bluuebunny/crossref_metadata_2025 huggingface dataset:
A bifunctional enzyme of gluthathione‐dependent formaldehyde dehydrogenase and S‐nitrosogluthathione reductase from Antrodia camphorata
Authors: Wen Lisa , Huang Chih‐Yu , Ken Chuian‐Fu , Lin Chi‐Tsai
Published: June 2021

Glutathione‐dependent formaldehyde dehydrogenase (GFD) plays important roles in formaldehyde detoxification and antioxidation. A gene encoding GFD from A. camphorate was identified based on sequence homology. The deduced amino acid sequence of 378 residues is conserved among the reported GFDs. To characterize the Ac‐GFD, the coding region was subcloned into a vector pET‐20b(+) and transformed into E. coli. The recombinant GFD was expressed and purified by Ni2+‐nitrilotriacetic acid Sepharose. This purified enzyme showed a single band on a 10 % SDS‐PAGE. The enzyme retained 50% GFD activity after heating at 50 o C for 5 min. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S‐nitrosoglutathione reductase (GSNOR) presumably to safeguard against nitrosative stress. The Km values for S‐hydroxymethylglutathione and S‐ nitrosoglutathione were 1.197 mM and 0.276 mM, respectively.

Supported by the National Science Council of the Republic of China under grant NSC 96‐2313‐B‐019‐001 to C‐T. L.
Retinol esterification in the hepatopancreas of the crayfish (Procambarus clarkii)
Authors: Lindhorst Rebecca , Fioret Bryan , Palisch Chase , Canelas Anna , Dew Stephanie
Published: June 2021

Vitamin A (retinol) is a necessary nutrient for all higher animals, including many invertebrates which use it as their visual chromophore. All of these animals must get vitamin A in their diet and must be able not only to transport this hydrophobic vitamin to target cells but must also convert it into active forms of the vitamin, such as retinal and retinoic acid. In mammals, retinol is converted into retinyl esters for export from the small intestine, for storage in the liver, and as part of the visual cycle. Little is known about these processes in invertebrates. Mammals have two enzymes that can catalyze the conversion of retinol to retinyl‐esters: lecithin:retinol acyltransferase (LRAT) and acyl‐coA:retinol acyltransferase (ARAT). LRAT is the physiologically important one in mammals. We have identified an ARAT activity in microsomes from the hepatopancrease of the freshwater crayfish, Procambarus clarkii. We were unable to detect any LRAT activity, suggesting crayfish only have one enzyme that can catalyze this reaction. This activity shows a linear dependence on time and microsome concentration. It shows temperature dependence and an optimal pH of 6.5. We have characterized its acyl‐CoA preference and determined Km and Vmax's for both retinol and the acyl‐CoA.
Kinetics of Elastase Inhibition
Authors: Lazari Mark , Todd Erik , Firestone Gary , Stone Koni
Published: June 2021

The proteolytic enzyme elastase is naturally inhibited by alpha‐1 Protease Inhibitor (α‐1PI). In an oxidative environment, α‐1PI is inactivated and this leads to elastase activation. Naturally occurring H2O2 provides the needed oxidative environment for tissue recycling, however, tar from cigarette smoke produces an unnatural oxidative environment within the body. Cigarette smoke inactivates α‐1PI inhibition of elastase and then undamaged elastin is broken down faster than it is resynthesized. This leads to Chronic Obstructive Pulmonary Diseases (COPDs) such as emphysema. This study focuses on the kinetics of inhibition of elastase by several small molecule inhibitors that are found in broccoli and other cruciferous vegetables. The substrate used was n‐succinyl‐ala‐ala‐ala‐p‐nitroanilide, and the kinetics were measured by UV‐Vis spectroscopy.
Characterization of propionic acidemia mutations A113T and I139T using E. coli biotin carboxylase
Authors: Broussard Tyler C , Waldrop Grover L
Published: June 2021

Propionic Acidemia (PA) is an inborn error of metabolism caused by a deficiency of propionyl‐CoA carboxylase (PCC). PCC is a biotin dependent carboxylase involved in odd chain fatty acid oxidation and branched chain amino acid catabolism. Two mutations in the αsubunit of PCC that result in PA are A113T and I139T. The homodimeric biotin carboxylase (BC) subunit of E. coli acetyl‐CoA carboxylase is a model system for studying these mutations. Sequence alignments revealed the mutations A113T and I139T in PCC are homologous to A77T and I103T in BC. These residues are very close in proximity lying in adjacent β‐sheets far from the active site. Kinetic analysis was carried out for WT and both mutants holding biotin and bicarbonate constant while varying ATP. The results showed the I103T had no change in Km for ATP and only a 3‐fold lower Vmax with respect to WT. In contrast, the A77T mutant exhibited negative cooperativity with respect to ATP. This result provides further evidence for communication between the two subunits of E. coli BC and insight into the molecular pathogenesis of this mutation.
Cell‐specific ATP7A transport sustains copper‐dependent tyrosinase activity in melanosomes
Authors: Gangi Setty Subba Rao , Tenza Daniele , Sviderskaya Elena V. , Bennett Dorothy C. , Raposo Graca , Marks Michael S.
Published: June 2021

