bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning, Expression and Characterization of Novel Aldo‐Keto Reductase 1B (AKR1B) Proteins ‐ Human AKR1B11 and Murine Akr1b16
Authors: Salabei Joshua Kuimeta , Bhatnagar Aruni , Barski Oleg A 
Published: June 2021

The Aldo‐Keto reductase (AKR) superfamily comprises enzymes that catalyze redox transformations. A novel gene locus, tcag7.1260 (AKR1B11) has been predicted to cluster with human members of the AKR1B subfamily, AKR1B1 and AKR1B10. AKR1B11 is predicted to have 10 exons and an open reading frame having 91% and 67% identities with AKR1B10 and AKR1B1 respectively. Cross genome comparison of human and murine AKRs reveals the existence of a murine AKR gene, 2310005E10Rik (Akr1b16), having an open reading frame with 82.9% identity with human AKR1B10 protein. We test the hypothesis that AKR1B11 and Akr1b16 are expressed into functional proteins. Using whole human/mouse tissue mRNA, we demonstrated the existence of spliced AKR1B11 and Akr1b16 mRNAs in human brain and testes, and in multiple murine tissues. cDNAs were cloned into pET28a and pIRES‐hrGFP‐1α vectors for bacterial and mammalian expression respectively. Both genes were expressed as 37 kDA proteins. COS 7 cells transfected with pIRES‐hr‐GFP‐1α/AKR1B11 showed a 4.8 fold (with 500μM p‐NB) and 3.3 fold (with 10mM DL‐glyceraldehyde) increase in enzymatic activity. We raised specific antibody against AKR1B11. Immunoprecipitation and Western blot showed the presence of AKR1B11 protein in the ovary, testes and kidney. AKR1B11 protein was also detected in human ovary sections by immunohistochemistry. We conclude that AKR1B11 is present in human tissues. Further confirmation of its activity in these and other tissues is needed. Protein activity measurements with a broad variety of AKR substrates are required for the characterization of AKR1B11 and Akr1b16.Research support: COBRE: P20RR024489
Molecular and functional characterization of a new tumor suppressor protein ZNF509 in the regulation of cell growth
Authors: Jeon Bu‐Nam , Yoon Jae‐Hyeon , Hur Man‐Wook 
Published: June 2021

A variety of cellular biological functions like cell proliferation are precisely controlled by a number of protein‐protein interactions among regulatory proteins. In cancer cells, aberrant expression of oncogenes or/and tumor suppressor genes causes dramatic changes in the cellular regulatory program governing cell proliferation. Recently, POK family transcription factors were characterized as critical oncogenes or tumor suppressors that could regulate expression of cell cycle control genes.We found that a novel POK family transcription factor, ZNF509, is induced by p53 and may be a tumor suppressor. ZNF509 is a potent transcriptional activator of CDKN1A encoding cell cycle arrest gene, p21. EMSA, oligo pull‐down, and ChIP assays showed that ZNF509 directly binds to proximal promoter region of p21 in vitro and in vivo. Also ZNF509 and MIZ‐1 interact with each other and synergistically activate transcription of p21 gene. The molecular interaction recruits more co‐activator p300 to the p21 proximal promoter. The ZNF509‐MIZ‐1‐p300 complex acetylates histone Ac‐H3 and ‐H4 at the p21 proximal promoter. Knockdown of endogenous ZNF509 expression results in decrease in the transcription of p21 gene, which increases cell proliferation and DNA synthesis. Overall, our data suggest that ZNF509 is a potent transcription activator of the cell cycle arrest gene p21 and may be a tumor suppressor gene.
Small molecule inhibitors of fatty acid uptake in 3T3‐L1, primary human adipocytes, INS‐1E and C2C12 cell lines
Authors: Jesch Elliot Denver , Wang Zhigang , Black Paul N , DiRusso Concetta C 
Published: June 2021

