bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycosylation as a novel stabilization approach preventing aggregation and inactivation of a‐chymotrypsin in the solid‐state and upon encapsulation in bioerodible nanosheperes
Authors: Flores Giselle Marie , Pagan Miraida , Griebenow Kai
Published: June 2021

The complex physicochemical nature of proteins that enhances their potential as pharmaceuticals, also adds to their fragility. Typically, proteins are relatively stable in the solid‐state; however, exposure of lyophilized proteins to excess moisture has been reported to cause undesirable physical and chemical changes. These effects will have a direct impact on solid therapeutic protein formulations and on sustained‐release devises. Therefore, it is important to examine the stability of solid proteins under pharmaceutical relevant conditions. The goal of our study was to examine the stability and structural changes that a protein undergoes after moisture sorption, develop a rational stabilization strategy and prove this in a sustain release. The protein α‐chymotrypsin was used to study the effect of water sorption after incubation at different relative humidities. FTIR, circular dichroism, H/D exchange, enzyme kinetics and aggregation studies were perform. These studies demonstrated that moisture causes deleterious processes to occur to the solid‐state protein. Chemical modification with glycans was performed to test if glycosylation could decrease protein unfolding and aggregation. Results showed that chemical glycosylation reduced and/or prevent these deleterious processes. These glyco‐conjugates are been used to develop sustained release devices; initial results show high encapsulation efficiency and an increase in protein stability.
CHEMICAL PROTEIN GLYCOSYLATION: A METHOD TO PREVENT PROTEIN INSTABILITIES ON BIOSENSOR APPLICATION
Authors: Pagan Miraida , Griebenow Kai
Published: June 2021

The development of small and robust sensors with low electrical power requirement will be essential to monitor astronaut physical fitness and condition in order to prevent and minimize adverse health effects that could result from space flight. Even though there are many advances reported recently using enzymes (e.g., alcohol dehydrogenase, glucose oxidase) as the principal sensing element to monitor specific biochemical analytes, an efficient methodology to prevent short lifetime and poor loading of the protein onto the sensor is lacking. These limitations are related to enzyme stability problems. Herein, we seek to improve the understanding of enzyme stabilization in order to increase the lifetime and performance of enzyme biosensors by employing the glycosylation technique. Covalent chemical glycosylation consists in decorating the enzyme with glycans which will increase protein thermodynamic stability and prevent unfolding by caging effects. Furthermore, attachment of the modified protein to a nano‐material will increase the enzyme loading and improved reaction kinetics In order to study the effect of chemical glycosylation it was used a simple enzyme model, Subtilisin. The central hypothesis of the research is that covalent chemical modification of the enzyme with glycans will reduce conformational motions and increase its thermodynamic stability thus positively affecting long‐term stability
Cloning a Histidine‐rich Loop of the CrMTP1 Protein of Chlamydomonas reinhardtii
Authors: Yang Huidong , Gingrich David J
Published: June 2021

Zinc plays several roles in cellular metabolism. To accomplish this, the amount and forms of zinc must be controled within the cell. One important aspect of zinc homeostatis is the transport of zinc ions across cell membranes via transport proteins. This research involves the cloning of a loop of a putative zinc‐transport protein CrMTP1from the organism Chlamydomonas reinhartdii. This histidine‐rich region of the protein, between putative transmembrane helicies 4 and 5, may provide zinc binding sites involved directly in zinc transport or in regulation. PCR has been utilized to amplify the complete gene from a Chlamydomonas cDNA library and subsequently the sequence of the gene corresponding to only the histidine loop region. The PCR amplification is designed to incorporate the DNA into an expression vector in order to produce the protein in E. coli. After purification, putative zinc binding to the purified protein will be studied in order to investigate its possible role in the function of the putative zinc transport protein.
Reconstitution of macrolide pump MacAB‐TolC into lipid nanodiscs
Authors: Lu Shuo , Zgurskaya Helen I
Published: June 2021

In Gram‐negative bacteria, intrinsic antibiotic resistance is provided by three component efflux transporters. In Escherichia coli, MacAB‐TolC is an ATP‐driven tripartite efflux pump involved in the extrusion of macrolide antibiotics. In this complex, MacA is a periplasmic membrane fusion protein, MacB is an ABC‐type transporter and TolC is an outer membrane protein required for MacAB function. The goal of this project is to investigate the assembly of MacAB‐TolC complex in vitro and characterize the affect of substrates and ATP on the assembly of MacAB‐TolC. To achieve this goal, we reconstituted MacB, TolC and MacAB into lipid nanodiscs. First, we cloned, expressed and purified the intein‐apoA‐I fusion protein. We then optimized the reconstitution conditions for all these proteins. The protein associated nanodiscs were purified from empty nanodiscs by His‐tag binding column. To check the functionality of reconstituted nanodiscs, we measured the ATPase activity of MacB and MacAB containing nanodiscs. Upon reconstitution into lipid nanodiscs, MacB and MacAB displayed basal ATPase activity. MacA stimulated MacB ATPase activity by 40% and the prescence of TolC had no effect to the ATPase activity of MacB. Thus, we successfully reconstituted MacB, TolC and MacAB into lipid nanodiscs and these nanodiscs are functionally active. (supported by National Institutes of Health Grant AI052293 )
Mechanism of the stimulation of MacB ATPase by the periplasmic Membrane Fusion Protein (MFP) MacA
Authors: Modali Sita D , Zgurskaya Helen I
Published: June 2021

