bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of Cytochrome P450 Reductase in an Open Conformation Capable of Reducing Cytochrome P450
Authors: Waskell Lucy , Kim Jung‐Ja P. , Hamdane Djemel , Xia Chuanwu , Im Sang‐Choul , Zhang Haoming
Published: June 2021

NADPH‐cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450 (cyt P450). A CYPOR variant, with a four amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three extended conformations. The variant donates an electron to cytochrome P450 at the same rate as wild type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that by pivoting on the C‐terminus of the hinge, the FMN domain is able to execute a large movement in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner. Both NIH and VA Merit grants supported this research.
Dietary Garlic Prevents Development of Diabesity in Mice
Authors: Tu Chen‐Pei David , Akgul Bunyamin , Lin Kai‐Wei , Pan Huei‐Ju , Chen Yen‐Hui , Lu Tzu‐Huan , Hsieh Li‐Lin , Chen Chien‐Hsiun , Kikuchi Tateki , Chen Yuan‐Tsong
Published: June 2021

Garlic (Allium sativum) has been an important nutrient supplement worldwide for millennia but its various health benefits lacks a unifying mechanism. We found that dietary garlic can prevent the development of obesity and type II diabetes in a mouse model. Relative to the controls, which were obese and diabetic, garlic‐fed mice had normal levels of blood glucose, cholesterol, triglycerides, and lower levels of insulin (~15‐fold) and leptin (~7‐fold). Further studies demonstrated that garlic accelerated RBC turnover by shortening RBC half‐lives, and increased expression of splenic erythropoiesis and related genes to avoid anemia. The increased RBC degradation in the garlic‐fed mouse spleen induced heme oxygenase‐1 to generate more iron, bilirubin, and CO, a pleiotropic gasotransmitter. Thus, the various health benefits of garlic are the result of a biological amplification mechanism beginning with the formation of darkly‐stained RBC, through accelerated RBC turnover, and ending with the second messenger CO in a general homeostatic response to garlic's phytochemicals. We propose that garlic may serve as a natural remedy in the management of the diabesity epidemics through a combination of the energy‐intensive process of accelerated RBC turnover and the pleiotropic effect of heme‐derived CO. (Supported by NSC‐95‐3112‐B‐001‐011 and Academia Sinica, Taiwan)
Unexpected metabolic alterations in VLCAD (very long chain acyl‐CoA dehydrogenase) deficient mouse hearts
Authors: Gélinas Roselle , Bouchard Bertrand , Thompson‐Legault Julie , Robillard‐Frayne Isabelle , Charron Guy , Labarthe François , Des Rosiers Christine
Published: June 2021

Patients with fatty acid oxidation (FAO) defects develop a cardiomyopathy, yet the underlying mechanism is unclear. Using our established working mouse heart model and 13C‐methodology, we have compared the metabolic phenotype of hearts from VLCAD deficient (VLCAD−/−) mice, which is the most common FAO disorder in human, and their control VLCAD+/+ counterparts. The expression of selected metabolic genes was also assessed using qPCR. Unexpectedly, 3 month‐old fed or fasted, or 7 month‐old fed VLCAD−/− mouse hearts displayed values for exogenous long chain FAO that were similar to controls, suggesting a compensatory mechanism, although the partitioning of these FA between oxidation and triglycerides was altered in these hearts. Gene expression profiling data revealed little or no difference in the transcript levels for other long chain FAO enzymes (Acadl and Acad9) in 3 or 7 month‐old VLCAD−/− hearts, but that of hormone sensitive lipase (Hsl) was significantly decreased (p<0.001) at 7 months following fasting. Finally, VLCAD−/− hearts showed additional lipid alterations, namely a 30% (p<0.001) decline in docosahexaenoic acid levels in cardiac phospholipids at 3 and 7 months. Collectively, our data highlight unexpected age‐dependent metabolic alterations in VLCAD deficient mouse hearts, which may contribute to cardiomyopathy development. (Supported by NIH & CIHR)
Connexin 43 is present in subsarcolemmal but not in interfibrillar mitochondria of rat cardiac myocytes
Authors: Boengler Kerstin , Stahlhofen Sabine , van de Sand Anita , Gres Petra , Buechert Astrid , Garcia‐Dorado David , Heusch Gerd , Schulz Rainer
Published: June 2021

In cardiac myocytes, connexin 43 (Cx43) is present in mitochondria, and mitochondrial Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). Cardiac myocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ functionally. Variations in the protein distribution between SSM and IFM have not yet been identified. The aim of the present study was to characterize the function of SSM and IFM, and to investigate whether Cx43 is present in SSM or IFM. The ADP‐stimulated respiration was greater in IFM than in SSM from rat ventricles using substrates for complex I and II. The calcium‐induced loss of membrane potential occurred at lower concentrations in SSM than in IFM. Cx43 was exclusively detected in SSM by Western blot analysis (n=4).

Taken together, our data confirm that SSM and IFM differ in respiration and calcium handling. Cx43 is localized in SSM. Since loss of mitochondrial Cx43 abolishes IP's cardioprotection, SSM and IFM might serve different functions in the signal transduction of IP.
Potential role of connexin43 in carbenoxolone‐promoted opening of the permeability transition pore in isolated brain and heart mitochondria
Authors: Reiser Georg , Azarashvili Tamara , Baburina Yulia , Grachev Dmitry , Krestinina Olga
Published: June 2021