Copper is a cofactor for many cellular enzymes and transporters. To be loaded onto secreted and endomembrane cuproproteins, copper is translocated from the cytosol into membrane‐bound organelles by one of two transporters, ATP7A or ATP7B, the genes for which are mutated in the copper imbalance syndromes, Menkes and Wilson disease, respectively. Endomembrane cuproproteins are thought to stably incorporate copper upon transit through the trans Golgi network (TGN), within which ATP7A and ATP7B accumulate in most cells by dynamic cycling through early endocytic compartments. Here we show that the pigment cell‐specific cuproenzyme tyrosinase acquires copper only transiently within the TGN of melanocytes. To catalyze melanin synthesis, tyrosinase must be subsequently reloaded with copper within specialized pigment organelles called melanosomes. Copper is supplied to melanosomes by ATP7A, a cohort of which localizes to melanosomes in a Biogenesis of Lysosome‐related Organelles Complex‐1 (BLOC‐1)‐dependent manner. These results indicate that cell type‐specific localization of a metal transporter is required to sustain metallation of an endomembrane cuproenzyme, providing a mechanism for exquisite cell type‐specific spatial control of metalloenzyme activity. Moreover, as BLOC‐1 subunits are mutated in subtypes of the genetic disease, Hermansky‐Pudlak syndrome (HPS), these results also show that defects in copper transporter localization contribute to hypopigmentation, and hence perhaps other systemic defects, in HPS.
18beta‐glycyrrhetinic acid prevents free fatty acid‐induced lipotoxicity by inhibiting ER stress and oxidative stress
Authors: Wu Xudong , Zha Weibin , Sun Lixin , Pecora Beth S , Cao Risheng , Gurley Emily C , Studer Elaine J , Hylemon Phillip B , Pandak William M , Zhang Luyong , Zhou Huiping
Published: June 2021

The endoplasmic reticulum (ER) stress signal pathway has been linked to free fatty acid (FFA)‐induced inflammatory response and disease progression of nonalcoholic fatty liver disease in clinic. We have previously shown that 18?β‐glycyrrhetinic acid (GA) prevents FFA‐induced hepatic lipotoxicity by inhibiting cathepsin B expression and activity. But the underlying cellular mechanism remains unclear. This study determined if GA prevents FFA‐induced lipotoxicity by regulating ER stress signaling pathways.

Methods

Mouse macrophages, primary rat hepatocytes and Kupffer cells were used in this study. Cells were treated with palmitic acid (PA) and oleic acid (OA) in the presence or absence of GA. Activation of the UPR was determined by real time RT‐PCR and Western Blot analysis. The apoptosis was detected by fluorescence microscopy using Annexin V‐FITC/propidium iodide. Reactive oxygen species (ROS) was measured using dichlorofluorescein diacetate (DCFH‐DA).

Results

PA dose‐dependently induced UPR activation, ROS formation, and apoptosis both in macrophages and hepatocytes, while OA had no effect. GA not only inhibited PA‐induced UPR activation, but also inhibited PA‐induced ROS formation. PA‐induced ROS formation and cathepsin B expression were inhibited in CHOP knockout macrophages and hepatocytes.

Conclusion

GA prevents FFA‐induced lipotoxicity by inhibiting ER stress and oxidative stress.
Membrane penetration of the FYVE domain is modulated by pH
Authors: He Ju , Vora Mohsin , Filonov Grigory S. , Musselman Catherine A. , Verkhusha Vladislav V. , Stahelin Robert V. , Kutateladze Tatiana G.
Published: June 2021

The FYVE domain (Fab1, YOTB, Vac1, EEA1) binds phosphatidylinositol 3‐phosphate [PtdIns(3)P] in membranes of early endosomes and penetrates bilayers. Here, we investigate the membrane anchoring mechanism by NMR, liposome binding, monolayer surface tension, mutagenesis, and in‐vivo localization experiment. Our results show that the extent of the FYVE domain insertion into PtdIns(3)P‐enriched membranes is substantially enhanced by acidic conditions and contacts with anionic lipids. The EEA1 FYVE domain associates with POPC/POPE/PtdIns(3)P vesicles with a Kd of 49 nM at pH 6.0, however binding is ~~24 fold weaker at pH 8.0. The decrease of the binding affinity is primarily due to much faster dissociation of the FYVE domain from membranes in basic media. The membrane penetration is modulated by the tandem His residues in the RRHHCRXCG signature motif, mutations of which abolish the pH‐sensitivity. The increased membrane association in vitro and in vivo by the Hrs, RUFY1, Vps27p and WDFY1 FYVE domains in a low pH environment demonstrates that pH‐dependency is a general function of the FYVE protein family.

Supported by NIH grants GM071424 and CA113472 (TGK).
Purification and characterization of Drs2p, a P4‐ATPase and potential flippase in yeast
Authors: Zhou Xiaoming , Graham Todd R.
Published: June 2021

Phospholipids are usually asymmetrically distributed across the plasma membranes of eukaryotic cells. Type‐IV P‐type ATPases (P4‐ATPases) appear to be phospholipid translocases, or flippases, and are proposed to play a major role in the establishment and maintenance of membrane asymmetry. Several lines of evidence strongly suggest that Drs2p, a yeast P4‐ATPase, is a flippase with specificity for phosphatidylserine and phosphatidylethanolamine. However, a flippase activity has not been reconstituted with a purified P4‐ATPase. Toward this goal, we report here the purification of Drs2p from the yeast Saccharomyces cerevisiae using the tandem affinity purification (TAP) technique, and characterization of its ATPase activity in vitro. The TAP tag was fused to either the N‐terminus or the C‐terminus of Drs2p (TAP‐Drs2p and Drs2p‐TAP, respectively). About 10 ug of Drs2p were typically recovered from 1L culture containing ~1011 yeast cells, and Drs2p was the major protein in the final eluate. The purified TAP‐Drs2p showed a specific ATPase activity of ~0.4 umol Pi/mg/min. However, no ATP hydrolysis could be detected with Drs2p‐TAP. The ATPase activity of TAP‐Drs2p is Mg2+‐dependent, sensitive to orthovanadate with an IC50 of ~5 uM, and has a KM of ~1.5 mM for ATP and optimal pH of ~7.5. Reconstitution of Drs2p in proteoliposomes to test for flippase activity in vitro is underway. Supported by NIH Grant 5R01GM062367 to T.R.G..
Characterization of Novel PtdIns(4,5)P2 Effector Domains
Authors: Moravcevic Katarina , Lemmon Mark A
Published: June 2021