Drug efflux pumps are the main contributors to the failure of antibiotic therapy. In Gram‐negative bacteria, these pumps function as tripartite complexes that extrude drugs out of the cells. In Escherichia coli, MacA‐MacB‐TolC is the first experimentally characterized macrolide specific efflux pump. Here, MacB is the ABC type inner membrane transporter, TolC is the outer membrane channel and MacA is the periplasmic Membrane Fusion Protein. MacA physically connects MacB to TolC and also stimulates the ATPase activity of MacB. The C‐terminal domain of MacA is essential for the MacB ATPase stimulatory activity. However, the biochemical mechanism of MacA mediated stimulation of MacB ATPase is unknown. In this study, using site directed mutagenesis we identified G353 and G357 residues in MacA which are essential for the stimulation of MacB ATPase in vitro. Trypsin proteolysis of the wild type MacA and mutants showed that mutations in these positions affect the conformation of MacA both in vivo and in vitro. Protein co‐purification and proteolytic digests indicate that MacA mutants assemble into the tripartite complex MacAB‐TolC. However, mutant complexes are different from those of the wild type MacA. We conclude that the amino acid residues G353 and G357 are essential for the function of MacA in stimulating the activity of MacB ATPase and also for the assembly of the proper MacA‐MacB‐TolC efflux pump.

NIH Grant AI052293
How does V‐ATPase Subunit d Couple ATP Hydrolysis and Proton Transport in Yeast?
Authors: Sheak Joshua , Parra Karlett J
Published: June 2021

Acidification of intracellular vesicles and the extracellular environment is paramount to various cellular processes—bone resorption, late endosome formation, sperm maturation, renal acidification, and protein degradation. The protein central to pH homeostasis is named V‐ATPase. V‐ATPase is a large multisubunit protein that couples the hydrolysis of ATP with pumping protons against a concentration gradient. V‐ATPase has two domains: an intramembranous domain (Vo) and a peripheral domain (V1). V‐ATPase activity is regulated by reversible disassembly of V1 and Vo. One of the subunits that could be critical for reversible disassembly and activity is V0 subunit d (Vma6). Subunit d is located on top of a proteolipid ring of other V0 subunits, c′, c″, c‴. Subunit d contributes to the connection of V0 and V1; and the mutation F94A in subunit d stops proton translocation, but not V‐ATPase activity in yeast cells. To further investigate the role of F94 and neighboring amino acids for function, we introduced site‐directed mutations on F94 (F94H and F94W), Y100 (Y100A), and M103 (M103A). The effect of the mutations on V‐ATPase expression, function (proton transport and ATP hydrolysis), and assembly are currently being assessed. These studies will lead novel information about the mechanisms by which V‐ATPase subunit d couples ATP hydrolysis to the transport of protons.
Hypertension affects the regulatory effect of nitric oxide on the respiratory chain
Authors: Calderón‐Cortés Elizabeth , Cortés‐Rojo Christian , Servín‐Campuzano Luisa , Clemente‐Guerrero Mónica , Noriega‐Cisneros Ruth , Saavedra‐Molina Alfredo
Published: June 2021

Nitric oxide (NO) synthesized from mitochondrial nitric oxide synthase (NOSmt) contributes to the regulation of oxidative phosphorylation by inhibiting ATP synthesis through the binding of NO to cytochrome c oxidase complex (complex IV) of the electron transport chain (ETC). We hypothesize that during hypertension the regulation of the ETC exerted by NO could be affected and could lead to a disruption in the mitochondrial energetic metabolism. Basal oxygen consumption during state 3 was increased in liver and kidney mitochondria from SHR of 1 and 7‐m.o. as compared with WKY animals of the same age. The sensitivity of complex IV to SNAP inhibition was lower in liver and kidney mitochondria from 7‐m.o. SHR as compared with mitochondria from WKY animals of the same age. Differential susceptibility to inhibition by SNAP was observed in complex IV activity from SHR rats of 7 months old for both organs. Activity of complex IV was inhibited with SNAP in a concentration‐dependent fashion in both WKY and SHR kidney and liver mitochondria from 1‐m.o. rats. The results suggest that hypertension lead to changes in ETC activity at the level of complex IV, which may be related with assembly defects of this complex during hypertension reported by others and contributes to mitochondrial dysfunction in kidney and liver during hypertension progression.
Novel Mechanism for Sympathetically‐Mediated Hypertension by Naturally Occurring Human Alpha1aAR Genetic Variant
Authors: Oganesian Anush , Schwinn Debra
Published: June 2021

α1a‐Adrenergic receptors (α1aARs) control human blood pressure by mediating vasoconstriction in small arterial resistance vessels; this has important implications in regulation of hypertension. A recent finding is that matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAMs) play key roles in development of hypertension by shedding growth factors and activating some signaling pathways. We previously identified a naturally occurring single nucleotide polymorphism (SNP) G247R in the third intracellular loop of α1aAR (α1aAR‐247R) in a patient with severe hypertension. Expression of this SNP in Rat‐1 fibroblasts results in unique, striking 2‐fold increased proliferation compared with wild type (α1aAR‐WT) or other α1AR subtypes, α1b/α1d. In this study we elucidate the molecular mechanisms and signaling pathways responsible for this unique proliferation triggered by α1aAR‐247R. We show for the first time that elevated levels of MMP‐7 and ADAM‐12 in cells expressing α1aAR‐247R are responsible for EGFR transactivation, ERK activation and increased cell proliferation; a pathway confirmed using EGFR, MMP and ERK specific inhibitors. Importantly, we show that accelerated proliferation triggered by α1aAR‐247R is serum and agonist independent. Thus, these new data suggest a novel mechanism of sympathetically‐mediated hypertension in humans with this naturally occurring SNP.
Hypertension Mechanisms: RGS2 Coordinates Gq/11 and Gi Signaling in Endothelium and Smooth Muscle of the Resistance Vasculature
Authors: Osei‐Owusu Patrick , Dietrich Hans H. , Blumer Kendall J.
Published: June 2021