Carbenoxolone (Cbx) was reported to induce permeability transition pore (PTP) opening in isolated rat liver mitochondria (RLM), although the mitochondrial molecular target of Cbx has yet to be identified. Here, we investigated the effects of Cbx on Ca2+‐induced PTP opening in RLM, rat heart (RHM), and rat brain (RBM) mitochondria. In RLM, RHM, and RBM, threshold Ca2+ load induced PTP opening, as seen by sudden Ca2+efflux from the mitochondrial matrix and a drop in membrane potential. At about 1 μM, Cbx enhanced Ca2+‐induced CyclosporinA‐sensitive PTP opening. Since in isolated heart mitochondria Connexin43 (Cx43) was found, we suggested that Cbx might be target for Cx43 in mitochondria. We detected Cx43 by Western blot in the outer RBM membrane and in mitoplasts, but not in RLM. Anti‐Cx43 antibody abolished Cbx‐enhanced PTP opening in RBM but not in RLM, supposing possible involvement of another connexin type in RLM. Besides being a gap junction inhibitor, Cbx bears structural similarity to corticosteroids, indicating possible translocator protein (TSPO)/ peripheral‐type benzodiazepine receptor (PBR) targeting. Cbx‐enhanced PTP opening, which correlated with increased protein phosphorylation, was suppressed by an anti‐TSPO antibody. All these results indicate that Cbx facilitates Ca2+‐induced PTP opening having as possible targets in RHM and RBM both TSPO and Cx43.
The acyl‐CoA synthetase ACSVL3 maintains the malignant phenotype of human gliomas
Authors: Watkins Paul A. , Pei Zhengtong , Sun Peng , Huang Ping , Lal Bachchu , Laterra John
Published: June 2021

The contribution of lipid metabolic pathways to malignancies is poorly understood. Expression of the fatty acyl‐CoA synthetase, ACSVL3, was markedly elevated in malignant gliomas but nearly undetectable in normal glia. ACSVL3 knockdown (KD) in cultured glioma cells using RNA interference significantly reduced both their in vitro malignant growth properties and in vivo tumorigenicity. Subcutaneous xenografts produced from ACSVL3 KD cells grew ~60% slower than control tumors and intracranial KD xenografts were ~85% smaller than controls. KD glioma cells had decreased capacity to activate fatty acids containing 16‐24 carbons. Incorporation of labeled palmitate (C16:0) into signaling lipids such as diacylglycerol and phosphatidylinositol, but not bulk membrane lipids, was reduced in KD cells. ACSVL3 expression was induced by activating oncogenic receptor tyrosine kinases (RTK) c‐Met and EGFR. ACSVL3 KD disrupted Akt responses to RTK activation, with a transient loss of both total and phosphorylated Akt via a caspase‐dependent mechanism. Our results show that ACSVL3 maintains oncogenic properties of malignant glioma cells via a mechanism that involves, in part, the regulation of Akt function and synthesis of signaling lipids. Targeting ACSVL3 could be therapeutically beneficial in malignant glioma and perhaps other cancers. Supported by NIH grants NS37355 (PAW) and NS43987 (JL).
Structural characterization of the PtdIns(4,5)P2‐PKCα‐C2 domain interaction
Authors: Corbalan‐Garcia Senena , Guerrero‐Valero Marta , Ferrer‐Orta Cristina , Querol‐Audi Jordi , Marin‐Vicente Consuelo , Gomez‐Fernandez Juan C , Fita Ignacio , Verdaguer Nuria
Published: June 2021

Classical PKCs contain two structural modules, the C1 and the C2 domains, in their regulatory region. These domains, in turn, dictate the cofactor‐dependence of the isoenzymes, being the C1 domain the diacylglycerol sensor and the C2 domain the Ca2+ and acidic phospholipids sensor. Here we show that a polybasic region located in the β3‐β4 strands of the C2 domain of classical PKCs, interacts specifically with PtdIns(4,5)P2 rather than with other polyphosphoinositides or phosphatidylserine. We have used X‐ray crystallography to analyze the structure of the C2 domain of PKCα in complex with PtdIns(4,5)P2, which was found at the conserved polybasic cluster. Site‐directed mutagenesis, on the residues from this cluster that interact directly with the phosphoinositide, revealed an important decrease in the ability of PKCα to interact with the plasma membrane of living cells. Furthermore, the mutant proteins were not able to translocate to the plasma membrane when the cells were stimulated with ionomycin and diacylglycerol. Instead, the protein localized in vesicles and Golgi apparatus, suggesting that the residues mutated are involved in recognizing the proper target of the C2 domain in the plasma membrane. Together, these data reveal the importance of the PtdIns(4,5)P2‐C2 domain interaction for the plasma membrane localization of PKCα.
Estradiol Stimulates Export of Sphingosine‐1‐Phosphate From MCF7 Breast Cancer Cells By Specific ABC Transporters
Authors: Takabe Kazuaki , Mitra Poulami , Allegood Jeremy C , Kim Roger H , Hait Nitai C , Milstien Sheldon , Spiegel Sarah
Published: June 2021

Sphingosine 1‐phosphate (S1P), produced by two sphingosine kinase isoenzymes (SphK1 and SphK2), regulates many cellular processes important for breast cancer progression by binding to specific S1P receptors. SphK1 is overexpressed in breast cancer, and both estradiol (E2) and epidermal growth factor (EGF) activate SphK1. In this study, we examined how intracellularly produced S1P is secreted from breast cancer cells. Overexpression of SphK1, but not SphK2, increased S1P export from MCF7 cells, and downregulation of expression of SphK1, but not SphK2, decreased its export. Although both E2 and EGF activated SphK1 and increased intracellular S1P, only E2 significantly stimulated S1P secretion. Export of S1P was inhibited by MK571, an inhibitor of ABCC1 (multi‐drug resistant protein 1), and fumitrimorgin C, an inhibitor of ABCG2 (breast cancer resistance protein), but not by the ABCB1 inhibitor, verapamil. In addition, E2‐induced secretion of S1P was blunted by downregulation of ABCC1 or ABCG2. These findings suggest that E2‐induced export of S1P is mediated by specific ABC transporters and may play an important role in multidrug resistance in breast cancer. This work was supported by NIH grants R37 GM043880 and R01CA61774 to SS and 5K12HD055881 to KT.
The cholesterol homeostatic protein Niemann‐Pick Type C‐2 (NPC2) is required for HIV‐1 assembly and release
Authors: Coleman Ebony Michelle , Hildreth James E.K.
Published: June 2021

