA core complex to reveal the function of these other proteins. The complex contains two spatially separate FANCL molecules that are dimerized by FANCB and FAAP100. FANCC and FANCE act as substrate receptors and restrict monoubiquitination to the FANCD2:FANCI heterodimer in only a DNA-bound form. FANCA and FANCG are dispensable for maximal in vitro ubiquitination. Finally, we show that the reversal of this reaction by the USP1:UAF1 deubiquitinase only occurs when DNA is disengaged. Our work reveals the mechanistic basis for temporal and spatial control of FANCD2:FANCI monoubiquitination that is critical for chemotherapy responses and prevention of Fanconi anemia. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis and Biological Evaluation of Novel Folic Acid Receptor-Targeted, β-Cyclodextrin-Based Drug Complexes for Cancer Treatment

PubMed Central

Yin, Juan-Juan; Sharma, Sonali; Shumyak, Stepan P.; Wang, Zhi-Xin; Zhou, Zhi-Wei; Zhang, Yangde; Guo, Peixuan; Li, Chen-Zhong; Kanwar, Jagat R.; Yang, Tianxin; Mohapatra, Shyam S.; Liu, Wanqing; Duan, Wei; Wang, Jian-Cheng; Li, Qi; Zhang, Xueji; Tan, Jun; Jia, Lee; Liang, Jun; Wei, Ming Q.; Li, Xiaotian; Zhou, Shu-Feng

2013-01-01

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5–2.5 nm. The host-guest association constant K a was 1,639 M−1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer. PMID:23658721

Talin determines the nanoscale architecture of focal adhesions.

PubMed

Liu, Jaron; Wang, Yilin; Goh, Wah Ing; Goh, Honzhen; Baird, Michelle A; Ruehland, Svenja; Teo, Shijia; Bate, Neil; Critchley, David R; Davidson, Michael W; Kanchanawong, Pakorn

2015-09-01

Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.

Ferulic acid alleviates symptoms of preeclampsia in rats by upregulating vascular endothelial growth factor.

PubMed

Gong, Weiyan; Wan, Jipeng; Yuan, Qing; Man, Quanzhan; Zhang, Xiaojing

2017-10-01

Preeclampsia is a complication affecting pregnant women worldwide, which leads to maternal and fetal morbidity and mortality. In this study, we evaluated the efficacy of ferulic acid (FA) on an N ω -nitro-L-arginine methyl ester hydrochloride (L-NAME) induced rat model of preeclampsia. L-NAME was administered to pregnant rats to induce preeclampsia. 48 rats were divided into three experimental groups (n=16 each): control group, preeclampsia group and preeclampsia with FA treatment (preeclampsia+FA). Physiological characteristics such as urine volume, total urine protein and blood pressure were assessed. Expressions levels of urinary nephrin and podocin mRNAs were analyzed by RT-PCR. Levels of renal vascular endothelial growth factor (VEGF), renal soluble fms-like tyrosine kinase-1 (sFlt-1) and serum placenta growth factor (PlGF) were also examined. Urine volume, total urine protein and blood pressure were markedly increased in preeclampsia group rats compared to control (P<.05), which were then significantly reduced in preeclampsia+FA group (P<.05). Expressions of urinary nephrin and podocin mRNAs, levels of VEGF, sFlt-1 and PlGF were also reversed in preeclampsia+FA group compared to preeclampsia rats (P<.05). We hereby report for the first time, FA alleviates preeclampsia symptoms in a rat preeclampsia model, supporting its potential value in treating preeclampsia. © 2017 John Wiley & Sons Australia, Ltd.

Genetic variants in a lipid regulatory pathway as potential tools for improving the nutritional quality of grass-fed beef.

PubMed

Baeza, M C; Corva, P M; Soria, L A; Pavan, E; Rincon, G; Medrano, J F

2013-04-01

The aim of this study was to evaluate the effect of genetic variants on candidate genes corresponding to the sterol recognition element-binding protein-1 (SREBP-1) signaling pathway and stearoyl-CoA desaturases (SCD1 and SCD5) on muscle fatty acid (FA) composition of Brangus steers fattened on grass. FA profiles were measured on Longissimus lumborum muscle samples using a gas chromatography-flame ionization detection technique. A total of 43 tag single-nucleotide polymorphisms on the SCD1, SCD5, SREBP-1, SCAP, INSIG1, INSIG2, MBTPS1, MBTPS2, and SRPR genes were genotyped on 246 steers to perform a marker-trait association study. To evaluate the influence of the Indicine breed in the composite breed, additional groups of 48 Angus, 18 Hereford, 75 Hereford x Angus, and 36 Limousin x Hereford-Angus steers were also genotyped. To perform the association analysis, FA data were grouped according to the number of carbon atoms and/or number of double bonds (i.e. SFA, MUFA, PUFA, etc.). In addition, different indexes that reflect the activity of FA desaturase and elongase enzymes were calculated. SCD1 markers significantly affected C14:1/(C14:0 + C14:1) and C18:1/(C18:0 + C18:1) indexes, whereas one SNP in SCD5 was correlated with the C16:1/(C16:0 + C16:1) index. Polymorphisms in the signal recognition particle receptor (SRPR) gene were associated with all the estimated desaturase indexes. Because the evaluated markers showed no effect on total lipid content of beef, this work supports the potential utilization of these markers for the improvement of grass-fed beef without undesirable side effects. © 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.

Fanconi anemia: a disorder defective in the DNA damage response.

PubMed

Kitao, Hiroyuki; Takata, Minoru

2011-04-01

Fanconi anemia (FA) is a cancer predisposition disorder characterized by progressive bone marrow failure, congenital developmental defects, chromosomal abnormalities, and cellular hypersensitivity to DNA interstrand crosslink (ICL) agents. So far mutations in 14 FANC genes were identified in FA or FA-like patients. These gene products constitute a common ubiquitin-phosphorylation network called the "FA pathway" and cooperate with other proteins involved in DNA repair and cell cycle control to repair ICL lesions and to maintain genome stability. In this review, we summarize recent exciting discoveries that have expanded our view of the molecular mechanisms operating in DNA repair and DNA damage signaling.

Water and Temperature Stresses Impact Canola (Brassica napus L.) Fatty Acid, Protein, and Yield over Nitrogen and Sulfur.

PubMed

Hammac, W Ashley; Maaz, Tai M; Koenig, Richard T; Burke, Ian C; Pan, William L

2017-12-06

Interactive effects of weather and soil nutrient status often control crop productivity. An experiment was conducted to determine effects of nitrogen (N) and sulfur (S) fertilizer rate, soil water, and atmospheric temperature on canola (Brassica napus L.) fatty acid (FA), total oil, protein, and grain yield. Nitrogen and sulfur were assessed in a 4-yr study with two locations, five N rates (0, 45, 90, 135, and 180 kg ha -1 ), and two S rates (0 and 17 kg ha -1 ). Water and temperature were assessed using variability across 12 site-years of dryland canola production. Effects of N and S were inconsistent. Unsaturated FA, oleic acid, grain oil, protein, and theoretical maximum grain yield were highly related to water and temperature variability across the site-years. A nonlinear model identified water and temperature conditions that enabled production of maximum unsaturated FA content, oleic acid content, total oil, protein, and theoretical maximum grain yield. Water and temperature variability played a larger role than soil nutrient status on canola grain constituents and yield.

Novel FANCI mutations in Fanconi anemia with VACTERL association.

PubMed

Savage, Sharon A; Ballew, Bari J; Giri, Neelam; Chandrasekharappa, Settara C; Ameziane, Najim; de Winter, Johan; Alter, Blanche P

2016-02-01

Fanconi anemia (FA) is an inherited bone marrow failure syndrome caused by mutations in DNA repair genes; some of these patients may have features of the VACTERL association. Autosomal recessive mutations in FANCI are a rare cause of FA. We identified FANCI mutations by next generation sequencing in three patients in our FA cohort among several whose mutated gene was unknown. Four of the six mutations are novel and all mutations are likely deleterious to protein function. There are now 16 reported cases of FA due to FANCI of whom 7 have at least 3 features of the VACTERL association (44%). This suggests that the VACTERL association in patients with FA may be seen in patients with FANCI mutations more often than previously recognized. © 2015 Wiley Periodicals, Inc.

Modeling the binding of fulvic acid by goethite: the speciation of adsorbed FA molecules

NASA Astrophysics Data System (ADS)

Filius, Jeroen D.; Meeussen, Johannes C. L.; Lumsdon, David G.; Hiemstra, Tjisse; van Riemsdijk, Willem H.

2003-04-01

Under natural conditions, the adsorption of ions at the solid-water interface may be strongly influenced by the adsorption of organic matter. In this paper, we describe the adsorption of fulvic acid (FA) by metal(hydr)oxide surfaces with a heterogeneous surface complexation model, the ligand and charge distribution (LCD) model. The model is a self-consistent combination of the nonideal competitive adsorption (NICA) equation and the CD-MUSIC model. The LCD model can describe simultaneously the concentration, pH, and salt dependency of the adsorption with a minimum of only three adjustable parameters. Furthermore, the model predicts the coadsorption of protons accurately for an extended range of conditions. Surface speciation calculations show that almost all hydroxyl groups of the adsorbed FA molecules are involved in outer sphere complexation reactions. The carboxylic groups of the adsorbed FA molecule form inner and outer sphere complexes. Furthermore, part of the carboxylate groups remain noncoordinated and deprotonated.

Hindlimb unweighting decreases endothelium-dependent dilation and eNOS expression in soleus not gastrocnemius

NASA Technical Reports Server (NTRS)

Woodman, C. R.; Schrage, W. G.; Rush, J. W.; Ray, C. A.; Price, E. M.; Hasser, E. M.; Laughlin, M. H.

2001-01-01

We tested the hypothesis that hindlimb unweighting (HLU) decreases endothelium-dependent vasodilation and expression of endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) in arteries of skeletal muscle with reduced blood flow during HLU. Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 15) or control (n = 15) conditions for 14 days. ACh-induced dilation was assessed in muscle with reduced [soleus (Sol)] or unchanged [gastrocnemius (Gast)] blood flow during HLU. eNOS and SOD-1 expression were measured in feed arteries (FA) and in first-order (1A), second-order (2A), and third-order (3A) arterioles. Dilation to infusion of ACh in vivo was blunted in Sol but not Gast. In arteries of Sol muscle, HLU decreased eNOS mRNA and protein content. eNOS mRNA content was significantly less in Sol FA (35%), 1A arterioles (25%) and 2A arterioles (18%). eNOS protein content was less in Sol FA (64%) and 1A arterioles (65%) from HLU rats. In arteries of Gast, HLU did not decrease eNOS mRNA or protein. SOD-1 mRNA expression was less in Sol 2A arterioles (31%) and 3A arterioles (29%) of HLU rats. SOD-1 protein content was less in Sol FA (67%) but not arterioles. SOD-1 mRNA and protein content were not decreased in arteries from Gast. These data indicate that HLU decreases endothelium-dependent vasodilation, eNOS expression, and SOD-1 expression primarily in arteries of Sol muscle where blood flow is reduced during HLU.

Regulation of Chromatin Assembly and Cell Transformation by Formaldehyde Exposure in Human Cells

PubMed Central

Chen, Danqi; Fang, Lei; Mei, Shenglin; Li, Hongjie; Xu, Xia; Des Marais, Thomas L.; Lu, Kun; Liu, X. Shirley

2017-01-01

Background: Formaldehyde (FA) is an environmental and occupational chemical carcinogen. Recent studies have shown that exogenous FA causes only a modest increase in DNA adduct formation compared with the amount of adducts formed by endogenous FA, raising the possibility that epigenetic mechanisms may contribute to FA-mediated carcinogenicity. Objectives: We investigated the effects of FA exposure on histone modifications and chromatin assembly. We also examined the role of defective chromatin assembly in FA-mediated transcription and cell transformation. Methods: Cellular fractionation and Western blot analysis were used to measure the levels of histone modifications in human bronchial epithelial BEAS-2B cells and human nasal RPMI2650 cells in the presence of FA. Chromatin immunoprecipitation (ChIP) and micrococcal nuclease (MNase) digest assays were performed to examine the changes in chromatin assembly and accessibility after FA exposure. RNA sequencing (RNA-seq) and real-time polymerase chain reaction (PCR) were used to examine transcriptional dysregulation. Finally, anchorage-independent cell growth ability was tested by soft agar assay following FA exposure. Results: Exposure to FA dramatically decreased the acetylation of the N-terminal tails of cytosolic histones. These modifications are important for histone nuclear import and subsequent chromatin assembly. Histone proteins were depleted in both the chromatin fraction and at most of the genomic loci tested following FA exposure, suggesting that FA compromises chromatin assembly. Moreover, FA increased chromatin accessibility and altered the expression of hundreds of cancer-related genes. Knockdown of the histone H3.3 gene (an H3 variant), which mimics inhibition of chromatin assembly, facilitated FA-mediated anchorage-independent cell growth. Conclusions: We propose that the inhibition of chromatin assembly represents a novel mechanism of cell transformation induced by the environmental and occupational chemical carcinogen FA. https://doi.org/10.1289/EHP1275 PMID:28937961

Exploiting Tumor-Activated Testes Proteins to Enhance Efficacy of First-Line Chemotherapeutics in NSCLC

DTIC Science & Technology

2015-10-01

TERMS Cancer Testis Antigen (CTA), Fanconia- Anemia (FA), DNA Damage, Genomic Instability, DNA Double Strand Break (DSB) 16. SECURITY CLASSIFICATION OF...Cancer Testis Antigen (CTA) o Fanconia- Anemia (FA) o DNA Damage o Genomic Instability o DNA Double Strand Break (DSB) 3. Accomplishments • What

[Fanconi anemia: genes and function(s) revisited].

PubMed

Papadopoulo, Dora; Moustacchi, Ethel

2005-01-01

Fanconi anemia (FA), a rare inherited disorder, exhibits a complex phenotype including progressive bone marrow failure, congenital malformations and increased risk of cancers, mainly acute myeloid leukaemia. At the cellular level, FA is characterized by hypersensitivity to DNA cross-linking agents and by high frequencies of induced chromosomal aberrations, a property used for diagnosis. FA results from mutations in one of the eleven FANC (FANCA to FANCJ) genes. Nine of them have been identified. In addition, FANCD1 gene has been shown to be identical to BRCA2, one of the two breast cancer susceptibility genes. Seven of the FANC proteins form a complex, which exists in four different forms depending of its subcellular localisation. Four FANC proteins (D1(BRCA2), D2, I and J) are not associated to the complex. The presence of the nuclear form of the FA core complex is necessary for the mono-ubiquitinylation of FANCD2 protein, a modification required for its re-localization to nuclear foci, likely to be sites of DNA repair. A clue towards understanding the molecular function of the FANC genes comes from the recently identified connection of FANC to the BRCA1, ATM, NBS1 and ATR genes. Two of the FANC proteins (A and D2) directly interact with BRCA1, which in turn interacts with the MRE11/RAD50/NBS1 complex, which is one of the key components in the mechanisms involved in the cellular response to DNA double strand breaks (DSB). Moreover, ATM, a protein kinase that plays a central role in the network of DSB signalling, phosphorylates in vitro and in vivo FANCD2 in response to ionising radiations. Moreover, the NBS1 protein and the monoubiquitinated form of FANCD2 seem to act together in response to DNA crosslinking agents. Taken together with the previously reported impaired DSB and DNA interstrand crosslinks repair in FA cells, the connection of FANC genes to the ATM, ATR, NBS1 and BRCA1 links the FANC genes function to the finely orchestrated network involved in the sensing, signalling and repair of DNA replication-blocking lesions.

Extracellular fluid proteins of goldfish brain: evidence for the presence of proteases and esterases.

PubMed

Shashoua, V E; Holmquist, B

1986-09-01

Preparations of enriched fractions of extracellular fluid (ECF) proteins from goldfish brain were found to contain protease(s) and esterase(s). The N-substituted furanacryloyl (FA) peptides FA-Phe-Gly-Gly and FA-Phe-OMe were used as model substrates for determining protease and esterase activity, respectively, in a spectrophotometric assay. Studies of the profile of substrate specificity and identification of the types of compounds that were effective as inhibitors showed that these ECF enzymes have some distinctive properties. GSH, but not GSSG, and EDTA inhibited the protease(s) without influencing the esterase(s), whereas L-1-tosylamide-2-phenylethylchloromethyl ketone blocked both protease and esterase activities of ECF. Most of the protease and esterase properties of ECF could be bound to concanavalin A-Sepharose affinity chromatographic columns in association with ependymin--a brain extracellular protein. These observations indicate that ECF may contain a metalloprotease(s) and raise the possibility that the ependymins might be a substrate for these ECF enzymes.

Co-expression analysis identifies CRC and AP1 the regulator of Arabidopsis fatty acid biosynthesis.

PubMed

Han, Xinxin; Yin, Linlin; Xue, Hongwei

2012-07-01

Fatty acids (FAs) play crucial rules in signal transduction and plant development, however, the regulation of FA metabolism is still poorly understood. To study the relevant regulatory network, fifty-eight FA biosynthesis genes including de novo synthases, desaturases and elongases were selected as "guide genes" to construct the co-expression network. Calculation of the correlation between all Arabidopsis thaliana (L.) genes with each guide gene by Arabidopsis co-expression dating mining tools (ACT) identifies 797 candidate FA-correlated genes. Gene ontology (GO) analysis of these co-expressed genes showed they are tightly correlated to photosynthesis and carbohydrate metabolism, and function in many processes. Interestingly, 63 transcription factors (TFs) were identified as candidate FA biosynthesis regulators and 8 TF families are enriched. Two TF genes, CRC and AP1, both correlating with 8 FA guide genes, were further characterized. Analyses of the ap1 and crc mutant showed the altered total FA composition of mature seeds. The contents of palmitoleic acid, stearic acid, arachidic acid and eicosadienoic acid are decreased, whereas that of oleic acid is increased in ap1 and crc seeds, which is consistent with the qRT-PCR analysis revealing the suppressed expression of the corresponding guide genes. In addition, yeast one-hybrid analysis and electrophoretic mobility shift assay (EMSA) revealed that CRC can bind to the promoter regions of KCS7 and KCS15, indicating that CRC may directly regulate FA biosynthesis. © 2012 Institute of Botany, Chinese Academy of Sciences.

[Fanconi anemia: cellular and molecular features].

PubMed

Macé, G; Briot, D; Guervilly, J-H; Rosselli, F

2007-02-01

Fanconi anemia (FA) is a recessive human cancer prone syndrome featuring bone marrow failure, developmental abnormalities and hypersensitivity to DNA crosslinking agents exposure. 11 among 12 FA gene have been isolated. The biochemical functions of the FANC proteins remain poorly understood. Anyhow, to cope with DNA crosslinks a cell needs a functional FANC pathway. Moreover, the FANC proteins appear to be involved in cell protection against oxidative damage and in the control of TNF-alpha activity. In this review, we describe the current understanding of the FANC pathway and we present how it may be integrated in the complex networks of proteins involved in maintaining the cellular homeostasis.

Chalcones suppress fatty acid-induced lipid accumulation through a LKB1/AMPK signaling pathway in HepG2 cells.

PubMed

Zhang, Tianshun; Yamamoto, Norio; Ashida, Hitoshi

2014-06-01

Excessive lipid accumulation in the liver has been proposed to cause hyperlipidemia, diabetes and fatty liver disease. 4-Hydroxyderricin (4HD), xanthoangelol (XAG), cardamonin (CAR) and flavokawain B (FKB) are chalcones that have exhibited various biological effects against obesity, inflammation, and diabetes; however, little is known about the inhibitory effects of these chalcones on fatty liver disease. In the present study, we investigated the ability of 4HD, XAG, CAR, and FKB to reduce lipid accumulation in hepatocytes. When HepG2 cells were treated with a mixture of fatty acids (FAs; palmitic acid : oleic acid = 1 : 2 ratio), significant lipid accumulation was observed. Under the same experimental conditions, addition of chalcones at 5 μM significantly suppressed the FA-induced lipid accumulation. We found that the expression of sterol regulatory element-binding protein-1 (SREBP-1), a key molecule involved in lipogenesis, was decreased in these chalcone-treated cells. We also found that these chalcones increased the expression of peroxisome proliferator-activated receptor α (PPARα), which is involved in FA oxidation. Moreover, these chalcones increased phosphorylation of AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1), upstream regulators of SREBP-1 and PPARα. We confirmed that an AMPK inhibitor, compound C, reversed chalcone-induced changes in SREBP-1 and PPARα expression in the HepG2 cells. Collectively, we found that 4HD, XAG, CAR, and XAG attenuated lipid accumulation through activation of the LKB1/AMPK signaling pathway in HepG2 cells.

The nuclease hSNM1B/Apollo is linked to the Fanconi anemia pathway via its interaction with FANCP/SLX4.

PubMed

Salewsky, Bastian; Schmiester, Maren; Schindler, Detlev; Digweed, Martin; Demuth, Ilja

2012-11-15

The recessive genetic disorder Fanconi anemia (FA) is clinically characterized by congenital defects, bone marrow failure and an increased incidence of cancer. Cells derived from FA patients exhibit hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents. We have earlier reported a similar cellular phenotype for human cells depleted of hSNM1B/Apollo (siRNA). In fact, hSNM1B/Apollo has a dual role in the DNA damage response and in generation and maintenance of telomeres, the latter function involving interaction with the shelterin protein TRF2. Here we find that ectopically expressed hSNM1B/Apollo co-immunoprecipitates with SLX4, a protein recently identified as a new FA protein, FANCP, and known to interact with several structure-specific nucleases. As shown by immunofluorescence analysis, FANCP/SLX4 depletion (siRNA) resulted in a significant reduction of hSNM1B/Apollo nuclear foci, supporting the functional relevance of this new protein interaction. Interestingly, as an additional consequence of FANCP/SLX4 depletion, we found a reduction of cellular TRF2, in line with its telomere-related function. Finally, analysis of human cells following double knockdown of hSNM1B/Apollo and FANCP/SLX4 indicated that they function epistatically. These findings further substantiate the role of hSNM1B/Apollo in a downstream step of the FA pathway during the repair of DNA ICLs.

Semi-empirical proton binding constants for natural organic matter

NASA Astrophysics Data System (ADS)

Matynia, Anthony; Lenoir, Thomas; Causse, Benjamin; Spadini, Lorenzo; Jacquet, Thierry; Manceau, Alain

2010-03-01

Average proton binding constants ( KH,i) for structure models of humic (HA) and fulvic (FA) acids were estimated semi-empirically by breaking down the macromolecules into reactive structural units (RSUs), and calculating KH,i values of the RSUs using linear free energy relationships (LFER) of Hammett. Predicted log KH,COOH and log KH,Ph-OH are 3.73 ± 0.13 and 9.83 ± 0.23 for HA, and 3.80 ± 0.20 and 9.87 ± 0.31 for FA. The predicted constants for phenolic-type sites (Ph-OH) are generally higher than those derived from potentiometric titrations, but the difference may not be significant in view of the considerable uncertainty of the acidity constants determined from acid-base measurements at high pH. The predicted constants for carboxylic-type sites agree well with titration data analyzed with Model VI (4.10 ± 0.16 for HA, 3.20 ± 0.13 for FA; Tipping, 1998), the Impermeable Sphere model (3.50-4.50 for HA; Avena et al., 1999), and the Stockholm Humic Model (4.10 ± 0.20 for HA, 3.50 ± 0.40 for FA; Gustafsson, 2001), but differ by about one log unit from those obtained by Milne et al. (2001) with the NICA-Donnan model (3.09 ± 0.51 for HA, 2.65 ± 0.43 for FA), and used to derive recommended generic values. To clarify this ambiguity, 10 high-quality titration data from Milne et al. (2001) were re-analyzed with the new predicted equilibrium constants. The data are described equally well with the previous and new sets of values ( R2 ⩾ 0.98), not necessarily because the NICA-Donnan model is overparametrized, but because titration lacks the sensitivity needed to quantify the full binding properties of humic substances. Correlations between NICA-Donnan parameters are discussed, but general progress is impeded by the unknown number of independent parameters that can be varied during regression of a model fit to titration data. The high consistency between predicted and experimental KH,COOH values, excluding those of Milne et al. (2001), gives faith in the proposed semi-empirical structural approach, and its usefulness to assess the plausibility of proton stability constants derived from simulations of titration data.

BLM promotes the activation of Fanconi Anemia signaling pathway.

PubMed

Panneerselvam, Jayabal; Wang, Hong; Zhang, Jun; Che, Raymond; Yu, Herbert; Fei, Peiwen

2016-05-31

Mutations in the human RecQ helicase, BLM, causes Bloom Syndrome, which is a rare autosomal recessive disorder and characterized by genomic instability and an increased risk of cancer. Fanconi Anemia (FA), resulting from mutations in any of the 19 known FA genes and those yet to be known, is also characterized by chromosomal instability and a high incidence of cancer. BLM helicase and FA proteins, therefore, may work in a common tumor-suppressor signaling pathway. To date, it remains largely unclear as to how BLM and FA proteins work concurrently in the maintenance of genome stability. Here we report that BLM is involved in the early activation of FA group D2 protein (FANCD2). We found that FANCD2 activation is substantially delayed and attenuated in crosslinking agent-treated cells harboring deficient Blm compared to similarly treated control cells with sufficient BLM. We also identified that the domain VI of BLM plays an essential role in promoting FANCD2 activation in cells treated with DNA crosslinking agents, especially ultraviolet B. The similar biological effects performed by ΔVI-BLM and inactivated FANCD2 further confirm the relationship between BLM and FANCD2. Mutations within the domain VI of BLM detected in human cancer samples demonstrate the functional importance of this domain, suggesting human tumorigenicity resulting from mtBLM may be at least partly attributed to mitigated FANCD2 activation. Collectively, our data show a previously unknown regulatory liaison in advancing our understanding of how the cancer susceptibility gene products act in concert to maintain genome stability.

Purification and characterization of peptides from Capsicum annuum fruits which are α-amylase inhibitors and exhibit high antimicrobial activity against fungi of agronomic importance.

PubMed

Dos Santos, Layrana de Azevedo; Taveira, Gabriel Bonan; Ribeiro, Suzanna de Fátima Ferreira; Pereira, Lídia da Silva; Carvalho, André de Oliveira; Rodrigues, Rosana; Oliveira, Antônia Elenir Amâncio; Machado, Olga Lima Tavares; Araújo, Jucélia da Silva; Vasconcelos, Ilka Maria; Gomes, Valdirene Moreira

2017-04-01

Proteins extracted from Capsicum annuum L. fruits were initially subjected to reversed-phase chromatography on HPLC, resulting in eight peptide-rich fractions. All the fractions obtained were tested for their ability to inhibit porcine trypsin and amylase from both human saliva and from larval insect in vitro. All fractions were also tested for their ability to inhibit growth of the phytopathogenic fungi. Several fractions inhibited the activity of human salivary amylase and larval insect amylase, especially fraction Fa5. No fraction tested was found to inhibit trypsin activity, being Fa2 fraction an exception. Interestingly fraction Fa5 also displayed high antimicrobial activity against the species of the Fusarium genus. Fraction Fa5 was found to have two major protein bands of 17 and 6.5 kDa, and these were sequenced by mass spectrometry. Two peptides were obtained from the 6.5-kDa band, which showed similarity to antimicrobial peptides. Fraction Fa5 was also tested for its ability to permeabilize membranes and induce ROS. Fraction Fa5 was able to permeabilize the membranes of all the fungi tested. Fungi belonging to the genus Fusarium also showed an increase in the endogenous production of ROS when treated with this fraction. Antimicrobial peptides were also identified in the fruits from other Capsicum species. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic parameters of milk production traits and fatty acid contents in milk for Holstein cows in parity 1-3.

PubMed

Bastin, C; Soyeurt, H; Gengler, N

2013-04-01

The objective of this study was to estimate genetic parameters of milk, fat, and protein yields, fat and protein contents, somatic cell count, and 17 groups and individual milk fatty acid (FA) contents predicted by mid-infrared spectrometry for first-, second- and third-parity Holstein cows. Edited data included records collected in the Walloon region of Belgium from 37,768 cows in parity 1,22,566 cows in parity 2 and 8221 in parity 3. A total of 69 (23 traits for three parities) single-trait random regression animal test-day models were run. Approximate genetic correlations among traits were inferred from pairwise regressions among estimated breeding values of cow having observations. Heritability and genetic correlation estimates from this study reflected the origins of FA: de novo synthetized or originating from the diet and the body fat mobilization. Averaged daily heritabilities of FA contents in milk ranged between 0.18 and 0.47. Average daily genetic correlations (averaged across days in milk and parities) among groups and individual FA contents in milk ranged between 0.31 and 0.99. The genetic variability of FAs in combination with the moderate to high heritabilities indicated that FA contents in milk could be changed by genetic selection; however, desirable direction of change in these traits remains unclear and should be defined with respect to all issues of importance related to milk FA. © 2012 Blackwell Verlag GmbH.

Milk from cows grazing on cool-season pastures provides an enhanced profile of bioactive fatty acids compared to those grazed on a monoculture of pearl millet.

PubMed

Bainbridge, Melissa L; Egolf, Emily; Barlow, John W; Alvez, Juan P; Roman, Joe; Kraft, Jana

2017-02-15

The demand for dairy products from grass-fed cows is driven, in part, by their more desirable fatty acid (FA) profile, containing more n-3 FA and conjugated linoleic acids (CLA) than conventionally produced dairy products. This study investigated the effects of pearl millet (PM) vs. cool-season pasture (CSP) on animal performance and milk FA in a grazing system. Eight Holstein dairy cows were used in a repeated measures design with four-week periods. Forage type had no effect on animal performance (estimated dry matter intake, milk production, fat, or protein). The contents of CLA and n-3 FA in a serving of whole milk (3.25% fat) increased when cows grazed CSP compared to PM. A serving of whole milk from cows grazing PM had a higher content of saturated FA and branched-chain FA. In conclusion, the contents of various bioactive FA were higher in milk fat of cows grazing a CSP compared to PM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Fanconi anemia links reactive oxygen species to insulin resistance and obesity.

PubMed

Li, Jie; Sipple, Jared; Maynard, Suzette; Mehta, Parinda A; Rose, Susan R; Davies, Stella M; Pang, Qishen

2012-10-15

Insulin resistance is a hallmark of obesity and type 2 diabetes. Reactive oxygen species (ROS) have been proposed to play a causal role in insulin resistance. However, evidence linking ROS to insulin resistance in disease settings has been scant. Since both oxidative stress and diabetes have been observed in patients with the Fanconi anemia (FA), we sought to investigate the link between ROS and insulin resistance in this unique disease model. Mice deficient for the Fanconi anemia complementation group A (Fanca) or Fanconi anemia complementation group C (Fancc) gene seem to be diabetes-prone, as manifested by significant hyperglycemia and hyperinsulinemia, and rapid weight gain when fed with a high-fat diet. These phenotypic features of insulin resistance are characterized by two critical events in insulin signaling: a reduction in tyrosine phosphorylation of the insulin receptor (IR) and an increase in inhibitory serine phosphorylation of the IR substrate-1 in the liver, muscle, and fat tissues from the insulin-challenged FA mice. High levels of ROS, spontaneously accumulated or generated by tumor necrosis factor alpha in these insulin-sensitive tissues of FA mice, were shown to underlie the FA insulin resistance. Treatment of FA mice with the natural anti-oxidant Quercetin restores IR signaling and ameliorates the diabetes- and obesity-prone phenotypes. Finally, pairwise screen identifies protein-tyrosine phosphatase (PTP)-α and stress kinase double-stranded RNA-dependent protein kinase (PKR) that mediate the ROS effect on FA insulin resistance. These findings establish a pathogenic and mechanistic link between ROS and insulin resistance in a unique human disease setting. ROS accumulation contributes to the insulin resistance in FA deficiency by targeting both PTP-α and PKR.

Effects of Male Hypogonadism on Regional Adipose Tissue Fatty Acid Storage and Lipogenic Proteins

PubMed Central

Santosa, Sylvia; Jensen, Michael D.

2012-01-01

Testosterone has long been known to affect body fat distribution, although the underlying mechanisms remain elusive. We investigated the effects of chronic hypogonadism in men on adipose tissue fatty acid (FA) storage and FA storage factors. Twelve men with chronic hypogonadism and 13 control men matched for age and body composition: 1) underwent measures of body composition with dual energy x-ray absorptiometry and an abdominal CT scan; 2) consumed an experimental meal containing [3H]triolein to determine the fate of meal FA (biopsy-measured adipose storage vs. oxidation); 3) received infusions of [U-13C]palmitate and [1-14C]palmitate to measure rates of direct free (F)FA storage (adipose biopsies). Adipose tissue lipoprotein lipase, acyl-CoA synthetase (ACS), and diacylglycerol acetyl-transferase (DGAT) activities, as well as, CD36 content were measured to understand the mechanism by which alterations in fat storage occur in response to testosterone deficiency. Results of the study showed that hypogonadal men stored a greater proportion of both dietary FA and FFA in lower body subcutaneous fat than did eugonadal men (both p<0.05). Femoral adipose tissue ACS activity was significantly greater in hypogonadal than eugonadal men, whereas CD36 and DGAT were not different between the two groups. The relationships between these proteins and FA storage varied somewhat between the two groups. We conclude that chronic effects of testosterone deficiency has effects on leg adipose tissue ACS activity which may relate to greater lower body FA storage. These results provide further insight into the role of androgens in body fat distribution and adipose tissue metabolism in humans. PMID:22363653

Fanconi Anemia Links Reactive Oxygen Species to Insulin Resistance and Obesity

PubMed Central

Li, Jie; Sipple, Jared; Maynard, Suzette; Mehta, Parinda A.; Rose, Susan R.; Davies, Stella M.

2012-01-01

Abstract Aims: Insulin resistance is a hallmark of obesity and type 2 diabetes. Reactive oxygen species (ROS) have been proposed to play a causal role in insulin resistance. However, evidence linking ROS to insulin resistance in disease settings has been scant. Since both oxidative stress and diabetes have been observed in patients with the Fanconi anemia (FA), we sought to investigate the link between ROS and insulin resistance in this unique disease model. Results: Mice deficient for the Fanconi anemia complementation group A (Fanca) or Fanconi anemia complementation group C (Fancc) gene seem to be diabetes-prone, as manifested by significant hyperglycemia and hyperinsulinemia, and rapid weight gain when fed with a high-fat diet. These phenotypic features of insulin resistance are characterized by two critical events in insulin signaling: a reduction in tyrosine phosphorylation of the insulin receptor (IR) and an increase in inhibitory serine phosphorylation of the IR substrate-1 in the liver, muscle, and fat tissues from the insulin-challenged FA mice. High levels of ROS, spontaneously accumulated or generated by tumor necrosis factor alpha in these insulin-sensitive tissues of FA mice, were shown to underlie the FA insulin resistance. Treatment of FA mice with the natural anti-oxidant Quercetin restores IR signaling and ameliorates the diabetes- and obesity-prone phenotypes. Finally, pairwise screen identifies protein-tyrosine phosphatase (PTP)-α and stress kinase double-stranded RNA-dependent protein kinase (PKR) that mediate the ROS effect on FA insulin resistance. Innovation: These findings establish a pathogenic and mechanistic link between ROS and insulin resistance in a unique human disease setting. Conclusion: ROS accumulation contributes to the insulin resistance in FA deficiency by targeting both PTP-α and PKR. Antioxid. Redox Signal. 00, 000–000. PMID:22482891

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis.

PubMed

Lung, Shiu-Cheung; Liao, Pan; Yeung, Edward C; Hsiao, An-Shan; Xue, Yan; Chye, Mee-Len

2017-07-01

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis ( Arabidopsis thaliana ) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1 Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 ( GL2 ), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1 , smo1-1 , and ACBP1 +/- smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1 +/- smo1-1 Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling. © 2017 American Society of Plant Biologists. All Rights Reserved.

Exploiting Tumor Activated Testes Proteins To Enhance Efficacy of First Line Chemotherapeutics in NSCLC

DTIC Science & Technology

2016-10-01

Antigen (CTA), Fanconia- Anemia (FA), DNA Damage, Genomic Instability, DNA Double Strand Break (DSB) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...Fanconia- Anemia (FA) o DNA Damage o Genomic Instability o DNA Double Strand Break (DSB) 3. Accomplishments • What were the major goals and objectives of

Air pollution is associated with the development of atherosclerosis via the cooperation of CD36 and NLRP3 inflammasome in ApoE-/- mice.

PubMed

Du, Xihao; Jiang, Shuo; Zeng, Xuejiao; Zhang, Jia; Pan, Kun; Zhou, Ji; Xie, Yuquan; Kan, Haidong; Song, Weimin; Sun, Qinghua; Zhao, Jinzhuo

2018-06-15

Previous studies have indicated that the main air pollutant fine particulate matter (≤2.5 μm; PM 2.5 ) exposure is associated with the development of atherosclerosis. Although the mechanism is not fully illustrated, the inflammatory responses play an important role. The present study aimed to explore whether PM 2.5 -exacerbated atherosclerosis was mediated by the cooperation of cluster of differentiation 36 (CD36) and nucleotide-binding oligomerization domain-like receptor protein (NLRP3) inflammasome in apolipoprotein E -/- (ApoE -/- ) mice. Thirty-two ApoE -/- mice were randomly divided into two groups. One group was fed with high fat chow (HFC) for 10 weeks to establish atherosclerotic model, and the other was fed with normal chow (NC). From week 11, the mice were exposed to concentrated PM 2.5 (PM) or filtered air (FA) using Shanghai Meteorological and Environmental Animal Exposure System for 16 weeks. In both NC and HFC groups, PM 2.5 exposure induced the formation of atherosclerosis plaque. Similarly, PM mice appeared higher lipid content in the aortic root than that in the FA mice. Compared with the FA mice, PM mice appeared a decrease in high density lipoprotein-cholesterol (HDL-C) and apolipoprotein A1 along with an increase in apolipoprotein B, low density lipoprotein-cholesterol (LDL-C) and oxidized low-density lipoprotein (ox-LDL). Moreover, PM 2.5 exposure induced increase of CD36 in serum and aorta. In both NC and HFC groups, NLRP3 inflammasome activation-related indicators were activated or increased in the aorta of the PM mice when compared with the FA mice. The cooperation of CD36 and NLRP3 inflammasome activation may be the potential mechanisms linkixposed to concentrated PM 2.5 (PM) or filtered air (FA) using Shanghai Meteorological and Environmental Animal Exposure System for 16 weeks. In both NC and HFC groups, PM 2.5 exposure induced the formation of atherosclerosis plaque. Similarly, PM mice appeared higher lipid content in the aortic root than that in the FA mice. Compared with the FA mice, PM mice appeared a decrease in high density lipoprotein-cholesterol (HDL-C) and apolipoprotein A1 along with an increase in apolipoprotein B, low density lipoprotein-cholesterol (LDL-C) and oxidized low-density lipoprotein (ox-LDL). Moreover, PM 2.5 exposure induced increase of CD36 in serum and aorta. In both NC and HFC groups, NLRP3 inflammasome activation-related indicators were activated or increased in the aorta of the PM mice when compared with the FA mice. The cooperation of CD36 and NLRP3 inflammasome activation may be the potential mechanisms linking air pollution and HFC-induced atherosclerosis even in the mice with NC intake. Copyright © 2018. Published by Elsevier B.V.