RGS2 attenuates signaling by Gq/11α which mediate the action of many vasoactive substances, Gi/oα and adenylyl cyclases. RGS2 is expressed in tissues involved in blood pressure (BP) control and is induced by angiotensin II. We showed that RGS2−/− mice are hypertensive, and others identified RGS2 polymorphisms in hypertension patients. To determine how RGS2 regulates BP, we are analyzing mice with global or tissue/cell type‐specific deletion of RGS2. In vascular smooth muscle (VSM), RGS2 functions as a novel effector of NO‐cGMP‐PKG pathway since the absence of RGS2 impairs the ability of PKG to attenuate Gq‐coupled vasoconstrictor signaling and promote VSM relaxation. Furthermore, PKG phosphorylation of RGS2 augments its GAP activity and association with the plasma membrane. RGS2 also functions in endothelium. We found that ACh‐elicited dilation of mesenteric arteries (MA) from RGS2−/− mice is strikingly impaired. In contrast, ACh‐induced vasodilation was nearly normal in MA from mice lacking RGS2 only in VSM. In RGS2−/− MA, ACh‐induced vasodilation was sensitive to L‐NAME but insensitive to EDHF inhibitors, suggesting that EDHF but not NO production is impaired due to the absence of RGS2. RGS2 deficiency therefore contributes to hypertension by impairing VSM response to NO and blunting endothelial EDHF production. Financial Support from AHA 09POST2260099 to P.O. and NIH GM‐44592 and HL‐075632 to K. J. Blumer.
Optimization of stripping/reprobing for Li‐COR Western blot analysis
Authors: McCauley Michael C , Nalbandian Ari , Brum Gabby , Lu Shan , Wan Yinsheng
Published: June 2021

The Li‐Cor Odyssey Infrared Imaging System is pioneering the way for standard Western blot analysis by using direct infrared fluorescence detection. An advantage of the Li‐COR Odyssey Infrared Imaging System is that it provides a safer and cleaner method to detect proteins compared to traditional darkroom scans because each membrane does not have to be soaked in toxic detection reagents before the scan is initiated. However, one key problem with the Odyssey imaging system is that its standard stripping protocol fails to remove all of the antibodies from each membrane. In order to completely strip each membrane, some elements of the standard protocol had to be altered. We chose to manipulate the amount of time the membranes were exposed to the Newblot Stripping Buffer, the temperature at which the membranes were soaked in the buffer solution, and the concentration of the stripping buffer. We concluded that the optimal stripping procedure involved soaking the membranes in a 1:5 diluted buffer solution at 50°C for 30 minutes. Removing antibodies from a membrane is critical and our data suggests that these modifications to the original protocol offer a more effective stripping procedure.
Potential Antitoxin effects of Green Tea Polyphenols on Human Lung Cells Affected by World Trade Center Dust
Authors: Polozani Miraxh , Lambroussis Constantino , Dilorenzo Ann Marie , Lee Lee
Published: June 2021

Green tea polyphenols are antioxidants frequently used to promote good health. In this study green tea extracts were used to study the cytotoxicity and wound healing effect on human lung cells. Epigallocatechin (EGCG), a water soluble polyphenol in pure form, a group of water soluble polyphenols (GTP), a group of non water soluble polyphenols (LTP), and a mixture of all three (combo333) were used at 12.5, 25, 50, 75 and 100 μM concentrations. Results suggested that EGCG promoted lung cell proliferation at concentrations of 12.5 to 25 μM. GTP results suggest promoted lung cell proliferation at 12.5–50 μM. Combo333 shows increased proliferation and no morphology alteration. The results suggest that particulate green tea polyphenols may possess antitoxin abilities. Tests on the potency of antitoxin abilities of green tea against World Trade Center dust exposed human lung cells were conducted based on these findings. Previous studies indicated that WTC dust decreases cell proliferation and increases apoptosis of human lung cells. In the presence of green tea extracts, EGCG at 12.5 μM was the only concentration that showed some proliferation during the presence of the dust solution. All other extracts displayed no re‐growth into the wound region, however no morphological changes were detected. This suggests green tea polyphenols may protect the integrity of the cell.
Arrestin1 self‐associates to prevent cell death induced by the cytotoxic monomer
Authors: Gurevich Vsevolod V , Song Xiufeng , Vishnivetskiy Sergey A , Kim Miyeon , Hanson Susan M , Hubbell Wayne L , Chen Jeannie , Gurevich Eugenia V
Published: June 2021

Arrestin1 ensures timely signal shutoff by binding light‐activated phosphorhodopsin. Genetic defects in rhodopsin phosphorylation result in excessive signaling leading to photoreceptor death. Engineered arrestin1 mutant with increased affinity for light‐activated unphosphorylated rhodopsin partially compensates for lack of phosphorylation, improving rod function. However, this “enhanced” mutant has reduced propensity to oligomerize. Its high expression results in progressive light‐independent rod degeneration via apoptosis involving the activation of caspases‐9 and –3. In contrast, similar expression levels of wild type (WT) arrestin1 do not affect rod survival. Moreover, co‐expression of WT arrestin1 alleviates the damage induced by enhanced mutant. Since WT arrestin1 promotes self‐association of mutant by forming mixed oligomers, our data indicate that excess of monomeric arrestin1 induces apoptosis. This finding explains why arrestin1 acquired propensity to oligomerize, even though only the monomer is the active receptor‐binding species. It may also explain oligomerization of non‐visual arrestin2 and arrestin3.