PtdIns(4,5)P2 plays a major role in a wide variety of cellular activities. The influence of PtdIns(4,5)P2 on the many processes that it regulates is typically mediated by its direct interaction with a variety of different protein domains. Characterization of these PtdIns(4,5)P2 'effector' domains has been central in beginning to understand how PtdIns(4,5)P2 can regulate such a wide range of cellular functions. However, it is clear that the number of currently known candidate PtdIns(4,5)P2 effector domains cannot explain the diversity of PtdIns(4,5)P2 functions. To understand the biochemical basis for the effects of PtdIns(4,5)P2 on additional key cellular processes, we have analyzed candidate novel PtdIns(4,5)P2 effector proteins found in a previous genome‐wide study of potential phosphoinositide‐binding proteins in S. cerevisiae. Using cellular and biochemical approaches, we have discovered several previously‐unappreciated PtdIns(4,5)P2‐binding domains that provide new insight into the biology of this phosphoinositide.
Heme oxygenase‐1 exists as dimers in endoplasmic reticulum
Authors: Hwang Hsuan‐Wen , Lee Jay‐Ron , Chou Kuan‐Yu , Suen Ching‐Shu , Hwang Ming‐Jing , Shieh Ru‐Chi , Chau Lee‐Young
Published: June 2021

Heme oxygenase‐1 (HO‐1) catalyzes the oxidative degradation of heme to biliverdin. Previous studies suggest that HO‐1 exists as a monomer in ER. In the present study, we performed chemical cross‐linking and fluorescence resonance energy transfer (FRET) experiments to show that HO‐1 forms dimers in ER. However, the dimerization was not observed with a truncated HO‐1 which lacks the C‐terminal transmembrane segment (TMS) (aa 266‐285) and exhibits cytosolic and nuclear localization, indicating that the TMS is essential for the self‐assembly of HO‐1 in native membrane. To identify the interface of TMS‐TMS interaction, W270 predicted by molecular modeling with stronger hydrophobic force in the interface was mutated and the effect on protein subcellular localization and dimerization was assessed. The results showed that the W270A mutant was present exclusively in ER and formed dimers as the wild type HO‐1. Interestingly, W270N mutant was localized not only in ER but also in cytosol and nucleus as well, suggesting that it is susceptible to proteolytic cleavage. Moreover, W270N mutation appeared to interfere with dimeric state as revealed by the lower FRET efficiency. Collectively, these data suggest that the dimerization driven by the hydrophobic interaction in the TMS is implicated in the stabilization of HO‐1 in ER. This work was supported by a grant from National Science Council of Taiwan (NSC‐95‐2320‐B‐001‐008).
Structure of an activated G protein‐coupled receptor kinase reveals its receptor‐docking domain
Authors: Tesmer John JG , Singh Puja , Huang Chih‐chin
Published: June 2021

G protein‐coupled receptor (GPCR) kinases (GRKs) play a key role in the desensitization of GPCRs by phosphorylating the cytoplasmic loops and tails of activated receptors. How GRKs recognize and are in turn activated by these receptors is not understood. High resolution crystal structures have been determined for GRK1, GRK2, and GRK6, but in each of these structures the kinase domain assumes an open, inactive conformation, and key structural elements believed to be involved in the recognition of GPCRs are missing or poorly ordered. We have now determined the crystal structure of a GRK bound to a nucleotide substrate analog wherein the kinase domain assumes a closed, catalytically active conformation. In this structure, missing structural elements have coalesced to form a receptor‐docking domain that explains how receptor binding promotes kinase activation. Based on this structure we can model a GRK‐GPCR complex, leading us to propose that GRKs and heterotrimeric G proteins recognize activated receptors via similar mechanisms.

Support for this research was provided by NIH grants HL071818 and HL086865.
The five transmembrane vasoactive intestinal peptide receptor 1 isoform is expressed in human blood CD14+ monocyte cells
Authors: Hermann Rebecca J , Van der Steen Travis , Failing Jarrett , Branch Donald R , Dorsam Glenn
Published: June 2021

Vasoactive intestinal peptide receptor 1 (VPAC1) is a 7 transmembrane (TM) G protein coupled receptor that couples to Gαs and increases intracellular cAMP levels in response to external stimuli. Recently, a novel VPAC1 isoform was identified in human peripheral blood mononuclear cells (PBMC) that lacks exons 10‐12 to form a 5TM receptor that lacks Gαs coupling, but elicits significant tyrosine phosphorylation when treated with exogenous ligand in ectopically overexpressed CHO cells. VPAC1 is a deactivating factor for peripheral blood monocytes by inhibiting the production of many proinflammatory cytokines, but fails to increase intracellular cAMP levels. We hypothesize dual expression of 5 and 7TM VPAC1 may act antagonistically with regard to intracellular cAMP elevation. Uncovering the expression profile of the 5TM VPAC1 splice variant is a necessary first step at understanding the biological role of this novel receptor isoform. To this end, mRNA was collected from CD14+ cells separated from human PBMC by magnetic beads. First strand cDNA was synthesized with anchored primers and the expression profile of the 7TM and 5TM assessed by RT‐PCR, sequence analysis, and Southern hybridization. Our findings suggest preferential 5TM expression in blood monocytes (CD14+). Research supported by NIH‐KO1 1K01DK064828 and COBRE 2P20RR05566.
Investigating the role of cyclic AMP signaling in mushroom development in the spilt gill fungus, Schizophyllum commune
Authors: Hyatt Colette E.L. , Horton J. Stephen
Published: June 2021