Effect of modified atmosphere packaging for keeping gel-forming ability of lizardfish

NASA Astrophysics Data System (ADS)

Oka, Hiroyasu; Wang, Xi-Chang

In order to make clear the deterioration and its control for the gel-forming ability of lizardfish, formaldehyde (FA) and tyrosine (Tyr) accumulated in the fillets packaged with air, nitrogen (N2) and oxygen (O2) were determined during storage at 5°C. Amount of Tyr had no increase, however, th at of FA increased especially in the samples packed with N2 on the earlier periods of storage. It was presumed that an enzyme trimethylamine N-oxide demethylase (TMAOase) in the fillets acts sufficiently under oxygen-deficient storage, and the produced FA is rapidly combined with fish protein, causing the deterioration of the gel-forming ability, before protease of fillets act on the fish protein. These findings suggested that avoidance of oxygen deficiency during storage is eftective to keep gelforming ability of lizardfish.

Influence of Fat/Carbohydrate Ratio on Progression of Fatty Liver Disease and on Development of Osteopenia in Male Rats Fed Alcohol via Total Enteral Nutrition (TEN)

PubMed Central

Ronis, Martin J. J.; Mercer, Kelly; Suva, Larry J.; Vantrease, Jamie; Ferguson, Matthew; Hogue, William R.; Sharma, Neha; Cleves, Mario A.; Blackburn, Michael L.; Badger, Thomas M.

2014-01-01

Alcohol abuse is associated with the development of fatty liver disease and also with significant osteopenia in both genders. In this study, we examined ethanol-induced pathology in response to diets with differing fat/carbohydrate ratios. Male Sprague-Dawley rats were fed intragastrically with isocaloric liquid diets. Dietary fat content was either 5% (high carbohydrate, HC) or 45% (high fat, HF), with or without ethanol (12–13 g/kg/day). After 14, 28, or 65 days, livers were harvested and analyzed. In addition, bone morphology was analyzed after 65 days. HC rats gained more weight and had larger fat pads than HF rats with or without ethanol. Steatosis developed in HC + ethanol (HC+EtOH) compared to HF + ethanol (HF+EtOH) rats, accompanied by increased fatty acid (FA) synthesis and increased nuclear carbohydrate response element binding protein (ChREBP) (p < 0.05), but in the absence of effects on hepatic silent mating type information regulation 2 homolog (SIRT-1) or nuclear sterol regulatory binding element protein (SREBP-1c). Ethanol reduced serum leptin (p < 0.05) but not adiponectin. Over time, HC rats developed fatty liver independent of ethanol. FA degradation was significantly elevated by ethanol in both HC and HF groups (p < 0.05). HF+EtOH rats had increased oxidative stress from 28 days, increased necrosis compared to HF controls and higher expression of cytochromes P450, CYP2E1, and CYP4A1 compared to HC+EtOH rats (p < 0.05). In contrast, HC+EtOH rats had no significant increase in oxidative stress until day 65 with no observed increase in necrosis. Unlike liver pathology, no dietary differences were observed on ethanol-induced osteopenia in HC compared to HF groups. These data demonstrate that interactions between diet composition and alcohol are complex, dependent on the length of exposure, and are an important influence in development of fatty liver injury. Importantly, it appears that diet composition does not affect alcohol-associated skeletal toxicity. PMID:24581955

Urea free and more efficient sample preparation method for mass spectrometry based protein identification via combining the formic acid-assisted chemical cleavage and trypsin digestion.

PubMed

Wu, Shuaibin; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2011-10-30

A formic acid (FA)-assisted sample preparation method was presented for protein identification via mass spectrometry (MS). Detailedly, an aqueous solution containing 2% FA and dithiothreitol was selected to perform protein denaturation, aspartic acid (D) sites cleavage and disulfide linkages reduction simultaneously at 108°C for 2h. Subsequently, FA wiped off via vacuum concentration. Finally, iodoacetamide (IAA) alkylation and trypsin digestion could be performed ordinally. A series of model proteins (BSA, β-lactoglobulin and apo-Transferrin) were treated respectively using such method, followed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The identified peptide number was increased by ∼ 80% in comparison with the conventional urea-assisted sample preparation method. Moreover, BSA identification was achieved efficiently down to femtomole (25 ± 0 sequence coverage and 16 ± 1 peptides) via such method. In contrast, there were not peptides identified confidently via the urea-assisted method before desalination via the C18 zip tip. The absence of urea in this sample preparation method was an advantage for the more favorable digestion and MALDI-TOF MS analysis. The performances of two methods for the real sample (rat liver proteome) were also compared, followed by a nanoflow reversed-phase liquid chromatography with electrospray ionization tandem mass spectrometry system analysis. As a result, 1335 ± 43 peptides were identified confidently (false discovery rate <1%) via FA-assisted method, corresponding to 295 ± 12 proteins (of top match=1 and requiring 2 unique peptides at least). In contrast, there were only 1107 ± 16 peptides (corresponding to 231 ± 10 proteins) obtained from the conventional urea-assisted method. It was serving as a more efficient protein sample preparation method for researching specific proteomes better, and providing assistance to develop other proteomics analysis methods, such as, peptide quantitative analysis. Copyright © 2011 Elsevier B.V. All rights reserved.

Δ(9)-THC modulation of fatty acid 2-hydroxylase (FA2H) gene expression: possible involvement of induced levels of PPARα in MDA-MB-231 breast cancer cells.

PubMed

Takeda, Shuso; Ikeda, Eriko; Su, Shengzhong; Harada, Mari; Okazaki, Hiroyuki; Yoshioka, Yasushi; Nishimura, Hajime; Ishii, Hiroyuki; Kakizoe, Kazuhiro; Taniguchi, Aya; Tokuyasu, Miki; Himeno, Taichi; Watanabe, Kazuhito; Omiecinski, Curtis J; Aramaki, Hironori

2014-12-04

We recently reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), a major cannabinoid component in Cannabis Sativa (marijuana), significantly stimulated the expression of fatty acid 2-hydroxylase (FA2H) in human breast cancer MDA-MB-231 cells. Peroxisome proliferator-activated receptor α (PPARα) was previously implicated in this induction. However, the mechanisms mediating this induction have not been elucidated in detail. We performed a DNA microarray analysis of Δ(9)-THC-treated samples and showed the selective up-regulation of the PPARα isoform coupled with the induction of FA2H over the other isoforms (β and γ). Δ(9)-THC itself had no binding/activation potential to/on PPARα, and palmitic acid (PA), a PPARα ligand, exhibited no stimulatory effects on FA2H in MDA-MB-231 cells; thus, we hypothesized that the levels of PPARα induced were involved in the Δ(9)-THC-mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ(9)-THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ(9)-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ(9)-THC-induced up-regulation of FA2H was further stimulated by the co-treatment with L-663,536 (a known PPARα inducer). Taken together, these results support the concept that the induced levels of PPARα may be involved in the Δ(9)-THC up-regulation of FA2H in MDA-MB-231 cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Role of Fatty Acid Kinase in Cellular Lipid Homeostasis and SaeRS-Dependent Virulence Factor Expression in Staphylococcus aureus.

PubMed

Ericson, Megan E; Subramanian, Chitra; Frank, Matthew W; Rock, Charles O

2017-08-01

The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus , but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nonesterified FA pool in S. aureus that is elevated in strains lacking FA kinase activity. SaeRS-mediated transcription is restored in FA kinase-negative strains when the intracellular FA pool is reduced either by growth with FA-depleted bovine serum albumin to extract the FA into the medium or by the heterologous expression of Neisseria gonorrhoeae acyl-acyl carrier protein synthetase to activate FA for phospholipid synthesis. These data show that FAs act as negative regulators of SaeRS signaling, and FA kinase activates SaeRS-dependent virulence factor production by lowering inhibitory FA levels. Thus, FA kinase plays a role in cellular lipid homeostasis by activating FA for incorporation into phospholipid, and it indirectly regulates SaeRS signaling by maintaining a low intracellular FA pool. IMPORTANCE The SaeRS two-component system is a master transcriptional activator of virulence factor production in response to the host environment in S. aureus , and strains lacking FA kinase have severely attenuated SaeRS-dependent virulence factor transcription. FA kinase is required for the activation of exogenous FAs, and it plays a role in cellular lipid homeostasis by recycling cellular FAs into the phospholipid biosynthetic pathway. Activation of the sensor kinase, SaeS, is mediated by its membrane anchor domain, and the FAs which accumulate in FA kinase knockout strains are potent inhibitors of SaeS-dependent signaling. This work identifies FAs as physiological effectors for the SaeRS system and reveals a connection between cellular lipid homeostasis and the regulation of virulence factor transcription. FA kinase is widely distributed in Gram-positive bacteria, suggesting similar roles for FA kinase in these organisms. Copyright © 2017 Ericson et al.

Multi-substrate flavonol O-glucosyltransferases from strawberry (Fragaria×ananassa) achene and receptacle

PubMed Central

Griesser, Markus; Vitzthum, Florian; Fink, Barbara; Bellido, Mari Luz; Raasch, Constanze; Munoz-Blanco, Juan; Schwab, Wilfried

2008-01-01

In an effort to characterize fruit ripening-related genes functionally, two glucosyltransferases, FaGT6 and FaGT7, were cloned from a strawberry (Fragaria×ananassa) cDNA library and the full-length open reading frames were amplified by rapid amplification of cDNA ends. FaGT6 and FaGT7 were expressed heterologously as fusion proteins in Escherichia coli and target protein was purified using affinity chromatography. Both recombinant enzymes exhibited a broad substrate tolerance in vitro, accepting numerous flavonoids, hydroxycoumarins, and naphthols. FaGT6 formed 3-O-glucosides and minor amounts of 7-O-, 4′-O-, and 3′-O-monoglucosides and one diglucoside from flavonols such as quercetin. FaGT7 converted quercetin to the 3-O-glucoside and 4′-O-glucoside and minor levels of the 7- and 3′-isomers but formed no diglucoside. Gene expression studies showed that both genes are strongly expressed in achenes of small-sized green fruits, while the expression levels were generally lower in the receptacle. Significant levels of quercetin 3-O-, 7-O-, and 4′-O-glucosides, kaempferol 3-O- and 7-O-glucosides, as well as isorhamnetin 7-O-glucoside, were identified in achenes and the receptacle. In the receptacle, the expression of both genes is negatively controlled by auxin which correlates with the ripening-related gene expression in this tissue. Salicylic acid, a known signal molecule in plant defence, induces the expression of both genes. Thus, it appears that FaGT6 and FaGT7 are involved in the glucosylation of flavonols and may also participate in xenobiotic metabolism. The latter function is supported by the proven ability of strawberries to glucosylate selected unnatural substrates injected in ripe fruits. This report presents the first biochemical characterization of enzymes mainly expressed in strawberry achenes and provides the foundation of flavonoid metabolism in the seeds. PMID:18487633

Dietary fatty acid composition and the homeostatic regulation of mitochondrial phospholipid classes in red muscle of rainbow trout (Oncorhynchus mykiss).

PubMed

Martin, Nicolas; Kraffe, Edouard; Le Grand, Fabienne; Marty, Yanic; Bureau, Dominique P; Guderley, Helga

2015-01-01

Although dietary lipid quality markedly affects fatty acid (FA) composition of mitochondrial membranes from rainbow trout red muscle (Oncorhynchus mykiss), mitochondrial processes are relatively unchanged. As certain classes of phospholipids interact more intimately with membrane proteins than others, we examined whether specific phospholipid classes from these muscle mitochondria were more affected by dietary FA composition than others. To test this hypothesis, we fed trout with two diets differing only in their FA composition: Diet 1 had higher levels of 18:1n-9 and 18:2n-6 than Diet 2, while 22:6n-3 and 22:5n-6 were virtually absent from Diet 1 and high in Diet 2. After 5 months, trout fed Diet 2 had higher proportions of phosphatidylcholine (PC) and less phosphatidylethanolamine (PE) in mitochondrial membranes than those fed Diet 1. The FA composition of PC, PE and cardiolipin (CL) showed clear evidence of regulated incorporation of dietary FA. For trout fed Diet 2, 22:6n-3 was the most abundant FA in PC, PE and CL. The n-6 FA were consistently higher in all phospholipid classes of trout fed Diet 1, with shorter n-6 FA being favoured in CL than in PC and PE. Despite these marked changes in individual FA levels with diet, general characteristics such as total polyunsaturated FA, total monounsaturated FA and total saturated FA were conserved in PE and CL, confirming differential regulation of the FA composition of PC, PE and CL. The regulated changes of phospholipid classes presumably maintain critical membrane characteristics despite varying nutritional quality. We postulate that these changes aim to protect mitochondrial function. © 2014 Wiley Periodicals, Inc.

Delta-like 1/Fetal Antigen-1 (Dlk1/FA1) Is a Novel Regulator of Chondrogenic Cell Differentiation via Inhibition of the Akt Kinase-dependent Pathway*

PubMed Central

Chen, Li; Qanie, Diyako; Jafari, Abbas; Taipaleenmaki, Hanna; Jensen, Charlotte H.; Säämänen, Anna-Marja; Sanz, Maria Luisa Nueda; Laborda, Jorge; Abdallah, Basem M.; Kassem, Moustapha

2011-01-01

Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis. PMID:21724852

Association of complementation group and mutation type with clinical outcome in fanconi anemia. European Fanconi Anemia Research Group.

PubMed

Faivre, L; Guardiola, P; Lewis, C; Dokal, I; Ebell, W; Zatterale, A; Altay, C; Poole, J; Stones, D; Kwee, M L; van Weel-Sipman, M; Havenga, C; Morgan, N; de Winter, J; Digweed, M; Savoia, A; Pronk, J; de Ravel, T; Jansen, S; Joenje, H; Gluckman, E; Mathew, C G

2000-12-15

Fanconi anemia (FA) is a clinically and genetically heterogeneous disorder. Clinical care is complicated by variable age at onset and severity of hematologic symptoms. Recent advances in the molecular biology of FA have allowed us to investigate the relationship between FA genotype and the nature and severity of the clinical phenotype. Two hundred forty-five patients from all 7 known complementation groups (FA-A to FA-G) were studied. Mutations were detected in one of the cloned FANC genes in 169 patients; in the remainder the complementation group was assigned by cell fusion or Western blotting. A range of qualitative and quantitative clinical parameters was compared for each complementation group and for different classes of mutation. Significant phenotypic differences were found. FA-G patients had more severe cytopenia and a higher incidence of leukemia. Somatic abnormalities were less prevalent in FA-C, but more common in the rare groups FA-D, FA-E, and FA-F. In FA-A, patients homozygous for null mutations had an earlier onset of anemia and a higher incidence of leukemia than those with mutations producing an altered protein. In FA-C, there was a later age of onset of aplastic anemia and fewer somatic abnormalities in patients with the 322delG mutation, but there were more somatic abnormalities in patients with IVS4 + 4A --> T. This study indicates that FA patients with mutations in the FANCG gene and patients homozygous for null mutations in FANCA are high-risk groups with a poor hematologic outcome and should be considered as candidates both for frequent monitoring and early therapeutic intervention. (Blood. 2000;96:4064-4070)

Differential Ratios of Omega Fatty Acids (AA/EPA+DHA) Modulate Growth, Lipid Peroxidation and Expression of Tumor Regulatory MARBPs in Breast Cancer Cell Lines MCF7 and MDA-MB-231

PubMed Central

Mansara, Prakash P.; Deshpande, Rashmi A.; Vaidya, Milind M.; Kaul-Ghanekar, Ruchika

2015-01-01

Omega 3 (n3) and Omega 6 (n6) polyunsaturated fatty acids (PUFAs) have been reported to exhibit opposing roles in cancer progression. Our objective was to determine whether different ratios of n6/n3 (AA/EPA+DHA) FAs could modulate the cell viability, lipid peroxidation, total cellular fatty acid composition and expression of tumor regulatory Matrix Attachment Region binding proteins (MARBPs) in breast cancer cell lines and in non-cancerous, MCF10A cells. Low ratios of n6/n3 (1:2.5, 1:4, 1:5, 1:10) FA decreased the viability and growth of MDA-MB-231 and MCF7 significantly compared to the non-cancerous cells (MCF10A). Contrarily, higher n6/n3 FA (2.5:1, 4:1, 5:1, 10:1) decreased the survival of both the cancerous and non-cancerous cell types. Lower ratios of n6/n3 selectively induced LPO in the breast cancer cells whereas the higher ratios induced in both cancerous and non-cancerous cell types. Interestingly, compared to higher n6/n3 FA ratios, lower ratios increased the expression of tumor suppressor MARBP, SMAR1 and decreased the expression of tumor activator Cux/CDP in both breast cancer and non-cancerous, MCF10A cells. Low n6/n3 FAs significantly increased SMAR1 expression which resulted into activation of p21WAF1/CIP1 in MDA-MB-231 and MCF7, the increase being ratio dependent in MDA-MB-231. These results suggest that increased intake of n3 fatty acids in our diet could help both in the prevention as well as management of breast cancer. PMID:26325577

Effect of unsaturated fatty acids and triglycerides from soybeans on milk fat synthesis and biohydrogenation intermediates in dairy cattle.

PubMed

Boerman, J P; Lock, A L

2014-11-01

Increased rumen unsaturated fatty acid (FA) load is a risk factor for milk fat depression. This study evaluated if increasing the amount of unsaturated FA in the diet as triglycerides or free FA affected feed intake, yield of milk and milk components, and feed efficiency. Eighteen Holstein cows (132 ± 75 d in milk) were used in a replicated 3 × 3 Latin square design. Treatments were a control (CON) diet, or 1 of 2 unsaturated FA (UFA) treatments supplemented with either soybean oil (FA present as triglycerides; TAG treatment) or soybean FA distillate (FA present as free FA; FFA treatment). The soybean oil contained a higher concentration of cis-9 C18:1 (26.0 vs. 11.8 g/100g of FA) and lower concentrations of C16:0 (9.6 vs. 15.0 g/100g of FA) and cis-9,cis-12 C18:2 (50.5 vs. 59.1g/100g of FA) than the soybean FA distillate. The soybean oil and soybean FA distillate were included in the diet at 2% dry matter (DM) to replace soyhulls in the CON diet. Treatment periods were 21 d, with the final 4 d used for sample and data collection. The corn silage- and alfalfa silage-based diets contained 23% forage neutral detergent fiber and 17% crude protein. Total dietary FA were 2.6, 4.2, and 4.3% of diet DM for CON, FFA, and TAG treatments, respectively. Total FA intake was increased 57% for UFA treatments and was similar between FFA and TAG. The intakes of individual FA were similar, with the exception of a 24 g/d lower intake of C16:0 and a 64 g/d greater intake of cis-9 C18:1 for the TAG compared with the FFA treatment. Compared with CON, the UFA treatments decreased DM intake (1.0 kg/d) but increased milk yield (2.2 kg/d) and milk lactose concentration and yield. The UFA treatments reduced milk fat concentration, averaging 3.30, 3.18, and 3.11% for CON, FFA, and TAG treatments, respectively. Yield of milk fat, milk protein, and 3.5% fat-corrected milk remained unchanged when comparing CON with the UFA treatments. No differences existed in the yield of milk or milk components between the FFA and TAG treatments. The UFA treatments increased feed efficiency (energy-corrected milk/DM intake), averaging 1.42, 1.53, and 1.48 for CON, FFA, and TAG treatments, respectively. Although milk fat yield was not affected, the UFA treatments decreased the yield of de novo (<16-carbon) synthesized FA (40 g/d) and increased the yield of preformed (>16-carbon) FA (134 g/d). Yield of FA from both sources (16-carbon FA) was reduced by the UFA treatments but to a different extent for FFA versus TAG (72 vs. 100g/d). An increase was detected in the concentration of trans-10 C18:1 and a trend for an increase in trans-10,cis-12 C18:2 and trans-9,cis-11 C18:2 for the UFA treatments compared with CON. Under the dietary conditions tested, UFA treatments supplemented at 2% diet DM as either soybean FA distillate or soybean oil increased milk yield but did not effectively cause a reduction in milk fat yield, with preformed FA replacing de novo synthesized FA in milk fat. Further research is required to determine if the response to changes in dietary free and esterified FA concentrations is different in diets that differ in their risk for milk fat depression. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Changes in behavioural parameters, oxidative stress and neurotrophins in the brain of adult offspring induced to an animal model of schizophrenia: The effects of FA deficient or FA supplemented diet during the neurodevelopmental phase.

PubMed

Canever, L; Freire, T G; Mastella, G A; Damázio, L; Gomes, S; Fachim, I; Michels, C; Carvalho, G; Godói, A K; Peterle, B R; Gava, F F; Valvassori, S S; Budni, J; Quevedo, J; Zugno, A I

2018-05-18

A deficiency of maternal folic acid (FA) can compromise the function and development of the brain, and may produce a susceptibility to diseases such as schizophrenia (SZ) in the later life of offspring. The aim of this study was to evaluate the effects of both FA deficient and FA supplemented diets during gestation and lactation on behavioural parameters, the markers of oxidative stress and neurotrophic factors in adult offspring which had been subjected to an animal model of SZ. Female mother rats (Dam's) were separated into experimental maternal groups, which began receiving a special diet (food) consisting of the AIN-93 diet, a control diet, or an FA deficient diet during the periods of pregnancy and lactation. Dam's receiving the control diet were further subdivided into four groups: one group received only control diet, while three groups to receive supplementation with FA at different doses (5, 10 and 50 mg/kg). Adult offspring bred from the Dam's were divided into ten groups for induction of the animal model of SZ through the administration of ketamine (Ket) (25 mg/kg). After the last administration of the drug, the animals were subjected to the behavioural tests and were then euthanized. The frontal cortex (FC) and hippocampus (Hip) were then dissected for later biochemical analysis. Our data demonstrates that Ket induced the model of SZ by altering the behavioural parameters (e.g. hyperlocomotion, social impairment, deficits in the sensory-motor profile and memory damage in the adult animals); and also caused changes in the parameters of oxidative stress (lipid hydroperoxide - LPO; 8-isoprostane - 8-ISO; 4-hydroxynonenal - 4-HNE; protein carbonyl content; superoxide dismutase - SOD and catalase - CAT) as well as in the levels of neurotrophic factors (brain-derived neurotrophic factor - BDNF and nerve growth factor - NGF) particularly within the FC of adult offspring. A deficiency in maternal FA, alone or in combination with ket, was able to induce hyperlocomotion and social impairment in the offspring with increased levels of lipid and protein damage (LPO, 8-ISO, 4-HNE, carbonylation of protein) within the FC, increased activity of antioxidant enzymes (SOD and CAT) in both of the brain structures studied, and also reduced the levels of neurotrophins (BDNF and NGF), particularly within the Hip of the adult offspring. Supplementation of FA (5, 10 and 50 mg/kg) to the Dam's was mostly able to prevent the cognitive damage which was induced by Ket in the adult animals. FA (10 and 50 mg/kg) attenuated the action of Ket in the animals in relation to the biochemical parameters, proving the possible neuroprotective effect of FA in the adulthood of offspring that were subjected to the animal model of SZ. Our study indicates that the intake of maternal FA during pregnancy and lactation plays an important role, particularly in the regulation of markers of oxidative stress and neurotrophins. Copyright © 2018 Elsevier Inc. All rights reserved.

Indigenous lactobacilli strains of food and human sources reverse enteropathogenic E. coli O26:H11-induced damage in intestinal epithelial cell lines: effect on redistribution of tight junction proteins.

PubMed

Jariwala, Ruchi; Mandal, Hemanti; Bagchi, Tamishraha

2017-09-01

The aim of the study was to investigate the neutralizing effect of lactobacilli isolated from indigenous food and human sources on enteropathogenic Escherichia coli (EPEC) O26 : H11-induced epithelial barrier dysfunction in vitro. This was assessed by transepithelial electrical resistance (TEER) and permeability assays using intestinal cell lines, HT-29 and Caco-2. Furthermore, the expression and distribution of tight junction (TJ) proteins were analysed by qRT-PCR and immunofluorescence assay, respectively. The nine strains used in the study were from different species viz. Lactobacillus fermentum, Lactobacillushelveticus, Lactobacillus salivarius and Lactobacillus plantarum. All strains were able to reverse the decrease in TEER and corresponding increase in permeability across E. coli-infected monolayers. Maximum reversal was observed after 18 h [up to 93.8±2.0 % by L. rhamnosus GG followed by L. fermentum IIs11.2 (92.6±2.2 %) and L. plantarum GRI-2 (91.9±0.9 %)] of lactobacilli exposure following EPEC O26 : H11 infection. All strains were able to redistribute the TJ proteins to the cell periphery either partially or completely. Moreover, L. helveticus FA-7 was also able to significantly increase the mRNA expression of ZO-1 and claudin-1 (2.5-fold and 3.0-fold, respectively; P<0.05). The rapid reversal observed by these strains could be mostly because of the redistribution rather than increased mRNA expression of TJ proteins. In conclusion, L. helveticus FA-7, L. fermentum FA-1 and L. plantarum GRI-2 were good in all the aspects studied, and the other strains were good in some aspects. L. helveticus FA-7, L. fermentum FA-1 and L. plantarum GRI-2 can therefore be used for potential therapeutic purpose against intestinal epithelial dysfunction.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Yanwu; Wang, Xiaoxia; Hawkins, Cheryl A.

The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK {center_dot} PINCH complex (28 kDa, K{sub D} {approx} 68 nm) involving the N-terminal ankyrin repeatmore » domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication.« less

Response of MiRNA-22-3p and MiRNA-149-5p to Folate Deficiency and the Differential Regulation of MTHFR Expression in Normal and Cancerous Human Hepatocytes

PubMed Central

Li, Chao; Ni, Juan; Liu, Yao-Xian; Wang, Han; Liang, Zi-Qing; Wang, Xu

2017-01-01

Background/Aims Folic acid (FA) is a core micronutrient involved in DNA synthesis/methylation, and the metabolism of FA is responsible for genomic stability. MicroRNAs may affect gene expression during folate metabolism when cellular homeostasis is changed. This study aimed to reveal the relationship between FA deficiency and the expression of miR-22-p/miR-149-5p and the targeted regulation of miR-22-3p/miR-149-5p on the key folate metabolic gene Methylenetetrahydrofolate reductase (MTHFR). Methods Normal (HL-7702 cells) and cancerous (QGY-7703 cells) human hepatocytes were intervened in modified RPMI 1640 with FA deficiency for 21 days. The interaction between MTHFR and the tested miRNAs was verified by Dual-Luciferase Reporter Assays. The changes in the expression of miR-22-3p/miR-149-5p in response to FA deficiency were detected by Poly (A) Tailing RT-qPCR, and the expression of MTHFR at both the transcriptional and translational levels was determined by RT-qPCR and Western blotting, respectively. Result MiR-22-3p/miR-149-5p directly targeted the 3’UTR sequence of the MTHFR gene. FA deficiency led to an upregulation of miR-22-3p/miR-149-5p expression in QGY-7703/HL-7702 cells, while the transcription of MTHFR was decreased in QGY-7703 cells but elevated in HL-7702 cells. Western blotting showed that FA deficiency resulted in a decline of the MTHFR protein in QGY-7703 cells, whereas in HL-7702 cells, the MTHFR protein level remained constant. Conclusion The results suggested that miR-22-3p/miR-149-5p exert different post-transcriptional effects on MTHFR under conditions of FA deficiency in normal and cancerous human hepatocytes. The results also implied that miR-22-3p/miR-149-5p might exert anticancer effects in cases of long-term FA deficiency. PMID:28045918

Sorption of Eu(III) on humic acid or fulvic acid bound to hydrous alumina studied by SEM-EDS, XPS, TRLFS, and batch techniques.

PubMed

Tan, X L; Wang, X K; Geckeis, H; Rabung, Th

2008-09-01

To identify the effect of humic acid (HA) and fulvic acid (FA) on the sorption mechanism of Eu(III) on organic--inorganic colloids in the environment at a molecular level, surface adsorbed/ complexed Eu(III) on hydrous alumina, HA-, and FA-hydrous alumina hybrids were characterized by using X-ray photoelectron spectroscopy (XPS) and time-resolved laser fluorescence spectroscopy (TRLFS). The experiments were performed in 0.1 mol/L KNO3 or 0.1 mol/L NaClO4 under ambient conditions. The pH values were varied between 2 and 11 at a fixed Eu(III) concentration of 6.0 x 10(-7) mol/L and 4.3 x 10(-5) mol/L. The different Eu(III)/FA(HA)/hydrous alumina complexes were characterized by their fluorescence emission spectra ((5D0-F1)/ (5D0 --> 7F2)) and binding energy of Eu(III). Inner-sphere surface complexation may contribute mainly to Eu(III) sorption on hydrous alumina, and a ternary surface complex is formed at the HA/ FA-hydrous alumina hybrid surfaces. The sorption and species of Eu(III) in ternary Eu-HA/FA-hydrous alumina systems are not dominated by either HA/FA or hydrous alumina, but are dominated by both HA/FA and hydrous alumina. The results are important for understanding the sorption mechanisms and the nature of surface adsorbed Eu(III) species and trivalent chemical homologues of Eu(III) in the natural environment.

Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro

PubMed Central

Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

2010-01-01

Abstract Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell–cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase. PMID:19382912

Recombinant vaccinia/Venezuelan equine encephalitis (VEE) virus expresses VEE structural proteins.

PubMed

Kinney, R M; Esposito, J J; Johnson, B J; Roehrig, J T; Mathews, J H; Barrett, A D; Trent, D W

1988-12-01

cDNA molecules encoding the structural proteins of the virulent Trinidad donkey and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four E1-specific epitopes were demonstrated by FA.

Targeting and transport: How microtubules control focal adhesion dynamics

PubMed Central

Stehbens, Samantha

2012-01-01

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge. PMID:22908306

Deletion of Fanca or Fancd2 dysregulates Treg in mice.

PubMed

Du, Wei; Erden, Ozlem; Wilson, Andrew; Sipple, Jared M; Schick, Jonathan; Mehta, Parinda; Myers, Kasiani C; Steinbrecher, Kris A; Davies, Stella M; Pang, Qishen

2014-03-20

Fanconi anemia (FA) is a genetic disorder associated with bone marrow (BM) failure and leukemia. Recent studies demonstrate variable immune defects in FA. However, the cause for FA immunodeficiency is unknown. Here we report that deletion of Fanca or Fancd2 dysregulates the suppressive activity of regulatory T cells (Tregs), shown functionally as exacerbation of graft-vs-host disease (GVHD) in mice. Recipient mice of Fanca(-/-) or Fancd2(-/-) BM chimeras exhibited severe acute GVHD after allogeneic BM transplantation (BMT). T cells from Fanca(-/-) or Fancd2(-/-) mice induced higher GVHD lethality than those from wild-type (WT) littermates. FA Tregs possessed lower proliferative suppression potential compared with WT Tregs, as demonstrated by in vitro proliferation assay and BMT. Analysis of CD25(+)Foxp3(+) Tregs indicated that loss of Fanca or Fancd2 dysregulated Foxp3 target gene expression. Additionally, CD25(+)Foxp3(+) Tregs of Fanca(-/-) or Fancd2(-/-) mice were less efficient in suppressing the production of GVHD-associated inflammatory cytokines. Consistently, aberrant NF-κB activity was observed in infiltrated T cells from FA GVHD mice. Conditional deletion of p65 in FA Tregs decreased GVHD mortality. Our study uncovers an essential role for FA proteins in maintaining Treg homeostasis, possibly explaining, at least in part, the immune deficiency reported in some FA patients.

Opuntia ficus indica (nopal) attenuates hepatic steatosis and oxidative stress in obese Zucker (fa/fa) rats.

PubMed

Morán-Ramos, Sofía; Avila-Nava, Azalia; Tovar, Armando R; Pedraza-Chaverri, José; López-Romero, Patricia; Torres, Nimbe

2012-11-01

Nonalcoholic fatty liver disease (NAFLD) is associated with multiple factors such as obesity, insulin resistance, and oxidative stress. Nopal, a cactus plant widely consumed in the Mexican diet, is considered a functional food because of its antioxidant activity and ability to improve biomarkers of metabolic syndrome. The aim of this study was to assess the effect of nopal consumption on the development of hepatic steatosis and hepatic oxidative stress and on the regulation of genes involved in hepatic lipid metabolism. Obese Zucker (fa/fa) rats were fed a control diet or a diet containing 4% nopal for 7 wk. Rats fed the nopal-containing diet had ∼50% lower hepatic TG than the control group as well as a reduction in hepatomegaly and biomarkers of hepatocyte injury such as alanine and aspartate aminotransferases. Attenuation of hepatic steatosis by nopal consumption was accompanied by a higher serum concentration of adiponectin and a greater abundance of mRNA for genes involved in lipid oxidation and lipid export and production of carnitine palmitoyltransferase-1 and microsomal TG transfer proteins in liver. Hepatic reactive oxygen species and lipid peroxidation biomarkers were significantly lower in rats fed nopal compared with the control rats. Furthermore, rats fed the nopal diet had a lower postprandial serum insulin concentration and a greater liver phosphorylated protein kinase B (pAKT):AKT ratio in the postprandial state. This study suggests that nopal consumption attenuates hepatic steatosis by increasing fatty acid oxidation and VLDL synthesis, decreasing oxidative stress, and improving liver insulin signaling in obese Zucker (fa/fa) rats.

Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway.

PubMed

Das, Ujjal; Manna, Krishnendu; Khan, Amitava; Sinha, Mahuya; Biswas, Sushobhan; Sengupta, Aaveri; Chakraborty, Anindita; Dey, Sanjit

2017-01-01

The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50 mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10 Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.

The hemoglobins of the trematodes Fasciola hepatica and Paramphistomum epiclitum: A molecular biological, physico-chemical, kinetic, and vaccination study

PubMed Central

Dewilde, Sylvia; Ioanitescu, A. Iulia; Kiger, Laurent; Gilany, Kambiz; Marden, Michael C.; Van Doorslaer, Sabine; Vercruysse, Jozef; Pesce, Alessandra; Nardini, Marco; Bolognesi, Martino; Moens, Luc

2008-01-01

The trematode Fasciola hepatica (Fa.he.) is a common parasite of human and livestock. The hemoglobin (Hb) of Fa.he., a potential immunogen, was chosen for characterization in the search for an effective vaccine. Characterization of trematode Hbs show that they are intracellular single-domain globins with the following remarkable features: (1) Fa.he. expresses two Hb isoforms that differ at two amino acid sites (F1: 119Y/123Q; F2: 119F/123L). Both isoforms are monoacetylated at their N-termini; (2) the genes coding for Fa.he. and Paramphistomum epiclitum (Pa.ep.) Hbs are interrupted by two introns at the conserved positions B12.2 and G7.0.; (3) UV/VIS and resonance Raman spectroscopy identify the recombinant Fa.he. HbF2 as a pentacoordinated high-spin ferrous Hb; (4) electron paramagnetic resonance spectroscopy of cyano-met Fa.he. HbF2 proves that the endogenously bound imidazole has no imidazolate character; (5) the major structural determinants of the globin fold are present, they contain a TyrB10/TyrE7 residue pair on the distal side. Although such distal-site pair is a signature for high oxygen affinity, as shown for Pa.ep. Hb, the oxygen-binding rate parameters for Fa.he. Hb are intermediate between those of myoglobin and those of other trematode Hbs; (6) the three-dimensional structure of recombinant Fa.he. HbF2 from this study closely resembles the three-dimensional structure of Pa.ep. determined earlier. The set of distal-site polar interactions observed in Pa.ep. Hb is matched with small but significant structural adjustments; (7) despite the potential immunogenic character of the fluke Hb, vaccination of calves with recombinant Fa.he. HbF2 failed to promote protection against parasitic infection. PMID:18621914

Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells.

PubMed

Renaud, Emilie; Barascu, Aurelia; Rosselli, Filippo

2016-01-29

To rescue collapsed replication forks cells utilize homologous recombination (HR)-mediated mechanisms to avoid the induction of gross chromosomal abnormalities that would be generated by non-homologous end joining (NHEJ). Using DNA interstrand crosslinks as a replication barrier, we investigated how the Fanconi anemia (FA) pathway promotes HR at stalled replication forks. FA pathway inactivation results in Fanconi anemia, which is associated with a predisposition to cancer. FANCD2 monoubiquitination and assembly in subnuclear foci appear to be involved in TIP60 relocalization to the chromatin to acetylates histone H4K16 and prevents the binding of 53BP1 to its docking site, H4K20Me2. Thus, FA pathway loss-of-function results in accumulation of 53BP1, RIF1 and RAP80 at damaged chromatin, which impair DNA resection at stalled replication fork-associated DNA breaks and impede HR. Consequently, DNA repair in FA cells proceeds through the NHEJ pathway, which is likely responsible for the accumulation of chromosome abnormalities. We demonstrate that the inhibition of NHEJ or deacetylase activity rescue HR in FA cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Tailored coating of gold nanostars: rational approach to prototype of theranostic device based on SERS and photothermal effects at ultralow irradiance.

PubMed

Bassi, B; Dacarro, G; Galinetto, P; Giulotto, E; Marchesi, N; Pallavicini, P; Pascale, A; Perversi, S; Taglietti, A

2018-06-08

The last decade has come across an increasing demand for theranostic biocompatible nanodevices possessing the double ability of diagnosis and therapy. In this work, we report the design, synthesis and step-by-step characterization of rationally coated gold nanostars (GNSs) for the SERS imaging and photothermal therapy of HeLa cancer cells. The nanodevices were realized by synthesizing GNSs with a seed growth approach, coating them with a controlled mixture of thiols composed of a Raman reporter and a polyethylene glycol with a terminal amino group, and then reacting these amino groups with folic acid (FA), in order to impart selectivity towards cancer cells which overexpress folate receptors on their membranes. After a complete characterization, we demonstrate that these FA-functionalized GNSs (FA-GNSs) are able to bind selectively to the membranes of HeLa cells, acting as SERS tags and allowing SERS imaging. Moreover, we demonstrate that once bound to HeLa cell membranes, FA-GNSs exhibit photothermal effect which can be exploited to kill the same cells in vitro using laser irradiation in the NIR at a very low and safe irradiance. We thus demonstrate that the FA-GNSs designed following the described approach are an efficient prototype of theranostic nanodevices.

Tailored coating of gold nanostars: rational approach to prototype of theranostic device based on SERS and photothermal effects at ultralow irradiance

NASA Astrophysics Data System (ADS)

Bassi, B.; Dacarro, G.; Galinetto, P.; Giulotto, E.; Marchesi, N.; Pallavicini, P.; Pascale, A.; Perversi, S.; Taglietti, A.

2018-06-01

The last decade has come across an increasing demand for theranostic biocompatible nanodevices possessing the double ability of diagnosis and therapy. In this work, we report the design, synthesis and step-by-step characterization of rationally coated gold nanostars (GNSs) for the SERS imaging and photothermal therapy of HeLa cancer cells. The nanodevices were realized by synthesizing GNSs with a seed growth approach, coating them with a controlled mixture of thiols composed of a Raman reporter and a polyethylene glycol with a terminal amino group, and then reacting these amino groups with folic acid (FA), in order to impart selectivity towards cancer cells which overexpress folate receptors on their membranes. After a complete characterization, we demonstrate that these FA-functionalized GNSs (FA-GNSs) are able to bind selectively to the membranes of HeLa cells, acting as SERS tags and allowing SERS imaging. Moreover, we demonstrate that once bound to HeLa cell membranes, FA-GNSs exhibit photothermal effect which can be exploited to kill the same cells in vitro using laser irradiation in the NIR at a very low and safe irradiance. We thus demonstrate that the FA-GNSs designed following the described approach are an efficient prototype of theranostic nanodevices.

Human FAN1 promotes strand incision in 5'-flapped DNA complexed with RPA.

PubMed

Takahashi, Daisuke; Sato, Koichi; Hirayama, Emiko; Takata, Minoru; Kurumizaka, Hitoshi

2015-09-01

Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endonuclease that is considered to be involved in DNA incision at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been determined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 exhibited robust endonuclease activity with 5'-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 5'-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 5'-flapped DNA covered by RPA. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

In-situ coupling between kinase activities and protein dynamics within single focal adhesions

NASA Astrophysics Data System (ADS)

Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

2016-07-01

The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells.