HIV‐1 utilizes host proteins for productive viral replication. It is well established that HIV‐1 buds from cholesterol‐rich microdomains called lipid rafts, and that cholesterol is required for HIV‐1 entry, infectivity, and structural integrity. NPC2 protein binds cholesterol with high affinity and regulates cholesterol efflux from the late endosomal/ lysosomal (LE/L) compartments. NPC2 mutations result in NPC disease (NPCD), a cholesterol storage disorder characterized by an accumulation of cholesterol in LE/L compartments. We hypothesize that NPC2 is required for HIV‐1 assembly and release. Our findings show that in the absence of functional NPC2, HIV‐1 release is decreased. In fibroblasts from NPC2‐deficient patients infected with VSVG pseudotyped HIV‐1, virus release is reduced by 80%. Moreover, HIV‐1 Gag accumulated in the cholesterol laden LE/L compartments of the fibroblasts. Lysosomal targeting of NPC2 is mannose receptor dependent. Addition of mannose 6‐phosphate to chronically‐infected Jurkat cells disrupts both lysosomal targeting of NPC2 and HIV‐1 egress. Similarly, siRNA‐mediated silencing of NPC2 in these cells inhibited virus release. Collectively, these data indicate that NPC2 is required for HIV‐1 assembly and release.

Supported by NIH grant 5732HL‐007737‐15
Cloning and expression of thiolase enzymes in freshwater molluscs
Authors: Dyer James H. , Prezant Robert S.
Published: June 2021

Many species of freshwater molluscs exist in North America. Among these are the common gastropods Helisoma trivolvis, Physa acuta, and Goniobasis virginica. Additionally, the Asian clam, Corbicula fluminea, which found its way from China to North America some 70 years ago, is a very common bivalve. A considerable gap remains in the knowledge of lipid metabolism and lipid biochemistry in molluscs. The purpose of this research is to clone and express thiolase enzymes in various freshwater molluscs.
A novel fluorimetric assay for detection of aggrecanase‐1 (ADAMTS‐4) activity using a long wavelength FRET peptide substrate
Authors: Rakhmanova Vera A , Lui Siu Kei , Fan Weizhen , Bai Yuehong , He Jianjun , Hong Anita , Tong Xiaohe
Published: June 2021

Aggrecanase‐1, also called ADAMTS‐4, is a member of the ADAMTS (A disintegrin and metalloprotease with thrombospondin motif) family of proteases. Aggrecanase cleaves specific peptide bonds in aggrecan, the main proteoglycan of articular cartilage. The high activity of aggrecanase results in cartilage degradation in diseases such as arthritis. To develop homogeneous high throughput screening of ADAMTS‐4 inhibitors, we synthesized a novel internally quenched peptide substrate for aggrecanase‐1 consisting of 5‐FAM/TAMRA as the FRET pair. Active ADAMTS‐4 cleaves this FRET substrate into two separate fragments resulting in an increase of 5‐FAM fluorescence, monitored at excitation/emission = 490 nm/520 nm. The new FRET substrate is more sensitive to aggrecanase‐1 cleavage than the previously described Abz/Dnp substrate and can detect subnanogram amounts of ADAMTS‐4. Its cleavage by other members of aggrecanase family, such as ADAMTS‐1 and ADAMTS‐5, is negligible. Inhibitor screening assays with several known metalloprotease inhibitors were validated using this substrate.

This novel FRET substrate provides a convenient homogeneous format for aggrecanase‐1 activity assay where signal is less interfered by the autofluorescence of test compounds due to the long wavelength excitation and emission of 5‐FAM, resulting in an overall increase of signal to background ratio.
Role of ADAM17 in bradykinin‐induced EGF receptor activation in glomerular podocytes
Authors: Dey Mamon , Raymond John R , Gooz Monika
Published: June 2021

Beneficial effect of ACE inhibitors was attributed partially by upregulating bradykinin (BK) production. We have shown previously that BK cross‐activates the epidermal growth factor receptor (EGFR) and decreases albumin permeability of mouse podocyte monolayer. We hypothesized that the crosstalk between BK receptor (BKR) and EGFR through a metalloenzyme‐disintegrin (possibly through ADAM17) is responsible for the permeability change in the podocytes. We treated human and mouse podocytes with 10−10‐10−6 M BK and measured ERK activation as a read‐out of the inter‐receptor crosstalk. We found maximum activation of ERK1/2 at 10 min by 10−8 M BK. We pretreated the cells for 30 min with 100 nM AG1478 (inhibitor of EGFR) or 10 µM GM6001 (inhibitor for metalloenzymes) followed by BK treatment and found that ERK1/2 phosphorylation was inhibited. To identify the metalloenzyme involved in this process we transfected the podocytes with small interfering RNA against ADAM17. After successfully silencing the enzyme expression we found that BK induced ERK1/2 activation was attenuated. We conclude that (1) ADAM17 is involved in the crosstalk between BKR and EGFR; (2) ADAM17 is a potential contributor to BK‐induced permeability changes and a potential therapeutic target in kidney fibrosis.
Prostaglandin F2α (PGF2α) stimulation of tumor necrosis factor‐α (TNF‐α) promoter activity by sequential activation of protein kinase C (PKC) and nuclear factor‐κB (NF‐κB) signaling
Authors: Chen David C. , Chou Chih‐Ling , Hutchinson Anthony J. , Regan John W.
Published: June 2021