NIH grants EY11500 (VVG), EY005216 (WLH), NS065868 (EVG), EY012155 (JC)
Identification of arrestin elements mediating c‐Raf‐1 binding
Authors: Coffa Sergio , Song Xiufeng , Gurevich Vsevolod V
Published: June 2021

Arrestins regulate signaling and trafficking of G protein‐coupled receptor (GPCR). GPCR‐arrestin complexes initiate several signaling pathways, including the activation of mitogen activated protein kinases (MAPKs), such as extracellular signal regulated kinase 1/2 (ERK1/2) and c‐Jun N‐terminal kinase 3 (JNK3). Different MAPKs are involved in proliferation, survival, and apoptosis. Understanding of the structure and dynamics of arrestin‐MAPK complexes is necessary to design signaling‐biased arrestins to re‐direct MAPK signaling. We showed that c‐Raf1, MEK1, ERK2, ASK1, MKK4, and JNK3 interact with both domains of arrestin2 and arrestin3. Thus, three kinases in each cascade (c‐Raf‐1/MEK1/ERK2 or ASK1/MKK4/JNK3) are oriented in the same way parallel to the long axis of the arrestin molecule. We mutated residues on the non receptor‐binding surface conserved between arrestin2 and arrestin3. We found that the binding of arrestin2‐Arg307Ala mutant to c‐Raf‐1 is reduced by ~50%. Arg307Ala mutation does not affect the binding to phosphorylated receptor, MEK1, ERK2, and other MAPKs, ruling out gross folding defects. Thus, Arg307 plays a specific role in arrestin interaction with c‐Raf‐1. Arg307Ala mutant can disrupt the arrestin‐dependent ERK activation and will be a useful tool to study arrestin‐dependent MAPK activation.

NIH grants GM077561 and GM081756 (VVG).
C1q/WOX1 signaling for superinduction of microvillus cluster formation
Authors: He Ruei‐Yu , Chang Jean‐Yun , Lin Sing‐Ru , Lee Ming‐Hui , Chen Shean‐Jen , Chang Nan‐Shan
Published: June 2021

Complement C1q induces apoptosis of prostate DU145 and neuroblastoma cells overexpressing tumor suppressor WOX1 (known as WWOX or FOR), but not GFP control protein. By total internal reflection fluorescence microscopy, C1q signals with WOX1 for inducing microvillus formation in clusters in between cells, followed by fragmentation. This results in thousands of fragments containing WOX1 and amyloid beta adhered on the extracellular matrix. The induction is blocked by dominant negative WOX1, anti‐C1q receptor antibody, and inhibitors for G protein/Rho/ROCK signaling and metalloproteinases. Meanwhile, cytosolic WOX1 undergoes self‐aggregation, leading to apoptosis. Aggregated WOX1 is shown with amyloid beta plaques in the hippocampus of patients with Alzheimer's disease, suggesting a role of C1q/WOX1 signaling in contributing to neurodegeneration. (Supported in part by NSC and NHRI, Taiwan, and DoD, USA)
Epigenetic regulations of the Sall genes in embryonic and trophoblast stem cells and involvement of non‐coding RNA
Authors: Ohgane Jun , Kumagai Katsuyoshi , Takamatsu Takeshi , Yagi Shintaro , Tanaka Satoshi , Shiota Kunio
Published: June 2021

Epigenetics including DNA methylation is involved in gene silencing and fundamental to embryonic development in mammals. Genome‐wide DNA methylation analyses have indicated that there are numerous tissue‐dependent and differentially methylated regions (T‐DMRs). Mammals have four Spalt‐like genes (Sall1–4), which encode C2H2 type transcription factors and are indispensable for normal embryonic development. We found that all the Sall family genes contained T‐DMRs that were hypomethylated in embryonic stem (ES) cells but hypermethylated in trophoblast stem (TS) cells, together with specific histone methylation patterns and promoter‐associated antisense non‐coding RNAs (ASncRNAs). Among them, Sall4 is required for self‐renewal of ES cells and prevents differentiation into trophoblast lineages. We identified an ASncRNA overlapping with the Sall4 promoter T‐DMR, and the Sall4 ASncRNA was expressed in ES cells but barely detected in TS cells. The expression pattern of the Sall4 ASncRNA was similar to that of sense Sall4 mRNA and inversely correlated with the DNA methylation pattern. In conclusion, we have found T‐DMRs in all the Sall genes, and promoter‐associated antisense RNA is likely involved in epigenetic regulation of Sall family genes.

This work was supported by KAKENHI (21248039) from MEXT, Japan.
Microbial hydroxylation of steroids
Authors: Afzal Mohammad , Al‐Awadhi Sameera , Al‐Tamimi Safa , Oommen Sushama
Published: June 2021

Current study was initiated to investigate steroid hydroxylation potential of a new food born thermophile Geobacillus kaustophilus. Incubation of progesterone and testosterone with G. kaustophilus resulted in stereospecific hydroxylation of progesterone and testosterone giving monohydroxylated and dihydroxylated products. A cleavage of ring B of the steroid nucleus was also obtained which is a novel metabolite. Geobacillus kaustophilus has not been used before for steroid hydroxylations. Thermophilic enzymes have a significant advantage for their isolation at elevated temperatures and can be used for cell free transformations offering a convenient method for introduction of hydroxyl groups in a stereospecific and regiospecific manner into steroid nuclei. Stereochemical oxygenation of a steroid nucleus is not possible with chemical reactions and microbial hydroxylation of steroids may have a direct impact on drug development.

Conclusion

G. kaustophilus has an excellent potential for stereospecific hydroxylation of natural steroid nucleus at elevated temperature. This study was supported by Research Administration, Kuwait University, Kuwait.
Evidence for cross‐talk between PKC1 and TOR signaling pathways in the cell wall stress response of the myo1Δ cytokinesis mutant of Saccharomyces cerevisiae
Authors: Pagán‐Mercado Glorivee , Rodríguez‐Medina José R.
Published: June 2021