The life cycle of S. commune consists of both haploid and dikaryotic phases. In normal circumstances, only the dikaryotic phase is able to produce fruiting bodies or mushrooms. Previous research in other related fungi suggests that a cAMP signalling pathway is likely to be involved in the sexual development of fruiting bodies in S. commune. The present study examines the role of members of the cAMP signaling pathway in the sexual development of this fungus. Specifically, we are studying the roles of protein kinase A and adenylyl cyclase on the formation of mushrooms by performing separate gene knockouts in S. commune. Currently, we are in the process of constructing the necessary recombinant DNA molecules necessary for use in DNA‐mediated transformation experiments. The phenotypic differences between the "knocked out" or null mutant strains generated and the wild type progenitors will be compared in order to gain insight into the role these proteins play in the life cycle of the organism. We have hypothesize that S. commune null mutants for the genes encoding protein kinase A and adenylyl cyclase will likely cause a decrease in mushroom production and/or perturbations in the normal development of these reproductive structures. Supported by a Union College IEF Grant to C.E.H.
Molecular mechanisms underlying transient cGMP signals
Authors: Rich Thomas C , Britain Andrea L , Zhu Bing , Henesy Michelle B
Published: June 2021

We have examined the relationships between natriuretic peptide receptor‐A (NPR‐A) desensitization and phosphodiesterase (PDE) activity in regulating ANP‐induced cGMP signals in a murine Leydig tumor (MA‐10) cells. We have observed that prolonged exposure to ANP leads to an increase and subsequent decrease in cGMP levels. This suggests that following exposure to ANP NPR‐A desensitizes or the rate of cGMP hydrolysis increases with time. We have presented data suggesting that calcineurin underlies the desensitization of NPR‐A in these cells. We have also measured PDE activity and cGMP accumulation in the presence of a variety of type‐specific PDE inhibitors and found that PDE5 is primarily responsible for the hydrolysis of cGMP. In addition, we have observed an ANP‐dependent increase in PDE5 activity. These data suggest that the decrease in cGMP levels is due to both calcineurin‐mediated desensitization of NPR‐A and an ANP‐induced increase in PDE5 activity. To discern the relative roles of desensitization and PDE activity we have developed a quantitative model of the cGMP signaling pathway. Simulations of the model suggest that both calcineurin‐mediated desensitization of NPRA and ANP‐induced stimulation of PDE5 activity are critical processes in the shaping of cGMP signals in MA‐10 cells. Supported by AHA 0715101B, 0335084N, and NIH HL074278.
Bioaccessibility of beta‐carotene in orange fleshed sweet potato
Authors: Thakkar Sagar K. , Kim J. Y. , Failla M. L.
Published: June 2021

Orange fleshed variety of sweet potatoes (OFSP) was recently shown to alleviate vitamin A deficiency in children in Africa. However, the relationship between β‐carotene (BC) content of different cultivars of OFSP and its bioavailability is unknown. Here, we investigated the bioaccessibility of BC from eight cultivars of boiled OFSP containing different amounts of BC using three phase (oral, gastric and small intestinal) in vitro digestion. All trans BC was the only isomer of BC in cultivars of OFSP with amounts ranging from 112‐281 µg/g in tubers. Boiling OFSP decreased all trans BC content by 11% with generation of 13‐cis BC. The efficiency of BC micellarization was only 0.6‐3% during digestion of boiled OFSP. Addition of soybean oil (2% v/w) to boiled OFSP prior to in vitro digestion more than doubled partitioning of all trans BC in the micelle fraction for all cultivars. The relatively poor bioaccessibility of all trans BC was minimally altered for digestion of 0.5‐4 g OFSP or by increasing the amount of bile salts during digestion, suggesting that the food matrix and not the model system was limiting. Moreover, micellarization of all trans BC from boiled fresh OFSP and commercially processed OFSP was significantly less than that from processed carrots handled identically. Examination of physical form and subcellular localization of BC in the tuber is warranted. Supported by HarvestPlus and OARDC.
Impact of d‐δ‐Tocotrienol on Human A2058 and A375 Melanoma Cells
Authors: Fernandes Nicolle V , Mo Huanbiao
Published: June 2021

The rate‐limiting activity of the mevalonate pathway, 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, provides essential intermediates for the prenylation or dolichylation of growth‐related proteins including nuclear lamins, Ras and growth factor receptors. d‐δ‐Tocotrienol, a post‐transcriptional down‐regulator of HMG CoA reductase, suppresses the proliferation of murine B16 melanoma cells and human blood, breast, cervix, colon, liver, lung, lymph gland, nerve, pancreas, and prostate tumor cells. Dietary d‐δ‐tocotrienol suppresses the growth of implanted B16 melanomas without toxicity to host mice. We evaluated d‐δ‐tocotrienol in human A2058 and A375 melanoma cells. d‐δ‐Tocotrienol induced dose‐dependent suppression of the cell proliferation following 72 h incubation in 96‐well plates with IC50 values of 48 (A2058) and 22 (A375) μmol/L, respectively. Morphological changes detected by fluorescence microscopy following acridine orange and ethidium bromide dual staining showed d‐δ‐tocotrienol‐induced apoptosis in A2058 cells. The impact of d‐δ‐tocotrienol on A2058 cell proliferation was potentiated by lovastatin (IC50=3 μmol/L), a competitive inhibitor of HMG CoA reductase. d‐δ‐Tocotrienol may have potential application in melanoma chemoprevention and/or therapy. TDA, TWU REP & Summer Stipend Award.
Higher Dietary Calcium Intake is Associated with Lower Risk for Cervical Intraepithelial Neoplasia in Women Infected with Carcinogenic Human Papillomaviruses
Authors: Thomas Dana‐Marie , Badiga Suguna , Rahman Nuzhat , Neeley Monica R. , Kimball Kristopher J , Frederick Peter J , Piyathilake Chandrika J
Published: June 2021