TNF‐α is a cytokine that promotes inflammation and apoptosis. Over production of TNF‐α in microglia, the resident macrophages in the brain, causes neuronal death and has been linked to inflammatory neurological disorders such as Alzheimer's disease. Another signaling molecule up regulated during inflammation is PGF2α, which activates intracellular Ca2+ and PKC signaling through the Gq‐coupled FP prostanoid receptor. The specific role, however, of PGF2α in the promotion of inflammation by TNF‐α is not well understood. In this study, we hypothesized that activation of the human FP receptor by PGF2α can stimulate the activity of the TNF‐α gene promoter. In support of this hypothesis PGF2α stimulated TNF‐α promoter activity in cultured microglia transfected with a reporter plasmid controlled by the TNF‐α promoter; similar results were obtained with HEK‐293 cells stably expressing the FP receptor. The TNF‐α promoter contains a NF‐κB binding site and inhibition of NF‐κB reduced PGF2α stimulated TNF‐α promoter activity. Additionally, inhibition of PKC decreased the activity of reporter genes under the control of either NF‐κB or TNF‐α, respectively. These results indicate that PGF2α acts through the FP receptor to induce TNF‐α promoter activity by a mechanism involving the PKC dependent activation of NF‐κB. We believe this mechanism helps provide a better understanding of the role of PGF2α in inflammatory diseases.
Vasoactive intestinal peptide induces apoptosis in naïve but not in activated HuT 78 cells
Authors: Failing Jarrett , Dorsam Sheri , Van der Steen Travis , Dorsam Glenn
Published: June 2021

Activation induced cell death (AICD) of T lymphocytes plays a vital role in the regulation of the immune response. Cellular resistance to apoptosis can contribute to cancer and autoimmune diseases. Vasoactive intestinal peptide receptor 1 and 2 (VPAC1/2) are G‐protein coupled receptors highly expressed in the immune system that bind vasoactive intestinal peptide (VIP). It has been previously shown that inducible VPAC2 inhibits apoptosis in Th2 polarized CD4 T cells. However, the effect of VPAC1 signaling is not well understood. Naïve and activated HuT 78 cells, a transformed human T lymphocyte cell line predominantly expressing VPAC1, were treated +/‐ VIP. The extent of apoptosis was measured using a histone fragmentation‐based ELISA. In activated cells, VIP had little effect on apoptosis (10‐6‐10‐10M), but caused a significant increase in apoptosis in naïve cells (10‐8M). VIP/VPAC1 signaling induces apoptosis in naïve T cells but has little effect on activated cells. Furthermore, it appears that the VPAC1 and VPAC2 receptors affect apoptosis in opposing ways, most likely due to subtle differences in intracellular signaling. Research supported by NIH‐K01 1K01DK064828 and COBRE 2P20RR05566.
Nuclear factor erythroid derived‐2 (NF‐E2)‐mediated regulation of NF‐kB activation in neutrophils: A potential mechanism of NF‐E2‐mediated apoptosis
Authors: Johnson Paul R , Cummins Timothy D , Manning Terri R , Wu Rui , Sachleben Leroy R , Gozal Evelyne , Rane Madhavi J
Published: June 2021

Neutrophils (PMNs) are short‐lived cells that constitutively undergo apoptosis to prevent excessive tissue damage. We previously identified NF‐E2 as an Akt substrate in human PMNs, and showed that antibody depletion of NF‐E2 delayed PMN apoptosis at 24 hours as assessed by propidium iodide flow cytometry. Additionally, NF‐E2 depletion of PMNs increased IκB phosphorylation, a marker of NFκB activation with concomitant increases in pro‐survival Mcl‐1 expression. These studies led us to hypothesize that NF‐E2 promotes constitutive PMN apoptosis by inhibiting NFκB activation. Preliminary EMSA revealed increased PMN NFκB DNA binding after NF‐E2 depletion. Furthermore, co‐transfection of HEK‐293 cells with NF‐E2 and NFκB luciferase reporter resulted in significantly lower NFκB luciferase promoter activity after TNF‐α stimulation as compared to vector and NFκB luciferase reporter transfected cells. These results collectively demonstrate that NF‐E2 regulates PMN apoptosis by inhibiting NFκB activity. In our future studies, we will utilize NFκB inhibitors to further delineate a role for NFκB in promoting PMN survival and Mcl‐1 expression as a consequence of NF‐E2 depletion. Taken together, our results suggest that NF‐E2 is a pro‐apoptotic protein that promotes constitutive PMN apoptosis by inhibiting NFκB‐mediated transcription of pro‐survival genes.

Support for this work was provided by AHA #0815433D (PRJ) and AHA #0335278N, NIH/NIAID #1R56AI059165‐01A2, RO1AI075212‐01A1 (MJR).

ASBMMB abstract, EB2009, Deadline November 5, 2008
Calcium signaling in beating human embryonic stem cell‐derived cardiomyocytes
Authors: Apati Agota , Paszty Katalin , Hegedus Luca , Orban Tamas , Homolya Laszlo , Enyedi Agnes , Sarkadi Balazs
Published: June 2021