Previous work in this lab identified 5 chemically distinct small molecules that inhibit the uptake of fatty acid (C1‐BODIPY‐C12) through a series of screenings in yeast expressinghomo sapiens fatty acid transport protein (FATP) 2 and Caco2 cells. Subsequently, we looked to examine the effectiveness and toxicity of each compound in a variety of cells that accumulate lipid. We examined two adipocyte cell lines (mouse 3T3‐L1 and primary human), one pancreatic beta cell line (INS‐1E) and one skeletal muscle cell line (mouse C2C12). Cells were seeded into 96 well (black wall/optically clear bottom) plates and differentiated or allowed to incubate until firmly attached. Cells were subsequently starved for one hour in serum free MEM, one hour incubation with a dilution series of each compound (1nM‐1mM, range dependent on individual cell line), a 5–15 minute incubation with the fluorescent fatty acid C1‐BODIPY‐C12 and finally measured fluorescence intensity using 485/20 nm excitation and 530/20 nm emission filters. Sigmoid dose‐response curves for C1‐BODIPY‐C12uptake were generated in each cell line and EC50 values were from the low nM to high μM range, depending on the particular compound and cell line. The compounds were determined to benon‐toxic to each cell line by utilizing the MTT cytotoxicity assay. Specificity to fatty acid uptake was confirmed by measuring acyl‐CoA synthestase activity and glucose uptake.[Supported by NIH DK071076]
Role of the transcription factor Opi1p and phosphatidic acid in the formation of lipid droplets in Saccharomyces cerevisiae
Authors: Chang Yu‐Fang , Henry Susan A 
Published: June 2021

Phosphatidic acid (PA) is a signaling molecule that plays an important role in mediating yeast phospholipid biosynthesis. In cells grown in the absence of inositol, the transcriptional repressor Opi1p is localized to the endoplasmic reticulum (ER) through interactions with Scs2p via its FFAT motif (two phenylalanines in an acidic tract) and the binding with PA. Under these conditions, the UASINO‐containing phospholipid biosynthetic genes are expressed at the maximum level through activation via the Ino2p‐Ino4p activation complex. When inositol is added to the medium, the increased synthesis of phosphatidylinositol causes a rapid depletion of PA in the ER. Opi1p then translocates to the nucleus and represses the transcription of target genes (Loewen et al. 2004. Science 304, 1644–1647). Here we present studies utilizing Opi1p reporter constructs to monitor the change in PA metabolism. After the cells grew under inositol starvation for 2 hrs, novel punctate structures were recognized by Opi1p‐GFP in the FFAT and scs2 null mutants. Interestingly, these structures are highly associated with ER. Furthermore, by using Tgl4p‐RFP as a lipid droplet marker to identify these structures, we observed colocalization of Tgl4p‐RFP with the punctate structures. These results suggest a role for both Opi1p and PA in the formation of lipid droplets.Supported by NIH grant GM019629.
Yeast diacylglycerol kinase is essential for the resumption of growth from stationary phase
Authors: Fakas Stylianos , Carman George 
Published: June 2021

The Saccharomyces cerevisiae DGK1 gene encodes a novel diacylglycerol (DAG) kinase that catalyzes the formation of phosphatidate (PA) from DAG. This enzyme is unique in that it uses CTP, instead of ATP, as the phosphate donor. DGK1 is a major regulator of PA levels in yeast and its overexpression causes PA overproduction and a nuclear membrane expansion by inducing the formation of PA‐enriched membranes around the nucleus. In this work, we discovered that DGK1 is essential for the resumption of growth from stationary phase when de novo fatty acid synthesis is blocked with cerulenin. Lipid analysis showed that under these conditions wild type cells mobilized triacylglycerol (TAG) reserves to release fatty acids needed for phospholipid synthesis and growth, but dgk1Δ cells were unable to mobilize TAG. Supplementation with choline or ethanolamine resulted in the synthesis of phospholipids, the hydrolysis of TAG, and suppression of the growth inhibition phenotype of dgk1Δ cells.Supported by NIH grant GM‐28140.
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
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).
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.ConclusionG. 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.
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
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
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
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

IntroductionDifferent studies have shown that nutritional pathology induces depression of the immune system.ObjectiveIn this preliminary study we measured Adenosine Deaminase‐enzyme related with lymphocytes T, and complement fractions (C3, C4), in patients suffering diabetes (DTB).MethodsTwenty 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.ConclusionsThese 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