Persistent infections with carcinogenic or high‐risk human papillomaviruses (HR‐HPVs) are a necessary step in the genesis of higher grades of cervical intraepithelial neoplasia (CIN), precursor lesions for cervical cancer (CC). However, micronutrients are known to modify HR‐HPV associated risk of CIN or CC. There is increasing interest in calcium (Ca) as a cancer‐protective micronutrient for several cancers including cervical. We investigated whether dietary (Ca) intake is an independent risk factor for higher grades of CIN (CIN2+) in 376 HR‐HPV positive women who are free of precancerous CIN (=CIN1) or diagnosed with CIN2+. Demographic and lifestyle data were obtained from risk factor questionnaires. Dietary intake of nutrients and Healthy Eating Index score were obtained from the Block FFQ. Logistic regression analyses tested associations between dietary (Ca) and risk of CIN2+ after adjusting for cancer‐protective micronutrients (folate), (Ca) related minerals/vitamins (magnesium, phosphorous, vit. D) and other factors. Micronutrients were adjusted for total energy intake, and the highest tertile was compared to the middle, and middle to the lowest. Higher dietary (Ca) intake was associated with 37% lower risk of CIN2+ (OR: 0.63, 95% CI: 0.4‐0.9, p=0.03).Other micronutrients were not significant. Smoking and higher parity were associated with higher risk for CIN2+. (Ca) participates in cell proliferation, differentiation, and apoptosis. This study is the first, to our knowledge, to show that dietary (Ca) intake may exert a protective effect against risk for CIN2+ in women with HR‐HPVs.
The blood cobalamin levels were associated with liver injuries and tumour progression: a prognostic indicator in patients with hepatocellular carcinoma
Authors: Kuo Chang Sheng , Lin Ching Yih , Lu Chin Li , Wu Meng Ying , Syu Huang Rwei Fen
Published: June 2021

The aims of the study were to investigate the potential factors to influence serum vitamin B12 levels which may serve as a prognostic indicator in patients with hepatocellular carcinoma (HCC). Ninety patients with HCC and 90 healthy controls provided a blood specimen for B12 analysis. Demographic and disease‐related variables were previously recorded. Compared with the controls, HCC patients had a significantly higher level of mean serum B12 (1355 ± 1365 vs. 717 ± 589 pg/mL, respectively, P = 0.0001). There were significant correlations between serum B12, liver injuries (GOT, GPT and ALT levels, P < 0.05), alpha‐fetal protein levels (r = 0.382, P = 0.001), and tumour size (r = 0.300, P = 0.004). As patients were followed up for at least 30 months or until death, HCC cases in the highest terile of serum B12 levels (mean survival 13.5 months) had a significantly worse survival than those in the second (mean survival 25.0 months) and lowest tertile groups (mean survival 30.7 months; P for trend = 0.001). Thus, the data suggest that elevated blood vitamin B12 levels of HCC patients were associated with liver injuries and tumour progression. High B12 levels predict poor survival in patients with HCC.

The study was supported by the grants (DOH95, 96‐TD‐F‐113‐008) to RF Huang.
Suppressive effects of Schizandra chinensis Baillon water extract on Th2 cytokines expression in IgE‐stimulated mast cell
Authors: Chung Mi Ja , Kim Jeong Mi , Choe Myeon , Kim Dae Jung , Seo Dong Joo , You Jin Kyun
Published: June 2021

The mast cell‐mediated immediate‐type allergic reaction is involved asthma and asthma is a common chronic inflammatory disease regulated by coordination of T‐helper cell type 2 (Th2) cytokines and inflammatory signal molecules. The effect of water extract from Schizandra chinensis Baillon (SCB), known as a traditional folk medicine in Korea, on several cytokines such as interleukin (IL)‐4, IL‐13, tumor necrosis factor‐alpha (TNF‐α), IL‐2 and IL‐3 was investigated in the rat mast cell line (RBL2H3)‐mediated allergy model. Suppressive effects of SCB on Th2 cytokines (IL‐4, IL‐13) was compared with that of digested SCB. The RBL2H3 sensitized with monoclonal anti‐dinitrophenol (DNP) IgE was challenged with the multivalent antigen DNP‐human serum albumin (DNP‐HAS) in the presence or absence of SCB or digested SCB. RT‐PCR and ELISA were performed to measure mRNA and protein expression of cytokines. SCB and digested SCB inhibited the expression of IL‐4 and IL‐13 mRNA and protein in RBL2H3 cells stimulated by IgE‐antigen complex. The result suggested that SCB might suppress the Th2‐mediated response through the down‐regulation of the production of Th2 cytokines.
Real‐time dynamics of pre‐mRNA transcription and splicing
Authors: Carmo‐Fonseca Maria
Published: June 2021

The spliceosome is a dynamic RNA‐protein macromolecular machine that is responsible for the splicing of intronic sequences from pre‐mRNA. The spliceosome undergoes major structural changes during the splicing reaction and its components must be recycled for each new round of splicing. Although the spliceosome cycle has been extensively studied at the molecular level, little is known about the dynamics of splicing and spliceosome assembly in living cells.