Intelligent transportation systems field operational test cross-cutting study : emergency notification and response

DOT National Transportation Integrated Search

2000-08-01

Transportation is vital to the social and economic health of the nation. Surface transportation systems ? the networks of highways, local streets, bus routes, and rail lines ? are the ties that bind communities and facilitate commerce. Despite the fa...

The Evaluation of Folic Acid-Deficient or Folic Acid-Supplemented Diet in the Gestational Phase of Female Rats and in Their Adult Offspring Subjected to an Animal Model of Schizophrenia.

PubMed

Canever, L; Alves, C S V; Mastella, G; Damázio, L; Polla, J V; Citadin, S; De Luca, L A; Barcellos, A S; Garcez, M L; Quevedo, J; Budni, J; Zugno, A I

2018-03-01

Although folic acid (FA) supplementation is known to influence numerous physiological functions, especially during pregnancy, little is known about its direct effects on the mothers' health. However, this vitamin is essential for the health of the mother and for the normal growth and development of the fetus. Thus, the aim of this study was (1) to evaluate the cognitive effects and biochemical markers produced by the AIN-93 diet (control), the AIN-93 diet supplemented with different doses of FA (5, 10, and 50 mg/kg), and a FA-deficient diet during pregnancy and lactation in female mother rats (dams) and (2) to evaluate the effect of maternal diets on inflammatory parameters in the adult offspring which were subjected to an animal model of schizophrenia (SZ) induced by ketamine (Ket). Our study demonstrated through the Y-maze test that rats subjected to the FA-deficient diet showed significant deficits in spatial memory, while animals supplemented with FA (5 and 10 mg/kg) showed no deficit in spatial memory. Our results also suggest that the rats subjected to the FA-deficient diet had increased levels of carbonylated proteins in the frontal cortex and hippocampus and also increased plasma levels of homocysteine (Hcy). Folate was able to prevent cognitive impairments in the rats supplemented with FA (5 and 10 mg/kg), data which may be attributed to the antioxidant effect of the vitamin. Moreover, FA prevented protein damage and elevations in Hcy levels in the rats subjected to different doses of this vitamin (5, 10, and 50 mg/kg). We verified a significant increase of the anti-inflammatory cytokine (interleukin-4 (IL-4)) and a reduction in the plasma levels of proinflammatory cytokines (interleukin-6 (IL-6)) and TNF-α) in the dams that were subjected to the diets supplemented with FA (5, 10, and 50 mg/kg), showing the possible anti-inflammatory effects of FA during pregnancy and lactation. In general, we also found that in the adult offspring that were subjected to an animal model of SZ, FA had a protective effect in relation to the levels of IL-4, IL-6, and TNF-α, which indicates that the action of FA persisted in the adult offspring, since FA showed a lasting effect on the inflammatory response, which was similar in both the dams and their offspring. In conclusion, the importance of supplementation with FA during pregnancy and lactation should be emphasized, not only for the benefit of the offspring but also for the health of the mother. All this is due to the considerable protective effect of this vitamin against oxidative damage, cognitive impairment, hyperhomocysteinemia, immune function, and also its ability in preventing common processes in post-pregnancy stages, as well as in reducing the risks of neurodevelopmental disorders and enhancing fetal immune development.

Adaptive firefly algorithm: parameter analysis and its application.

PubMed

Cheung, Ngaam J; Ding, Xue-Ming; Shen, Hong-Bin

2014-01-01

As a nature-inspired search algorithm, firefly algorithm (FA) has several control parameters, which may have great effects on its performance. In this study, we investigate the parameter selection and adaptation strategies in a modified firefly algorithm - adaptive firefly algorithm (AdaFa). There are three strategies in AdaFa including (1) a distance-based light absorption coefficient; (2) a gray coefficient enhancing fireflies to share difference information from attractive ones efficiently; and (3) five different dynamic strategies for the randomization parameter. Promising selections of parameters in the strategies are analyzed to guarantee the efficient performance of AdaFa. AdaFa is validated over widely used benchmark functions, and the numerical experiments and statistical tests yield useful conclusions on the strategies and the parameter selections affecting the performance of AdaFa. When applied to the real-world problem - protein tertiary structure prediction, the results demonstrated improved variants can rebuild the tertiary structure with the average root mean square deviation less than 0.4Å and 1.5Å from the native constrains with noise free and 10% Gaussian white noise.

Adaptive Firefly Algorithm: Parameter Analysis and its Application

PubMed Central

Shen, Hong-Bin

2014-01-01

As a nature-inspired search algorithm, firefly algorithm (FA) has several control parameters, which may have great effects on its performance. In this study, we investigate the parameter selection and adaptation strategies in a modified firefly algorithm — adaptive firefly algorithm (AdaFa). There are three strategies in AdaFa including (1) a distance-based light absorption coefficient; (2) a gray coefficient enhancing fireflies to share difference information from attractive ones efficiently; and (3) five different dynamic strategies for the randomization parameter. Promising selections of parameters in the strategies are analyzed to guarantee the efficient performance of AdaFa. AdaFa is validated over widely used benchmark functions, and the numerical experiments and statistical tests yield useful conclusions on the strategies and the parameter selections affecting the performance of AdaFa. When applied to the real-world problem — protein tertiary structure prediction, the results demonstrated improved variants can rebuild the tertiary structure with the average root mean square deviation less than 0.4Å and 1.5Å from the native constrains with noise free and 10% Gaussian white noise. PMID:25397812

Mass Measurements of Focal Adhesions in Single Cells Using High Resolution Surface Plasmon Resonance Microscopy.

PubMed

Peterson, Alexander W; Halter, Michael; Tona, Alessandro; Plant, Anne L; Elliott, John T

2018-01-01

Surface plasmon resonance microscopy (SPRM) is a powerful label-free imaging technique with spatial resolution approaching the optical diffraction limit. The high sensitivity of SPRM to small changes in index of refraction at an interface allows imaging of dynamic protein structures within a cell. Visualization of subcellular features, such as focal adhesions (FAs), can be performed on live cells using a high numerical aperture objective lens with a digital light projector to precisely position the incident angle of the excitation light. Within the cell-substrate region of the SPRM image, punctate regions of high contrast are putatively identified as the cellular FAs. Optical parameter analysis is achieved by application of the Fresnel model to the SPRM data and resulting refractive index measurements are used to calculate protein density and mass. FAs are known to be regions of high protein density that reside at the cell-substratum interface. Comparing SPRM with fluorescence images of antibody stained for vinculin, a component in FAs, reveals similar measurements of FA size. In addition, a positive correlation between FA size and protein density is revealed by SPRM. Comparing SPRM images for two cell types reveals a distinct difference in the protein density and mass of their respective FAs. Application of SPRM to quantify mass can greatly aid monitoring basic processes that control FA mass and growth and contribute to accurate models that describe cell-extracellular interactions.

Low and high dietary folic acid levels perturb postnatal cerebellar morphology in growing rats.

PubMed

Partearroyo, Teresa; Pérez-Miguelsanz, Juliana; Peña-Melián, Ángel; Maestro-de-Las-Casas, Carmen; Úbeda, Natalia; Varela-Moreiras, Gregorio

2016-06-01

The brain is particularly sensitive to folate metabolic disturbances, because methyl groups are critical for brain functions. This study aimed to investigate the effects of different dietary levels of folic acid (FA) on postnatal cerebellar morphology, including the architecture and organisation of the various layers. A total of forty male OFA rats (a Sprague-Dawley strain), 5 weeks old, were classified into the following four dietary groups: FA deficient (0 mg/kg FA); FA supplemented (8 mg/kg FA); FA supra-supplemented (40 mg/kg FA); and control (2 mg/kg FA) (all n 10 per group). Rats were fed ad libitum for 30 d. The cerebellum was quickly removed and processed for histological and immunohistochemical analysis. Slides were immunostained for glial fibrillary acidic protein (to label Bergmann glia), calbindin (to label Purkinje cells) and NeuN (to label post-mitotic neurons). Microscopic analysis revealed two types of defect: partial disappearance of fissures and/or neuronal ectopia, primarily in supra-supplemented animals (incidence of 80 %, P≤0·01), but also in deficient and supplemented groups (incidence of 40 %, P≤0·05), compared with control animals. The primary fissure was predominantly affected, sometimes accompanied by defects in the secondary fissure. Our findings show that growing rats fed an FA-modified diet, including both deficient and supplemented diets, have an increased risk of disturbances in cerebellar corticogenesis. Defects caused by these diets may have functional consequences in later life. The present study is the first to demonstrate that cerebellar morphological defects can arise from deficient, as well as high, FA levels in the diet.

Folic Acid Targeting for Efficient Isolation and Detection of Ovarian Cancer CTCs from Human Whole Blood Based on Two-Step Binding Strategy.

PubMed

Nie, Liju; Li, Fulai; Huang, Xiaolin; Aguilar, Zoraida P; Wang, Yongqiang Andrew; Xiong, Yonghua; Fu, Fen; Xu, Hengyi

2018-04-25

Studies regarding circulating tumor cells (CTCs) have great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis. However, due to their extremely low concentration in peripheral blood, isolation and enrichment of CTCs are the key steps for early detection. To this end, targeting the folic acid receptors (FRs) on the CTC surface for capture with folic acid (FA) using bovine serum albumin (BSA)-tether for multibiotin enhancement in combination with streptavidin-coated magnetic nanoparticles (MNPs-SA) was developed for ovarian cancer CTC isolation. The streptavidin-biotin-system-mediated two-step binding strategy was shown to capture CTCs from whole blood efficiently without the need for a pretreatment process. The optimized parameters for this system exhibited an average capture efficiency of 80%, which was 25% higher than that of FA-decorated magnetic nanoparticles based on the one-step CTC separation method. Moreover, the isolated cells remained highly viable and were cultured directly without detachment from the MNPs-SA-biotin-CTC complex. Furthermore, when the system was applied for the isolation and detection of CTCs in ovarian cancer patients' peripheral blood samples, it exhibited an 80% correlation with clinical diagnostic criteria. The results indicated that FA targeting, in combination with BSA-based multibiotin enhancement magnetic nanoparticle separation, is a promising tool for CTC enrichment and detection of early-stage ovarian cancer.

Effects of dietary fat on the saturated and monounsaturated fatty acid metabolism in growing pigs.

PubMed

Raj, Stanisława; Skiba, Grzegorz; Sobol, Monika; Pastuszewska, Barbara

2017-08-01

The effect of dietary fats differing in fatty acid (FA) composition on the metabolism of saturated FA (SFA) and monounsaturated FA (MUFA) in growing pigs was investigated. The deposition of FA in the body and the fate of individual dietary FA were assessed after slaughter. Gilts with an initial body weight (BW) of 60 kg were used as experimental animals. Six pigs were slaughtered at 60 kg BW, while further 18 pigs received three isoenergetic and isonitrogen experimental diets containing linseed oil, rapeseed oil or beef tallow at 50 g/kg diet until they reached 105 kg (six pigs per group). The chemical composition and the content of FA in the whole body were determined and compared across groups. Regardless of dietary treatment, the whole body contained similar amounts of protein, fat and total FA. The total accumulation (percentage of net intake and de novo production) of SFA and MUFA was similar in all groups, but the processes of elongation and desaturation of SFA and MUFA depended upon the type of FA added to the diet. A high dietary content and intake of MUFA inhibits desaturation compared to SFA- and PUFA-rich diets, whereas a high SFA content and intake lowers elongation rate. The increasing net intake of total SFA and MUFA was associated with a lower total de novo production of these FA in the whole body of pigs.

Effect of Folic Acid and Vitamin B12 on Pemetrexed Antifolate Chemotherapy in Nutrient Lung Cancer Cells

PubMed Central

Yang, Tsung-Ying; Chang, Gee-Chen; Hsu, Shih-Lan; Huang, Yi-Rou; Chiu, Ling-Yen

2013-01-01

Pemetrexed (MTA) is a multitargeted antifolate drug approved for lung cancer therapy. Clinically, supplementation with high doses of folic acid (FA) and vitamin B12 (VB12) lowers MTA cytotoxicities. An antagonistic effect of FA/VB12 on MTA efficacy has been proposed. However, patients who receive FA/VB12 show better tolerance to MTA with improved survival. The aims of this study are to investigate the modulation of FA and VB12 on MTA drug efficacy in human nonsmall cell lung cancer (NSCLC) cell lines. The sensitivities of cells, apoptosis, and MTA-regulated proteins were characterized to determine the possible effects of high doses of FA and VB12 on MTA efficacy. MTA has the lowest efficacy under 10% serum conditions. However, supplementation with FA and VB12 individually and additively reversed the insensitivity of NSCLC cells to MTA treatment with 10% serum. The enhanced sensitivities of cells following FA/VB12 treatment were correlated with increasing apoptosis and were specific to MTA but not to 5-fluorouracil (5-FU). Enhanced sensitivity was also associated with p21WAF1/Cip1 expression level. Our results revealed no antagonistic effect of high doses of FA/VB12 on MTA efficacy in cancer cells grown in nutrient medium. Furthermore, these data may partially explain why supplementation of FA and VB12 resulted in better survival in MTA-treated patients. PMID:23984356

Minimalism in fabrication of self-organized nanogels holding both anti-cancer drug and targeting moiety.

PubMed

Kim, Sungwon; Park, Kyong Mi; Ko, Jin Young; Kwon, Ick Chan; Cho, Hyeon Geun; Kang, Dongmin; Yu, In Tag; Kim, Kwangmeyung; Na, Kun

2008-05-01

Recent researches to develop nano-carrier systems in anti-cancer drug delivery have focused on more complicated design to improve therapeutic efficacy and to reduce side effects. Although such efforts have great impact to biomedical science and engineering, the complexity has been a huddle because of clinical and economic problems. In order to overcome the problems, a simplest strategy to fabricate nano-carriers to deliver doxorubicin (DOX) was proposed in the present study. Two significant subjects (i) formation of nanoparticles loading and releasing DOX and (ii) binding specificity of them to cells, were examined. Folic acid (FA) was directly coupled with pullulan (Pul) backbone by ester linkage (FA/Pul conjugate) and the degree of substitution (DS) was varied, which were confirmed by 1H NMR and UV spectrophotometry. Light scattering results revealed that the nanogels possessed two major size distributions around 70 and 270 nm in an aqueous solution. Their critical aggregation concentrations (CACs) were less than 10 microg/mL, which are lower than general critical micelle concentrations (CMCs) of low-molecular-weight surfactants. Transmission electron microscopy (TEM) images showed well-dispersed nanogel morphology in a dried state. Depending on the DS, the nanogels showed different DOX-loading and releasing profiles. The DOX release rate from FA8/Pul (with the highest DS) for 24h was slower than that from FA4/or FA6/Pul, indicating that the FA worked as a hydrophobic moiety for drug holding. Cellular uptake of the nanogels (KB cells) was also monitored by confocal microscopy. All nanogels were internalized regardless of the DS of FA. Based on the results, the objectives of this study, to suggest a new method overcoming the complications in the drug carrier design, were successfully verified.

FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis

PubMed Central

Kato, Yasuko; Alavattam, Kris G.; Sin, Ho-Su; Meetei, Amom Ruhikanta; Pang, Qishen; Andreassen, Paul R.; Namekawa, Satoshi H.

2015-01-01

Fanconi anemia (FA) is a recessive X-linked and autosomal genetic disease associated with bone marrow failure and increased cancer, as well as severe germline defects such as hypogonadism and germ cell depletion. Although deficiencies in FA factors are commonly associated with germ cell defects, it remains unknown whether the FA pathway is involved in unique epigenetic events in germ cells. In this study, we generated Fancb mutant mice, the first mouse model of X-linked FA, and identified a novel function of the FA pathway in epigenetic regulation during mammalian gametogenesis. Fancb mutant mice were infertile and exhibited primordial germ cell (PGC) defects during embryogenesis. Further, Fancb mutation resulted in the reduction of undifferentiated spermatogonia in spermatogenesis, suggesting that FANCB regulates the maintenance of undifferentiated spermatogonia. Additionally, based on functional studies, we dissected the pathway in which FANCB functions during meiosis. The localization of FANCB on sex chromosomes is dependent on MDC1, a binding partner of H2AX phosphorylated at serine 139 (γH2AX), which initiates chromosome-wide silencing. Also, FANCB is required for FANCD2 localization during meiosis, suggesting that the role of FANCB in the activation of the FA pathway is common to both meiosis and somatic DNA damage responses. H3K9me2, a silent epigenetic mark, was decreased on sex chromosomes, whereas H3K9me3 was increased on sex chromosomes in Fancb mutant spermatocytes. Taken together, these results indicate that FANCB functions at critical stages of germ cell development and reveal a novel function of the FA pathway in the regulation of H3K9 methylation in the germline. PMID:26123487

Deletion of Fanca or Fancd2 dysregulates Treg in mice

PubMed Central

Du, Wei; Erden, Ozlem; Wilson, Andrew; Sipple, Jared M.; Schick, Jonathan; Mehta, Parinda; Myers, Kasiani C.; Steinbrecher, Kris A.; Davies, Stella M.

2014-01-01

Fanconi anemia (FA) is a genetic disorder associated with bone marrow (BM) failure and leukemia. Recent studies demonstrate variable immune defects in FA. However, the cause for FA immunodeficiency is unknown. Here we report that deletion of Fanca or Fancd2 dysregulates the suppressive activity of regulatory T cells (Tregs), shown functionally as exacerbation of graft-vs-host disease (GVHD) in mice. Recipient mice of Fanca−/− or Fancd2−/− BM chimeras exhibited severe acute GVHD after allogeneic BM transplantation (BMT). T cells from Fanca−/− or Fancd2−/− mice induced higher GVHD lethality than those from wild-type (WT) littermates. FA Tregs possessed lower proliferative suppression potential compared with WT Tregs, as demonstrated by in vitro proliferation assay and BMT. Analysis of CD25+Foxp3+ Tregs indicated that loss of Fanca or Fancd2 dysregulated Foxp3 target gene expression. Additionally, CD25+Foxp3+ Tregs of Fanca−/− or Fancd2−/− mice were less efficient in suppressing the production of GVHD-associated inflammatory cytokines. Consistently, aberrant NF-κB activity was observed in infiltrated T cells from FA GVHD mice. Conditional deletion of p65 in FA Tregs decreased GVHD mortality. Our study uncovers an essential role for FA proteins in maintaining Treg homeostasis, possibly explaining, at least in part, the immune deficiency reported in some FA patients. PMID:24501220

Fatty acid profile of the initial oral biofilm (pellicle): an in-situ study.

PubMed

Reich, Marco; Kümmerer, Klaus; Al-Ahmad, Ali; Hannig, Christian

2013-09-01

The first step of bioadhesion on dental surfaces is the formation of the acquired pellicle. This mainly acellular layer is formed instantaneously on all solid surfaces exposed to oral fluids. It is composed of proteins, glycoproteins and lipids. However, information on the lipid composition is sparse. The aim of the present study was to evaluate the fatty acid (FA) profile of the in-situ pellicle for the first time. Furthermore, the impact of rinses with safflower oil on the pellicle's FA composition was investigated. Pellicles were formed in situ on bovine enamel slabs mounted on individual upper jaw splints. The splints were carried by ten subjects over durations of 3-240 min. After comprehensive sample preparation, gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI/MS) was used in order to characterize qualitatively and quantitatively a wide range of FA (C12-C24). The relative FA profiles of the pellicle samples gained from different subjects were remarkably similar, whereas the amount of FA showed significant interindividual variability. An increase in FA in the pellicle was observed over time. The application of rinses with safflower oil resulted in an accumulation of its specific FA in the pellicle. Pellicle formation is a highly selective process that does not correlate directly with salivary composition, as shown for FA.

Acute alcohol exposure during mouse gastrulation alters lipid metabolism in placental and heart development: Folate prevention

PubMed Central

Han, Mingda

2016-01-01

Background Embryonic acute exposure to ethanol (EtOH), lithium, and homocysteine (HCy) induces cardiac defects at the time of exposure; folic acid (FA) supplementation protects normal cardiogenesis (Han et al., 2009, 2012; Serrano et al., 2010). Our hypothesis is that EtOH exposure and FA protection relate to lipid and FA metabolism during mouse cardiogenesis and placentation. Methods On the morning of conception, pregnant C57BL/6J mice were placed on either of two FA‐containing diets: a 3.3 mg health maintenance diet or a high FA diet of 10.5 mg/kg. Mice were injected a binge level of EtOH, HCy, or saline on embryonic day (E) 6.75, targeting gastrulation. On E15.5, cardiac and umbilical blood flow were examined by ultrasound. Embryonic cardiac tissues were processed for gene expression of lipid and FA metabolism; the placenta and heart tissues for neutral lipid droplets, or for medium chain acyl‐dehydrogenase (MCAD) protein. Results EtOH exposure altered lipid‐related gene expression on E7.5 in comparison to control or FA‐supplemented groups and remained altered on E15.5 similarly to changes with HCy, signifying FA deficiency. In comparison to control tissues, the lipid‐related acyl CoA dehydrogenase medium length chain gene and its protein MCAD were altered with EtOH exposure, as were neutral lipid droplet localization in the heart and placenta. Conclusion EtOH altered gene expression associated with lipid and folate metabolism, as well as neutral lipids, in the E15.5 abnormally functioning heart and placenta. In comparison to controls, the high FA diet protected the embryo and placenta from these effects allowing normal development. Birth Defects Research (Part A) 106:749–760, 2016. © 2016 The Authors Birth Defects Research Part A: Clinical and Molecular Teratology Published by Wiley Periodicals, Inc. PMID:27296863

The Elicitor Protein AsES Induces a Systemic Acquired Resistance Response Accompanied by Systemic Microbursts and Micro-Hypersensitive Responses in Fragaria ananassa.

PubMed

Hael-Conrad, Verónica; Perato, Silvia Marisa; Arias, Marta Eugenia; Martínez-Zamora, Martín Gustavo; Di Peto, Pía de Los Ángeles; Martos, Gustavo Gabriel; Castagnaro, Atilio Pedro; Díaz-Ricci, Juan Carlos; Chalfoun, Nadia Regina

2018-01-01

The elicitor AsES (Acremonium strictum elicitor subtilisin) is a 34-kDa subtilisin-like protein secreted by the opportunistic fungus Acremonium strictum. AsES activates innate immunity and confers resistance against anthracnose and gray mold diseases in strawberry plants (Fragaria × ananassa Duch.) and the last disease also in Arabidopsis. In the present work, we show that, upon AsES recognition, a cascade of defense responses is activated, including: calcium influx, biphasic oxidative burst (O 2 ⋅- and H 2 O 2 ), hypersensitive cell-death response (HR), accumulation of autofluorescent compounds, cell-wall reinforcement with callose and lignin deposition, salicylic acid accumulation, and expression of defense-related genes, such as FaPR1, FaPG1, FaMYB30, FaRBOH-D, FaRBOH-F, FaCHI23, and FaFLS. All these responses occurred following a spatial and temporal program, first induced in infiltrated leaflets (local acquired resistance), spreading out to untreated lateral leaflets, and later, to distal leaves (systemic acquired resistance). After AsES treatment, macro-HR and macro-oxidative bursts were localized in infiltrated leaflets, while micro-HRs and microbursts occurred later in untreated leaves, being confined to a single cell or a cluster of a few epidermal cells that differentiated from the surrounding ones. The differentiated cells initiated a time-dependent series of physiological and anatomical changes, evolving to idioblasts accumulating H 2 O 2 and autofluorescent compounds that blast, delivering its content into surrounding cells. This kind of systemic cell-death process in plants is described for the first time in response to a single elicitor. All data presented in this study suggest that AsES has the potential to activate a wide spectrum of biochemical and molecular defense responses in F. ananassa that may explain the induced protection toward pathogens of opposite lifestyle, like hemibiotrophic and necrotrophic fungi.

AMPKα2 deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle.

PubMed

Abbott, Marcia J; Bogachus, Lindsey D; Turcotte, Lorraine P

2011-07-01

AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple downstream signaling targets that may be triggered by increases in intracellular Ca(2+) concentration ([Ca(2+)]). The purpose of this study was to determine whether increases in intracellular [Ca(2+)] induced by caffeine act solely via AMPKα(2) and whether AMPKα(2) is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPKα(2) dominant-negative (DN) transgene mice were perfused during rest (n = 11), treatment with 3 mM caffeine (n = 10), or muscle contraction (n = 11). Time-dependent effects on glucose and FA uptake were uncovered throughout the 20-min muscle contraction perfusion period (P < 0.05). Glucose uptake rates did not increase in DN mice during muscle contraction until the last 5 min of the protocol (P < 0.05). FA uptake rates were elevated at the onset of muscle contraction and diminished by the end of the protocol in DN mice (P < 0.05). FA oxidation rates were abolished in the DN mice during muscle contraction (P < 0.05). The DN transgene had no effect on caffeine-induced FA uptake and oxidation (P > 0.05). Glucose uptake rates were blunted in caffeine-treated DN mice (P < 0.05). The DN transgene resulted in a greater use of intramuscular triglycerides as a fuel source during muscle contraction. The DN transgene did not alter caffeine- or contraction-mediated changes in the phosphorylation of Ca(2+)/calmodulin-dependent protein kinase I or ERK1/2 (P > 0.05). These data suggest that AMPKα(2) is involved in the regulation of substrate uptake in a time-dependent manner in contracting muscle but is not necessary for regulation of FA uptake and oxidation during caffeine treatment.

Identification of differentially expressed genes associated with the enhancement of X-ray susceptibility by RITA in a hypopharyngeal squamous cell carcinoma cell line (FaDu).

PubMed

Luan, Jinwei; Li, Xianglan; Guo, Rutao; Liu, Shanshan; Luo, Hongyu; You, Qingshan

2016-06-01

Next generation sequencing and bio-informatic analyses were conducted to investigate the mechanism of reactivation of p53 and induction of tumor cell apoptosis (RITA)-enhancing X-ray susceptibility in FaDu cells. The cDNA was isolated from FaDu cells treated with 0 X-ray, 8 Gy X-ray, or 8 Gy X-ray + RITA. Then, cDNA libraries were created and sequenced using next generation sequencing, and each assay was repeated twice. Subsequently, differentially expressed genes (DEGs) were identified using Cuffdiff in Cufflinks and their functions were predicted by pathway enrichment analyses. Genes that were constantly up- or down-regulated in 8 Gy X-ray-treated FaDu cells and 8 Gy X-ray + RITA-treated FaDu cells were obtained as RITA genes. Afterward, the protein-protein interaction (PPI) relationships were obtained from the STRING database and a PPI network was constructed using Cytoscape. Furthermore, ClueGO was used for pathway enrichment analysis of genes in the PPI network. Total 2,040 and 297 DEGs were identified in FaDu cells treated with 8 Gy X-ray or 8 Gy X-ray + RITA, respectively. PARP3 and NEIL1 were enriched in base excision repair, and CDK1 was enriched in p53 signaling pathway. RFC2 and EZH2 were identified as RITA genes. In the PPI network, many interaction relationships were identified (e.g., RFC2-CDK1, EZH2-CDK1 and PARP3-EZH2). ClueGO analysis showed that RFC2 and EZH2 were related to cell cycle. RFC2, EZH2, CDK1, PARP3 and NEIL1 may be associated, and together enhance the susceptibility of FaDu cells treated with RITA to the deleterious effects of X-ray.

Isolation and characterization of Chinese standard fulvic acid sub-fractions separated from forest soil by stepwise elution with pyrophosphate buffer.

PubMed

Bai, Yingchen; Wu, Fengchang; Xing, Baoshan; Meng, Wei; Shi, Guolan; Ma, Yan; Giesy, John P

2015-03-04

XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants.

Isolation and Characterization of Chinese Standard Fulvic Acid Sub-fractions Separated from Forest Soil by Stepwise Elution with Pyrophosphate Buffer

PubMed Central

Bai, Yingchen; Wu, Fengchang; Xing, Baoshan; Meng, Wei; Shi, Guolan; Ma, Yan; Giesy, John P.

2015-01-01

XAD-8 adsorption technique coupled with stepwise elution using pyrophosphate buffers with initial pH values of 3, 5, 7, 9, and 13 was developed to isolate Chinese standard fulvic acid (FA) and then separated the FA into five sub-fractions: FApH3, FApH5, FApH7, FApH9 and FApH13, respectively. Mass percentages of FApH3-FApH13 decreased from 42% to 2.5%, and the recovery ratios ranged from 99.0% to 99.5%. Earlier eluting sub-fractions contained greater proportions of carboxylic groups with greater polarity and molecular mass, and later eluting sub-fractions had greater phenolic and aliphatic content. Protein-like components, as well as amorphous and crystalline poly(methylene)-containing components were enriched using neutral and basic buffers. Three main mechanisms likely affect stepwise elution of humic components from XAD-8 resin with pyrophosphate buffers including: 1) the carboxylic-rich sub-fractions are deprotonated at lower pH values and eluted earlier, while phenolic-rich sub-fractions are deprotonated at greater pH values and eluted later. 2) protein or protein-like components can be desorbed and eluted by use of stepwise elution as progressively greater pH values exceed their isoelectric points. 3) size exclusion affects elution of FA sub-fractions. Successful isolation of FA sub-fractions will benefit exploration of the origin, structure, evolution and the investigation of interactions with environmental contaminants. PMID:25735451

Changes in amino acid profile and metal content in seeds of Cicer arietinum L. (chickpea) grown under various fly-ash amendments.

PubMed

Gupta, D K; Tripathi, R D; Rai, U N; Dwivedi, S; Mishra, Seema; Srivastava, S; Inouhe, M

2006-11-01

Seeds of Cicer arietinum L. plants are edible and a valuable source of protein. Accumulation of toxic metals in the edible part of the plant, grown in fields close to fly-ash (FA) landfills, may pose a threat to human health. In the present study, the effects of FA and its amendments with different ameliorants viz., garden soil (GS), press mud (PM) and saw dust (SD), on total soluble protein contents, amino acid composition and metal accumulation in seeds were investigated in var. CSG-8962 and var. C-235 of C. arietinum. Plants accumulated adequate amounts of essential metals viz. Fe, Cu, Zn in seeds, while the toxic metals such as Cd and Cr were taken up in smaller quantities. The accumulation of Cr and Cd was less in var. C-235 than var. CSG-8962. Amendment of FA with PM enhanced the amount of soluble protein and amino acids in both varieties and was found to be superior among all tested ameliorants. Both quantitative and qualitative analysis of amino acids showed better response in var. C-235 as compared to var. CSG-8962. Thus var. C-235 seems to be suitable for cultivation in FA contaminated areas due to more accumulation of essential metals and less accumulation of toxic metals in seeds. Application of PM may further improve the growth of plants and nutritional quality of seeds.

Evidence for complete epistasis of null mutations in murine Fanconi anemia genes Fanca and Fancg.

PubMed

van de Vrugt, Henri J; Koomen, Mireille; Bakker, Sietske; Berns, Mariska A D; Cheng, Ngan Ching; van der Valk, Martin A; de Vries, Yne; Rooimans, Martin A; Oostra, Anneke B; Hoatlin, Maureen E; Te Riele, Hein; Joenje, Hans; Arwert, Fré

2011-12-10

Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely. 2011 Elsevier B.V. All rights reserved.

The Fanconi anemia family of genes and its correlation with breast cancer susceptibility and breast cancer features.

PubMed

Barroso, E; Pita, G; Arias, J I; Menendez, P; Zamora, P; Blanco, M; Benitez, J; Ribas, G

2009-12-01

Fanconi anemia (FA) family of proteins participates in the DNA repair pathway by homologous recombination, and it is currently formed by 13 genes. Some of these proteins also confer susceptibility to hereditary breast and ovarian cancer (HBOC), since FANCD1 is the BRCA2 breast cancer susceptibility gene, and FANCN/PALB2 and FANCJ/BRIP1 explain 2% of non-BRCA1/2 HBOC families. Thus, there is an important connection between FA and BRCA pathways. In a previous case-control association study analysing FANCA, FANCD2 and FANCL, we reported an association between FANCD2 and sporadic breast cancer (BC) risk (OR = 1.35). In order to know whether variants in other FA genes could also be involved in this association, we have extended our study with the rest of FA genes and some others implicated in the BRCA pathway. We have also analyzed the correlation with survival, nodal metastasis and hormonal receptors (ER- and PR-). A total of 61 SNPs in ten FA genes (FANC-B, -C, -D1, -E, -F, -G, -I, -J, -M, -N) and five FA related genes (ATM, ATR, BRCA1, H2AX and USP1) were studied in a total of 547 consecutive and nonrelated sporadic BC cases and 552 unaffected controls from the Spanish population. Association analyses reported marginal statistically significant results with the minor allele of intronic SNPs in three genes: BRCA1, BRCA2/FANCD1, and ATM. Survival association with SNPs on FANCC and BRCA2/FANCD1 genes were also reported. Sub-group analyses revealed associations between SNPs on FANCI and ATM and nodal metastasis status and between FANCJ/BRIP1 and FANCN/PALB2 and PR- status.

TGF-Alpha Expression During Breast Tumorigenesis

DTIC Science & Technology

1999-07-01

contributors to breast tumorigenesis. Therefore, we examined TGFa expression in normal and transformed epithelium. We also examined TOFa and EGFR protein in...patient samples it is difficult to determine what effect tissue culture has on normal T(3Fa protein synthesis and processing. TOFa protein expression

2-methoxylated fatty acids in marine sponges: defense mechanism against mycobacteria?

PubMed

Carballeira, Néstor M; Cruz, Heidyleen; Kwong, Cecil D; Wan, Baojie; Franzblau, Scott

2004-07-01

A series of saturated 2-methoxylated FA having even-numbered chains with 8-14 carbons were synthesized, and their spectroscopic data are presented for the first time. The 2-methoxylated C10-C14 acids were prepared from the corresponding 2-hydroxylated FA, whereas the 2-methoxyoctanoic acid was synthesized starting with heptaldehyde. All of the methoxylated FA displayed some degree of inhibition (between 2 and 99%) of Mycobacterium tuberculosis H(37)Rv at 6.25 microg/mL. The most inhibitory FA was 2-methoxydecanoic acid, with a minimum inhibitory concentration of 200-239 microM against M. tuberculosis H(37)Rv as determined by both the microplate Alamar Blue assay and the green fluorescent protein microplate assay. These results are discussed in terms of the possible role of the 2-methoxylated FA as antimicrobial lipids produced either by marine sponges, or the associated marine symbiotic bacteria, as a defense mechanism in a highly competitive environment.

Production responses of Holstein dairy cows when fed supplemental fat containing saturated free fatty acids: a meta-analysis

PubMed Central

Hu, Wenping; Boerman, Jacquelyn P.; Aldrich, James M.

2017-01-01

Objective A meta-analysis was conducted to evaluate the effects of supplemental fat containing saturated free fatty acids (FA) on milk performance of Holstein dairy cows. Methods A database was developed from 21 studies published between 1991 and 2016 that included 502 dairy cows and a total of 29 to 30 comparisons between dietary treatment and control without fat supplementation. Only saturated free FA (>80% of total FA) was considered as the supplemental fat. Concentration of the supplemental fat was not higher than 3.5% of diet dry matter (DM). Dairy cows were offered total mixed ration, and fed individually. Statistical analysis was conducted using random- or mixed-effects models with Metafor package in R. Results Sub-group analysis showed that there were no differences in studies between randomized block design and Latin square/crossover design for dry matter intake (DMI) and milk production responses to the supplemental fat (all response variables, p≥0.344). The supplemental fat across all studies improved milk yield, milk fat concentration and yield, and milk protein yield by 1.684 kg/d (p<0.001), 0.095 percent unit (p = 0.003), 0.072 kg/d (p<0.001), and 0.036 kg/d (p<0.001), respectively, but tended to decrease milk protein concentration (mean difference = −0.022 percent unit; p = 0.063) while DMI (mean difference = 0.061 kg/d; p = 0.768) remained unchanged. The assessment of heterogeneity suggested that no substantial heterogeneity occurred among all studies for DMI and milk production responses to the supplemental fat (all response variables, I2≤24.1%; p≥0.166). Conclusion The effects of saturated free FA were quantitatively evaluated. Higher milk production and yields of milk fat and protein, with DMI remaining unchanged, indicated that saturated free FA, supplemented at ≤3.5% dietary DM from commercially available fat sources, likely improved the efficiency of milk production. Nevertheless, more studies are needed to assess the variation of production responses to different saturated free FA, either C16:0 or C18:0 alone, or in combination with potentially optimal ratio, when supplemented in dairy cow diets. PMID:28183166

Prediction of microalgae hydrothermal liquefaction products from feedstock biochemical composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leow, Shijie; Witter, John R.; Vardon, Derek R.

Hydrothermal liquefaction (HTL) uses water under elevated temperatures and pressures (200–350 °C, 5–20 MPa) to convert biomass into liquid “biocrude” oil. Despite extensive reports on factors influencing microalgae cell composition during cultivation and separate reports on HTL products linked to cell composition, the field still lacks a quantitative model to predict HTL conversion product yield and qualities from feedstock biochemical composition; the tailoring of microalgae feedstock for downstream conversion is a unique and critical aspect of microalgae biofuels that must be leveraged upon for optimization of the whole process. This study developed predictive relationships for HTL biocrude yield and othermore » conversion product characteristics based on HTL of Nannochloropsis oculata batches harvested with a wide range of compositions (23–59% dw lipids, 58–17% dw proteins, 12–22% dw carbohydrates) and a defatted batch (0% dw lipids, 75% dw proteins, 19% dw carbohydrates). HTL biocrude yield (33–68% dw) and carbon distribution (49–83%) increased in proportion to the fatty acid (FA) content. A component additivity model (predicting biocrude yield from lipid, protein, and carbohydrates) was more accurate predicting literature yields for diverse microalgae species than previous additivity models derived from model compounds. FA profiling of the biocrude product showed strong links to the initial feedstock FA profile of the lipid component, demonstrating that HTL acts as a water-based extraction process for FAs; the remainder non-FA structural components could be represented using the defatted batch. These findings were used to introduce a new FA-based model that predicts biocrude oil yields along with other critical parameters, and is capable of adjusting for the wide variations in HTL methodology and microalgae species through the defatted batch. Lastly, the FA model was linked to an upstream cultivation model (Phototrophic Process Model), providing for the first time an integrated modeling framework to overcome a critical barrier to microalgae-derived HTL biofuels and enable predictive analysis of the overall microalgal-to-biofuel process.« less

Prediction of microalgae hydrothermal liquefaction products from feedstock biochemical composition

DOE PAGES

Leow, Shijie; Witter, John R.; Vardon, Derek R.; ...