Down‐regulation of ribosomal protein (RP) genes was observed in myosin II null strains (myo1Δ) of the yeast Saccharomyces cerevisiae, which is likely to be a result of repression of the TOR pathway. We have shown the PKC1 Cell Wall Integrity Pathway (CWIP) is activated in this mutant, the Slt2p/Mpk1p is phosphorylated and it's expression is essential for survival. The objective of this study was to determine whether there is cross‐talk between the PKC1 and TOR signaling pathways in the CWIP response in a myo1Δ strain. To test this, we deleted the ROM1, TOR1 and SIT4 genes in a myo1Δ strain and analyzed the levels of phosphorylated Slt2p/Mpk1p by western blot in single and double mutant strains. Viability assays and growth curves were performed to test for genetic interactions. Preliminary results show that upon rapamycin treatment, rom1Δmyo1Δ, sit4Δmyo1Δ and tor1Δmyo1Δ strains presented decreased levels of phosphorylated Slt2p relative to the treated myo1Δ single mutant. Also, there was a synthetic growth defect in rom1Δmyo1Δ and tor1Δmyo1Δ but not in sit4Δmyo1Δ strains. We concluded that ROM1, TOR1 and SIT4 gene products modulate cross‐talk between the PKC1 and the TOR signaling pathways, and that Rom1p and Tor1p are important to maintain optimal activation of the PKC1 CWIP under cell wall stress conditions. Supported by NIGMS‐NIAID (5SC1AI081658‐02), NCRR‐RCMI (G12RR03051) and MBRS‐RISE (R25GM061838).
Zucker fatty rat hepatocytes have impaired regulation of gene expression mediated by insulin and retinoids
Authors: Chen Wei , Chen Guoxun
Published: June 2021

The mechanism of insulin resistance at gene expression level is still unrevealed. We demonstrated retinoids synergize with insulin (INS) to induce glucokinase (Gck) expression in primary hepatocytes, showing their roles in modulating insulin action. Herein, we compared the responses of Gck, Srebf‐1c, Pck1 and Pklr in hepatocytes isolated from Zucker lean (ZL) and fatty (ZF) rats to INS (0 to 100nM) without or with 5 uM all trans retinoic acid (RA). The mRNA levels of these genes were analyzed by real time PCR. For Gck, RA started to synergize with INS at 1nM to induce its expression in ZL and ZF hepatocytes, but the induction folds by INS were higher in ZL than in ZF cells with or without RA. The Srebf‐1c mRNA was only induced in ZL, but not ZF hepatocytes. The Pck1 mRNA levels were decreased more in ZL than in ZF cells by 1 and 10nM INS without RA, and by 1nM INS with RA. The Pklr mRNA level was higher in ZF cells at basal state. It was not responsive to INS, RA or INS + RA in both ZL and ZF hepatocytes. We showed here ZL hepatocytes respond to INS and RA more robustly in regulation of gene expression than ZF cells, suggesting a potential model to study insulin resistance at gene expression level.

Grant Funding Source: American Heart Association
Changes in Rat Liver Proteome Following Acute In Vivo Iron Overload
Authors: Bakirci Aynur , Elamin Ashraf , McAbee Douglas
Published: June 2021

The mammalian liver is a primary site for iron processing. Excess iron consumption or iron uptake/storage disorders alter hepatic gene expression and correlate with various liver diseases. The general patterns of change in the hepatic proteome following acute iron overload, however, are poorly understood. To address this, we injected rats with iron dextran to induce liver iron accumulation. After 24, 48, and 96 hours post‐injection, whole livers were isolated and flash‐frozen. Liver non‐heme iron levels increased 30‐fold at all post‐injection times. Liver proteins were solubilized from frozen liver powders with thiourea‐containing buffer and subjected to two‐dimensional gel electrophoresis. Digital spot quantitation of gel images and analysis of protein tryptic digests by MALDI‐TOF/TOF mass spectrometry identified 38 proteins whose expression changed ≥ 1.5 fold following acute iron overload. The majority of identified proteins increased during iron overload including urea cycle enzymes and various enzymes involved in anti‐oxidant defense, lipid metabolism, and mitochondrial ATP formation. Fewer proteins decreased during iron overloading and included specific lipid binding proteins. These results provide a foundation for more detailed analyses on the sequential patterns of changes in liver protein expression during acute iron overload. (Support: NIH DK‐61984)
The Effects of Transformation and ZnT‐1 Silencing on Zinc Homeostasis in Cultured Cells
Authors: Sankavaram Kavitha , Freake Hedley C
Published: June 2021

We have shown that the extracellular chelator DTPA (diethylenetriaminepentaaceticacid) promotes efflux of Zn65 from prelabeled rat primary hepatocytes, but increases retention of label in H4IIE hepatoma cells. To further understand this difference between primary cells and the corresponding cancer cell line, we investigated the effects of immortalizing primary cells on their zinc homeostasis. Freshly isolated rat hepatocytes were electroporated with the SV40 large T‐antigen‐coding‐plasmid, pSV3‐neo. Successful transformation was demonstrated by continued growth in the presence of neomycin. When these cells were pre‐labeled with Zn65, DTPA decreased efflux of Zn65, similarly to hepatoma cells and differently from primary hepatocytes. This homeostatic change may be required to account for the greater zinc requirements of rapidly dividing cells. To further understand the mechanism of DTPA inducedzinc retention we down‐regulated the expression of the plasma membrane located zinc efflux transporter, ZnT‐1, in rat hepatoma cells using vector‐based shRNA interference. Expression of ZnT‐1 protein was reduced to approximately 50%. Down‐regulation of ZnT‐1 resulted in greater retention of Zn65 in control cells. However, DTPA increased rather than decreased efflux of label from knockdown cells, suggesting that functional ZnT‐1 is required for the decreased efflux seen with DTPA in normal cells.