We have been using Photobleaching and Photoactivation microscopy techniques to study how splicing factors are recruited to nascent transcripts in the living cell nucleus. Currently, we are developing tools to visualize in real‐time the dynamics of RNA Pol II transcription, splicing and spliceosome assembly.
Effects of Monster Energy drink on cardiovascular and renal function
Authors: Denzer Dustin L , Ragsdale Frances R , Wilson Ted
Published: June 2021

Recent anecdotal reports have linked energy drink consumption with adverse cardiovascular side effects, especially among college‐aged persons. Drinks, such as Monster Energy (ME), contain a wide variety of ingredients, including but not limited to caffeine, whose physiological effects are not completely understood. This study sought to determine if ME was associated with physiologically significant changes. Following an overnight fast, human subjects (age 19.39±1.03 years; 52 female; 23 male) were randomized to ME (2 servings; 480ml; 200 Cal), Isocaloric sugar water (480 ml; 200 Calories), low Cal sugar water (480 ml; 100 Calories), Water (480 ml; 0 calories), or nothing. Heart rate (HR), hematocrit (Hct%), SpO2, blood glucose, urine specific gravity (USG), urine volume (UV), and urine formation rate (UFR) were measured before beverage administration (0‐minutes), then 30‐, 60‐, and 120‐min postprandially. Visual reaction times (VRT) were measured at 0‐ and 60 min. No significant changes in HR, Hct%, SpO2, UV, and UFR were observed within or between groups. Blood glucose did increase significantly for subjects receiving 200 and 100 calorie treatments. USG decreased significantly in all subjects receiving any beverage. VRT improved significantly following ME consumption. This study suggests that ME consumption improves reaction time and was not associated with any adverse effects.
Ability of English and black walnut phenolics to inhibit cupric‐ion induced LDL oxidation in vitro and following human nut consumption
Authors: Wilson Ted , Freeman Michelle R , Olson Rachel M , Rorabaugh Jacki M , Singh Ajay P , Vorsa Nicholi , Maher Margaret A , Fitschen Peter
Published: June 2021

Walnut consumption may reduce the risk of cardiovascular disease, possibly by improving the plasma lipid profile or providing antioxidant protection to low density lipoproteins (LDL). This study sought to compare the phenolic profile and antioxidant activity of English walnut (EW) to its less well known relative, the black walnut (BW). EW and BW samples were extracted in ethanol, methanol, or acetone, prior to analysis using LC‐MS‐MS. EW contained more phenolic compounds (ug/g wet weight) than BW. In vitro studies of LDL oxidation (5uM Cupric ions) demonstrated that EW phenolics extended lag‐time (A234) and reduced TBARS formation, providing significantly better antioxidant protection than BW at 1.0 ug/ml. Furthermore, acetone extracts (1.0 ug/ml) of either EW or BW provided greater antioxidant capacity than paired ethanol extracts. When EW or BW (30 g nuts/day) was administered to humans for 28 days, LDL antioxidant capacity (A234 or TBARS) did not change significantly between groups or within groups between day 0 and 28. This study suggests that EW polyphenolic profile and in vitro antioxidant capacity is better than BW, but that LDL resistance to oxidation does not improve following nut consumption.
Survey of biological active molecule in the Chinese herbal formula‐ Suan Zao Ren Tang in treating insomnia
Authors: Chuang Gloria , Mao Sena , Tam W , Tam G , Tam Chick
Published: June 2021

Objective

Identify biological active molecules found in ancient Chinese herbal formula Suan Zao Ren Tang (SZRT) in treating insomnia.

Methods

Use various sources of textbooks, e.g., Handbook of Active Constituents from Chinese Materia Medica & Phytomedicine (ISBN: 9620731549) by Y. H. Wing and X. P. Gen, peer‐reviewed journals, clinical studies and internet research to identify biological molecules found in the SZRT.

Result

The main herbs of Suan Zao Ren Tang are Sour Jujube Seed or Suan Zao Ren (semen Zizyphi spinosae), Fu‐Ling (sclerotium Poriae cocos) and licorice (Glycyrrhiza glabra). The active molecules found in these herbs for example are triterpene saponins , tumulosic acid and glycyrrhetic acid which are molecules important to treat people suffered from insomnia, restlessness and chronic fatigue without significant side effects of Western medications.

Conclusion

The biologic active molecules found in SZRT can help people suffered from insomnia. The use of SZRT can reduce the cost when compared to the use of Western medicine, and minimize side effects of the prescriptive medication. Thus it could promote overall patients' quality of life.
Impacts of Piglet Age and Route of Delivery on Ileal Lactobacillus Diversity
Authors: Chen Long , Monaco Marcia H , Wang Mei , Donovan Sharon M , Miller Michael J
Published: June 2021

Lactobacilli are inhabitants of the human and porcine gastrointestinal tract. Herein, Lactobacillus diversity in piglets that were either Cesarean ‐ (CD, n = 11) or vaginally delivered (VD, n=9) was characterized. Piglets were weaned on d21 and samples obtained on d3, 14, 21 and 28. Total lactobacilli increased from d3 to 14, were stable until d21 and decreased at d28. Ten random isolates per piglet were identified by partial (499‐bp) hsp60 gene sequencing using Lactobacillus specific primers and 7 species were identified: L. johnsonii, L. thermotolerans, L. reurteri, L. amylovorus, L. delbrueckii, L. salivarius and L. vaginalis. At d3, VD and CD piglets had 3‐5 Lactobacillus species, but the composition differed between the groups. At d14 and 21, L. johnsonii dominated in both CD and VD piglets (>75%). At 28 days, L. johnsonii remained dominant with VD (91%) piglets, whereas CD piglets had both L. johnsonii (72%) and L. thermotolerans (21%). Carbohydrate utilization profiles of 200 Lactobacillus isolates demonstrated intraspecies diversity confirming that several strains may be present for each Lactobacillus species. Terminal restriction fragment length polymorphism patterns (T‐RFLP) generated from ileal contents confirmed the Lactobacillus species composition identified by cultivation. Thus, piglet age and route of delivery impacts the ileal Lactobacillus community. (BMS Freedom to Discover Award).