2015-05-11

Hydrothermal liquefaction (HTL) uses water under elevated temperatures and pressures (200–350 °C, 5–20 MPa) to convert biomass into liquid “biocrude” oil. Despite extensive reports on factors influencing microalgae cell composition during cultivation and separate reports on HTL products linked to cell composition, the field still lacks a quantitative model to predict HTL conversion product yield and qualities from feedstock biochemical composition; the tailoring of microalgae feedstock for downstream conversion is a unique and critical aspect of microalgae biofuels that must be leveraged upon for optimization of the whole process. This study developed predictive relationships for HTL biocrude yield and othermore » conversion product characteristics based on HTL of Nannochloropsis oculata batches harvested with a wide range of compositions (23–59% dw lipids, 58–17% dw proteins, 12–22% dw carbohydrates) and a defatted batch (0% dw lipids, 75% dw proteins, 19% dw carbohydrates). HTL biocrude yield (33–68% dw) and carbon distribution (49–83%) increased in proportion to the fatty acid (FA) content. A component additivity model (predicting biocrude yield from lipid, protein, and carbohydrates) was more accurate predicting literature yields for diverse microalgae species than previous additivity models derived from model compounds. FA profiling of the biocrude product showed strong links to the initial feedstock FA profile of the lipid component, demonstrating that HTL acts as a water-based extraction process for FAs; the remainder non-FA structural components could be represented using the defatted batch. These findings were used to introduce a new FA-based model that predicts biocrude oil yields along with other critical parameters, and is capable of adjusting for the wide variations in HTL methodology and microalgae species through the defatted batch. Lastly, the FA model was linked to an upstream cultivation model (Phototrophic Process Model), providing for the first time an integrated modeling framework to overcome a critical barrier to microalgae-derived HTL biofuels and enable predictive analysis of the overall microalgal-to-biofuel process.« less

Chitinase Expression Due to Reduction in Fusaric Acid Level in an Antagonistic Trichoderma harzianum S17TH.

PubMed

Sharma, Vivek; Bhandari, Pamita; Singh, Bikram; Bhatacharya, Amita; Shanmugam, Veerubommu

2013-06-01

To study the effect of reduction in phytotoxin level on fungal chitinases, antagonistic Trichoderma spp. were screened for their ability to reduce the level of fusaric acid (FA), the phytotoxin produced by Fusarium spp. A T. harzianum isolate S17TH was able to tolerate high levels of FA (up to 500 ppm) but was unable to reduce the toxin to a significant level (non-toxic) added to minimal synthetic broth (MSB). However, the isolate was able to reduce 400 ppm FA in the liquid medium after 7 days to a non-toxic level and displayed similar level of antagonism over the control (without FA). In studies of the effect of the reduction in FA (400 ppm) level on chitinase gene expression in PCR assays, nag1 was significantly repressed but ech42 expression was only slightly repressed. Chitinase activity was either reduced or absent in the extracellular proteins of MSB supplemented with 400 ppm FA, which could be attributed to the effect of residual FA or its breakdown products through unknown mechanisms. Selection of S17TH as a toxin insensitive isolate that could commensurate the negative effect on chitinase activity makes it a potential antagonist against Fusarium spp.

FANCD2 re-expression is associated with glioma grade and chemical inhibition of the Fanconi Anaemia pathway sensitises gliomas to chemotherapeutic agents.

PubMed

Patil, Abhijit A; Sayal, Parag; Depondt, Marie-Lise; Beveridge, Ryan D; Roylance, Anthony; Kriplani, Deepti H; Myers, Katie N; Cox, Angela; Jellinek, David; Fernando, Malee; Carroll, Thomas A; Collis, Spencer J

2014-08-15

Brain tumours kill more children and adults under 40 than any other cancer. Around half of primary brain tumours are glioblastoma multiforme (GBMs) where treatment remains a significant challenge, where survival rates have improved little over the last 40 years, thus highlighting an unmet need for the identification/development of novel therapeutic targets and agents to improve GBM treatment. Using archived and fresh glioma tissue, we show that in contrast to normal brain or benign schwannomas GBMs exhibit re-expression of FANCD2, a key protein of the Fanconi Anaemia (FA) DNA repair pathway, and possess an active FA pathway. Importantly, FANCD2 expression levels are strongly associated with tumour grade, revealing a potential exploitable therapeutic window to allow inhibition of the FA pathway in tumour cells, whilst sparing normal brain tissue. Using several small molecule inhibitors of the FA pathway in combination with isogenic FA-proficient/deficient glioma cell lines as well as primary GBM cultures, we demonstrate that inhibition of the FA pathway sensitises gliomas to the chemotherapeutic agents Temozolomide and Carmustine. Our findings therefore provide a strong rationale for the development of novel and potent inhibitors of the FA pathway to improve the treatment of GBMs, which may ultimately impact on patient outcome.

FANCD2 re-expression is associated with glioma grade and chemical inhibition of the Fanconi Anaemia pathway sensitises gliomas to chemotherapeutic agents

PubMed Central

Patil, Abhijit A.; Sayal, Parag; Depondt, Marie-Lise; Beveridge, Ryan D.; Roylance, Anthony; Kriplani, Deepti H.; Myers, Katie N.; Cox, Angela; Jellinek, David; Fernando, Malee; Carroll, Thomas A.; Collis, Spencer J.

2014-01-01

Brain tumours kill more children and adults under 40 than any other cancer. Around half of primary brain tumours are glioblastoma multiforme (GBMs) where treatment remains a significant challenge. GBM survival rates have improved little over the last 40 years, thus highlighting an unmet need for the identification/development of novel therapeutic targets and agents to improve GBM treatment. Using archived and fresh glioma tissue, we show that in contrast to normal brain or benign schwannomas GBMs exhibit re-expression of FANCD2, a key protein of the Fanconi Anaemia (FA) DNA repair pathway, and possess an active FA pathway. Importantly, FANCD2 expression levels are strongly associated with tumour grade, revealing a potential exploitable therapeutic window to allow inhibition of the FA pathway in tumour cells, whilst sparing normal brain tissue. Using several small molecule inhibitors of the FA pathway in combination with isogenic FA-proficient/deficient glioma cell lines as well as primary GBM cultures, we demonstrate that inhibition of the FA pathway sensitises gliomas to the chemotherapeutic agents Temozolomide and Carmustine. Our findings therefore provide a strong rationale for the development of novel and potent inhibitors of the FA pathway to improve the treatment of GBMs, which may ultimately impact on patient outcome. PMID:25071006

Growth performance and biochemical responses of three rice (Oryza sativa L.) cultivars grown in fly-ash amended soil.

PubMed

Dwivedi, S; Tripathi, R D; Srivastava, S; Mishra, S; Shukla, M K; Tiwari, K K; Singh, R; Rai, U N

2007-02-01

The disposal of fly-ash (FA) from coal-fired power stations causes significant economic and environmental problems. Use of such contaminated sites for crop production and use of contaminated water for irrigation not only decreases crop productivity but also poses health hazards to humans due to accumulation of toxic metals in edible grains. In the present investigation, three rice cultivars viz., Saryu-52, Sabha-5204, and Pant-4 were grown in garden soil (GS, control) and various amendments (10%, 25%, 50%, 75% and 100%) of FA for a period of 90 days and effect on growth and productivity of plant was evaluated vis-a-vis metal accumulation in the plants. The toxicity of FA at higher concentration (50%) was reflected by the reduction in photosynthetic pigments, protein and growth parameters viz., plant height, root biomass, number of tillers, grain and straw weight. However, at lower concentrations (10-25%), FA enhanced growth of the plants as evident by the increase of studied growth parameters. The cysteine and non-protein thiol (NP-SH) content showed increase in their levels up to 100% FA as compared to control, however, maximum content was found at 25% FA in Saryu-52 and Pant-4 and at 50% FA in Sabha-5204. Accumulation of Fe, Si, Cu, Zn, Mn, Ni, Cd and As was investigated in roots, leaves and seeds of the plants. Fe accumulation was maximum in all the parts of plant followed by Si and both showed more translocation to leaves while Mn, Zn, Cu, Ni and Cd showed lower accumulation and most of the metal was confined to roots in all the three cultivars. As was accumulated only in leaves and was not found to be in detectable levels in roots and seeds. The metal accumulation order in three rice cultivars was Fe > Si > Mn > Zn > Ni > Cu > Cd > As in all the plant parts. The results showed that rice varieties Saryu-52 and Sabha-5204 were more tolerant and could show improved growth and yield in lower FA application doses as compared to Pant-4. Thus, Sabha-5204 and Saryu-52 are found suitable for cultivation in FA amended agricultural soils for better crop yields.

The Effect of Coloring and Compacting Pressure Paving Block by Adding 5 Wt.% Fly Ash in The Compressive Strength

NASA Astrophysics Data System (ADS)

Nurzal; Nursyuhada, Aries

2017-12-01

This research aims based on SNI 03-0691-1996 to investigate the effect of coloring and compacting pressure with the addition of 5 wt.% fly ash (Fa) on compressive strength. Fa derived from waste material coal-fired Sijantang Sawahlunto thermal power plant. The growing production of Fa caused negative environmental impact. So, one of the solutions to overcome that effects is to use the Fa as a raw material for paving block mixture that can reduce the cost of raw material and increase its strength. Paving blocks are gray and red with 0 wt.%, 5 wt.% Fa + Pb composition. Compaction pressure variations 55, 65, 75, 85 and 95 Kg/cm2. The drying time for 35 days. Specimens were produced in the form of rectangular bar (length, L = 20 cm, width, B = 10 cm, thickness, W = 6 cm). The test results showed that the addition of 5 wt% FA has a compressive strength value higher than 0 wt%. The red color has a compressive strength lower than the gray color paving block caused the red color (Iron Oxide) is less binding at the time of mixing the material. Gray and red Paving blocks both increase in each additional compaction pressure, because the higher the compaction pressure will increase the bond between the particles so porosity is reduced increased compressive strength. The overall data, the gray paving block with the composition of 5 wt% FA at compaction pressure 95 kg/cm2 with the optimal compressive strength value of 36.1 MPa and the lowest value is found in the red color paving block at 0 wt% FA at a pressure of 55 kg/cm2 with a value of 6.5 MPa. Gray and red Color paving blocks has a compressive strength quality based on SNI 03-0691-1996.

pH-sensitive Au–BSA–DOX–FA nanocomposites for combined CT imaging and targeted drug delivery

PubMed Central

Huang, He; Yang, Da-Peng; Liu, Minghuan; Wang, Xiangsheng; Zhang, Zhiyong; Zhou, Guangdong; Liu, Wei; Cao, Yilin; Zhang, Wen Jie; Wang, Xiansong

2017-01-01

Albumin-based nanoparticles (NPs) as a drug delivery system have attracted much attention owing to their nontoxicity, non-immunogenicity, great stability and ability to bind to many therapeutic drugs. Herein, bovine serum albumin (BSA) was utilized as a template to prepare Au–BSA core/shell NPs. The outer layer BSA was subsequently conjugated with cis-aconityl doxorubicin (DOX) and folic acid (FA) to create Au–BSA–DOX–FA nanocomposites. A list of characterizations was undertaken to identify the successful conjugation of drug molecules and targeted agents. In vitro cytotoxicity using a cell counting kit-8 (CCK-8) assay indicated that Au–BSA NPs did not display obvious cytotoxicity to MGC-803 and GES-1 cells in the concentration range of 0–100 μg/mL, which can therefore be used as a safe drug delivery carrier. Furthermore, compared with free DOX, Au–BSA–DOX–FA nanocomposites exhibited a pH-sensitive drug release ability and superior antitumor activity in a drug concentration-dependent manner. In vivo computed tomography (CT) imaging experiments showed that Au–BSA–DOX–FA nanocomposites could be used as an efficient and durable CT contrast agent for targeted CT imaging of the folate receptor (FR) overexpressed in cancer tissues. In vivo antitumor experiments demonstrated that Au–BSA–DOX–FA nanocomposites have selective antitumor activity effects on FR-overexpressing tumors and no adverse effects on normal tissues and organs. In conclusion, the Au–BSA–DOX–FA nanocomposite exhibits selective targeting activity, X-ray attenuation activity and pH-sensitive drug release activity. Therefore, it can enhance CT imaging and improve the targeting therapeutic efficacy of FR-overexpressing gastric cancers. Our findings suggest that Au–BSA–DOX–FA nanocomposite is a novel drug delivery carrier and a promising candidate for cancer theranostic applications. PMID:28435261

Covalent modification of reduced graphene oxide by means of diazonium chemistry and use as a drug-delivery system.

PubMed

Wei, Guangcheng; Yan, Miaomiao; Dong, Renhao; Wang, Dong; Zhou, Xiangzhu; Chen, Jingfei; Hao, Jingcheng

2012-11-12

Under acidic conditions, reduced graphene oxide (rGO) was functionalized with p-aminobenzoic acid, which formed the diazonium ions through the diazotization with a wet-chemical method. Surfactants or stabilizers were not applied during the diazotization. After the functionalized rGO was treated through mild sonication in aqueous solution, these functionalized rGO sheets were less than two layers, which was determined by atomic force microscopy (AFM) imaging. The water solubility of functionalized rGO after the introduction of polyethyleneimine (PEI) was improved significantly; it was followed by covalent binding of folic acid (FA) molecules to the functionalized rGO to allow us to specifically target CBRH7919 cancer cells by using FA as a receptor. The loading and release behaviors of elsinochrome A (EA) and doxorubicin (DOX) on the functionalized rGO sheets were investigated. The EA loading ratio onto rGO-C(6)H(4)-CO-NH-PEI-NH-CO-FA (abbreviated rGO-PEI-FA, the weight ratio of drug loaded onto rGO-PEI-FA) was approximately 45.56 %, and that of DOX was approximately 28.62 %. It was interesting that the drug release from rGO-PEI-FA was pH- and salt-dependent. The results of cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry (FCM) assays, as well as cell morphology observations) clearly showed that the concentration of rGO-PEI-FA as the drug-delivery composite should be less than 12.5 mg L(-1). The conjugation of DOX and rGO-PEI-FA can enhance the cancer-cell apoptosis effectively and can also push the cancer cells to the vulnerable G2 phase of the cell cycle, which is most sensitive and susceptible to damage by drugs or radiation. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of a FANCE Splice Isoform in Regard to DNA Repair.

PubMed

Bouffard, Frédérick; Plourde, Karine; Bélanger, Simon; Ouellette, Geneviève; Labrie, Yvan; Durocher, Francine

2015-09-25

The FANC-BRCA DNA repair pathway is activated in response to interstrand crosslinks formed in DNA. A homozygous mutation in 1 of the 17 Fanconi anemia (FA) genes results in malfunctions of this pathway and development of FA syndrome. The integrity of this protein network is essential for good maintenance of DNA repair process and genome stability. Following the identification of an alternatively splice isoform of FANCE (Fanconi anemia complementation group E) significantly expressed in breast cancer individuals from high-risk non-BRCA1/2 families, we studied the impact of this FANCE splice isoform (FANCEΔ4) on DNA repair processes. We have demonstrated that FANCEΔ4 mRNA was efficiently translated into a functional protein and expressed in normal and breast cancer cell lines. Following treatment with the crosslinking agent mitomycin C, EUFA130 (FANCE-deficient) cells infected with FANCEΔ4 were blocked into G2/M phase, while cell survival was significantly reduced compared with FANCE-infected EUFA130 cells. In addition, FANCEΔ4 did not allow FANCD2 and FANCI monoubiquitination, which represents a crucial step of the FANC-BRCA functional pathway. As observed for FANCE wild-type protein, localization of FANCEΔ4 protein was confined to the nucleus following mitomycin C treatment. Although FANCEΔ4 protein showed interaction with FANCE, FANCEΔ4 did not support normal function of FANCE protein in this pathway and could have deleterious effects on FANCE protein activity. We have demonstrated that FANCEΔ4 seems to act as a regulator of FANCD2 protein expression level by promoting its degradation. This study highlights the importance of an efficient regulation of alternative splicing expression of FA genes for proper DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

Silver nanoparticles exhibit size-dependent differential toxicity and induce expression of syncytin-1 in FA-AML1 and MOLT-4 leukaemia cell lines.

PubMed

Alqahtani, Sultan; Promtong, Pawika; Oliver, Anthony W; He, Xiaotong T; Walker, Thomas D; Povey, Andrew; Hampson, Lynne; Hampson, Ian N

2016-11-01

Human endogenous retrovirus (HERV) sequences make up ~8% of the human genome and increased expression of some HERV proteins has been observed in various pathologies including leukaemia and multiple sclerosis. However, little is known about the function of these HERV proteins or environmental factors which regulate their expression. Silver nanoparticles (AgNPs) are used very extensively as antimicrobials and antivirals in numerous consumer products although their effect on the expression of HERV gene products is unknown. Cell proliferation and cell toxicity assays were carried out on human acute T lymphoblastic leukaemia (MOLT-4) and Fanconi anaemia associated acute myeloid leukaemia (FA-AML1) cells treated with two different sizes of AgNPs (7nm and 50nm diameter). Reverse-transcriptase polymerase chain reaction and western blotting were then used to the assess expression of HERV-W syncytin-1 mRNA and protein in these cells. FA-AML1 cells were more sensitive overall than MOLT-4 to treatment with the smaller 7nm sized AgNp's being the most toxic in these cells. MOLT-4 cell were more resistant and showed no evidence of differential toxicity to the different sized particles. Syncytin-1 mRNA and protein were induced by both 7 and 50nm AgNPs in both cell types yet with different kinetics. In summary, the observation that AgNPs induce expression of syncytin-1 in FA-AML1 and MOLT-4 cells at doses as little as 5 µg/ml is grounds for concern since this protein is up-regulated in both malignant and neurodegenerative diseases. Considering the widespread use of AgNPs in the environment it is clear that their ability to induce syncytin-1 should be investigated further in other cell types. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Dual hydrogen-bonding motifs in complexes formed between tropolone and formic acid

NASA Astrophysics Data System (ADS)

Nemchick, Deacon J.; Cohen, Michael K.; Vaccaro, Patrick H.

2016-11-01

The near-ultraviolet π*←π absorption system of weakly bound complexes formed between tropolone (TrOH) and formic acid (FA) under cryogenic free-jet expansion conditions has been interrogated by exploiting a variety of fluorescence-based laser-spectroscopic probes, with synergistic quantum-chemical calculations built upon diverse model chemistries being enlisted to unravel the structural and dynamical properties of the pertinent ground [X˜ 1A'] and excited [A˜ 1A'(" separators="π*π )] electronic states. For binary TrOH ṡ FA adducts, the presence of dual hydrogen-bond linkages gives rise to three low-lying isomers designated (in relative energy order) as INT, EXT1, and EXT2 depending on whether docking of the FA ligand to the TrOH substrate takes place internal or external to the five-membered reaction cleft of tropolone. While the symmetric double-minimum topography predicted for the INT potential surface mediates an intermolecular double proton-transfer event, the EXT1 and EXT2 structures are interconverted by an asymmetric single proton-transfer process that is TrOH-centric in nature. The A ˜ -X ˜ origin of TrOH ṡ FA at ν˜ 00=27 484 .45 cm-1 is displaced by δ ν˜ 00=+466 .76 cm-1 with respect to the analogous feature for bare tropolone and displays a hybrid type - a/b rotational contour that reflects the configuration of binding. A comprehensive analysis of vibrational landscapes supported by the optically connected X˜ 1A' and A˜ 1A'(" separators="π*π ) manifolds, including the characteristic isotopic shifts incurred by partial deuteration of the labile TrOH and FA protons, has been performed leading to the uniform assignment of numerous intermolecular (viz., modulating hydrogen-bond linkages) and intramolecular (viz., localized on monomer subunits) degrees of freedom. The holistic interpretation of all experimental and computational findings affords compelling evidence that an external-binding motif (attributed to EXT1), rather than the thermodynamically more stable cleft-bound (INT) form, was the carrier of fluorescence signals observed during the present work.

Lycopene and Apo-10′-lycopenoic Acid Have Differential Mechanisms of Protection against Hepatic Steatosis in β-Carotene-9′,10′-oxygenase Knockout Male Mice123

PubMed Central

Ip, Blanche C; Liu, Chun; Lichtenstein, Alice H; von Lintig, Johannes; Wang, Xiang-Dong

2015-01-01

Background: Nonalcoholic fatty liver disease is positively associated with obesity and cardiovascular disease risk. Apo-10′-lycopenoic acid (APO10LA), a potential oxidation product of apo-10′-lycopenal that is generated endogenously by β-carotene-9′,10′-oxygenase (BCO2) cleavage of lycopene, inhibited hepatic steatosis in BCO2-expressing mice. Objective: The present study evaluated lycopene and APO10LA effects on hepatic steatosis in mice without BCO2 expression. Methods: Male and female BCO2-knockout (BCO2-KO) mice were fed a high saturated fat diet (HSFD) with or without APO10LA (10 mg/kg diet) or lycopene (100 mg/kg diet) for 12 wk. Results: Lycopene or APO10LA supplementation reduced hepatic steatosis incidence (78% and 72%, respectively) and severity in BCO2-KO male mice. Female mice did not develop steatosis, had greater hepatic total cholesterol (3.06 vs. 2.31 mg/g tissue) and cholesteryl ester (1.58 vs. 0.86 mg/g tissue), but had lower plasma triglyceride (TG) (229 vs. 282 mg/dL) and cholesterol (97.1 vs. 119 mg/dL) than male mice. APO10LA-mitigated steatosis in males was associated with reduced hepatic total cholesterol (18%) and activated sirtuin 1 signaling, which resulted in reduced fatty acids (FAs) and TG synthesis markers [stearoyl-coenzyme A (CoA) desaturase protein, 71%; acetyl-CoA carboxylase phosphorylation, 79%; AMP-activated protein kinase phosphorylation, 67%], and elevated cholesterol efflux genes (cytochrome P450 family 7A1, 65%; ATP-binding cassette transporter G5/8, 11%). These APO10LA-mediated effects were not mimicked by lycopene supplementation. Intriguingly, steatosis inhibition by lycopene induced peroxisome proliferator–activated receptor (PPAR)α- and PPARγ-related genes in mesenteric adipose tissue (MAT) that increases mitochondrial uncoupling [cell death–inducing DNA fragmentation factor, α subunit-like effector a, 55%; PR domain-containing 16, 47%; uncoupling protein 3 (Ucp3), 55%], FA β-oxidation (PPARα, 53%; very long chain acyl-CoA dehydrogenase, 38%), and uptake (FA transport protein 4, 29%; lipoprotein lipase 43%). Expressions of 10 MAT PPAR-related genes were inversely correlated with steatosis score, suggesting that lycopene reduced steatosis by increasing MAT FA utilization. Conclusions: Our data suggest that lycopene and APO10LA inhibit HSFD-induced steatosis in BCO2-KO male mice through differential mechanisms. Sex disparity of BCO2-KO mice was observed in the outcomes of HSFD-induced liver steatosis and plasma lipids. PMID:25644347

Smart IR780 Theranostic Nanocarrier for Tumor-Specific Therapy: Hyperthermia-Mediated Bubble-Generating and Folate-Targeted Liposomes.

PubMed

Guo, Fang; Yu, Meng; Wang, Jinping; Tan, Fengping; Li, Nan

2015-09-23

The therapeutic effectiveness of chemotherapy was hampered by dose-limiting toxicity and was optimal only when tumor cells were subjected to a maximum drug exposure. The purpose of this work was to design a dual-functional thermosensitive bubble-generating liposome (BTSL) combined with conjugated targeted ligand (folate, FA) and photothermal agent (IR780), to realize enhanced therapeutic and diagnostic functions. This drug carrier was proposed to target tumor cells owing to FA-specific binding, followed by triggering drug release due to the decomposition of encapsulated ammonium bicarbonate (NH4HCO3) (generated CO2 bubbles) by being subjected to near-infrared (near-IR) laser irradiation, creating permeable defects in the lipid bilayer that rapidly release drug. In vitro temperature-triggered release study indicated the BTSL system was sensitive to heat triggering, resulting in rapid drug release under hyperthermia. For in vitro cellular uptake experiments, different results were observed on human epidermoid carcinoma cells (KB cells) and human lung cancer cells (A549 cells) due to their different (positive or negative) response to FA receptor. Furthermore, in vivo biodistribution analysis and antitumor study indicated IR780-BTSL-FA could specifically target KB tumor cells, exhibiting longer circulation time than free drug. In the pharmacodynamics experiments, IR780-BTSL-FA efficiently inhibited tumor growth in nude mice with no evident side effect to normal tissues and organs. Results of this study demonstrated that the constructed smart theranostic nanocarrier IR780-BTSL-FA might contribute to establishment of tumor-selective and effective chemotherapy.

Fractionation of fulvic acid by iron and aluminum oxides: influence on copper toxicity to Ceriodaphnia dubia

USGS Publications Warehouse

Smith, Kathleen S.; Ranville, James F.; Lesher, Emily K.; Diedrich, Daniel J.; McKnight, Diane M.; Sofield, Ruth M.

2014-01-01

This study examines the effect on aquatic copper toxicity of the chemical fractionation of fulvic acid (FA) that results from its association with iron and aluminum oxyhydroxide precipitates. Fractionated and unfractionated FAs obtained from streamwater and suspended sediment were utilized in acute Cu toxicity tests on ,i>Ceriodaphnia dubia. Toxicity test results with equal FA concentrations (6 mg FA/L) show that the fractionated dissolved FA was 3 times less effective at reducing Cu toxicity (EC50 13 ± 0.6 μg Cu/L) than were the unfractionated dissolved FAs (EC50 39 ± 0.4 and 41 ± 1.2 μg Cu/L). The fractionation is a consequence of preferential sorption of molecules having strong metal-binding (more aromatic) moieties to precipitating Fe- and Al-rich oxyhydroxides, causing the remaining dissolved FA to be depleted in these functional groups. As a result, there is more bioavailable dissolved Cu in the water and hence greater potential for Cu toxicity to aquatic organisms. In predicting Cu toxicity, biotic ligand models (BLMs) take into account dissolved organic carbon (DOC) concentration; however, unless DOC characteristics are accounted for, model predictions can underestimate acute Cu toxicity for water containing fractionated dissolved FA. This may have implications for water-quality criteria in systems containing Fe- and Al-rich sediment, and in mined and mineralized areas in particular. Optical measurements, such as specific ultraviolet absorbance at 254 nm (SUVA254), show promise for use as spectral indicators of DOC chemical fractionation and inferred increased Cu toxicity.

A combined behavioral and morphological study on the effects of fetal asphyxia on the nigrostriatal dopaminergic system in adult rats.

PubMed

Strackx, E; Van den Hove, D L A; Steinbusch, H P; Steinbusch, H W M; Vles, J S H; Blanco, C E; Gavilanes, A W D

2008-06-01

Fetal asphyxic insults in the brain are known to be associated with developmental neurological problems like neuromotor disorders. However, little is known about the long-term consequences of fetal asphyxia (FA). For that reason, the present study investigated the long-term effects of FA on motor behavior and dopaminergic circuitry. FA was induced at embryonic day 17 by 75-minute clamping of the uterine circulation. SHAM animals underwent the same procedure except for the clamping. This was followed by full-term vaginal delivery of animals in all groups (FA, SHAM and untreated controls). At 6 months, basal and amphetamine-induced locomotor activity was measured during open field testing. Brain sections were stained for tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP). TH-positive cells and GFAP-positive cells in substantia nigra pars compacta (SN(C)) and striatum were counted using design-based stereology. Moreover, TH-immunoreactivity in the striatum was assessed by grey value measurements. Behavioral analysis demonstrated that SHAM and FA showed less basal and amphetamine-induced activity than controls. Histochemically, FA decreased the number of TH-positive neurons in the SN(C) and lowered TH-positive in the striatum. Furthermore, more GFAP-positive cells were found in the SN(C) and striatum in FA than in either control and SHAM groups. Additionally, FA animals showed ventriculomegaly associated with smaller white matter as well as grey matter volumes. The data show that FA was associated with deficits in both dopamine-related motor behavior and biochemistry. These alterations were associated with nigrostriatal astrogliosis. The present study demonstrates the sensitivity of the dopaminergic system towards FA.

A genome-wide association study of red-blood cell fatty acids and ratios incorporating dietary covariates: Framingham Heart Study Offspring Cohort.

PubMed

Kalsbeek, Anya; Veenstra, Jenna; Westra, Jason; Disselkoen, Craig; Koch, Kristin; McKenzie, Katelyn A; O'Bott, Jacob; Vander Woude, Jason; Fischer, Karen; Shearer, Greg C; Harris, William S; Tintle, Nathan L

2018-01-01

Recent analyses have suggested a strong heritable component to circulating fatty acid (FA) levels; however, only a limited number of genes have been identified which associate with FA levels. In order to expand upon a previous genome wide association study done on participants in the Framingham Heart Study Offspring Cohort and FA levels, we used data from 2,400 of these individuals for whom red blood cell FA profiles, dietary information and genotypes are available, and then conducted a genome-wide evaluation of potential genetic variants associated with 22 FAs and 15 FA ratios, after adjusting for relevant dietary covariates. Our analysis found nine previously identified loci associated with FA levels (FADS, ELOVL2, PCOLCE2, LPCAT3, AGPAT4, NTAN1/PDXDC1, PKD2L1, HBS1L/MYB and RAB3GAP1/MCM6), while identifying four novel loci. The latter include an association between variants in CALN1 (Chromosome 7) and eicosapentaenoic acid (EPA), DHRS4L2 (Chromosome 14) and a FA ratio measuring delta-9-desaturase activity, as well as two loci associated with less well understood proteins. Thus, the inclusion of dietary covariates had a modest impact, helping to uncover four additional loci. While genome-wide association studies continue to uncover additional genes associated with circulating FA levels, much of the heritable risk is yet to be explained, suggesting the potential role of rare genetic variation, epistasis and gene-environment interactions on FA levels as well. Further studies are needed to continue to understand the complex genetic picture of FA metabolism and synthesis.

A genome-wide association study of red-blood cell fatty acids and ratios incorporating dietary covariates: Framingham Heart Study Offspring Cohort

PubMed Central

Veenstra, Jenna; Westra, Jason; Disselkoen, Craig; Koch, Kristin; McKenzie, Katelyn A.; O’Bott, Jacob; Vander Woude, Jason; Fischer, Karen; Shearer, Greg C.; Harris, William S.; Tintle, Nathan L.

2018-01-01

Recent analyses have suggested a strong heritable component to circulating fatty acid (FA) levels; however, only a limited number of genes have been identified which associate with FA levels. In order to expand upon a previous genome wide association study done on participants in the Framingham Heart Study Offspring Cohort and FA levels, we used data from 2,400 of these individuals for whom red blood cell FA profiles, dietary information and genotypes are available, and then conducted a genome-wide evaluation of potential genetic variants associated with 22 FAs and 15 FA ratios, after adjusting for relevant dietary covariates. Our analysis found nine previously identified loci associated with FA levels (FADS, ELOVL2, PCOLCE2, LPCAT3, AGPAT4, NTAN1/PDXDC1, PKD2L1, HBS1L/MYB and RAB3GAP1/MCM6), while identifying four novel loci. The latter include an association between variants in CALN1 (Chromosome 7) and eicosapentaenoic acid (EPA), DHRS4L2 (Chromosome 14) and a FA ratio measuring delta-9-desaturase activity, as well as two loci associated with less well understood proteins. Thus, the inclusion of dietary covariates had a modest impact, helping to uncover four additional loci. While genome-wide association studies continue to uncover additional genes associated with circulating FA levels, much of the heritable risk is yet to be explained, suggesting the potential role of rare genetic variation, epistasis and gene-environment interactions on FA levels as well. Further studies are needed to continue to understand the complex genetic picture of FA metabolism and synthesis. PMID:29652918

Studies on potential effects of fumaric acid on rumen microbial fermentation, methane production and microbial community.

PubMed

Riede, Susanne; Boguhn, Jeannette; Breves, Gerhard

2013-01-01

The greenhouse gas methane (CH4) contributes substantially to global climate change. As a potential approach to decrease ruminal methanogenesis, the effects of different dosages of fumaric acid (FA) on ruminal microbial metabolism and on the microbial community (archaea, bacteria) were studied using a rumen simulation technique (RUSITEC). FA acts as alternative hydrogen acceptor diverting 2H from methanogenesis of archaea towards propionate formation of bacteria. Three identical trials were conducted with 12 fermentation vessels over a period of 14 days. In each trial, four fermentation vessels were assigned to one of the three treatment groups differing in FA dosage: low fumaric acid (LFA), high fumaric acid (HFA) and without FA (control). FA was continuously infused with the buffer. Grass silage and concentrate served as substrate. FA led to decreases in pH and to higher production rates of total short chain fatty acids (SCFA) mediated by increases in propionate for LFA of 1.69 mmol d(-1) and in propionate and acetate production for HFA of 4.49 and 1.10 mmol d(-1), respectively. Concentrations of NH3-N, microbial crude protein synthesis, their efficiency, degradation of crude nutrients and detergent fibre fraction were unchanged. Total gas and CH4 production were not affected by FA. Effects of FA on structure of microbial community by means of single strand conformation polymorphism (SSCP) analyses could not be detected. Given the observed increase in propionate production and the unaffected CH4 production it can be supposed that the availability of reduction equivalents like 2H was not limited by the addition of FA in this study. It has to be concluded from the present study that the application of FA is not an appropriate approach to decrease the ruminal CH4 production.

Preparation and characterization of (-)-Epigallocatechin-3-gallate (EGCG)-loaded nanoparticles and their inhibitory effects on Human breast cancer MCF-7 cells.

PubMed

Zeng, Liang; Yan, Jingna; Luo, Liyong; Ma, Mengjun; Zhu, Huiqun

2017-03-28

We were employing nanotechnology to improve the targeting ability of (-)-Epigallocatechin-3-gallate (EGCG) towards MCF-7 cells, and two kinds of EGCG nanoparticles (FA-NPS-PEG and FA-PEG-NPS) were obtained, besides, their characteristics and effects on MCF-7 cells were studied. The results indicated that (i) both FA-NPS-PEG and FA-PEG-NPS have high stabilities; (ii) their particles sizes were 185.0 ± 13.5 nm and 142.7 ± 7.2 nm, respectively; (iii) their encapsulation efficiencies of EGCG were 90.36 ± 2.20% and 39.79 ± 7.54%, respectively. (iv) there was no cytotoxicity observed in EGCG, FA-NPS-PEG and FA-PEG-NPS toward MCF-7 cells over all concentrations (0~400 μg/mL) tested; (v) EGCG, FA-NPS-PEG and FA-PEG-NPS inhibited MCF-7 cells proliferation in dose-dependent manners, with the average IC 50 of 470.5 ± 33.0, 65.9 ± 0.4 and 66.6 ± 0.6 μg/mL; (vi) EGCG, FA-NPS-PEG and FA-PEG-NPS could modulated the expressions of several key regulatory proteins in PI3K-Akt pathway such as up-regulation of PTEN, p21 and Bax, and down-regulation of p-PDK1, p-AKT, CyclinD1 and Bcl-2, which gave an illustration about the mechanism by which EGCG nanoparticles inhibited MCF-7 cells proliferation. In this study, EGCG nanoparticles can significantly enhance the targeting ability and efficacy of EGCG, which is considered to an experimental foundation for further research on its activity, targeting ability and metabolism in vivo.

The Fanconi Anemia Pathway: Repairing the Link Between DNA Damage and Squamous Cell Carcinoma

PubMed Central

Romick-Rosendale, Lindsey E.; Lui, Vivian W. Y.; Grandis, Jennifer R.; Wells, Susanne I.

2013-01-01

Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in one of fifteen genes known to encode FA pathway components. In response to DNA damage, nuclear FA proteins associate into high molecular weight complexes through a cascade of post-translational modifications and physical interactions, followed by the repair of damaged DNA. Hematopoietic cells are particularly sensitive to the loss of these interactions, and bone marrow failure occurs almost universally in FA patients. FA as a disease is further characterized by cancer susceptibility, which highlights the importance of the FA pathway in tumor suppression, and will be the focus of this review. Acute myeloid leukemia is the most common cancer type, often subsequent to bone marrow failure. However, FA patients are also at an extreme risk of squamous cell carcinoma (SCC) of the head and neck and gynecological tract, with an even greater incidence in those individuals who have received a bone marrow transplant and recovered from hematopoietic disease. FA tumor suppression in hematopoietic versus epithelial compartments could be mechanistically similar or distinct. Definition of compartment specific FA activities is now critical to assess the effects of today’s bone marrow failure treatments on tomorrow’s solid tumor development. It is our hope that current therapies can then be optimized to decrease the risk of malignant transformation in both hematopoietic and epithelial cells. Here we review our current understanding of the mechanisms of action of the Fanconi anemia pathway as it contributes to stress responses, DNA repair and squamous cell carcinoma susceptibility. PMID:23333482

Identification of novel target genes involved in Indian Fanconi anemia patients using microarray.

PubMed

Shyamsunder, Pavithra; Ganesh, Kripa S; Vidyasekar, Prasanna; Mohan, Sheila; Verma, Rama Shanker

2013-12-01

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure and a predisposition to cancers. Mutations have been documented in 15 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its characteristic symptoms. Certain symptoms such as oxygen sensitivity, hematological abnormalities and impaired immunity suggest that FA proteins could participate in or independently control other pathways as well. In this study, we identified 9 DNA repair genes that were down regulated in a genome wide analysis of 6 Indian Fanconi anemia patients. Functional clustering of a total of 233 dysregulated genes identified key biological processes that included regulation of transcription, DNA repair, cell cycle and chromosomal organization. Microarray data revealed the down regulation of ATXN3, ARID4A and ETS-1, which were validated by RTPCR in a subsequent sample set of 9 Indian FA patients. Here we report for the first time a gene expression profile of Fanconi anemia patients from the Indian population and a pool of genes that might aid in the acquisition and progression of the FA phenotype. © 2013 Elsevier B.V. All rights reserved.

The Fanconi anemia pathway and ICL repair: implications for cancer therapy

PubMed Central

Wang, Lily C; Gautier, Jean

2011-01-01

Fanconi anemia (FA) is an inherited disease caused by mutations in at least 13 genes and characterized by genomic instability. In addition to displaying strikingly heterogenous clinical phenotypes, FA patients are exquisitely sensitive to treatments with crosslinking agents that create interstrand crosslinks (ICL). In contrast to bacteria and yeast, in which ICLs are repaired through replication-dependent and –independent mechanisms, it is thought that ICLs are repaired primarily during DNA replication in vertebrates (Moldovan and D’Andrea, 2009). However, recent data indicate that replication-independent ICL repair also operates in vertebrates. While the precise role of the FA pathway in ICL repair remains elusive, increasing evidence suggests that FA proteins function at different steps in the sensing, recognition and processing of ICLs, as well as in signaling from these very toxic lesions, which can be generated by a wide variety of cancer chemotherapeutic drugs. Here, we discuss some of the recent findings that have shed light on the role of the FA pathway in ICL repair with special emphasis on the implications of these findings for cancer therapy since disruption of FA genes have been associated with cancer predisposition. PMID:20807115

Characterization of isolated fractions of dissolved organic matter derived from municipal solid waste compost.

PubMed

Yu, Minda; He, Xiaosong; Liu, Jiaomei; Wang, Yuefeng; Xi, Beidou; Li, Dan; Zhang, Hui; Yang, Chao

2018-04-14

Understanding the heterogeneous evolution characteristics of dissolved organic matter fractions derived from compost is crucial to exploring the composting biodegradation process and the possible applications of compost products. Herein, two-dimensional correlation spectroscopy integrated with reversed-phase high performance liquid chromatography and size exclusion chromatography were utilized to obtain the molecular weight (MW) and polarity evolution characteristics of humic acid (HA), fulvic acid (FA), and the hydrophilic (HyI) fractions during composting. The high-MW humic substances and building blocks in the HA fraction degraded faster during composting than polymers, proteins, and organic colloids. Similarly, the low MW acid FA factions transformed faster than the low weight neutral fractions, followed by building blocks, and finally polymers, proteins, and organic colloids. The evolutions of HyI fractions during composting occurred first for building blocks, followed by low MW acids, and finally low weight neutrals. With the progress of composting, the hydrophobic properties of the HA and FA fractions were enhanced. The degradation/humification process of the hydrophilic and transphilic components was faster than that of the hydrophobic component. Compared with the FA and HyI fractions, the HA fraction exhibited a higher MW and increased hydrophobicity. Copyright © 2018 Elsevier B.V. All rights reserved.

The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway.

PubMed

Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D; Kee, Younghoon

2014-03-07

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.