In cardiomyocytes calcium has a crucial role in maintaining rhytmic contractions through the excitation‐contraction coupling process. To obtain spontaneously contracting cardiomyocytes, human embryonic stem (HuES) cells were differentiated through embryoid body formation. Clumps of beating cells were mechanically separated, seeded onto microscope chambers, and loaded with Fluo‐4 Ca2+‐sensitive dye. We showed simultaneous contractions and Ca2+ transients of the HuES‐derived beating clumps. We demonstrated that epinephrine increased, whereas verapamil, a drug that blocks calcium channel, stopped the contractions. Thus the basic pharmacological characteristics of the beating heart were preserved. In these cells PMCAs play a special role by lowering Ca2+ locally in selected membrane areas, therefore the subcellular localization of specific PMCA pumps were tested by confocal laser scanning microscopy, following the presence of PMCA1 and PMCA4 expression. Our HuES derived cardiomyocyte model helps to develop functional human heart tissue pharmacological test systems in connection with Ca2+ signaling.
Pharmacologic evidence for stabilization of a nucleotide‐bound, stimulatory conformation of SUR1Q1178R, an ABCC8 neonatal diabetes mutation
Authors: Ortiz David Javier , Rüdiger Benjamin , Bryan Joseph , Quast Ulrich
Published: June 2021

Mutations in ABCC8/SUR1 that generate hyperactive neuroendocrine‐type Kir6.2/SUR1 KATP channels are a cause of neonatal diabetes. Analysis of one mutant, SUR1Q1178R, is consistent with the MgATP‐dependent accumulation of a post‐hydrolytic, ADP‐bound enzymatic intermediate that stimulates channel opening. SUR1 is the target for KATP channel inhibitors, e.g., glibenclamide, and openers, e.g., diazoxide and NCC. In the Kir6.2/SUR1 channel, the mutation potentiated the negative allosteric effect of MgATP on binding of [3H]‐glibenclamide reducing the number of specific channel binding sites by ~50% whereas in wild‐type, the KD increased 4.5x. This is consistent with a greater depopulation of antagonist binding sites in the R1178 channels at steady‐state. MgATP is required for opener function and high‐affinity agonist binding. SUR1R1178 increased the apparent number of agonist binding sites, without affecting their Ki, implying these agonists interact with the stimulatory, post‐hydrolytic conformation stabilized by the R1178 substitution. The data suggest the stimulatory, post‐hydrolytic conformation could accumulate either because R1178 increases the rate of ATP hydrolysis or reduces the rate of product dissociation, and rationalize the need for a higher glibenclamide dose to reach metabolic control in ABCC8 neonatal diabetes patients.

Supported by NIH DK44311(JB); DFG QU_100/4‐1(UQ).
Role of caspase‐8 ubiquitination and degradation by the 26S proteasome in regulating extrinsic cell death
Authors: Fiandalo Michael V. , Schwarze Steven R.
Published: June 2021

Studying the extrinsic and intrinsic pathways of apoptosis has led to new anticancer strategies. Caspase‐8 activation is critical for extrinsic apoptosis induction. Our data have shown that proteasome inhibition, with the chemotherapeutic agent Velcade, sensitizes prostate cancer cells to apoptosis and stabilizes the caspase‐8 p18 subunit. The goal of this study is to identify the amino acids that regulate caspase‐8 stability. Using a HA‐tagged gene approach, several caspase‐8 fragments were expressed and tested for stability and ubiquitin incorporation to define which region was a target for ubiquitination. Consistent with endogenous pro‐caspase‐8, catalytically inactive unprocessed caspase‐8 was readily expressed and protein levels were not enhanced by proteasome inhibition, while expression of the active processed p18 and p10 subunits required Velcade. Furthermore, only the p18 subunit was covalently associated with polyubiquitin. Site‐directed mutagenesis studies are currently underway to mutate lysine residues to arginine in the p18 subunit in an attempt to generate a degradation‐resistant caspase‐8. This study has demonstrated that active caspase‐8 and more specifically the p18 subunit, is a target for ubiquitination and degradation by the 26S proteasome. Future studies will test the extent to which non‐ubiquitinated caspase‐8 can modulate extrinsic apoptosis.
A new signaling pathway that is involved in the BMP signaling
Authors: Hagihara Meiko , Yamashita Toshihide
Published: June 2021

Bone morphogenetic proteins (BMPs) are multifunctional proteins that comprise the largest subfamily of the transforming growth factors‐beta. BMPs transduce the signal through BMP type I and type II receptors. Here we identify a new protein that directly binds to BMPs. This protein has a transmembrane domain that localizes at the plasma membrane. Although BMP‐2 as well as BMP‐4 induces osteoblast differentiation of ST2 cells, knockdown of this protein results in suppression of the osteoblast differentiation and phosphorylation of smad1/5/8 induced by BMPs, suggesting that the protein enhances the BMPs signals. The osteoblast differentiation maintained by BMPs binding to the protein appears to be dependent on the activity of RhoA. Thus, we uncover a new mechanism that regulates the signaling pathway from BMPs to smads and osteoblast differentiation.
AHNAK expression and subcellular localization in gastric surface epithelial cells
Authors: Alli Abdel A , Rideout Drew A , Carter Gay M , Gower William R
Published: June 2021

We have previously demonstrated the expression of AHNAK protein in normal human organ donor stomach tissues. In order to identify the specific cells that express AHNAK in human stomach, we performed immunohistochemistry. We evaluated the subcellular localization and movement of AHNAK protein by immunohistochemistry, sucrose density gradient centrifugation, and Western blot analysis using specific AHNAK and subcellular marker antibodies. AHNAK immunoreactivity was localized to PAS, Alcian Blue, MUC5A positive gastric surface epithelial cells in normal human organ donor stomachs. Normal rat gastric mucosa (RGM1) cells demonstrated strong cytoplasmic staining for AHNAK. AHNAK protein was found predominately in the cytoplasmic fraction under standard culture conditions of RGM1 cells. AHNAK is often described as a nuclear phosphoprotein that translocates to the plasma membrane where it is involved in signal transduction, cell membrane differentiation, and repair. Further investigation and understanding of the potential recruitment of AHNAK to the plasma membrane may allow for novel strategies to regulate specific signaling cascades and induce membrane repair in gastric surface epithelial cells. This research was supported by an American Heart Association Predoctoral Fellowship and a Veterans Affairs Merit grant.
Proteasome Inactivation Promotes p38 MAPK‐Dependent PI3K Activation and Increases IL‐8 Production
Authors: Shang Fu , Fernandes Alexandre F , Bian Qingning , Taylor Allen , Pereira Paulo
Published: June 2021