Grant Funding Source

FSHN
Effect of circulating insulin concentration on whole body and muscle protein metabolism in mature thoroughbred mares
Authors: Urschel Kristine L. , Escobar Jeffery , McCutcheon L. Jill , Geor Raymond J.
Published: June 2021

The role of insulin (INS) in carbohydrate metabolism and the development of certain metabolic diseases has been well studied in horsse; however, there has been minimal research with regards to the role that INS plays in equine protein metabolism. The objective of this study was to determine the effect of circulating INS concentration on whole‐body protein metabolism and the activation of translation initiation factors in muscle. Mature thoroughbred mares (n=8) underwent euglycemic clamp procedures at each of 4 different rates of INS infusion: 0, 1.2, 3 and 6 mU/kg/min. Blood samples were taken during the clamp and at the end of the clamp a biopsy from the gluteal muscle was taken. Western blotting was used to examine the phosphorylation of protein synthesis initiation factors Akt, rpS6 and 4E‐BP1 in the gluteal muscle, and HPLC was used to determine plasma and muscle free amino acid concentrations. INS infusion resulted in a decrease (P < 0.05) in the plasma concentration of all indispensable amino acids, with greater decreases seen with increasing rates of INS infusion. Muscle free valine, leucine and isoleucine concentrations were also lower (P < 0.05) following INS infusion, in a dose dependent manner. Akt phosphorylation was maximized at 6 mU/kg/min INS, while rpS6 and 4E‐BP1 phosphorylation was maximized at 3 mU/kg/min INS. INS infusion appeared to promote both whole‐body amino acid utilization and activation of translation initiation factors in muscle of mature horses. Future research should focus on the effects of altered INS sensitivity on equine protein metabolism. Funded by estate of the late Paul Mellon.
All‐trans retinoic acid up regulates matrix metalloproteinase‐9 activity by murine C57BL/6J myeloid dendritic cells
Authors: Lackey Denise Elizabeth , Hoag Kathleen Ann
Published: June 2021

Myeloid dendritic cells (DC) are specialized cells for antigen presentation. Upon activation in peripheral tissues, DC migrate to lymph nodes to present antigen to and activate T cells. Matrix metalloproteinase (MMP)‐9 is a gelatinase essential for DC migration to sites of infection and inflammation as well as to lymph nodes. We have previously shown that all‐trans retinoic acid (atRA), a bioactive metabolite of vitamin A, is important for release of DC from the surface of the tissue culture dish in vitro and that it significantly augments MMP‐9 mRNA and protein production. In the current studies, we investigated the effects of atRA on MMP‐9 activity related to DC adhesion in vitro. Mouse bone marrow cells cultured under myeloid DC generating conditions in the presence of a retinoic acid receptor antagonist demonstrated decreased gelatinase activity compared to cells rescued with atRA treatment for the last 48 hours of the culture period. DC cultured in the presence of atRA and a specific MMP‐9 inhibitor showed significant increase in the adherent cell yield and percentage of total cells and DC that are adherent in a dose dependent manner compared to cultures treated with atRA only for the full culture period. These data suggest that atRA regulates DC adhesion in vitro through MMP‐9 activity.

Grant Funding Source

Department of Food Science and Human Nutrition, Michigan State University
Inhibition of adhesion molecules expression by allicin in TNF‐α‐ and LPS‐treated MOVAS via downregulation of NF‐κB
Authors: Kim Ji‐Hye , Jung Da‐Hye , Sohn Eun‐Hwa , Pyo Suhneung
Published: June 2021

Allicin is a precursor of a number of secondary products formed in garlic preparations. Since the upregulation of adhesion molecules in response to numerous inducing factors is associated with inflammation, interfering with the expression of adhesion molecules may be an important therapeutic target of inflammatory diseases. We examined the effect of allicin on the expression of adhesion molecules induced by TNF‐α or LPS in cultured mouse vascular smooth muscle cells (MOVAS) by ELISA, Western blot and Luciferase analysis, respectively. MOVAS were pretreated for 20 h with allicin (0.01‐1 μg/ml) and then exposed to TNF‐α or LPS for 4 and 8 h to measure the expression of E‐selectin, VCAM‐1 and ICAM‐1, respectively. Allicin significantly inhibited surface expression of adhesion molecules in a dose dependent manner. Specifically, the most significant reduction was observed in ICAM‐1 expression. In addition, allicin treatment resulted in the decrease of NF‐κB activation, whereas phosphorylation of AKT and ERK induced by LPS but not TNF‐α was inhibited. These results suggest that allicin downregulates TNF‐α‐ or LPS‐induced adhesion molecules expression via inhibition of NF‐κB activity and LPS‐mediated induction of adhesion molecules by inhibiting AKT and ERK signaling. Moreover, the data indicate that allicin may be considered in therapeutic strategies for the management of vascular inflammatory diseases
Effects of quercetin on skeletal muscle mitochondrial biogenesis and performance in untrained males
Authors: Williams Ashley Silberman , Nieman David C. , Jin Fuxia , Sobieraj James , Shanely Andrew , McAnulty Steve R. , Triplett N. Travis , Austin Melanie
Published: June 2021

We determined the influence of 2‐weeks quercetin supplementation (1000 mg/day) compared to placebo on exercise performance and markers of skeletal muscle mitochondrial biogenesis in 26 untrained, physically inactive, young adult males. Utilizing a randomized, crossover design with a 2‐week washout period, subjects provided blood and muscle biopsy samples pre‐ and post‐supplementation periods, and were given 12‐minute time trials on 15% graded treadmills following 60‐min moderate exercise pre‐loads at 60% VO2max. Plasma quercetin levels rose significantly during the two‐week quercetin compared to placebo supplementation period. During the 12‐minute trial (15% treadmill grade, self‐selected speed), the net change in distance achieved was significantly greater with quercetin compared to placebo (29.5±11.5 vs. ‐11.9±16.0 m, respectively, P=0.038). Skeletal muscle mRNA expression tended to increase during supplementation with quercetin compared to placebo for SIRT‐1 (P‐value=0.152), PGC‐1α (P=0.192), cytochrome c oxidase (P=0.081), and citrate synthase (P=0.166). In summary, 1000 mg/d quercetin supplementation for 2 weeks by untrained males was associated with an improvement in 12‐minute treadmill time trial performance and 16% to 25% increases above placebo levels for mRNA expression for four genes related to mitochondrial biogenesis.