Grant Funding Source: UConn Research Foundation
Identification of Novel Menkes Copper Atpase (Atp7a) Protein Variants in the Cytoplasm and Nucleus of Intestinal Epithelial Cells
Authors: Lu Yan , Kim Changae , Collins James F.
Published: June 2021

We previously noted alternative 5′ splice variants of the Atp7a transcript. To fully understand the splicing of this gene, we sought to identify additional “downstream” splice variants and to test the hypothesis that different protein variants exist. RT‐PCR was utilized with combinations of Atp7a primers spanning exons 1–23 and amplified products were subcloned and sequenced. Novel splice products which were missing various internal exons were identified. To identify Atp7a proteins potentially encoded by these splice variants, we utilized 3 polyclonal Atp7a antisera raised against different antigens: 1) against 2 C‐terminal peptides (called 54‐10), 2) against a fusion protein of the six, N‐terminal copper binding domains (called R17; a gift from Dr. Julian Mercer), and 3) against 2 peptides within the first 68 N‐terminal amino acids (called N‐term). The 54‐10 and R17 antisera, both proven to specifically react with Atp7a, detected a presumably full length Atp7a protein of ~180 kDa in rat intestine and IEC‐6 cells. The 54‐10 antiserum also detected a smaller protein band of ~160 kDa and a specific, ~97 kDa protein in the nuclear fraction of IEC‐6 cells. Moreover, a nuclear protein was detected by the N‐term antiserum, which was similar in size to the protein detected by the 54‐10 antiserum (~97 kDa). IP experiments and siRNA knockdowns provide additional evidence that these proteins may be novel Atp7a variants.

Grant Funding Source: NIH
The PSM or “Science‐Plus”
Authors: Tobias Sheila
Published: June 2021

The Afred P. Sloan Foundation initiated a program in 1997 to promote development of “science‐plus” degree programs (PSM) in science, mathematics, and interdisciplinary science/math‐based areas. The intention is to prepare graduates for all‐level professional positions in business, government and non‐profit employment sectors. Together with a core of advanced work in the discipline/interdisciipline, augmented by information technology, PSM programs include short courses or modules that expose students to fundamental elements of business/government/nonprofit environments, such as business basics, intellectual property law, teamwork, regulatory affairs, communication/presentation skills, and project management; also required in most programs is an internship in a professional environment. New curricula (especially in biotechnology, bioinformatics) and new pedagogy are emerging from the more than 150 programs in 64 universities now in place.
Ganodermanontriol inhibits growth and invasiveness of human breast cancer cells by the down‐regulation of CDC20 signaling
Authors: Jiang Jiahua , Jedinak Andrej , Sliva Daniel
Published: June 2021

In the present study, we investigated the effects of Ganodermanontriol (GT), natural triterpene isolated from Ganoderma lucidum, on the growth and invasiveness of human breast cancer cells. GT suppressed cell proliferation, colony formation and invasiveness (cell migration, cell adhesion and invasion of cancer cells through Matrigel) of MDA‐MB‐231 cells. The growth inhibition by GT was mediated through the down‐regulation of expression of cell cycle regulatory proteins: Cdc20, Cdc25, Cdk2, PCNA, and cyclin E. The inhibition of invasive behavior was mediated by the suppression of secretion of urokinase plasminogen activator (uPA) and inhibition of uPA receptor expression. CDC20 is a key player in the Spindle Assembly Checkpoint (SAC) and was implicated in cell cycle and tumor progression. We found higher expression of CDC20 protein in human breast cancer tissues than in normal human mammary tissues. The down‐regulation of expression of CDC20 by GT in breast cancer cells was mediated through the transcriptional regulation as we demonstrated by the DNA‐binding and reporter gene assays. Initial analysis of the CDC20 promoter suggested the presence of the GT sensitive regulatory elements in the ‐400‐1259 region of the CDC20 promoter. Our data demonstrate that GT inhibits growth and invasive behavior of breast and suggest CDC20 as a new potential therapeutic target for the treatment of breast cancer.
Impact of dietary Ellagic acid on cholesterol efflux in lipid‐laden murine macrophages
Authors: Park Sin‐Hye , Li Jing , Sung Hye‐Young , Kang Young‐Hee
Published: June 2021

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by scavenger receptor in macrophages initiates the chronic pro‐inflammatory cascade that characterizes atherosclerosis. Ellagic acid is a polyphenolic compound in fruits and vegetables including raspberries, strawberries, and walnuts. This study investigated that Ellagic acid enhanced expression of ATP‐binding cassette transporter A1 (ABCA1) involved in cholesterol efflux from macrophage‐derived foam cells. J774A.1 murine macrophages were treated with 50 μg/mL oxidized LDL (oxLDL) for 24 h in DMEM containing 0.4% fatty acid free BSA. ABCA1 expression was boosted in lipid‐laden foam cells treated with 10 μM Ellagic acid that was cytoprotective against toxicity of free cholesterol accumulation. In addition, the heavy Oil Red O staining observed in oxLDL‐alone macrophages was substantially diminished in macrophage foam cells exposed to Ellagic acid, whereas the ABCA1‐mediated cholesterol efflux was enhanced. These results demonstrate that Ellagic acid may be a dietary anti‐atherogenic agent enhancing disposal of harmful lipoproteins from lipid‐laden foam cells. Supported by the Ministry of Knowledge Economy through Technological Development Project for Regional Strategic Planning.
Metabolism of chokeberry anthocyanins in saliva
Authors: Kamonpatana Kom , Chitchumroonchokchai Chureeporn , Giusti Monica M. , Failla Mark L.
Published: June 2021