Oxidative stress and inflammation are implicated in the pathogenesis of many age‐related diseases. The objective of this work is to elucidate the molecular link between oxidative stress and expression of pro‐inflammatory cytokines. We demonstrated that oxidative inactivation of the proteasome is a molecular link between oxidative stress and over‐expression of IL‐8. We also elucidated a novel molecular mechanism underlying the upregulation of IL‐8 induced by proteasome inactivation. Proteasome inactivation in retinal pigment epithelial cells resulted in activation of p38 MAPK, EGFR and PI3Ksignaling pathways, and a dramatic increase in the expression of IL‐8. Blocking any of these signaling pathways abolished the proteasome‐inactivation‐induced production of IL‐8. Using chemical inhibitors and constitutively active and mutant forms of MKK3, MKK6 and Akt, we elucidated a cascade of sequential activation of these signaling pathways in response to proteasome inactivation. The sequential events of this cascade are: proteasome inactivation, activation of MKK3/MKK6, activation of p38 MAPK, EGFR phosphorylation, PI3K activation and increased IL‐8 expression. Proteasome inactivation also induced a sustained Akt activation, but inhibition of Akt activity did not block proteasome‐inactivation‐induced IL‐8 production. Together, these data elucidate a novel cross‐talk of signaling pathways that lead to increased IL‐8 production in response to proteasome inactivation.
Preliminary investigation to the synthesis of gamma amino acids
Authors: McCormick Casey Jerome , Swartling Daniel J
Published: June 2021

Cyclic and acyclic precursors are being prepared for conversion in several steps to gamma amino acid analogs, which may possess biological activity. Cyclic and acyclic aldehydes and ketones were coupled to Meldrum's acid using the Knoevenagel condensation method to yield the corresponding adducts. These will be reacted with nitromethane to form the corresponding Michael addition products, which will be reduced and decarboxylated to form gamma amino acid analogs.
Antibacterial And Antithrombic Activities Of Agrowastes Culture Extract Of P.Chrysogenum
Authors: Mina Onyegeme‐Okerenta Blessing , Ihuaku Okochi Veronica , Albert Ebuehi O , Uzoma Okafor Anthony
Published: June 2021

Penicillium chrysogenum is a microfungus with a potent antibiotic production activity. It is also well known for its agrowastes biodegradation and enzyme production. This study investigated the use of agro waste based culture extract of a novel P. chrysogenum PCL 501 in antibacterial and antithrombic activities (thrombin, a serine protease is involved in blood clotting). The fungus was cultivated for a period of 240 h on cassava shavings and sugar cane pulp media in submerged fermentation and the culture extract was analyzed for its antibacterial and antithrombic activities, using benzyl penicillin as reference drug. The results revealed that the two agrowastes were well utilized by the fungus for growth and the culture extract had a potent antibacterial activity against E.coli and B.subtilis. Extract from cassava shavings show potency indices of 11.76 and 10.432 against E.coli and B.subtilis respectively and that from sugarcane pulps 17.95 and 11.41 against E.coli and B.subtilis respectively. The extract inhibited thrombin (serine protease) action which explains its antithrombic activities observed in terms of delayed clotting time. A similar pattern of inhibition was also observed with the reference drug (Benzyl penicillin).
Characterization of the anthrone oxidase in aflatoxin biosynthesis
Authors: Ehrlich Kenneth C. , Scharfenstein Leslie L , Chang Perng‐Kuang , Li Ping , Bhatnagar Deepak
Published: June 2021

The first stable intermediate in aflatoxin biosynthesis is the red anthraquinone, norsolorinic acid (NA), even though the polyketide synthase (PKSA) product is predicted to be the norsolorinic acid anthrone (NAA). Recently, NAA was obtained in low yields in vitro when hexanoyl‐ and malonylCoA were fed to re‐constituted PKSA. We now show that NAA is converted to NA by an anthrone oxidase encoded by the gene hypB1 (aflC1) a 548 bp gene situated adjacent to pksA in the aflatoxin gene cluster. Insertional mutagenesis of hypB1 gave A. parasiticus transformants that accumulated excess amounts of NA and a greenish‐fluorescent compound that migrated slightly faster on TLC. Upon standing in air this compound slowly oxidized to NA. The new compound was not present after incubation of the extract from hypB1 disruptant colonies with an extract from wild‐type A. parasiticus. Attempts were made to repeat this result using extracts of IPTG‐induced E. coli BL21(DE3)plysS harboring the expression vector pEThypB1. The active site motif NXXQW, where the W is required for the oxygen transfer, catalyzes anthrone oxidation in known bacterial anthrone oxidases. In HypB1 the motif QLVHQW may be acting in a similar capacity. In the A. nidulans sterigmatocystin biosynthesis cluster, the HypB1 homolog, StcM, contains a comparable motif (QLVRHW) and the HypB1 homolog in the emodin biosynthesis cluster contains the motif AHQPW.
Ketogenic diets increase hepatic insulin resistance by the attenuation of hypothalamic leptin signaling and hepatic insulin signaling in diabetic rats
Authors: Park Sunmin , Hong Sang Mee , Ahn Il Sung
Published: June 2021