The Carboxyl Terminus of FANCE Recruits FANCD2 to the Fanconi Anemia (FA) E3 Ligase Complex to Promote the FA DNA Repair Pathway*

PubMed Central

Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D.; Kee, Younghoon

2014-01-01

Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair. PMID:24451376

Differential protection of Cry1Fa toxin against Spodoptera frugiperda larval gut proteases by cadherin orthologs correlates with increased synergism.

PubMed

Rahman, Khalidur; Abdullah, Mohd Amir F; Ambati, Suresh; Taylor, Milton D; Adang, Michael J

2012-01-01

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin.

The impact of human breast milk components on the infant metabolism

PubMed Central

Hellmuth, Christian; Uhl, Olaf; Demmelmair, Hans; Grunewald, Maria; Auricchio, Renata; Castillejo, Gemma; Korponay-Szabo, Ilma R.; Polanco, Isabel; Roca, María; Vriezinga, Sabine L.; Werkstetter, Katharina J.; Koletzko, Berthold; Mearin, M. Luisa

2018-01-01

Background & aims Breastfeeding is beneficial for mothers and infants. Underlying mechanisms and biochemical mediators thus need to be investigated to develop and support improved infant nutrition practices promoting the child health. We analysed the relation between maternal breast milk composition and infant metabolism. Methods 196 pairs of mothers and infants from a European research project (PreventCD) were studied. Maternal milk samples collected at month 1 and month 4 after birth were analysed for macronutrient classes, hormone, and fatty acid (FA) content. Phospholipids, acylcarnitines, and amino acids were measured in serum samples of 4-month old infants. Associations between milk components and infant metabolites were analysed with spearman correlation and linear mixed effect models (LME). P-values were corrected for multiple testing (PLME). Results Month 1 milk protein content was strongly associated with infant serum lyso-phosphatidylcholine (LPC) 14:0 (PLME = 0.009). Month 1 milk insulin was associated to infant acetylcarnitine (PLME = 0.01). There were no associations between milk protein content and serum amino acids and milk total fat content and serum polar lipids. Middle- and odd-chain FA% in breast milk at both ages were significantly related to serum LPC and sphingomyelins (SM) species in infant serum (all PLME<0.05), while FA% 20:5n-3 and 22:6n-3 percentages were significantly associated to serum LPC 22:6 (PLME = 1.91×10−4/7.93×10−5) in milk only at month 4. Other polyunsaturated fatty acids and hormones in milk showed only weak associations with infant serum metabolites. Conclusions Infant serum LPC are influenced by breast milk FA composition and, intriguingly, milk protein content in early but not late lactation. LPC 14:0, previously found positively associated with obesity risk, was the serum metabolite which was the most strongly associated to milk protein content. Thus, LPC 14:0 might be a key metabolite not only reflecting milk protein intake in infants, but also relating high protein content in milk or infant formula to childhood obesity risk. PMID:29856767

Diamagnetic levitation causes changes in the morphology, cytoskeleton, and focal adhesion proteins expression in osteocytes.

PubMed

Qian, A R; Wang, L; Gao, X; Zhang, W; Hu, L F; Han, J; Li, J B; Di, S M; Shang, Peng

2012-01-01

Diamagnetic levitation technology is a novel simulated weightless technique and has recently been applied in life-science research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely, μg (diamagnetic levitation), 1g, and 2g for diamagnetic materials. In this study, the effects of LG-HMF on the activity, morphology, and cytoskeleton (actin filament, microtubules, and vimentin intermediate filaments) in osteocyte - like cell line MLO-Y4 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, hematoxylin-eosin (HE) staining, and laser scanning confocal microscopy (LSCM), respectively. The changes induced by LG-HMF in distribution and expression of focal adhesion (FA) proteins, including vinculin, paxillin, and talin in MLO-Y4 were determined by LSCM and Western blotting. The results showed that LG-HMF produced by superconducting magnet had no lethal effects on MLO-Y4. Compared to control, diamagnetic levitation (μg) affected MLO-Y4 morphology, nucleus size, cytoskeleton architecture, and FA proteins distribution and expression. The study indicates that osteocytes are sensitive to altered gravity and FA proteins (vinculin, paxillin, and talin) may be involved in osteocyte mechanosensation. The diamagnetic levitation may be a novel ground-based space-gravity simulator and can be used for biological experiment at cellular level. © 2011 IEEE

Diagnosis of Fanconi Anemia by Diepoxybutane Analysis

PubMed Central

Auerbach, Arleen D.

2015-01-01

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous disorder characterized by congenital malformations, progressive bone marrow failure, and predisposition to cancer, particularly hematological malignancies and solid tumors of the head and neck. The main role of FA proteins is in the repair of DNA interstrand crosslinks (ICLs). FA results from pathogenic variants in at least 16 distinct genes, causing genomic instability. Although the highly variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, diagnosis based on a profound sensitivity to DNA crosslinking agents can be used to identify the pre-anemia patient as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with the syndrome. Diepoxybutane (DEB) analysis is the preferred test for FA because other agents have higher rates of false-positive and false-negative results. PMID:25827349

Influence of fat/carbohydrate ratio on progression of fatty liver disease and on development of osteopenia in male rats fed alcohol via total enteral nutrition (TEN).

PubMed

Ronis, Martin J J; Mercer, Kelly; Suva, Larry J; Vantrease, Jamie; Ferguson, Matthew; Hogue, William R; Sharma, Neha; Cleves, Mario A; Blackburn, Michael L; Badger, Thomas M

2014-03-01

Alcohol abuse is associated with the development of fatty liver disease and also with significant osteopenia in both genders. In this study, we examined ethanol-induced pathology in response to diets with differing fat/carbohydrate ratios. Male Sprague-Dawley rats were fed intragastrically with isocaloric liquid diets. Dietary fat content was either 5% (high carbohydrate, HC) or 45% (high fat, HF), with or without ethanol (12-13 g/kg/day). After 14, 28, or 65 days, livers were harvested and analyzed. In addition, bone morphology was analyzed after 65 days. HC rats gained more weight and had larger fat pads than HF rats with or without ethanol. Steatosis developed in HC + ethanol (HC + EtOH) compared to HF + ethanol (HF + EtOH) rats, accompanied by increased fatty acid (FA) synthesis and increased nuclear carbohydrate response element binding protein (ChREBP) (p < 0.05), but in the absence of effects on hepatic silent mating type information regulation 2 homolog (SIRT-1) or nuclear sterol regulatory binding element protein (SREBP-1c). Ethanol reduced serum leptin (p < 0.05) but not adiponectin. Over time, HC rats developed fatty liver independent of ethanol. FA degradation was significantly elevated by ethanol in both HC and HF groups (p < 0.05). HF + EtOH rats had increased oxidative stress from 28 days, increased necrosis compared to HF controls and higher expression of cytochromes P450, CYP2E1, and CYP4A1 compared to HC + EtOH rats (p < 0.05). In contrast, HC + EtOH rats had no significant increase in oxidative stress until day 65 with no observed increase in necrosis. Unlike liver pathology, no dietary differences were observed on ethanol-induced osteopenia in HC compared to HF groups. These data demonstrate that interactions between diet composition and alcohol are complex, dependent on the length of exposure, and are an important influence in development of fatty liver injury. Importantly, it appears that diet composition does not affect alcohol-associated skeletal toxicity. Copyright © 2014 Elsevier Inc. All rights reserved.

A Novel Hydroxamate-Based Compound WMJ-J-09 Causes Head and Neck Squamous Cell Carcinoma Cell Death via LKB1-AMPK-p38MAPK-p63-Survivin Cascade.

PubMed

Yen, Chia-Sheng; Choy, Cheuk-Sing; Huang, Wei-Jan; Huang, Shiu-Wen; Lai, Pin-Ye; Yu, Meng-Chieh; Shiue, Ching; Hsu, Ya-Fen; Hsu, Ming-Jen

2018-01-01

Growing evidence shows that hydroxamate-based compounds exhibit broad-spectrum pharmacological properties including anti-tumor activity. However, the precise mechanisms underlying hydroxamate derivative-induced cancer cell death remain incomplete understood. In this study, we explored the anti-tumor mechanisms of a novel aliphatic hydroxamate-based compound, WMJ-J-09, in FaDu head and neck squamous cell carcinoma (HNSCC) cells. WMJ-J-09 induced G2/M cell cycle arrest and apoptosis in FaDu cells. These actions were associated with liver kinase B1 (LKB1), AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (p38MAPK) activation, transcription factor p63 phosphorylation, as well as modulation of p21 and survivin. LKB1-AMPK-p38MAPK signaling blockade reduced WMJ-J-09's enhancing effects in p63 phosphorylation, p21 elevation and survivin reduction. Moreover, WMJ-J-09 caused an increase in α-tubulin acetylation and interfered with microtubule assembly. Furthermore, WMJ-J-09 suppressed the growth of subcutaneous FaDu xenografts in vivo . Taken together, WMJ-J-09-induced FaDu cell death may involve LKB1-AMPK-p38MAPK-p63-survivin signaling cascade. HDACs inhibition and disruption of microtubule assembly may also contribute to WMJ-J-09's actions in FaDu cells. This study suggests that WMJ-J-09 may be a potential lead compound and warrant the clinical development in the treatment of HNSCC.

Gap Junction Intercellular Communication in Bone Marrow Failure

DTIC Science & Technology

2012-10-01

enzyme systems, making individuals with these syndromes highly sensitive to DNA-damaging events. However, researchers suspect that modifier genes or...associated with a single gene defect. A major example of the progress in this area is Fanconi Anemia (FA), where mutations in up to 15 different...proteins have been associated to this disease, being FA-A the most frequent (1, 2). Single mutated genes in the DNA repair or ribosome biogenesis of HSC

BLM and the FANC proteins collaborate in a common pathway in response to stalled replication forks

PubMed Central

Pichierri, Pietro; Franchitto, Annapaola; Rosselli, Filippo

2004-01-01

Fanconi anaemia (FA) and Bloom syndrome (BS) are autosomal recessive diseases characterised by chromosome fragility and cancer proneness. Here, we report that BLM and the FA pathway are activated in response to both crosslinked DNA and replication fork stall. We provide evidence that BLM and FANCD2 colocalise and co-immunoprecipitate following treatment with either DNA crosslinkers or agents inducing replication arrest. We also find that the FA core complex is necessary for BLM phosphorylation and assembly in nuclear foci in response to crosslinked DNA. Moreover, we show that knock-down of the MRE11 complex, whose function is also under the control of the FA core complex, enhances cellular and chromosomal sensitivity to DNA interstrand crosslinks in BS cells. These findings suggest the existence of a functional link between BLM and the FA pathway and that BLM and the MRE11 complex are in two separated branches of a pathway resulting in S-phase checkpoint activation, chromosome integrity and cell survival in response to crosslinked DNA. PMID:15257300

FAN1 acts with FANCI-FANCD2 to promote DNA interstrand cross-link repair.

PubMed

Liu, Ting; Ghosal, Gargi; Yuan, Jingsong; Chen, Junjie; Huang, Jun

2010-08-06

Fanconi anemia (FA) is caused by mutations in 13 Fanc genes and renders cells hypersensitive to DNA interstrand cross-linking (ICL) agents. A central event in the FA pathway is mono-ubiquitylation of the FANCI-FANCD2 (ID) protein complex. Here, we characterize a previously unrecognized nuclease, Fanconi anemia-associated nuclease 1 (FAN1), that promotes ICL repair in a manner strictly dependent on its ability to accumulate at or near sites of DNA damage and that relies on mono-ubiquitylation of the ID complex. Thus, the mono-ubiquitylated ID complex recruits the downstream repair protein FAN1 and facilitates the repair of DNA interstrand cross-links.

Ferulic acid lowers body weight and visceral fat accumulation via modulation of enzymatic, hormonal and inflammatory changes in a mouse model of high-fat diet-induced obesity.

PubMed

de Melo, T S; Lima, P R; Carvalho, K M M B; Fontenele, T M; Solon, F R N; Tomé, A R; de Lemos, T L G; da Cruz Fonseca, S G; Santos, F A; Rao, V S; de Queiroz, M G R

2017-01-05

Previous studies have reported on the glucose and lipid-lowering effects of ferulic acid (FA) but its anti-obesity potential has not yet been firmly established. This study investigated the possible anti-obesitogenic effects of FA in mice fed a high-fat diet (HFD) for 15 weeks. To assess the antiobesity potential of FA, 32 male Swiss mice, weighing 20-25 g (n=6-8 per group) were fed a normal diet (ND) or HFD, treated orally or not with either FA (10 mg/kg) or sibutramine (10 mg/kg) for 15 weeks and at the end of this period, the body weights of animals, visceral fat accumulation, plasma levels of glucose and insulin hormone, amylase and lipase activities, the satiety hormones ghrelin and leptin, and tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCH-1) were analyzed. Results revealed that FA could effectively suppress the HFD-associated increase in visceral fat accumulation, adipocyte size and body weight gain, similar to sibutramine, the positive control. FA also significantly (P<0.05) decreased the HFD-induced elevations in serum lipid profiles, amylase and lipase activities, and the levels of blood glucose and insulin hormone. The markedly elevated leptin and decreased ghrelin levels seen in HFD-fed control mice were significantly (P<0.05) reversed by FA treatment, almost reaching the values seen in ND-fed mice. Furthermore, FA demonstrated significant (P<0.05) inhibition of serum levels of inflammatory mediators TNF-α, and MCH-1. These results suggest that FA could be beneficial in lowering the risk of HFD-induced obesity via modulation of enzymatic, hormonal and inflammatory responses.

Omega-3 fatty acid therapy dose-dependently and significantly decreased triglycerides and improved flow-mediated dilation, however, did not significantly improve insulin sensitivity in patients with hypertriglyceridemia.

PubMed

Oh, Pyung Chun; Koh, Kwang Kon; Sakuma, Ichiro; Lim, Soo; Lee, Yonghee; Lee, Seungik; Lee, Kyounghoon; Han, Seung Hwan; Shin, Eak Kyun

2014-10-20

Experimental studies demonstrate that higher intake of omega-3 fatty acids (n-3 FA) improves insulin sensitivity, however, we reported that n-3 FA 2g therapy, most commonly used dosage did not significantly improve insulin sensitivity despite reducing triglycerides by 21% in patients. Therefore, we investigated the effects of different dosages of n-3 FA in patients with hypertriglyceridemia. This was a randomized, single-blind, placebo-controlled, parallel study. Age, sex, and body mass index were matched among groups. All patients were recommended to maintain a low fat diet. Forty-four patients (about 18 had metabolic syndrome/type 2 diabetes mellitus) in each group were given placebo, n-3 FA 1 (O1), 2 (O2), or 4 g (O4), respectively daily for 2 months. n-3 FA therapy dose-dependently and significantly decreased triglycerides and triglycerides/HDL cholesterol and improved flow-mediated dilation, compared with placebo (by ANOVA). However, each n-3 FA therapy did not significantly decrease high-sensitivity C-reactive protein and fibrinogen, compared with placebo. O1 significantly increased insulin levels and decreased insulin sensitivity (determined by QUICKI) and O2 significantly decreased plasma adiponectin levels relative to baseline measurements. Of note, when compared with placebo, each n-3 FA therapy did not significantly change insulin, glucose, adiponectin, glycated hemoglobin levels and insulin sensitivity (by ANOVA). We observed similar results in a subgroup of patients with the metabolic syndrome. n-3 FA therapy dose-dependently and significantly decreased triglycerides and improved flow-mediated dilation. Nonetheless, n-3 FA therapy did not significantly improve acute-phase reactants and insulin sensitivity in patients with hypertriglyceridemia, regardless of dosages. Copyright © 2014. Published by Elsevier Ireland Ltd.

Filaggrin loss-of-function mutations are associated with food allergy in childhood and adolescence.

PubMed

Venkataraman, Devasmitha; Soto-Ramírez, Nelís; Kurukulaaratchy, Ramesh J; Holloway, John W; Karmaus, Wilfried; Ewart, Susan L; Arshad, S Hasan; Erlewyn-Lajeunesse, Mich

2014-10-01

Filaggrin is an epidermal protein that has a role in skin barrier function. Filaggrin loss-of-function (FLG-LOF) mutations are a significant risk factor for eczema and atopy, but their association with food allergy (FA) is less clear. We explored the longitudinal relationship between 3 common FLG-LOF mutations and FA using the Isle of Wight birth cohort. FA diagnosis was based on recognized allergic reactions within 4 hours after exposure to known food allergens. Food allergen sensitization (FAS) was identified by using skin prick tests conducted between 1 and 18 years of age to a range of food allergens. Three FLG mutations were genotyped in 1150 (79%) of 1456 children. The temporal relationships between FA, FAS, and eczema in children with FLG mutations were explored by using path analysis with total, direct, and indirect effect models. There was a significant total effect of FLG-LOF mutations on the risk of FA in later childhood at the ages of 10 (odds ratio, 31.46; 95% CI, 2.86 to >100) and 18 (odds ratio, 4.25; 95% CI, 1.55-11.61) years. Path analysis showed that there was no direct effect of FLG-LOF mutations on FA at any age; however, an indirect effect was found on FA at all ages through eczema and FAS in the earlier years. FLG-LOF mutations are associated with FA in older children through eczema and FAS during early childhood. Our results highlight a biologically plausible pathway, which suggests that skin barrier function is important in the development and persistence of FA. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Ferulic acid lowers body weight and visceral fat accumulation via modulation of enzymatic, hormonal and inflammatory changes in a mouse model of high-fat diet-induced obesity

PubMed Central

de Melo, T.S.; Lima, P.R.; Carvalho, K.M.M.B.; Fontenele, T.M.; Solon, F.R.N.; Tomé, A.R.; de Lemos, T.L.G.; da Cruz Fonseca, S.G.; Santos, F.A.; Rao, V.S.; de Queiroz, M.G.R.

2017-01-01

Previous studies have reported on the glucose and lipid-lowering effects of ferulic acid (FA) but its anti-obesity potential has not yet been firmly established. This study investigated the possible anti-obesitogenic effects of FA in mice fed a high-fat diet (HFD) for 15 weeks. To assess the antiobesity potential of FA, 32 male Swiss mice, weighing 20–25 g (n=6–8 per group) were fed a normal diet (ND) or HFD, treated orally or not with either FA (10 mg/kg) or sibutramine (10 mg/kg) for 15 weeks and at the end of this period, the body weights of animals, visceral fat accumulation, plasma levels of glucose and insulin hormone, amylase and lipase activities, the satiety hormones ghrelin and leptin, and tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCH-1) were analyzed. Results revealed that FA could effectively suppress the HFD-associated increase in visceral fat accumulation, adipocyte size and body weight gain, similar to sibutramine, the positive control. FA also significantly (P<0.05) decreased the HFD-induced elevations in serum lipid profiles, amylase and lipase activities, and the levels of blood glucose and insulin hormone. The markedly elevated leptin and decreased ghrelin levels seen in HFD-fed control mice were significantly (P<0.05) reversed by FA treatment, almost reaching the values seen in ND-fed mice. Furthermore, FA demonstrated significant (P<0.05) inhibition of serum levels of inflammatory mediators TNF-α, and MCH-1. These results suggest that FA could be beneficial in lowering the risk of HFD-induced obesity via modulation of enzymatic, hormonal and inflammatory responses. PMID:28076453

Milk fatty acid profile is modulated by DGAT1 and SCD1 genotypes in dairy cattle on pasture and strategic supplementation.

PubMed

Carvajal, A M; Huircan, P; Dezamour, J M; Subiabre, I; Kerr, B; Morales, R; Ungerfeld, E M

2016-05-09

Milk fat composition is important to consumer health. During the last decade, some fatty acids (FA) have received attention because of their functional and beneficial effects on human health. The milk FA profile is affected by both diet and genetics. Differences in milk fat composition are based on biochemical pathways, and candidate genes have been proposed to explain FA profile variation. Here, the association between DGAT1 K232A, SCD1 A293V, and LEPR T945M markers with milk fat composition in southern Chile was evaluated. We selected five herds of Holstein-Friesian, Jersey, Frisón Negro, Montbeliarde, and Overo Colorado cows (pasture-grazed) that received strategic supplementation with concentrates and conserved forages. We genotyped the SNPs and calculated allele frequencies and Hardy-Weinberg equilibrium. Milk fat composition was determined for individual milk samples over a year, and associations between genotypes and milk composition were studied. The most frequent variants for DGAT1, SCD1, and LEPR polymorphisms were GC/GC, C, and C, respectively. The DGAT1 GC/GC allele was associated with lower milk fat and protein content, lower saturated fatty acid levels, and higher polyunsaturated FA (PUFA), n-3 and n-6 FA, and a linolenic acid to cholesterolemic FA ratios, which implied a healthier FA profile. The SCD1 CC genotype was associated with a low cholesterolemic FA content, a high ratio of linolenic acid to cholesterolemic FA, and lower conjugated-linolenic acid and PUFA content. These results suggest the possible modulation of milk fat profiles, using specific genotypes, to improve the nutritional quality of dairy products.

Sorption of Th (IV) to silica as a function of pH, humic/fulvic acid, ionic strength, electrolyte type.

PubMed

Chen, Changlun; Wang, Xiangke

2007-02-01

The removal behavior of thorium (Th(IV)) has been investigated in multicomponent systems containing silica (SiO2) as the model of inorganic particles because of its widespread presence in the earth's crust and soil humic acid (HA)/fulvic acid (FA) by batch experiments. The influence of pH from 2 to 12, ionic strength from 0.02 to 0.2 M KNO3, soil HA/FA concentration from 8.3 to 22.5 mg/L, and foreign cations (Li+, Na+, K+) and anions (NO3(-), Cl-) on the sorption of Th(IV) onto SiO2 was also tested. The sorption isotherms of Th(IV) at approximately constant pH (3.50+/-0.02) were determined and analyzed regressively with three kinds of sorption isotherm models, i.e., linear, Langmuir, and Freundlich models. The results demonstrated that the sorption of Th(IV) onto SiO2 increased steeply with increasing pH from 2 to 4. Generally, humic substances (HSs) were shown to enhance Th(IV) sorption at low pH, but to reduce Th(IV) sorption at intermediate and high pH. It was a hypothesis that the significantly positive influence of HA/FA at pH from 2 to 4 on the sorption of Th(IV) onto SiO2 was attributed to strong surface binding of HA/FA on SiO2 and subsequently the formation of ternary surface complexes such as [triple bond]MO-O-HA-Th or [triple bond]MO-O-FA-Th. The results also demonstrated that the sorption was strongly dependent on the concentration of HA/FA, and independent of ionic strength and foreign ions under our experimental conditions.

Comparison of erythrocyte membrane fatty acid contents in renal transplant recipients and dialysis patients.

PubMed

Oh, J S; Kim, S M; Sin, Y H; Kim, J K; Park, Y; Bae, H R; Son, Y K; Nam, H K; Kang, H J; An, W S

2012-12-01

Alterations of erythrocyte membrane fatty acid (FA) composition play important roles in cellular function because they change the membrane microenvironment, including transmembrane receptors. The erythrocyte membrane oleic acid content is higher among patients with acute coronary syndrome and also in dialysis patients. However, available data are limited concerning erythrocyte membrane FA content in kidney transplant recipients (KTP). We sought to test the hypothesis that erythrocyte membrane FA content among KTP were different from those in dialysis patients. In this cross-sectional study, we recruited 35 hemodialysis, 33 peritoneal dialysis 49 KTP, and 33 normal control subjects (CTL). Their erythrocyte membrane FA content were measured by gas chromatography. The mean ages of the enrolled dialysis patients, KTP, and CTL were 56.4 ± 10.1, 48.9 ± 10.4, and 49.5 ± 8.3 years, respectively. Mean kidney transplant duration was 89.8 ± 64.8 months and mean dialysis duration, 49.0 ± 32.6 months. The intakes of vegetable lipid and vegetable protein including total calories were significantly increased among KTP versus dialysis patients. Total cholesterol (P < .001) and high density lipoprotein cholesterol (HDL; P < .001) levels were significantly higher and C-reactive protein was significantly lower among KTP compared with dialysis patients. The erythrocyte membrane content of palmitoleic acid (P < .001) was significantly higher but oleic acid (P < .001) significantly lower in KTP compared with dialysis patients. The erythrocyte membrane contents of arachidonic acid and docosahexaenoic acid were significantly higher, and linoleic acid and the omega-6 FA to omega-3 FA ratio (P < .001) significantly lower in KTP compared with dialysis patients. The erythrocyte membrane content of oleic acid was independently associated with monounsaturated fatty acid (beta = 0.771, P < .001), eicosapentaeonic acid (beta = -0.244, P = .010), and HDL (beta = -0.139, P = .049) in KTP. FA contents of erythrocyte membranes were significantly different in KTP compared with dialysis patients. These differences may have been associated with improved dietary intake and immunosuppression after kidney transplantation. Copyright © 2012 Elsevier Inc. All rights reserved.

Reassessment of the Genetic Regulation of Fatty Acid Synthesis in Escherichia coli: Global Positive Control by the Dual Functional Regulator FadR

PubMed Central

My, L.; Ghandour Achkar, N.; Viala, J. P.

2015-01-01

ABSTRACT In Escherichia coli, the FadR transcriptional regulator represses the expression of fatty acid degradation (fad) genes. However, FadR is also an activator of the expression of fabA and fabB, two genes involved in unsaturated fatty acid synthesis. Therefore, FadR plays an important role in maintaining the balance between saturated and unsaturated fatty acids in the membrane. We recently showed that FadR also activates the promoter upstream of the fabH gene (L. My, B. Rekoske, J. J. Lemke, J. P. Viala, R. L. Gourse, and E. Bouveret, J Bacteriol 195:3784–3795, 2013, doi:10.1128/JB.00384-13). Furthermore, recent transcriptomic and proteomic data suggested that FadR activates the majority of fatty acid (FA) synthesis genes. In the present study, we tested the role of FadR in the expression of all genes involved in FA synthesis. We found that FadR activates the transcription of all tested FA synthesis genes, and we identified the FadR binding site for each of these genes. This necessitated the reassessment of the transcription start sites for accA and accB genes described previously, and we provide evidence for the presence of multiple promoters driving the expression of these genes. We showed further that regulation by FadR impacts the amount of FA synthesis enzymes in the cell. Our results show that FadR is a global regulator of FA metabolism in E. coli, acting both as a repressor of catabolism and an activator of anabolism, two directly opposing pathways. IMPORTANCE In most bacteria, a transcriptional regulator tunes the level of FA synthesis enzymes. Oddly, such a global regulator still was missing for E. coli, which nonetheless is one of the prominent model bacteria used for engineering biofuel production using the FA synthesis pathway. Our work identifies the FadR functional dual regulator as a global activator of almost all FA synthesis genes in E. coli. Because FadR also is the repressor of FA degradation, FadR acts both as a repressor and an activator of the two opposite pathways of FA degradation and synthesis. Our results show that there are still discoveries waiting to be made in the understanding of the genetic regulation of FA synthesis, even in the very well-known bacterium E. coli. PMID:25802297

Glycosylation of random IgG distinguishes seropositive and seronegative rheumatoid arthritis.

PubMed

Magorivska, I; Döncző, B; Dumych, T; Karmash, A; Boichuk, M; Hychka, K; Mihalj, M; Szabó, M; Csánky, E; Rech, J; Guttman, A; Vari, S G; Bilyy, R

2018-05-01

The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n = 15) and seronegative RA (n = 12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p = .006) and FABG2S1 (p = .005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p = .074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p = .055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.

Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica.

PubMed

Dulermo, Rémi; Gamboa-Meléndez, Heber; Ledesma-Amaro, Rodrigo; Thévenieau, France; Nicaud, Jean-Marc

2015-09-01

Fatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica. Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae. While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, ΔYlpxa1 ΔYlpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the ΔYlpxa1 ΔYlpxa2 ΔYlant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast. Copyright © 2015. Published by Elsevier B.V.

Orientation-specific responses to sustained uniaxial stretching in focal adhesion growth and turnover

PubMed Central

Chen, Yun; Pasapera, Ana M.; Koretsky, Alan P.; Waterman, Clare M.

2013-01-01

Cells are mechanosensitive to extracellular matrix (ECM) deformation, which can be caused by muscle contraction or changes in hydrostatic pressure. Focal adhesions (FAs) mediate the linkage between the cell and the ECM and initiate mechanically stimulated signaling events. We developed a stretching apparatus in which cells grown on fibronectin-coated elastic substrates can be stretched and imaged live to study how FAs dynamically respond to ECM deformation. Human bone osteosarcoma epithelial cell line U2OS was transfected with GFP-paxillin as an FA marker and subjected to sustained uniaxial stretching. Two responses at different timescales were observed: rapid FA growth within seconds after stretching, and delayed FA disassembly and loss of cell polarity that occurred over tens of minutes. Rapid FA growth occurred in all cells; however, delayed responses to stretch occurred in an orientation-specific manner, specifically in cells with their long axes perpendicular to the stretching direction, but not in cells with their long axes parallel to stretch. Pharmacological treatments demonstrated that FA kinase (FAK) promotes but Src inhibits rapid FA growth, whereas FAK, Src, and calpain 2 all contribute to delayed FA disassembly and loss of polarity in cells perpendicular to stretching. Immunostaining for phospho-FAK after stretching revealed that FAK activation was maximal at 5 s after stretching, specifically in FAs oriented perpendicular to stretch. We hypothesize that orientation-specific activation of strain/stress-sensitive proteins in FAs upstream to FAK and Src promote orientation-specific responses in FA growth and disassembly that mediate polarity rearrangement in response to sustained stretch. PMID:23754369

Induction of mitophagy-mediated antitumor activity with folate-appended methyl-β-cyclodextrin.

PubMed

Kameyama, Kazuhisa; Motoyama, Keiichi; Tanaka, Nao; Yamashita, Yuki; Higashi, Taishi; Arima, Hidetoshi

2017-01-01

Mitophagy is the specific autophagic elimination system of mitochondria, which regulates cellular survival via the removal of damaged mitochondria. Recently, we revealed that folate-appended methyl-β-cyclodextrin (FA-M-β-CyD) provides selective antitumor activity in folate receptor-α (FR-α)-expressing cells by the induction of autophagy. In this study, to gain insight into the detailed mechanism of this antitumor activity, we focused on the induction of mitophagy by the treatment of FR-α-expressing tumor cells with FA-M-β-CyD. In contrast to methyl-β-cyclodextrin, FA-M-β-CyD entered KB cells, human epithelial cells from a fatal cervical carcinoma (FR-α (+)) through FR-α-mediated endocytosis. The transmembrane potential of isolated mitochondria after treatment with FA-M-β-CyD was significantly elevated. In addition, FA-M-β-CyD lowered adenosine triphosphate (ATP) production and promoted reactive oxygen species production in KB cells (FR-α (+)). Importantly, FA-M-β-CyD enhanced light chain 3 (LC3) conversion (LC3-I to LC3-II) in KB cells (FR-α (+)) and induced PTEN-induced putative kinase 1 (PINK1) protein expression, which is involved in the induction of mitophagy. Furthermore, FA-M-β-CyD had potent antitumor activity in BALB/c nu/nu mice xenografted with KB cells (FR-α (+)) without any significant side effects. Taken together, these findings demonstrate that the autophagic cell death elicited by FA-M-β-CyD could be associated with mitophagy induced by an impaired mitochondrial function.

Methanoculleus sediminis sp. nov., a methanogen from sediments near a submarine mud volcano.

PubMed

Chen, Sheng-Chung; Chen, Mei-Fei; Lai, Mei-Chin; Weng, Chieh-Yin; Wu, Sue-Yao; Lin, Saulwood; Yang, Tsanyao F; Chen, Po-Chun

2015-07-01

A mesophilic, hydrogenotrophic methanogen, strain S3Fa(T), was isolated from sediments collected by Ocean Researcher I cruise ORI-934 in 2010 near the submarine mud volcano MV4 located at the upper slope of south-west Taiwan. The methanogenic substrates utilized by strain S3Fa(T) were formate and H2/CO2 but not acetate, secondary alcohols, methylamines, methanol or ethanol. Cells of strain S3Fa(T) were non-motile, irregular cocci, 0.5-1.0 μm in diameter. The surface-layer protein showed an Mr of 128,000.The optimum growth conditions were 37 °C, pH 7.1 and 0.17 M NaCl. The DNA G+C content of the genome of strain S3Fa(T) was 62.3 mol%. Phylogenetic analysis revealed that strain S3Fa(T) was most closely related to Methanoculleus marisnigri JR1(T) (99.3% 16S rRNA gene sequence similarity). Genome relatedness between strain S3Fa(T) and Methanoculleus marisnigri JR1(T) was computed using both genome-to-genome distance analysis (GGDA) and average nucleotide identity (ANI) with values of 46.3-55.5% and 93.08%, respectively. Based on morphological, phenotypic, phylogenetic and genomic relatedness data, it is evident that strain S3Fa(T) represents a novel species of the genus Methanoculleus, for which the name Methanoculleus sediminis sp. nov. is proposed. The type strain is S3Fa(T) ( = BCRC AR10044(T) = DSM 29354(T)).

Differential Protection of Cry1Fa Toxin against Spodoptera frugiperda Larval Gut Proteases by Cadherin Orthologs Correlates with Increased Synergism

PubMed Central

Rahman, Khalidur; Abdullah, Mohd Amir F.; Ambati, Suresh; Taylor, Milton D.

2012-01-01

The Cry proteins produced by Bacillus thuringiensis (Bt) are the most widely used biopesticides effective against a range of crop pests and disease vectors. Like chemical pesticides, development of resistance is the primary threat to the long-term efficacy of Bt toxins. Recently discovered cadherin-based Bt Cry synergists showed the potential to augment resistance management by improving efficacy of Cry toxins. However, the mode of action of Bt Cry synergists is thus far unclear. Here we elucidate the mechanism of cadherin-based Cry toxin synergism utilizing two cadherin peptides, Spodoptera frugiperda Cad (SfCad) and Manduca sexta Cad (MsCad), which differentially enhance Cry1Fa toxicity to Spodoptera frugiperda neonates. We show that differential SfCad- and MsCad-mediated protection of Cry1Fa toxin in the Spodoptera frugiperda midgut correlates with differential Cry1Fa toxicity enhancement. Both peptides exhibited high affinity for Cry1Fa toxin and an increased rate of Cry1Fa-induced pore formation in S. frugiperda. However, only SfCad bound the S. frugiperda brush border membrane vesicle and more effectively prolonged the stability of Cry1Fa toxin in the gut, explaining higher Cry1Fa enhancement by this peptide. This study shows that cadherin fragments may enhance B. thuringiensis toxicity by at least two different mechanisms or a combination thereof: (i) protection of Cry toxin from protease degradation in the insect midgut and (ii) enhancement of pore-forming ability of Cry toxin. PMID:22081566

Measurement of the alpha4beta2* nicotinic acetylcholine receptor ligand 2-[(18)F]Fluoro-A-85380 and its metabolites in human blood during PET investigation: a methodological study.

PubMed

Sorger, Dietlind; Becker, Georg A; Patt, Marianne; Schildan, Andreas; Grossmann, Udo; Schliebs, Reinhard; Seese, Anita; Kendziorra, Kai; Kluge, Magnus; Brust, Peter; Mukhin, Alexey G; Sabri, Osama

2007-04-01

2-[(18)F]fluoro-A-85380 (2-[(18)F]FA) is a new radioligand for noninvasive imaging of alpha4beta2* nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET) in human brain. In most cases, quantification of 2-[(18)F]FA receptor binding involves measurement of free nonmetabolized radioligand concentration in blood. This requires an efficient and reliable method to separate radioactive metabolites from the parent compound. In the present study, three analytical methods, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and solid phase extraction (SPE) have been tested. Reversed-phase TLC of deproteinized aqueous samples of plasma provides good estimates of 2-[(18)F]FA and its metabolites. However, because of the decreased radioactivity in plasma samples, this method can be used in humans over the first 2 h after radioligand injection only. Reliable quantification of the parent radioligand and its main metabolites was obtained using reversed-phase HPLC, followed by counting of eluted fractions in a well gamma counter. Three main and five minor metabolites of 2-[(18)F]FA were detected in human blood using this method. On average, the unchanged 2-[(18)F]FA fraction in plasma of healthy volunteers measured at 14, 60, 120, 240 and 420 min after radioligand injection was 87.3+/-2.2%, 74.4+/-3%, 68.8+/-5%, 62.3+/-8% and 61.0+/-8%, respectively. In patients with neurodegenerative disorders, the values corresponding to the three last time points were significantly lower. The fraction of nonmetabolized 2-[(18)F]FA in plasma determined using SPE did not differ significantly from that obtained by HPLC (+gamma counting) (n=73, r=.95). Since SPE is less time-consuming than HPLC and provides comparable results, we conclude that SPE appears to be the most suitable method for measurement of 2-[(18)F]FA parent fraction during PET investigations.

Decreased cerebral α4β2* nicotinic acetylcholine receptor availability in patients with mild cognitive impairment and Alzheimer's disease assessed with positron emission tomography.

PubMed

Kendziorra, Kai; Wolf, Henrike; Meyer, Philipp Mael; Barthel, Henryk; Hesse, Swen; Becker, Georg Alexander; Luthardt, Julia; Schildan, Andreas; Patt, Marianne; Sorger, Dietlind; Seese, Anita; Gertz, Herman-Josef; Sabri, Osama

2011-03-01

Postmortem studies indicate a loss of nicotinic acetylcholine receptor (nAChRs) in Alzheimer's disease (AD). In order to establish whether these changes in the cholinergic system occur at an early stage of AD, we carried out positron emission tomography (PET) with a specific radioligand for the α4β2* nicotinic acetylcholine receptor (α4β2* nAChR) in patients with mild to moderate AD and in patients with amnestic mild cognitive impairment (MCI), who have a high risk to progress to AD. Nine patients with moderate AD, eight patients with MCI and seven age-matched healthy controls underwent 2-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[(18)F]FA-85380) PET. After coregistration with individual magnetic resonance imaging the binding potential (BP(ND)) of 2-[(18)F]FA-85380 was calculated using either the corpus callosum or the cerebellum as reference regions. PET data were analysed by region of interest analysis and by voxel-based analysis. Both patients with AD and MCI showed a significant reduction in 2-[(18)F]FA-85380 BP(ND) in typical AD-affected brain regions. Thereby, the corpus callosum was identified as the most suitable reference region. The 2-[(18)F]FA-85380 BP(ND) correlated with the severity of cognitive impairment. Only MCI patients that converted to AD in the later course (n = 5) had a reduction in 2-[(18)F]FA-85380 BP(ND). 2-[(18)F]FA-85380 PET appears to be a sensitive and feasible tool for the detection of a reduction in α4β2* nAChRs which seems to be an early event in AD. In addition, 2-[(18)F]FA-85380 PET might give prognostic information about a conversion from MCI to AD.

Nitro-fatty acid metabolome: saturation, desaturation, beta-oxidation, and protein adduction.

PubMed

Rudolph, Volker; Schopfer, Francisco J; Khoo, Nicholas K H; Rudolph, Tanja K; Cole, Marsha P; Woodcock, Steven R; Bonacci, Gustavo; Groeger, Alison L; Golin-Bisello, Franca; Chen, Chen-Shan; Baker, Paul R S; Freeman, Bruce A

2009-01-16

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via beta-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO2 hydration or nitric oxide (*NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added beta-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and beta-oxidation reactions that terminate at the site of acyl-chain nitration.

Nitro-fatty Acid Metabolome: Saturation, Desaturation, β-Oxidation, and Protein Adduction*

PubMed Central

Rudolph, Volker; Schopfer, Francisco J.; Khoo, Nicholas K. H.; Rudolph, Tanja K.; Cole, Marsha P.; Woodcock, Steven R.; Bonacci, Gustavo; Groeger, Alison L.; Golin-Bisello, Franca; Chen, Chen-Shan; Baker, Paul R. S.; Freeman, Bruce A.