Supported by a Grant from Quercegen Pharma
Dietary Habits, Media Usage, and Physical Activity in Preschoolers Born to Overweight Mothers
Authors: Bowman Anne Eyre , Lovelady Cheryl , Ostbye Truls
Published: June 2021

The purpose of this study was to describe dietary habits, media use, and physical activity in 2 to 5‐year‐olds born to overweight (BMI = 25) mothers. Data were collected on 174 preschoolers, of whom 72.8% were white, 23.1% African‐American, and 4.1% other races. Mothers reported the time their preschooler spent watching TV and videos and using the computer, the number of servings of certain food groups eaten, and the duration of outdoor play. Preschoolers spent 3.4 ± 1.0 hours watching TV and videos and 1.4 ± 0.7 hours using the computer each day. They spent 4.4 ± 3.1 hours each week playing outdoors. In bivariate analyses, there was a positive relationship between hours of TV watched and servings of soda, sweetened beverages, fruit juice, fast food, and French fries; and a negative relationship between hours of TV and milk servings. Time spent using the computer was positively related to sweetened beverage and fast food intake. Outdoor play was positively related to fruit and sweetened beverage intake, but not to soda, fruit juice, fast food, or French fries. Media use and physical activity were not related to yogurt, dessert, or vegetable consumption. Results of this exploratory analysis suggest that there is a relationship between increased media use by preschoolers and a higher intake of less healthy foods. Higher levels of physical activity are related to increased sweetened beverage intake. Supported by R01‐DK075439.
The prevalence of natural and fortified calcium food sources in the self‐selected diets of adolescent girls
Authors: Rafferty Karen Ann , Lappe Joan M
Published: June 2021

Fortification of foods with calcium may provide an opportunity for improving dietary calcium intake; however, quantifying the nutritional impact of calcium fortified foods in the U.S. population remains difficult. This analysis reports on the penetration of calcium from natural and fortified food sources in the self‐selected diets of teenage girls participating in two randomized controlled trials at our research center from 2002 ‐ 2005. One‐hundred‐forty‐nine girls were randomized to either a high calcium group (HC), instructed to consume 1500 mg Ca/d from dairy and/or fortified foods (mean 1494 mg Ca/d), or to a control group instructed to consume their usual calcium (UC) diet (mean 765 mg Ca/d.) Dairy foods contributed 69% of the total calcium in the HC group and calcium fortified foods contributed the calcium equivalent of 250 g milk (300 mg Ca). Compared with the nutrient profile of milk, the calcium‐fortified food profile contributed 192% the calories, 362% the carbohydrate, and 200% the sugar of milk, with 62% less protein, 50% less phosphorus, 40% less potassium, 35% less vitamin A, and 12% less vitamin D. In the UC group, dairy foods contributed 70% of the total calcium with breakfast cereals, the top calcium fortified food, providing only 2.5% of total daily calcium. In this cohort of teenage girls following their usual diet, calcium‐fortified foods had little impact on overall calcium nutrition.
The impact of vegetable intake on food diversity and dietary adequacy of obese adolescent girls
Authors: Yoon Jin‐Sook , Lee Nan‐Jo , Ju Hyun‐Ok , Jang , Heekyung
Published: June 2021

Adolescence is the critical period of hormonal changes and rapid growth which demands balanced intake of nutrients. This study was conducted to investigate the relationship between obesity and dietary patterns in adolescent girls, mainly focusing on the impact of vegetable intake level on improving the quality of their diet. The subjects were 306 female high school students (normal 180, obese 126) from low income families. Dietary intakes were measured by 24‐hour recall method. We classified the subjects into high and low vegetable intake group from their vegetable intake data. The extent of dietary adequacy was evaluated by the estimated average requirement (EAR) and nutrient adequacy ratio (NAR). Food diversity was compared by dietary variety score (DVS). Average intake of Ca, Folate, vitamin C and riboflavin were below the level of EAR in all subjects. Higher risk of nutritional inadequacy was observed among obese girls compared to normal girls. Significantly higher intake of meat and fish group and higher DVS were observed for subjects with high vegetable intake. It appeared that adolescent female students with high vegetable intake had better dietary quality and nutritional status. These results suggest that acquiring sufficient intake of vegetables would be the most essential step in improving the nutritional adequacy of obese adolescent girls. Supported by KRF # ‐2007‐531‐C00065.
Examine the Over Time Shifts of Under‐ to Overnutrition Problems In India
Authors: Wang Youfa , Chen Hsin‐Jen , Shaikh Saijuddin , Mathur Prashant
Published: June 2021

It is speculated obesity has increased in India in recent years thank to economic development and to follow trends observed in many other courtiers. Based on a systematic literature search on PubMed and other data sources, we examined the trends in overweight, obesity and undernutrition in recent decades in India. Most published studies were based on regional or local surveys in urban areas, while good representative data from the India National Family Health Surveys (NFHS, 1992‐93, 1998‐99, and 2005‐06) allowed for examining the shifts at national level. Overall, available data showed that in India, prevalence of overweight was low while that of undernutrition remained high, in children and adults. Overweight was more prevalent in female, urban, and high‐socioeconomic status (SES) groups. NFHS showed that overweight rates in women and preschool children did not increase much in the last decade: 10.6% and 1.6% in 1998‐99 to 12.6% and 1.5% in 2005‐06, respectively. As for underweight, NFHS 2005‐06 showed high prevalence among ever married women (about 35%) and preschool children (about 42%). Thus, India's nationwide overweight/obesity prevalence has been relatively stable in past decade, but in some urban‐ and high‐SES groups it has increased and reached a high level. Factors associated with undernutrition need closer examination; prevention of obesity should be targeted at the high‐risk groups simultaneously.

This study is supported by Johns Hopkins University.