We determined the effects of ketogenic diet (KTD) on insulin resistance and insulin secretion in 90% pancreatectomized (Px) diabetic rats. KTD (0 En% carbohydrates and 78 En% fat) or control diet (COD; 58 En% carbohydrates and 20 En% fat) was given to diabetic rats for 4 weeks. Serum glucagon and leptin levels were higher in rats fed KTD (p<0.05). During oral glucose tolerance test, serum glucose levels slowly increased until 80 min in KTD, compared to COD, and they decreased slowly. Insulin secretion capacity at hyperglycemic clamp did not significantly differ between two groups. However, euglycemic hyperinsulinemic clamp revealed KTD decreased glucose infusion rates and increased hepatic glucose output at hyperinsulinemic states. KTD attenuated hypothalamic STAT‐3 phosphorylation in comparison with the control diet. The increased hepatic glucose output in KTD was associated with increased hepatic PEPCK expression through attenuated tyrosine phosphorylation of IRS2 and serine473 phosphporylation of Akt (p<0.05). Additionally, hepatic serine phosphorylation of AMPK and ACC was reduced in KTD (p<0.05), suggesting decreased fatty acid oxidation and decreased triglyceride synthesis. In conclusion, KTD suppressed hepatic insulin sensitivity through attenuated AMPK phosphorylation and insulin signaling in diabetic rats.
Effects of vitamin A deficiency on plasma glucose, insulin and leptin in rats
Authors: Zhang Yan , Gao Yifei , Chen Guoxun
Published: June 2021

We have observed that vitamin A and its catalytic metabolites synergize with insulin to induce hepatic glucokinase gene expression. It has been known that retinoic acid (RA) can stimulate the expression of phosphoenolpyruvate carboxyl kinase gene. To better understand the role of vitamin A in glucose and lipid homeostasis, Zucker lean (female) rats were fed with vitamin A deficient (VAD) diet (TestDiet, #5822) and vitamin A sufficient (VAS) control diet (TestDiet, #5755) after weaning. When body weight gain stops in VAD group (a sign of vitamin A deficiency), animals were used to access the effect of vitamin A deficiency on glucose and lipid metabolism. All animals were fasted for six hours before their plasma and tissue samples were collected for analysis. To this end, levels of plasma glucose (153 ± 35.1 mg/dl), insulin (0.6 ±0.35 ng/ml), and leptin (1.3 ± 0.64 ng/ml) of VAD rats were all significantly lower than those (glucose, 176 ± 23.1 mg/dl; insulin, 1.3 ± 0.6 ng/ml; 4.0 ± 2.1 ng/ml, all P<0.04)) of VAS fed rats. When VAD rats were fed with VAS diet, the plasma glucose level (150 ± 40 mg/dl) of VAD rats was significantly increased at three days (207 ± 62 mg/dl) and seven days (221 ± 45 mg/dl) after initiation of VAS diet (all P<0.05). At the same time, plasma cholesterol level (49.6 ± 4.4 mg/dl) of VAD rats was also significantly increased at three days (74.6 ± 8.8 mg/dl) and seven days (83.9 ± 9.2 mg/dl) after resuming the VAS diet (all P<0.004). These results demonstrated that vitamin A deficiency can result in the changes of plasma glucose and lipid homeostasis. Further work is warranted to understand the underlying molecular mechanisms.
Negligible long‐chain omega‐3 intake among vegetarians is not associated with negative emotion
Authors: Beezhold Bonnie Lynn , Johnston Carol S. , Daigle Deanna
Published: June 2021

Considering the low eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) status widely reported for vegetarians, and the evidence that EPA+DHA promotes mental health, we examined the relationship between dietary EPA+DHA and depression in healthy vegetarian (VEG) and omnivore (OMN) adults from Seventh‐Day Adventist populations in the Southwest. Participants (60 VEG; 78 OMN) completed a survey including a food frequency questionnaire and Depression Anxiety Stress (DASS) and Profile of Mood States (POMS) scales. VEG consumed less EPA+DHA and arachidonic acid (AA), but significantly more a‐linoleic acid and linoleic acid, compared to OMN. VEG reported significantly less negative emotion than OMN as assessed by DASS scores (8.3±0.9 and 17.5±1.9, p=.003, adjusted for the confounding variable, physical activity). VEG tended to report less mood disturbance than OMN as assessed by POMS scores (0.1±2.0 and 15.3±3.1, p=.083, adjusted for age and physical activity); however, VEG females reported significantly less mood disturbance than OMN females (1.6±2.9 vs 22.2±4.6, p=.006). Moreover, dietary AA, but not EPA+DHA, was significantly related to mood disturbance among omnivores (r=.256, p=.024). We conclude that VEG reported less negative emotion than OMN due perhaps to lower intakes of AA which may alter brain eicosanoid profile and inflammatory potential. This research was supported by the ASU Nutrition Research Fund.
Dietary resistant starch and acarbose reduce body weight gain in rodents
Authors: McCutcheon Kathleen L , Keenan Michael J , Zhou Jun , Tulley Richard T , Raggio Anne M , Vidrine Kirk , Goita Mfamara , Greenway Frank , Martin Roy J
Published: June 2021

Resistant starch (RS) resists digestion in the small intestine thus increasing the amount of starch available for microbial fermentation producing short chain fatty acids in the colon. Additionally, RS has been shown to reduce body fat in rodent models through mechanisms possibly related to colonic fermentation pathways. Acarbose, an α‐glucosidase inhibitor used to treat Type 2 diabetes, inhibits starch digestion in the small intestines, thus undigested starch reaches the colon mimicking the effects of RS. In this study, we compared changes in fermentation and body fat in rats fed diets with RS or acarbose (AC). Thirty female retired breeder rats were placed in 3 treatment groups (control, 30% RS, 0.4% AC) and fed energy balanced diets (20% fat, 3.7kcal/g) for 12 weeks. Dietary RS and AC reduced body weight and body fat gain. The control group gained 20g, compared to no gain for AC and a 20g loss for RS. Both RS and AC had larger full GI tracts, indicating reduced starch digestion; however, only RS had lower pH of cecal contents indicating only RS increased colonic fermentation. Acarbose seems to prevent starch digestion in the small intestine and starch fermentation in the large intestine. At the doses used, both RS and AC were effective in preventing weight gain, but the greater effect of weight loss with RS may be due to fermentation. Funding by LSU AgCenter.
Characterization of protein intakes in older adults in the US
Authors: Berner Louise , Prakash Priya , Becker Gabe , Doi Jimmy
Published: June 2021