2009-01-01

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via β-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO2 hydration or nitric oxide (•NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added β-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet identified. In aggregate, these findings show that electrophilic FA nitroalkene derivatives (a) acquire an extended half-life by undergoing reversible and exchangeable electrophilic reactions with nucleophilic targets and (b) are metabolized predominantly via saturation of the double bond and β-oxidation reactions that terminate at the site of acyl-chain nitration. PMID:19015269

Cloning and functional characterization of HKT1 and AKT1 genes of Fragaria spp.-Relationship to plant response to salt stress.

PubMed

Garriga, Miguel; Raddatz, Natalia; Véry, Anne-Aliénor; Sentenac, Hervé; Rubio-Meléndez, María E; González, Wendy; Dreyer, Ingo

2017-03-01

Commercial strawberry, Fragaria x ananassa Duch., is a species sensitive to salinity. Under saline conditions, Na + uptake by the plant is increased, while K + uptake is significantly reduced. Maintaining an adequate K + /Na + cytosolic ratio determines the ability of the plant to survive in saline environments. The goal of the present work was to clone and functionally characterize the genes AKT1 and HKT1 involved in K + and Na + transport in strawberry and to determine the relationship of these genes with the responses of three Fragaria spp. genotypes having different ecological adaptations to salt stress. FaHKT1 and FcHKT1 proteins from F. x ananassa and F. chiloensis have 98.1% of identity, while FaAKT1 and FcAKT1 identity is 99.7%. FaHKT1 and FaAKT1 from F. x ananassa, were functionally characterized in Xenopus oocytes. FaHKT1, belongs to the group I of HKT transporters and is selective for Na + . Expression of FaAKT1 in oocytes showed that the protein is a typical inward-rectifying and highly K + -selective channel. The relative expression of Fragaria HKT1 and AKT1 genes was studied in roots of F. x ananassa cv. Camarosa and of F. chiloensis (accessions Bau and Cucao) grown under salt stress. The expression of AKT1 was transiently increased in 'Camarosa', decreased in 'Cucao' and was not affected in 'Bau' upon salt stress. HKT1 expression was significantly increased in roots of 'Cucao' and was not affected in the other two genotypes. The increased relative expression of HKT1 and decreased expression of AKT1 in 'Cucao' roots correlates with the higher tolerance to salinity of this genotype in comparison with 'Camarosa' and 'Bau'. Copyright © 2016 Elsevier GmbH. All rights reserved.

Association of desaturase activity and C-reactive protein in European children.

PubMed

Wolters, Maike; Börnhorst, Claudia; Schwarz, Heike; Risé, Patrizia; Galli, Claudio; Moreno, Luis A; Pala, Valeria; Russo, Paola; Veidebaum, Toomas; Tornaritis, Michael; Fraterman, Arno; De Henauw, Stefaan; Eiben, Gabriele; Lissner, Lauren; Molnár, Dénes; Ahrens, Wolfgang

2017-01-01

Desaturase enzymes influence the fatty acid (FA) composition of body tissues and their activity affects the conversion rate of saturated to monounsaturated FA and of polyunsaturated FA (PUFA) to long-chain PUFA. Desaturase activity has further been shown to be associated with inflammation. We investigate the association between delta-9 (D9D), delta-6 (D6D) and delta-5 desaturase (D5D) activity and high-sensitive C-reactive protein (CRP) in young children. In the IDEFICS (Identification and prevention of dietary- and lifestyle-induced health effects in children and infants) cohort study children were examined at baseline (T0) and after 2 y (T1). D9D, D6D, and D5D activities were estimated from T0 product-precursor FA ratios. CRP was measured at T0 and T1. In a subsample of 1,943 children with available information on FA, CRP, and covariates, the cross-sectional and longitudinal associations of desaturase activity and CRP were analyzed. Cross-sectionally, a D9D increase of 0.01 units was associated with a 11% higher risk of having a serum CRP ≥ Percentile 75 (P75) (OR, 99% CI: 1.11 (1.01; 1.22)) whereas D6D and D5D were not associated with CRP. No significant associations were observed between baseline desaturase activity and CRP 2 y later. Cross-sectionally, our results indicate a positive association of D9D and CRP independent of weight status. High D9D activity may increase the risk of subclinical inflammation which is associated with metabolic disorders. As D9D expression increases with higher intake of saturated FA and carbohydrates, dietary changes may influence D9D activity and thus CRP. However, it remains to be investigated whether there is a causal relationship between D9D activity and CRP.

Number of independent parameters in the potentiometric titration of humic substances.

PubMed

Lenoir, Thomas; Manceau, Alain

2010-03-16

With the advent of high-precision automatic titrators operating in pH stat mode, measuring the mass balance of protons in solid-solution mixtures against the pH of natural and synthetic polyelectrolytes is now routine. However, titration curves of complex molecules typically lack obvious inflection points, which complicates their analysis despite the high-precision measurements. The calculation of site densities and median proton affinity constants (pK) from such data can lead to considerable covariance between fit parameters. Knowing the number of independent parameters that can be freely varied during the least-squares minimization of a model fit to titration data is necessary to improve the model's applicability. This number was calculated for natural organic matter by applying principal component analysis (PCA) to a reference data set of 47 independent titration curves from fulvic and humic acids measured at I = 0.1 M. The complete data set was reconstructed statistically from pH 3.5 to 9.8 with only six parameters, compared to seven or eight generally adjusted with common semi-empirical speciation models for organic matter, and explains correlations that occur with the higher number of parameters. Existing proton-binding models are not necessarily overparametrized, but instead titration data lack the sensitivity needed to quantify the full set of binding properties of humic materials. Model-independent conditional pK values can be obtained directly from the derivative of titration data, and this approach is the most conservative. The apparent proton-binding constants of the 23 fulvic acids (FA) and 24 humic acids (HA) derived from a high-quality polynomial parametrization of the data set are pK(H,COOH)(FA) = 4.18 +/- 0.21, pK(H,Ph-OH)(FA) = 9.29 +/- 0.33, pK(H,COOH)(HA) = 4.49 +/- 0.18, and pK(H,Ph-OH)(HA) = 9.29 +/- 0.38. Their values at other ionic strengths are more reliably calculated with the empirical Davies equation than any existing model fit.

Adaptations to excess choline in insulin resistant and Pcyt2 deficient skeletal muscle.

PubMed

Taylor, Adrian; Schenkel, Laila Cigana; Yokich, Maiya; Bakovic, Marica

2017-04-01

It was hypothesized that choline supplementation in insulin resistant (IR) CTP:phosphoethanolamine cytidylyltransferase deficient (Pcyt2 +/- ) mice would ameliorate muscle function by remodeling glucose and fatty acid (FA) metabolism. Pcyt2 +/- mice either received no treatment or were allowed access to 2 mg/mL choline in drinking water for 4 weeks. Skeletal muscle was harvested from choline treated and untreated mice. Lipid analysis and metabolic gene expression and signaling pathways were compared between untreated Pcyt2 +/- mice, treated Pcyt2 +/- mice, and Pcyt2 +/+ mice. The major positive effect of choline supplementation on IR muscle was the reduction of glucose utilization for FA and triglyceride (TAG) synthesis and increased muscle glucose storage as glycogen. Choline reduced the expression of genes for FA and TAG formation (Scd1, Fas, Srebp1c, Dgat1/2), upregulated the genes for FA oxidation (Cpt1, Pparα, Pgc1α), and had minor effects on phospholipid and lipolysis genes. Pcyt2 +/- muscle had reduced insulin signaling (IRS1), autophagy (LC3), and choline transport (CTL1) proteins that were restored by choline treatment. Additionally, choline activated AMPK and Akt while inhibiting mTORC1 phosphorylation. These data established that choline supplementation could restore muscle glucose metabolism by reducing lipogenesis and improving mitochondrial and intracellular signaling for protein and energy metabolism in insulin resistant Pcyt2 deficient mice.

Preparation and characterization of (−)-Epigallocatechin-3-gallate (EGCG)-loaded nanoparticles and their inhibitory effects on Human breast cancer MCF-7 cells

PubMed Central

Zeng, Liang; Yan, Jingna; Luo, Liyong; Ma, Mengjun; Zhu, Huiqun

2017-01-01

We were employing nanotechnology to improve the targeting ability of (−)-Epigallocatechin-3-gallate (EGCG) towards MCF-7 cells, and two kinds of EGCG nanoparticles (FA-NPS-PEG and FA-PEG-NPS) were obtained, besides, their characteristics and effects on MCF-7 cells were studied. The results indicated that (i) both FA-NPS-PEG and FA-PEG-NPS have high stabilities; (ii) their particles sizes were 185.0 ± 13.5 nm and 142.7 ± 7.2 nm, respectively; (iii) their encapsulation efficiencies of EGCG were 90.36 ± 2.20% and 39.79 ± 7.54%, respectively. (iv) there was no cytotoxicity observed in EGCG, FA-NPS-PEG and FA-PEG-NPS toward MCF-7 cells over all concentrations (0~400 μg/mL) tested; (v) EGCG, FA-NPS-PEG and FA-PEG-NPS inhibited MCF-7 cells proliferation in dose-dependent manners, with the average IC50 of 470.5 ± 33.0, 65.9 ± 0.4 and 66.6 ± 0.6 μg/mL; (vi) EGCG, FA-NPS-PEG and FA-PEG-NPS could modulated the expressions of several key regulatory proteins in PI3K-Akt pathway such as up-regulation of PTEN, p21 and Bax, and down-regulation of p-PDK1, p-AKT, CyclinD1 and Bcl-2, which gave an illustration about the mechanism by which EGCG nanoparticles inhibited MCF-7 cells proliferation. In this study, EGCG nanoparticles can significantly enhance the targeting ability and efficacy of EGCG, which is considered to an experimental foundation for further research on its activity, targeting ability and metabolism in vivo. PMID:28349962

Docosahexaenoic acid, G protein-coupled receptors, and melanoma: is G protein-coupled receptor 40 a potential therapeutic target?

PubMed

Nehra, Deepika; Pan, Amy H; Le, Hau D; Fallon, Erica M; Carlson, Sarah J; Kalish, Brian T; Puder, Mark

2014-05-15

To determine the effect of docosahexaenoic acid (DHA) on the growth of human melanoma in vitro and in vivo and to better understand the potential role of the G protein-coupled receptors (GPRs) in mediating this effect. For in vitro studies, human melanoma and control fibroblast cells were treated with DHA and TAK-875 (selective GPR40 agonist) and a cell viability assay was performed to determine cell counts. A murine subcutaneous xenograft model of human melanoma was used to test the effect of dietary treatment with an omega-3 fatty acid (FA) rich diet compared with an omega-6 FA rich diet on the growth of human melanoma in vivo. A similar animal model was used to test the effect of oral TAK-875 on the growth of established melanoma tumors in vivo. DHA has an inhibitory effect on the growth of human melanoma both in vitro and in vivo. Tumors from animals on the omega-3 FA rich diet were 69% smaller in weight (P = 0.005) and 76% smaller in volume compared with tumors from animals on the omega-6 FA rich diet. TAK-875 has an inhibitory effect on the growth of human melanoma both in vitro and in vivo. Tumors from animals treated with TAK-875 were 46% smaller in weight (P = 0.07), 62% smaller in volume (P = 0.03), and grew 77% slower (P = 0.04) compared with the placebo group. DHA and TAK-875 have a profound and selective inhibitory effect on the growth of human melanoma both in vitro and in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

Stromal interaction molecule 1 (STIM1) silencing inhibits tumor growth and promotes cell cycle arrest and apoptosis in hypopharyngeal carcinoma.

PubMed

Sun, Yuanhao; Cui, Xiaobo; Wang, Jun; Wu, Shuai; Bai, Yunfei; Wang, Yaping; Wang, Boqian; Fang, Jugao

2015-05-01

As an important pathway maintaining the balance of intracellular calcium (Ca(2+)), store-operated Ca(2+) entry (SOCE) is critical for cellular functions. Stromal interaction molecule 1 (STIM1), a key component of SOCE, plays a dual role as an endoplasmic reticulum Ca(2+) receptor and an SOCE exciter. Aberrant expression of STIM1 could be discovered in several human cancer cells. However, the role of STIM1 in regulating human hypopharyngeal carcinoma still remains unclear. Real-time polymerase chain reaction (PCR) was used to detect expression of STIM1 in human hypopharyngeal carcinoma cell line FaDu. STIM1 on FaDu cells was knocked down by lentiviral transduction method. The biological impacts after knocking down of STIM1 on FaDu cells were investigated in vitro and in vivo. The result of real-time PCR showed that STIM1 was expressed in FaDu cells. Lentiviral transduction efficiently downregulated the expression of STIM1 in FaDu cells at both mRNA and protein levels. Significant downregulation of STIM1 on FaDu cells inhibited cell proliferation, induced cell cycle arrest in G0/G1 phase, promoted cell apoptosis, and restrained cell growth rate. The antigrowth effect of STIM1 silencing was also discovered in FaDu hypopharyngeal tumor model. Our findings indicate that STIM1 is likely to become a new therapeutic target for hypopharyngeal carcinoma treatment.

Role of Ferulic Acid in the Amelioration of Ionizing Radiation Induced Inflammation: A Murine Model

PubMed Central

Das, Ujjal; Manna, Krishnendu; Sinha, Mahuya; Datta, Sanjukta; Das, Dipesh Kr; Chakraborty, Anindita; Ghosh, Mahua; Saha, Krishna Das; Dey, Sanjit

2014-01-01

Ionizing radiation is responsible for oxidative stress by generating reactive oxygen species (ROS), which alters the cellular redox potential. This change activates several redox sensitive enzymes which are crucial in activating signaling pathways at molecular level and can lead to oxidative stress induced inflammation. Therefore, the present study was intended to assess the anti-inflammatory role of ferulic acid (FA), a plant flavonoid, against radiation-induced oxidative stress with a novel mechanistic viewpoint. FA was administered (50 mg/kg body wt) to Swiss albino mice for five consecutive days prior to exposing them to a single dose of 10 Gy 60Co γ-irradiation. The dose of FA was optimized from the survival experiment and 50 mg/kg body wt dose showed optimum effect. FA significantly ameliorated the radiation induced inflammatory response such as phosphorylation of IKKα/β and IκBα and consequent nuclear translocation of nuclear factor kappa B (NF-κB). FA also prevented the increase of cycloxygenase-2 (Cox-2) protein, inducible nitric oxide synthase-2 (iNOS-2) gene expression, lipid peroxidation in liver and the increase of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in serum. It was observed that exposure to radiation results in decreased activity of superoxide dismutase (SOD), catalase (CAT) and the pool of reduced glutathione (GSH) content. However, FA treatment prior to irradiation increased the activities of the same endogenous antioxidants. Thus, pretreatment with FA offers protection against gamma radiation induced inflammation. PMID:24854039

Effect of different feeding strategies in intensive dairy farming systems on milk fatty acid profiles, and implications on feeding costs in Italy.

PubMed

Borreani, G; Coppa, M; Revello-Chion, A; Comino, L; Giaccone, D; Ferlay, A; Tabacco, E

2013-01-01

The aim of this work was to characterize the fatty acid (FA) profile of milk from intensive dairy farming systems in the Po Plain (Italy) to estimate the costs of the adopted feeding strategies and to simulate the effect of supplementary premiums on the basis of milk FA composition on milk income. Twenty dairy farms with 5 different feeding strategies were studied: 3 corn silage-based systems in which cows were supplemented with a great proportion (CCH), a medium proportion (CCM), or without commercial concentrate mix (CC0), and 2 systems in which part of corn silage was replaced with grass or legume silage (HF) or with fresh herbage (G), cut and fed indoors. Bulk milk was sampled and lactating cow performance, feeding strategies and forage characteristics were recorded through a survey, 3 times during a year. The milk FA supplementary premium was calculated considering C18:3n-3 and saturated FA (SFA) concentrations, and ratio of total cis C18:1 isomers to C16:0. The CCH, CCM, and CC0 systems bought most of their dairy cow feeds off farm, which allowed them to increase milk production to 35,000 L/yr per hectare. Their low dry matter and crude protein self-sufficiency led to higher feeding costs per liter of milk (from €0.158 to €0.184), and highest income over feed cost was achieved only for milk yield performance greater than 10,000 kg/cow per year. The use of homegrown forages in HF and G increased dry matter and crude protein self-sufficiency and reduced the feeding costs per liter of milk from 9 to 22%, compared with the other studied systems, making HF and G feeding economically competitive, even for a lower milk yield per cow. The studied systems highlighted a remarkable variation in FA profiles. The concentrations of C16:0 and SFA were the highest in CCH (31.53 and 67.84 g/100g of FA) and G (31.23 and 68.45 g/100g of FA), because of the larger proportion of commercial concentrate mix in the cow diet. The concentrations of C16:0 and SFA were the lowest in CCM (27.86 and 63.10 g/100g of FA), because of low roughage-to-concentrate ratio in the cow diet, which is known to favor milk fat depression, affecting particularly these FA. The calculated supplementary premium was the highest in the CCM system, based on milk FA profiles from those herds. The HF diet was rich in forages and resulted in greater concentration of C18:3n-3 in milk (0.57 g/100g of FA) than the other systems and thus led to an increase in milk FA supplementary premium. Milk from G and HF milk had the lowest ratio of Σn-6:Σn-3 FA compared with milk from the systems based on higher corn silage proportion in the cow diet (3.71, and 3.25, respectively, vs. 4.58 to 4.78), with the lower ratios being closer to recommendation for human nutrition. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[Impact of bariatric surgery on vitamin metabolisms in obese patients].

PubMed

Bodunova, N A; Askerkhanov, R G; Khatkov, I E; Sabelnikova, E A; Parfenov, A I; Tkachenko, E V; Varvanina, G G; Feydorov, I Yu

2015-01-01

To investigate the impact of bariatric surgery (BS) on the level of vitamins in obese patients. One hundred obese patients (78 women and 22 men) aged 19 to 61 years were examined. Controlled gastric banding (CGB) was carried out in 20 patients (mean body mass index (BMI), 41.3 ± 8.2 kg/m2); gastric sleeve resection (GSR) in 40 patients, and gastric shunting (GS) in 40 (the mean BMI in these groups were 41.1 ± 17.8 and 45.9 ± 6.2 kg/m2, respectively). A control group consisted of 10 apparently healthy individuals (BMI, 24.9 ± 3.2 kg/m2). An enzyme immunoassay was used to determine the serum concentrations of vitamins B1, B2, B5, B6, B9, B12, C, and D, niacin, biotin, and retinol-binding protein (RBP) before and 1 year after surgery. All the three groups showed a considerable decrease in the levels of vitamins C, B5, B6, and D, and RBP both prior to and following BS. More than 50% of the patients who had undergone GSR had also a lower baseline niacin level. A year after CGB, GSR, and GS, the number of patents with deficiency of these vitamins remained the same or increased. The majority of patients with the same level of vitamin B2, niacin, and folic acid (FA) were observed to have its decrease a year postsurgery. The concentration of the other test vitamins was also reduced a year after all operations; however, it remained within the normal range. GS had no substantial impact on the concentrations of FA, vitamins B2, B12, B1, and biotin. The lower serum vitamin levels were not accompanied by clinical symptoms in most patients following BS. In 80% of the patients with obesity, the levels of vitamins C, B6, and D were decreased to a variable degree. After BS, there was a rise in the number of patients with low serum vitamin C, D, B6, B5, niacin, FA, and RBP concentrations, at the same time the number of patients with FA deficiency increased by more than twice. BS did not significantly affect the metabolism of vitamins B1 B2 B12, and biotin.

Targeted disruption of exons 1 to 6 of the Fanconi Anemia group A gene leads to growth retardation, strain-specific microphthalmia, meiotic defects and primordial germ cell hypoplasia.

PubMed

Wong, Jasmine C Y; Alon, Noa; Mckerlie, Colin; Huang, Jun R; Meyn, M Stephen; Buchwald, Manuel

2003-08-15

Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene (Fanca), gene-targeting techniques were used to generate Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice. Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth retardation, microphthalmia and craniofacial malformations that are not found in other Fanca mouse models, and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism, and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells, implicating a role for Fanca in meiotic recombination. However, the localization of Rad51, Brca1, Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic chromosomes. Taken together, our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination.

Regulation of L-ascorbic acid content in strawberry fruits

PubMed Central

Cruz-Rus, Eduardo; Amaya, Iraida; Sánchez-Sevilla, José F.; Botella, Miguel A.; Valpuesta, Victoriano

2011-01-01

Plants have several L-ascorbic acid (AsA) biosynthetic pathways, but the contribution of each one to the synthesis of AsA varyies between different species, organs, and developmental stages. Strawberry (Fragaria×ananassa) fruits are rich in AsA. The pathway that uses D-galacturonate as the initial substrate is functional in ripe fruits, but the contribution of other pathways to AsA biosynthesis has not been studied. The transcription of genes encoding biosynthetic enzymes such as D-galacturonate reductase (FaGalUR) and myo-inositol oxygenase (FaMIOX), and the AsA recycling enzyme monodehydroascorbate reductase (FaMDHAR) were positively correlated with the increase in AsA during fruit ripening. Fruit storage for 72 h in a cold room reduced the AsA content by 30%. Under an ozone atmosphere, this reduction was 15%. Ozone treatment increased the expression of the FaGalUR, FaMIOX, and L-galactose-1-phosphate phosphatase (FaGIPP) genes, and transcription of the L-galactono-1,4-lactone dehydrogenase (FaGLDH) and FAMDHAR genes was higher in the ozone-stored than in the air-stored fruits. Analysis of AsA content in a segregating population from two strawberry cultivars showed high variability, which did not correlate with the transcription of any of the genes studied. Study of GalUR protein in diverse cultivars of strawberry and different Fragaria species showed that a correlation between GalUR and AsA content was apparent in most cases, but it was not general. Three alleles were identified in strawberry, but any sequence effect on the AsA variability was eliminated by analysis of the allele-specific expression. Taken together, these results indicate that FaGalUR shares the control of AsA levels with other enzymes and regulatory elements in strawberry fruit. PMID:21561953

Formaldehyde as hypothetical primer of biohomochirality

NASA Astrophysics Data System (ADS)

Goldanskii, Vitalii I.

1996-07-01

One of the most intriguing and crucial problems of the prebiotic evolution and the origin of life is the explanation of the origin of biohomochirality. A scheme of conversions originated by formaldehyde (FA) as hypothetical primer of biohomochirality is proposed. The merit of FA as executor of this function is based -inter alia - on the distinguished role of FA as one of the earliest and simplest molecules in both warm, terrestrial and cold, extraterrestrial scenarios of the origin of life. The confirmation of the role of FA as primer of biohomochirality would support the option of an RNA world as an alternative to the protein world. The suggested hypothesis puts forward for the first time a concrete sequence of chemical reactions which can lead to biohomochirality. The spontaneous breaking of the mirror symmetry is secured by the application of the well-known Frank scheme (combination of autocatalysis and ``annihilation'' of L and D enantiomers) to the series of interactions of FA ``trimers'' (i.e. C3H6O3 compounds) of (aaa), (apa) and (app) types, where the monomeric groups (a) means ``achirons'' (a=CHn, n>=2 and C=M, M=C,O) and (p) mean ``prochirons'' (p=HC*OM, M=H,C).

Heterozygote FANCD2 mutations associated with childhood T Cell ALL and testicular seminoma.

PubMed

Smetsers, Stephanie; Muter, Joanne; Bristow, Claire; Patel, Leena; Chandler, Kate; Bonney, Denise; Wynn, Robert F; Whetton, Anthony D; Will, Andrew M; Rockx, Davy; Joenje, Hans; Strathdee, Gordon; Shanks, Jonathan; Klopocki, Eva; Gille, Johan J P; Dorsman, Josephine; Meyer, Stefan

2012-12-01

Fanconi anaemia (FA) is an inherited disease with congenital and developmental abnormalities characterised by cellular cross linker hypersensitivity. FA is caused by mutations in any of so far 15 identified FANC genes, which encode proteins that interact in a common DNA damage response (DDR) pathway. Individuals with FA have a high risk of developing acute myeloid leukaemia (AML) and squamous cell carcinoma. An increased cancer risk has been firmly established for carriers of mutations in FANCD1/BRCA2, FANCJ/BRIP1, FANCN/PALB2, RAD51C/FANCO and link the FA pathway to inherited breast and ovarian cancer. We describe a pedigree with FANCD2 mutations c.458T > C (p.Leu153Ser) and c.2715 + 1G > A (p.Glu906LeufsX4) with mild phenotype FA in the index case, T cell ALL in the Leu153Ser heterozygous brother and testicular seminoma in the p.Glu906LeufsX4 heterozygous father. Both FANCD2 alleles were present in the T Cell ALL and the seminoma. This links specific FANCD2 mutations to T cell ALL and seminoma without evidence of allelic loss in the tumour tissue.

Seed-specific overexpression of AtFAX1 increases seed oil content in Arabidopsis.

PubMed

Tian, Yinshuai; Lv, Xueyan; Xie, Guilan; Zhang, Jing; Xu, Ying; Chen, Fang

2018-06-02

Biosynthesis of plant seed oil is accomplished through the coordinate action of multiple enzymes in multiple subcellular compartments. Fatty acid (FA) has to be transported from plastid to endoplasmic reticulum (ER) for TAG synthesis. However, the role of plastid FA transportation during seed oil accumulation has not been evaluated. AtFAX1 (Arabidopsis fatty acid export1) mediated the FA export from plastid. In this study, we overexpressed AtFAX1 under the control of a seed specific promoter in Arabidopsis. The resultant overexpression lines (OEs) produced seeds which contained 21-33% more oil and 24-30% more protein per seed than those of the wild type (WT). The increased oil content was probably because of the enhanced FA and TAG synthetic activity. The seed size and weight were both increased accordingly. In addition, the seed number per silique and silique number per plant had no changes in transgenic plants. Taken together, our results demonstrated that seed specific overexpression of AtFAX1 could promote oil accumulation in Arabidopsis seeds and manipulating FA transportation is a feasible strategy for increasing the seed oil content. Copyright © 2018 Elsevier Inc. All rights reserved.

Fatty acid profiles and adipogenic gene expression of various fat depots in Japanese Black and Holstein steers.

PubMed

Shirouchi, Bungo; Albrecht, Elke; Nuernberg, Gerd; Maak, Steffen; Olavanh, Samadmanivong; Nakamura, Yoshinori; Sato, Masao; Gotoh, Takafumi; Nuernberg, Karin

2014-01-01

Objective of the study was to assess the breed effect on fatty acid (FA) composition of different adipose tissues and on mRNA expression of genes involved in adipogenesis and fat metabolism. Japanese Black (JB) and Holstein (HS) steers were kept under equivalent conditions with high energy intake resulting in large differences in intramuscular fat (IMF) accumulation in longissimus muscle (LM). The relative FA composition of muscle, intermuscular fat, visceral fat, and perirenal fat was comparable between JB and HS steers. Circulating fatty acids were also similar in both breeds. Most relevant breed effects were identified in IMF, underlining the uniqueness of this adipose tissue site. JB steers had more monounsaturated FA and less saturated FA. Perilipin 1 and adipose differentiation-related protein (ADFP) mRNA levels were higher in IMF of JB. The results suggest advanced maturity of IMF cells in JB and altered local conditions in muscle influencing IMF accumulation and composition. © 2013.

Zein nanoparticles as delivery systems for covalently linked and physically entrapped folic acid

NASA Astrophysics Data System (ADS)

Chuacharoen, Thanida; Sabliov, Cristina M.

2017-02-01

Zein nanoparticles covalently linked to folic acid were hypothesized to sustain the release of the folic acid in addition to targeting cancer cells overexpressing folate-binding receptors, whereas zein nanoparticles with physically entrapped folic acid would only be able to control the release of the bioactive without targeting of cancer cells. The two types of particles, folic acid covalently linked zein nanoparticles (ZN-FA nps) and zein nanoparticles with entrapped folic acid (ZN(FA) nps), were synthesized and the covalent link between folic acid and zein was assessed by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (1H NMR). Their size, polydispersity index, zeta potential, morphology, and loading capacity were evaluated by dynamic light scattering (DLS), transmission electron microscopy (TEM), and spectrophotometric technique. The release studies of the folic acid preformed in phosphate-buffered saline (PBS) at 37 °C for 7 days concluded that the release of the loaded folic acid was sustained over 7 days for both systems. The cytotoxicity was investigated using a methyl thiazolyl tetrazolium (MTT) assay, and the results showed that zein nanoparticles were biocompatible to HeLa (an overexpressing folate receptor cells) and A549 (a deficient folate receptor cells) cells, which have different levels of folate receptors on surface and both folic acid nanoparticle systems were able to diminish the adverse toxic effect of folic acid to cells. The increased uptake of ZN-FA nps relative to ZN(FA) nps supported the use of ZN-FA nps as targeting nanoagents to cells overexpressing folate receptors.

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

PubMed

Botthof, Jan Gregor; Bielczyk-Maczyńska, Ewa; Ferreira, Lauren; Cvejic, Ana

2017-05-30

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

Mechanism and DNA-based detection of field-evolved resistance to transgenic Bt corn in fall armyworm (Spodoptera frugiperda)

USDA-ARS?s Scientific Manuscript database

Evolution of resistance threatens sustainability of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). The fall armyworm is a devastating pest controlled by transgenic Bt corn producing the Cry1Fa insecticidal protein. However, fall armyworm populations ...

Resveratrol Enhances Palmitate-Induced ER Stress and Apoptosis in Cancer Cells

PubMed Central

Rojas, Cristina; Pan-Castillo, Belén; Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Arola, Lluis; Mulero, Miquel

2014-01-01

Background Palmitate, a saturated fatty acid (FA), is known to induce toxicity and cell death in various types of cells. Resveratrol (RSV) is able to prevent pathogenesis and/or decelerate the progression of a variety of diseases. Several in vitro and in vivo studies have also shown a protective effect of RSV on fat accumulation induced by FAs. Additionally, endoplasmic reticulum (ER) stress has recently been linked to cellular adipogenic responses. To address the hypothesis that the RSV effect on excessive fat accumulation promoted by elevated saturated FAs could be partially mediated by a reduction of ER stress, we studied the RSV action on experimentally induced ER stress using palmitate in several cancer cell lines. Principal Findings We show that, unexpectedly, RSV promotes an amplification of palmitate toxicity and cell death and that this mechanism is likely due to a perturbation of palmitate accumulation in the triglyceride form and to a less important membrane fluidity variation. Additionally, RSV decreases radical oxygen species (ROS) generation in palmitate-treated cells but leads to enhanced X-box binding protein-1 (XBP1) splicing and C/EBP homologous protein (CHOP) expression. These molecular effects are induced simultaneously to caspase-3 cleavage, suggesting that RSV promotes palmitate lipoapoptosis primarily through an ER stress-dependent mechanism. Moreover, the lipotoxicity reversion induced by eicosapentaenoic acid (EPA) or by a liver X receptor (LXR) agonist reinforces the hypothesis that RSV-mediated inhibition of palmitate channeling into triglyceride pools could be a key factor in the aggravation of palmitate-induced cytotoxicity. Conclusions Our results suggest that RSV exerts its cytotoxic role in cancer cells exposed to a saturated FA context primarily by triglyceride accumulation inhibition, probably leading to an intracellular palmitate accumulation that triggers a lipid-mediated cell death. Additionally, this cell death is promoted by ER stress through a CHOP-mediated apoptotic process and may represent a potential anticancer strategy. PMID:25436452

Molecular-receptor-specific, non-toxic, near-infrared-emitting Au cluster-protein nanoconjugates for targeted cancer imaging

NASA Astrophysics Data System (ADS)

Retnakumari, Archana; Setua, Sonali; Menon, Deepthy; Ravindran, Prasanth; Muhammed, Habeeb; Pradeep, Thalappil; Nair, Shantikumar; Koyakutty, Manzoor

2010-02-01

Molecular-receptor-targeted imaging of folate receptor positive oral carcinoma cells using folic-acid-conjugated fluorescent Au25 nanoclusters (Au NCs) is reported. Highly fluorescent Au25 clusters were synthesized by controlled reduction of Au+ ions, stabilized in bovine serum albumin (BSA), using a green-chemical reducing agent, ascorbic acid (vitamin-C). For targeted-imaging-based detection of cancer cells, the clusters were conjugated with folic acid (FA) through amide linkage with the BSA shell. The bioconjugated clusters show excellent stability over a wide range of pH from 4 to 14 and fluorescence efficiency of ~5.7% at pH 7.4 in phosphate buffer saline (PBS), indicating effective protection of nanoclusters by serum albumin during the bioconjugation reaction and cell-cluster interaction. The nanoclusters were characterized for their physico-chemical properties, toxicity and cancer targeting efficacy in vitro. X-ray photoelectron spectroscopy (XPS) suggests binding energies correlating to metal Au 4f7/2~83.97 eV and Au 4f5/2~87.768 eV. Transmission electron microscopy and atomic force microscopy revealed the formation of individual nanoclusters of size ~1 nm and protein cluster aggregates of size ~8 nm. Photoluminescence studies show bright fluorescence with peak maximum at ~674 nm with the spectral profile covering the near-infrared (NIR) region, making it possible to image clusters at the 700-800 nm emission window where the tissue absorption of light is minimum. The cell viability and reactive oxygen toxicity studies indicate the non-toxic nature of the Au clusters up to relatively higher concentrations of 500 µg ml-1. Receptor-targeted cancer detection using Au clusters is demonstrated on FR+ve oral squamous cell carcinoma (KB) and breast adenocarcinoma cell MCF-7, where the FA-conjugated Au25 clusters were found internalized in significantly higher concentrations compared to the negative control cell lines. This study demonstrates the potential of using non-toxic fluorescent Au nanoclusters for the targeted imaging of cancer.

Intake of specific fatty acids and fat alters growth, health, and titers following vaccination in dairy calves.

PubMed

Esselburn, K M; O'Diam, K M; Hill, T M; Bateman, H G; Aldrich, J M; Schlotterbeck, R L; Daniels, K M

2013-09-01

Typical fatty acid profiles of milk and milk replacer (MR) differ. Calf MR in the United States are made from animal fat, which are low in short- and medium-chain fatty acids and linolenic acid. Two 56-d trials compared a control MR containing 27% crude protein and formulated with 3 fat and fatty acid compositions. The 3 MR treatments were (1) only animal fat totaling 17% fat (CON), (2) animal fat supplemented with butyrate, medium-chain fatty acids, and linolenic acid using a commercial product (1.25% NeoTec4 MR; Provimi North America, Brookville, OH) totaling 17% fat (fatty acid-supplemented; FA-S), and (3) milk fat totaling 33% fat (MF). The MR were fed at 660 g of dry matter from d 0 to 42 and weaned. Starter (20% crude protein) and water were fed ad libitum for 56 d. Trial 1 utilized Holstein calves (24 female, 24 male) during summer months and trial 2 utilized Holstein calves (48 male) during fall months. Calves (41±1 kg of initial body weight; 2 to 3d of age) were sourced from a single farm and housed in a naturally ventilated nursery without added heat. Calves were in individual pens with straw bedding. Calf was the experimental unit. Data for each trial were analyzed as a completely randomized design with a 3 (MR treatment) × 2 (sex) factorial arrangement of treatments in trial 1 with repeated measures and as a completely randomized design with 3 MR treatments in trial 2 with repeated measures. Preplanned contrast statements of treatments CON versus FA-S and CON versus MF were used to separate means. We found no interactions of MR treatment by sex. Calf average daily gain, hip width change, and feed efficiency differed (CONFA-S). Titers to bovine respiratory parainfluenza-3 and bovine virus diarrhea type 1 (vaccinations to these pathogens were on d 7 and 28) in serum samples taken on d 49 and 56 differed (CONFA-S; CONFA-S; CON>MF). Calves fed FA-S and MF had improved growth and feed efficiency compared with calves fed CON, whereas calves fed FA-S also had improved measurements related to health and immunity. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The characterization of protein release from sericin film in the presence of an enzyme: towards fibroblast growth factor-2 delivery.

PubMed

Nishida, Ayumu; Naganuma, Tsuyoshi; Kanazawa, Takanori; Takashima, Yuuki; Yamada, Masaki; Okada, Hiroaki

2011-07-29

Aqueous preparations of silk protein (sericin) films were prepared to evaluate their biodegradation properties. In the absence of trypsin, sericin film swelled rapidly, kept its shape, and remained unaltered for 28 days or longer due to form β-sheet structures. In the presence of trypsin, sericin film gradually degraded; since the rate depended on the concentration of trypsin, the films likely underwent enzymatic hydrolysis. Sericin film incorporating the model protein drug fluorescein isothiocyanate-albumin (FA) also gradually degraded in the presence of trypsin and resulted in the sustained release of FA for 2 weeks or longer; in contrast, FA release was quite slow in the absence of trypsin. It is expected that sericin film has potential as a biodegradable and drug-releasing carrier. To evaluate the practical applicability of sericin film for the repair of defective tissues, fibroblast growth factor-2 (FGF-2) was incorporated into sericin films and the films were implanted on skull defects in rats. Whereas FGF-2 release was suppressed in the absence of trypsin in vitro, it appears that FGF-2, immobilized by ionic interactions between sericin and FGF-2, can be sustained-released in vivo from films incorporating 2500 or 250 ng of FGF-2 to support the growth of tissue around wounds. Copyright © 2011 Elsevier B.V. All rights reserved.

Allergen-Specific Immunotherapies for Food Allergy

PubMed Central

Feuille, Elizabeth

2018-01-01

With rising prevalence of food allergy (FA), allergen-specific immunotherapy (AIT) for FA has become an active area of research in recent years. In AIT, incrementally increasing doses of inciting allergen are given with the goal to increase tolerance, initially through desensitization, which relies on regular exposure to allergen. With prolonged therapy in some subjects, AIT may induce sustained unresponsiveness, in which tolerance is retained after a period of allergen avoidance. Methods of AIT currently under study in humans include oral, sublingual, epicutaneous, and subcutaneous delivery of modified allergenic protein, as well as via DNA-based vaccines encoding allergen with lysosomal-associated membrane protein I. The balance of safety and efficacy varies by type of AIT, as well as by targeted allergen. Age, degree of sensitization, and other comorbidities may affect this balance within an individual patient. More recently, AIT with modified proteins or combined with immunomodulatory therapies has shown promise in making AIT safer and/or more effective. Though methods of AIT are neither currently advised by experts (oral immunotherapy [OIT]) nor widely available, AIT is likely to become a part of recommended management of FA in the coming years. Here, we review and compare methods of AIT currently under study in humans to prepare the practitioner for an exciting new phase in the care of food allergic patients in which improved tolerance to inciting foods will be a real possibility. PMID:29676066

Cyanidin-3-glucoside and its phenolic acid metabolites attenuate visible light-induced retinal degeneration in vivo via activation of Nrf2/HO-1 pathway and NF-κB suppression.