Anthocyanin‐rich extracts (ARE) have been shown to suppress proliferation of human oral squamous cell carcinoma and reduce the number of oral tumors when fed in diet to hamsters. These data suggest the potential use of anthocyanins (ACN) for oral chemoprevention. However, the metabolism and uptake of anthocyanins into the epithelial lining of oral tissues remains unknown. Here, we investigated the fate of ARE from chokeberry in unstimulated saliva. HPLC‐PDA and HPLC‐MS showed that the extract contained cyanidin‐3‐galactoside, cyanidin‐3‐glucoside, cyanidin‐3‐arabinoside and cyanidin‐3‐xyloside. Ex‐vivo incubation (37°C) of ARE with saliva collected from human subjects showed similar extent of decrease in the amounts of all four ACNs without detection of cyanidin aglycone. Heat treatment of intact saliva (80°C for 15 min), as well as incubation with saliva at 0°C, inhibited degradation of cyanidin glycosides. Degradation of cyanidin glycosides also was markedly reduced by removing shed epithelial cells and bacteria from saliva. These results suggest that cyanidin glycosides can be enzymatically converted to phenolic acids in human saliva and that the primary origin of the glycosidase activity is not salivary secretions. There also was marked variation in the extent of degradation of the cyanidin glycosides in saliva from different subjects. Outcomes from ongoing ex‐vivo and in‐vivo studies examining the influence of ACN structure on susceptibility to metabolism in the oral cavity will be also presented. Supported in part by OARDC.
High flavanol cocoa and dark chocolate enhance vasodilation and reduce arterial stiffness
Authors: West Sheila G , Skulas‐Ray Ann C , Piotrowski Matthew J , Crispell Molly D , Groves Lisa Fuqua , Poupin Nathalie , Wagner Paul R
Published: June 2021

Consumption of cocoa and dark chocolate has been shown to increase flow mediated dilation (FMD), but less is known about its effects on arterial compliance. We examined whether vascular effects could be sustained over 4 weeks. We enrolled 30 middle‐aged, overweight adults in a randomized, placebo‐controlled, crossover study. At baseline and end of each treatment, we measured brachial artery diameter and augmentation index (AI), a measure of arterial stiffness. During the active treatment, participants consumed 37 g/d dark chocolate and a sugar free cocoa beverage (CHOC) [total flavanols (TF) = 332mg/d] in place of a snack. Color‐matched controls included a low‐flavanol chocolate bar and a sugar‐free, non‐cocoa beverage (CONT) (TF = 0 mg/d). Treatments were roughly equivalent in total calories and fatty acid content. After CHOC treatment, artery diameter was significantly increased under resting conditions (+0.2 mm) and at the post‐deflation peak (+0.2 mm). CHOC significantly reduced AI (−1.0 ± 2.5 vs. 6.1 ± 2.5, CHOC vs. CONT, respectively). FMD and body weight did not differ across treatments. These results indicate that cocoa and dark chocolate increase arterial diameter, primarily through a reduction in vascular stiffness. Future work should examine dose‐response relationships to determine the threshold polyphenol intake to produce optimal vascular responses. Funded by: The Hershey Company
Effects of Opuntia ficus indica on Glucacon like peptide (GLP‐1), Glucose‐dependent insulinotropic peptide (GIP), blood glucose and insulin after the consumption of a high carbohydrate meal
Authors: Lopez‐Romero Patricia , Moran‐Ramos Sofia , Tovar Armando R , Torres Nimbe
Published: June 2021

Opuntia ficus‐indica (nopal), a plant widely consumed in Mexico has shown to be rich in bioactive compounds such as dietary fiber, polyphenols and calcium with antihyperglycemic properties in diabetic subjects; however, the mechanism of action has not been elucidated. The physicochemical characteristics of Opuntia cladodes (OC) are mainly responsible for these effects, however these can be modified by OC processing. The aim of this work was to evaluate whether the consumption of different preparations of OC with a high carbohydrate meal (HCM) modify the blood glucose, insulin, GIP and GLP‐1 response. Eight healthy volunteers were given a HCM in the presence of five different cladode preparations after a 12 h fast. Blood samples were drawn before and 15, 30, 45, 60, 90, 120 min following the HCM. The results showed that consumption of OC with the HCM modulates the area under the curve (AUC) for GIP and GLP‐1 and that this is influenced by the cladode preparation. The AUC for insulin, shows a reducing tendency (P = 0.06) during the two hour period. The latter reduction is lost when OC are given dehydrated in aqueous solution. There was no significant difference in glucose AUC in the normoglycemic subjects. These results suggest that one possible mechanism for the physiological effect of Opuntia may be partly mediated by incretins and depended of the OC processing.

Supported by ICyT DF (PICDS08‐6) and Grupo Produce
Hypoglycemic effect of quercetin in animal models of diabetes
Authors: Choi Haneul , Kim Jungin , Kim Jihye , Joo Heejeong , Kang Younghee
Published: June 2021

Tight control of fasting and postprandial hyperglycemia is crucial for the management of diabetes mellitus. Quercetin was reported to have strong alpha‐glucosidase inhibitory activity in vitro. The purpose of this study was to investigate the hypoglycemic effect of quercetin in animal models of diabetes. The effect of the quercetin on the postprandial hyperglycemia was studied in streptozotocin‐induced diabetic rats (n=12) using a carbohydrate load test. Oral administration of quercetin (100 mg/kg) significantly decreased incremental serum glucose levels at 60 and 90 min (P < 0.05) after an oral ingestion of starch (1 g/kg). The area under the glucose response curve of the quercetin group was significantly reduced compared with that of the control group (p<0.01). The effect of chronic feeding of quercetin was studied in an animal model of type 2 diabetes. Four‐week‐old db/db mice (n=14) were fed AIN‐93G diet with and without quercetin (100 mg/kg) for 7 weeks. Consumption of quercetin significantly decreased serum glucose and blood glycated hemoglobin levels compared with the control group (P < 0.05). These results indicate that quercetin is effective in controlling hyperglycemia in animal models of diabetes mellitus. This study was supported by the National Research Foundation of Korea (NRF).
Antiobesity and antidiabetic effects of Jerusalem artichoke and purple sweet potato in the diet‐induced obese rats
Authors: Cho Kyung‐Dong , Kim Eun Jin , Kim Mi Young , Kim Jin Soo , Han Chan‐Kyu , Lee Bog‐Hieu
Published: June 2021