We analyzed data from NHANES 2003‐04 to determine adequacy of protein; contribution of animal foods/ingredients to protein; and meal patterns of protein intake in older adults. SAS, CSIDE, and SUDAAN software were used for data management/analyses. Results showed ~9 or 11% of women 65+ but fewer men consumed <EAR of 0.66 g/kg healthy body weight (defined as actual or closest weight to be within BMI range of 18.5‐24.9 or 22‐27 kg/m2). With a revised EAR of 1.0 g/kg, in line with higher proposed protein RDAs advocated by some, >50% had intakes <EAR. Using the FNDDS 2.0 and SR 18 datasets, we devised a strategy to determine the contribution of animal protein (total and specific categories such as beef, dairy, poultry, etc.) to total protein intakes. Animal foods/ingredients contributed, on average, 61.5 % of protein intake in older adults; the highest contributors were dairy (17.5%), beef (13%), and poultry (12%). In regression models, with covariables including energy, the % protein from animal foods (or any animal category) predicted protein intake (P<0.001). Logistic regression analyses showed that the odds of meeting the current protein RDA were significantly higher as % protein from animal foods increased. These results may have practical implications for dietary guidance in older adults. Supported by grants from CSU Ag Research Initiative, National Cattlemen's Beef Association, and Dairy Council of California.
Current Status of Nutrition Transition in South Korea based on the 2005 Korean National Health and Nutrition Survey
Authors: Park Hyun Ah , Lee Jung Sun , Lee Sung Hee , Kim Ok Hyun , Oh Sang Woo , Lee Soo Kyung
Published: June 2021

South Korea has undergone a unique nutrition transition pattern, and shown relatively lower level of total dietary fat and obesity compared with other Asian countries with commensurate income level. This study reports the current status of energy and macronutrient intake and weight in South Korea using the latest 2005 Korean National Health and Nutrition Survey (n=6436, ≥3 yr, 45% women). The energy intake from macronutrients in South Koreans peaked at 15‐19 yr old and averaged 2,208±24 kcal in men and 1,783±15 kcal in women. The mean percentages of energy from carbohydrate, protein, and fat were 65%, 15%, and 20%, respectively. The energy intake from fat was highest in 3‐5yr old boys and 15‐19 yr old girls, and decreased with age. Body Mass Index (BMI) of adults was 23.9±0.1 kg/m2 in men and 23.3±0.1 kg/m2 in women. The prevalence of obesity (BMI≥25) in adult men and women were 27.4% and 22.5%, respectively. The energy intake from fat and the prevalence of obesity in South Korea are still lower than those of other countries at comparable economic development level, however they have been increasing over the last decade with differential rates across age groups. Better understanding on the unique trends related to energy and macronutrient intake is essential to address current and future nutritional health issues in South Korea as well as other developing countries undergoing similar economic and nutritional transitions.
Relationships between body mass index, diabetes, and blood glucose level in South Korean adults: 2005 Korea National Health and Nutrition Survey
Authors: Lee Soo‐Kyung
Published: June 2021

How to define overweight and obesity among Asian individuals has not yet been firmly decided. Western countries have been using the general criteria for overweight (BMI > 25) and obesity (BMI > 30) with Asian populations in their country, while WHO and Asian countries have recommended or adopted lower BMI cutoff points for overweight and obesity. More data on relationships between BMI, morbidity, and mortality in Asian populations have been called for to decide which criteria are more appropriate. This study examined relationships between BMI, diabetes, and fasting blood glucose level in Koreans using 2005 Korea National Health and Nutrition Survey. The relationships found in the 2005 dataset were compared to those in 1998 dataset to determine whether the relationships were affected by nutrition transition occurred in Korea. The average BMI of Korean adult (age > 20) men and women were 23.9 ±0.1 kg/m2 and 23.3±0.1 kg/m2, respectively. According to the WHO Asian Pacific criteria for overweight (23kg/m2=BMI=25kg/m2) and obesity (25kg/m2 =BMI), 23% of men and 19.9% of women were grouped as overweight, while 27.4% of men and 22.5% of women as obese. The diabetes prevalence was on average 51.7 per 1000 persons with men showing higher prevalence than women. BMI and the diabetes prevalence showed a positive relationship, and the nature of the relationship appeared slightly different from those found in 1998 dataset. The difference, albeit small, should be examined further and followed up to determine whether the relationships are fixed.
Predicting Body Fat in College Students
Authors: Notte Dana Renae , Tyzbir Robert S.
Published: June 2021

The purpose of this study is to determine which anthropometric variables can be used to develop equations that best predict body fatness in college age students. The body mass index (BMI), waist to hip ratio (W/H), and conicity index (Ci), was calculated for men and women participating in an elective nutrition course from 1992 to 2007. These calculations were used to develop the following linear regression prediction equations using SPSS:

These equations were then used to predict body fat for male and female students in 2008 and results were compared to the percent body fat measured using bioelectrical impedance (BEI). Our results indicate that in males the equation using W/H ratio is the best predictor of body fat followed by equations using BMI and Ci. However, in females, the BMI equation is the best predictor of body fat followed by W/H and Ci respectively. We conclude that equations derived from different anthropometric variables should be used when predicting body fat in males verses females. In males, the equation using W/H is best at predicting body fat while, in females, the equation using BMI is the best.