PubMed

Wang, Yong; Huo, Yazhen; Zhao, Liang; Lu, Feng; Wang, Ou; Yang, Xue; Ji, Baoping; Zhou, Feng

2016-07-01

Cyanidin-3-glucoside (C3G) is a major anthocyanin in berries and a potential nutritional supplement for preventing retinal degeneration. However, the protective mechanism of C3G and its metabolites, protocatechuic acid (PCA) and ferulic acid (FA), remain unclear. The molecular mechanisms of C3G and its metabolites against retinal photooxidative damage in vivo are investigated. Pigmented rabbits were orally administered C3G, PCA, and FA (0.11 mmol/kg/day) for 3 weeks. Electroretinography, histological analysis, and TUNEL assay showed that C3G and its metabolites attenuated retinal cell apoptosis. The expression of oxidative stress markers were upregulated after light exposure but attenuated by C3G and FA, which may be attributed to the elevated secretion and expression of heme oxygenase (HO-1) and nuclear factor erythroid-2 related factor 2 (Nrf2). C3G, PCA, and FA attenuated the secretion or expression of inflammation-related genes; FA suppressed nuclear factor kappa B (NF-κB) activation. The treatments attenuated the light-induced changes on certain apoptotic proteins and angiogenesis-related cytokines. C3G and FA reduced light-induced retinal oxidative stress by activating the Nrf2/HO-1 antioxidant pathway. FA attenuated the light-induced retinal inflammation by suppressing NF-κB activation. C3G and its metabolites attenuated the photooxidation-induced apoptosis and angiogenesis in the retina. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The ubiquitin family meets the Fanconi anemia proteins.

PubMed

Renaudin, Xavier; Koch Lerner, Leticia; Menck, Carlos Frederico Martins; Rosselli, Filippo

2016-01-01

Fanconi anaemia (FA) is a hereditary disorder characterized by bone marrow failure, developmental defects, predisposition to cancer and chromosomal abnormalities. FA is caused by biallelic mutations that inactivate genes encoding proteins involved in replication stress-associated DNA damage responses. The 20 FANC proteins identified to date constitute the FANC pathway. A key event in this pathway involves the monoubiquitination of the FANCD2-FANCI heterodimer by the collective action of at least 10 different proteins assembled in the FANC core complex. The FANC core complex-mediated monoubiquitination of FANCD2-FANCI is essential to assemble the heterodimer in subnuclear, chromatin-associated, foci and to regulate the process of DNA repair as well as the rescue of stalled replication forks. Several recent works have demonstrated that the activity of the FANC pathway is linked to several other protein post-translational modifications from the ubiquitin-like family, including SUMO and NEDD8. These modifications are related to DNA damage responses but may also affect other cellular functions potentially related to the clinical phenotypes of the syndrome. This review summarizes the interplay between the ubiquitin and ubiquitin-like proteins and the FANC proteins that constitute a major pathway for the surveillance of the genomic integrity and addresses the implications of their interactions in maintaining genome stability. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of major and trace minerals, fatty acid composition, and cholesterol content of Protected Designation of Origin cheeses.

PubMed

Manuelian, C L; Currò, S; Penasa, M; Cassandro, M; De Marchi, M

2017-05-01

Cheese provides essential nutrients for human nutrition and health, such as minerals and fatty acids (FA). Its composition varies according to milk origin (e.g., species and breed), rearing conditions (e.g., feeding and management), and cheese-making technology (e.g., coagulation process, addition of salt, ripening period). In recent years, cheese production has increased worldwide. Italy is one of the main producers and exporters of cheese. This study aimed to describe mineral, FA, and cholesterol content of 133 samples from 18 commercial cheeses from 4 dairy species (buffalo, cow, goat, and sheep) and from 3 classes of moisture content (hard, <35% moisture; semi-hard, 35-45%; and soft, >45%). Mineral concentrations of cheese samples were determined by inductively coupled plasma optical emission spectrometry, and FA and cholesterol contents were determined by gas chromatography. Moisture and species had a significant effect on almost all traits: the highest levels of Na, Ca, and Fe were found in cheeses made from sheep milk; the greatest level of Cu was found in cow milk cheese, the lowest amount of K was found in buffalo milk cheese, and the lowest amount of Zn was found in goat cheeses. In all samples, Cr and Pb were not detected (below the level of detection). In general, total fat, protein, and minerals significantly increased when the moisture decreased. Buffalo and goat cheeses had the highest saturated FA content, and sheep cheeses showed the highest content of unsaturated and polyunsaturated FA, conjugated linoleic acid, and n-3 FA. Goat and sheep cheeses achieved higher proportions of minor FA than did cow and buffalo cheeses. Buffalo cheese exhibited the lowest cholesterol level. Our results confirm that cheese mineral content is mainly affected by the cheese-making process, whereas FA profile mainly reflects the FA composition of the source milk. This study allowed the characterization of mineral and FA composition and cholesterol content and revealed large variability among different commercial cheeses. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Effects of pistachio by-products on digestibility, milk production, milk fatty acid profile and blood metabolites in Saanen dairy goats.

PubMed

Sedighi-Vesagh, R; Naserian, A A; Ghaffari, M H; Petit, H V

2015-08-01

The objective of this study was to investigate the effects of pistachio by-products (PBP) on nutrient digestibility, blood metabolites and milk fatty acid (FA) profile in Saanen dairy goats. Nine multiparous lactating Saanen goats (on day 90 post-partum, 45 ± 2/kg BW) were randomly assigned to a 3 × 3 Latin square design with three treatment diets: 1) control diet (alfalfa hay based), 2) 32% PBP and 3) 32% PBP + polyethylene glycol (PEG-4000; 1 g/kg dry matter). Each period lasted 21 days, including 14 day for treatment adaptation and 7 day for data collection. Pistachio by-products significantly decreased (p < 0.01) crude protein (CP) digestibility compared with the control diet (64.4% vs. 58.7%), but PEG addition did not differ for CP digestibility of goats fed 32% PBP + PEG and those fed the two other diets. The digestibility of NDF tended (p = 0.06) to decrease for goats fed PBP compared with those fed the control diet. Yields of milk and 4% fat-corrected milk were not affected by dietary treatments. Compared with the control diet, PBP supplementation appreciably changed the proportions of almost all the milk FA measured; the main effects were decreases (p < 0.01) in FA from 8:0 to 16:0 and increases (p < 0.01) proportions of cis-9, trans-11 18:2 and trans-11 18:1, monounsaturated FA, polyunsaturated FA and long-chain FA. The saturated FA, short-chain FA and medium-chain FA proportions were lower (p < 0.01) in goats fed the two PBP supplemented diet than in those fed the control diet and PEG addition led to intermediate proportions of saturated FA, unsaturated and monounsaturated FA. Inclusion of PBP in the diet decreased (p < 0.01) plasma concentrations of glucose and urea nitrogen compared with the control diet. It was concluded that PBP can be used as forage in the diet of dairy goats without interfering with milk yield. Inclusion of 32% PBP in the diet of dairy goats had beneficial effects on milk FA profile but PEG addition to PBP did not contribute to enhance further milk FA profile. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Modulation of C-reactive protein and plasma omega-6 fatty acid levels by phospholipase A2 gene polymorphisms following a 6-week supplementation with fish oil.

PubMed

Tremblay, B L; Rudkowska, I; Couture, P; Lemieux, S; Julien, P; Vohl, M C

2015-12-01

This clinical trial investigated the impact of a six-week supplementation with fish oil and single nucleotide polymorphisms (SNPs) in PLA2G4A and PLA2G6 genes on total omega-6 fatty acid (n-6 FA) levels in plasma phospholipids (PL) and plasma C-reactive protein (CRP) levels in 191 subjects. Interaction effects between SNPs and supplementation modulated total n-6 FAs and CRP levels in both men and women. Associations between SNPs and total n-6 FA levels and between SNPs and CRP levels were identified in men, independently of supplementation. Supplementation decreased total n-6 FAs without affecting plasma CRP levels. Changes in CRP levels correlated positively with changes in total n-6 FAs in men (r=0.25 p=0.01), but not in women. In conclusion, total n-6 FA levels in plasma PL and plasma CRP levels are modulated by SNPs within PLA2G4A and PLA2G6 genes alone or in combination with fish oil supplementation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of partly replacing dietary starch with fiber and fat on milk production and energy partitioning.

PubMed

Boerman, J P; Potts, S B; VandeHaar, M J; Lock, A L

2015-10-01

The effects of partly replacing dietary starch with fiber and fat to provide a diet with similar net energy for lactation (NEL) density on yields of milk and milk components and on energy partitioning were evaluated in a crossover design experiment. Holstein cows (n = 32; 109 ± 22 d in milk, mean ± standard deviation) were randomly assigned to treatment sequence. Treatments were a high-starch diet containing 33% corn grain (mixture of dry ground and high-moisture corn; HS) or a high-fiber, high-fat diet containing 2.5% palmitic acid-enriched fatty acid (FA) supplement (HFF). Diets contained corn silage, alfalfa silage, and wheat straw as forage sources; HS contained 32% starch, 3.2% FA, and 25% neutral detergent fiber, whereas HFF contained 16% starch, 5.4% FA, and 33% neutral detergent fiber. Compared with HS, the HFF treatment reduced milk yield, milk protein concentration, and milk protein yield, but increased milk fat concentration, milk fat yield, milk energy output, and milk to feed ratio (energy-corrected milk/dry matter intake). The HFF treatment reduced the yield of de novo synthesized (< 16-carbon) milk FA and increased the yield of 16-carbon milk FA. Yield of preformed (> 16-carbon) milk FA was not different. The HFF treatment increased plasma concentrations of triglycerides and nonesterified fatty acids, but decreased plasma concentration of insulin. Compared with HS, the HFF treatment reduced body weight gain, change in body condition score, and fat thickness over the rump and rib. Calculated body energy gain, as a fraction of NEL use, was less for HFF than HS, whereas milk energy as a fraction of NEL use was increased for HFF. We concluded that the 2 treatments resulted in similar apparent NEL densities and intakes, but the HS treatment partitioned more energy toward body gain whereas the HFF treatment partitioned more energy toward milk. A high-fiber, high-fat diet might diminish the incidence of over conditioning in mid-lactation cows while maintaining high milk production. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Natural organic matter and the event horizon of mass spectrometry.

PubMed

Hertkorn, N; Frommberger, M; Witt, M; Koch, B P; Schmitt-Kopplin, Ph; Perdue, E M

2008-12-01

Soils, sediments, freshwaters, and marine waters contain natural organic matter (NOM), an exceedingly complex mixture of organic compounds that collectively exhibit a nearly continuous range of properties (size-reactivity continuum). NOM is composed mainly of carbon, hydrogen, and oxygen, with minor contributions from heteroatoms such as nitrogen, sulfur, and phosphorus. Suwannee River fulvic acid (SuwFA) is a fraction of NOM that is relatively depleted in heteroatoms. Ultrahigh resolution Fourier transform ion cyclotron (FTICR) mass spectra of SuwFA reveal several thousand molecular formulas, corresponding in turn to several hundred thousand distinct chemical environments of carbon even without accountancy of isomers. The mass difference deltam among adjoining C,H,O-molecules between and within clusters of nominal mass is inversely related to molecular dissimilarity: any decrease of deltam imposes an ever growing mandatory difference in molecular composition. Molecular formulas that are expected for likely biochemical precursor molecules are notably absent from these spectra, indicating that SuwFA is the product of diagenetic reactions that have altered the major components of biomass beyond the point of recognition. The degree of complexity of SuwFA can be brought into sharp focus through comparison with the theoretical limits of chemical complexity, as constrained and quantized by the fundamentals of chemical binding. The theoretical C,H,O-compositional space denotes the isomer-filtered complement of the entire, very vast space of molecular structures composed solely of carbon, hydrogen, and oxygen. The molecular formulas within SuwFA occupy a sizable proportion of the theoretical C,H,O-compositional space. A 100 percent coverage of the theoretically feasible C,H,O-compositional space by SuwFA molecules is attained throughout a sizable range of mass and H/C and O/C elemental ratios. The substantial differences between (and complementarity of) the SuwFA molecular formulas that are observed using six different modes of ionization (APCI, APPI, and ESI in positive and negative modus) imply considerable selectivity of the ionization process and suggest that the observed mass spectra represent simplified projections of still more complex mixtures.

In situ crosslinked smart polypeptide nanoparticles for multistage responsive tumor-targeted drug delivery

NASA Astrophysics Data System (ADS)

Yi, Huqiang; Liu, Peng; Sheng, Nan; Gong, Ping; Ma, Yifan; Cai, Lintao

2016-03-01

Smart tumor-targeted drug delivery is crucial for improving the effect of chemotherapy and reducing the adverse effects. Here, we synthesized a smart polypeptide copolymer based on n-butylamine-poly(l-lysine)-b-poly(l-cysteine) (PLL-PLC) with functionalization of folic acid (FA) and 1,2-dicarboxylic-cyclohexene anhydride (DCA) for multistage responsive tumor-targeted drug delivery. The copolymers (FA-PLL(DCA)-PLC) spontaneously crosslinked in situ to form redox and pH dual responsive FA-PLL(DCA)-PLC nanoparticles (FD-NPs), which had a reversible zeta potential around -30 mV at pH 7.4, but switched to +15 mV at pH 5.0. Moreover, FD-NPs effectively loaded DOX with a loading capacity at 15.7 wt%. At pH 7.4, only 24.5% DOX was released within 60 h. However, at pH 5.0, the presence of 10 mM DTT dramatically accelerated DOX release with over 90% of DOX released within 10 h. Although the FD-NPs only enhanced DOX uptake in FA receptor positive (FR+) cancer cells at pH 7.4, a weak acidic condition promoted FD-NP-facilitated DOX uptake in both FR+ HeLa and FR- A549 cells, as well as significantly improving cellular binding and end/lysosomal escape. In vivo studies in a HeLa cancer model demonstrated that the charge-reversible FD-NPs delivered DOX into tumors more effectively than charge-irreversible nanoparticles. Hence, these multistage responsive FD-NPs would serve as highly efficient drug vectors for targeted cancer chemotherapy.Smart tumor-targeted drug delivery is crucial for improving the effect of chemotherapy and reducing the adverse effects. Here, we synthesized a smart polypeptide copolymer based on n-butylamine-poly(l-lysine)-b-poly(l-cysteine) (PLL-PLC) with functionalization of folic acid (FA) and 1,2-dicarboxylic-cyclohexene anhydride (DCA) for multistage responsive tumor-targeted drug delivery. The copolymers (FA-PLL(DCA)-PLC) spontaneously crosslinked in situ to form redox and pH dual responsive FA-PLL(DCA)-PLC nanoparticles (FD-NPs), which had a reversible zeta potential around -30 mV at pH 7.4, but switched to +15 mV at pH 5.0. Moreover, FD-NPs effectively loaded DOX with a loading capacity at 15.7 wt%. At pH 7.4, only 24.5% DOX was released within 60 h. However, at pH 5.0, the presence of 10 mM DTT dramatically accelerated DOX release with over 90% of DOX released within 10 h. Although the FD-NPs only enhanced DOX uptake in FA receptor positive (FR+) cancer cells at pH 7.4, a weak acidic condition promoted FD-NP-facilitated DOX uptake in both FR+ HeLa and FR- A549 cells, as well as significantly improving cellular binding and end/lysosomal escape. In vivo studies in a HeLa cancer model demonstrated that the charge-reversible FD-NPs delivered DOX into tumors more effectively than charge-irreversible nanoparticles. Hence, these multistage responsive FD-NPs would serve as highly efficient drug vectors for targeted cancer chemotherapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07348k

Functional assignment to JEV proteins using SVM.

PubMed

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

Functional assignment to JEV proteins using SVM

PubMed Central

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP). PMID:19052658

Invariant natural killer T cells from children with versus without food allergy exhibit differential responsiveness to milk-derived sphingomyelin.

PubMed

Jyonouchi, Soma; Abraham, Valsamma; Orange, Jordan S; Spergel, Jonathan M; Gober, Laura; Dudek, Emily; Saltzman, Rushani; Nichols, Kim E; Cianferoni, Antonella

2011-07-01

A key immunologic feature of food allergy (FA) is the presence of a T(h)2-type cytokine bias. Ligation of the invariant natural killer T cell (iNKT) T-cell receptor (TCR) by sphingolipids presented via the CD1d molecule leads to copious secretion of T(h)2-type cytokines. Major food allergens (eg, milk, egg) are the richest dietary source of sphingolipids (food-derived sphingolipids [food-SLs]). Nonetheless, the role of iNKTs in FA is unknown. To investigate the role of iNKTs in FA and to assess whether food-SL-CD1d complexes can engage the iNKT-TCR and induce iNKT functions. PBMCs from 15 children with cow's milk allergy (MA), 12 children tolerant to cow's milk but with allergy to egg, and 13 healthy controls were incubated with α-galactosylceramide (αGal), cow's milk-sphingomyelin, or hen's egg-ceramide. iNKTs were quantified, and their cytokine production and proliferation were assessed. Human CD1d tetramers loaded with milk-sphingomyelin or egg-ceramide were used to determine food-SL binding to the iNKT-TCR. Milk-sphingomyelin, but not egg-ceramide, can engage the iNKT-TCR and induce iNKT proliferation and T(h)2-type cytokine secretion. Children with FA, especially those with MA, had significantly fewer peripheral blood iNKTs and their iNKTs exhibited a greater T(h)2 response to αGal and milk-sphingomyelin than iNKTs of healthy controls. iNKTs from children with FA, especially those with MA, are reduced in number and exhibit a T(h)2 bias in response to αGal and milk-sphingomyelin. These data suggest a potential role for iNKTs in FA. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Acid-base properties of humic and fulvic acids formed during composting.

PubMed

Plaza, César; Senesi, Nicola; Polo, Alfredo; Brunetti, Gennaro

2005-09-15

The soil acid-base buffering capacity and the biological availability, mobilization, and transport of macro- and micronutrients, toxic metal ions, and xenobiotic organic cations in soil are strongly influenced by the acid-base properties of humic substances, of which humic and fulvic acids are the major fractions. For these reasons, the proton binding behavior of the humic acid-like (HA) and fulvic acid-like (FA) fractions contained in a compost are believed to be instrumental in its successful performance in soil. In this work, the acid-base properties of the HAs and FAs isolated from a mixture of the sludge residue obtained from olive oil mill wastewater (OMW) evaporated in an open-air pond and tree cuttings (TC) at different stages of composting were investigated by a current potentiometric titration method and the nonideal competitive adsorption (NICA)-Donnan model. The NICA-Donnan model provided an excellent description of the acid-base titration data, and pointed out substantial differences in site density and proton-binding affinity between the HAs and FAs examined. With respect to FAs, HAs were characterized by a smaller content of carboxylic- and phenolic-type groups and their larger affinities for proton binding. Further, HAs featured a greater heterogeneity in carboxylic-type groups than FAs. The composting process increased the content and decreased the proton affinity of carboxylic- and phenolic-type groups of HAs and FAs, and increased the heterogeneity of phenolic-type groups of HAs. As a whole, these effects indicated that the composting process could produce HA and FA fractions with greater cation binding capacities. These results suggest that composting of organic materials improves their agronomic and environmental value by increasing their potential to retain and exchange macro- and micronutrients, and to reduce the bioavailability of organic and inorganic pollutants.

Myoinhibiting peptides are the ancestral ligands of the promiscuous Drosophila sex peptide receptor

USDA-ARS?s Scientific Manuscript database

Male insects change behaviors of female partners by co-transferring accessory gland proteins (Acps) like sex peptide (SP), with their sperm. The Drosophila sex peptide receptor (SPR) is a G protein-coupled receptor expressed in the female’s nervous system and genital tract. While most Acps show a fa...

Fanconi anemia FANCD2 and FANCI proteins regulate the nuclear dynamics of splicing factors.

PubMed

Moriel-Carretero, María; Ovejero, Sara; Gérus-Durand, Marie; Vryzas, Dimos; Constantinou, Angelos

2017-12-04

Proteins disabled in the cancer-prone disorder Fanconi anemia (FA) ensure the maintenance of chromosomal stability during DNA replication. FA proteins regulate replication dynamics, coordinate replication-coupled repair of interstrand DNA cross-links, and mitigate conflicts between replication and transcription. Here we show that FANCI and FANCD2 associate with splicing factor 3B1 (SF3B1), a key spliceosomal protein of the U2 small nuclear ribonucleoprotein (U2 snRNP). FANCI is in close proximity to SF3B1 in the nucleoplasm of interphase and mitotic cells. Furthermore, we find that DNA replication stress induces the release of SF3B1 from nuclear speckles in a manner that depends on FANCI and on the activity of the checkpoint kinase ATR. In chromatin, both FANCD2 and FANCI associate with SF3B1, prevent accumulation of postcatalytic intron lariats, and contribute to the timely eviction of splicing factors. We propose that FANCD2 and FANCI contribute to the organization of functional domains in chromatin, ensuring the coordination of DNA replication and cotranscriptional processes. © 2017 Moriel-Carretero et al.

Milk production and composition, nitrogen utilization, and grazing behavior of late-lactation dairy cows as affected by time of allocation of a fresh strip of pasture.

PubMed

Vibart, R E; Tavendale, M; Otter, D; Schwendel, B H; Lowe, K; Gregorini, P; Pacheco, D

2017-07-01

Eighty late-lactation dairy cows were used to examine the effects of allocating a new pasture strip of a sward based on ryegrass (Lolium perenne L.) in the morning (a.m.; ∼0730 h) or in the afternoon (p.m.; ∼1530 h) on milk production and composition, nitrogen (N) utilization, and grazing behavior. Cows grazed the same pasture strips for 24 h and were offered the same daily herbage allowance. Herbage composition differed among treatments; p.m. herbage had greater dry matter (DM; 22.7 vs. 19.9%), organic matter (OM; 89.5 vs. 88.9%), and water-soluble carbohydrate (10.9 vs. 7.6%) concentrations and lesser crude protein (20.5 vs. 22.2%) and neutral detergent fiber (48.8 vs. 50.4%) concentrations compared with a.m. herbage. Total fatty acids (FA), α-linolenic acid, and polyunsaturated FA (PUFA) were greater in a.m. herbage, whereas monounsaturated FA were greater in p.m. herbage. Estimates of herbage DM intake did not differ among treatments. Daily milk yields and milk fat and milk protein concentrations were similar among treatments, whereas milk fat (684 vs. 627 g/cow), milk protein (545 vs. 505 g/cow), and milk solids (milk fat + milk protein) yields (1,228 vs. 1,132 g/cow) tended to be greater for cows on p.m. herbage. Rumenic acid and total PUFA in milk were greater for cows on a.m. herbage, whereas oleic acid was greater for cows on p.m. herbage. Estimates of urinary N excretion (g/d) did not differ among treatments, but urinary N concentrations were greater for cows on a.m. herbage (5.85 vs. 5.36 g/L). Initial herbage mass (HM) available (kg of DM/ha) and instantaneous HM disappearance rates (kg of DM/ha and kg of DM/h) did not differ, but fractional disappearance rates (0.56 vs. 0.74 per hour for a.m. vs. p.m., respectively) differed. Under the current conditions, timing of pasture strip allocation altered the herbage nutrient supply to cows; allocating a fresh strip of pasture later in the day resulted in moderate increases in milk and milk solids yields in late-lactation dairy cows. Conversely, a greater concentration of precursor FA in a.m. herbage resulted in a greater concentration of beneficial FA in milk, compared with cows on p.m. herbage. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system.

PubMed

Reuter, T; Herterich, S; Bernhard, O; Hoehn, H; Gross, H J

2000-01-15

Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown. Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins. They also obtained evidence for a weak interaction between FANCA and FANCC. Neither FANCA nor FANCC was found to interact with itself. These results support the notion of a functional association between the FA gene products. (Blood. 2000;95:719-720)

Novel Biocatalysts Combining the Special Assembly Properties of S-Layer Proteins and the Functionality of Enzymes of Extremophiles (BIOCAT)

DTIC Science & Technology

2010-04-14

THE FUNCTIONALITY OF ENZYMES OF EXTREMOPHILES 5a. CONTRACT NUMBER 5b. GRANT NUMBER FA9550-07-1-0313 5c. PROGRAM ELEMENT NUMBER 61102F 6. AUTHOR... Enzymes of Extremophiles (BIOCAT) Agreement Award No.: FA9550-07-1-0313 Reporting period: April 15th. 2008 to April 14th, 2010 3Lo\\*p<\\\\%\\%, Prof...Status of effort 4 4. Nanobiotechnologie of S-layers 4 5. Biocat accomplishments 6 5.1. The S-layer-based enzyme immobilization system 6 5.2. S

21 CFR 866.5765 - Retinol-binding protein immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...

21 CFR 866.5765 - Retinol-binding protein immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...

21 CFR 866.5765 - Retinol-binding protein immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...

21 CFR 866.5765 - Retinol-binding protein immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Retinol-binding protein immunological test system....5765 Retinol-binding protein immunological test system. (a) Identification. A retinol-binding protein... the retinol-binding protein that binds and transports vitamin A in serum and urine. Measurement of...

The Fanconi anemia DNA damage repair pathway in the spotlight for germline predisposition to colorectal cancer.

PubMed

Esteban-Jurado, Clara; Franch-Expósito, Sebastià; Muñoz, Jenifer; Ocaña, Teresa; Carballal, Sabela; López-Cerón, Maria; Cuatrecasas, Miriam; Vila-Casadesús, Maria; Lozano, Juan José; Serra, Enric; Beltran, Sergi; Brea-Fernández, Alejandro; Ruiz-Ponte, Clara; Castells, Antoni; Bujanda, Luis; Garre, Pilar; Caldés, Trinidad; Cubiella, Joaquín; Balaguer, Francesc; Castellví-Bel, Sergi

2016-10-01

Colorectal cancer (CRC) is one of the most common neoplasms in the world. Fanconi anemia (FA) is a very rare genetic disease causing bone marrow failure, congenital growth abnormalities and cancer predisposition. The comprehensive FA DNA damage repair pathway requires the collaboration of 53 proteins and it is necessary to restore genome integrity by efficiently repairing damaged DNA. A link between FA genes in breast and ovarian cancer germline predisposition has been previously suggested. We selected 74 CRC patients from 40 unrelated Spanish families with strong CRC aggregation compatible with an autosomal dominant pattern of inheritance and without mutations in known hereditary CRC genes and performed germline DNA whole-exome sequencing with the aim of finding new candidate germline predisposition variants. After sequencing and data analysis, variant prioritization selected only those very rare alterations, producing a putative loss of function and located in genes with a role compatible with cancer. We detected an enrichment for variants in FA DNA damage repair pathway genes in our familial CRC cohort as 6 families carried heterozygous, rare, potentially pathogenic variants located in BRCA2/FANCD1, BRIP1/FANCJ, FANCC, FANCE and REV3L/POLZ. In conclusion, the FA DNA damage repair pathway may play an important role in the inherited predisposition to CRC.

Constitutive role of the Fanconi anemia D2 gene in the replication stress response.

PubMed

Tian, Yanyan; Shen, Xi; Wang, Rui; Klages-Mundt, Naeh L; Lynn, Erica J; Martin, Sara K; Ye, Yin; Gao, Min; Chen, Junjie; Schlacher, Katharina; Li, Lei

2017-12-08

In response to DNA cross-linking damage, the Fanconi anemia (FA) core complex activates the FA pathway by monoubiquitinating Fanconi anemia complementation group D2 (FANCD2) for the initiation of the nucleolytic processing of the DNA cross-links and stabilization of stalled replication forks. Given that all the classic FA proteins coordinately monoubiquitinate FANCD2, it is unclear why losses of individual classic FA genes yield varying cellular sensitivities to cross-linking damage. To address this question, we generated cellular knock-out models of FA core complex components and FANCD2 and found that FANCD2-null mutants display higher levels of spontaneous chromosomal damage and hypersensitivity to replication-blocking lesions than Fanconi anemia complementation group L (FANCL)-null mutants, suggesting that FANCD2 provides a basal level of DNA protection countering endogenous lesions in the absence of monoubiquitination. FANCD2's ubiquitination-independent function is likely involved in optimized recruitment of nucleolytic activities for the processing and protection of stressed replication forks. Our results reveal that FANCD2 has a ubiquitination-independent role in countering endogenous levels of replication stress, a function that is critical for the maintenance of genomic stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Food allergy in dogs and cats: a review.

PubMed

Verlinden, A; Hesta, M; Millet, S; Janssens, G P J

2006-01-01

Food allergy (FA) is defined as "all immune-mediated reactions following food intake," in contrast with food intolerance (FI), which is non-immune-mediated. Impairment of the mucosal barrier and loss of oral tolerance are risk factors for the development of FA. Type I, III, and IV hypersensitivity reactions are the most likely immunologic mechanisms. Food allergens are (glyco-)proteins with a molecular weight from 10-70 kDa and are resistant to treatment with heat, acid, and proteases. The exact prevalence of FA in dogs and cats remains unknown. There is no breed, sex or age predilection, although some breeds are commonly affected. Before the onset of clinical signs, the animals have been fed the offending food components for at least two years, although some animals are less than a year old. FA is a non-seasonal disease with skin and/or gastrointestinal disorders. Pruritus is the main complaint and is mostly corticoid-resistant. In 20-30% of the cases, dogs and cats have concurrent allergic diseases (atopy/flea-allergic dermatitis). A reliable diagnosis can only be made with dietary elimination-challenge trials. Provocation testing is necessary for the identification of the causative food component(s). Therapy of FA consists of avoiding the offending food component(s).

Monitoring methotrexate in clinical samples from cancer patients during chemotherapy with a LSPR-based competitive sensor.

PubMed

Zhao, Sandy Shuo; Bichelberger, Mathilde A; Colin, Damien Y; Robitaille, Robert; Pelletier, Joelle N; Masson, Jean-François

2012-10-21

A competitive binding assay based on localized surface plasmon resonance (LSPR) of folic acid-functionalized gold nanoparticles (FA-AuNPs) and human dihydrofolate reductase enzyme (hDHFR) was developed to detect nanomolar to micromolar concentrations of the widely applied anti-cancer drug, methotrexate (MTX). By the nature of the competitive assay for MTX, the LSPR shift from specific binding between FA-AuNPs and the free enzyme was inversely proportional to the concentration of MTX. In addition, the dynamic range for MTX was tuned from 10(-11) to 10(-6) M by varying the concentration of hDHFR from 1 to 100 nM. Inter-day reproducibility and recovery of MTX spiked in phosphate buffer saline (PBS) were excellent. Potential interferents such as FA, trimethoprim (TMP) and 4-amino-4-deoxy-N-methylpteroic acid (DAMPA) did not occur in the concentration range of interest for MTX. Clinical samples of human serum from patients undergoing MTX chemotherapy were analyzed following a simple solid-phase extraction step to isolate MTX from the serum matrix, with a limit of detection of 155 nM. Validation of the LSPR method was carried out in comparison to Fluorescence Polarization Immunoassay (FPIA), a commonly used method in clinical settings, and LC-MS/MS, a reference technique. The results of the LSPR competitive assay compared well to FPIA and LC-MS/MS, with a slope of 2.4 and 1.1, respectively, for the correlation plots. The method established herein is intended for therapeutic drug monitoring (TDM) of MTX levels in patients undergoing chemotherapy to ensure safety and efficacy of the treatment.

Rational design of cancer-targeted selenium nanoparticles to antagonize multidrug resistance in cancer cells.

PubMed

Liu, Ting; Zeng, Lilan; Jiang, Wenting; Fu, Yuanting; Zheng, Wenjie; Chen, Tianfeng

2015-05-01

Multidrug resistance is one of the greatest challenges in cancer therapy. Herein we described the synthesis of folate (FA)-conjugated selenium nanoparticles (SeNPs) as cancer-targeted nano-drug delivery system for ruthenium polypyridyl (RuPOP) exhibits strong fluorescence, which allows the direct imaging of the cellular trafficking of the nanosystem. This nanosystem could effectively antagonize against multidrug resistance in liver cancer. FA surface conjugation significantly enhanced the cellular uptake of SeNPs by FA receptor-mediated endocytosis through nystain-dependent lipid raft-mediated and clathrin-mediated pathways. The nanomaterials overcame the multidrug resistance in R-HepG2 cells through inhibition of ABC family proteins expression. Internalized nanoparticles triggered ROS overproduction and induced apoptosis by activating p53 and MAPKs pathways. Moreover, FA-SeNPs exhibited low in vivo acute toxicity, which verified the safety and application potential of FA-SeNPs as nanodrugs. This study provides an effective strategy for the design of cancer-targeted nanodrugs against multidrug resistant cancers. In the combat against hepatocellular carcinoma, multidrug resistance remains one of the obstacles to be overcome. The authors designed and synthesized folate (FA)-conjugated selenium nanoparticles (SeNPs) with enhanced cancer-targeting capability. This system carried ruthenium polypyridyl (RuPOP), an efficient metal-based anti-cancer drug with strong fluorescence. It was shown that this combination was effective in antagonizing against multidrug resistance in vitro. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of ferulic acid and γ-oryzanol on high-fat and high-fructose diet-induced metabolic syndrome in rats.

PubMed

Wang, Ou; Liu, Jia; Cheng, Qian; Guo, Xiaoxuan; Wang, Yong; Zhao, Liang; Zhou, Feng; Ji, Baoping

2015-01-01

The high morbidity of metabolic dysfunction diseases has heightened interest in seeking natural and safe compounds to maintain optimal health. γ-Oryzanol (OZ), the ferulic acid (FA) ester with phytosterols, mainly present in rice bran has been shown to improve markers of metabolic syndrome. This study investigates the effects of FA and OZ on alleviating high-fat and high-fructose diet (HFFD)-induced metabolic syndrome parameters. Male SD rats were fed with a regular rodent diet, HFFD, or HFFD supplemented with 0.05% FA or 0.16% OZ (equimolar concentrations) for 13 weeks. Food intake, organ indices, serum lipid profiles, glucose metabolism, insulin resistance (IR) index and cytokine levels were analyzed. The mechanisms were further investigated in oleic acid-stimulated HepG2 cells by analyzing triglyceride (TG) content and lipogenesis-related gene expressions. In the in vivo study, FA and OZ exhibited similar effects in alleviating HFFD-induced obesity, hyperlipidemia, hyperglycemia, and IR. However, only OZ treatment significantly decreased liver index and hepatic TG content, lowered serum levels of C-reactive protein and IL-6, and increased serum concentration of adiponectin. In the in vitro assay, only OZ administration significantly inhibited intracellular TG accumulation and down-regulated expression of stearoyl coenzyme-A desaturase-1, which might facilitate OZ to enhance its hepatoprotective effect. OZ is more effective than FA in inhibiting hepatic fat accumulation and inflammation. Thus, FA and OZ could be used as dietary supplements to alleviate the deleterious effects of HFFD.

Fitness costs and stability of Cry1Fa resistance in Brazilian populations of Spodoptera frugiperda.

PubMed

Santos-Amaya, Oscar F; Tavares, Clébson S; Rodrigues, João Victor C; Campos, Silverio O; Guedes, Raul Narciso C; Alves, Analiza P; Pereira, Eliseu José G

2017-01-01

The presence of fitness costs of resistance to Bacillus thuringiensis (Bt) insecticidal proteins in insect populations may delay or even reverse the local selection of insect resistance to Bt transgenic crops, and deserves rigorous investigation. Here we assessed the fitness costs associated with Cry1Fa resistance in two strains of fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), derived from field collections in different Brazilian regions and further selected in the laboratory for high levels of resistance to Cry1Fa using leaves of TC1507 corn. Fitness components were compared using paired resistant and susceptible strains with similar genetic backgrounds and F 1 generations from reciprocal crosses, all of them reared on non-transgenic corn leaves. No apparent life history costs in the larval stage were observed in the Bt-resistant strains. Moreover, the resistance remained stable for seven generations in the absence of selection, with no decrease in the proportion of resistant individuals. Larval respiration rates were also similar between resistant and susceptible homozygotes, and heterozygotes displayed respiration rates and demographic performance equal or superior to those of susceptible homozygotes. In combination, these results indicate the lack of strong fitness costs associated with resistance to Cry1Fa in the fall armyworm strains studied. These findings suggest that Cry1Fa resistance in S. frugiperda populations is unlikely to be counterselected in Cry1Fa-free environments. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Synergistic effect of pH-responsive folate-functionalized poloxamer 407-TPGS-mixed micelles on targeted delivery of anticancer drugs.

PubMed

Butt, Adeel Masood; Mohd Amin, Mohd Cairul Iqbal; Katas, Haliza

2015-01-01

Doxorubicin (DOX), an anthracycline anticancer antibiotic, is used for treating various types of cancers. However, its use is associated with toxicity to normal cells and development of resistance due to overexpression of drug efflux pumps. Poloxamer 407 (P407) and vitamin E TPGS (D-α-tocopheryl polyethylene glycol succinate, TPGS) are widely used polymers as drug delivery carriers and excipients for enhancing the drug retention times and stability. TPGS reduces multidrug resistance, induces apoptosis, and shows selective anticancer activity against tumor cells. Keeping in view the problems, we designed a mixed micelle system encapsulating DOX comprising TPGS for its selective anticancer activity and P407 conjugated with folic acid (FA) for folate-mediated receptor targeting to cancer cells. FA-functionalized P407 was prepared by carbodiimide crosslinker chemistry. P407-TPGS/FA-P407-TPGS-mixed micelles were prepared by thin-film hydration method. Cytotoxicity of blank micelles, DOX, and DOX-loaded micelles was determined by alamarBlue(®) assay. The size of micelles was less than 200 nm with encapsulation efficiency of 85% and 73% for P407-TPGS and FA-P407-TPGS micelles, respectively. Intracellular trafficking study using nile red-loaded micelles indicated improved drug uptake and perinuclear drug localization. The micelles show minimal toxicity to normal human cell line WRL-68, enhanced cellular uptake of DOX, reduced drug efflux, increased DOX-DNA binding in SKOV3 and DOX-resistant SKOV3 human ovarian carcinoma cell lines, and enhanced in vitro cytotoxicity as compared to free DOX. FA-P407-TPGS-DOX micelles show potential as a targeted nano-drug delivery system for DOX due to their multiple synergistic factors of selective anticancer activity, inhibition of multidrug resistance, and folate-mediated selective uptake.

Synergistic effect of pH-responsive folate-functionalized poloxamer 407-TPGS-mixed micelles on targeted delivery of anticancer drugs

PubMed Central

Butt, Adeel Masood; Mohd Amin, Mohd Cairul Iqbal; Katas, Haliza

2015-01-01

Background Doxorubicin (DOX), an anthracycline anticancer antibiotic, is used for treating various types of cancers. However, its use is associated with toxicity to normal cells and development of resistance due to overexpression of drug efflux pumps. Poloxamer 407 (P407) and vitamin E TPGS (D-α-tocopheryl polyethylene glycol succinate, TPGS) are widely used polymers as drug delivery carriers and excipients for enhancing the drug retention times and stability. TPGS reduces multidrug resistance, induces apoptosis, and shows selective anticancer activity against tumor cells. Keeping in view the problems, we designed a mixed micelle system encapsulating DOX comprising TPGS for its selective anticancer activity and P407 conjugated with folic acid (FA) for folate-mediated receptor targeting to cancer cells. Methods FA-functionalized P407 was prepared by carbodiimide crosslinker chemistry. P407-TPGS/FA-P407-TPGS-mixed micelles were prepared by thin-film hydration method. Cytotoxicity of blank micelles, DOX, and DOX-loaded micelles was determined by alamarBlue® assay. Results The size of micelles was less than 200 nm with encapsulation efficiency of 85% and 73% for P407-TPGS and FA-P407-TPGS micelles, respectively. Intracellular trafficking study using nile red-loaded micelles indicated improved drug uptake and perinuclear drug localization. The micelles show minimal toxicity to normal human cell line WRL-68, enhanced cellular uptake of DOX, reduced drug efflux, increased DOX–DNA binding in SKOV3 and DOX-resistant SKOV3 human ovarian carcinoma cell lines, and enhanced in vitro cytotoxicity as compared to free DOX. Conclusion FA-P407-TPGS-DOX micelles show potential as a targeted nano-drug delivery system for DOX due to their multiple synergistic factors of selective anticancer activity, inhibition of multidrug resistance, and folate-mediated selective uptake. PMID:25709451

Effects of Chain Length and Degree of Unsaturation of Fatty Acids on Structure and in Vitro Digestibility of Starch-Protein-Fatty Acid Complexes.