The study was conducted to investigate the antiobesity effect of Jerusalem artichoke and purple sweet potato supplementation in diet‐induced obese rats. High fat diet was given for 4 weeks and experimental diets for 5 weeks. Thirty‐two 3‐week‐old male Sprague‐Dawley rats were assigned to four groups (n=8/group); normal diet(ND), high‐fat diet(HFD), high fat diet with 10% Jerusalem artichoke(JA), or high fat diet with 10% purple sweet potato(SP). Final body weight, total and average body weight gain of JA and Sp groups were significantly lower or similar to ND group. Total adipose tissue weight was the lowest in ND and JA group, followed by SP and HFD group(p<0.05), and similar tendency was observed in epididymal, perirenal, and visceral white adipose tissue (p<0.05) without difference in interscapular brown adipose tissue. Adipocyte size of epdidymis in JA and SP groups were lower than HFD group and similar to ND group(p<0.05), although the subcutaneous adipose tissue did not differ among the groups. Serum insulin level did not differ, but serum glucose level and HOMA‐IR were lower in JA and SP groups than those of HFD group(p<0.05). Taken together, the supplementation of JA and SP exerted the antiobesity and antidiabetic effects in diet‐induced obese rats and may be a good source for preventing obesity and diabetes. Further research should be conducted to elucidate the mechanism involved in those effects.
Effect of Puerariae radix extract supplementation on the hippocampus NGF in galactose –induced aged rats
Authors: Lee Okhee , Woo Jeungwha
Published: June 2021

Isoflavone are known to prevent decline of cognitive function in aged rats. In the previous report, Pueraria radix extract was isoflavone‐rich and contained especially puerarin showing high antioxidative capacity. This study was aimed to determine the effect of isoflavone‐rich Pueraria radix extract on the cognitive function in ageing rats. Male Sprague‐Dawley rats were fed an AIN93M‐based diet, and D‐galactose administered and fed with diets added with Pueraria radix extract to contain 0%(=ageing‐control), 0.05%(=P0.05), 0,1%(=P0.1) isoflavone. The Pueraria radix ethanol‐extract was shown to contain 8476 ㎎ isoflavone/100g dried extract, with 69.6% puerarin. After 42 days of Pueararia extract intake, weight of rats was significantly low in P0.05% and P0.1% group compared to control and aged control groups. The feed efficiency rate was significantly low in P0.1 group compared to control and aged control groups. Organ weights such as hippocampus, cerebrum and cellebrum did not show significant difference according to experimental treatment (p<0.05). The NGF and CREB expression in hippocampus increased significantly in P0.1% group. Conclusively, this study suggest that isoflavone‐rich, Puerariae radix extract may improve the decline of cognitive function in ageing rats.
SERUM ADENOSINE DEAMINASE ACTIVITY, C3c and C4c FRACTIONS IN CHILDREN SUFFERING DIABETES. PRELIMINARY STUDY
Authors: Feliu María Susana , Perris Paula Daniela , Silva Carolina , Slobodianik Nora
Published: June 2021

Introduction

Different studies have shown that nutritional pathology induces depression of the immune system.

Objective

In this preliminary study we measured Adenosine Deaminase‐enzyme related with lymphocytes T, and complement fractions (C3, C4), in patients suffering diabetes (DTB).

Methods

Twenty five persons, between 12 and 18 years of age, assisted by the Nutrition Service, were evaluated. Samples of whole blood were collected from fasting patients. Serum ADA(U/L), was determined by Giusti and Galanti method and C3c and C4c (mg/dL), were measured by single radial immunodiffusion techniques with commercially available kits (Diffuplate, Biocientifica, Buenos Aires, Argentina). The results expressed as X± SD were compared with reference values obtained from children of the same range of age (* p < 0001) It was observed, statistical diminution of C3c and C4c with a concomitant increase of activity of ADA sera concentration.

Conclusions

These preliminary results suggest that the mechanisms of defense of these children was altered. On the other hand, these fractions might be included in the periodic evaluation of patients suffering diabetes.

Group
C3c
C4c
ADA

DBT
68.6±24.0
14.6±7.5
39.1±15.5

Refence
126.3±45.5
27.5±7.7
23.0±5.6
Do alterations in temperature regulation contribute to protein‐energy malnutrition‐induced increase in reactive gliosis after global ischemia?
Authors: Smith Shari Elaine , Prosser‐Loose Erin J , Paterson Phyllis G
Published: June 2021

Protein‐energy malnutrition (PEM) impairs functional outcome after global ischemia, and this is associated with an increase in reactive gliosis. Since temperature is a key determinant of brain damage following an ischemic insult, the objective of this study was to determine if PEM modifies post‐ischemic temperature regulation. Bio‐electrical sensor transmitters were implanted into the peritoneal cavity of male, Sprague‐Dawley rats (31d) for continuous monitoring of core temperature. Rats were randomized for 7d to modified AIN‐93G control diet (CON; 18% protein) or PEM (2% protein). Animals were exposed to global ischemia (I) induced by 10 min bilateral carotid artery occlusion and hypotension (38 ± 0.2 mm Hg) or sham surgery (S) with tympanic temperature maintained at 37.4 ± 0.2°C. Physiological values were as follows: pH 7.29–7.41, pO2 119–144 mm Hg, pCO2 34–51 mm Hg, blood glucose 3.8–6.7 mmol/L. Preliminary results indicate mean (± SEM) core and daily temperature fluctuation from 5d of postsurgical monitoring as follows: CON‐S: 37.4 ± 0.04°C, 2.0 ± 0.1°C (n=3); PEM‐S: 37.4 ± 0.1°C, 2.6 ± 0.1°C (n=3); CON‐I: 37.7 ± 0.1°C, 1.9 ± 0.2 °C (n=3); PEM‐I: 37.5 ± 0.2°C, 2.3 ± 0.3°C (n=2). These data suggest that although PEM does not cause marked hypothermia or hyperthermia during the postsurgical period, it reduces thermoregulatory function in both sham and ischemic animals. Funded by the Heart and Stroke Foundation of SK.