PubMed

Zheng, Mengge; Chao, Chen; Yu, Jinglin; Copeland, Les; Wang, Shuo; Wang, Shujun

2018-02-28

The effects of chain length and degree of unsaturation of fatty acids (FAs) on structure and in vitro digestibility of starch-protein-FA complexes were investigated in model systems. Studies with the rapid visco analyzer (RVA) showed that the formation of ternary complex resulted in higher viscosities than those of binary complex during the cooling and holding stages. The results of differential scanning calorimetry (DSC), Raman, and X-ray diffraction (XRD) showed that the structural differences for ternary complexes were much less than those for binary complexes. Starch-protein-FA complexes presented lower in vitro enzymatic digestibility compared with starch-FAs complexes. We conclude that shorter chain and lower unsaturation FAs favor the formation of ternary complexes but decrease the thermal stability of these complexes. FAs had a smaller effect on the ordered structures of ternary complexes than on those of binary complexes and little effect on enzymatic digestibility of both binary and ternary complexes.

Non-IgE-mediated gastrointestinal food allergies in children.

PubMed

Caubet, Jean-Christoph; Szajewska, Hania; Shamir, Raanan; Nowak-Węgrzyn, Anna

2017-02-01

Non-IgE-mediated gastrointestinal food allergic disorders (non-IgE-GI-FA) including food protein-induced enterocolitis syndrome (FPIES), food protein-induced enteropathy (FPE), and food protein-induced allergic proctocolitis (FPIAP) are relatively uncommon in infants and young children, but are likely under-diagnosed. Non-IgE-GI-FA have a favorable prognosis, with majority resolving by age 3-5 years. Diagnosis relies on the recognition of symptoms pattern in FPIAP and FPIES and biopsy in FPE. Further studies are needed for a better understanding of the pathomechanism, which will lead eventually to the development of diagnostic tests and treatments. Limited evidence supports the role of food allergens in subsets of constipation, gastroesophageal reflux disease, irritable bowel syndrome, and colic. The immunologic pathomechanism is not fully understood and empiric prolonged avoidance of food allergens should be limited to minimize nutrient deficiency and feeding disorders/food aversions in infants. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Role of blue green algae biofertilizer in ameliorating the nitrogen demand and fly-ash stress to the growth and yield of rice (Oryza sativa L.) plants.

PubMed

Tripathi, R D; Dwivedi, S; Shukla, M K; Mishra, S; Srivastava, S; Singh, R; Rai, U N; Gupta, D K

2008-02-01

Rice is a major food crop throughout the world; however, accumulation of toxic metals and metalloids in grains in contaminated environments is a matter of growing concern. Field experiments were conducted to analyze the growth performance, elemental composition (Fe, Si, Zn, Mn, Cu, Ni, Cd and As) and yield of the rice plants (Oryza sativa L. cv. Saryu-52) grown under different doses of fly-ash (FA; applied @ 10 and 100 tha(-1) denoted as FA(10) and FA(100), respectively) mixed with garden soil (GS) in combination with nitrogen fertilizer (NF; applied @ 90 and 120 kg ha(-1) denoted as NF(90) and NF(120), respectively) and blue green algae biofertilizer (BGA; applied @ 12.5 kg ha(-1) denoted as BGA(12.5)). Significant enhancement of growth was observed in the plants growing on amended soils as compared to GS and best response was obtained in amendment of FA(10)+NF(90)+BGA(12.5). Accumulation of Si, Fe, Zn and Mn was higher than Cu, Cd, Ni and As. Arsenic accumulation was detected only in FA(100) and its amendments. Inoculation of BGA(12.5) caused slight reduction in Cd, Ni and As content of plants as compared to NF(120) amendment. The high levels of stress inducible non-protein thiols (NP-SH) and cysteine in FA(100) were decreased by application of NF and BGA indicating stress amelioration. Study suggests integrated use of FA, BGA and NF for improved growth, yield and mineral composition of the rice plants besides reducing the high demand of nitrogen fertilizers.

Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus.

PubMed

Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji

2012-01-01

Thioredoxin binding protein -2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein -2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein -2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein -2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein -2 in metabolic control. Enhancement of thioredoxin binding protein -2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein -2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein -2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β(2)-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus.

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

PubMed Central

Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S.; Højrup, Peter; Poulsen, Steen S.; Nexo, Ebba

2012-01-01

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins. PMID:22872637

The Walker B motif in avian FANCM is required to limit sister chromatid exchanges but is dispensable for DNA crosslink repair

PubMed Central

Rosado, Ivan V.; Niedzwiedz, Wojciech; Alpi, Arno F.; Patel, Ketan J.

2009-01-01

FANCM, the most highly conserved component of the Fanconi Anaemia (FA) pathway can resolve recombination intermediates and remodel synthetic replication forks. However, it is not known if these activities are relevant to how this conserved protein activates the FA pathway and promotes DNA crosslink repair. Here we use chicken DT40 cells to systematically dissect the function of the helicase and nuclease domains of FANCM. Our studies reveal that these domains contribute distinct roles in the tolerance of crosslinker, UV light and camptothecin-induced DNA damage. Although the complete helicase domain is critical for crosslink repair, a predicted inactivating mutation of the Walker B box domain has no impact on FA pathway associated functions. However, this mutation does result in elevated sister chromatid exchanges (SCE). Furthermore, our genetic dissection indicates that FANCM functions with the Blm helicase to suppress spontaneous SCE events. Overall our results lead us to reappraise the role of helicase domain associated activities of FANCM with respect to the activation of the FA pathway, crosslink repair and in the resolution of recombination intermediates. PMID:19465393

Aerosol delivery of folate-decorated hyperbranched polyspermine complexes to suppress lung tumorigenesis via Akt signaling pathway.

PubMed

Luo, Cheng-Qiong; Jang, Yoonjeong; Xing, Lei; Cui, Peng-Fei; Qiao, Jian-Bin; Lee, Ah Young; Kim, Hyeon-Jeong; Cho, Myung-Haing; Jiang, Hu-Lin

2016-11-20

Lung cancer has been a leading cause of cancer mortality worldwide and aerosol-mediated gene therapy endows numerous advantages compared to other traditional modalities. Here, we reported a folic acid (FA)-modified hyperbranched polyspermine (HPSPE) with prominent biocompatibility for lung cancer cell targeted gene therapy. FA was decorated to the HPSPE via an amidation reaction and the physicochemical properties of nanoplexes formed with DNA were characterized. Gel electrophoresis study elucidated that the designed polymer was capable to condense DNA and protect it from degradation by DNase I. Cell viability and transfection efficiency assay in vitro and in vivo indicated its increased transfection performance with lower toxicity. Furthermore, reduced tumor numbers and down-regulation of Akt1 protein after aerosol treatment containing FA-HPSPE/shAkt1 complexes proved its therapeutic potential for lung cancer suppression. Results obtained in this study suggested that FA-HPSPE with highly biocompatibility and targeting capability while forming complexes with shAkt1 and administrated through noninvasive aerosol could be prospective for inhibiting lung tumorigenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, So-Hee; Moon, Jeonghee; Lee, Myungkyu

2013-09-13

Highlights: •PTPRT is a brain-specific, expressed, protein tyrosine phosphatase. •PTPRT regulated the interaction of Syntaxin-binding protein 1 with Syntaxin 1. •PTPRT dephosphorylated the specific tyrosine residue of Syntaxin-binding protein 1. •Dephosphorylation of Syntaxin-binding protein 1 enhanced the interaction with Syntaxin 1. •PTPRT appears to regulate the fusion of synaptic vesicle through dephosphorylation. -- Abstract: PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified asmore » a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1.« less

Comparative Proteomic Study of Fatty Acid-treated Myoblasts Reveals Role of Cox-2 in Palmitate-induced Insulin Resistance

PubMed Central

Chen, Xiulan; Xu, Shimeng; Wei, Shasha; Deng, Yaqin; Li, Yiran; Yang, Fuquan; Liu, Pingsheng

2016-01-01

Accumulated studies demonstrate that saturated fatty acids (FAs) such as palmitic acid (PA) inhibit insulin signaling in skeletal muscle cells and monounsaturated fatty acids such as oleic acid (OA) reverse the effect of PA on insulin signaling. The detailed molecular mechanism of these opposite effects remains elusive. Here we provide a comparative proteomic study of skeletal myoblast cell line C2C12 that were untreated or treated with PA, and PA plus OA. A total of 3437 proteins were quantified using SILAC in this study and 29 proteins fall into the pattern that OA reverses PA effect. Expression of some these proteins were verified using qRT-PCR and Western blot. The most significant change was cyclooxygenase-2 (Cox-2). In addition to whole cell comparative proteomic study, we also compared lipid droplet (LD)-associated proteins and identified that Cox-2 was one of three major altered proteins under the FA treatment. This finding was then confirmed using immunofluorescence. Finally, Cox-2 selective inhibitor, celecoxib protected cells from PA-reduced insulin signaling Akt phosphorylation. Together, these results not only provide a dataset of protein expression change in FA treatment but also suggest that Cox-2 and lipid droplets (LDs) are potential players in PA- and OA-mediated cellular processes. PMID:26899878

Monoclonal antibodies to human vitamin D-binding protein.

PubMed Central

Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F

1985-01-01

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035

Computer modeling of obesity links theoretical energetic measures with experimental measures of fuel use for lean and obese men.

PubMed

Rossow, Heidi A; Calvert, C Chris

2014-10-01

The goal of this research was to use a computational model of human metabolism to predict energy metabolism for lean and obese men. The model is composed of 6 state variables representing amino acids, muscle protein, visceral protein, glucose, triglycerides, and fatty acids (FAs). Differential equations represent carbohydrate, amino acid, and FA uptake and output by tissues based on ATP creation and use for both lean and obese men. Model parameterization is based on data from previous studies. Results from sensitivity analyses indicate that model predictions of resting energy expenditure (REE) and respiratory quotient (RQ) are dependent on FA and glucose oxidation rates with the highest sensitivity coefficients (0.6, 0.8 and 0.43, 0.15, respectively, for lean and obese models). Metabolizable energy (ME) is influenced by ingested energy intake with a sensitivity coefficient of 0.98, and a phosphate-to-oxygen ratio by FA oxidation rate and amino acid oxidation rate (0.32, 0.24 and 0.55, 0.65 for lean and obese models, respectively). Simulations of previously published studies showed that the model is able to predict ME ranging from 6.6 to 9.3 with 0% differences between published and model values, and RQ ranging from 0.79 to 0.86 with 1% differences between published and model values. REEs >7 MJ/d are predicted with 6% differences between published and model values. Glucose oxidation increases by ∼0.59 mol/d, RQ increases by 0.03, REE increases by 2 MJ/d, and heat production increases by 1.8 MJ/d in the obese model compared with lean model simulations. Increased FA oxidation results in higher changes in RQ and lower relative changes in REE. These results suggest that because fat mass is directly related to REE and rate of FA oxidation, body fat content could be used as a predictor of RQ. © 2014 American Society for Nutrition.

Complementation of hypersensitivity to DNA interstrand crosslinking agents demonstrates that XRCC2 is a Fanconi anaemia gene.

PubMed

Park, Jung-Young; Virts, Elizabeth L; Jankowska, Anna; Wiek, Constanze; Othman, Mohamed; Chakraborty, Sujata C; Vance, Gail H; Alkuraya, Fowzan S; Hanenberg, Helmut; Andreassen, Paul R

2016-10-01

Fanconi anaemia (FA) is a heterogeneous inherited disorder clinically characterised by progressive bone marrow failure, congenital anomalies and a predisposition to malignancies. Determine, based on correction of cellular phenotypes, whether XRCC2 is a FA gene. Cells (900677A) from a previously identified patient with biallelic mutation of XRCC2, among other mutations, were genetically complemented with wild-type XRCC2. Wild-type XRCC2 corrects each of three phenotypes characteristic of FA cells, all related to the repair of DNA interstrand crosslinks, including increased sensitivity to mitomycin C (MMC), chromosome breakage and G2-M accumulation in the cell cycle. Further, the p.R215X mutant of XRCC2, which is harboured by the patient, is unstable. This provides an explanation for the pathogenesis of this mutant, as does the fact that 900677A cells have reduced levels of other proteins in the XRCC2-RAD51B-C-D complex. Also, FANCD2 monoubiquitination and foci formation, but not assembly of RAD51 foci, are normal in 900677A cells. Thus, XRCC2 acts late in the FA-BRCA pathway as also suggested by hypersensitivity of 900677A cells to ionising radiation. These cells also share milder sensitivities towards olaparib and formaldehyde with certain other FA cells. XRCC2/FANCU is a FA gene, as is another RAD51 paralog gene, RAD51C/FANCO. Notably, similar to a subset of FA genes that act downstream of FANCD2, biallelic mutation of XRCC2/FANCU has not been associated with bone marrow failure. Taken together, our results yield important insights into phenotypes related to FA and its genetic origins. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Development and characterization of an effective food allergy model in Brown Norway rats.

PubMed

Abril-Gil, Mar; Garcia-Just, Alba; Pérez-Cano, Francisco J; Franch, Àngels; Castell, Margarida

2015-01-01

Food allergy (FA) is an adverse health effect produced by the exposure to a given food. Currently, there is no optimal animal model of FA for the screening of immunotherapies or for testing the allergenicity of new foods. The aim of the present study was to develop an effective and rapid model of FA in Brown Norway rats. In order to establish biomarkers of FA in rat, we compared the immune response and the anaphylactic shock obtained in this model with those achieved with only intraperitoneal immunization. Rats received an intraperitoneal injection of ovalbumin (OVA) with alum and toxin from Bordetella pertussis, and 14 days later, OVA by oral route daily for three weeks (FA group). A group of rats receiving only the i.p. injection (IP group) were also tested. Serum anti-OVA IgE, IgG1, IgG2a, IgG2b and IgA antibodies were quantified throughout the study. After an oral challenge, body temperature, intestinal permeability, motor activity, and mast cell protease II (RMCP-II) levels were determined. At the end of the study, anti-OVA intestinal IgA, spleen cytokine production, lymphocyte composition of Peyer's patches and mesenteric lymph nodes, and gene expression in the small intestine were quantified. Serum OVA-specific IgG1, IgG2a and IgG2b concentrations rose with the i.p. immunization but were highly augmented after the oral OVA administration. Anti-OVA IgE increased twofold during the first week of oral OVA gavage. The anaphylaxis in both IP and FA groups decreased body temperature and motor activity, whereas intestinal permeability increased. Interestingly, the FA group showed a much higher RMCP II serum protein and intestinal mRNA expression. These results show both an effective and relatively rapid model of FA assessed by means of specific antibody titres and the high production of RMCP-II and its intestinal gene expression.

Development and Characterization of an Effective Food Allergy Model in Brown Norway Rats

PubMed Central

Abril-Gil, Mar; Garcia-Just, Alba; Pérez-Cano, Francisco J.; Franch, Àngels; Castell, Margarida

2015-01-01

Background Food allergy (FA) is an adverse health effect produced by the exposure to a given food. Currently, there is no optimal animal model of FA for the screening of immunotherapies or for testing the allergenicity of new foods. Objective The aim of the present study was to develop an effective and rapid model of FA in Brown Norway rats. In order to establish biomarkers of FA in rat, we compared the immune response and the anaphylactic shock obtained in this model with those achieved with only intraperitoneal immunization. Methods Rats received an intraperitoneal injection of ovalbumin (OVA) with alum and toxin from Bordetella pertussis, and 14 days later, OVA by oral route daily for three weeks (FA group). A group of rats receiving only the i.p. injection (IP group) were also tested. Serum anti-OVA IgE, IgG1, IgG2a, IgG2b and IgA antibodies were quantified throughout the study. After an oral challenge, body temperature, intestinal permeability, motor activity, and mast cell protease II (RMCP-II) levels were determined. At the end of the study, anti-OVA intestinal IgA, spleen cytokine production, lymphocyte composition of Peyer’s patches and mesenteric lymph nodes, and gene expression in the small intestine were quantified. Results Serum OVA-specific IgG1, IgG2a and IgG2b concentrations rose with the i.p. immunization but were highly augmented after the oral OVA administration. Anti-OVA IgE increased twofold during the first week of oral OVA gavage. The anaphylaxis in both IP and FA groups decreased body temperature and motor activity, whereas intestinal permeability increased. Interestingly, the FA group showed a much higher RMCP II serum protein and intestinal mRNA expression. Conclusions These results show both an effective and relatively rapid model of FA assessed by means of specific antibody titres and the high production of RMCP-II and its intestinal gene expression. PMID:25923134

The Fanconi anemia/BRCA gene network in zebrafish: embryonic expression and comparative genomics.

PubMed

Titus, Tom A; Yan, Yi-Lin; Wilson, Catherine; Starks, Amber M; Frohnmayer, Jonathan D; Bremiller, Ruth A; Cañestro, Cristian; Rodriguez-Mari, Adriana; He, Xinjun; Postlethwait, John H

2009-07-31

Fanconi anemia (FA) is a genetic disease resulting in bone marrow failure, high cancer risks, and infertility, and developmental anomalies including microphthalmia, microcephaly, hypoplastic radius and thumb. Here we present cDNA sequences, genetic mapping, and genomic analyses for the four previously undescribed zebrafish FA genes (fanci, fancj, fancm, and fancn), and show that they reverted to single copy after the teleost genome duplication. We tested the hypothesis that FA genes are expressed during embryonic development in tissues that are disrupted in human patients by investigating fanc gene expression patterns. We found fanc gene maternal message, which can provide Fanc proteins to repair DNA damage encountered in rapid cleavage divisions. Zygotic expression was broad but especially strong in eyes, central nervous system and hematopoietic tissues. In the pectoral fin bud at hatching, fanc genes were expressed specifically in the apical ectodermal ridge, a signaling center for fin/limb development that may be relevant to the radius/thumb anomaly of FA patients. Hatching embryos expressed fanc genes strongly in the oral epithelium, a site of squamous cell carcinomas in FA patients. Larval and adult zebrafish expressed fanc genes in proliferative regions of the brain, which may be related to microcephaly in FA. Mature ovaries and testes expressed fanc genes in specific stages of oocyte and spermatocyte development, which may be related to DNA repair during homologous recombination in meiosis and to infertility in human patients. The intestine strongly expressed some fanc genes specifically in proliferative zones. Our results show that zebrafish has a complete complement of fanc genes in single copy and that these genes are expressed in zebrafish embryos and adults in proliferative tissues that are often affected in FA patients. These results support the notion that zebrafish offers an attractive experimental system to help unravel mechanisms relevant not only to FA, but also to breast cancer, given the involvement of fancj (brip1), fancn (palb2) and fancd1 (brca2) in both conditions.

The Fanconi anemia/BRCA gene network in zebrafish: Embryonic expression and comparative genomics

PubMed Central

Titus, Tom A.; Yan, Yi-Lin; Wilson, Catherine; Starks, Amber M.; Frohnmayer, Jonathan D.; Canestro, Cristian; Rodriguez-Mari, Adriana; He, Xinjun; Postlethwait, John H.

2008-01-01

Fanconi anemia (FA) is a genic disease resulting in bone marrow failure, high cancer risks, and infertility, and developmental anomalies including microphthalmia, microcephaly, hypoplastic radius and thumb. Here we present cDNA sequences, genetic mapping, and genomic analyses for the four previously undescribed zebrafish FA genes (fanci, fancj, fancm, and fancn, and show that they reverted to single copy after the teleost genome duplication. We tested the hypothesis that FA genes are expressed during embryonic development in tissues that are disrupted in human patients by investigating fanc gene expression patterns. We found fanc gene maternal message, which can provide Fanc proteins to repair DNA damage encountered in rapid cleavage divisions. Zygotic expression was broad but especially strong in eyes, central nervous system and hematopoietic tissues. In the pectoral fin bud at hatching, fanc genes were expressed specifically in the apical ectodermal ridge, a signaling center for fin/limb development that may be relevant to the radius/thumb anomaly of FA patients. Hatching embryos expressed fanc genes strongly in the oral epithelium, a site of squamous cell carcinomas in FA patients. Larval and adult zebrafish expressed fanc genes in proliferative regions of the brain, which may be related to microcephaly in FA. Mature ovaries and testes expressed fanc genes in specific stages of oocyte and spermatocyte development, which may be related to DNA repair during homologous recombination in meiosis and to infertility in human patients. The intestine strongly expressed some fanc genes specifically in proliferative zones. Our results show that zebrafish has a complete complement of fanc genes in single copy and that these genes are expressed in zebrafish embryos and adults in proliferative tissues that are often affected in FA patients. These results support the notion that zebrafish offers an attractive experimental system to help unravel mechanisms relevant not only to FA, but also to breast cancer, given the involvement of fancj (brip1), fancn (palb2) and fancd1 (brca2) in both conditions. PMID:19101574

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

PubMed

Putta, Priya; Rankenberg, Johanna; Korver, Ruud A; van Wijk, Ringo; Munnik, Teun; Testerink, Christa; Kooijman, Edgar E

2016-11-01

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins. Copyright © 2016. Published by Elsevier B.V.

In Vivo Evaluation of ¹⁸F-SiFAlin-Modified TATE: A Potential Challenge for ⁶⁸Ga-DOTATATE, the Clinical Gold Standard for Somatostatin Receptor Imaging with PET.

PubMed

Niedermoser, Sabrina; Chin, Joshua; Wängler, Carmen; Kostikov, Alexey; Bernard-Gauthier, Vadim; Vogler, Nils; Soucy, Jean-Paul; McEwan, Alexander J; Schirrmacher, Ralf; Wängler, Björn

2015-07-01

Radiolabeled peptides for tumor imaging with PET that can be produced with kits are currently in the spotlight of radiopharmacy and nuclear medicine. The diagnosis of neuroendocrine tumors in particular has been a prime example for the usefulness of peptides labeled with a variety of different radionuclides. Among those, (68)Ga and (18)F stand out because of the ease of radionuclide introduction (e.g., (68)Ga isotope) or optimal nuclide properties for PET imaging (slightly favoring the (18)F isotope). The in vivo properties of good manufacturing practice-compliant, newly developed kitlike-producible (18)F-SiFA- and (18)F-SiFAlin- (SiFA = silicon-fluoride acceptor) modified TATE derivatives were compared with the current clinical gold standard (68)Ga-DOTATATE for high-quality imaging of somatostatin receptor-bearing tumors. SiFA- and SiFAlin-derivatized somatostatin analogs were synthesized and radiolabeled using cartridge-based dried (18)F and purified via a C18 cartridge (radiochemical yield 49.8% ± 5.9% within 20-25 min) without high-performance liquid chromatography purification. Tracer lipophilicity and stability in human serum were tested in vitro. Competitive receptor binding affinity studies were performed using AR42J cells. The most promising tracers were evaluated in vivo in an AR42J xenograft mouse model by ex vivo biodistribution and in vivo PET/CT imaging studies for evaluation of their pharmacokinetic profiles, and the results were compared with those of the current clinical gold standard (68)Ga-DOTATATE. Synthetically easily accessible (18)F-labeled silicon-fluoride acceptor-modified somatostatin analogs were developed. They exhibited high binding affinities to somatostatin receptor-positive tumor cells (1.88-14.82 nM). The most potent compound demonstrated comparable pharmacokinetics and an even slightly higher absolute tumor accumulation level in ex vivo biodistribution studies as well as higher tumor standardized uptake values in PET/CT imaging than (68)Ga-DOTATATE in vivo. The radioactivity uptake in nontumor tissue was higher than for (68)Ga-DOTATATE. The introduction of the novel SiFA building block SiFAlin and of hydrophilic auxiliaries enables a favorable in vivo biodistribution profile of the modified TATE peptides, resulting in high tumor-to-background ratios although lower than those observed with (68)Ga-DOTATATE. As further advantage, the SiFA methodology enables a kitlike labeling procedure for (18)F-labeled peptides advantageous for routine clinical application. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Nutritional composition of raw fresh cashew (Anacardium occidentale L.) kernels from different origin.

PubMed

Rico, Ricard; Bulló, Mònica; Salas-Salvadó, Jordi

2016-03-01

The total dietary fiber, sugar, protein, lipid profile, sodium, and energy contents of 11 raw cashew kernel (Anacardium occidentale L.) samples from India, Brazil, Ivory Coast, Kenya, Mozambique, and Vietnam were determined. Total fat was the major component accounting for 48.3% of the total weight, of which 79.7% were unsaturated FA (fatty acids), 20.1% saturated FA, and 0.2% trans FA. Proteins, with 21.3 g/100 g, were ranked second followed by carbohydrates (20.5 g/100 g). The average sodium content was 144 mg/kg. Fourteen FA were identified among which oleic acid was the most abundant with a contribution of 60.7% to the total fat, followed by linoleic (17.77%), palmitic (10.2%), and stearic (8.93%) acids. The mean energy content was 2525 kJ/100g. Furthermore, the sterol profile and content, amino acids, vitamins, and minerals of four raw cashew kernel samples from Brazil, India, Ivory Coast, and Vietnam were determined. β-Sitosterol with 2380 ± 4 mg/kg fat was the most occurring sterol. Glutamic acid, with 4.60 g/100 g, was the amino acid with highest presence, whereas tryptophan with 0.32 g/100 g was the one with lower presence. Vitamin E with an average contribution of 5.80 mg/100 g was the most abundant vitamin. Potassium with a mean value of 6225 mg/kg was the mineral with highest amount in cashew samples.

Cell proliferation and progesterone synthesis depend on lipid metabolism in bovine granulosa cells.

PubMed

Elis, Sebastien; Desmarchais, Alice; Maillard, Virginie; Uzbekova, Svetlana; Monget, Philippe; Dupont, Joëlle

2015-03-15

In dairy cows, lipids are essential to support energy supplies for all biological functions, especially during early lactation. Lipid metabolism is crucial for sustaining proper reproductive function. Alteration of lipid metabolism impacts follicular development and affects oocyte developmental competence. Indeed, nonesterified fatty acids are able to decrease granulosa cell (GC) proliferation and affect estradiol synthesis, thus potentially affecting follicular growth and viability. The objective of this study was to assess the impact of lipid metabolism on bovine GCs, through the use of the lipid metabolism inhibitors etomoxir, an inhibitor of fatty acid (FA) oxidation through inhibition of carnitine palmitoyl transferase 1 (CPT1), and C75, an inhibitor of FA synthesis through inhibition of fatty acid synthase. We showed that etomoxir and C75 significantly inhibited DNA synthesis in vitro; C75 also significantly decreased progesterone synthesis. Both inhibitors significantly reduced AMPK (5' adenosine monophosphate-activated protein kinase) and acetyl-CoA carboxylase phosphorylation. Etomoxir also affected the AKT (protein kinase B) signaling pathway. Combined, these data suggest that both FA oxidation and synthesis are important for the bovine GCs to express a proliferative and steroidogenic phenotype and, thus, for sustaining follicular growth. Despite these findings, it is important to note that the changes caused by the inhibitors of FA metabolism on GCs in vitro are globally mild, suggesting that lipid metabolism is not as critical in GCs as was observed in the oocyte-cumulus complex. Further studies are needed to investigate the detailed mechanisms by which lipid metabolism interacts with GC functions. Copyright © 2015 Elsevier Inc. All rights reserved.

Water and temperature stresses impact canola (Brassica napus L.) fatty acid, protein and yield over nitrogen and sulfur

USDA-ARS?s Scientific Manuscript database

Interactive effects of weather and soil nutrient status often control crop productivity. An experiment was conducted to determine effects of N and S fertilizer rate, soil water, and atmospheric temperature on canola fatty acid (FA), total oil, protein and grain yield. Nitrogen and S were assessed in...

Spodoptera frugiperda (J.E. Smith) with field-evolved resistance to Bt maize are susceptible to Bt pesticides.

PubMed

Jakka, S R K; Knight, V R; Jurat-Fuentes, J L

2014-10-01

Field-evolved resistance to maize event TC1507 expressing the Cry1Fa toxin from Bacillus thuringiensis (Bt) was detected in populations of Spodoptera frugiperda from Puerto Rico. We tested for cross-resistance to purified Cry1A toxins and commercial Bt pesticides in susceptible (Benzon) and TC1507-resistant (456) strains of S. frugiperda. Larvae from the 456 strain exhibited cross-resistance to Cry1Ab and Cry1Ac toxins, while no differences in susceptibility to XenTari WG and DiPel ES pesticides were detected. These data support cross-resistance to toxins that share binding sites with Cry1Fa and no cross-resistance to Bt pesticides in S. frugiperda with field-evolved resistance to Bt maize. Copyright © 2014 Elsevier Inc. All rights reserved.

Thioredoxin binding protein (TBP)-2/Txnip and α-arrestin proteins in cancer and diabetes mellitus

PubMed Central

Masutani, Hiroshi; Yoshihara, Eiji; Masaki, So; Chen, Zhe; Yodoi, Junji

2012-01-01

Thioredoxin binding protein −2/ thioredoxin interacting protein is an α-arrestin protein that has attracted much attention as a multifunctional regulator. Thioredoxin binding protein −2 expression is downregulated in tumor cells and the level of thioredoxin binding protein is correlated with clinical stage of cancer. Mice with mutations or knockout of the thioredoxin binding protein −2 gene are much more susceptible to carcinogenesis than wild-type mice, indicating a role for thioredoxin binding protein −2 in cancer suppression. Studies have also revealed roles for thioredoxin binding protein −2 in metabolic control. Enhancement of thioredoxin binding protein −2 expression causes impairment of insulin sensitivity and glucose-induced insulin secretion, and β-cell apoptosis. These changes are important characteristics of type 2 diabetes mellitus. Thioredoxin binding protein −2 regulates transcription of metabolic regulating genes. Thioredoxin binding protein −2-like inducible membrane protein/ arrestin domain containing 3 regulates endocytosis of receptors such as the β2-adrenergic receptor. The α-arrestin family possesses PPXY motifs and may function as an adaptor/scaffold for NEDD family ubiquitin ligases. Elucidation of the molecular mechanisms of α-arrestin proteins would provide a new pharmacological basis for developing approaches against cancer and type 2 diabetes mellitus. PMID:22247597

Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

PubMed

Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

2014-10-01

Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

Inhibited Carnitine Synthesis Causes Systemic Alteration of Nutrient Metabolism in Zebrafish

PubMed Central

Li, Jia-Min; Li, Ling-Yu; Qin, Xuan; Degrace, Pascal; Demizieux, Laurent; Limbu, Samwel M.; Wang, Xin; Zhang, Mei-Ling; Li, Dong-Liang; Du, Zhen-Yu

2018-01-01

Impaired mitochondrial fatty acid β-oxidation has been correlated with many metabolic syndromes, and the metabolic characteristics of the mammalian models of mitochondrial dysfunction have also been intensively studied. However, the effects of the impaired mitochondrial fatty acid β-oxidation on systemic metabolism in teleost have never been investigated. In the present study, we established a low-carnitine zebrafish model by feeding fish with mildronate as a specific carnitine synthesis inhibitor [0.05% body weight (BW)/d] for 7 weeks, and the systemically changed nutrient metabolism, including carnitine and triglyceride (TG) concentrations, fatty acid (FA) β-oxidation capability, and other molecular and biochemical assays of lipid, glucose, and protein metabolism, were measured. The results indicated that mildronate markedly decreased hepatic carnitine concentrations while it had no effect in muscle. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial β-oxidation, increased the hepatic mRNA expression of genes related to FA β-oxidation and lipolysis, and decreased the expression of lipogenesis genes. Mildronate decreased whole body glycogen content, increased glucose metabolism rate, and upregulated the expression of glucose uptake and glycolysis genes. Mildronate also increased whole body protein content and hepatic mRNA expression of mechanistic target of rapamycin (mtor), and decreased the expression of a protein catabolism-related gene. Liver, rather than muscle, was the primary organ targeted by mildronate. In short, mildronate-induced hepatic inhibited carnitine synthesis in zebrafish caused decreased mitochondrial FA β-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid β-oxidation in nutrient metabolism in fish, and this low-carnitine zebrafish model could also be used as a novel fish model for future metabolism studies. PMID:29867554

Inhibited Carnitine Synthesis Causes Systemic Alteration of Nutrient Metabolism in Zebrafish.

PubMed

Li, Jia-Min; Li, Ling-Yu; Qin, Xuan; Degrace, Pascal; Demizieux, Laurent; Limbu, Samwel M; Wang, Xin; Zhang, Mei-Ling; Li, Dong-Liang; Du, Zhen-Yu

2018-01-01

Impaired mitochondrial fatty acid β-oxidation has been correlated with many metabolic syndromes, and the metabolic characteristics of the mammalian models of mitochondrial dysfunction have also been intensively studied. However, the effects of the impaired mitochondrial fatty acid β-oxidation on systemic metabolism in teleost have never been investigated. In the present study, we established a low-carnitine zebrafish model by feeding fish with mildronate as a specific carnitine synthesis inhibitor [0.05% body weight (BW)/d] for 7 weeks, and the systemically changed nutrient metabolism, including carnitine and triglyceride (TG) concentrations, fatty acid (FA) β-oxidation capability, and other molecular and biochemical assays of lipid, glucose, and protein metabolism, were measured. The results indicated that mildronate markedly decreased hepatic carnitine concentrations while it had no effect in muscle. Liver TG concentrations increased by more than 50% in mildronate-treated fish. Mildronate decreased the efficiency of liver mitochondrial β-oxidation, increased the hepatic mRNA expression of genes related to FA β-oxidation and lipolysis, and decreased the expression of lipogenesis genes. Mildronate decreased whole body glycogen content, increased glucose metabolism rate, and upregulated the expression of glucose uptake and glycolysis genes. Mildronate also increased whole body protein content and hepatic mRNA expression of mechanistic target of rapamycin ( mtor ), and decreased the expression of a protein catabolism-related gene. Liver, rather than muscle, was the primary organ targeted by mildronate. In short, mildronate-induced hepatic inhibited carnitine synthesis in zebrafish caused decreased mitochondrial FA β-oxidation efficiency, greater lipid accumulation, and altered glucose and protein metabolism. This reveals the key roles of mitochondrial fatty acid β-oxidation in nutrient metabolism in fish, and this low-carnitine zebrafish model could also be used as a novel fish model for future metabolism studies.

The Fanconi anemia pathway sensitizes to DNA alkylating agents by inducing JNK-p53-dependent mitochondrial apoptosis in breast cancer cells.

PubMed

Zhao, Lin; Li, Yanlin; He, Miao; Song, Zhiguo; Lin, Shu; Yu, Zhaojin; Bai, Xuefeng; Wang, Enhua; Wei, Minjie

2014-07-01

The Fanconi anemia/BRCA (FA/BRCA) DNA damage repair pathway plays a pivotal role in the cellular response to DNA alkylating agents and greatly influences drug response in cancer treatment. However, the molecular mechanisms underlying the FA/BRCA pathway reversed resistance have received limited attention. In the present study, we investigated the effect of Fanconi anemia complementation group F protein (FANCF), a critical factor of the FA/BRCA pathway, on cancer cell apoptosis induced by DNA alkylating agents such as mitomycin c (MMC). We found that FANCF shRNA potentiated MMC-induced cytotoxicity and apoptosis in MCF-7 and MDA-MB-231 breast cancer cells. At a mechanistic level, FANCF shRNA downregulated the anti-apoptotic protein Bcl-2 and upregulated the pro-apoptotic protein Bax, accompanied by release of cyt-c and smac into the cytosol in MMC-treated cells. Furthermore, activation of caspase-3 and -9, other than caspase-8, cleavage of poly(ADP ribose) polymerase (PARP), and a decrease of mitochondrial membrane potential (MMP) indicated that involvement of the mitochondrial apoptotic pathway in FANCF silencing of MMC-treated breast cancer cells. A decrease in IAP family proteins XIAP and survivin were also observed following FANCF silencing in MMC-treated breast cancer cells. Notably, FANCF shRNA was able to increase p53 levels through activation of the JNK pathway in MMC-treated breast cancer cells. Furthermore, p53 inhibition using pifithrin-α abolished the induction of caspase-3 and PARP by FANCF shRNA and MMC, indicating that MMC-induced apoptosis is substantially enhanced by FANCF shRNA via p53-dependent mechanisms. To our knowledge, we provide new evidence for the potential application of FANCF as a chemosensitizer in breast cancer therapy.

Cortactin modulates RhoA activation and expression of Cip/Kip cyclin-dependent kinase inhibitors to promote cell cycle progression in 11q13-amplified head and neck squamous cell carcinoma cells.

PubMed

Croucher, David R; Rickwood, Danny; Tactacan, Carole M; Musgrove, Elizabeth A; Daly, Roger J

2010-11-01

The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21(WAF1/Cip1), p27(Kip1), and p57(Kip2) and inhibition of S-phase entry. These effects were associated with increased binding of p21(WAF1/Cip1) and p27(Kip1) to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21(WAF1/Cip1) and p27(Kip1) at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21(WAF1/Cip1), p27(Kip1), and p57(Kip2) downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27(Kip1) and p57(Kip2) for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.

Restricted access magnetic materials prepared by dual surface modification for selective extraction of therapeutic drugs from biological fluids

NASA Astrophysics Data System (ADS)

Wang, Yu; Wang, Yuxia; Chen, Lei; Wan, Qian-Hong

2012-02-01

Magnetic porous particles with dual functionality have been prepared by a two-step procedure and evaluated as novel restricted access materials for extraction of therapeutic agents from biological fluids. The magnetic silica particles served as scaffolds were first modified with diol groups, which were then converted to octadecyl esters through reaction with stearoyl chloride. In the second step, the octadecyl esters on the exterior surface were hydrolyzed by the action of lipase to yield magnetic particles with hydrophobic reversed-phase ligands on the inner surface and biocompatible diol groups on the outer surface. The restricted access behavior of the resulting materials was confirmed by differential binding of small molecules such as methotrexate (MTX), leucovorin (LV) and folic acid (FA) relative to bovine serum albumin. While MTX, LV and FA were all bound to the magnetic particles with high affinity, the adsorption of the protein was markedly reduced due to size exclusion effect. The utility of the magnetic particles for sample preparation was tested in solid-phase extraction of MTX, LV and FA from spiked human serum and the effects of the SPE conditions on the recovery of the analytes were systematically studied. Moreover, the magnetic particle-based sample preparation procedure coupled with reversed-phase liquid chromatography analysis was validated in terms of specificity, linearity and reproducibility. The method was shown to be free from interference of endogenous compounds and linear over the concentration range of 0.5-10 μg/mL for the three drugs studied. The limits of detection for the three drugs in serum were in the range of 0.160-0.302 μg/mL. Reproducibility expressed as the RSD of the recovery for ten replicated extractions at three different concentrations was found to be less than 8.93%. With a unique combination of surface functionality with magnetic cores, the restricted access magnetic particles may be adapted in automated and high throughput protocols for routine analysis of a large volume of clinical samples.

Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giulliani, S. E.; Frank, A. E.; Collart, F. R.

2008-12-08

We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity andmore » to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.« less

The Influence of 8 Weeks of Whey-Protein and Leucine Supplementation on Physical and Cognitive Performance

DTIC Science & Technology

2010-01-01

Influence of 8 Weeks of Whey Protein and Leucine Supplementation on Physical and Cognitive Performance 5a. GONTRAGT NUMBER FA8650-04-D-6472 5b. GRANT NUMBER...investigate the ability of whey -protein and leucine supplementation to enhance physical and cognitive performance and body composition. Thirty moderately fit...composition before and after supplementing their daily diet for 8 wk with either 19.7 g of whey protein and 6.2 g leucine (WPL) or a calorie-equivalent placebo